Compound for detecting amyloid-beta oligomer and method for diagnosing degenerative brain disease using same

Abstract

The present invention relates to a novel compound for detecting amyloid-beta and a method for diagnosing degenerative brain disease using same, the compound represented by chemical formula 1 or a salt thereof, according to the present invention, being capable of specifically binding to amyloid-beta. The compound or a salt thereof has high selectivity and thus may detect amyloid-beta in plasma with high sensitivity despite interference in the detection due to the presence of other proteins in plasma, and may be utilized for diagnosing degenerative brain disease or screening a therapeutic agent for degenerative brain disease.

Description

Compounds for detecting amyloid-beta oligomers and methods for diagnosing degenerative brain diseases using the same

[0001] The present invention relates to a novel compound for detecting amyloid-beta oligomers and a method for diagnosing degenerative brain diseases using the same.

[0002]

[0003] Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by the deposition of amyloid beta (Aβ) peptides in the brain. Aβ peptides are produced by enzymatic cleavage of amyloid precursor protein, and the produced peptides aggregate into oligomers and plaques in AD patients. Although there is no clear correlation between Aβ plaque burden and cognitive dysfunction, Aβ oligomers are known to be involved in the development of AD symptoms. Furthermore, Aβ oligomers induce neurotoxicity, neuronal cell death, and synaptic dysfunction in the brains of AD patients. Therefore, the detection of Aβ oligomers is essential for the development of AD therapeutics and diagnostic methods.

[0004] Considering that the major Aβ species in plasma are soluble Aβ oligomers, detecting plasma Aβ can be utilized to monitor changes in Aβ oligomer levels and predict the onset of AD. Moreover, blood Aβ testing can be a noninvasive and cost-effective method for detecting Aβ oligomers in clinical trials, which may be a beneficial approach for AD patients. However, accurate measurement of Aβ oligomer levels in the bloodstream has long been problematic in terms of the accuracy and sensitivity of plasma Aβ detection. The controversy over accurate plasma Aβ measurement is known to be due to the heterogeneous and polymorphic properties of Aβ oligomers. Specifically, unlike Aβ plaques with a β-sheet-rich structure, Aβ oligomers are unstable intermediates with diverse shapes, structures, and compositions. The transient and polymorphic nature of Aβ oligomers can mask Aβ oligomer-targeting compounds and create steric hindrance, resulting in low diagnostic accuracy and sensitivity. Consequently, there is an urgent need to develop novel imaging agents that selectively target Aβ oligomers.

[0005] Accordingly, the inventors of the present invention seek to provide a compound that not only has the effect of significantly improving detection efficiency by interacting with Aβ aggregates, but also has the effect of maintaining the detection efficiency without decreasing for a modified complex form of Aβ.

[0006]

[0007] One aspect is to provide a compound represented by the following chemical formula 1 or a salt thereof:

[0008] [Chemical Formula 1]

[0009] .

[0010] Another aspect is to provide a method for preparing a compound represented by the above chemical formula 1.

[0011] Another aspect is to provide a composition for detecting amyloid-beta (Aβ) comprising a compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0012] Another aspect provides a method for detecting amyloid-beta (Aβ), comprising the step of contacting a separated biological sample with a compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0013] Another aspect is to provide a composition for diagnosing a degenerative brain disease comprising a compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0014] Another aspect provides a method for providing information regarding the diagnosis of a degenerative brain disease, comprising the step of contacting a separated biological sample with a compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0015] Another aspect provides a method for diagnosing a degenerative brain disease, comprising administering to a biological sample a compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0016]

[0017] One aspect is to provide a compound represented by the following chemical formula 1 or a salt thereof:

[0018] [Chemical Formula 1]

[0019]

[0020] In the above chemical formula 1,

[0021] The above R 1a and R 1b are each independently hydrogen, deuterium, halogen, amino, hydroxy, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C2-10 Alkenyl, substituted or unsubstituted C 2-10 Alkynyl, substituted or unsubstituted C 1-10 an alkoxy, a substituted or unsubstituted 6- to 10-membered aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl;

[0022] The above R 1c and R 1d are each independently hydrogen, deuterium, halogen, amino, hydroxy, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 2-10 Alkenyl, substituted or unsubstituted C 2-10 Alkynyl, substituted or unsubstituted C 1-10 an alkoxy, a substituted or unsubstituted 6- to 10-membered aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl,

[0023] The above R 1c and R 1d are linked to each other to form a compound represented by the following chemical formula 2,

[0024] [Chemical Formula 2]

[0025]

[0026] In the above chemical formula 2, the X 1 , X 2 , X 3 and X 4 are each independently C(R 4 ), nitrogen, oxygen, sulfur or phosphorus,

[0027] The above R 4 is hydrogen, deuterium, halogen, amino, hydroxy, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 2-10 Alkenyl, substituted or unsubstituted C 2-10 Alkynyl, substituted or unsubstituted C 1-10 an alkoxy, a substituted or unsubstituted 6- to 10-membered aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl;

[0028] The above R 2a and R 2b are each independently hydrogen, deuterium, halogen, amino, hydroxy, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 2-10 Alkenyl, substituted or unsubstituted C 2-10 Alkynyl, substituted or unsubstituted C 1-10 an alkoxy, a substituted or unsubstituted 6- to 10-membered aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl;

[0029] The above R 3a and R 3b are each independently hydrogen, deuterium, halogen, amino, hydroxy, -CN, -C(O)NH2, -C(O)O(C 1-10 alkyl), substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted 6- to 10-membered aryl, or substituted or unsubstituted 5- to 10-membered heteroaryl.

[0030] In one specific example, the R 1a and R 1b are each independently hydrogen, deuterium or halogen;

[0031] The above R 1c and R 1d are each independently hydrogen, deuterium, halogen, amino, hydroxy, substituted or unsubstituted C 1-5 Alkyl, substituted or unsubstituted C 2-5 Alkenyl, substituted or unsubstituted C 2-5 Alkynyl, substituted or unsubstituted C 1-5 an alkoxy, a substituted or unsubstituted 6-membered aryl, or a substituted or unsubstituted 6-membered heteroaryl,

[0032] The above R 1c and R 1d are linked to each other to form a compound represented by the chemical formula 2,

[0033] Above X1 , X 2 , X 3 and X 4 are each independently C(R 4 ), nitrogen, oxygen, sulfur or phosphorus,

[0034] The above R 4 is hydrogen, deuterium or halogen;

[0035] The above R 2a and R 2b are each independently hydrogen, deuterium, halogen, substituted or unsubstituted C 1-5 Alkyl, substituted or unsubstituted C 2-5 Alkenyl, substituted or unsubstituted C 2-5 an alkynyl, a substituted or unsubstituted 6- to 10-membered aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl;

[0036] The above R 3a and R 3b are each independently hydrogen, deuterium, halogen, amino, hydroxy, -CN, -C(O)NH2, -C(O)O(C 1-5 alkyl), substituted or unsubstituted C 1-5 It may be an alkyl or a substituted or unsubstituted 6- to 10-membered aryl.

[0037] In another specific example, the R 1a and R 1b are each independently hydrogen or halogen;

[0038] The above R 1c and R 1d are each independently hydrogen, halogen, substituted or unsubstituted C 1-5 Alkyl, substituted or unsubstituted C 1-5 or alkoxy or a substituted or unsubstituted 6-membered aryl,

[0039] The above R 1c and R 1d are linked to each other to form a compound represented by the chemical formula 2,

[0040] Above X1 , X 2 , X 3 and X 4 are each independently C(R 4 ) and,

[0041] The above R 4 is hydrogen;

[0042] The above R 2a and R 2b are each independently hydrogen, halogen, substituted or unsubstituted C 1-5 alkyl, substituted or unsubstituted 6- to 10-membered aryl, or substituted or unsubstituted 5- to 6-membered heteroaryl;

[0043] The above R 3a and R 3b are each independently hydrogen, -CN, -C(O)NH2, -C(O)O(C 1-5 alkyl), substituted or unsubstituted C 1-5 It may be an alkyl or a substituted or unsubstituted 6-membered aryl.

[0044] The above term "alkyl" means a functional group in which one hydrogen atom is removed from one carbon atom in a saturated hydrocarbon group connected by a single bond between carbon atoms. The above alkyl is a functional group having 1 to 20 carbon atoms (C 1-20 ), 1 to 10 carbon atoms (C 1-10 ) or 1 to 5 carbon atoms (C 1-5 ) may be a straight-chain or chain hydrocarbon. For example, it may be methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, etc.

[0045] The above term "Cm - n" (where m and n are each independently integers greater than or equal to 1) means having m to n carbon atoms.

[0046] The term "alkenyl" refers to a functional group in which one hydrogen atom is removed from one carbon atom in an unsaturated hydrocarbon group, including one or more double bonds between carbon atoms. The alkenyl group has 2 to 20 carbon atoms (C 2-20 ), 2 to 10 carbon atoms (C 2-10 ) or 2 to 5 carbon atoms (C 2-5 ) may be a straight or chain hydrocarbon. For example, it may be vinyl, propenyl, butenyl, etc.

[0047] The term "alkynyl" refers to a functional group in which one hydrogen atom is removed from one carbon atom in an unsaturated hydrocarbon group, including one or more triple bonds between carbon atoms. The alkynyl group has 2 to 20 carbon atoms (C 2-20 ), 2 to 10 carbon atoms (C 2-10 ) or 2 to 5 carbon atoms (C 2-5 ) may be a straight or chain hydrocarbon. For example, it may be ethynyl, propynyl, butinyl, etc.

[0048] The term "alkoxy" above means alkyl (-O-alkyl) bonded to oxygen. The alkoxy above has 2 to 20 carbon atoms (C 2-20 ), 2 to 10 carbon atoms (C 2-10 ) or 2 to 5 carbon atoms (C 2-5 ) may include, for example, methoxy, ethoxy, propoxy, butoxy, etc.

[0049] The term "aryl" above refers to a functional group in which one hydrogen atom is removed from one carbon atom in a parent aromatic hydrocarbon. The aryl may be a 5- to 18-membered, 5- to 14-membered, 5- to 10-membered, or 6- to 10-membered group, depending on the number of atoms constituting the ring. For example, it may be phenyl, biphenyl, naphthalenyl, fluorenyl, indenyl, indanyl, etc.

[0050] The term "heteroaryl" above refers to an aryl in which one or more carbon atoms constituting the aromatic ring are replaced with an atom selected from the group consisting of nitrogen, oxygen, and sulfur. The heteroaryl may be a 5- to 18-membered, 5- to 14-membered, or 5- to 10-membered group, depending on the number of atoms constituting the ring. For example, it may be thiopheneyl, pyrrolyl, pyrazolyl, pyridinyl, imidazolyl, triazolyl, oxazolyl, etc.

[0051] The above alkyl, alkenyl, alkynyl, alkoxy, aryl, or heteroaryl may each be substituted or unsubstituted. The term "substituted" means that one or more hydrogens attached to carbon are replaced by another atom or functional group, and "unsubstituted" means that all hydrogens attached to carbon are not replaced by another atom or functional group. Examples of other atoms that can be substituted include fluorine, chlorine, bromine or iodine, and examples of functional groups that can be substituted include hydroxyl, thiol, nitro, oxo, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted cycloalkoxy, substituted or unsubstituted alkylthio, substituted or unsubstituted aryloxy, substituted or unsubstituted arylthio, substituted or unsubstituted heteroaryloxy or substituted or unsubstituted heteroarylthio.

[0052] Examples of cases where the above alkyl is substituted include trifluoromethyl (-CF3) or (2-(2-(2-fluoroethoxy)ethoxy)ethoxy)methyl (2-(2-(2-fluoroethoxy)ethoxy)ethoxy)methyl; -CH2OC2H4OC2H4OC2H5F).

[0053] Examples of cases where the above aryl is substituted include methoxy-phenyl, fluoro-phenyl, chloro-phenyl, or tolyl.

[0054] In one specific example, the compound represented by the chemical formula 1 may be a compound represented by one chemical formula selected from the group consisting of chemical formulas 3 to 43:

[0055] [Chemical Formula 3]

[0056] ,

[0057] [Chemical Formula 4]

[0058] ,

[0059] [Chemical Formula 5]

[0060] ,

[0061] [Chemical Formula 6]

[0062] ,

[0063] [Chemical Formula 7]

[0064] ,

[0065] [Chemical Formula 8]

[0066] ,

[0067] [Chemical Formula 9]

[0068] ,

[0069] [Chemical Formula 10]

[0070] ,

[0071] [Chemical Formula 11]

[0072] ,

[0073] [Chemical Formula 12]

[0074] ,

[0075] [Chemical Formula 13]

[0076] ,

[0077] [Chemical Formula 14]

[0078] ,

[0079] [Chemical Formula 15]

[0080] ,

[0081] [Chemical Formula 16]

[0082] ,

[0083] [Chemical Formula 17]

[0084] ,

[0085] [Chemical Formula 18]

[0086] ,

[0087] [Chemical Formula 19]

[0088] ,

[0089] [Chemical Formula 20]

[0090] ,

[0091] [Chemical Formula 21]

[0092] ,

[0093] [Chemical Formula 22]

[0094] ,

[0095] [Chemical Formula 23]

[0096] ,

[0097] [Chemical Formula 24]

[0098] ,

[0099] [Chemical Formula 25]

[0100] ,

[0101] [Chemical Formula 26]

[0102] ,

[0103] [Chemical Formula 27]

[0104] ,

[0105] [Chemical Formula 28]

[0106] ,

[0107] [Chemical Formula 29]

[0108] ,

[0109] [Chemical Formula 30]

[0110] ,

[0111] [Chemical Formula 31]

[0112] ,

[0113] [Chemical Formula 32]

[0114] ,

[0115] [Chemical Formula 33]

[0116] ,

[0117] [Chemical Formula 34]

[0118] ,

[0119] [Chemical Formula 35]

[0120] ,

[0121] [Chemical Formula 36]

[0122] ,

[0123] [Chemical Formula 37]

[0124] ,

[0125] [Chemical Formula 38]

[0126] ,

[0127] [Chemical Formula 39]

[0128] ,

[0129] [Chemical Formula 40]

[0130] ,

[0131] [Chemical Formula 41]

[0132] ,

[0133] [Chemical Formula 42]

[0134] and

[0135] [Chemical Formula 43]

[0136] .

[0137] The above salt may be an acid addition salt formed by a free acid. When the compound is used for the diagnosis of a disease, it may be in the form of a pharmaceutically acceptable salt or a diagnostically acceptable salt. The term pharmaceutically or diagnostically acceptable salt refers to any organic or inorganic addition salt of a base compound of an active substance at a concentration that is relatively non-toxic and harmless to the patient and has an effective effect, and the side effects caused by the salt do not diminish the beneficial effects of the base compound of the active substance. Inorganic acids and organic acids can be used as free acids forming acid addition salts. Inorganic acids include hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, perchloric acid, phosphoric acid, etc., and organic acids include citric acid, acetic acid, lactic acid, maleic acid, fumaric acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, tartaric acid, galacturonic acid, embonic acid, glutamic acid, aspartic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethanesulfonic acid, 4-toluenesulfonic acid, salicylic acid, citric acid, benzoic acid, or malonic acid. In addition, the salts include alkali metal salts (sodium salts, potassium salts, etc.) and alkaline earth metal salts (calcium salts, magnesium salts, etc.).For example, acid addition salts include acetate, aspartate, benzoate, besylate, bicarbonate / carbonate, bisulfate / sulfate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hybenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulfate, naphthylate, 2-naphthylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogen phosphate / dihydrogen phosphate, saccharate, stearate, succinate, It may include tartrate, tosylate, trifluoroacetate, aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, zinc salts, and the like, and may be specifically hydrochloride or trifluoroacetate.

[0138] The above acid addition salt can be prepared by a conventional method, for example, by dissolving the active substance in an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile, etc., adding an organic acid or inorganic acid, filtering and drying the resulting precipitate, or by distilling the solvent and an excess of acid under reduced pressure and then drying or crystallizing the same in an organic solvent.

[0139] Additionally, metal salts can be prepared using bases. Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving a compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering out the undissolved compound salt, and evaporating and drying the filtrate. In this case, sodium, potassium, or calcium salts are pharmaceutically suitable as metal salts. Furthermore, the corresponding silver salts are obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

[0140] Furthermore, the compound represented by the above chemical formula 1 or its salt may include all solvates, hydrates, isomers, etc. that can be prepared therefrom.

[0141] The term "isomer" above refers to compounds that have the same chemical formula or molecular formula but are structurally or stereochemically different. Examples of the 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).

[0142]

[0143] Another aspect is as shown in the following reaction scheme 1:

[0144] A step of obtaining compound 1003 by reacting compound 1001 and compound 1002 in an organic solvent (step 1);

[0145] A step of reacting compound 1003 and NH4OAc in an organic solvent to obtain compound 1004 (step 2);

[0146] A step of obtaining compound 1006 by reacting compound 1005-1, compound 1005-2 or compound 1005-3; and compound 1004 in an organic solvent (step 3); and

[0147] A method for producing a compound represented by the following chemical formula 1 is provided, which comprises a step (step 4) of reacting compound 1006 and a catalyst in an organic solvent to obtain a compound (compound 1007) represented by the following chemical formula 1:

[0148] [Reaction Formula 1]

[0149]

[0150] In the above reaction formula 1,

[0151] The above R is R 1c or halogen;

[0152] Above X p and X q are each independently hydrogen, deuterium or halogen;

[0153] The above R 1a and R 1b are each independently hydrogen, deuterium, halogen, amino, hydroxy, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 2-10 Alkenyl, substituted or unsubstituted C 2-10 Alkynyl, substituted or unsubstituted C 1-10 an alkoxy, a substituted or unsubstituted 6- to 10-membered aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl;

[0154] The above R 1c and R 1d are each independently hydrogen, deuterium, halogen, amino, hydroxy, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 2-10 Alkenyl, substituted or unsubstituted C 2-10 Alkynyl, substituted or unsubstituted C 1-10 an alkoxy, a substituted or unsubstituted 6- to 10-membered aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl,

[0155] The above R 1c and R 1dare linked to each other to form a compound represented by the following chemical formula 2,

[0156] [Chemical Formula 2]

[0157]

[0158] In the above chemical formula 2, the X 1 , X 2 , X 3 and X 4 are each independently C(R 4 ), nitrogen, oxygen, sulfur or phosphorus,

[0159] The above R 4 is hydrogen, deuterium, halogen, amino, hydroxy, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 2-10 Alkenyl, substituted or unsubstituted C 2-10 Alkynyl, substituted or unsubstituted C 1-10 an alkoxy, a substituted or unsubstituted 6- to 10-membered aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl;

[0160] The above R 2a and R 2b are each independently hydrogen, deuterium, halogen, amino, hydroxy, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 2-10 Alkenyl, substituted or unsubstituted C 2-10 Alkynyl, substituted or unsubstituted C 1-10 an alkoxy, a substituted or unsubstituted 6- to 10-membered aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl;

[0161] The above R 3a and R 3b are each independently hydrogen, deuterium, halogen, amino, hydroxy, -CN, -C(O)NH2, -C(O)O(C 1-10 alkyl), substituted or unsubstituted C 1-10Alkyl, substituted or unsubstituted 6- to 10-membered aryl, or substituted or unsubstituted 5- to 10-membered heteroaryl.

[0162] The above terms “alkyl”, “alkoxy”, “aryl”, “heteroaryl”, “salt”, etc. may be within the above-mentioned range.

[0163] In one specific example, the manufacturing method may further include a step of reacting the compound of Chemical Formula 1 with sulfuric acid after step 4. For example, a reaction such as the following reaction scheme 2 may be performed:

[0164] [Reaction Formula 2]

[0165] .

[0166] In one specific example, the manufacturing method may further include a step of reacting the intermediate produced after reacting compound 1004 and compound 1005-2 in the organic solvent in step 3 and before obtaining compound 1006, with an oxidizing agent. For example, a reaction such as the following reaction scheme 3 may be performed:

[0167] [Reaction Formula 3]

[0168] .

[0169] In one specific example, the manufacturing method may further include a step of reacting the intermediate produced after reacting compound 1004 and compound 1005-3 in the organic solvent in step 3 and before obtaining compound 1006, with an oxidizing agent. For example, a reaction such as the following reaction scheme 4 may be performed:

[0170] [Reaction Formula 4]

[0171] .

[0172] In one specific example, the oxidizing agent may be at least one selected from the group consisting of Pyridinium chlorochromate (PCC), Pyridinium dichromate (PDC), Dess-Martin periodinane (DMP), 2-Iodoxybenzoic acid (IBX), Swern oxidizing agent, and Parikh-Doering.

[0173] In one specific example, when R is halogen, the manufacturing method may further include a step of reacting the intermediate produced after reacting compound 1006 and the catalyst in an organic solvent in step 4, and before obtaining the compound represented by chemical formula 1, with a Stille coupling starting material. Specifically, the Stille coupling starting material may be (Tributylstannyl)methanol.

[0174] In one specific example, when R is a halogen, after the step of reacting with the Stille coupling starting material, R 1c (OTs) may further include a step of reacting with. For example, a reaction such as the following reaction scheme 5 may be performed:

[0175] [Reaction Formula 5]

[0176] .

[0177] In one specific example, the organic solvents of steps 1 to 4 may be independently one or more selected from the group consisting of methanol, ethanol, propanol, acetonitrile, DCE (Dichloroethane), ethyl acetate, dimethyl sulfoxide, and dimethyl formamide.

[0178] In one specific example, the catalyst of step 4 may be at least one selected from the group consisting of trifluoromethanesulfonic acid (TfOH), methanesulfonic acid (MsOH), p-toluenesulfonic acid (p-TsOH), and scandium triflate (Sc(OTf)3), but is not limited thereto.

[0179] Another aspect is to provide a composition for detecting amyloid-beta (Aβ) comprising a compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0180] The above term "amyloid-beta" or "amyloid-beta protein" refers to a protein or fragment thereof that is capable of forming amyloid deposits by entangling and aggregating together in the form of a beta plate structure. In the present invention, amyloid-beta protein may refer to an individual protein that constitutes an amyloid-beta protein aggregate.

[0181] In one specific example, the amyloid-beta may include an oligomer, a protofibril, a fibril, a plaque, a protein monomer associated with a homologous protein, and an aggregate, which are aggregates of the amyloid-beta protein. Specifically, the amyloid-beta may be at least one selected from the group consisting of an Aβ oligomer, an Aβ protofibril, an Aβ fibril, and an Aβ plaque. More specifically, the amyloid-beta may be an Aβ oligomer.

[0182] In one specific example, the amyloid-beta may be mutated.

[0183] In one specific example, the compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof may interact with one or more domains selected from the group consisting of a domain comprising an amino acid sequence of SEQ ID NO: 1, a domain comprising an amino acid sequence of SEQ ID NO: 2, and a domain comprising an amino acid sequence of SEQ ID NO: 3.

[0184] The above “domain” refers to amyloid beta (Aβ) 1-42 ) refers to the amino acid sequence or structural site to which a specific compound binds within the amino acid sequence encoding a protein.

[0185] Specifically, the above “domain comprising an amino acid sequence of sequence number n (wherein n is 1, 2 or 3)” may further comprise 0 to 5 (0, 1, 2, 3, 4 or 5) amino acid sequences from the N-terminal amino acid and / or C-terminal amino acid sequence of the above amino acid.

[0186] For example, in the case of a domain including the amino acid sequence of SEQ ID NO: 1, it may be a domain consisting of SEQ ID NO: 1, a domain consisting of the amino acid sequence of SEQ ID NO: 1 and a sequence in which 1 to 5 amino acids are further added to the N-terminal position of the amino acid of SEQ ID NO: 1, a domain consisting of the amino acid sequence of SEQ ID NO: 1 and a sequence in which 1 to 5 amino acids are further added to the C-terminal position of the amino acid of SEQ ID NO: 1, or a domain consisting of the amino acid sequence of SEQ ID NO: 1 and a sequence in which 1 to 5 amino acids are further added to the N-terminal and C-terminal positions of the amino acid of SEQ ID NO: 1.

[0187] If it is a domain including the amino acid sequence of the above sequence number 2, it may be a domain consisting of the above sequence number 2 or a domain consisting of the amino acid sequence of the above sequence number 2 and a sequence in which 1 to 5 amino acids are added to the N-terminal position of the amino acid of the above sequence number 2.

[0188] Furthermore, in the case of a domain including the amino acid sequence of the above sequence number 3, it may be a domain consisting of the above sequence number 3 or a domain consisting of the amino acid sequence of the above sequence number 3 and a sequence in which 1 to 5 amino acids are further added to the C-terminal position of the amino acid of the above sequence number 3.

[0189] The amino acid sequences of the above sequence numbers 1 to 3 are as follows:

[0190] Sequence number 1: FFAEDVG, Sequence number 2: MVGGVVIA Sequence number 3: DAEFRHDS.

[0191] In one specific example, the compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof comprises a domain consisting of an amino acid sequence having a sequence identity of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% with the amino acid sequence of SEQ ID NO: 1, a domain consisting of an amino acid sequence having a sequence identity of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% with the amino acid sequence of SEQ ID NO: 2, and a domain consisting of an amino acid sequence having a sequence identity of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% with the amino acid sequence of SEQ ID NO: 3. It may interact with one or more domains selected from the group consisting of domains consisting of amino acid sequences having a sequence identity of at least about 92%, at least about 95%, at least about 97%, at least about 98%, or at least about 99%.

[0192] In one specific example, the compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof may interact with one or more domains selected from the group consisting of a domain consisting of an amino acid sequence of SEQ ID NO: 1, a domain consisting of an amino acid sequence of SEQ ID NO: 2, and a domain consisting of an amino acid sequence of SEQ ID NO: 3.

[0193] The above detection composition may detect amyloid-beta by combining a compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof with an indicator substance. The indicator substance may be for measuring the degree of binding between amyloid-beta and the compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof, and an example of the indicator substance may be a fluorescent substance or a fluorescent protein fragment.

[0194] The above fluorescent material may be a fluorescent dye having a basic skeleton of rhodamine, coumarin, EvoBlue, oxazine, carbopyronine, naphthalene, biphenyl, anthracene, phenanthrene, pyrene or carbazole, or a derivative of the above fluorescent dye. Specifically, the fluorescent material is Fluorescein, CR110: Carboxyrhodamine 110: Rhodamine Green (trade name), TAMRA: Carboxytetramethylrhodamine: TMR, Carboxyrhodamine 6G: CR6G, BODIPY FL (trade name): 4,4-Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, BODIPY 493 / 503 (trade name): 4,4-Difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-propionic acid, BODIPY R6G (trade name): 4,4-Difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, BODIPY 558 / 568 (trade name): 4,4-Difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, BODIPY 564 / 570 (trade name): 4,4-Difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, BODIPY 576 / 589 (trade name): 4,4-Difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, BODIPY 581 / 591 (trade name): 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, EvoBlue10 (trade name), EvoBlue30 (trade name), MR121, ATTO 655 (trade name), ATTO 680 (trade name), ATTO 700 (trade name), ATTO MB2 (trade name), Alexa Fluor 350 (trade name), Alexa Fluor405 (trade name), Alexa Fluor 430 (trade name), Alexa Fluor 488 (trade name), Alexa Fluor 532 (trade name), Alexa Fluor546 (trade name), Alexa Fluor 555 (trade name),Alexa Fluor 568 (trade name), Alexa Fluor 594 (trade name), Alexa Fluor633 (trade name), Alexa Fluor 680 (trade name), Alexa Fluor 700 (trade name), Alexa Fluor 750 (trade name), Alexa Fluor790 (trade name), Flamma 496 (trade name), Flamma 507 (trade name), Flamma 530 (trade name), Flamma 552 (trade name), Flamma 560 (trade name), Flamma 575 (trade name), Flamma 581 (trade name), Flamma 648 (trade name), Flamma 675 (trade name), Flamma749 (trade name), Flamma 774 (trade name), Flamma 775 (trade name), Rhodamine Red-X (trade name), Texas Red-X (trade name), 5(6)-TAMRA-X (trade name), 5TAMRA (trade name), Indocyanine green (ICG) and 2-((E)-2-((E)-2-(4-(2-carboxyethyl)phenoxy)-3-((E)-2-(3,3-dimethyl-5-sulfonato-1-(3-(tri-methyl ammonio)-propyl)indolin-2-ylidene)ethylidene)cyclohex-1-enyl)vinyl)-3,3-dimethyl-1-(3-(trimethyl ammonio)-propyl)-3H-indolium-5-sulfonate disodium bromide (ZW800-1) may be selected from the group consisting of

[0195] The above fluorescent protein fragments include venus, Cerulean, Citrine, and mKate, and may be fluorescent proteins with different colors or parts thereof.

[0196] In one specific example, the detection composition may be used to determine whether amyloid-beta protein is detected from a sample isolated from an individual, and the isolated sample may be a biologically isolated sample.

[0197] The subject may be a mammal, for example a mouse, a human, a pig, a cow, a horse, or a sheep, and may be specifically a human, and more specifically a human suspected of having a disease mediated by amyloid-beta accumulation.

[0198] The above biologically isolated sample may be isolated from a tissue in which amyloid-beta is distributed, and may be specifically one or more selected from the group consisting of blood, plasma, cerebrospinal fluid, brain tissue lysate, cortical lysate, and hippocampal lysate.

[0199]

[0200] Another aspect provides a method for detecting amyloid-beta (Aβ), comprising the step of contacting a separated biological sample with a compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0201] The above amyloid-beta (Aβ) detection method may further include a step of confirming the presence of amyloid-beta in a biological sample treated with the compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof, or quantifying the amount of amyloid-beta.

[0202] The step of confirming the presence or quantifying the amount of amyloid-beta may include a process of confirming the binding of the compound represented by the chemical formula 1 or a pharmaceutically acceptable salt thereof to the amyloid-beta, and the confirmation of the binding may be performed by measuring the emission spectrum emitted by the compound represented by the chemical formula 1 or a pharmaceutically acceptable salt thereof and the amyloid-beta that are bound. Specifically, when an excitation source is irradiated into a biological sample treated with the compound represented by the chemical formula 1 or a pharmaceutically acceptable salt thereof, the presence or quantification of the amount of amyloid-beta may be performed by measuring the fluorescence generated from the compound represented by the chemical formula 1 or a salt thereof to which the amyloid-beta is bound.

[0203] In one specific example, the measurement of the fluorescence signal may be performed using a microplate reader, a spectrophotometer, a microscope-based fluorescence detector, or a flow cytometer. Additionally, the measurement may be performed using, but is not limited to, a commercial or publicly known fluorescence detection system.

[0204] The above terms “pharmaceutically acceptable salt” and “amyloid-beta” are within the scope described above.

[0205]

[0206] Another aspect is to provide a composition for diagnosing a degenerative brain disease comprising a compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0207] The term "degenerative brain disease" above refers to any disease associated with degenerative changes in the brain, particularly any brain disease that can be caused by factors such as the aggregation or accumulation of amyloid-beta in the brain and / or neuronal cells. Furthermore, prodromal symptoms that are not generally recognized as diseases are also included in the amyloid-beta-related diseases. Examples of the above degenerative brain diseases include mild cognitive impairment, dementia, Alzheimer's disease, hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D), preclinical alzheimer's disease, Parkinson's disease, Huntington's disease, mild cognitive impairment, mild cognitive impairment due to Alzheimer's disease, cerebral amyloid angiopathy, Down syndrome, tauopathy, amyloid stroke, Lou Gehrig's disease, systemic amyloid disease, Dutch amyloidosis, Niemann-Pick disease, senile dementia, amyotrophic lateral sclerosis, It may be a disease selected from the group consisting of spinocerebellar atrophy, Tourette's syndrome, Friedrich's ataxia, Machado-Joseph's disease, Lewy body dementia, dystonia, progressive supranuclear palsy, and frontotemporal dementia.

[0208] The term "Alzheimer's disease" is used interchangeably with senile dementia and refers to a disease involving mental deterioration associated with a specific degenerative brain disorder characterized by senile plaques, neuroinflammatory tangles, and progressive neuronal loss.

[0209] The term "Parkinson's disease" refers to a chronic and progressive degenerative disease of the central nervous system, often characterized by symptoms that impair motor function and speech ability.

[0210] The above term "Huntington's disease" refers to a neurodegenerative disease caused by a triplet repeat expansion in the gene encoding the Huntington protein, which is accompanied by chorea, mental disorder, dementia, etc.

[0211] The above term "Lou Gehrig's disease" refers to a disease in which only motor neurons are selectively killed, and both the upper motor neurons of the cerebral cortex and the lower motor neurons of the brainstem and spinal cord are gradually destroyed.

[0212] The above term "Niemann-Pick disease" refers to a disease that exhibits symptoms indicating progressive destruction of nerve cells in the brain.

[0213] The above term "tauopathy" may refer to a neurodegenerative disease in which brain nerves are damaged due to abnormal accumulation of tau protein (a family of closely related intracellular microtubule-associated proteins) in brain tissue.

[0214] The above-mentioned degenerative brain disease may be characterized by a higher amyloid-beta level in a group of disease-carrying individuals compared to a group of disease-free individuals. Specifically, the amyloid-beta may be at least one selected from the group consisting of Aβ oligomers, Aβ protofibrils, Aβ fibrils, and Aβ plaques. More specifically, the amyloid-beta may be an Aβ oligomer. In addition, the high amyloid-beta level may be present in at least one selected from the group consisting of plasma, blood, cerebrospinal fluid, cortex, and hippocampus.

[0215] The above terms “pharmaceutically acceptable salt”, “amyloid-beta”, etc. are within the scope mentioned above.

[0216]

[0217] Another aspect provides a method for diagnosing a degenerative brain disease, comprising the step of contacting a biological sample with a compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0218] The above terms “pharmaceutically acceptable salt” and “degenerative brain disease” are within the above-mentioned scope.

[0219]

[0220] Another aspect provides a method for providing information regarding the diagnosis of an amyloid-beta related disease, for example, a degenerative brain disease, comprising the step of contacting a separated biological sample with a compound represented by the above formula 1 or a pharmaceutically acceptable salt thereof.

[0221] The method for providing the above information may include a step of confirming the presence of amyloid-beta in a biological sample contacted with a compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof, or quantifying the amount of amyloid-beta.

[0222] In addition, the method for providing the above information may further include a step of comparing the quantified amount of amyloid-beta with the amount of amyloid-beta in a biological sample isolated from an individual who does not have the degenerative brain disease (hereinafter, a normal control group), and, if the amount of the quantified amyloid-beta is greater than the amount of amyloid-beta in the normal control group, the method may further include a step of determining that the individual has the degenerative brain disease.

[0223] The above terms “pharmaceutically acceptable salt”, “amyloid-beta related disease”, “degenerative brain disease” and “amyloid-beta” are within the scope mentioned above.

[0224]

[0225] Another aspect provides a method for screening for an amyloid-beta protein aggregation inhibitor or aggregation-dissolving agent, comprising the step of contacting a sample containing amyloid-beta protein with a compound represented by chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0226] The sample containing the amyloid-beta protein may be, for example, plasma, blood, cerebrospinal fluid, cortical lysate, or hippocampal lysate derived from a degenerative brain disease model animal, may be cultured cells derived from a degenerative brain disease model animal, or may be a solution containing purified amyloid-beta protein.

[0227] The above screening method can be used to screen for a therapeutic agent for the amyloid-beta related disease or a therapeutic agent for a degenerative brain disease. For example, the method can be used by administering a test substance and a compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof to an Alzheimer's disease model animal (in vivo); or treating a sample derived from an Alzheimer's disease model animal (ex vivo) or a test tube containing amyloid-beta protein (in vitro) with a test substance and a compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof, quantifying the amount of amyloid-beta protein, and selecting the test substance as a disease treatment agent if the experimental group administered or treated with a specific test substance has a lower amyloid-beta quantitative value than the control group that was not administered or treated.

[0228] The above terms “amyloid-beta” and “pharmaceutically acceptable salt” are within the scope described above.

[0229]

[0230] The compound represented by chemical formula 1 according to the present invention or a salt thereof can specifically bind to amyloid-beta (Aβ). The compound or a pharmaceutically acceptable salt thereof can detect amyloid-beta in plasma with high sensitivity due to high selectivity despite detection interference caused by the presence of other proteins in plasma, and can be utilized for the diagnosis of degenerative brain diseases or screening for therapeutic agents for degenerative brain diseases.

[0231]

[0232] Figures 1A to 1F are diagrams measuring the fluorescence intensity according to the binding of compounds and Aβ monomers (mAβ), Aβ oligomers (oAβ), or Aβ fibrils (fAβ) according to one aspect (Figures 1A and 1B: IK15 compound; Figures 1C to 1F: AMD compound).

[0233] Figures 2A and 2B are diagrams showing the fluorescence intensity over time after binding of a compound to an Aβ monomer (mAβ), an Aβ oligomer (oAβ), or an Aβ fibril (fAβ) according to one aspect.

[0234] Figure 3A is a diagram showing the fluorescence intensity measured according to the binding of compounds and Aβ monomers (mAβ), Aβ oligomers (oAβ), and Aβ fibrils (fAβ) according to one aspect.

[0235] Figure 3B is a diagram showing the fluorescence intensity over time after binding of a compound to Aβ monomer (mAβ), Aβ oligomer (oAβ), or Aβ fibril (fAβ) according to one aspect.

[0236] Figure 3C is a diagram confirming the binding of a compound and Aβ oligomers (oAβ) according to one aspect.

[0237] Figure 3D is a diagram confirming the Aβ binding site of the compound according to the aspect.

[0238] Figure 3E shows the compound and tau aggregates or Aβ oligomers (Aβ) according to one aspect. 1-42 ) is a diagram that confirms the combination of

[0239] Figure 3F is a schematic diagram of an experiment to identify the Aβ oligomer targeting site of a compound according to one aspect.

[0240] Figures 3G to 3J are diagrams showing the binding affinity of compounds according to one aspect for Aβ fragments or Aβ complexes.

[0241] Figure 4A is a schematic diagram of an experiment to confirm the Aβ detection efficacy of a compound according to one aspect in a 5XFAD transgenic mouse model.

[0242] Figure 4B is a diagram confirming whether Aβ is detected in the cerebral cortex lysate of the 5XFAD transgenic mouse model of the IK15j compound according to one aspect.

[0243] Figure 4C is a diagram confirming whether Aβ is detected in the hippocampal lysate of the 5XFAD transgenic mouse model of the IK15j compound according to one aspect.

[0244] Figure 4D is a diagram confirming whether Aβ is detected in the cerebrospinal fluid (CSF) of the 5XFAD transgenic mouse model of the IK15j compound according to one aspect.

[0245] Figure 4E is a diagram confirming whether Aβ is detected in the plasma of a 5XFAD transgenic mouse model of the IK15j compound according to one aspect.

[0246] Figure 4F is a schematic diagram showing the brain regions of mice subjected to immunofluorescence with compounds and anti-Aβ antibodies according to one aspect.

[0247] Figure 4G shows immunofluorescence analysis performed on the brain of a 5XFAD transgenic mouse model with compounds and anti-Aβ antibodies according to one aspect.

[0248] Figures 5A and 5B are diagrams showing whether Aβ is detected in the cerebral cortex lysate of a 5XFAD transgenic mouse model of a compound according to one aspect.

[0249] Figures 6A and 6B are diagrams showing whether Aβ is detected in the hippocampal lysate of a 5XFAD transgenic mouse model of a compound according to one aspect.

[0250] Figures 7A and 7B are diagrams confirming whether Aβ is detected in the cerebrospinal fluid (CSF) of a 5XFAD transgenic mouse model of a compound according to one aspect.

[0251] Figure 8 is a diagram confirming whether Aβ is detected in the plasma of a 5XFAD transgenic mouse model of a compound according to one aspect.

[0252] Figure 9A is a diagram confirming the sensitivity of a compound to Aβ oligomers according to one aspect.

[0253] Figure 9B is a diagram confirming the binding of a compound to fibrinogen or albumin according to one aspect.

[0254] Figure 9C is a diagram confirming the binding of a compound to Aβ oligomers in the presence of albumin according to one aspect.

[0255] Figure 9D is a schematic diagram of an experiment to confirm the binding of a compound to Aβ oligomers in the presence of albumin according to one aspect.

[0256] Figure 9E is a diagram confirming the binding of a compound to Aβ oligomers in the presence of albumin according to one aspect.

[0257]

[0258] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following examples.

[0259]

[0260] <Reference example>

[0261] <Reference Example 1> Fluorescence spectrum scanning

[0262] A microplate reader (SpectraMax M5, Molecular devices) was used to perform fluorescence spectral scans of IK15. For excitation scans, compounds of the present invention were dissolved in DMSO to prepare 1 mM stocks, which were then serially diluted with deionized water. Then, 150 μL of sample (100 μM) was aliquoted into a 96-well clear round-bottom plate, and data points of the compound excitation spectra were measured from 200 nm to 900 nm in 5 nm increments. The peak of each compound was recorded as the excitation wavelength. For emission scans, 150 μL of the same sample prepared above was transferred to a 96-well opaque round-bottom plate. The excitation wavelength was set according to the compound in question, and emission was scanned in 5 nm increments across various spectral ranges. Next, the emission spectra of the candidate compounds mixed with Aβ were required to determine whether these compounds exhibited fluorescence shifts in the presence of Aβ.

[0263] <Reference Example 2> Manufacturing of MAP

[0264] Aβ 1-42 fragments or Aβ 1-42 A mapping amyloid plate (MAP) for investigating the target site of the compound of the present invention in an oligomer was prepared with reference to Korean Patent Application No. 10-2023-0133625, the entire contents of which are incorporated herein by reference.

[0265] Briefly, each well of the plate was washed three times with 200 μL of wash buffer (0.1 M sodium phosphate, 0.15 M sodium chloride, 0.05% Tween-20, pH 7.2) to remove the bovine serum albumin coated on the plate. Aβ 1-42Full-length and fragmented peptides were individually dissolved in DMSO to prepare 1.0 M stock solutions, which were then diluted with binding buffer (0.1 M sodium phosphate, 0.15 M sodium chloride, 10 mM EDTA, pH 7.2) to prepare 50 μg / mL peptide solutions (5% DMSO). 100 μL of the peptide solution was added to each well and reacted with maleimide overnight at room temperature. After peptide immobilization, unbound peptides were washed three times with 200 μL of wash buffer. To inactivate the residual maleimide groups, 200 μL of cysteine ​​solution (10 μg / mL in binding buffer) was added to each well and incubated for 1 h at room temperature. After cysteine ​​capping, all wells of the plate were washed three times with 200 μL of wash buffer.

[0266] <Reference Example 3> Aβ 1-42 Investigation of the targeting site of IK15 for oligomers

[0267] To determine whether the compound of the present invention targets the structure or sequence of Aβ oligomers, two conditions were prepared. First, the compound of the present invention (50 μM) was prepared in binding buffer (10% DMSO), and 100 μL of the compound was added to MAP and treated at room temperature for 24 hours. After incubation, all wells were washed three times with 200 μL of washing buffer. Second, full-length Aβ 1-42 Peptides were added to MAP and incubated at 37°C for 8 hours to obtain full-length Aβ 1-42 -Fragmented Aβ 1-42 Complexes were formed. After incubation, all wells were washed three times with 200 μL of wash buffer, and 100 μL of the compound of the present invention (50 μM) was treated in MAP at room temperature for 24 hours. After treatment, all wells were washed three times with 200 μL of wash buffer. The fluorescence intensities of both plates were measured using a microplate reader (λex = 285 nm / λem = 475 nm).

[0268] <Reference Example 4> Animals

[0269] To investigate the interaction between the compound of the present invention and Aβ oligomers in brain lysate, cerebrospinal fluid (CSF), and plasma of an AD mouse model, 6-month-old female 5XFAD transgenic mice (Strain name: B6SJL-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas / -Mmjax expressing the Swedish (K670N / M671L), Florida (I716V), and London (V717I) mutations of APP and the M146L and L286V mutations of PSEN1) and wild-type mice (C57BL / 6 x SJL) were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). All animals were handled in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. The study protocol was approved by the Yonsei University Institutional Animal Care and Use Committee (IACUC-202103-1221-01, Seoul, Republic of Korea). All animal studies followed the ARRIVE reporting guidelines.

[0270] <Reference Example 5> Brain lysate analysis

[0271] To prepare brain lysates, mice were sacrificed, and the hippocampus and cortex regions of the mouse brain were dissected separately. Each brain region was homogenized in ice-cold RIPA buffer (20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.5% NP-40, 4 mM EDTA, 0.1% SDS, 0.5% sodium deoxycholate) containing 1X protease inhibitor cocktail (Roche Diagnostics, Switzerland). The homogenized brain tissue was incubated on ice for 20 min, then centrifuged at 14,000 rpm at 4°C for 30 min, and the supernatant (soluble fraction) of the brain lysate was collected. To analyze the interaction between the compound of the present invention and the mouse brain lysate sample, diluted brain lysate samples were mixed with the compound of the present invention (50 μM). Lysate samples containing compounds were loaded into wells of a 96-well half-area black microplate (Corning, USA). Samples were distributed and detected at λex = 285 nm / λem = 475 nm.

[0272] <Reference Example 6> Cerebrospinal fluid (CSF) analysis

[0273] To analyze the interaction between the compounds of the present invention and mouse CSF, sacrificed mouse CSF samples were used. Each CSF sample was treated with the compounds of the present invention (50 μM). Individual samples and the compounds of the present invention were added to wells of a 96-well half-field black microplate. The total volume of each sample was 50 μL. The signal of each sample was detected using a microplate reader (λex = 285 nm / λem = 475 nm).

[0274] <Reference Example 7> Plasma analysis

[0275] To analyze the interaction between the compounds of the present invention and mouse plasma, sacrificed mouse plasma samples were used. Individual samples and the compounds of the present invention were added to wells of a 96-well, half-field black microplate. The total volume of each sample was 50 μL. The signal of each sample was detected using a microplate reader (λex = 285 nm / λem = 475 nm).

[0276] <Reference Example 8> Brain staining of AD mouse model

[0277] Mouse brain tissues were fixed in 4% paraformaldehyde (Biosesang, Korea) overnight at 4°C and then cryoprotected in 30% sucrose for 48 hours. Brain sections (25 μm) were cut with a cryostat (CM1860, Leica) and attached to slides. The brain tissues were treated with the compound of the present invention (500 μM) overnight at room temperature. After washing with 1X PBS, antigen retrieval for the fixed brain sections was performed with 1% SDS (Biosesang, Korea) in 1X PBS (Gibco, Korea) for 10 minutes, followed by blocking with 20% horse serum in 1X PBS. After 1 hour, the slides were incubated with mouse monoclonal antibody 6E10 (1:200, BioLegend, USA) for 1 hour at room temperature. After washing with 1X PBS, the slides were incubated with goat anti-mouse IgG conjugated with Alexa Fluor 555 (1:200, Invitrogen, USA) for 1 hour at room temperature. As a local marker, brain slides were stained with Hoechst 33342 (10 μg / mL in PBS) for 3 minutes at room temperature. After staining the cortex and hippocampal regions of the fixed brains, each brain slide was visualized under a fluorescence microscope (DM500, Leica) with filter cubes containing excitation and emission filters: an N2.1 filter cube (excitation filter: BP 515-560; dichromatic mirror: 580; emission filter: LP 590) for detection of 6E10 staining and an L5 filter cube (excitation filter: BP 480 / 40; dichromatic mirror: 505; emission filter: BP 527 / 30) for detection of the compounds of the present invention.

[0278] <Reference Example 9> Detection of plasma Aβ oligomers

[0279] Aβ 1-42Peptides (1000, 100, 10, 1, 0.1, 0.01, 0.001 pg / mL) were aggregated at 37°C for 8 hours and added to human plasma buffer (Sigma-Aldrich, USA). Then, the compound of the present invention (100 μM) was treated with each plasma sample at room temperature for 8 hours. The signal of each sample was detected with a microplate reader (λex = 285 nm / λem = 475 nm).

[0280] <Reference Example 10> Investigation of IK15 selectivity for blood proteins

[0281] Aβ 1-42 The peptide was aggregated at 37°C for 8 hours and 25 μM Aβ 1-42 Oligomers, albumin (Sigma-Aldrich, USA), and fibrinogen (Sigma-Aldrich, USA) were seeded in black microplates. IK15j (100 μM) was treated with each plasma sample for 8 h at room temperature. The signal of each sample was detected using a microplate reader (λex = 285 nm / λem = 475 nm).

[0282] <Reference Example 11> Investigation of IK15 selectivity for albumin-Aβ oligomer complexes

[0283] Full-length Aβ in maleimide-activated microplates 1-42 The peptide was fixed. Then, additional Aβ 1-42The peptide (10 μM) was treated at 37°C for 8 hours. After peptide immobilization, unbound peptide was washed three times with 200 μL of washing buffer. To form albumin-Aβ oligomer complexes, albumin (10 μM, Sigma-Aldrich, USA) was treated in the plate at room temperature for 24 hours, and the unbound residue was washed three times with 200 μL of washing buffer. The Aβ oligomer wells, which served as the control, were not treated with albumin. The compound of the present invention (100 μM) was treated to each well, and the signal of each sample was detected with a microplate reader (λex = 285 nm / λem = 475 nm).

[0284] <Reference Example 12> Statistical Analysis

[0285] All graphical data were analyzed using GraphPad Prism 9.0 software, and statistical analysis was performed using one-way ANOVA followed by Bonferroni post hoc comparisons or Student's unpaired t-test. Error bars represent the standard error of the mean (SEM).

[0286]

[0287] <Manufacturing Example>

[0288] <Manufacturing Example 1> Manufacturing of Examples 1 to 13

[0289] Examples for compounds of one aspect (hereinafter, Examples 1 to 13) were synthesized according to the following reaction scheme 6.

[0290] [Reaction Formula 6]

[0291]

[0292] Compound 2001 was obtained by treating various commercially available α-bromoketones with NaCN, and then β was subjected to base-mediated alkylation with 2-chloroacetone to obtain 1,4-diketo compound 2002, which was then converted to the corresponding 2-arylpyrrole compound 2003 by treatment with NH4OAc. Subsequently, compound 2004 was generated by N-alkylation of various α-haloketones and 2-arylpyrrole compound 13. Subsequently, under optimized conditions, compound 2004 was synthesized into pyrrolo[2,1-a]isoquinoline compound 2005, which is a variety of N-substituted 2-arylpyrrole derivative compound 2004. By changing the α-bromoketone or the α-haloketone, 13 exemplary compounds (Example 1 (a compound represented by the chemical formula 3; IK15a), Example 2 (a compound represented by the chemical formula 4; IK15b), Example 3 (a compound represented by the chemical formula 5; IK15c), Example 4 (a compound represented by the chemical formula 6; IK15d), Example 5 (a compound represented by the chemical formula 7; IK15e), Example 6 (a compound represented by the chemical formula 8; IK15f), Example 7 (a compound represented by the chemical formula 9; IK15l), Example 8 (a compound represented by the chemical formula 10; IK15n), Example 9 (a compound represented by the chemical formula 11; IK15o), Example 10 (a compound represented by the chemical formula 12; IK15p), Example 11 (a compound represented by the chemical formula 13; IK15q), Example 12 (a compound represented by the chemical formula A compound represented by chemical formula 14; IK15r), Example 13 (a compound represented by chemical formula 15; IK15s)) was prepared.

[0293] For the compounds of the above synthesized examples 1 to 13, H-NMR (400 MHz, CDCl3) and C-NMR (100 MHz, CdCl3) were performed according to known NMR experimental methods to analyze the structure of the material, and the results are as follows:

[0294] [Example 1 (compound represented by the above chemical formula 3; IK15a; 3-methyl-6-phenylpyrrolo[2,1-a]isoquinoline-1-carbonitrile)]

[0295] 1 H NMR (400 MHz, CDCl3)δ8.91(d,J=8.0 Hz, 1H), 7.62 (s, 3H), 7.54-7.46 (m, 6H), 6.76 (s, 1H), 2.49 (s, 3H),

[0296] 13 C{1H} NMR (100 MHz, CDCl3) δ136.5, 133.3, 130.2, 128.7, 128.3, 128.2, 127.6, 127.5, 126.7, 125.9, 125.0, 123.6, 123.1, 120.1, 118.9, 114.3, 82.4, 11.5; HRMS (ESI-QTOF) m / z [M+H]+ calcd for C 20 H 15 N2283.1230, found 280.1239.

[0297] [Example 2 (compound represented by the chemical formula 4; IK15b; 6-(4-methoxyphenyl)-3-methylpyrrolo[2,1-a]isoquinoline-1-carbonitrile)]

[0298] 1H NMR (400 MHz, CDCl3)δ8.89 (d, J = 7.6 Hz, 1H), 7.67-7.62 (m, 2H), 7.59 (s, 1H), 7.50-7.45 (m, 1H), 7.40 (d, J = 7.6 Hz, 2H), 7.06 (d, J = 7.6 Hz, 2H), 6.75 (s, 1H), 3.91 (s, 3H), 2.48 (s, 3H),

[0299] 13 C{1H} NMR (100 MHz, CDCl3) δ159.6, 133.2, 131.3, 128.6, 128.2, 127.8, 127.6, 126.3, 125.9, 125.0, 123.5, 123.1, 120.0, 119.0, 114.2, 114.1, 82.3, 55.4, 11.5; HRMS (ESI-QTOF) m / z [M+H]+ calcd for C 21 H 17 N 2O 313.1336, found 313.1340.

[0300] [Example 3 (compound represented by the above chemical formula 5; IK15c; 3-methyl-6-(p-tolyl)pyrrolo[2,1-a]isoquinoline-1-carbonitrile)]

[0301] 1 H NMR (400 MHz, CDCl3)δ8.90 (d, J = 7.6 Hz, 1H), 7.66-7.62 (m, 2H), 7.60 (s, 1H), 7.50-7.43 (m, 1H), 7.39-7.33 (m, 4H), 6.75 (s, 1H), 2.47 (s, 6H),

[0302] 13C{1H} NMR (100 MHz, CDCl3) δ138.1, 133.5, 133.2, 130.0, 129.4, 128.3, 127.7, 127.6, 126.6, 125.9, 125.0, 123.6, 123.1, 120.0, 119.0, 114.2, 82.4, 21.3, 11.5; HRMS (ESI-QTOF) m / z [M+H]+ calcd for C 21 H 17 N2297.1386, found 297.1392.

[0303] [Example 4 (compound represented by the above chemical formula 6; IK15d; 6-(4-chlorophenyl)-3-methylpyrrolo[2,1-a]isoquinoline-1-carbonitrile)]

[0304] 1 H NMR (400 MHz, CDCl3) δ8.89 (d, J = 8.0 Hz, 1H), 7.65-7.60 (m, 1H), 7.59 (s, 1H), 7.56 (s, 1H), 7.52-7.47 (m, 3H), 7.42 (d, J = 8.4 Hz, 2H), 6.76 (s, 1H), 2.49 (s, 3H),

[0305] 13 C{1H} NMR (100 MHz, CDCl3) δ134.9, 134.4, 133.2, 131.5, 129.0, 128.5, 127.7, 127.2, 125.6, 125.5, 125.0, 123.7, 123.2, 120.2, 118.8, 114.4, 82.7, 11.5; HRMS (ESI-QTOF) m / z [M+H]+ calcd for C 20 H 14 N2ClN2317.0840, found 317.0828.

[0306] [Example 5 (compound represented by the above chemical formula 7; IK15e; 3-methyl-6-(naphthalen-2-yl)pyrrolo[2,1-a]isoquinoline-1-carbonitrile)]

[0307] 1 H NMR (400 MHz, CDCl3)δ8.93 (d, J = 8.0 Hz, 1H), 8.01-7.91 (m, 4H), 7.71 (s, 1H), 7.68-7.63 (m, 2H), 7.59 (s, 3H), 7.50-7.43 (m, 1H), 6.78 (s, 1H), 2.51 (s, 3H),

[0308] 13 C{1H} NMR (100 MHz, CDCl3) δ134.0, 133.4, 133.3, 132.9, 129.0, 128.4, 128.2, 128.1, 128.0, 127.8, 127.7, 127.6, 126.69, 126.65, 126.6, 126.0, 125.1, 123.7, 123.1, 120.4, 118.9, 114.3, 82.5, 11.6; HRMS (ESI-QTOF) m / z [M+H]+ calcd for C 24 H 17 N2333.1386, found 333.1396.

[0309] [Example 6 (compound represented by the above chemical formula 8; IK15f; 3,6-dimethylpyrrolo[2,1-a]isoquinoline-1-carbonitrile)]

[0310] 1H NMR (400 MHz, CDCl3) δ8.83 (d,J= 7.2 Hz, 1H), 7.77 (d,J= 6.8 Hz, 1H), 7.63-7.56 (m, 2H), 7.51 (s, 1H), 6.67 (s, 1H), 2.50 (s, 3H), 2.46 (s, 3H),

[0311] 13 C{1H} NMR (100 MHz, CDCl3) δ133.2, 128.3, 128.0, 127.6, 124.8, 123.8, 123.1, 123.0, 119.5, 119.2, 119.1, 113.6, 82.0, 16.7, 11.5; HRMS (ESI-QTOF) m / z [M+H]+ calcd for C 15 H 13 N2221.1073, found 221.1084.

[0312] [Example 7 (compound represented by the above chemical formula 9; IK15l; 5-(4-methoxyphenyl)-8-methylbenzo[f]pyrrolo[2,1-a]isoquinoline-10-carbonitrile)]

[0313] 1 H NMR (400 MHz, CDCl3) δ8.95 (d, J = 7.2 Hz, 1H), 8.80 (d, J = 7.6 Hz, 1H), 7.89 (d, J = 7.2 Hz, 1H), 7.65 (s, 2H), 7.45 (s, 1H), 7.34 (d, J = 6.4 Hz, 2H), 7.15 (s, 1H), 7.04 (d, J = 6.4 Hz, 2H), 6.85 (s, 1H), 3.93 (s, 3H), 2.50 (s, 3H),

[0314] 13C{1H} NMR (100 MHz, CDCl3) δ159.4, 133.6, 133.2, 130.4, 130.1, 129.5, 128.8, 127.5, 126.2, 125.5, 124.5, 123.6, 122.6, 120.7, 119.3, 115.7, 114.5, 81.7, 55.4, 11.5; HRMS (ESI-QTOF) m / z [M+H]+ calcd for C 25 H 19 N 2O 363.1492, found 363.1502.

[0315] [Example 8 (compound represented by the chemical formula 10; IK15n; 3-methyl-6,8-diphenylpyrrolo[2,1-a]isoquinoline-1-carbonitrile)]

[0316] 1 H NMR (400 MHz, CDCl3) δ8.97 (d, J = 8.4 Hz, 1H), 7.88 (d, J = 8.4 Hz, 1H), 7.84 (s, 1H), 7.64 (s, 1H), 7.59-7.52 (m, 7H), 7.43 (t, J = 7.2 Hz, 2H), 7.36 (d, J = 6.0 Hz, 1H), 6.78 (s, 1H), 2.50 (s, 3H),

[0317] 13 C{1H} NMR (100 MHz, CDCl3) δ140.3, 135.4, 133.2, 130.1, 128.9, 128.8, 128.3, 128.0, 127.7, 127.5, 127.2, 126.8, 124.1, 123.9, 123.8, 123.6, 120.5, 118.9, 114.4, 93.3, 82.5, 11.6; HRMS (ESI-QTOF) m / z [M+H]+ calcd for C 26 H 19 N2359.1543, found 359.2311.

[0318] [Example 9 (compound represented by the above chemical formula 11; IK15o; 6-(4-methoxyphenyl)-3-methyl-8-phenylpyrrolo[2,1-a]isoquinoline-1-carbonitrile)]

[0319] 1 H NMR (400 MHz, CDCl3) δ8.94 (d, J = 8.4 Hz, 1H), 7.88-7.84 (m, 2H), 7.61 (s, 1H), 7.57 (d, J = 7.6 Hz, 2H), 7.46-7.41 (m, 4H), 7.37 (d, J) = 6.8 Hz, 1H), 7.06 (d, J = 8.4 Hz, 2H), 6.75 (s, 1H), 3.91 (s, 3H), 2.49 (s, 3H),

[0320] 13 C{1H} NMR (100 MHz, CDCl3) δ159.6, 140.3, 130.2, 133.1, 131.3, 128.9, 128.6, 128.3, 127.7, 127.3, 127.2, 126.4, 124.03, 123.96, 123.7, 123.6, 120.4, 119.0, 114.3, 114.2, 82.4, 55.4, 11.6; HRMS (ESI-QTOF) m / z [M+H]+ calcd for C 27 H 21 N 2O 389.1649, found 389.1658.

[0321] [Example 10 (Compound represented by the above chemical formula 12; IK15p; 3-methyl-8-phenyl-6-(p-tolyl)pyrrolo[2,1-a]isoquinoline-1-carbonitrile)]

[0322] 1H NMR (400 MHz, CDCl3) δ8.96 (d, J = 8.8 Hz, 1H), 7.89-7.84 (m, 2H), 7.62 (s, 1H), 7.57 (d, J = 7.6 Hz, 2H), 7.46-7.41 (m, 3H), 7.40 (s, 1H), 7.37-7.33 (m, 3H), 6.77 (s, 1H), 2.49 (s, 3H), 2.48 (s, 3H),

[0323] 13 C{1H} NMR (100 MHz, CDCl3) δ140.4, 140.3, 138.1, 133.4, 133.1, 130.0, 129.5, 128.9, 128.1, 127.7, 127.4, 127.3, 126.7, 124.1, 124.0, 123.7, 123.6, 120.4, 119.0, 114.3, 82.4, 21.3, 11.6; HRMS (ESI-QTOF) m / z [M+H]+ calcd for C 27 H 21 N2373.1699, found 373.1693.

[0324] [Example 11 (compound represented by the above chemical formula 13; IK15q; 3,6-dimethyl-8-phenylpyrrolo[2,1-a]isoquinoline-1-carbonitrile)]

[0325] 1 H NMR (400 MHz, CDCl3) δ8.87 (d, J = 8.4 Hz, 1H), 7.93 (s, 1H), 7.85 (d, J = 6.8 Hz, 1H), 7.70 (d, J = 6.8 Hz, 2H), 7.52 (d, J = 6.0 Hz, 2H), 7.49 (s, 1H), 7.42 (d, J = 6.4 Hz, 1H), 6.67 (s, 1H), 2.54 (s, 3H), 2.47 (s, 3H),

[0326] 13C{1H} NMR (100 MHz, CDCl3) δ140.5, 140.3, 133.1, 129.0, 128.7, 127.8, 127.3, 127.2, 123.9, 123.6, 131.2, 122.0, 119.7, 119.5, 119.1, 113.8, 82.1, 16.8, 11.5; HRMS (ESI-QTOF) m / z [M+H]+ calcd for C 21 H 17 N2297.1386, found 297.1391.

[0327] [Example 12 (Compound represented by the above chemical formula 14; IK15r; 8-chloro-6-(4-methoxyphenyl)-3-methylpyrrolo[2,1-a]isoquinoline-1-carbonitrile)]

[0328] 1 H NMR (400 MHz, CDCl3) δ8.80 (d, J = 6.4 Hz, 1H), 7.60 (d, J = 4.8 Hz, 2H), 7.55 (d, J = 7.6 Hz, 1H), 7.38 (d, J = 6.0 Hz, 2H), 7.07 (d, J = 6.0) Hz, 2H), 6.75 (s, 1H), 3.91 (s, 3H), 2.48 (s, 3H),

[0329] 13 C{1H} NMR (100 MHz, CDCl3) δ159.8, 133.5, 132.5, 131.2, 129.3, 128.6, 127.8, 125.4, 125.3, 124.5, 123.9, 123.4, 121.0, 118.6, 114.4, 114.3, 82.7, 55.4, 11.5; HRMS (ESI-QTOF) m / z [M+H]+ calcd for C 21 H 16 ClN 2O 347.0946, found 347.0952.

[0330] [Example 13 (compound represented by the above chemical formula 15; IK15s; 8-bromo-6-(4-methoxyphenyl)-3-methylpyrrolo[2,1-a]isoquinoline-1-carbonitrile)]

[0331] 1 H NMR (400 MHz, CDCl3) δ8.74 (d, J = 8.8 Hz, 1H), 7.75 (s, 1H), 7.69 (d, J = 7.2 Hz, 1H), 7.60 (s, 1H), 7.37 (d, J = 8.4 Hz, 2H), 7.07 (d, J = 8.0 Hz, 2H), 6.76 (s, 1H), 3.91 (s, 3H), 2.48 (s, 3H),

[0332] 13 C{1H} NMR (100 MHz, CDCl3) δ159.8, 132.6, 131.3, 131.2, 129.5, 128.4, 127.8, 125.3, 123.6, 123.9, 123.7, 121.7, 121.0, 118.6, 114.5, 114.3, 82.9, 55.4, 11.5; HRMS (ESI-QTOF) m / z [M+H]+ calcd for C 21 H 16 BrN 2O 391.0441, found 391.0444.

[0333]

[0334] <Manufacturing Example 2> Manufacture of Example 14 (compound represented by the chemical formula 16; AMD-D-253)

[0335] The compound of Example 14 was synthesized according to the following reaction scheme 7.

[0336] [Reaction Formula 7]

[0337]

[0338] Step 1. Preparation of 2-(4-fluorobenzoyl)-4-oxopentanenitrile

[0339] 3-(4-Fluorophenyl)-3-oxopropanenitrile (5 g, 30.60 mmol) and 1-chloropropan-2-one (3.1 g, 33.66 mmol) were mixed in ethanol (80 mL), potassium carbonate (10.5 g, 76.50 mmol) was added, and the mixture was stirred at 25°C under nitrogen for 2 hours. Water (50 mL) was added to the reaction mixture, and extracted with ethyl acetate (120 mL × 3). The organic layer was combined, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The concentrate was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio = 5:95) to obtain 2-(4-fluorobenzoyl)-4-oxopentanenitrile (3 g, yield 44%).

[0340]

[0341] Step 2. Preparation of 2-(4-fluorophenyl)-5-methyl-1H-pyrrole-3-carbonitrile

[0342] 2-(4-Fluorobenzoyl)-4-oxopentanenitrile (3 g, 13.70 mmol) and ammonium acetate (2.1 g, 27.40 mmol) were mixed in ethanol (50 mL) and stirred at 80°C for 5 hours. Water (50 mL) was added to the reaction mixture and extracted with dichloromethane (100 mL × 3). The organic layer was combined, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The concentrate was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 12:88) to give 2-(4-fluorophenyl)-5-methyl-1H-pyrrole-3-carbonitrile (2.2 g, yield 80%) as a yellow solid.

[0343]

[0344] Step 3. Preparation of 2-(4-fluorophenyl)-5-methyl-1-(2-oxo-2-phenylethyl)-1H-pyrrole-3-carbonitrile

[0345] 2-(4-Fluorophenyl)-5-methyl-1H-pyrrole-3-carbonitrile (200 mg, 0.99 mmol) and 2-bromo-1-phenylethan-1-one (238 mg, 1.20 mmol) were mixed in acetonitrile (60 mL), and cesium carbonate (651 mg, 1.99 mmol) was added. The mixture was stirred at 60°C for 24 h. Water (40 mL) was added to the reaction mixture, and dichloromethane (80 mL × 3) was extracted. The organic layer was combined, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The concentrate was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 10:90) to obtain 2-(4-fluorophenyl)-5-methyl-1-(2-oxo-2-phenylethyl)-1H-pyrrole-3-carbonitrile (220 mg, yield 69%) as a white solid.

[0346]

[0347] Step 4. Preparation of 8-fluoro-3-methyl-6-phenylpyrrolo[2,1-a]isoquinoline-1-carbonitrile (Example 14)

[0348] 2-(4-Fluorophenyl)-5-methyl-1-(2-oxo-2-phenylethyl)-1H-pyrrole-3-carbonitrile (200 mg, 0.63 mmol), trifluoromethanesulfonic acid (471 mg, 3.14 mmol), and 1,2-dichloroethane (30 mL) were mixed and stirred at 60°C for 2 hours. Water (25 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane (50 mL × 3). The organic layer was combined, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The concentrate was purified by Prep-HPLC (25% - 95% acetonitrile / 0.1% ammonia water) to give 8-fluoro-3-methyl-6-phenylpyrrolo[2,1-a]isoquinoline-1-carbonitrile (19.8 mg, yield 10%) as a white solid.

[0349] LCMS (ESI-MS): Calculated molecular weight (mass calcd) C 20 H 13 FN2300.1 m / z, observed value (found) 301.1 [M+H] + .

[0350] 1 H NMR (400 MHz, CDCl3) δ8.91 (dd, J = 9.2, 5.6 Hz, 1H), 7.65 (s, 1H), 7.57-7.50 (m, 3H), 7.49-7.45 (m, 2H), 7.39-7.33 (m, 1H), 7.30-7.27 (m, 1H), 6.76 (s, 1H), 2.49 (s, 3H).

[0351]

[0352] <Manufacturing Example 3> Manufacture of Example 15 (compound represented by the chemical formula 17; AMD-D-254)

[0353] The compound of Example 15 was synthesized according to the following reaction scheme 8.

[0354] [Reaction Formula 8]

[0355]

[0356] Step 1. Preparation of 2-(4-fluorophenyl)-1-(2-(4-methoxyphenyl)-2-oxoethyl)-5-methyl-1H-pyrrole-3-carbonitrile

[0357] 2-(4-fluorophenyl)-5-methyl-1H-pyrrole-3-carbonitrile (200 mg, 0.99 mmol) and 2-bromo-1-(4-methoxyphenyl)ethan-1-one (275 mg, 1.20 mmol), which are intermediates of step 2 of Example 14, were mixed in acetonitrile (60 mL), and cesium carbonate (651 mg, 1.99 mmol) was added. The mixture was stirred at 60°C for 24 hours. Water (40 mL) was added to the reaction mixture, and extracted with dichloromethane (80 mL × 3). The organic layer was collected, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The concentrate was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 10:90) to obtain 2-(4-fluorophenyl)-1-(2-(4-methoxyphenyl)-2-oxoethyl)-5-methyl-1H-pyrrole-3-carbonitrile (220 mg, yield 63%) as a white solid.

[0358]

[0359] Step 2. Preparation of 8-fluoro-6-(4-methoxyphenyl)-3-methylpyrrolo[2,1-a]isoquinoline-1-carbonitrile (Example 15)

[0360] 2-(4-Fluorophenyl)-1-(2-(4-methoxyphenyl)-2-oxoethyl)-5-methyl-1H-pyrrole-3-carbonitrile (100 mg, 0.29 mmol), trifluoromethanesulfonic acid (430 mg, 2.87 mmol), and 1,2-dichloroethane (30 mL) were mixed and stirred at 80°C for 2 hours. Water (25 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane (50 mL × 3). The organic layer was combined, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The concentrate was purified using Prep-HPLC (25% - 95% acetonitrile / ammonia 0.1%) to obtain 8-fluoro-6-(4-methoxyphenyl)-3-methylpyrrolo[2,1-a]isoquinoline-1-carbonitrile (25.9 mg, yield 27%) as a white solid.

[0361] LCMS (ESI-MS): Calculated molecular weight C 21 H 15 FN 2O 330.1 m / z, observed value 330.9 [M+H] + .

[0362] 1 H NMR (400 MHz, CDCl3) δ8.89 (dd, J = 8.8, 5.2 Hz, 1H), 7.62 (s, 1H), 7.40-7.37 (m, 2H), 7.36-7.27 (m, 2H), 7.08-7.05 (m, 2H), 6.75 (s, 1H), 3.91 (s, 3H), 2.48 (s, 3H).

[0363]

[0364] <Manufacturing Example 4> Manufacture of Example 16 (compound represented by the chemical formula 18; AMD-D-255)

[0365] The compound of Example 16 was synthesized according to the following reaction scheme 9.

[0366] [Reaction Formula 9]

[0367]

[0368] Step 1. Preparation of 2-(4-fluorophenyl)-1-(2-(4-fluorophenyl)-2-oxoethyl)-5-methyl-1H-pyrrole-3-carbonitrile

[0369] 2-(4-fluorophenyl)-5-methyl-1H-pyrrole-3-carbonitrile (300 mg, 1.50 mmol) and 2-bromo-1-(4-fluorophenyl)ethan-1-one (390 mg, 1.80 mmol), which are intermediates of step 2 of Example 14, were mixed in acetonitrile (40 mL), and cesium carbonate (976 mg, 3.00 mmol) was added. The mixture was stirred at 60°C for 24 hours. Water (40 mL) was added to the reaction mixture, and extracted with dichloromethane (80 mL × 3). The organic layer was collected, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The concentrate was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio = 10:90) to obtain 2-(4-fluorophenyl)-1-(2-(4-fluorophenyl)-2-oxoethyl)-5-methyl-1H-pyrrole-3-carbonitrile (150 mg, yield 29%) as a white solid.

[0370]

[0371] Step 2. Preparation of 8-fluoro-6-(4-fluorophenyl)-3-methylpyrrolo[2,1-a]isoquinoline-1-carbonitrile (Example 16)

[0372] 2-(4-Fluorophenyl)-1-(2-(4-fluorophenyl)-2-oxoethyl)-5-methyl-1H-pyrrole-3-carbonitrile (100 mg, 0.29 mmol), trifluoromethanesulfonic acid (223 mg, 1.49 mmol), and 1,2-dichloroethane (30 mL) were mixed and stirred at 80°C for 1 h. Water (25 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane (50 mL × 3). The organic layer was combined, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The concentrate was purified by Prep-HPLC (25% to 65% acetonitrile / 0.1% ammonia water) to give 8-fluoro-6-(4-fluorophenyl)-3-methylpyrrolo[2,1-a]isoquinoline-1-carbonitrile (20.1 mg, yield 21%) as a white solid.

[0373] LCMS (ESI-MS): Calculated molecular weight C 20 H 12 F2N2318.1 m / z, observed value 318.6 [M+H] + .

[0374] 1 H NMR (400 MHz, CDCl3) δ8.91 (dd, J = 9.2, 5.6 Hz, 1H), 7.63 (s, 1H), 7.46-7.42 (m, 2H), 7.39-7.34 (m, 1H), 7.25-7.19 (m, 3H), 6.77 (s, 1H), 2.49 (s, 3H).

[0375]

[0376] <Manufacturing Example 5> Manufacture of Example 17 (compound represented by the chemical formula 19; AMD-D-257)

[0377] The compound of Example 17 was synthesized according to the following reaction scheme 10.

[0378] [Reaction Formula 10]

[0379]

[0380] Step 1. Preparation of 2-(4-fluorophenyl)-5-methyl-1-(2-oxo-2-(thiophen-2-yl) ethyl)-1H-pyrrole-3-carbonitrile

[0381] 2-(4-Fluorophenyl)-5-methyl-1H-pyrrole-3-carbonitrile (300 mg, 1.50 mmol) and 2-bromo-1-(thiophen-2-yl)ethan-1-one (369 mg, 1.80 mmol), which are intermediates of step 2 of Example 14, were mixed in acetonitrile (50 mL), and cesium carbonate (976 mg, 3.00 mmol) was added. The mixture was stirred at 60°C for 24 hours. Water (40 mL) was added to the reaction mixture, and extracted with dichloromethane (80 mL × 3). The organic layer was collected, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The concentrate was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio = 10:90) to obtain 2-(4-fluorophenyl)-5-methyl-1-(2-oxo-2-(thiophen-2-yl)ethyl)-1H-pyrrole-3-carbonitrile (160 mg, yield 33%) as a white solid.

[0382]

[0383] Step 2. Preparation of 8-fluoro-3-methyl-6-(thiophen-2-yl) pyrrolo[2,1-a] isoquinoline-1-carbonitrile (Example 17)

[0384] 2-(4-Fluorophenyl)-5-methyl-1-(2-oxo-2-(thiophen-2-yl)ethyl)-1H-pyrrole-3-carbonitrile (100 mg, 0.31 mmol), trifluoromethanesulfonic acid (231 mg, 1.54 mmol), and 1,2-dichloroethane (30 mL) were mixed and stirred at 80°C for 2 h. Water (25 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane (50 mL × 3). The organic layer was combined, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The concentrate was purified by Prep-HPLC (25% - 70% acetonitrile / 0.1% ammonia water) to give 8-fluoro-3-methyl-6-(thiophen-2-yl)pyrrolo[2,1-a]isoquinoline-1-carbonitrile (AMD-D-257) (18.5 mg, yield 19%) as a white solid.

[0385] LCMS (ESI-MS): Calculated molecular weight C 18 H 11 FN2S 306.1 m / z, observed value 307.0 [M+H] + .

[0386] 1 H NMR (400 MHz, CDCl₃) δ8.89 (dd, J = 8.8, 5.2 Hz, 1H), 7.79 (s, 1H), 7.58-7.56 (m, 1H), 7.51-7.48 (m, 1H), 7.41-7.35 (m, 1H), 7.25-7.21 (m, 2H), 6.76 (s, 1H), 2.50 (s, 3H).

[0387]

[0388] <Manufacturing Example 6> Manufacture of Example 18 (compound represented by the chemical formula 20; AMD-D-258)

[0389] The compound of Example 18 was synthesized according to the following reaction scheme 11.

[0390] [Reaction Formula 11]

[0391]

[0392] Step 1. Preparation of 4-oxo-2-(4-(trifluoromethyl)benzoyl)pentanenitrile

[0393] 3-Oxo-3-[4-(trifluoromethyl)phenyl]propanenitrile (5 g, 0.023 mol) and potassium carbonate (8.12 g, 0.058 mol) were mixed in ethanol (150 mL), and 1-chloropropan-2-one (2.39 g, 0.025 mol) was added. The mixture was stirred at 25°C for 2 h. Water (100 mL) was added to the reaction mixture, and extracted with dichloromethane (100 mL × 3). The organic layers were combined, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The concentrate was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 15:85) to give 4-oxo-2-(4-(trifluoromethyl)benzoyl)pentanenitrile (5 g, yield 79%) as a yellow solid.

[0394]

[0395] Step 2. Preparation of 5-methyl-2-(4-(trifluoromethyl)phenyl)-1H-pyrrole-3-carbonitrile

[0396] 4-Oxo-2-(4-(trifluoromethyl)benzoyl)pentanenitrile (2.5 g, 0.009 mol) and ammonium acetate (1.51 g, 0.019 mol) were mixed in ethanol (50 mL) and stirred at 80°C for 2 hours. Water (50 mL) was added to the reaction mixture, and extracted with dichloromethane (100 mL × 3). The organic layers were combined, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The concentrate was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 15:85) to obtain 5-methyl-2-(4-(trifluoromethyl)phenyl)-1H-pyrrole-3-carbonitrile (1.1 g, yield 43%) as a white solid.

[0397]

[0398] Step 3. Preparation of 5-methyl-1-(2-oxo-2-phenylethyl)-2-(4-(trifluoromethyl)phenyl)-1H-pyrrole-3-carbonitrile

[0399] 5-Methyl-2-(4-(trifluoromethyl)phenyl)-1H-pyrrole-3-carbonitrile (200 mg, 0.799 mmol), 2-bromo-1-phenylethan-1-one (190.92 mg, 0.959 mmol), and acetonitrile (9 mL) were mixed, and cesium carbonate (520.86 mg, 1.598 mmol) was added. The mixture was stirred at 60°C for 16 h. Water (10 mL) was added to the reaction mixture, and it was extracted with dichloromethane (20 mL × 3). The organic layer was combined, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The concentrate was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 8:92) to obtain 5-methyl-1-(2-oxo-2-phenylethyl)-2-(4-(trifluoromethyl)phenyl)-1H-pyrrole-3-carbonitrile (168 mg, yield 45%) as a yellow solid.

[0400]

[0401] Step 4. Preparation of 3-methyl-6-phenyl-8-(trifluoromethyl)pyrrolo[2,1-a]isoquinoline-1-carbonitrile (Example 18)

[0402] 5-Methyl-1-(2-oxo-2-phenylethyl)-2-(4-(trifluoromethyl)phenyl)-1H-pyrrole-3-carbonitrile (100 mg, 0.271 mmol), trifluoromethanesulfonic acid (407.47 mg, 2.715 mmol), and 1,2-dichloroethane (5 mL) were mixed and stirred at 60°C for 1 h. Water (10 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane (15 mL × 3). The organic layer was combined, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The concentrate was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 5:95) to obtain 3-methyl-6-phenyl-8-(trifluoromethyl)pyrrolo[2,1-a]isoquinoline-1-carbonitrile (27 mg, yield 27%) as a white solid.

[0403] LCMS (ESI-MS): Calculated molecular weight C 21 H 13 F3N2 350.1 m / z, observed value 350.7 [M+H] + .

[0404] 1 H NMR (400 MHz, CDCl3) δ9.00 (d, J = 8.4 Hz, 1H), 7.89 (s, 1H), 7.84-7.81 (m, 1H), 7.70 (s, 1H), 7.58-7.51 (m, 3H), 7.48-7.46 (m, 2H), 6.84 (s, 1H), 2.52 (s, 3H).

[0405]

[0406] <Manufacturing Example 7> Manufacture of Example 19 (compound represented by the chemical formula 21; AMD-D-259)

[0407] The compound of Example 19 was synthesized according to the following reaction scheme 12.

[0408] [Reaction Formula 12]

[0409]

[0410] Step 1. Preparation of 1-(2-(4-methoxyphenyl)-2-oxoethyl)-5-methyl-2-(4-(trifluoromethyl)phenyl)-1H-pyrrole-3-carbonitrile

[0411] 5-Methyl-2-(4-(trifluoromethyl)phenyl)-1H-pyrrole-3-carbonitrile (200 mg, 0.799 mmol) and 2-bromo-1-(4-methoxyphenyl)ethan-1-one (219.72 mg, 0.959 mmol), which are intermediates of step 2 of Example 18, were mixed in acetonitrile (9 mL), and cesium carbonate (520.86 mg, 1.598 mmol) was added. The mixture was stirred at 60°C for 16 hours. Water (10 mL) was added to the reaction mixture, and extracted with dichloromethane (20 mL × 3). The organic layer was collected, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The concentrate was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 8:92) to obtain 1-(2-(4-methoxyphenyl)-2-oxoethyl)-5-methyl-2-(4-(trifluoromethyl)phenyl)-1H-pyrrole-3-carbonitrile (150 mg, yield 37%) as a yellow solid.

[0412]

[0413] Step 2. Preparation of 6-(4-methoxyphenyl)-3-methyl-8-(trifluoromethyl)pyrrolo[2,1-a]isoquinoline-1-carbonitrile (Example 19)

[0414] 1-(2-(4-methoxyphenyl)-2-oxoethyl)-5-methyl-2-(4-(trifluoromethyl)phenyl)-1H-pyrrole-3-carbonitrile (100 mg, 0.251 mmol), trifluoromethanesulfonic acid (376.7 mg, 2.51 mmol), and 1,2-dichloroethane (5 mL) were mixed and stirred at 60°C for 1 h. Water (10 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane (15 mL × 3). The organic layer was combined, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The concentrate was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 5:95) to obtain 6-(4-methoxyphenyl)-3-methyl-8-(trifluoromethyl)pyrrolo[2,1-a]isoquinoline-1-carbonitrile (14 mg, yield 14%) as a white solid.

[0415] LCMS (ESI-MS): Calculated molecular weight C 22 H 15 F3N 2O 380.1 m / z, observed value 380.7 [M+H] + .

[0416] 1 H NMR (400 MHz, CDCl3) δ8.99 (d, J = 8.4 Hz, 1H), 7.91 (s, 1H), 7.83-7.80 (m, 1H), 7.67 (s, 1H), 7.43-7.36 (m, 2H), 7.12-7.05 (m, 2H), 6.83 (s, 1H), 3.92 (s, 3H), 2.51 (s, 3H).

[0417]

[0418] <Manufacturing Example 8> Manufacture of Example 20 (compound represented by the chemical formula 22; AMD-D-260)

[0419] The compound of Example 20 was synthesized according to the following reaction scheme 13.

[0420] [Reaction Formula 13]

[0421]

[0422] Step 1. Preparation of 1-(2-(4-fluorophenyl)-2-oxoethyl)-5-methyl-2-(4-(trifluoromethyl)phenyl)-1H-pyrrole-3-carbonitrile

[0423] 5-Methyl-2-(4-(trifluoromethyl)phenyl)-1H-pyrrole-3-carbonitrile (200 mg, 0.799 mmol) and 2-bromo-1-(4-fluorophenyl)ethan-1-one (208.17 mg, 0.959 mmol), which are intermediates of step 2 of Example 18, were mixed in acetonitrile (9 mL), and cesium carbonate (520.86 mg, 1.598 mmol) was added. The mixture was stirred at 60°C for 16 hours. Water (10 mL) was added to the reaction mixture, and extracted with dichloromethane (20 mL × 3). The organic layer was collected, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The concentrate was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 10:90) to obtain 1-(2-(4-fluorophenyl)-2-oxoethyl)-5-methyl-2-(4-(trifluoromethyl)phenyl)-1H-pyrrole-3-carbonitrile (100 mg, yield 29%) as a yellow solid.

[0424]

[0425] Step 2. Preparation of 6-(4-fluorophenyl)-3-methyl-8-(trifluoromethyl)pyrrolo[2,1-a]isoquinoline-1-carbonitrile (Example 20)

[0426] 1-(2-(4-Fluorophenyl)-2-oxoethyl)-5-methyl-2-(4-(trifluoromethyl)phenyl)-1H-pyrrole-3-carbonitrile (100 mg, 0.258 mmol), trifluoromethanesulfonic acid (388.41 mg, 2.588 mmol), and 1,2-dichloroethane (5 mL) were mixed and stirred at 60°C for 1 h. Water (10 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane (15 mL × 3). The organic layer was combined, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The concentrate was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 6:94) to obtain 6-(4-fluorophenyl)-3-methyl-8-(trifluoromethyl)pyrrolo[2,1-a]isoquinoline-1-carbonitrile (10 mg, yield 10%) as a white solid.

[0427] LCMS (ESI-MS): Calculated molecular weight C 21 H 12 F4N2368.1 m / z, observed value 368.7 [M+H] + .

[0428] 1 H NMR (400 MHz, CDCl3) δ9.00 (d, J = 8.4 Hz, 1H), 7.84-7.82 (m, 2H), 7.68 (s, 1H), 7.48-7.40 (m, 2H), 7.28-7.27 (m, 1H), 7.24 (s, 1H), 6.85 (s, 1H), 2.53 (s, 3H).

[0429]

[0430] <Manufacturing Example 9> Manufacture of Example 21 (compound represented by the chemical formula 23; AMD-D-262)

[0431] The compound of Example 21 was synthesized according to the following reaction scheme 14.

[0432] [Reaction Formula 14]

[0433]

[0434] Step 1. Preparation of 5-methyl-1-(2-oxo-2-(thiophen-2-yl)ethyl)-2-(4-(trifluoromethyl)phenyl)-1H-pyrrole-3-carbonitrile

[0435] 5-Methyl-2-(4-(trifluoromethyl)phenyl)-1H-pyrrole-3-carbonitrile (200 mg, 0.799 mmol) and 2-bromo-1-(thiophen-2-yl)ethan-1-one (196.7 mg, 0.959 mmol), which are intermediates of step 2 of Example 18, were mixed in acetonitrile (9 mL), and cesium carbonate (520.86 mg, 1.598 mmol) was added. The mixture was stirred at 60°C for 16 hours. Water (10 mL) was added to the reaction mixture, and extracted with dichloromethane (20 mL × 3). The organic layer was collected, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The concentrate was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 10:90) to obtain 5-methyl-1-(2-oxo-2-(thiophen-2-yl)ethyl)-2-(4-(trifluoromethyl)phenyl)-1H-pyrrole-3-carbonitrile (80 mg, yield 24%) as a yellow solid.

[0436]

[0437] Step 2. Preparation of 3-methyl-6-(thiophen-2-yl)-8-(trifluoromethyl)pyrrolo[2,1-a]isoquinoline-1-carbonitrile (Example 21)

[0438] 5-Methyl-1-(2-oxo-2-(thiophen-2-yl)ethyl)-2-(4-(trifluoromethyl)phenyl)-1H-pyrrole-3-carbonitrile (80 mg, 0.213 mmol), trifluoromethanesulfonic acid (320.72 mg, 2.137 mmol), and 1,2-dichloroethane (4 mL) were mixed and stirred at 60°C for 1 h. Water (10 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane (15 mL × 3). The organic layer was combined, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The concentrate was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 5:95) to obtain 3-methyl-6-(thiophen-2-yl)-8-(trifluoromethyl)pyrrolo[2,1-a]isoquinoline-1-carbonitrile (11 mg, yield 13%) as a white solid.

[0439] LCMS (ESI-MS): Calculated molecular weight C 19 H 11 F3N2S 356.1 m / z, observed value 356.7 [M+H] + .

[0440] 1 H NMR (400 MHz, CDCl3) δ8.99 (d, J = 8.8 Hz, 1H), 8.18 (s, 1H), 7.85-7.84 (m, 2H), 7.52-7.51 (m, 1H), 7.27 (s, 1H), 7.24-7.23 (m, 1H), 6.84 (s, 1H), 2.53 (s, 3H).

[0441]

[0442] <Manufacturing Example 10> Manufacture of Example 22 (compound represented by the chemical formula 24; IK15g)

[0443] The compound of Example 22 was synthesized according to the following reaction scheme 15.

[0444] [Reaction Formula 15]

[0445]

[0446] Step 1. Preparation of 2-(4-methoxybenzoyl)-4-oxopentanenitrile

[0447] 3-(4-Methoxyphenyl)-3-oxopropanenitrile (2 g, 0.0114 mol), 1-chloropropan-2-one (1.16 g, 0.0125 mol), and potassium carbonate (3.94 g, 0.0285 mol) were mixed in ethanol (24 mL), and the mixture was stirred at 25°C for 2 h. Water (100 mL) was added to the reaction mixture, and extracted with dichloromethane (100 mL × 3). The organic layers were combined, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The concentrate was purified by silica gel column chromatography (ethyl acetate / hexane, volume ratio 40:60) to give 2-(4-methoxybenzoyl)-4-oxopentanenitrile (2.3 g, yield 78%) as a white solid.

[0448]

[0449] Step 2. Preparation of 2-(4-methoxyphenyl)-5-methyl-1H-pyrrole-3-carbonitrile

[0450] 2-(4-Methoxybenzoyl)-4-oxopentanenitrile (1.8 g, 0.0078 mol) and ammonium acetate (1.26 g, 0.0164 mol) were mixed in ethanol (15 mL) and stirred at 80°C for 5 hours. Water (200 mL) was added to the reaction mixture and extracted with ethyl acetate (200 mL × 3). The organic layer was combined, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The concentrate was purified by silica gel column chromatography (ethyl acetate / hexane, volume ratio 30:70) to obtain 2-(4-methoxyphenyl)-5-methyl-1H-pyrrole-3-carbonitrile (1.7 g, yield 92%) as a yellow solid.

[0451]

[0452] Step 3. Preparation of 2-(4-methoxyphenyl)-5-methyl-1-(2-oxo-2-phenylethyl)-1H-pyrrole-3-carbonitrile

[0453] 2-(4-Methoxyphenyl)-5-methyl-1H-pyrrole-3-carbonitrile (200 mg, 0.9423 mmol), 2-bromo-1-phenylethan-1-one (225.07 mg, 1.1307 mmol), and cesium carbonate (614.04 mg, 1.8846 mmol) were mixed in acetonitrile (5 mL) and stirred at 60°C for 24 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate / hexane, volume ratio 30:70) to obtain 2-(4-methoxyphenyl)-5-methyl-1-(2-oxo-2-phenylethyl)-1H-pyrrole-3-carbonitrile (140 mg, yield 40%) as a yellow solid.

[0454]

[0455] Step 4. Preparation of 8-methoxy-3-methyl-6-phenylpyrrolo[2,1-a]isoquinoline-1-carbonitrile (Example 22)

[0456] 2-(4-Methoxyphenyl)-5-methyl-1-(2-oxo-2-phenylethyl)-1H-pyrrole-3-carbonitrile (70 mg, 0.2119 mmol), trifluoromethanesulfonic acid (318.02 mg, 2.119 mmol), and 1,2-dichloroethane (6 mL) were mixed and stirred at 25°C for 1 h. The reaction was quenched with saturated aqueous sodium bicarbonate solution and extracted with dichloromethane. The organic layer was combined, washed with saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The residue was purified by Prep-HPLC (Gemini 5 μm C18, 150 × 21.2 mm, 70–95% acetonitrile / 0.1% formic acid) to give 8-methoxy-3-methyl-6-phenylpyrrolo[2,1-a]isoquinoline-1-carbonitrile (AMD-D-263) (23 mg, yield 33%) as a white solid.

[0457] LCMS (ESI-MS): Calculated molecular weight C 21 H 16 N2O 312.1 m / z, observed value 312.7 [M+H] + .

[0458] 1 H NMR (400 MHz, CDCl3) δ8.84 (d, J = 9.0 Hz, 1H), 7.59 (s, 1H), 7.56-7.49 (m, 5H), 7.26-7.22 (m, 1H), 7.05 (s, 1H), 6.71 (s, 1H), 3.79 (s, 3H), 2.46 (s, 3H).

[0459]

[0460] <Manufacturing Example 11> Manufacture of Example 23 (compound represented by the chemical formula 25; AMD-D-264)

[0461] The compound of Example 23 was synthesized according to the following reaction scheme 16.

[0462] [Reaction Formula 16]

[0463]

[0464] Step 1. Preparation of 2-(4-methoxyphenyl)-1-(2-(4-methoxyphenyl)-2-oxoethyl)-5-methyl-1H-pyrrole-3-carbonitrile

[0465] 2-(4-methoxyphenyl)-5-methyl-1H-pyrrole-3-carbonitrile (200 mg, 0.9423 mmol), 2-bromo-1-(4-methoxyphenyl)ethan-1-one (259.0 mg, 1.1307 mmol), and cesium carbonate (614.04 mg, 1.8846 mmol), which are intermediates of step 2 of Example 22, were mixed in acetonitrile (5 mL) and stirred at 60°C for 24 hours. After concentrating the reaction mixture under reduced pressure, the residue was purified by silica gel column chromatography (ethyl acetate / hexane, volume ratio 30:70) to obtain 2-(4-methoxyphenyl)-1-(2-(4-methoxyphenyl)-2-oxoethyl)-5-methyl-1H-pyrrole-3-carbonitrile (340 mg, yield 80%) as a yellow solid.

[0466]

[0467] Step 2. Preparation of 8-methoxy-6-(4-methoxyphenyl)-3-methylpyrrolo[2,1-a]isoquinoline-1-carbonitrile (Example 23)

[0468] 2-(4-methoxyphenyl)-1-(2-(4-methoxyphenyl)-2-oxoethyl)-5-methyl-1H-pyrrole-3-carbonitrile (100 mg, 0.2775 mmol), trifluoromethanesulfonic acid (416.47 mg, 2.775 mmol), and 1,2-dichloroethane (6 mL) were mixed and stirred at 25°C for 3 h. The reaction was quenched with saturated aqueous sodium bicarbonate solution and extracted with dichloromethane. The organic layer was combined, washed with saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The residue was purified by Prep-HPLC (Gemini 5 μm C18, 150 × 21.2 mm, 70–95% acetonitrile / 0.1% formic acid) to give 8-methoxy-6-(4-methoxyphenyl)-3-methylpyrrolo[2,1-a]isoquinoline-1-carbonitrile (AMD-D-264) (17 mg, yield 17%) as a white solid.

[0469] LCMS (ESI-MS): Calculated molecular weight C 22 H 18 N2O2 342.1 m / z, observed value 342.7 [M+H] + .

[0470] 1 H NMR (400 MHz, CDCl3) δ8.83 (d, J = 9.0 Hz, 1H), 7.57 (s, 1H), 7.43-7.37 (m, 2H), 7.24 (m, 1H), 7.09-7.02 (m, 3H), 6.70 (s, 1H), 3.91 (s, 3H), 3.79 (s, 3H), 2.46 (s, 3H).

[0471]

[0472] <Manufacturing Example 12> Manufacture of Example 24 (compound represented by the chemical formula 26; AMD-D-265)

[0473] The compound of Example 24 was synthesized according to the following reaction scheme 17.

[0474] [Reaction Formula 17]

[0475]

[0476] Step 1. Preparation of 1-(2-(4-fluorophenyl)-2-oxoethyl)-2-(4-methoxyphenyl)-5-methyl-1H-pyrrole-3-carbonitrile

[0477] 2-(4-methoxyphenyl)-5-methyl-1H-pyrrole-3-carbonitrile (200 mg, 0.9423 mmol), 2-bromo-1-(4-fluorophenyl)ethan-1-one (245.41 mg, 1.1307 mmol), and cesium carbonate (614.04 mg, 1.8846 mmol), which are intermediates of step 2 of Example 22, were mixed in acetonitrile (15 mL) and stirred at 60°C for 24 hours. After concentrating the reaction mixture under reduced pressure, the residue was purified by silica gel column chromatography (ethyl acetate / hexane, volume ratio 30:70) to obtain 1-(2-(4-fluorophenyl)-2-oxoethyl)-2-(4-methoxyphenyl)-5-methyl-1H-pyrrole-3-carbonitrile (100 mg, yield 27%) as a yellow oily substance.

[0478]

[0479] Step 2. Preparation of 6-(4-fluorophenyl)-8-methoxy-3-methylpyrrolo[2,1-a]isoquinoline-1-carbonitrile (Example 24)

[0480] 1-(2-(4-Fluorophenyl)-2-oxoethyl)-2-(4-methoxyphenyl)-5-methyl-1H-pyrrole-3-carbonitrile (80 mg, 0.2296 mmol), trifluoromethanesulfonic acid (344.58 mg, 2.296 mmol), and 1,2-dichloroethane (6 mL) were mixed and stirred at 25°C for 2 h. The reaction was quenched with saturated aqueous sodium bicarbonate solution and extracted with dichloromethane. The organic layer was combined, washed with saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The residue was purified by Prep-HPLC (Gemini 5 μm C18, 150 × 21.2 mm, 70–95% acetonitrile / 0.1% formic acid) to give 6-(4-fluorophenyl)-8-methoxy-3-methylpyrrolo[2,1-a]isoquinoline-1-carbonitrile (14.3 mg, yield 18%) as a white solid.

[0481] LCMS (ESI-MS): Calculated molecular weight C 21 H 15 FN2O 330.1 m / z, observed value 330.7 [M+H] + .

[0482] 1 H NMR (400 MHz, CDCl3) δ8.84 (d, J = 9.0 Hz, 1H), 7.57 (s, 1H), 7.48-7.42 (m, 2H), 7.24-7.18 (m, 3H), 6.97 (s, 1H), 6.71 (s, 1H), 3.80 (s, 3H), 2.47 (s, 3H).

[0483]

[0484] <Manufacturing Example 13> Manufacture of Example 25 (compound represented by the chemical formula 27; AMD-D-267)

[0485] The compound of Example 25 was synthesized according to the following reaction scheme 18.

[0486] [Reaction Formula 18]

[0487]

[0488] Step 1. Preparation of 2-(4-methoxyphenyl)-5-methyl-1-(2-oxo-2-(thiophen-2-yl)ethyl)-1H-pyrrole-3-carbonitrile

[0489] 2-(4-methoxyphenyl)-5-methyl-1H-pyrrole-3-carbonitrile (350 mg, 1.649 mmol), 2-bromo-1-(thiophen-2-yl)ethan-1-one (405.8 mg, 1.979 mmol), and cesium carbonate (1074.5 mg, 3.298 mmol), which are intermediates of step 2 of Example 22, were mixed in acetonitrile (15 mL) and stirred at 60°C for 24 hours. After concentrating the reaction mixture under reduced pressure, the residue was purified by silica gel column chromatography (ethyl acetate / hexane, volume ratio 30:70) to obtain 2-(4-methoxyphenyl)-5-methyl-1-(2-oxo-2-(thiophen-2-yl)ethyl)-1H-pyrrole-3-carbonitrile (100 mg, yield 16%) as a yellow oily substance.

[0490]

[0491] Step 2. Preparation of 8-methoxy-3-methyl-6-(thiophen-2-yl)pyrrolo[2,1-a]isoquinoline-1-carbonitrile (Example 25)

[0492] 2-(4-Methoxyphenyl)-5-methyl-1-(2-oxo-2-(thiophen-2-yl)ethyl)-1H-pyrrole-3-carbonitrile (90 mg, 0.2675 mmol), trifluoromethanesulfonic acid (401.5 mg, 2.675 mmol), and 1,2-dichloroethane (6 mL) were mixed and stirred at 25°C for 3 hours. The reaction was quenched with saturated aqueous sodium bicarbonate solution, and extracted with dichloroethane. The organic layer was combined, washed with saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The residue was purified by Prep-HPLC (Gemini 5 μm C18, 150 × 21.2 mm, 60–95% acetonitrile / 0.1% formic acid) to give 8-methoxy-3-methyl-6-(thiophen-2-yl)pyrrolo[2,1-a]isoquinoline-1-carbonitrile (15 mg, yield 17%) as a white solid.

[0493] LCMS (ESI-MS): Calculated molecular weight C 19 H 14 N2OS 318.1 m / z, observed value 319.1 [M+H] + .

[0494] 1 H NMR (400 MHz, CDCl3) δ8.82 (d, J = 9.0 Hz, 1H), 7.74 (s, 1H), 7.49-7.47 (m, 1H), 7.35 (s, 1H), 7.27 (s, 1H), 7.25-7.21 (m, 2H), 6.70 (s, 1H), 3.84 (s, 3H), 2.47 (s, 3H).

[0495]

[0496] <Manufacturing Example 14> Manufacture of Example 26 (compound represented by the chemical formula 28; IK15j)

[0497] The compound of Example 26 was synthesized according to the following reaction scheme 19.

[0498] [Reaction Formula 19]

[0499]

[0500] Step 1. Preparation of 2-(4-methylbenzoyl)-4-oxopentanenitrile

[0501] 3-Oxo-3-(p-tolyl)propanenitrile (5 g, 31.44 mmol) and 1-chloropropan-2-one (4.3 g, 47.17 mmol) were mixed in ethanol (80 mL), and potassium carbonate (8.7 g, 62.88 mmol) was added. The mixture was stirred at 25°C under nitrogen for 2 h. Water (50 mL) was added to the reaction mixture, and extracted with ethyl acetate (120 mL × 3). The organic layer was combined, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 5:95) to obtain 2-(4-methylbenzoyl)-4-oxopentanenitrile (3.1 g, yield 49%).

[0502]

[0503] Step 2. Preparation of 5-methyl-2-(p-tolyl)-1H-pyrrole-3-carbonitrile

[0504] 2-(4-Methylbenzoyl)-4-oxopentanenitrile (3.1 g, 14.42 mmol) and ammonium acetate (2.2 g, 28.83 mmol) were mixed in ethanol (50 mL) and stirred at 80°C for 5 h. Water (50 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane (100 mL × 3). The organic layer was combined, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 10:90) to obtain 5-methyl-2-(p-tolyl)-1H-pyrrole-3-carbonitrile (2.0 g, yield 71%) as a yellow solid.

[0505]

[0506] Step 3. Preparation of 5-methyl-1-(2-oxo-2-phenylethyl)-2-(p-tolyl)-1H-pyrrole-3-carbonitrile

[0507] 5-Methyl-2-(p-tolyl)-1H-pyrrole-3-carbonitrile (1.0 g, 5.1 mmol) and 2-bromo-1-phenylethan-1-one (2.0 g, 10.2 mmol) were mixed in acetonitrile (60 mL), and cesium carbonate (3.3 g, 10.2 mmol) was added. The mixture was stirred at 60°C for 24 h. Water (40 mL) was added to the reaction mixture, and dichloromethane (80 mL × 3) was extracted. The organic layer was combined, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 15:85) to obtain 5-methyl-1-(2-oxo-2-phenylethyl)-2-(p-tolyl)-1H-pyrrole-3-carbonitrile (0.5 g, yield 31%) as a yellow solid.

[0508]

[0509] Step 4. Preparation of 3,8-dimethyl-6-phenylpyrrolo[2,1-a]isoquinoline-1-carbonitrile (Example 26)

[0510] 5-Methyl-1-(2-oxo-2-phenylethyl)-2-(p-tolyl)-1H-pyrrole-3-carbonitrile (500 mg, 1.6 mmol), trifluoromethanesulfonic acid (2.4 g, 16.0 mmol), and 1,2-dichloroethane (30 mL) were mixed and stirred at room temperature for 2 h. Water (25 mL) was added to the reaction mixture, and dichloromethane (50 mL × 3) was extracted. The organic layer was combined, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 8:92) to obtain 3,8-dimethyl-6-phenylpyrrolo[2,1-a]isoquinoline-1-carbonitrile (AMD-D-268) (300 mg, yield 63%) as a white solid.

[0511] LCMS (ESI-MS): Calculated molecular weight C 21 H 16 N2296.1 m / z, observed value 296.7 [M+H] + .

[0512] 1 H NMR (400 MHz, CDCl3) δ8.80 (d, J = 8.4 Hz, 1H), 7.66-7.35 (m, 8H), 6.73 (s, 1H), 2.47 (s, 3H), 2.42 (s, 3H).

[0513]

[0514] <Manufacturing Example 15> Manufacturing of Example 27 (compound represented by the chemical formula 29; IK15k)

[0515] The compound of Example 27 was synthesized according to the following reaction scheme 20.

[0516] [Reaction Formula 20]

[0517]

[0518] Step 1. Preparation of 1-(2-(4-methoxyphenyl)-2-oxoethyl)-5-methyl-2-(p-tolyl)-1H-pyrrole-3-carbonitrile

[0519] 5-Methyl-2-(p-tolyl)-1H-pyrrole-3-carbonitrile (250 mg, 1.28 mmol), 2-bromo-1-(4-methoxyphenyl)ethan-1-one (440 mg, 1.92 mmol), which are intermediates of step 2 of Example 26, were mixed in acetonitrile (15 mL), and cesium carbonate (832 mg, 2.56 mmol) was added. The mixture was stirred at 60°C for 24 hours. Water (10 mL) was added to the reaction mixture, and extracted with dichloromethane (20 mL × 3). The organic layer was collected, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 15:85) to obtain 1-(2-(4-methoxyphenyl)-2-oxoethyl)-5-methyl-2-(p-tolyl)-1H-pyrrole-3-carbonitrile (120 mg, yield 27%) as a yellow solid.

[0520]

[0521] Step 2. Preparation of 6-(4-methoxyphenyl)-3,8-dimethylpyrrolo[2,1-a]isoquinoline-1-carbonitrile (Example 27)

[0522] 1-(2-(4-Methoxyphenyl)-2-oxoethyl)-5-methyl-2-(p-tolyl)-1H-pyrrole-3-carbonitrile (50 mg, 0.15 mmol), trifluoromethanesulfonic acid (225 mg, 1.5 mmol), and 1,2-dichloroethane (2 mL) were mixed and stirred at room temperature for 2 h. Water (4 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane (10 mL × 3). The organic layer was combined, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 8:92) to obtain 6-(4-methoxyphenyl)-3,8-dimethylpyrrolo[2,1-a]isoquinoline-1-carbonitrile (10.7 mg, yield 22%) as a white solid.

[0523] LCMS (ESI-MS): Calculated molecular weight C 22 H 18 N2O 326.1 m / z, observed value 326.8 [M+H] + .

[0524] 1 H NMR (400 MHz, CDCl3) δ8.79 (d, J = 8.4 Hz, 1H), 7.56 (s, 1H), 7.48-7.33 (m, 4H), 7.09-7.01 (m, 2H), 6.72 (s, 1H), 3.91 (s, 3H), 3.00-2.25 (m, 6H).

[0525]

[0526] <Manufacturing Example 16> Manufacture of Example 28 (compound represented by the chemical formula 30; AMD-D-270)

[0527] The compound of Example 28 was synthesized according to the following reaction scheme 21.

[0528] [Reaction Formula 21]

[0529]

[0530] Step 1. Preparation of 1-(2-(4-fluorophenyl)-2-oxoethyl)-5-methyl-2-(p-tolyl)-1H-pyrrole-3-carbonitrile

[0531] 5-Methyl-2-(p-tolyl)-1H-pyrrole-3-carbonitrile (250 mg, 1.28 mmol), an intermediate of step 2 of Example 26, and 2-bromo-1-(4-fluorophenyl)ethan-1-one (416 mg, 1.92 mmol) were mixed in acetonitrile (15 mL), and cesium carbonate (832 mg, 2.56 mmol) was added. The mixture was stirred at 60°C for 24 hours. Water (10 mL) was added to the reaction mixture, and extracted with dichloromethane (20 mL × 3). The organic layer was collected, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 15:85) to obtain 1-(2-(4-fluorophenyl)-2-oxoethyl)-5-methyl-2-(p-tolyl)-1H-pyrrole-3-carbonitrile (110 mg, yield 26%) as a yellow solid.

[0532]

[0533] Step 2. Preparation of 6-(4-fluorophenyl)-3,8-dimethylpyrrolo[2,1-a]isoquinoline-1-carbonitrile (Example 28)

[0534] 1-(2-(4-Fluorophenyl)-2-oxoethyl)-5-methyl-2-(p-tolyl)-1H-pyrrole-3-carbonitrile (50 mg, 0.15 mmol), trifluoromethanesulfonic acid (225 mg, 1.5 mmol), and 1,2-dichloroethane (2 mL) were mixed and stirred at room temperature for 2 hours. Water (4 mL) was added to the reaction mixture, and extracted with dichloromethane (10 mL × 3). The organic layer was combined, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 9:91) to obtain 6-(4-fluorophenyl)-3,8-dimethylpyrrolo[2,1-a]isoquinoline-1-carbonitrile (10.9 mg, yield 23%) as a white solid.

[0535] LCMS (ESI-MS): Calculated molecular weight C 21 H 15 FN2314.1 m / z, observed value 314.7 [M+H] + .

[0536] 1 H NMR (400 MHz, CDCl3) δ8.80 (d, J = 8.4 Hz, 1H), 7.56 (s, 1H), 7.50-7.38 (m, 3H), 7.33 (s, 1H), 7.25-7.14 (m, 2H), 6.74 (s, 1H), 2.48-2.43 (m, 6H).

[0537]

[0538] <Manufacturing Example 17> Manufacture of Example 29 (compound represented by the chemical formula 31; AMD-D-272)

[0539] The compound of Example 29 was synthesized according to the following reaction scheme 22.

[0540] [Reaction Formula 22]

[0541]

[0542] Step 1. Preparation of 5-methyl-2-(4-methylcyclohexa-1,3-dien-1-yl)-1-(2-oxo-2-(thiophen-2-yl)ethyl)-1H-pyrrole-3-carbonitrile

[0543] 5-Methyl-2-(p-tolyl)-1H-pyrrole-3-carbonitrile (250 mg, 1.28 mmol), 2-bromo-1-(thiophen-2-yl)ethan-1-one (394 mg, 1.92 mmol), which are intermediates of step 2 of Example 26, were mixed in acetonitrile (15 mL), and cesium carbonate (832 mg, 2.56 mmol) was added. The mixture was stirred at 60°C for 24 hours. Water (10 mL) was added to the reaction mixture, and extracted with dichloromethane (20 mL × 3). The organic layer was collected, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 20:80) to obtain 5-methyl-2-(4-methylcyclohexa-1,3-dien-1-yl)-1-(2-oxo-2-(thiophen-2-yl) ethyl)-1H-pyrrole-3-carbonitrile (100 mg, yield 24%) as a yellow solid.

[0544]

[0545] Step 2. Preparation of 3,8-dimethyl-6-(thiophen-2-yl)pyrrolo[2,1-a]isoquinoline-1-carbonitrile (Example 29)

[0546] 5-Methyl-2-(4-methylcyclohexa-1,3-dien-1-yl)-1-(2-oxo-2-(thiophen-2-yl) ethyl)-1H-pyrrole-3-carbonitrile (50 mg, 0.16 mmol), trifluoromethanesulfonic acid (240 mg, 1.6 mmol), and 1,2-dichloroethane (2 mL) were mixed and stirred at room temperature for 2 h. Water (4 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane (10 mL × 3). The organic layer was combined, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 9:91) to obtain 3,8-dimethyl-6-(thiophen-2-yl)pyrrolo[2,1-a]isoquinoline-1-carbonitrile (10.4 mg, yield 22%) as a white solid.

[0547] LCMS (ESI-MS): Calculated molecular weight C 19 H 14 N2S 302.1 m / z, observed value 302.6 [M+H] + .

[0548] 1 H NMR (400 MHz, CDCl3) δ8.78 (d, J = 8.4 Hz, 1H), 7.73-7.67 (m, 2H), 7.53-7.42 (m, 2H), 7.27-7.11 (m, 2H), 6.73 (s, 1H), 2.63-2.22 (m, 6H).

[0549]

[0550] <Manufacturing Example 18> Manufacture of Example 30 (compound represented by the chemical formula 32; AMD-D-273)

[0551] The compound of Example 30 was synthesized according to the following reaction scheme 23.

[0552] [Reaction Formula 23]

[0553]

[0554] Step 1. Preparation of 2-(3-fluorobenzoyl)-4-oxopentanenitrile

[0555] 1-Chloropropan-2-one (2.19 g, 23.65 mmol) was added to 3-(3-fluorophenyl)-3-oxopropanenitrile (3.5 g, 21.5 mmol), potassium carbonate (7.42 g, 23.65 mmol), and ethanol (50 mL). The mixture was stirred at 25°C for 2 h. Water (100 mL) was added to the reaction mixture, and extracted with dichloromethane (60 mL × 3). The organic layer was combined, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 10:90) to give 2-(3-fluorobenzoyl)-4-oxopentanenitrile (4.6 g, yield 97%) as a yellow oily substance.

[0556]

[0557] Step 2. Preparation of 2-(3-fluorophenyl)-5-methyl-1H-pyrrole-3-carbonitrile

[0558] 2-(3-Fluorobenzoyl)-4-oxopentanenitrile (4.7 g, 21.4 mmol), ammonium acetate (3.3 g, 42.8 mmol), and ethanol (50 mL) were mixed and stirred at 80°C for 5 hours. The reaction mixture was concentrated, and the residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 10:90) to obtain 2-(3-fluorophenyl)-5-methyl-1H-pyrrole-3-carbonitrile (2.84 g, yield 66%) as a yellow solid.

[0559]

[0560] Step 3. Preparation of 2-(3-fluorophenyl)-5-methyl-1-(2-oxo-2-phenylethyl)-1H-pyrrole-3-carbonitrile

[0561] 2-(3-Fluorophenyl)-5-methyl-1H-pyrrole-3-carbonitrile (300 mg, 1.5 mmol), 2-bromo-1-phenylethan-1-one (450 mg, 2.25 mmol), and cesium carbonate (975 mg, 3.0 mmol) were mixed in acetonitrile (17 mL) and stirred at 60°C for 24 h. Water (20 mL) was added to the reaction mixture, and extracted with dichloromethane (40 mL × 3). The organic layer was combined, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 15:85) to obtain 2-(3-fluorophenyl)-5-methyl-1-(2-oxo-2-phenylethyl)-1H-pyrrole-3-carbonitrile (120 mg, yield 38%) as a yellow solid.

[0562]

[0563] Step 4. Preparation of 9-fluoro-3-methyl-6-phenylpyrrolo[2,1-a]isoquinoline-1-carbonitrile (Example 30)

[0564] 2-(3-Fluorophenyl)-5-methyl-1-(2-oxo-2-phenylethyl)-1H-pyrrole-3-carbonitrile (60 mg, 0.19 mmol), trifluoromethanesulfonic acid (285 mg, 1.9 mmol), and 1,2-dichloroethane (3 mL) were mixed and stirred at room temperature for 2 hours. Water (4 mL) was added to the reaction mixture, and extracted with dichloromethane (15 mL × 3). The organic layer was combined, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 10:90) to obtain 9-fluoro-3-methyl-6-phenylpyrrolo[2,1-a]isoquinoline-1-carbonitrile (10.9 mg, yield 19%) as a white solid.

[0565] LCMS (ESI-MS): Calculated molecular weight C 20 H 13 FN2300.1 m / z, observed value 300.7 [M+H] + .

[0566] 1 H NMR (400 MHz, CDCl3) δ8.55 (dd, J = 7.2, 2.4 Hz, 1H), 7.73-7.38 (m, 7H), 7.24-7.11 (m, 1H), 6.78 (s, 1H), 2.49 (s, 3H).

[0567]

[0568] <Manufacturing Example 19> Manufacture of Example 31 (compound represented by the chemical formula 33; AMD-D-274)

[0569] The compound of Example 31 was synthesized according to the following reaction scheme 24.

[0570] [Reaction Formula 24]

[0571]

[0572] Step 1. Preparation of 2-(3-fluorophenyl)-1-(2-(4-methoxyphenyl)-2-oxoethyl)-5-methyl-1H-pyrrole-3-carbonitrile

[0573] 2-(3-Fluorophenyl)-5-methyl-1H-pyrrole-3-carbonitrile (270 mg, 1.35 mmol), 2-bromo-1-(4-methoxyphenyl)ethan-1-one (462 mg, 2.03 mmol), which are intermediates of step 2 of Example 30, were mixed in acetonitrile (15 mL), and cesium carbonate (877 mg, 2.7 mmol) was added. The mixture was stirred at 60°C for 24 hours. Water (20 mL) was added to the reaction mixture, and extracted with dichloromethane (40 mL × 3). The organic layer was collected, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 16:84) to obtain 2-(3-fluorophenyl)-1-(2-(4-methoxyphenyl)-2-oxoethyl)-5-methyl-1H-pyrrole-3-carbonitrile (150 mg, yield 43%) as a yellow solid.

[0574]

[0575] Step 2. Preparation of 9-fluoro-6-(4-methoxyphenyl)-3-methylpyrrolo[2,1-a]isoquinoline-1-carbonitrile (Example 31)

[0576] 2-(3-Fluorophenyl)-1-(2-(4-methoxyphenyl)-2-oxoethyl)-5-methyl-1H-pyrrole-3-carbonitrile (100 mg, 0.29 mmol), trifluoromethanesulfonic acid (435 mg, 2.9 mmol), and 1,2-dichloroethane (5 mL) were mixed and stirred at room temperature for 2 h. Water (5 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane (15 mL × 3). The organic layer was combined, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 12:88) to obtain 9-fluoro-6-(4-methoxyphenyl)-3-methylpyrrolo[2,1-a]isoquinoline-1-carbonitrile (AMD-D-274) (10.6 mg, yield 11%) as a white solid.

[0577] LCMS (ESI-MS): Calculated molecular weight C 21 H 15 FN2O 330.1 m / z, observed value 330.7 [M+H] + .

[0578] 1 H NMR (400 MHz, CDCl3) δ8.53 (dd, J = 9.6, 2.4 Hz, 1H), 7.65-7.62 (m, 1H), 7.56 (s, 1H), 7.45-7.34 (m, 2H), 7.24-7.13 (m, 1H), 7.10-7.02 (m, 2H), 6.77 (s, 1H), 3.91 (s, 3H), 2.49 (s, 3H).

[0579]

[0580] <Manufacturing Example 20> Manufacture of Example 32 (compound represented by the chemical formula 34; AMD-D-275)

[0581] The compound of Example 32 was synthesized according to the following reaction scheme 25.

[0582] [Reaction Formula 25]

[0583]

[0584] Step 1. Preparation of 2-(3-fluorophenyl)-1-(2-(4-fluorophenyl)-2-oxoethyl)-5-methyl-1H-pyrrole-3-carbonitrile

[0585] 2-(3-Fluorophenyl)-5-methyl-1H-pyrrole-3-carbonitrile (270 mg, 1.35 mmol), 2-bromo-1-(4-fluorophenyl)ethan-1-one (440 mg, 2.03 mmol), which are intermediates of step 2 of Example 30, were mixed in acetonitrile (15 mL), and cesium carbonate (877 mg, 2.7 mmol) was added. The mixture was stirred at 60°C for 24 hours. Water (20 mL) was added to the reaction mixture, and extracted with dichloromethane (40 mL × 3). The organic layer was collected, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 16:84) to obtain 2-(3-fluorophenyl)-1-(2-(4-fluorophenyl)-2-oxoethyl)-5-methyl-1H-pyrrole-3-carbonitrile (100 mg, yield 22%) as a yellow solid.

[0586]

[0587] Step 2. Preparation of 9-fluoro-6-(4-fluorophenyl)-3-methylpyrrolo[2,1-a]isoquinoline-1-carbonitrile (Example 32)

[0588] 2-(3-Fluorophenyl)-1-(2-(4-fluorophenyl)-2-oxoethyl)-5-methyl-1H-pyrrole-3-carbonitrile (100 mg, 0.30 mmol), trifluoromethanesulfonic acid (450 mg, 3.0 mmol), and 1,2-dichloroethane (5 mL) were mixed and stirred at room temperature for 2 h. Water (5 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane (15 mL × 3). The organic layer was combined, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 13:87) to obtain 9-fluoro-6-(4-fluorophenyl)-3-methylpyrrolo[2,1-a]isoquinoline-1-carbonitrile (10.6 mg, yield 11%) as a white solid.

[0589] LCMS (ESI-MS): Calculated molecular weight C 20 H 12 F2N2318.1 m / z, observed value 318.7 [M+H] + .

[0590] 1 H NMR (400 MHz, CDCl3) δ8.54 (dd, J = 9.6, 2.4 Hz, 1H), 7.58-7.54 (m, 2H), 7.49-7.42 (m, 2H), 7.28-7.10 (m, 3H), 6.78 (s, 1H), 2.50 (s, 3H).

[0591]

[0592] <Manufacturing Example 21> Manufacture of Example 33 (compound represented by the chemical formula 35; AMD-D-277)

[0593] The compound of Example 33 was synthesized according to the following reaction scheme 26.

[0594] [Reaction Formula 26]

[0595]

[0596] Step 1. Preparation of 2-(3-fluorophenyl)-5-methyl-1-(2-oxo-2-(thiophen-2-yl)ethyl)-1H-pyrrole-3-carbonitrile

[0597] 2-(3-Fluorophenyl)-5-methyl-1H-pyrrole-3-carbonitrile (270 mg, 1.35 mmol), 2-bromo-1-(thiophen-2-yl)ethan-1-one (416 mg, 2.03 mmol), which are intermediates of step 2 of Example 30, were mixed in acetonitrile (15 mL), and cesium carbonate (877 mg, 2.7 mmol) was added. The mixture was stirred at 60°C for 24 hours. Water (20 mL) was added to the reaction mixture, and extracted with dichloromethane (40 mL × 3). The organic layer was collected, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 16:84) to obtain 2-(3-fluorophenyl)-5-methyl-1-(2-oxo-2-(thiophen-2-yl)ethyl)-1H-pyrrole-3-carbonitrile (150 mg, yield 46%) as a yellow solid.

[0598]

[0599] Step 2. Preparation of 9-fluoro-3-methyl-6-(thiophen-2-yl)pyrrolo[2,1-a]isoquinoline-1-carbonitril (Example 33)

[0600] 2-(3-Fluorophenyl)-5-methyl-1-(2-oxo-2-(thiophen-2-yl)ethyl)-1H-pyrrole-3-carbonitrile (80 mg, 0.25 mmol), trifluoromethanesulfonic acid (375 mg, 2.5 mmol), and 1,2-dichloroethane (3 mL) were mixed and stirred at room temperature for 2 h. Water (5 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane (15 mL × 3). The organic layer was combined, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 20:80) to obtain 9-fluoro-3-methyl-6-(thiophen-2-yl)pyrrolo[2,1-a]isoquinoline-1-carbonitrile (10.9 mg, yield 14%) as a white solid.

[0601] LCMS (ESI-MS): Calculated molecular weight C 18 H 11 FN2S 306.1 m / z, observed value 306.7 [M+H] + .

[0602] 1 H NMR (400 MHz, CDCl3) δ8.52 (dd, J = 9.6, 2.4 Hz, 1H), 7.92-7.88 (m, 1H), 7.73 (s, 1H), 7.49 (d, J = 4.8 Hz, 1H), 7.24-7.21 (m, 3H), 6.78 (s, 1H), 2.50 (s, 3H).

[0603]

[0604] <Manufacturing Example 22> Manufacture of Example 34 (compound represented by the chemical formula 36; AMD-D-278)

[0605] The compound of Example 34 was synthesized according to the following reaction scheme 27.

[0606] [Reaction Formula 27]

[0607]

[0608] Step 1. Preparation of 2-(2-fluorobenzoyl)-4-oxopentanenitrile

[0609] 1-Chloropropan-2-one (2.02 g, 21.78 mmol) was added to 3-(2-fluorophenyl)-3-oxopropanenitrile (3.23 g, 19.8 mmol), potassium carbonate (6.83 g, 49.5 mmol), and ethanol (50 mL). The mixture was stirred at 25°C for 2 h. Water (100 mL) was added to the reaction mixture, and extracted with ethyl acetate (100 mL × 3). The organic layer was combined, washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 10:90) to give 2-(2-fluorobenzoyl)-4-oxopentanenitrile (4.3 g, yield 98%) as a yellow oil.

[0610]

[0611] Step 2. Preparation of 2-(2-fluorophenyl)-5-methyl-1H-pyrrole-3-carbonitrile

[0612] 2-(2-Fluorobenzoyl)-4-oxopentanenitrile (4.66 g, 21.3 mmol), ammonium acetate (3.28 g, 42.6 mmol), and ethanol (50 mL) were mixed and stirred at 80°C for 5 hours. The reaction mixture was concentrated and purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 15:85) to obtain 2-(2-fluorophenyl)-5-methyl-1H-pyrrole-3-carbonitrile (3.04 g, yield 71%) as a yellow solid.

[0613]

[0614] Step 3. Preparation of 2-(2-fluorophenyl)-5-methyl-1-(2-oxo-2-phenylethyl)-1H-pyrrole-3-carbonitrile

[0615] 2-(2-Fluorophenyl)-5-methyl-1H-pyrrole-3-carbonitrile (200 mg, 0.999 mmol) and 2-bromo-1-phenylethan-1-one (238.6 mg, 1.20 mmol) were dissolved in acetonitrile (10 mL), and cesium carbonate (651 mg, 1.998 mmol) was added. The mixture was stirred at 60°C for 16 h. Water (30 mL) was added to the reaction mixture, and it was extracted with dichloromethane (30 mL × 3). The organic layer was washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 15:85) to obtain 2-(2-fluorophenyl)-5-methyl-1-(2-oxo-2-phenylethyl)-1H-pyrrole-3-carbonitrile (230 mg, yield 72%) as a yellow oil.

[0616]

[0617] Step 4. Preparation of 10-fluoro-3-methyl-6-phenylpyrrolo[2,1-a]isoquinoline-1-carbonitrile (Example 34)

[0618] 2-(2-Fluorophenyl)-5-methyl-1-(2-oxo-2-phenylethyl)-1H-pyrrole-3-carbonitrile (100 mg, 0.314 mmol) was dissolved in 1,2-dichloroethane (8 mL), and then trifluoromethanesulfonic acid (235.7 mg, 1.57 mmol) was added. The mixture was stirred at 60°C for 1 h. Water (20 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane (20 mL × 3). The organic layer was washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 8:92) to obtain 10-fluoro-3-methyl-6-phenylpyrrolo[2,1-a]isoquinoline-1-carbonitrile (10.6 mg, yield 11%) as a white solid.

[0619] LCMS (ESI-MS): Calculated molecular weight C 20 H 13 FN2300.1 m / z, observed value 301.1 [M+H] + .

[0620] 1 H NMR (400 MHz, CDCl3) δ7.64 (s, 1H), 7.56-7.46 (m, 5H), 7.43-7.39 (m, 2H), 7.38-7.33 (m, 1H), 6.90 (s, 1H), 2.50 (s, 3H).

[0621]

[0622] <Manufacturing Example 23> Manufacture of Example 35 (compound represented by the chemical formula 37; AMD-D-279)

[0623] The compound of Example 35 was synthesized according to the following reaction scheme 28.

[0624] [Reaction Formula 28]

[0625]

[0626] Step 1. Preparation of 2-(2-fluorophenyl)-1-(2-(4-methoxyphenyl)-2-oxoethyl)-5-methyl-1H-pyrrole-3-carbonitrile

[0627] 2-(2-fluorophenyl)-5-methyl-1H-pyrrole-3-carbonitrile (200 mg, 0.999 mmol), 2-bromo-1-(4-methoxyphenyl)ethan-1-one (343.23 mg, 1.50 mmol), which are intermediates of step 2 of Example 34, were dissolved in acetonitrile (10 mL), and cesium carbonate (651 mg, 1.998 mmol) was added. The mixture was stirred at 60°C for 16 hours. Water (30 mL) was added to the reaction mixture, and extracted with dichloromethane (30 mL × 3). The organic layer was washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 15:85) to obtain 2-(2-fluorophenyl)-1-(2-(4-methoxyphenyl)-2-oxoethyl)-5-methyl-1H-pyrrole-3-carbonitrile (330 mg, yield 94%) as a yellow oil.

[0628]

[0629] Step 2. Preparation of 10-fluoro-6-(4-methoxyphenyl)-3-methylpyrrolo[2,1-a]isoquinoline-1-carbonitrile (Example 35)

[0630] 2-(2-Fluorophenyl)-1-(2-(4-methoxyphenyl)-2-oxoethyl)-5-methyl-1H-pyrrole-3-carbonitrile (800 mg, 2.299 mmol), trifluoromethanesulfonic acid (3.448 g, 22.99 mmol), and 1,2-dichloroethane (50 mL) were mixed and stirred at 80°C for 1 h. Water (50 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane (70 mL × 3). The organic layer was washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 10:90) to obtain 10-fluoro-6-(4-methoxyphenyl)-3-methylpyrrolo[2,1-a]isoquinoline-1-carbonitrile (22 mg, yield 1%) as a white solid.

[0631] LCMS (ESI-MS): Calculated molecular weight C 21 H 15 FN2O 330.1 m / z, observed value 330.7 [M+H] + .

[0632] 1 H NMR (400 MHz, DMSO-d6) δ8.02 (s, 1H), 7.63-7.55 (m, 2H), 7.51-7.44 (m, 3H), 7.16-7.06 (m, 3H), 3.85 (s, 3H), 2.54 (s, 3H).

[0633]

[0634] <Manufacturing Example 24> Manufacture of Example 36 (compound represented by the chemical formula 38; AMD-D-280)

[0635] The compound of Example 36 was synthesized according to the following reaction scheme 29.

[0636] [Reaction Formula 29]

[0637]

[0638] Step 1. Preparation of 2-(2-fluorophenyl)-1-(2-(4-fluorophenyl)-2-oxoethyl)-5-methyl-1H-pyrrole-3-carbonitrile

[0639] 2-(2-Fluorophenyl)-5-methyl-1H-pyrrole-3-carbonitrile (200 mg, 0.999 mmol), 2-bromo-1-(4-fluorophenyl)ethan-1-one (325.19 mg, 1.50 mmol), which are intermediates of step 2 of Example 34, were dissolved in acetonitrile (10 mL), and cesium carbonate (651 mg, 1.998 mmol) was added. The mixture was stirred at 60°C for 16 hours. Water (30 mL) was added to the reaction mixture, and extracted with dichloromethane (30 mL × 3). The organic layer was washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 10:90) to obtain 2-(2-fluorophenyl)-1-(2-(4-fluorophenyl)-2-oxoethyl)-5-methyl-1H-pyrrole-3-carbonitrile (230 mg, yield 68%) as a yellow oil.

[0640]

[0641] Step 2. Preparation of 10-fluoro-6-(4-fluorophenyl)-3-methylpyrrolo[2,1-a]isoquinoline-1-carbonitrile (Example 36)

[0642] 2-(2-Fluorophenyl)-1-(2-(4-fluorophenyl)-2-oxoethyl)-5-methyl-1H-pyrrole-3-carbonitrile (100 mg, 0.297 mmol), trifluoromethanesulfonic acid (446.19 mg, 2.973 mmol), and 1,2-dichloroethane (5 mL) were mixed and stirred at 70°C for 1 h. Water (10 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane (15 mL × 3). The organic layer was washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 10:90) to obtain 10-fluoro-6-(4-fluorophenyl)-3-methylpyrrolo[2,1-a]isoquinoline-1-carbonitrile (15 mg, yield 15%) as a white solid.

[0643] LCMS (ESI-MS): Calculated molecular weight C 20 H 12 F2N2318.1 m / z, observed value 318.7 [M+H] + .

[0644] 1 H NMR (400 MHz, DMSO-d6) δ 8.08 (s, 1H), 7.62-7.57 (m, 4H), 7.46-7.35 (m, 3H), 7.08 (s, 1H), 2.54 (s, 3H).

[0645]

[0646] <Manufacturing Example 25> Manufacture of Example 37 (compound represented by the chemical formula 39; AMD-D-284)

[0647] The compound of Example 37 was synthesized according to the following reaction scheme 30.

[0648] [Reaction Formula 30]

[0649]

[0650] Step 1.2 Preparation of (4-methylbenzoyl)-4-oxo-4-phenylbutanenitrile (2-(4-methylbenzoyl)-4-oxo-4-phenylbutanenitrile)

[0651] 3-Oxo-3-(p-tolyl)propanenitrile (500 mg, 3.14 mmol) and 2-chloro-1-phenylethan-1-one (534 mg, 3.45 mmol) were mixed in ethanol (8 mL), and potassium carbonate (1085 mg, 7.85 mmol) was added under a nitrogen atmosphere. The mixture was stirred at 25°C for 2 h. Water (5 mL) was added to the reaction mixture, and extracted with ethyl acetate (15 mL × 3). The organic layer was washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 5:95) to obtain 2-(4-methylbenzoyl)-4-oxo-4-phenylbutanenitrile (260 mg, yield 29%).

[0652]

[0653] Step 2. Preparation of 5-phenyl-2-(p-tolyl)-1H-pyrrole-3-carbonitrile

[0654] 2-(4-Methylbenzoyl)-4-oxo-4-phenylbutanenitrile (260 mg, 0.94 mmol), ammonium acetate (145 mg, 1.88 mmol), and ethanol (5 mL) were mixed and stirred at 80°C for 5 h. Water (5 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane (10 mL × 3). The organic layer was washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 10:90) to give 5-phenyl-2-(p-tolyl)-1H-pyrrole-3-carbonitrile (166 mg, yield 66%) as a yellow solid.

[0655]

[0656] Step 3. Preparation of 1-(2-oxo-2-phenylethyl)-5-phenyl-2-(p-tolyl)-1H-pyrrole-3-carbonitrile

[0657] 5-Phenyl-2-(p-tolyl)-1H-pyrrole-3-carbonitrile (160 mg, 0.62 mmol) and 2-bromo-1-phenylethan-1-one (185 mg, 0.93 mmol) were dissolved in acetonitrile (6 mL), and cesium carbonate (404 mg, 1.24 mmol) was added. The mixture was stirred at 60°C for 24 h. Water (10 mL) was added to the reaction mixture, and it was extracted with dichloromethane (20 mL × 3). The organic layer was washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 15:85) to obtain 1-(2-oxo-2-phenylethyl)-5-phenyl-2-(p-tolyl)-1H-pyrrole-3-carbonitrile (160 mg, yield 68%) as a yellow solid.

[0658]

[0659] Step 4. Preparation of 8-methyl-3,6-diphenylpyrrolo[2,1-a]isoquinoline-1-carbonitrile (Example 37)

[0660] 1-(2-Oxo-2-phenylethyl)-5-phenyl-2-(p-tolyl)-1H-pyrrole-3-carbonitrile (100 mg, 0.26 mmol), trifluoromethanesulfonic acid (199 mg, 1.33 mmol), and 1,2-dichloroethane (10 mL) were mixed and stirred at 80°C for 0.5 h. Water (20 mL) was added to the reaction mixture, and extracted with dichloromethane (20 mL × 3). The organic layer was washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The residue was purified by Prep-HPLC (20 - 60% acetonitrile / 0.1% ammonia water) to give 8-methyl-3,6-diphenylpyrrolo[2,1-a]isoquinoline-1-carbonitrile (23.3 mg, yield 24%) as a white solid.

[0661] LCMS (ESI-MS): Calculated molecular weight C 26 H 18 N2358.1 m / z, observed value 359.0 [M+H] + .

[0662] 1 H NMR (400 MHz, CDCl3) δ8.11 (d, J = 7.6 Hz, 1H), 7.79 (s, 1H), 7.57-7.50 (m, 4H), 7.47-7.45 (m, 2H), 7.45-7.40 (m, 3H), 7.39-7.33 (m, 3H), 7.29 (s, 1H), 2.43 (s, 3H).

[0663]

[0664] <Manufacturing Example 26> Manufacture of Example 38 (compound represented by the chemical formula 40; AMD-D-286)

[0665] The compound of Example 38 was synthesized according to the following reaction scheme 31.

[0666] [Reaction Formula 31]

[0667]

[0668] Step 1. Preparation of 3,8-dimethyl-6-phenylpyrrolo[2,1-a]isoquinoline-1-carboxamide (Example 38)

[0669] 3,8-Dimethyl-6-phenylpyrrolo[2,1-a]isoquinoline-1-carbonitrile (Example 26, 300 mg, 1.01 mmol) and sulfuric acid (3 mL) were mixed and stirred at 25°C for 16 h. Water (10 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane (10 mL × 3). The mixture was washed, dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (methanol / dichloromethane, volume ratio 10:90) to obtain 3,8-dimethyl-6-phenylpyrrolo[2,1-a]isoquinoline-1-carboxamide (150 mg, yield 47%) as a white solid.

[0670] LCMS (ESI-MS): Calculated molecular weight C 21 H 18 N 2O 314.1 m / z, observed value 314.8 [M+H] + .

[0671] 1 H NMR (400 MHz, DMSO-d6) δ: 9.59 (d, J = 8.4 Hz, 1H), 7.80 (s, 1H), 7.62-7.45 (m, 5H), 7.35 (d, J = 8.4 Hz, 1H), 7.28 (s, 1H), 6.90 (s, 1H), 2.47 (s, 3H), 2.34 (s, 3H).

[0672]

[0673] <Manufacturing Example 27> Manufacture of Example 39 (compound represented by the chemical formula 41; AMD-D-287)

[0674] The compound of Example 39 was synthesized according to the following reaction scheme 32.

[0675] [Reaction Formula 32]

[0676]

[0677] Step 1. Preparation of ethyl 2-(4-methylbenzoyl)-4-oxopentanoate

[0678] Ethyl 3-(4-methylphenyl)-3-oxopropanoate (7 g, 33.9 mmol), sodium iodide (1.02 g, 6.70 mmol), and potassium carbonate (11.71 g, 84.7 mmol) were mixed in acetone (50 mL), and 1-chloropropan-2-one (3.45 g, 37.29 mmol) was added. The mixture was stirred at 60°C for 5 hours. The reaction mixture was diluted with water (150 mL) and extracted with ethyl acetate (100 mL × 3). The organic layer was combined, washed with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The product was purified by silica gel column chromatography (ethyl acetate / hexane, volume ratio 5:95) to obtain ethyl 2-(4-methylbenzoyl)-4-oxopentanoate (8 g, yield 90%) as a yellow solid.

[0679]

[0680] Step 2. Preparation of ethyl 5-methyl-2-(p-tolyl)-1H-pyrrole-3-carboxylate

[0681] Ethyl 2-(4-methylbenzoyl)-4-oxopentanoate (8 g, 30.5 mmol) was dissolved in ethanol (80 mL), and ammonium acetate (4.70 g, 61.0 mmol) was added. The mixture was stirred at 80°C for 5 hours. The reaction mixture was concentrated and purified by silica gel column chromatography (ethyl acetate / hexane, volume ratio 10:90) to obtain ethyl 5-methyl-2-(p-tolyl)-1H-pyrrole-3-carboxylate (7 g, yield 94%) as a yellow solid.

[0682]

[0683] Step 3. Preparation of ethyl 1-(2-hydroxy-2-phenylethyl)-5-methyl-2-(p-tolyl)-1H-pyrrole-3-carboxylate

[0684] Ethyl 5-methyl-2-(p-tolyl)-1H-pyrrole-3-carboxylate (2 g, 8 mmol), 2-phenyloxirane (1.48 g, 12 mmol), and potassium tert-butoxide (2.76 g, 24 mmol) were mixed in dimethylformamide (DMF, 60 mL) and heated at 80°C for 2 h. The reaction mixture was diluted with water (100 mL) and extracted with edyl acetate (100 mL × 3). The organic layer was washed with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated. Ethyl 1-(2-hydroxy-2-phenylethyl)-5-methyl-2-(p-tolyl)-1H-pyrrole-3-carboxylate (500 mg, yield 17%) was obtained as a white solid by crystallization with a mixed solvent of ethyl acetate / hexane (1:1), filtration, and vacuum drying.

[0685]

[0686] Step 4. Preparation of ethyl 5-methyl-1-(2-oxo-2-phenylethyl)-2-(p-tolyl)-1H-pyrrole-3-carboxylate

[0687] Ethyl 1-(2-hydroxy-2-phenylethyl)-5-methyl-2-(p-tolyl)-1H-pyrrole-3-carboxylate (500 mg, 1.375 mmol) and iodoxybenzoic acid (577.84 mg, 2.063 mmol) were dissolved in ethyl acetate (10 mL) and stirred at 70°C for 16 h. The reaction mixture was diluted with water (20 mL) and extracted with ethyl acetate (20 mL × 3). The organic layer was washed with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 15:85) to obtain ethyl 5-methyl-1-(2-oxo-2-phenylethyl)-2-(p-tolyl)-1H-pyrrole-3-carboxylate (300 mg, yield 60%) as a white solid.

[0688]

[0689] Step 5. Preparation of ethyl 3,8-dimethyl-6-phenylpyrrolo[2,1-a]isoquinoline-1-carboxylate (Example 39)

[0690] Ethyl 5-methyl-2-(4-methylphenyl)-1-(2-oxo-2-phenylethyl)-pyrrole-3-carboxylate (100 mg, 0.276 mmol), trifluoromethanesulfonic acid (415.27 mg, 2.767 mmol), and 1,2-dichloroethane (5 mL) were mixed and stirred at 60°C for 1 h. The reaction mixture was diluted with water (10 mL) and extracted with dichloromethane (15 mL × 3). The organic layer was washed with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 5:95) to obtain ethyl 3,8-dimethyl-6-phenylpyrrolo[2,1-a]isoquinoline-1-carboxylate (7 mg, yield 7%) as a white solid.

[0691] LCMS (ESI-MS): Calculated value C 23 H 21NO2343.2 m / z, measured 343.8 [M+H] + .

[0692] 1 H NMR (400 MHz, CDCl₃) δ9.85 (d, J = 8.4 Hz, 1H), 7.58 (s, 1H), 7.56-7.47 (m, 5H), 7.46-7.34 (m, 2H), 7.06-7.05 (m, 1H), 4.40 (q, J = 7.2 Hz, 2H), 2.47 (s, 3H), 2.40 (s, 3H), 1.44 (t, J = 7.2 Hz, 3H).

[0693]

[0694] <Manufacturing Example 28> Manufacture of Example 40 (compound represented by the chemical formula 42; AMD-D-289)

[0695] The compound of Example 40 was synthesized according to the following reaction scheme 33.

[0696] [Reaction Formula 33]

[0697]

[0698] Step 1. Preparation of 2-(2-(2-fluoroethoxy)ethoxy)ethyl 4-methylbenzenesulfonate

[0699] 2-(2-(2-fluoroethoxy)ethoxy)ethanol (300 mg, 1.97 mmol) and aqueous sodium hydroxide solution (5 N, 1 mL) were mixed in tetrahydrofuran (10 mL), and then 4-methylbenzenesulfonyl chloride (570 mg, 3.0 mmol) was added. The mixture was stirred at 0°C for 6 h. The reaction mixture was diluted with water (15 mL) and extracted with ethyl acetate (10 mL × 3). The organic layer was washed with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 20:80) to obtain the target compound (220 mg, yield 36%) as a yellow oil.

[0700]

[0701] Step 2. Preparation of 2-(4-bromobenzoyl)-4-oxopentanenitrile

[0702] 3-(4-Bromophenyl)-3-oxopropanenitrile (6.0 g, 26.8 mmol), 1-chloropropan-2-one (4.9 g, 53.6 mmol), and potassium carbonate (7.4 g, 53.6 mmol) were mixed in ethanol (100 mL) and stirred at room temperature (25°C) for 1 hour. The reaction mixture was diluted with water (100 mL) and extracted with dichloromethane (80 mL × 3). The organic layer was washed with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 13:87) to obtain a yellow solid (4.1 g, yield 55%).

[0703]

[0704] Step 3. Preparation of 2-(4-bromophenyl)-5-methyl-1H-pyrrole-3-carbonitrile

[0705] 2-(4-Bromobenzoyl)-4-oxopentanenenitrile (4.1 g, 14.6 mmol) and ammonium acetate (2.26 g, 29.3 mmol) were mixed in ethanol (60 mL) and stirred at 80°C for 5 hours. The reaction mixture was diluted with water (80 mL) and extracted with dichloromethane (60 mL × 3). The organic layer was washed, dried, concentrated, and purified by silica gel column chromatography (EtOAc / PE, volume ratio 20:80) to obtain a yellow solid (3.0 g, yield 79%).

[0706]

[0707] Step 4. Preparation of 2-(4-bromophenyl)-5-methyl-1-(2-oxo-2-phenylethyl)-1H-pyrrole-3-carbonitrile

[0708] 2-(4-Bromophenyl)-5-methyl-1H-pyrrole-3-carbonitrile (3.0 g, 11.5 mmol), 2-bromo-1-phenylethan-1-one (4.6 g, 23.0 mmol), and cesium carbonate (7.5 g, 23.0 mmol) were mixed in acetonitrile (150 mL) and stirred at 60°C for 16 h. The reaction mixture was diluted with water (200 mL) and extracted with dichloromethane (150 mL × 3). The residue was purified by silica gel column chromatography (ethyl acetate / petroleum ether, volume ratio 40:60) to obtain a yellow solid (2.0 g, yield 46%).

[0709]

[0710] Step 5. Preparation of 8-bromo-3-methyl-6-phenylpyrrolo[2,1-a]isoquinoline-1-carbonitrile

[0711] 2-(4-Bromophenyl)-5-methyl-1-(2-oxo-2-phenylethyl)-1H-pyrrole-3-carbonitrile (2.0 g, 7.9 mmol) and trifluoromethanesulfonic acid (11.9 g, 79.0 mmol) were dissolved in 1,2-dichloroethane (150 mL) and stirred at 60°C for 6 hours. The reaction mixture was diluted with water (100 mL) and extracted with dichloromethane (80 mL × 3). The residue was purified by silica gel column chromatography (methanol / dichloromethane, volume ratio 2:98) to obtain a yellow oil (1.2 g, yield 42%).

[0712]

[0713] Step 6. Preparation of 8-(hydroxymethyl)-3-methyl-6-phenylpyrrolo[2,1-a]isoquinoline-1-carbonitrile

[0714] 8-Bromo-3-methyl-6-phenylpyrrolo[2,1-a]isoquinoline-1-carbonitrile (300 mg, 0.83 mmol), (tributylstannyl)methanol (533 mg, 1.66 mmol), and tetrakis(triphenylphosphine)palladium (92 mg, 0.08 mmol) were dissolved in dioxane (10 mL) and stirred at 80°C for 16 h under a nitrogen atmosphere. The reaction mixture was diluted with water (20 mL), extracted with dichloromethane (20 mL × 3), and purified to obtain a red solid (170 mg, yield 66%).

[0715]

[0716] Step 7. Preparation of 8-((2-(2-(2-fluoroethoxy)ethoxy)ethoxy)methyl)-3-methyl-6-phenylpyrrolo[2,1-a]isoquinoline-1-carbonitrile (Example 40)

[0717] 8-(Hydroxymethyl)-3-methyl-6-phenylpyrrolo[2,1-a]isoquinoline-1-carbonitrile (60 mg, 0.19 mmol) and sodium hydride (38 mg, 0.95 mmol) were mixed in N,N-dimethylformamide (DMF, 5 mL), and 2-(2-(2-fluoroethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (116 mg, 0.38 mmol) was added. The mixture was stirred at 0°C for 2 h, diluted with water (10 mL), and extracted with ethyl acetate (10 mL × 3). The product was purified by prep-HPLC (Gemini 5 μm C18 column, 150×21.2 mm, acetonitrile / purified water 30-90% containing 0.1% formic acid) to obtain a yellow solid (10.3 mg, yield 12%).

[0718] LCMS (ESI-MS): Calculated value C 27 H 27 FN2O3446.2 m / z, measured 446.8 [M+H] + .

[0719] 1 H NMR (400 MHz, CDCl3): δ8.89 (d, J = 8.4 Hz, 1H), 7.75-7.43 (m, 8H), 6.76 (s, 1H), 4.70-4.55 (m, 3H), 4.53-4.44 (m, 1H), 3.82-3.74 (m, 1H), 3.74-3.54 (m, 9H), 2.49 (s, 3H).

[0720]

[0721] <Manufacturing Example 29> Manufacture of Example 41 (compound represented by the chemical formula 43; AMD-D-271)

[0722] The compound of Example 41 was synthesized according to the following reaction scheme 34.

[0723] [Reaction Formula 34]

[0724]

[0725] Step 1. Preparation of 2-bromo-1-(pyridin-3-yl)ethan-1-ol

[0726] Sodium borohydride (1.6 g, 42 mmol) was suspended in methanol (90 mL) under stirring, and a solution of 2-bromo-1-(pyridin-3-yl)ethan-1-one hydrobromide (3.0 g, 10.5 mmol) suspended in methanol (60 mL) was slowly added thereto at -60 °C over 1 h. After the addition was complete, the reaction mixture was diluted with water (150 mL) and extracted with ethyl acetate (100 mL × 3). The organic layer was concentrated under reduced pressure to obtain 600 mg (28% yield) of 2-bromo-1-(pyridin-3-yl)ethan-1-ol. Since this compound was unstable at 25 °C, it was used immediately in the next step without purification.

[0727]

[0728] Step 2. Preparation of 1-(2-hydroxy-2-(pyridin-3-yl)ethyl)-5-methyl-2-(p-tolyl)-1H-pyrrole-3-carbonitrile

[0729] 5-Methyl-2-(p-tolyl)-1H-pyrrole-3-carbonitrile (400 mg, 2.04 mmol) and tert-butoxypotassium (120 mg, 4.08 mmol) were dissolved in ethanol (10 mL), and 2-bromo-1-(pyridin-3-yl)ethan-1-ol (494 mg, 2.45 mmol) prepared in Step 1 was added. The reaction mixture was stirred at 80°C for 2 hours. After the reaction was completed, the reaction solution was diluted with water (10 mL) and extracted with ethyl acetate (10 mL × 3). The organic layer was washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography (ethyl acetate / petroleum ether = volume ratio 1:1) to obtain 160 mg (25% yield) of the target compound, 1-(2-hydroxy-2-(pyridin-3-yl)ethyl)-5-methyl-2-(p-tolyl)-1H-pyrrole-3-carbonitrile, in the form of a yellow oil.

[0730]

[0731] Step 3. Preparation of 5-methyl-1-(2-oxo-2-(pyridin-3-yl)ethyl)-2-(p-tolyl)-1H-pyrrole-3-carbonitrile

[0732] The compound prepared in Step 2 (140 mg, 0.38 mmol) and Dess-Martin oxidizer (240 mg, 0.57 mmol) were dissolved in dichloromethane (5 mL) and reacted at 0 °C for 2 hours. The reaction mixture was diluted with water (10 mL) and extracted with dichloromethane (10 mL × 3). The organic layer was washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography (ethyl acetate / petroleum ether = volume ratio 4:6) to obtain 60 mg (43% yield) of the target compound, 5-methyl-1-(2-oxo-2-(pyridin-3-yl)ethyl)-2-(p-tolyl)-1H-pyrrole-3-carbonitrile, as a white solid.

[0733]

[0734] Step 4. Preparation of 3,8-dimethyl-6-(pyridin-3-yl)pyrrolo[2,1-a]isoquinoline-1-carbonitrile (Example 41)

[0735] The compound (50 mg, 0.15 mmol) prepared in the above step 3 and trifluoromethanesulfonic acid (112 mg, 0.75 mmol) were dissolved in 1,2-dichloroethane (5 mL) and reacted at room temperature (25 ℃) for 2 hours. After the reaction, the reaction solution was diluted with water (10 mL) and extracted with dichloromethane (10 mL × 3). The organic layer was washed with a saturated sodium chloride solution, dried over sodium sulfate, and concentrated under reduced pressure. The concentrate was purified by prep-HPLC (Gemini 5 μm C18 column, 150 × 21.2 mm, mobile phase: 30–90% acetonitrile / water containing 0.1% formic acid) to give the final compound, 3,8-dimethyl-6-(pyridin-3-yl)pyrrolo[2,1-a]isoquinoline-1-carbonitrile (10.2 mg). (23% yield) Obtained in the form of a white solid.

[0736] LCMS (ESI-MS): Measured molecular weight C 20 H 15 N3, calculated 297.1, measured [M+H]+ = 297.6.

[0737] 1 H NMR (400 MHz, CDCl3): δ8.95-8.43 (m, 3H), 7.98-7.87 (m, 1H), 7.68-7.53 (m, 2H), 7.50-7.48 (m, 1H), 7.24 (s, 1H), 6.76 (s, 1H), 2.49 (s, 3H), 2.44 (s, 3H).

[0738]

[0739] <Experimental Example 1> Optical properties of IK15 for detection of Aβ oligomers

[0740] To evaluate the potential of the 41 compounds of the present invention (IK15 compounds: Examples 1 to 13, 22, 26 and 27; and AMD compounds: Examples 14 to 21, 23 to 25 and 28 to 41) as imaging agents for detecting Aβ oligomers, the changes in the IK15 fluorescence spectra in Aβ monomers (mAβ), Aβ oligomers (oAβ) and Aβ fibrils (fAβ) were analyzed, respectively.

[0741] To prepare monomeric Aβ samples, Aβ aggregates were treated with 20 mM 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS), which dissociates Aβ aggregates into monomeric forms. To prepare oligomeric and fibrillar Aβ, Aβ peptides were incubated according to a previously published Aβ aggregation protocol (Lee, HY et al. Amyloid Against Amyloid: Dimeric Amyloid Fragment Ameliorates Cognitive Impairments by Direct Clearance of Oligomers and Plaques. Angew Chem Int Ed Engl62, e202210209 (2023).).

[0742] Emission scan analysis results confirmed that the emission spectra of candidate compounds mixed with Aβ in IK15o or IK15p had lower fluorescence intensities than the emission spectra of the substances themselves. Among the IK15 compounds, for the remaining 14 IK15 substances, excluding the above substances, it was observed that the emission spectra further increased when combined with Aβ monomers, Aβ oligomers, or Aβ fibrils. Among them, it was confirmed that the emission spectra of IK15a, IK15b, IK15c, IK15d, IK15e, IK15g, IK15j, IK15k, IK15l, and IK15n substances specifically increased the most when combined with Aβ oligomers (Fig. 1A, Fig. 1B, and Fig. 3A).

[0743] In addition, among the AMD compounds, it was confirmed that the fluorescence emission intensity of AMD-D-255 specifically increased when bound to Aβ oligomers, similar to the IK-15j compound. Compounds AMD-D-253, AMD-D-254, AMD-D-257, AMD-D-264, AMD-D-272, AMD-D-273, AMD-D-277, AMD-D-278, AMD-D-279, and AMD-D-280 had low fluorescence emission intensities alone, but when bound to Aβ monomers, Aβ oligomers, and Aβ fibrils, the fluorescence signal specifically increased. In addition, it was confirmed that compound AMD-D-270 was an Aβ-selective fluorescent substance that showed almost no fluorescence when alone, but specifically fluoresced only when bound to Aβ monomers, Aβ oligomers, and Aβ fibrils (Figs. 1C to 1F).

[0744] In addition, to analyze the fluorescence changes of 16 IK15 substances over time, the fluorescence intensity of the candidate compounds mixed with Aβ monomers (mAβ), Aβ oligomers (oAβ), and Aβ fibrils (fAβ) was analyzed for 24 hours. Over time, the fluorescence intensity decreased due to the quenching phenomenon of the fluorescent substances, but when bound to Aβ, it was confirmed that the form was stably maintained and the fluorescence intensity decreased slightly. Consistent with the previous emission spectrum data, when IK15a, IK15b, IK15c, IK15d, IK15e, IK15g, IK15j, IK15k, IK15l, or IK15n substances were bound to Aβ oligomers, the fluorescence intensity decreased slightly and a stable fluorescence intensity was maintained (Fig. 2A, Fig. 2B, and Fig. 3B).

[0745] Furthermore, considering that EPPS may interfere with the interaction between IK15j and Aβ monomers, Aβ monomers and oligomers were immobilized on plates according to the Aβ-immobilization plate protocol of Reference Example 2, and then incubated with IK15. Briefly, 10 μM each of Aβ monomer (mAβ) and Aβ oligomer (oAβ) samples were immobilized on plates, and the fluorescence intensity was measured after treating with IK15j (100 μM) for 24 h, while blank wells were treated with only IK15j (100 μM) without immobilizing Aβ species. As a result, in contrast to Aβ monomers, the fluorescence signal of IK15j was significantly increased when Aβ oligomers were used (Fig. 3C). These results confirmed that there was a specific interaction between IK15j and Aβ oligomers.

[0746]

[0747] <Experimental Example 2> Aβ 1-42 Specificity of IK15 for detecting oligomers

[0748] Aβ peptides exhibit variations in length or N- / C-terminal modifications, and in the brains of AD patients, Aβ 1-40 , Aβ1-42 and pyroglutamate Aβ 3-42 (Aβ pE3-42 ) is the most commonly found. Aβ 1-40 and Aβ 1-42 The difference between Aβ 1-42 The presence of two additional residues at the C-terminus of Aβ. pE3-42 is Aβ 1-42 It is produced through a multistep protein modification, in which glutamate at the third position of the N-terminus is converted to pyroglutamate.

[0749] Here, Aβ mutant oligomers were prepared and incubated with IK15j to investigate the selectivity of the compounds toward Aβ mutants. Briefly, oligomeric Aβ 1-40 , Aβ 1-42 and Aβ pE3-42 Each 10 μM sample was treated with IK15j (10 μM), and the fluorescence intensity of each Aβ mutant sample was measured. As a result, the fluorescence intensity of IK15j was higher than that of Aβ 1-40 and Aβ pE3-42 Aβ compared to oligomers 1-42 There was a significant increase in oligomers (Fig. 3D).

[0750] Considering that aggregation of tau protein is one of the major pathological features of AD, Aβ 1-42And the selectivity of IK15j for the tau domain was further confirmed. Tau protein has four repeat domains, and repeat domain 3 is related to the microtubule-binding domain of tau protein (Le, L. et al. Self-Aggregating Tau Fragments Recapitulate Pathologic Phenotypes and Neurotoxicity of Alzheimer's Disease in Mice. Adv Sci (Weinh), e2302035 (2023). Repeat domain 3 is a core region of tau aggregates, so IK15j can be targeted to the tau domain or Aβ 1-42 were cultured with Aβ and the shift in fluorescence intensity was compared. Briefly, Aβ 1-42 Each of the tau aggregate samples was treated with IK15j, and the fluorescence intensity was measured. As a result, compared with the tau domain, the signal of IK15j was higher than that of Aβ 1-42 was increased in the presence of oligomers (Fig. 3E). These results indicate that IK15j inhibits Aβ 1-42 It indicates that it binds selectively to oligomers.

[0751]

[0752] <Experimental Example 3> Aβ 1-42 Identification of the IK15 binding site in oligomers

[0753] Aβ aggregates are considered a neuropathogenic biomarker of AD, but the transient and polymorphic nature of Aβ makes it difficult to identify target sites for Aβ imaging probes. Aβ 1-42 To identify the IK15 binding site of the oligomer, experiments were performed according to Reference Examples 2 and 3. Briefly, Aβ 1-42 , Aβ n-(n+5) Thirty-seven hexameric fragments (n = 1-37) were prepared and fixed on a plate to prepare MAP. IK15 is Aβ 1-42 A specific sequence or Aβ 1-42To investigate whether the structure of the oligomer is targeted, two conditions of MAP were prepared. First, to confirm the targeting sequence of IK15j, Aβ was prepared in MAP. n-(n+5) + treated with IK15j for 24 hours. Second, additional Aβ was added to MAP to induce Aβ-Aβ formation. 1-42 The peptides were incubated for 8 hours. Afterwards, Aβ 1-42 To understand the targeting site of the compound in the oligomer, IK15j was treated for 24 h (Fig. 3F).

[0754] As a result, Aβ 1-42 Compared to , 10 Aβ fragments (Aβ 14-19 Aβ in 23-28 ) was observed to increase the IK15j signal (Fig. 3G), and the heatmap using a two-color gradient of white and red showed the interaction between IK15j and Aβ 19-25 Although an interaction between the domains (SEQ ID NO: 1; FFAEDVG) was observed, the signal of IK15j was relatively low in all fragments (Fig. 3H), indicating that IK15j is Aβ 1-42 It indicates that IK15j does not bind to a specific sequence of Aβ. These results indicate that IK15j is a compound that 1-42 Monomeric Aβ does not form interactions with specific sequences of 1-42 You can see that it is not possible to identify .

[0755] Also, Aβ 1-42 fragments and Aβ 1-42 The fluorescence intensity change of IK15j for each complex with Aβ was measured. As a result, the total signal of IK15j was 1-42 -Aβ n-(n+5) + We observed an increase in the presence of IK15j, indicating that IK15j interacts with the structure of Aβ oligomers. This compound is Aβ 1-42 and two major domains, Aβ 19-25 (SEQ ID NO: 1; FFAEDVG) and Aβ 35-42It strongly bound to the complex with (SEQ ID NO: 2; MVGGVVIA) (Fig. 3I and Fig. 3J).

[0756] The central hydrophobic region containing the KLVFFA domain is Aβ 1-42 It is known to be the most aggregation-prone sequence in Aβ, and the GGVVIA domain is a hydrophobic C-terminal core that stabilizes hydrophobic interactions of Aβ. We confirmed that IK15j targets the hydrophobic core region of Aβ oligomers. In particular, this compound showed moderate binding strength to the N-terminus of Aβ. This hydrophilic region, Aβ 1-8 (SEQ ID NO: 3; DAEFRHDS) is known to alter the aggregation propensity of Aβ and affect the secondary structure of Aβ oligomers. IK15j can interact with both the hydrophobic and amphiphilic regions of Aβ oligomers, forming stable interactions with Aβ oligomers. Collectively, these results indicate that IK15j interacts with the structure of Aβ oligomers, and that the compound specifically targets the hydrophobic core of Aβ aggregates and the hydrophilic N-terminus of Aβ oligomers.

[0757]

[0758] <Experimental Example 4> Detection of soluble Aβ oligomers in brain lysate, CSF, and plasma of the 5XFAD mouse model.

[0759] 5XFAD, a transgenic AD mouse model, expresses human APP and presenilin-1 and exhibits key pathological features of AD, including Aβ aggregate formation in the brain.

[0760] The ex-vivo experimental protocol of IK15 using the 5XFAD mouse model is as shown in Fig. 4A. Specifically, as described in Reference Example 4, soluble Aβ species are detectable in the 5XFAD transgenic mouse model from 2 months after birth and the level increases significantly with age. Therefore, 6-month-old female 5XFAD transgenic mouse models (n=3) and age-matched female wild-type mice (n=3) were prepared. Each mouse was then sacrificed, and brain lysates (cortex and hippocampus), CSF, and plasma were obtained as described in Reference Examples 5 to 7.

[0761] Brain lysates (cortex and hippocampus) and CSF of 5XFAD mouse models or wild-type mice were treated with 16 IK15 example compounds at 50 μM each. Then, each sample was placed in a well of a 96-well half-area black microplate. At this time, the total volume of each sample was 100 μL. After treatment with the example compounds for 24 hours, the unique excitation / emission values ​​of each of the 16 substances were scanned to confirm the change in fluorescence signal, and the fluorescence scanning was detected with a microplate reader.

[0762] As a result, we observed a significant difference in fluorescence intensity between the 5XFAD transgenic mouse brain cortex group and the normal mouse brain cortex group in the brain cortex analysis treated with IK15d, IK15g, IK15j, IK15q, IK15r, or IK15s. In the analysis using IK15d, IK15j, IK15q, IK15r, or IK15s, the fluorescence intensity increased in the 5XFAD transgenic mouse brain cortex, and in the analysis using IK15g, the fluorescence intensity increased in the normal mouse brain cortex. Through this, we confirmed that the difference between the 5XFAD transgenic mouse and the normal mouse could be distinguished in the brain cortex analysis using a total of six IK15 substances, IK15d, IK15g, IK15j, IK15q, IK15r, or IK15s (Fig. 4B, Fig. 5A, and Fig. 5B).

[0763] In addition, we observed significant differences in fluorescence intensity between the 5XFAD transgenic mouse hippocampus group and the normal mouse hippocampus group in the brain hippocampus analysis treated with IK15a, IK15b, IK15j, IK15k, IK15n, IK15r, or IK15s. In the analysis using IK15a, IK15b, IK15j, IK15k, IK15r, or IK15s, the fluorescence intensity increased in the 5XFAD transgenic mouse brain hippocampus, and in the analysis using IK15n, the fluorescence intensity increased in the normal mouse brain hippocampus. Through this, it was confirmed that the difference between 5XFAD transgenic mice and normal mice could be distinguished in brain hippocampal analysis using a total of seven IK15 substances, IK15a, IK15b, IK15j, IK15k, IK15n, IK15r, or IK15s (Fig. 4C, Fig. 6A, and Fig. 6B).

[0764] In addition, we observed significant differences in fluorescence intensity between the 5XFAD transgenic mice and normal mice in cerebrospinal fluid analysis treated with IK15a, IK15j, IK15n, or IK15r. In the analysis using IK15a, IK15j, or IK15r, the fluorescence intensity increased in the cerebrospinal fluid of 5XFAD transgenic mice, and in the analysis using IK15n, the fluorescence intensity increased in the cerebrospinal fluid of normal mice. This confirmed that the differences between 5XFAD transgenic mice and normal mice could be distinguished in the cerebrospinal fluid analysis using a total of four IK15 substances, IK15a, IK15j, IK15n, or IK15r (Fig. 4D, Fig. 7A, and Fig. 7B).

[0765] In order to analyze the interaction between Aβ oligomers and IK15 substances in plasma, a total of four IK15 substances (IK15a, IK15j, IK15r, and IK15s) with high binding affinity for Aβ oligomers selected in the above experiment were used. Plasma samples were prepared from 6-month-old female 5XFAD transgenic (TG) mouse models (n = 3) and age-matched female normal (WT) mice (n = 3). After collecting each plasma sample, it was treated with EDTA and treated with each IK15 substance (50 μM) and placed into a well of a 96-well half-area black microplate. The total volume of each sample was 50 μL. After 24 hours, the unique excitation / emission values ​​of each of the 16 substances were scanned to confirm the change in fluorescence signal, and the fluorescence scanning was detected by a microplate reader.

[0766] As a result, we observed a significant difference in fluorescence intensity between the 5XFAD transgenic mouse group and the normal mouse group in plasma analysis using IK15a, IK15j, or IK15r among the four candidate substances. In the case of IK15a, the fluorescence intensity increased in the plasma of normal mice, whereas in the experiment using IK15j and IK15r, the fluorescence intensity increased in the plasma of 5XFAD transgenic mice. This confirmed that the difference between the 5XFAD transgenic mice and the normal mice could be distinguished in the plasma analysis using a total of three IK15 substances, IK15a, IK15j, or IK15r (Fig. 4E and Fig. 8).

[0767] Additionally, to confirm that IK15j precisely targets Aβ oligomers, brain staining was performed in the hippocampus and cortical regions using IK15j and the anti-Aβ antibody 6E10 as described in Reference Example 8 to visualize co-localization of signals (Fig. 4F).

[0768] As a result, as shown in Figures 4F and 4G, no signal of IK15j was observed in both the hippocampus and cortical regions, confirming that IK15j did not detect insoluble plaques in the 5XFAD mouse brain (Figure 4G).

[0769] Through this, we confirmed that IK15j selectively detects Aβ oligomers in brain lysate, CSF, and plasma of the 5XFAD mouse model.

[0770]

[0771] <Experimental Example 5> Confirmation of the interaction between IK15 and plasma Aβ

[0772] To investigate whether the compound of the present invention interacts with Aβ oligomers in plasma, human plasma (100, 10, 1, 0.1, 0.01, 0.001, 0 pg / mL) was spiked with Aβ oligomers and treated with IK15j (100 μM) for 8 hours, as described in Reference Example 9.

[0773] As a result, the signal of IK15j was significantly increased in the 1 pg / mL spiked sample compared to the blank human plasma (0 pg / mL). Compared to the control, the fluorescence intensity of the 10-100 pg / mL Aβ spiked sample showed a 52% increase (Fig. 9A).

[0774]

[0775] <Experimental Example 6> Confirmation of interaction between the compound of the present invention and plasma proteins

[0776] The solid portion of plasma, excluding water, is composed of coagulants and plasma proteins. Albumin is the most abundant protein in plasma, and fibrinogen is the main coagulant in plasma. Considering the possibility that fibrinogen or albumin may interact with AD diagnostic tools and interfere with the selectivity of the compounds, to analyze the selectivity of the compounds of the present invention toward these proteins, IK15j (25 μM) was used as described in Reference Example 10 for Aβ. 1-42, were incubated with albumin or fibrinogen, and the fluorescence signal of each sample was analyzed.

[0777] As a result, the strength of IK15j is higher than that of other proteins, Aβ 1-42 A significant increase was observed in oligomers. In contrast, IK15j was found to not interact with fibrinogen or albumin (Fig. 9B).

[0778] Additionally, albumin inhibits IK15j and Aβ 1-42 To investigate whether IK15j interferes with the binding between oligomers, as shown in Reference Example 11, Aβ 1-42 Co-culture with oligomers and albumin was performed, and the fluorescence signal of each sample was analyzed.

[0779] As a result, Aβ 1-42 Aβ compared to oligomer-only samples 1-42 Although the signals of the oligomer and albumin samples decreased, the difference was not significant (Fig. 9C).

[0780] Additionally, to confirm that albumin does not obscure the target site of IK15j, Aβ oligomer-albumin complexes and Aβ oligomers were prepared immobilized on plates as described in Reference Example 11. Subsequently, IK15j (25 μM) was treated in both wells and the signal difference was compared (Fig. 9D).

[0781] As a result, no significant difference in fluorescence intensity was observed between Aβ oligomer-albumin complexes and Aβ oligomers. Consequently, we confirmed that IK15j interacts with both Aβ oligomers and Aβ oligomer-albumin complexes in plasma, indicating that albumin does not obscure the target site of IK15j (Fig. 9E).

[0782] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. A compound represented by the following chemical formula 1 or a salt thereof: [Chemical Formula 1] In the above chemical formula 1, The above R 1a and R 1b are each independently hydrogen, deuterium, halogen, amino, hydroxy, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 2-10 Alkenyl, substituted or unsubstituted C 2-10 Alkynyl, substituted or unsubstituted C 1-10 an alkoxy, a substituted or unsubstituted 6- to 10-membered aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl; The above R 1c and R 1d are each independently hydrogen, deuterium, halogen, amino, hydroxy, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 2-10 Alkenyl, substituted or unsubstituted C 2-10 Alkynyl, substituted or unsubstituted C 1-10 an alkoxy, a substituted or unsubstituted 6- to 10-membered aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl, The above R 1c and R 1d are linked to each other to form a compound represented by the following chemical formula 2, [Chemical Formula 2] In the above chemical formula 2, the X 1 , X 2 , X 3 and X 4 are each independently C(R 4 ), nitrogen, oxygen, sulfur or phosphorus, The above R 4 is hydrogen, deuterium, halogen, amino, hydroxy, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 2-10 Alkenyl, substituted or unsubstituted C 2-10 Alkynyl, substituted or unsubstituted C 1-10 an alkoxy, a substituted or unsubstituted 6- to 10-membered aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl; The above R 2a and R 2b are each independently hydrogen, deuterium, halogen, amino, hydroxy, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 2-10 Alkenyl, substituted or unsubstituted C 2-10 Alkynyl, substituted or unsubstituted C 1-10 an alkoxy, a substituted or unsubstituted 6- to 10-membered aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl; The above R 3a and R 3b are each independently hydrogen, deuterium, halogen, amino, hydroxy, -CN, -C(O)NH2, -C(O)O(C 1-10 alkyl), substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted 6- to 10-membered aryl, or substituted or unsubstituted 5- to 10-membered heteroaryl.

2. In claim 1, The above R 1a and R 1b are each independently hydrogen, deuterium or halogen; The above R 1c and R 1d are each independently hydrogen, deuterium, halogen, amino, hydroxy, substituted or unsubstituted C 1-5 Alkyl, substituted or unsubstituted C 2-5 Alkenyl, substituted or unsubstituted C 2-5 Alkynyl, substituted or unsubstituted C 1-5 an alkoxy, a substituted or unsubstituted 6-membered aryl, or a substituted or unsubstituted 6-membered heteroaryl, The above R 1c and R 1d are linked to each other to form a compound represented by the chemical formula 2, Above X 1 , X 2 , X 3 and X 4 are each independently C(R 4 ), nitrogen, oxygen, sulfur or phosphorus, The above R 4 is hydrogen, deuterium or halogen; The above R 2a and R 2b are each independently hydrogen, deuterium, halogen, substituted or unsubstituted C 1-5 Alkyl, substituted or unsubstituted C 2-5 Alkenyl, substituted or unsubstituted C 2-5 an alkynyl, a substituted or unsubstituted 6- to 10-membered aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl; The above R 3a and R 3b are each independently hydrogen, deuterium, halogen, amino, hydroxy, -CN, -C(O)NH2, -C(O)O(C 1-5 alkyl), substituted or unsubstituted C 1-5 A compound or a salt thereof, which is an alkyl or a substituted or unsubstituted 6- to 10-membered aryl.

3. In claim 1, The above R 1a and R 1b are each independently hydrogen or halogen; The above R 1c and R 1d are each independently hydrogen, halogen, substituted or unsubstituted C 1-5 Alkyl, substituted or unsubstituted C 1-5 or alkoxy or a substituted or unsubstituted 6-membered aryl, The above R 1c and R 1d are linked to each other to form a compound represented by the chemical formula 2, Above X 1 , X 2 , X 3 and X 4 are each independently C(R 4 ) and, The above R 4 is hydrogen; The above R 2a and R 2b are each independently hydrogen, halogen, substituted or unsubstituted C 1-5 alkyl, substituted or unsubstituted 6- to 10-membered aryl, or substituted or unsubstituted 5- to 6-membered heteroaryl; The above R 3a and R 3b are each independently hydrogen, -CN, -C(O)NH2, -C(O)O(C 1-5 alkyl), substituted or unsubstituted C 1-5 A compound or a salt thereof, which is an alkyl or a substituted or unsubstituted 6-membered aryl.

4. As shown in the following reaction formula 1, A step of obtaining compound 1003 by reacting compound 1001 and compound 1002 in an organic solvent (step 1); A step of reacting compound 1003 and NH4OAc in an organic solvent to obtain compound 1004 (step 2); A step of obtaining compound 1006 by reacting compound 1005-1, compound 1005-2 or compound 1005-3; and compound 1004 in an organic solvent (step 3); and A method for producing a compound represented by the following chemical formula 1, comprising the step (step 4) of reacting compound 1006 and a catalyst in an organic solvent to obtain a compound (compound 1007) represented by the following chemical formula 1: [Reaction Formula 1] In the above reaction formula 1, The above R is R 1c or halogen; Above X p and X q are each independently hydrogen, deuterium or halogen; The above R 1a and R 1b are each independently hydrogen, deuterium, halogen, amino, hydroxy, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 2-10 Alkenyl, substituted or unsubstituted C 2-10 Alkynyl, substituted or unsubstituted C 1-10 an alkoxy, a substituted or unsubstituted 6- to 10-membered aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl; The above R 1c and R 1d are each independently hydrogen, deuterium, halogen, amino, hydroxy, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 2-10 Alkenyl, substituted or unsubstituted C 2-10 Alkynyl, substituted or unsubstituted C 1-10 an alkoxy, a substituted or unsubstituted 6- to 10-membered aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl, The above R 1c and R 1d are linked to each other to form a compound represented by the following chemical formula 2, [Chemical Formula 2] In the above chemical formula 2, the X 1 , X 2 , X 3 and X 4 are each independently C(R 4 ), nitrogen, oxygen, sulfur or phosphorus, The above R 4 is hydrogen, deuterium, halogen, amino, hydroxy, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 2-10 Alkenyl, substituted or unsubstituted C 2-10 Alkynyl, substituted or unsubstituted C 1-10 an alkoxy, a substituted or unsubstituted 6- to 10-membered aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl; The above R 2a and R 2b are each independently hydrogen, deuterium, halogen, amino, hydroxy, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 2-10 Alkenyl, substituted or unsubstituted C 2-10 Alkynyl, substituted or unsubstituted C 1-10 an alkoxy, a substituted or unsubstituted 6- to 10-membered aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl; The above R 3a and R 3b are each independently hydrogen, deuterium, halogen, amino, hydroxy, -CN, -C(O)NH2, -C(O)O(C 1-10 alkyl), substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted 6- to 10-membered aryl, or substituted or unsubstituted 5- to 10-membered heteroaryl.

5. A manufacturing method according to claim 4, wherein the organic solvents of steps 1 to 4 are each independently at least one selected from the group consisting of methanol, ethanol, propanol, acetonitrile, DCE (Dichloroethane), ethyl acetate, dimethyl sulfoxide, and dimethyl formamide.

6. A manufacturing method according to claim 4, wherein the catalyst of step 4 is at least one selected from the group consisting of TfOH (trifluoromethanesulfonic acid), MsOH (methanesulfonic acid), p-TsOH (p-toluenesulfonic acid), and Sc(OTf)3 (scandium triflate).

7. A composition for detecting amyloid-beta (Aβ) comprising a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] In the above chemical formula 1, The above R 1a and R 1b are each independently hydrogen, deuterium, halogen, amino, hydroxy, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 2-10 Alkenyl, substituted or unsubstituted C 2-10 Alkynyl, substituted or unsubstituted C 1-10 an alkoxy, a substituted or unsubstituted 6- to 10-membered aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl; The above R 1c and R 1d are each independently hydrogen, deuterium, halogen, amino, hydroxy, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 2-10 Alkenyl, substituted or unsubstituted C 2-10 Alkynyl, substituted or unsubstituted C 1-10 an alkoxy, a substituted or unsubstituted 6- to 10-membered aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl, The above R 1c and R 1d are linked to each other to form a compound represented by the following chemical formula 2, [Chemical Formula 2] In the above chemical formula 2, the X 1 , X 2 , X 3 and X 4 are each independently C(R 4 ), nitrogen, oxygen, sulfur or phosphorus, The above R 4 is hydrogen, deuterium, halogen, amino, hydroxy, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 2-10 Alkenyl, substituted or unsubstituted C 2-10 Alkynyl, substituted or unsubstituted C 1-10 an alkoxy, a substituted or unsubstituted 6- to 10-membered aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl; The above R 2a and R 2b are each independently hydrogen, deuterium, halogen, amino, hydroxy, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 2-10 Alkenyl, substituted or unsubstituted C 2-10 Alkynyl, substituted or unsubstituted C 1-10 an alkoxy, a substituted or unsubstituted 6- to 10-membered aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl; The above R 3a and R 3b are each independently hydrogen, deuterium, halogen, amino, hydroxy, -CN, -C(O)NH2, -C(O)O(C 1-10 alkyl), substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted 6- to 10-membered aryl, or substituted or unsubstituted 5- to 10-membered heteroaryl.

8. A composition for detecting amyloid-beta (Aβ) according to claim 7, wherein the Aβ is at least one selected from the group consisting of Aβ oligomers, Aβ protofibrils, Aβ fibrils, and Aβ plaques.

9. In claim 7, a composition for detecting amyloid-beta (Aβ) comprising a variant of Aβ.

10. A composition for detecting amyloid-beta (Aβ) according to claim 7, wherein the compound represented by the chemical formula 1 or a pharmaceutically acceptable salt thereof interacts with at least one domain selected from the group consisting of a domain comprising an amino acid sequence of SEQ ID NO: 1, a domain comprising an amino acid sequence of SEQ ID NO: 2, and a domain comprising an amino acid sequence of SEQ ID NO:

3.

11. A method for detecting amyloid-beta (Aβ), comprising the step of contacting a separated biological sample with a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] In the above chemical formula 1, The above R 1a and R 1b are each independently hydrogen, deuterium, halogen, amino, hydroxy, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 2-10 Alkenyl, substituted or unsubstituted C 2-10 Alkynyl, substituted or unsubstituted C 1-10 an alkoxy, a substituted or unsubstituted 6- to 10-membered aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl; The above R 1c and R 1d are each independently hydrogen, deuterium, halogen, amino, hydroxy, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 2-10 Alkenyl, substituted or unsubstituted C 2-10 Alkynyl, substituted or unsubstituted C 1-10 an alkoxy, a substituted or unsubstituted 6- to 10-membered aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl, The above R 1c and R 1d are linked to each other to form a compound represented by the following chemical formula 2, [Chemical Formula 2] In the above chemical formula 2, the X 1 , X 2 , X 3 and X 4 are each independently C(R 4 ), nitrogen, oxygen, sulfur or phosphorus, The above R 4 is hydrogen, deuterium, halogen, amino, hydroxy, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 2-10 Alkenyl, substituted or unsubstituted C 2-10 Alkynyl, substituted or unsubstituted C 1-10 an alkoxy, a substituted or unsubstituted 6- to 10-membered aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl; The above R 2a and R 2b are each independently hydrogen, deuterium, halogen, amino, hydroxy, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 2-10 Alkenyl, substituted or unsubstituted C 2-10 Alkynyl, substituted or unsubstituted C 1-10 an alkoxy, a substituted or unsubstituted 6- to 10-membered aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl; The above R 3a and R 3b are each independently hydrogen, deuterium, halogen, amino, hydroxy, -CN, -C(O)NH2, -C(O)O(C 1-10 alkyl), substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted 6- to 10-membered aryl, or substituted or unsubstituted 5- to 10-membered heteroaryl.

12. A method for detecting amyloid-beta (Aβ) according to claim 11, wherein the separated biological sample is at least one selected from the group consisting of blood, plasma, cerebrospinal fluid, cortical lysate, and hippocampal lysate.

13. A composition for diagnosing a degenerative brain disease comprising a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] In the above chemical formula 1, The above R 1a and R 1b are each independently hydrogen, deuterium, halogen, amino, hydroxy, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 2-10 Alkenyl, substituted or unsubstituted C 2-10 Alkynyl, substituted or unsubstituted C 1-10 an alkoxy, a substituted or unsubstituted 6- to 10-membered aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl; The above R 1c and R 1d are each independently hydrogen, deuterium, halogen, amino, hydroxy, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 2-10 Alkenyl, substituted or unsubstituted C 2-10 Alkynyl, substituted or unsubstituted C 1-10 an alkoxy, a substituted or unsubstituted 6- to 10-membered aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl, The above R 1c and R 1d are linked to each other to form a compound represented by the following chemical formula 2, [Chemical Formula 2] In the above chemical formula 2, the X 1 , X 2 , X 3 and X 4 are each independently C(R 4 ), nitrogen, oxygen, sulfur or phosphorus, The above R 4 is hydrogen, deuterium, halogen, amino, hydroxy, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 2-10 Alkenyl, substituted or unsubstituted C 2-10 Alkynyl, substituted or unsubstituted C 1-10 an alkoxy, a substituted or unsubstituted 6- to 10-membered aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl; The above R 2a and R 2b are each independently hydrogen, deuterium, halogen, amino, hydroxy, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 2-10 Alkenyl, substituted or unsubstituted C 2-10 Alkynyl, substituted or unsubstituted C 1-10 an alkoxy, a substituted or unsubstituted 6- to 10-membered aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl; The above R 3a and R 3b are each independently hydrogen, deuterium, halogen, amino, hydroxy, -CN, -C(O)NH2, -C(O)O(C 1-10 alkyl), substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted 6- to 10-membered aryl, or substituted or unsubstituted 5- to 10-membered heteroaryl.

14. A method for providing information on the diagnosis of a degenerative brain disease, comprising the step of contacting a separated biological sample with a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] In the above chemical formula 1, The above R 1a and R 1b are each independently hydrogen, deuterium, halogen, amino, hydroxy, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 2-10 Alkenyl, substituted or unsubstituted C 2-10 Alkynyl, substituted or unsubstituted C 1-10 an alkoxy, a substituted or unsubstituted 6- to 10-membered aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl; The above R 1c and R 1d are each independently hydrogen, deuterium, halogen, amino, hydroxy, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 2-10 Alkenyl, substituted or unsubstituted C 2-10 Alkynyl, substituted or unsubstituted C 1-10 an alkoxy, a substituted or unsubstituted 6- to 10-membered aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl, The above R 1c and R 1d are linked to each other to form a compound represented by the following chemical formula 2, [Chemical Formula 2] In the above chemical formula 2, the X 1 , X 2 , X 3 and X 4 are each independently C(R 4 ), nitrogen, oxygen, sulfur or phosphorus, The above R 4 is hydrogen, deuterium, halogen, amino, hydroxy, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 2-10 Alkenyl, substituted or unsubstituted C 2-10 Alkynyl, substituted or unsubstituted C 1-10 an alkoxy, a substituted or unsubstituted 6- to 10-membered aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl; The above R 2a and R 2b are each independently hydrogen, deuterium, halogen, amino, hydroxy, substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 2-10 Alkenyl, substituted or unsubstituted C 2-10 Alkynyl, substituted or unsubstituted C 1-10 an alkoxy, a substituted or unsubstituted 6- to 10-membered aryl, or a substituted or unsubstituted 5- to 10-membered heteroaryl; The above R 3a and R 3b are each independently hydrogen, deuterium, halogen, amino, hydroxy, -CN, -C(O)NH2, -C(O)O(C 1-10 alkyl), substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted 6- to 10-membered aryl, or substituted or unsubstituted 5- to 10-membered heteroaryl.

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