Lysosomal localized iron ion chelating agents, lysosomal localized ferroptosis inhibitors, antioxidants, and cell protection methods.

Abstract

To provide compounds with potential applications in lysosome-localized iron ion chelators, lysosome-localized ferroptosis inhibitors, antioxidants, and cell protection methods.SOLUTION: The present invention provides a dimethyl aniline derivative represented by a following formula. (R1 represents H, a C1-10 alkyl group, an allyl group, a heteroallyl group, an aralkyl group or the like; R2's independently represent H, a C1-10 alkyl group or the like; R3 represents H or a methyl group; R2 and R3 may bind to each other to form a ring, where R2 and R3 together constitute an ethylene group or a propane-1,3-diyl group; Y's independently represent an alkyl group, an alkoxy group, a nitro group, an acetylamino group, an aminoalkoxy group, an alkoxycarbonyl amino group or the like, attached at a para or meta position; l=0-3, m=1-3, n=1-3).SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a lysosomal localized iron ion chelating agent, a lysosomal localized ferroptosis inhibitor, and an antioxidant cell protection method. [Background technology]

[0002] Neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease are conditions in which cognitive and motor functions decline due to a decrease in nerve cells and the accumulation of abnormal proteins in the central nervous system. It has been suggested that nerve cell death in these neurodegenerative diseases may be caused by several mechanisms related to oxidative stress (Non-Patent Literature 1). Reactive oxygen species (ROS), such as superoxide anions, hydroxyl radicals, hydrogen peroxide, and singlet oxygen, are mainly produced in mitochondria as a byproduct of aerobic respiration and in the endoplasmic reticulum as a byproduct of oxidative protein folding. Normally, ROS are removed by the body's antioxidant mechanisms. However, when the function of these defense mechanisms declines due to various factors such as aging, the balance between ROS production and removal is disrupted, leading to their accumulation and exposing cells to oxidative stress. Cells exposed to excessive oxidative stress then undergo programmed cell death, of which two types have been reported: oxytosis and ferroptosis.

[0003] Oxytosis is cell death induced by excess glutamate, while ferroptosis is cell death induced by elastin. Glutamate and elastin are linked to the cystine / glutamate antiporter (System Xc - It is an inhibitor of the extracellular glutamate or elastin-mediated system Xc -This process is inhibited, leading to a decrease in intracellular glutathione (GSH). Glutathione (GSH) is the most common antioxidant in the body, and depletion of intracellular glutathione leads to the generation of reactive oxygen species and lipid peroxidation, ultimately causing oxytosis or ferroptosis. Oxytosis and ferroptosis are iron-dependent cell death mechanisms, induced by morphological, biochemical, and genetically distinct mechanisms from other cell death processes such as apoptosis, necrosis, and autophagy. Both oxytosis and ferroptosis are related to the Xc system. - Although oxidative stress-induced cell death occurs due to inhibition of oxytosis, it is thought that there are differences in the process of cell death. However, it has been reported that agents that suppress ferroptosis also suppress oxytosis, and agents that suppress oxytosis suppress ferroptosis, suggesting that oxytosis and ferroptosis have commonalities in their developmental mechanisms (Non-Patent Literature 2).

[0004] Currently, there is no effective treatment for neurodegenerative diseases. The clinical efficacy of antioxidant-based treatment strategies is minimal, and clinical trials attempting to treat neurodegenerative diseases with antioxidants such as tocopherol, ascorbic acid, and coenzyme Q10 have all yielded disappointing results. Therefore, it is hoped that small molecule compounds that suppress the generation of reactive oxygen species and protect nerve cells from oxidative stress will be used in the treatment of neurodegenerative diseases, and the present inventors have reported that the oxindole compound GIF-0726-r has a neuroprotective effect against oxidative stress (Non-Patent Documents 3, 4). In this study, we identified 15 compounds, including stereoisomers, and confirmed their protective effects against glutamate and elastin-induced cell death at submicromolar concentrations. Interestingly, while these compounds share N,N-dimethylaniline as a common structural element, they no longer contain the oxindole ring. We investigated the intracellular localization of the N,N-dimethylaniline derivatives GIF-2114 (compound of formula (2)) and GIF-2197-r (compound of formula (11)) using fluorescent probes. The results suggested that these compounds localize to late endosomes and lysosomes and may exhibit significant neuroprotective effects. Further testing and refinement are expected to lead to the clinical application of N,N-dimethylaniline derivatives with neuroprotective effects. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] LMSayre, G.Perry, MASmith, “Oxidative stress and neurotoxicity”, Chemical-research in Toxicology, 2008, 21, 1, 172-188 [Non-Patent Document 2] J. Lewerenz, G. Ates, A. Methner, M. Conrad, P. Maher, “Oxytosis / Ferroptosis-(Re-) emerging-roles for oxidative stress-dependent non-apoptotic cell death in diseases of the central nervous system”, Frontiers in Neuroscience, 2018, 12, 214 [Non-Patent Document 3] Y. Hirata, C. Yamada, Y. Ito, S. Yamamoto, H. Nagase, K. Oh-Hashi, K. Furuta, “Novel oxindole derivatives prevent oxidative stress-induced cell death in mouse hippocampal HT22 cells”, Neuropharmacology, 2018, 135, 242 - 252 [Non-Patent Document 3] Y. Hirata, Y. Ito, M. Takashima, K. Yagyu, K. Oh-Hashi, H. Suzuki, M. Sawada, “Novel oxindole-curcumin hybrid compound for antioxidative stress and neuroprotection”, ACS Chemical Neuroscience, 20, 2011, 1, 76 - 85 [Summary of the Invention] [Problems to be Solved by the Invention]

[0006] An object of the present invention is to provide a lysosome-localized iron ion chelating agent, a lysosome-localized ferroptosis inhibitor, an antioxidant, and a compound that can be expected to be involved in a cell protection method. [Means for Solving the Problems]

[0007] 1. A dimethylaniline derivative represented by the following formula (1). JPEG0007844284000001.jpg28170 (In formula (1), R1 represents hydrogen, an alkyl group having 1 to 10 carbon atoms, a hydroxyalkyl group, an aminoalkyl group, a carboxyalkyl group, an allyl group, an aralkyl group, or a carbamidomethyl group. R2 and R3 each independently represent hydrogen or an alkyl group having 1 to 10 carbon atoms (when R2 is an alkyl group having 1 to 10 carbon atoms, the carbon to which R2 is attached becomes an asymmetric carbon, but any of the (R)-form, (S)-form, and racemic form does not affect the activity). R2 and R3 may be bonded to each other to form a ring, and in that case, R2 and R3 together form an ethylene group or a propane-1,3-diyl group. Y is independently one or more selected from an alkyl group, a hydroxy group, an alkoxy group, an alkylamino group, a nitro group, an acylamino group, an aminoalkoxy group, an alkoxycarbonylamino group, and a halogen bonded to the p-position or m-position. And l = 0 to 3, m = 1 to 3, n = 0 to 3.) 2. The dimethylaniline derivative according to 1., which is represented by any one of the following formulas (2) to (5).

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Advantages of the Invention

[0008] According to the present invention, a lysosomal localized iron ion chelating agent, a lysosomal localized ferroptosis inhibitor, an antioxidant, and a cell protection method can be obtained. [Brief explanation of the drawing]

[0009] [Figure 1] Diagram showing cell death in the presence of glutamate (a) or elastin (b). [Figure 2-1] A diagram showing the effects of glutamic acid (a) or elastin (b) on reactive oxygen species generation and lipid peroxidation. [Figure 2-2] The numerical values ​​for each compound in Figure 2-1. [Figure 3] Diagram showing the effect on Fe2+ ions. [Figure 4] A figure showing the effect on glutamate or elastin using a fluorescent probe. [Figure 5] Diagram showing the intracellular localization of N,N-dimethylaniline derivatives. [Modes for carrying out the invention]

[0010] <Compound represented by formula (1)> The compound of the present invention is a compound represented by the following formula (1). The compound of the present invention may sometimes be simply referred to as a "dimethylaniline derivative." JPEG0007844284000006.jpg33170 (In formula (1), R1 represents hydrogen, an alkyl group having 1 to 10 carbon atoms, a hydroxyalkyl group, an aminoalkyl group, a carboxyalkyl group, an allyl group, an aralkyl group, or a carbamide methyl group. R2 and R3 independently represent hydrogen and an alkyl group having 1 to 10 carbon atoms. (Note that when R2 is an alkyl group having 1 to 10 carbon atoms, the carbon to which R2 is bonded becomes a chiral carbon, but this does not affect the activity in any of the (R), (S), or racemic mixtures.) (It does not resonate.) Note that R2 and R3 may bond to each other to form a ring, in which case R2 and R3 become one unit, forming an ethylene group or a propane-1,3-diyl group. Each Y is independently one or more selected from an alkyl group, hydroxyl group, alkoxy group, alkylamino group, nitro group, acylamino group, aminoalkoxy group, alkoxycarbonylaminoalkoxy group, or halogen, bonded at the p or m position. And l=0~3, m=1~3, n=0~3.

[0011] In the compound represented by formula (1) above, R1 may be a C1-C10 alkyl group, and may consist of a linear, branched, or alicyclic group. Examples include a methyl group, ethyl group, n-propyl group, i-propyl group, n-butyl group, sec-butyl group, t-butyl group, n-pentyl group, isopentyl group, neopentyl group, n-hexyl group, isohexyl group, sec-hexyl group, n-heptyl group, isoheptyl group, sec-heptyl group, n-octyl group, isooctyl group, sec-octyl group, nonyl group, decyl group, cyclopropyl group, cyclobutyl group, cyclopentyl group, cyclohexyl group, cycloheptyl group, cyclooctyl group, cyclononyl group, and cyclodecyl group. Aralkyl groups having 1 to 10 carbon atoms include benzyl, phenethyl, phenylpropyl, and naphthylmethyl groups.

[0012] In the compound represented by formula (1) above, R2 may be a C1-C10 alkyl group, and may consist of a linear, branched, or alicyclic group. Examples include a methyl group, ethyl group, n-propyl group, i-propyl group, n-butyl group, sec-butyl group, t-butyl group, n-pentyl group, isopentyl group, neopentyl group, n-hexyl group, isohexyl group, sec-hexyl group, n-heptyl group, isoheptyl group, sec-heptyl group, n-octyl group, isooctyl group, sec-octyl group, nonyl group, decyl group, cyclopropyl group, cyclobutyl group, cyclopentyl group, cyclohexyl group, cycloheptyl group, cyclooctyl group, cyclononyl group, and cyclodecyl group.

[0013] In the compound represented by formula (1) above, Y may be a C1-C10 alkyl group, and may consist of a linear, branched, or alicyclic group. Examples include methyl group, ethyl group, n-propyl group, i-propyl group, n-butyl group, sec-butyl group, t-butyl group, n-pentyl group, isopentyl group, neopentyl group, n-hexyl group, isohexyl group, sec-hexyl group, n-heptyl group, isoheptyl group, sec-heptyl group, n-octyl group, isooctyl group, sec-octyl group, nonyl group, decyl group, cyclopropyl group, cyclobutyl group, cyclopentyl group, cyclohexyl group, cycloheptyl group, cyclooctyl group, cyclononyl group, and cyclodecyl group. Examples of alkoxy groups having 1 to 10 carbon atoms include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, n-octyloxy, 2-ethylhexyloxy, and cyclohexyloxy groups. Examples of alkylamino groups having 1 to 10 carbon atoms include methylamino group, ethylamino group, n-propylamino group, i-propylamino group, n-butylamino group, sec-butylamino group, t-butylamino group, n-pentylamino group, isopentylamino group, neopentylamino group, n-hexylamino group, isohexylamino group, sec-hexylamino group, n-heptylamino group, isoheptylamino group, sec-heptylamino group, n-octylamino group, isooctylamino group, sec-octylamino group, nonylamino group, decylamino group, cyclopropylamino group, cyclobutylamino group, cyclopentylamino group, cyclohexylamino group, cycloheptylamino group, cyclooctylamino group, cyclononylamino group, and cyclodecylamino group. Dialkylamino groups having 1 to 10 carbon atoms include dimethylamino group, diethylamino group, di-n-propylamino group, di-i-propylamino group, di-n-butylamino group, and cyclic amino groups such as pyrrolidinyl group, methylpyrrolidinyl group, ethylpyrrolidinyl group, morphonyl group, methylmorphonyl group, ethylmorphonyl group, piperidinyl group, and piperazinyl group. Examples of aminoalkoxy groups having 1 to 10 carbon atoms include aminoethoxy group, n-aminopropoxy group, aminoisopropoxy group, aminobutoxy group, aminooctyloxy group, amino2-ethylhexyloxy group, aminocyclohexyloxy group, dimethylaminoethoxy group, diethylaminoethoxy group, pyrrolidinylethoxy group, piperidinylethoxy group, and morpholinoethoxy group. Examples of alkoxycarbonylaminoalkoxy groups include methoxycarbonylaminoethoxy group, ethoxycarbonylaminoethoxy group, propoxycarbonylaminoethoxy group, t-butoxycarbonylaminoethoxy group, pentyloxycarbonylaminoethoxy group, hexyloxycarbonylaminoethoxy group, and benzyloxycarbonylaminoethoxy group. Examples of acylamino groups include acetylamino group, propionylamino group, butanoylamino group, pentanoylamino group, benzoylamino group, N-methylacetylamino group, and 2-oxopyrrolidinyl group. Examples of halogens include fluorine, chlorine, bromine, and iodine. Regarding R2 and R3: When R2 and R3 combine to form an ethylene group or a propane-1,3-diyl group, R2 and R3 do not strictly become the alkyl groups described above.

[0014] The compounds represented by formulas (2) to (5) that are included in the compound represented by formula (1) above are shown below. <Compound represented by formula (2)> [ka] <Compound represented by formula (3)> [ka] <Compound represented by formula (4)> [ka] <Compound represented by formula (5)> [ka]

[0015] <Examples of other compounds> Other examples of compounds besides those represented by formulas (2) to (5) above include those represented by formulas (6) to (16) below. <Compound represented by formula (6)> [ka] <Compound represented by formula (7)> [ka] <Compound represented by formula (8)> [ka] <Compound represented by formula (9)> [ka] <Compound represented by formula (10)> [ka] <Compound represented by formula (11)> [ka] <Compound represented by formula (12)> [ka] <Compound represented by formula (13)> [ka] <Compound represented by formula (14)> [ka] <Compound represented by formula (15)> [ka] <Compound represented by formula (16)> [ka] [Examples]

[0016] <Example of synthesis> The above compounds have a secondary or tertiary amine structure and can be synthesized by general alkylamine synthesis methods. 1. Reductive amination reaction between aldehydes / ketones and amines 3. Nucleophilic substitution reaction between amines and haloalkanes / sulfonic acid esters 4. Reduction reactions of imines and amides

[0017] Synthesis of dimethylaniline derivatives represented by formulas (2) and (5) (hereinafter, these will be referred to as "compound of formula (2)" and "compound of formula (5)" as appropriate, and the same applies to other compounds.) (E)-N,N-dimethyl-4-[3-(2-phenylethylamino)-1-propenyl]aniline: To a methanol (14 mL) solution of 4-(dimethylamino)cinnamaldehyde (500 mg, 2.85 mmol), 2-phenylethylamine (359 μL, 2.85 mmol) and anhydrous magnesium sulfate (860 mg) were added, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was cooled to 0°C, sodium borohydride (215 mg, 5.70 mmol) was added, and the mixture was reacted at room temperature for 30 minutes. Saturated ammonium chloride aqueous solution was added to the reaction mixture, extracted with ethyl acetate, and dried with anhydrous sodium sulfate. The extract was concentrated under reduced pressure to obtain the marked compound (783 mg). This compound was used in the next reaction without purification.

[0018] Compound (E)-N,N-dimethyl-4-[3-(2-phenylethylamino)-1-propenyl]aniline (200 mg) of formula (2) was dissolved in methanol (2 mL), acetic acid (1 mL), paraformaldehyde (43 mg), and sodium borohydride cyanohydride (189 mg, 2.85 mmol) were added, and the mixture was stirred at room temperature for 18 hours. The reaction mixture was poured into a saturated sodium bicarbonate aqueous solution, extracted with ethyl acetate, washed with saturated brine, and dried over anhydrous sodium sulfate. The extract was concentrated under reduced pressure, and the residue (112 mg, 380 μmol) was dissolved in ethyl acetate (3 mL), Pd / C (10%, 12 mg) was added, and the mixture was reacted at room temperature under a hydrogen atmosphere of 1 atm (balloon) for 8 hours and 15 minutes. The reaction mixture was filtered through Celite, and the residue obtained by reducing pressure of the filtrate was purified by column chromatography (silica gel, ethyl acetate) to obtain a pale yellow oily compound of formula (4) (93 mg, 44%). 1 H NMR (400MHz, CDCl3) δ:1.98-2.08(m, 2H, ArCH2C H 2CH2), 2.58―2.66 and 2.78―3.08(complex, 8H, ArCH2and NCH2), 2.93(s, 6H, N(CH3)2), 6.69(d, J=8.2Hz, 2H, ArH), 7.05(d, J=8.2Hz, 2H, ArH), 7.14-7.34(complex, 5H, ArH)

[0019] Compound (E)-N,N-dimethyl-4-[3-(2-phenylethylamino)-1-propenyl]aniline (300 mg, 1.01 mmol) of formula (5) was dissolved in methanol (10 mL), Pd / C (10%, 30 mg) was added, and the mixture was reacted at room temperature for 17 hours under a hydrogen atmosphere at 1 atm (balloon). The reaction mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, ethyl acetate) to obtain a pale yellow oily compound of formula (5) (102 mg, 32%). 1 H NMR(400MHz, CDCl3) δ: 1.75(quin, J=7.4Hz, 2H, ArCH2C H 2CH2), 2.52(t, J=7.4Hz, 2H, Ar CH 2CH2CH2), 2.63(t, J=7.4Hz, 2H, ArCH2CH2C H 2), 2.76-2.9(complex, 4H, ArCH2and NCH2), 2.89(s, 6H, N(CH3)2), 6.67(d, J=8Hz, 2H, ArH), 7.02(d, J=8Hz, 2H, ArH), 7.17-7.31(complex, 5H, ArH) JPEG0007844284000022.jpg83170

[0020] <Synthesis of the compound in formula (3)> Compound (3) (30%) was obtained from compound (10) (170 mg) using the same method as the synthesis of compound (12) below. 1 H NMR (400MHz, CDCl3) δ:1.72-1.86(m, 2H, ArCH2C H 2CH2), 1.94―2.07(complex, 2H, ArCH2C H2CHN), 2.17(s, 3H, NCH3), 2.39―2.63(complex, 4H, ArCH2and NCH2), 2.72-2.82(m, 1H, ArCH2), 2.86-2.93(m, 1H, ArCH2), 2.89(s, 6H, N(CH3)2), 4.42(t, J=7.2Hz, 1H, ArCHN), 6.68(d, J=8.8Hz, ArH), 7.06(d, J=8.8Hz, 2H, ArH), 7.16-7.2(complex, 3H, ArH), 7.32-7.37(br m, 1H, ArH) JPEG0007844284000023.jpg33170

[0021] <Synthesis of the compound in formula (4)> 4-[3-(benzylamino)propyl]-N,N-dimethylaniline: 3-[4-(dimethylamino)phenyl]propanal (181 mg, 1.02 mmol) was dissolved in dichloromethane (5 mL), to which benzylamine (0.40 mL, 0.366 mmol) and sodium triacetoxyborohydride (622 mg, 2.93 mmol) were added, and the mixture was reacted at room temperature for 3 days. The reaction mixture was poured into a saturated sodium bicarbonate aqueous solution, extracted with ethyl acetate, washed with saturated saline solution, and dried over anhydrous sodium sulfate. The extract was concentrated under reduced pressure, and the resulting residue was purified by column chromatography (silica gel, ethyl acetate / hexane = 1 / 4) to obtain the yellow, oily compound (165 mg, 60%). 1 H NMR (400MHz, CDCl3) δ:1.79(quin, J=7.6Hz, 2H, ArCH2C H 2CH2), 2.56(t, J=7.6Hz, 2H, ArCH2CH2C H 2), 2.66(t, J=7.6Hz, 2H, ArC H 2CH2CH2), 2.90(s, 6H, N(CH3)2), 3.76(s, 2H, PhCH2N), 6.68(d, J=8.7Hz, 2H, ArH), 7.04(d, J=8.7Hz, 2H, ArH), 7.2-7.33(complex, 5H, ArH)

[0022] Compound of formula (4) To a 1 mL solution of 4-[3-(benzylamino)propyl]-N,N-dimethylaniline (49 mg, 0.183 mmol) in dichloromethane, sodium triacetoxyborohydride (58.3 mg, 0.275 mmol) and formalin (37% aqueous solution, 186 μL) were added and the mixture was stirred at room temperature for 3 hours. Further, sodium triacetoxyborohydride (61.7 mg) was added and the mixture was reacted for 1 hour and 40 minutes. The reaction mixture was then poured into a saturated aqueous solution of sodium bicarbonate, extracted with ethyl acetate, washed with saturated saline solution, and dried over anhydrous sodium sulfate. The residue obtained by concentrating the extract under reduced pressure was purified by column chromatography (silica gel, ethyl acetate / triethylamine = 100 / 3) to obtain a yellow, oily compound of formula (4) (20.7 mg, 40%). 1 H NMR (400MHz, CDCl3) δ:1.79(quin, J=7.6Hz, 2H, ArCH2C H 2CH2), 2.17(s, 3H, NCH3), 2.40(t, J=7.6Hz, 2H, ArCH2CH2C H 2), 2.54(t, J=7.6Hz, 2H, ArC H 2CH2CH2), 2.90(s, 6H, N(CH3)2), 3.47(s, 2H, PhCH2N), 6.68(d, J=8.7Hz, 2H, ArH), 7.05(d, J=8.7Hz, 2H, ArH), 7.26-7.31(complex, 5H, ArH) JPEG0007844284000024.jpg41170

[0023] <Synthesis of the compound in formula (6)> (E)-4-3-((3-methoxybenzyl)(methyl)amino)-1-propenyl)-N,N-dimethylaniline To a solution of 4-(dimethylamino)cinnamaldehyde (200 mg, 1.14 mmol) in methanol (5 mL) were added 3-methoxybenzylamine (146 μL, 1.14 mmol) and anhydrous magnesium sulfate (343 mg), and the mixture was stirred at room temperature for 45 minutes. The reaction solution was cooled to 0 °C, sodium borohydride (215 mg, 5.70 mmol) was added, and the cooling bath was removed and the reaction was carried out for 2 hours. The reaction solution was poured into a saturated aqueous ammonium chloride solution, extracted with ethyl acetate, washed with saturated brine, and dried over anhydrous sodium sulfate. A part (150 mg) of the residue obtained by concentrating the extract under reduced pressure was dissolved in methanol (4 mL), acetic acid (1 mL), paraformaldehyde (30 mg), and sodium cyanoborohydride (133 mg, 2.02 mmol) were added, and the mixture was stirred at room temperature for 6 hours. The reaction solution was poured into a saturated aqueous sodium hydrogen carbonate solution, extracted with ethyl acetate, washed with saturated brine, and dried over anhydrous sodium sulfate. The residue obtained by concentrating the extract under reduced pressure was purified by column chromatography (silica gel, ethyl acetate / hexane / triethylamine = 75 / 25 / 1) to obtain the title compound (87 mg, 55% in two steps) as a pale yellow oil. 1 H NMR (400 MHz, CDCl3) δ: 2.22 (s, 3H, NCH3), 2.93 (s, 6H, N(CH3)2), 3.1―3.18 (br, 2H, NC H 2CH), 3.50 (br s, 2H, ArCH2N), 3.80 (s, 3H, OCH3), 6.04―6.15 (m, 1H, CH2C H =CH), 6.42 (d, J = 15 Hz, 1H, CH2CH=C H Ar), 6.66 (d, J = 8.8 Hz, 2H, ArH), 6.75―6.8 (br d, J = 7.3 Hz, 1H, ArH), 6.85―6.95 (br, 2H, ArH), 7.16―7.3 (complex, 3H, ArH)

[0024] A solution of formula (6)(E)-4-(3-((3-methoxybenzyl)(methyl)amino)-1-propenyl)-N,N-dimethylaniline (70 mg, 225 μmol) in ethyl acetate (3 mL) was mixed with Pd / C (10%, 7 mg) and reacted at room temperature under a hydrogen atmosphere of 1 atm (balloon) for 1 hour and 20 minutes. The reaction mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, ethyl acetate / hexane = 3 / 1) to obtain a yellow, oily compound of formula (6) (45 mg, 63%). 1 H NMR (400MHz, CDCl3) δ:1.73(quin, J=7.5Hz, 2H, ArCH2C H 2CH2), 2.12(s, 3H, NCH3), 2.34(t, J=7.5Hz, 2H, ArC H 2CH2CH2), 2.48(t, J=7.5Hz, 2H, ArCH2CH2C H 2N), 2.84(s, 6H, N(CH3)2), 3.39(s, 2H, ArCH2N), 3.74(s, 3H, OCH3), 6.62(d, J=8.8Hz, 2H, ArH), 6.7-6.74(m, 1H, ArH), 6.83(d, J=8.1Hz, 1H, ArH), 6.83(s, 1H, ArH), 6.99(d, J=8.8Hz, 2H, ArH), 7.15(t, J=8.1Hz, 1H, ArH) JPEG0007844284000025.jpg39170

[0025] <Synthesis of the compound in formula (7)> 3-(4-(dimethylamino)phenyl)-1-propanol: 4-(dimethylamino)cinnamaldehyde (1.37 g, 7.82 mmol) was dissolved in ethyl acetate (28 mL) and Pd / C (10%, hydrated, 90 mg) was added. The mixture was reacted at room temperature under a hydrogen atmosphere for 3.5 hours. The reaction mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure. The residue was dissolved in methanol (22 mL), and sodium borohydride (132 mg, 3.49 mmol) was added at 0°C and the mixture was reacted for 15 minutes. The reaction mixture was poured into water, extracted with ethyl acetate, washed with saturated brine, and dried over anhydrous sodium sulfate. The residue obtained by concentrating the extract under reduced pressure was purified by column chromatography (silica gel, ethyl acetate / hexane = 2 / 1) to obtain the pale yellow oily compound (1.23 g, 88%). 1 H NMR (400MHz, CDCl3) δ:1.81-1.89(m, 2H, ArCH2C H 2CH2), 2.61(t, J=7.8Hz, 2H, ArC H 2CH2CH2), 2.90(s, 6H, N(CH3)2), 3.63―3.69(br m, 2H, C H 2OH), 6.69(d, J=8.8Hz, 2H, ArH), 7.07(d, J=8.8Hz, 2H, ArH) JPEG0007844284000026.jpg23170

[0026] 4-(3-bromopropyl)-N,N-dimethylaniline: To a solution of 3-(4-(dimethylamino)phenyl)-1-propanol (438 mg, 2.44 mmol) in dichloromethane (8 mL), carbon tetrabromide (1.22 g, 3.68 mmol) and triphenylphosphine (638 mg, 2.43 mmol) were sequentially added, and the mixture was reacted at 0°C for 3 hours. The reaction mixture was poured into water, extracted with dichloromethane, washed with saturated saline solution, and dried over anhydrous sodium sulfate. The extract was concentrated under reduced pressure, and the resulting residue was mixed with a diethyl ether / hexane mixture (1 / 4) and filtered. The residue obtained by concentrating the filtrate under reduced pressure was purified by column chromatography (silica gel, ethyl acetate / hexane = 1 / 10) to obtain the yellow, oily compound (384 mg, 65%). 1H NMR (400MHz, CDCl3) δ:2.11(quin, J=7Hz, 2H, ArCH2C H 2CH2), 2.67(t, J=7Hz, ArC H 2CH2CH2), 2.91(s, 6H, N(CH3)2), 3.38(t, J=7Hz, 2H, ArCH2CH2C H 2Br), 6.69(d, J=8.8Hz, 2H, ArH), 7.06(d, J=8.8Hz, 2H, ArH) JPEG0007844284000027.jpg20170

[0027] Compound of formula (7) To a solution of (S)-1-phenylethylamine (59 μL, 458 μmol) in THF (0.8 mL), butyllithium (2.63 M hexane solution, 200 μL, 526 μmol) was added under an argon atmosphere at 0°C and stirred for 10 minutes. Subsequently, 4-(3-bromopropyl)-N,N-dimethylaniline (111 mg, 458 μmol) dissolved in THF (1.2 mL) was added and reacted at 0°C for 1.5 hours. The reaction mixture was poured into water, extracted with ethyl acetate, washed with saturated saline solution, and dried over anhydrous sodium sulfate. The extract was concentrated under reduced pressure, and the residue was dissolved in diethyl ether (1.5 mL). Butyllithium (2.63 M hexane solution, 260 μL, 684 μmol) was added at 0°C and stirred for 30 minutes, then methyl iodide (46 μL, 733 μmol) was added dropwise and reacted for a further 30 minutes. The reaction mixture was poured into water, extracted with ethyl acetate, washed with saturated saline solution, and dried with anhydrous sodium sulfate. The extract was concentrated under reduced pressure, and the resulting residue was purified by column chromatography (silica gel, ethyl acetate) to obtain a yellow, oily compound of formula (7) (58.4 mg, 43% in two steps). 1 H NMR (400MHz, CDCl3) δ:1.33(d, J=6.6Hz, 3H, PhCH(C H 3)N), 1.73(quin, J=7.5Hz, 2H, ArCH2C H 2CH2), 2.16(s, 3H, NCH3), 2.28―2.36(m, 1H, ArC H 2CH2CH2), 2.4―2.54(complex, 3H, ArCH 2CH2C H 2), 2.89(s, 6H, N(CH3)2), 3.56(q, J=6.6Hz, PhC H (CH3)N), 6.67(d, J=8.6Hz, 2H, ArH), 7.02(d, J=8.6Hz, 2H, ArH), 7.19-7.26(m, 1H, ArH), 7.28-7.31(4H, ArH) JPEG0007844284000028.jpg26170

[0028] <Synthesis of the compound in formula (8)> The compound of formula (7) (48.7 mg, 37%) was obtained from (R)-1-phenylethylamine (58 μL, 448 μmol) using a method similar to that used for the synthesis of the dimethylaniline derivative compound represented by formula (7). 1 H NMR (400MHz, CDCl3) δ:1.33(d, J=6.6Hz, 3H, PhCH(C H 3)N), 1.73(quin, J=7.5Hz, 2H, ArCH2C H 2CH2), 2.16(s, 3H, NCH3), 2.27―2.35(m, 1H, ArC H 2CH2CH2), 2.4―2.54(complex, 3H, ArC H 2CH2C H 2), 2.89(s, 6H, N(CH3)2), 3.56(q, J=6.6Hz, PhC H (CH3)N), 6.67(d, J=8.6Hz, 2H, ArH), 7.02(d, J=8.6Hz, 2H, ArH), 7.19-7.26(m, 1H, ArH), 7.28-7.31(4H, ArH) JPEG0007844284000029.jpg26170

[0029] <Synthesis of the compound in formula (9)> To a methanol (0.5 mL) solution of 3-[4-(dimethylamino)phenyl]propanal (20 mg, 112 μmol), (R)-1-aminoindan hydrochloride (20 mg, 118 μmol) and triethylamine (16 μL, 115 μmol) were added, and the mixture was reacted at room temperature for 3 hours. The reaction mixture was cooled to 0°C, sodium borohydride (6.4 mg) was added, and the mixture was stirred for 30 minutes. Water was added to the reaction mixture, extracted with ethyl acetate, washed with saturated brine, and dried over anhydrous sodium sulfate. The extract was concentrated under reduced pressure, and the residue was purified by column chromatography (silica gel, ethyl acetate / triethylamine = 100 / 1) to obtain compound IX-5 (23 mg, 71%) of formula (9), a pale yellow oily substance. 1 H NMR (400MHz, CDCl3) δ: 1.76-1.88 (complex, 3H, ArCH2C H 2CH2and ArCH2C H 2CHN), 2.33―2.43(m, 1H, ArCH2C H 2CHN), 2.53-2.66(m, 2H, ArCH2), 2.72-3.04(complex, 4H, ArCH2and NCH2), 2.91(s, 6H, N(CH3)2), 4.22(t, J=6.6Hz, 1H, ArCHN), 6.70(d, J=8.6Hz, 2H, Ar H), 7.07(d, J=8.6Hz, 2H, ArH), 7.15-7.26(complex, 3H, ArH), 7.29-7.34(m, 1H, ArH) JPEG0007844284000030.jpg29170

[0030] <Synthesis of the compound of formula (10)> To a methanol (10 mL) solution of 4-(dimethylamino)cinnamaldehyde (400 mg, 2.28 mmol), (S)-1-aminoindan (301 μL, 2.28 mmol) and anhydrous magnesium sulfate (686 mg) were added, and the mixture was stirred at room temperature for 2 hours and 35 minutes. The reaction mixture was cooled to 0°C, sodium borohydride (172 mg, 4.56 mmol) was added, and the mixture was reacted at room temperature for 60 hours. Saturated ammonium chloride aqueous solution was added to the reaction mixture, extracted with ethyl acetate, washed with saturated brine, and dried with anhydrous sodium sulfate. The residue (653 mg) obtained by concentrating the extract under reduced pressure was dissolved in ethyl acetate (10 mL), Pd / C(en) (65 mg) was added, and the mixture was reacted at room temperature under a hydrogen atmosphere of 1 atm (balloon) for 17 hours and 30 minutes. The reaction mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography (silica gel, ethyl acetate / hexane / triethylamine = 400 / 100 / 1) to obtain a yellow, oily compound of formula (10) (520 mg, 79%). 1 H NMR (400MHz, CDCl3) δ: 1.75-1.91 (complex, 3H, ArCH2C H 2CH2and ArCH2C H 2CHN), 2.33―2.43(m, 1H, ArCH2C H 2CHN), 2.54-2.67(m, 2H, ArCH2), 2.72-3.04(complex, 4H, ArCH2and NCH2), 2.92(s, 6H, N(CH3)2), 4.23(t, J=6.6Hz, 1H, ArCHN), 6.70(d, J=8.6Hz, 2H, Ar H), 7.08(d, J=8.6Hz, 2H, ArH), 7.15-7.25(complex, 3H, ArH), 7.31-7.34(m, 1H, ArH) JPEG0007844284000031.jpg25170

[0031] <Synthesis of the compound of formula (11)> N-{3-[4-(dimethylamino)phenyl]propyl}-1-indanylamine: To a solution of 4-(3-aminopropyl)-N,N-dimethylaniline (226 mg, 1.27 mmol) in benzene (5 mL), 1-indanone (143 mg, 1.07 mmol), molecular sieve (3A, 153 mg), and tetraisopropyl orthotitanate (452 ​​μL) were added, and the mixture was reacted at 70°C for 5 hours and 40 minutes. The reaction mixture was cooled to 0°C, sodium borohydride (74 mg, 1.96 mmol) and methanol (5 mL) were added, and the mixture was stirred at room temperature for 22 hours. Ammonia water was added to the reaction mixture, extracted with ethyl acetate, washed with saturated brine, and dried over anhydrous sodium sulfate. The extract was concentrated under reduced pressure, and the residue was purified by column chromatography (silica gel, ethyl acetate / hexane / triethylamine = 400 / 100 / 1) to obtain the pale yellow oily compound (250 mg, 79%). 1 H NMR (400MHz, CDCl3) δ:1.76-1.87(complex, 3H, ArCH2C H 2CH2and ArCH2C H 2CHN), 2.32―2.42(m, 1H, ArCH2C H 2CHN), 2.52-2.66(m, 2H, ArCH2), 2.71-3.02(complex, 4H, ArCH2and NCH2), 2.90(s, 6H, N(CH3)2), 4.21(t, J=6.6Hz, 1H, ArCHN), 6.69(d, J=8.8Hz, 2H, Ar H), 7.06(d, J=8.8Hz, 2H, ArH), 7.14-7.23(complex, 3H, ArH), 7.28-7.33(m, 1H, ArH)

[0032] Compound of formula (11) To a methanol (1.5 mL) solution of N-{3-[4-(dimethylamino)phenyl]propyl}-1-indanylamine (127 mg, 433 μmol), paraformaldehyde (66 mg, 2.17 mmol) and sodium borohydride cyanohydride (45 mg, 650 μmol) were added, and the mixture was reacted at room temperature for 15 hours and 10 minutes. Hydrochloric acid (1 M) was added to the reaction mixture, and the mixture was extracted with ethyl acetate, washed with saturated brine, and dried over anhydrous sodium sulfate. The residue obtained by concentrating the extract under reduced pressure was purified by column chromatography (silica gel, ethyl acetate) to obtain a pale yellow oily compound of formula (11) (55.4 mg, 42%). 1 H NMR (400MHz, CDCl3) δ:1.72-1.85(m, 2H, ArCH2C H 2CH2), 1.96-2.08(complex, 2H, ArCH2C H 2CHN), 2.17(s, 3H, NCH3), 2.39―2.63(complex, 4H, ArCH2and NCH2), 2.72-2.82(m, 1H, ArCH2), 2.85-2.94(m, 1H, ArCH2), 2.89(s, 6H, N(CH3)2), 4.42(t, J=7.2Hz, 1H, ArCHN), 6.68(d, J=8.8Hz, ArH), 7.06(d, J=8.8Hz, 2H, ArH), 7.16-7.20(complex, 3H, ArH), 7.32-7.37(br m, 1H, ArH) JPEG0007844284000032.jpg52170

[0033] <Synthesis of the compound of formula (12)> Compound of formula (12): A solution of the compound of formula (9) (26 mg, 88 μmol) in methanol (0.35 mL) was mixed with paraformaldehyde (26 mg) and sodium borohydride cyanohydride (8.2 mg, 130 μmol), and the mixture was reacted at room temperature for 1 hour and 45 minutes. The reaction mixture was poured into a saturated aqueous solution of sodium bicarbonate, extracted with ethyl acetate, washed with saturated brine, and dried over anhydrous sodium sulfate. The extract was concentrated under reduced pressure, and the resulting residue was purified by column chromatography (silica gel, ethyl acetate / hexane / triethylamine = 83 / 17 / 3) to obtain a yellow, oily compound of formula (12) (15 mg, 56%). 1 H NMR (400MHz, CDCl3) δ:1.72-1.85(m, 2H, ArCH2C H 2CH2), 1.96-2.06(complex, 2H, ArCH2C H 2CHN), 2.17(s, 3H, NCH3), 2.38―2.63(complex, 4H, ArCH2and NCH2), 2.72-2.82(m, 1H, ArCH2), 2.86-2.93(m, 1H, ArCH2), 2.89(s, 6H, N(CH3)2), 4.41(t, J=7.4Hz, 1H, ArCHN), 6.68(d, J=8.8Hz, ArH), 7.06(d, J=8.8Hz, 2H, ArH), 7.16-7.21(complex, 3H, ArH), 7.32-7.37(br m, 1H, ArH) JPEG0007844284000033.jpg28170

[0034] <Synthesis of compound (13)> 6-nitro-1-indanone: 1-Indanone (420 mg, 3.44 mmol) was dissolved in concentrated sulfuric acid (3.5 mL) and cooled to 0°C. Potassium nitrate (348 mg, 3.44 mmol) dissolved in concentrated sulfuric acid (1 mL) was added, and the reaction was carried out at 0°C for 3 hours, followed by 15 hours at room temperature. The reaction mixture was neutralized by adding aqueous sodium bicarbonate dropwise, extracted with ethyl acetate, washed with saturated brine, and dried over anhydrous sodium sulfate. The extract was concentrated under reduced pressure, and the residue was purified by column chromatography (silica gel, ethyl acetate / hexane = 1 / 1) to obtain the marked compound (266 mg, 46%). 1 H NMR (400MHz, CDCl3) δ:2.82(t, J=6Hz, 2H, ArCOCH2), 3.26(t, J=6Hz, 2H, ArCH2), 7.65(d, J=8.3Hz, 1H, ArH), 8.43(br d, J=8.3Hz, 1H, ArH), 8.54(br s, 1H, ArH)

[0035] 6-acetamide-1-indanone: 6-nitro-1-indanone (103 mg, 581 μmol) was dissolved in methanol (1 mL) and Pd / C (10%, hydrated, 12 mg) was added. The reaction was carried out at room temperature for 14 hours under a hydrogen atmosphere at 1 atm (balloon). The reaction mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure. The residue was dissolved in acetic anhydride (1.5 mL) and stirred at room temperature for 28 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (silica gel, ethyl acetate / hexane = 1 / 1) to obtain the labeled compound (52 mg, 47%) as a white solid. 1 H NMR (400MHz, CDCl3) δ:2.20(s, 3H, CH3CO), 2.69(t, J=5.8Hz, 2H, ArCOCH2), 3.09(t, J=5.8Hz, 2H, ArCH2), 7.42(br d, J=8Hz, 1H, ArH), 7.65(s, 1H, ArH), 7.94(br s, 1H, ArNHCO), 8.04(dt, J=2 and 8Hz, 1H, ArH) JPEG0007844284000034.jpg27170

[0036] N-(1-{3-[4-(dimethylamino)phenyl]propyl}indan-6-yl)acetamide: 4-(3-aminopropyl)-N,N-dimethylaniline (49.1 mg, 275 μmol) was dissolved in benzene (1 mL), to which 6-acetamide-1-indanone (32.3 mg, 171 μmol) and tetraisopropyl orthotitanate (104 μL) were added, and the mixture was reacted at 70°C for 5 hours and 40 minutes. The reaction mixture was cooled to 0°C, sodium borohydride (22 mg, 581 μmol) and methanol (1 mL) were added, and the mixture was stirred at room temperature for 18 hours and 30 minutes. Ammonia water was added to the reaction mixture, extracted with ethyl acetate, washed with saturated brine, and dried over anhydrous sodium sulfate. The residue obtained by concentrating the extract under reduced pressure was purified by column chromatography (silica gel, acetone / ethyl acetate = 1 / 1) to obtain the pale yellow oily compound (29.1 mg, 48%). 1 H NMR (400MHz, CDCl3) δ:1.74-1.84(complex, 3H, ArCH2C H 2CH2and ArCH2C H 2CHN), 2.15(s, 3H, CH3CO), 2.3―2.41(m, 1H, ArCH2C H 2CHN), 2.5-2264(m, 2H, ArCH2), 2.67―2.78(complex, 3H, ArCH2and NCH2), 2.88-2.91(1H, NCH2), 2.90(s, 6H, N(CH3)2), 4.15(t, J=6.6Hz, 1H, ArCHN), 6. 70(d, J=8.8Hz, 2H, ArH), 7.07(d, J=8.8Hz, 2H, ArH), 7.13(d, J=8Hz, 1H, ArH), 7.19(br s, 1H, ArNHCO), 7.28 (s, 1H, ArH), 7.35 (d, J=8Hz, 1H, ArH)

[0037] Compound of formula (13) To a methanol (0.3 mL) solution of N-(1-{3-[4-(dimethylamino)phenyl]propyl}indan-6-yl)acetamide (28 mg, 80 μmol), paraformaldehyde (26.1 mg, 869 μmol) and sodium borohydride (6.3 mg, 100 μmol) were added, and the mixture was reacted at room temperature for 15 hours and 10 minutes. Saturated sodium bicarbonate aqueous solution was added to the reaction mixture, extracted with ethyl acetate, washed with saturated saline solution, and dried over anhydrous sodium sulfate. The extract was concentrated under reduced pressure, and the residue was purified by column chromatography (silica gel, acetone / ethyl acetate = 1 / 1) to obtain a pale yellow oily compound of formula (13) (13.3 mg, 45%). 1 H NMR (400MHz, CDCl3) δ:1.71-1.84(m, 2H, ArCH2C H 2CH2), 1.94―2.05(m, 2H, ArCH2C H 2CHN), 2.14 and 2.14(each s, each 3H, NCH3and CH3CO), 2.4―2.63(complex, 4H, ArCH2and NCH2), 2.68-2.77(m, 1H, NCH2), 2.8-2.88(m, 1H, NCH2), 2.89(s, 6H, N(CH3)2), 4.37(t, J=7Hz, 1H, ArCHN), 6.68(d, J=8.8Hz, 2H , ArH), 7.06(d, J=8.8Hz, 2H, ArH), 7.12(d, J=8.4Hz, 1H, ArH), 7.2(s,1H, ArH), 7.27(s, 1H, ArNHCO), 7.52(d, J=8.4Hz, 1H, ArH) JPEG0007844284000035.jpg51170

[0038] <Synthesis of compound (14)> N-{3-[4-(dimethylamino)phenyl]propyl}-6-nitro-1-indanylamine: To a solution of 4-(3-aminopropyl)-N,N-dimethylaniline (96.7 mg, 542 μmol) in benzene (1 mL), 6-nitro-1-indanone (80 mg, 452 μmol) and tetraisopropyl orthotitanate (160 μL, 542 μmol) were added, and the mixture was reacted at room temperature for 3 hours and 30 minutes. Sodium borohydride (25.6 mg, 678 μmol) and ethanol (1 mL) were added to the reaction mixture, and the mixture was stirred at room temperature for 2 hours and 30 minutes. Ammonia water (28%, 35 μL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate, washed with saturated saline solution, and dried over anhydrous sodium sulfate. The residue obtained by concentrating the extract under reduced pressure was purified by column chromatography (silica gel, ethyl acetate / hexane = 3 / 1, triethylamine 3%) to obtain the reddish-brown oily compound (71.2 mg, 47%). 1 H NMR (400MHz, CDCl3) δ:1.82(quin, J=7.2Hz, 2H, ArCH2C H 2CH2N), 1.84-1.92(m, 1H, ArCH2C H 2CHN), 2.42―2.51(m, 1H, ArCH2C H 2CHN), 2.55-2.64(m, 2H, ArCH2), 2.68-2.79(m, 2H, NCH2), 2.8-2.9(m, 1H, ArC H 2CH2CHN), 2.90(s, 6H, N(CH3)2), 3.0―3.09(m, 1H, ArC H 2CH2CHN), 4.25(t, J=6.8Hz, 1H, ArCHN), 6.68(d, J=8.6Hz, 2H, ArH), 7.06(d, J=8.6Hz, 2H, ArH), 7.32(d, J=8.4Hz, 1H, ArH), 8.07(dd, J=2.4 and 8.4Hz, 1H, ArH), 8.14(br d, J=2.4Hz, 1H, ArH)

[0039] Compound of formula (14) To a solution of N-{3-[4-(dimethylamino)phenyl]propyl}-6-nitro-1-indanylamine (41 mg, 121 μL) in dichloromethane (2 mL), sodium triacetoxyborohydride (76.9 mg, 363 μmol) and formalin (37% aqueous solution, 75 μL) were added and the mixture was reacted at room temperature for 17 hours. Water was added to the reaction mixture, extracted with dichloromethane, washed with saturated saline solution, and dried over anhydrous sodium sulfate. The extract was concentrated under reduced pressure, and the residue was purified by column chromatography (silica gel, ethyl acetate) to obtain a reddish-brown oily compound of formula (14) (30.3 mg, 71%). 1 H NMR (400MHz, CDCl3) δ:1.75-1.88(m, 2H, ArCH2C H 2CH2N), 2.05―2.14(m, 2H, ArCH2C H 2CHN), 2.17(s, 3H, NCH3), 2.38-2.48(m, 2H, ArCH2), 2.49-2.64(m, 2H, NCH2), 2.8-3.01(complex, 2H, ArC H 2CH2CHN), 2.89(s, 6H, N(CH3)2), 4.44(t, J=7.4Hz, 1H, ArCHN), 6.67(d, J=8.8Hz , 2H, ArH), 7.05(d, J=8.8Hz, 2H, ArH), 7.29(d, J=8.4Hz, 1H, ArH), 8.06(dd, J=2.4 and 8.4Hz, 1H, ArH), 8.17 (br s, 1H, ArH) JPEG0007844284000036.jpg50170

[0040] <Synthesis of compound (15)> N-{3-[4-(dimethylamino)phenyl]propyl}-5-methoxy-1-indanylamine: To a solution of 4-(3-aminopropyl)-N,N-dimethylaniline (52.8 mg, 296 μmol) in benzene (1 mL), 5-methoxy-1-indanone (38.5 mg, 237 μmol) and tetraisopropyl orthotitanate (104 μL, 350 μmol) were added, and the mixture was reacted at 70°C for 5 hours and 20 minutes. The reaction mixture was cooled to 0°C, sodium borohydride (21.1 mg, 558 μmol) and methanol (1 mL) were added, and the mixture was stirred at room temperature for 16 hours. Ammonia water was added to the reaction mixture, extracted with ethyl acetate, washed with saturated brine, and dried over anhydrous sodium sulfate. The residue obtained by concentrating the extract under reduced pressure was purified by column chromatography (silica gel, acetone / ethyl acetate = 1 / 1) to obtain the marked compound (47.7 mg, 62%), which is a pale yellow oily substance. 1 H NMR (400MHz, CDCl3) δ:1.76-1.87(complex, 3H, ArCH2C H 2CH2and ArCH2C H 2CHN), 2.31―2.41(m, 1H, ArCH2C H 2CHN), 2.5-2264(m, 2H, ArCH2), 2.72(t, J=7.4Hz, 2H, NCH2), 2.73-2.81(m, 1H, ArC H 2CH2CHN), 2.90(s, 6H, N(CH3)2), 2.9―3.0(m,1H, ArC H 2CH2CHN), 3.78(s, 3H, OCH3), 4.16(t, J=6.2Hz, 1H, ArCHN) 6.69(d, J=8.8Hz, 2H, ArH), 6.73( d, J=8.2Hz, 1H, ArH), 6.76(s, 1H, ArH), 7.06(d, J=8.8Hz, 2H, ArH), 7.20(d, J=8.2Hz, 1H, ArH)

[0041] To a methanol (0.3 mL) solution of formula (15) N-{3-[4-(dimethylamino)phenyl]propyl}-5-methoxy-1-indanylamine (28.8 mg, 88.8 μmol), paraformaldehyde (14.2 mg, 473 μmol) and sodium borohydride cyanohydride (8.4 mg, 134 μmol) were added, and the mixture was reacted at room temperature for 14 hours. Saturated sodium bicarbonate aqueous solution was added to the reaction mixture, extracted with ethyl acetate, washed with saturated brine, and dried over anhydrous sodium sulfate. The extract was concentrated under reduced pressure, and the residue was purified by column chromatography (silica gel, acetone / ethyl acetate = 1 / 1) to obtain the marked compound (24.6 mg, 82%), which is a pale yellow oily substance. 1 H NMR (400MHz, CDCl3) δ:1.71-1.83(m, 2H, ArCH2C H 2CH2), 1.97―2.06(m, 2H, ArCH2C H 2CHN), 2.16(s, 3H, NCH3), 2.37-2.63(complex, 4H, ArCH2and NCH2), 2.71-2.8(m, 1H, ArC H 2CH2CHN), 2.83-2.9(m,1H, ArC H 2CH2CHN), 2.90(s, 6H, N(CH3)2), 3.79(s, 3H, OCH3), 4.36(t, J=7Hz, 1H, ArCHN), 6.68(d,J=8.8Hz , 2H, ArH), 6.73(s, 1H, ArH), 6.73−6.76(1H, ArH), 7.05(d, J=8.8Hz, 2H, ArH), 7.21−7.26(1H, ArH) JPEG0007844284000037.jpg57170

[0042] <Synthesis of compound (16)> 5-(2-bromoethoxy)-1-indanone: 5-hydroxy-1-indanone (745 mg, 5.03 mmol) was dissolved in ethyl acetate (15 mL), and then potassium carbonate (2.45 g), 1,2-dibromoethane (3.27 mL), and benzyltriethylammonium chloride (121 mg), suspended in ethyl acetate (15 mL), were added sequentially, and the mixture was heated under reflux for 24 hours. After the reaction mixture was allowed to cool to room temperature, it was poured into ice water, extracted with ethyl acetate, washed with saturated brine, and dried over anhydrous sodium sulfate. The residue obtained by concentrating the extract under reduced pressure was purified by column chromatography (silica gel, hexane / ethyl acetate = 1 / 1) to obtain the marked compound (1.06 g, 83%) as a white solid. 1 H NMR (400MHz, CDCl3) δ:2.67(m, 2H, COCH2), 3.08(br t, J=6Hz, 2H, ArCH2), 3.66(t, J=6.4Hz, 2H, BrCH2), 4.35(t, J=6.4Hz, 2H, OCH2), 6.89-6.93(br m, 2H, ArH), 7.69 (d, J=9.2Hz, 1H, ArH)

[0043] 5-(2-azidoethoxy)-1-indanone: 5-(2-bromoethoxy)-1-indanone (410 mg, 1.61 mmol) was dissolved in N,N-dimethylformamide (5.0 mL), sodium azide (129 mg) was added, and the mixture was heated under reflux for 35 minutes. After the reaction mixture was allowed to cool to room temperature, it was poured into water, extracted with diethyl ether, washed with saturated saline solution, and dried over anhydrous sodium sulfate. The extract was concentrated under reduced pressure to obtain the reddish-brown solid of the marked compound (346 mg, 99%). 1 H NMR (400MHz, CDCl3) δ:2.66-2.7(m, 2H, COCH2), 3.09(br t, J=6Hz, 2H, ArCH2), 3.63(t, J=5Hz, 2H, N3CH2), 4.21(t, J=5Hz, 2H, OCH2), 6.90-6.94(br m, 2H, ArH), 7.70(d, J=9.2Hz, 1H, ArH)

[0044] 5-(2-azidoethoxy)-N-{3-[4-(dimethylamino)phenyl]propyl}-N-methyl-1-indanylamine: To a methanol (2 mL) solution of 5-(2-azidoethoxy)-1-indanone (109 mg, 0.5 mmol), methylamine hydrochloride (101 mg) and sodium acetate (123 mg) were added at room temperature and the mixture was stirred for 30 minutes. Subsequently, sodium borohydride cyanohydride (63 mg) was added to the reaction solution and the mixture was heated under reflux for 30 hours. After the reaction mixture was allowed to cool to room temperature, it was poured into water, extracted with dichloromethane, and dried over anhydrous sodium sulfate. The extract was concentrated under reduced pressure to obtain a light brown oily 5-(2-azidoethoxy)-N-methyl-1-indanylamine (118 mg). The obtained methylamine compound (118 mg) was dissolved in 1,2-dichloroethane (2 mL), and 3-[4-(dimethylamino)phenyl]propanal (88 mg) and sodium triacetoxyborohydride (185 mg) were added and the mixture was stirred at room temperature for 23 hours. The reaction mixture was poured into a saturated sodium bicarbonate aqueous solution, extracted with ethyl acetate, and dried over anhydrous sodium sulfate. The extract was concentrated under reduced pressure, and the resulting residue was purified by column chromatography (silica gel, acetone / ethyl acetate = 1 / 1) to obtain the pale yellow, oily compound (161 mg, 82%). 1 H NMR (400MHz, CDCl3) δ:1.72-1.85(m, 2H, ArCH2C H 2CH2), 2.03(br q, J=7.6Hz, 2H, ArCH2C H 2CH), 2.16(s, 3H, NCH3), 2.38―2.64(complex, 4H, ArCH2and NCH2), 2.72-2.9(complex, 2H, ArCH2), 2.90(s, 6H, N(CH3)2), 3.58(t, J=4.6Hz, 2H, CH2N3), 4.14(t, J=4.6Hz, 2H, ArOCH2), 4.36(t, J= 6.9Hz, 1H, ArCHN), 6.69(d, J=8.2Hz, 2H, ArH), 6.76(s, 1H, ArH), 6.76-6.78(1H, ArH), 7.06(d, J=8.2Hz, 2H, ArH), 7.23-7.26(1H, ArH)

[0045] To a methanol (0.4 mL) solution of formula (16) 5-(2-azidoethoxy)-N-{3-[4-(dimethylamino)phenyl]propyl}-N-methyl-1-indanylamine (23.5 mg, 60 μmol), Pd / C (10%, hydrated, 4.2 mg) was added and the mixture was reacted at room temperature for 3 hours and 50 minutes under a hydrogen atmosphere of 1 atm (balloon). Subsequently, di-tert-butyl dicarbonate (15.5 μL, 72 μmol) was added to the reaction mixture and the mixture was stirred at room temperature for 17 hours. The reaction mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, ethyl acetate) to obtain a pale yellow oily compound of formula (16) (20.1 mg, 72%). 1 H NMR (400MHz, CDCl3) δ:1.43(s, 9H, OC(CH3)3), 1.72-1.84(m, 2H, ArCH2C H 2CH2), 1.96―2.05(m, 2H, ArCH2C H 2CH), 2.15(s, 3H, NCH3), 2.36―2.62(complex, 4H, ArCH2and NCH2), 2.7-2.79(m, 1H, ArCH2), 2.8-2.9(m, 1H, ArCH2), 2.89(s, 6H, N(CH3)2), 3.46-3.55(m, 2H, BocNHC H 2), 3.99 (t, J=5Hz, 2H, ArOCH2), 4.35 (t, J=6.8Hz, 1H, ArCHN), 6.68 (d, J=8.6Hz, 2H, ArH), 6.72(s, 1H, ArH), 6.71-6.75(1H, ArH), 7.05(d, J=8.6Hz, 2H, ArH), 7.22(d, J=8Hz, 1H, ArH) JPEG0007844284000038.jpg83170

[0046] The cytoprotective activity of candidate compounds against oxytosis and ferroptosis was evaluated using mouse hippocampal HT22 cells. HT22 cells 2-3 x 10 4The cells were seeded in a 48-well plate and cultured in DMEM medium (Waco Pure Chemicals) (5% FBS (HyClone Laboratories)) at 37°C in a 5% CO2 incubator for 1 day. (Cell culture in subsequent experiments was carried out under the same conditions.) Subsequently, each compound was treated in the presence of glutamic acid or elastin. The rate of cell death was measured by assessing lactate dehydrogenase (LDH) activity in the culture medium using a Cytotoxicity Detection Kit (Takara Bio). The rate of cell death was calculated as 100 × (amount of LDH in the culture medium - blank) / (total amount of LDH when cells were treated with 1% TritonX-100 - blank). Of approximately 220 compounds, GIF-2114, -2115, -2232, -2108, -2212, -2211, -2027, -2112, -2197-r, -2196, -2239-r, -2226-r, -2241-r, and -2249-r significantly suppressed oxyposis at concentrations of 0.01, 0.1, 1.0, or 10 μM (Figure 1(a)). Furthermore, GIF-2114, -2115, -2196, and -2197-r also significantly suppressed ferroptosis at concentrations of 0.01, 0.1, or 1.0 μM (Figure 1(b)).

[0047] Furthermore, GIF-2011 is the following compound: [ka] GIF-2014 is the following compound: [ka] GIF-2071 is the following compound: [ka] GIF-2145 is the following compound: [ka] GIF-2071-r is the following compound: [ka]

[0048] GIF-2114 (compound of formula (2)), GIF-2115 (compound of formula (3)), GIF-2196 (compound of formula (12)), and GIF-2197-r (compound of formula (11)) exhibited the highest efficacy, with an effective concentration of 10 nM. Formulas (3), (11), and (12) are stereoisomers, with formula (3) being the R-type, formula (12) being the S-type, and formula (11) being the racemic mixture. Since the stereoisomers reduce glutamate and elastin-induced cell death to a similar degree, it was suggested that steric hindrance around the nitrogen atom does not affect cell protection.

[0049] To analyze the molecular basis of the cytoprotective effects of N,N-dimethylaniline derivatives, we investigated the effects of glutamate and elastin on reactive oxygen species generation and lipid peroxidation reactions.

[0050] <Reactive oxygen species generation> Place HT22 cells in a 12-well plate at a rate of 1.1 × 10⁶ 4 Cells were seeded in cell / wells and treated with glutamic acid or elastin in the presence of each compound, followed by incubation for 10 hours or 8 hours, respectively. Then, MitoSOX (Thermo Fisher Science, 5 μM), a reagent for detecting reactive oxygen species, was added, and the cells were incubated at 37°C for 15 minutes. The culture medium was then replaced with DMEM (Thermo Fisher Science) (serum-free, phenol red-free), and fluorescence was measured using a fluorescence microscope (BZ-X810, Keyence). Fluorescence intensity was quantified using the Keyence image measurement system and analysis software (BZ-X Analyzer, Keyence).

[0051] <Lipid peroxidation reaction> Fluorescence was measured in the same manner as above, except that BODIPY C11 (Thermo Fisher Science, 1 μM), a lipid peroxidation detection agent, was added instead of MitoSOX (concentration 5 μM), and the mixture was incubated at 37°C for 30 minutes. Glutamate and elastin increase mitochondrial reactive oxygen species production and lipid peroxidation reactions, but GIF-2011, -2014, -2071-r, -2145, and -2171 almost completely inhibited these reactions at 1 μM, while GIF-2114 (compound of formula (2)), GIF-2115 (compound of formula (3)), GIF-2196 (compound of formula (12)), and GIF-2197-r (compound of formula (11)) almost completely inhibited them at 0.1 μM (Figure 2-1). In a to d, the P value was 0.0001 or less compared to treatment with glutamate or elastin (specific values ​​for a to d in Figure 2-1 are shown in Figure 2-2).

[0052] Fe 2+ Ions are catalysts that generate the potent oxidizing agent hydroxyl radical from hydrogen peroxide via the Fenton reaction, and are important regulators of oxytosis / ferroptosis. Fe 2+ Using FerroOrange, an ion-specific fluorescent probe, the N,N-dimethylaniline derivative was found to be Fe 2+ We evaluated whether it could act on ions to reduce reactive oxygen species levels.

[0053] Place HT22 cells in a 12-well plate at a rate of 1.1 × 10⁶ 4 Cells were seeded in cell / wells and cultured for 1 day. Then, 1 μM GIF-2011, -2014, -2071-r, -2145, -2171, or GIF-2114 (compound of formula (2)), GIF-2115 (compound of formula (3)), GIF-2196 (compound of formula (12)), and GIF-2197-r (compound of formula (11)) at the concentrations shown in Figure 3b were added and cultured for 16 hours. Then, the Fe content in the cells was increased. 2+To visualize its behavior, cells were stained with HBSS (Hank's balanced salt solution) containing FerroOrange (Goryo Chemical Inc., 1 μM) in a CO2 incubator at 37°C for 30 minutes. After replacing the culture medium with HBSS, fluorescence was measured in the same manner as described above. Fe as a positive control 2+ Deferoxamine (DFO, 10 μM), known as a chelating agent, was used (Figure 3a).

[0054] GIF-2011, -2014, -2071-r, -2145, and -2171 suppressed FerroOrange fluorescence at 1 μM (Figure 3a). On the other hand, the most potent derivatives, GIF-2114 (compound of formula (2)), GIF-2115 (compound of formula (3)), GIF-2196 (compound of formula (12)), and GIF-2197-r (compound of formula (11)), were found to suppress FerroOrange fluorescence at 0.01 μM (Figures 3b, c). Since DFO suppressed oxytosis and ferroptosis (Figure 3d), to prevent glutamate-induced oxytosis and elastin-induced ferroptosis, Fe 2+ Since ion removal is shown to be effective, these results, combined with the above findings, suggest that each compound of the present invention is effective in suppressing oxytosis and elastin-induced ferroptosis, meaning that it functions as an iron ion chelating agent and a ferroptosis inhibitor.

[0055] Next, the effects of GIF-2115 (compound of formula (3)) and GIF-2228-r on glutamate-induced cell death of HT22 cells and Fe in vitro 2+ The effects of ions were compared and examined. GIF-2228-r has a similar structure to GIF-2115 (compound of formula (3)), but lacks the NMe2 group (Figure 3e). GIF-2228-r was not as effective at preventing glutamate-induced cell death as GIF-2115 (compound of formula (3)) (Figure 3f). Furthermore, the behavior of FerroOrange fluorescence in vitro in the presence of GIF-2228-r or GIF-2115 (compound of formula (3)) was measured. 100 μM of GIF-2228-r or GIF-2115 (compound of formula (3)), DFO, 100 μM of FeSO4, and 1 μM of FerroOrange were added to 50 mM HEPES buffer, and the intensity of light at 572 nm was measured every minute under excitation with 532 nm light. As a result, GIF-2115 (compound of formula (3)) suppressed FerroOrange fluorescence more effectively than DFO in vitro, while GIF-2228-r did not affect FerroOrange fluorescence (Figure 3g).

[0056] These results indicate that the NMe2 structure is Fe 2+ This suggests that it plays an important role in preventing oxidative stress by reducing the amount of reactive oxygen species through interaction with ions. All N,N-dimethylaniline derivatives are potent in vitro with Fe 2+ It demonstrated binding ability.

[0057] To analyze how cells transport GIF-2114 (compound of formula (2)) and GIF-2197-r (compound of formula (11)) and how this relates to cytoprotection, fluorescent probes of GIF-2114 (compound of formula (2)) and GIF-2197-r (compound of formula (11)), GIF-2264 and GIF-2250-r, were prepared, respectively. GIF-2264 and GIF-2250-r were synthesized by adding a fluorescent 4-methyla-mino-7-nitro-2,1,3-benzoxadiazole (NBD-NHMe) group to GGIF-2114 (compound of formula (2)) and GIF-2197-r (compound of formula (11), respectively).

[0058] To verify the cytoprotective properties of GIF-2264 and GIF-2250-r, HT22 cells were treated with glutamate or elastin for 24 hours in the presence of GIF-2264 and GIF-2250-r at the concentrations shown in Figure 4, using the method described above, and the rate of cell death was measured (Figure 4a, b, d, e). As a result, GIF-2264 and GIF-2250-r were found to have weaker protective effects than GIF-2114 (compound of formula (2)) and GIF-2197-r (compound of formula (11)), respectively. This suggests that the fluorescent tag may have some effect on anti-ferroptosis activity via steric hindrance. Furthermore, it was suggested that GIF-2250-r may be more useful than GIF-2264.

[0059] Next, HT22 cells 2 × 10 5 The cells were seeded in a 35mm / glass base dish and cultured in DMEM (5% FBS) for 24 hours. To stain the mitochondria of the cells, they were stained with MitoMM2 (0.5 μM), shown in the structural formula below, for 15 minutes, and then stained with 1 μM GIF-2264 and GIF-2250-r at 37°C for 15 minutes. Fluorescence images were then observed using the sectioning function of a fluorescence microscope (BZ-X810, Keyence). (MitoMM2) JPEG0007844284000044.jpg44170 To stain the endoplasmic reticulum, use ER-Tracker. TM After staining with Blue-White DPX (Thermo Fisher Science, 0.5 μM) for 30 minutes, the culture medium was replaced with DMEM (5% FBS), and then stained with 1 μM GIF-2264 and GIF-2250-r at 37°C for 15 minutes. Subsequently, the culture medium was replaced with DMEM (Thermo Fisher Science) (serum-free, phenol red-free), and fluorescence images were observed using the sectioning function of a fluorescence microscope (BZ-X810, Keyence). To stain lysosomes, use LysoBrite. TMSamples were stained with Red (ATT Bioquest, 1 μM) and 1 μM GIF-2264 and GIF-2250-r at 37°C for 30 minutes. The culture medium was then replaced with DMEM (Thermo Fisher Science) (serum-free, phenol red-free), and fluorescence images were observed using the sectioning function of a fluorescence microscope (BZ-X810, Keyence).

[0060] Since the culture medium containing GIF-2264 and GIF-2250-r did not fluoresce, it was possible to observe the green fluorescence of GIF-2264 and GIF-2250-r without changing the medium. HT22 cells treated with glutamate or elastin began to curl up and initiate cell death after 8 hours. However, when GIF-2264 and GIF-2250-r were added, these compounds localized to the perinuclear region, completely preventing morphological changes in the cells (Figure 4c, f).

[0061] Next, we investigated the co-localization of GIF-2264 or GIF-2250-r with the late endosome / lysosome markers CD63- or Rab7. Transfection of HT22 cells with CD63-mCherry or Rab7-mCherry resulted in punctate red fluorescence around the nucleus (Figure 5a, b). These signals overlapped with the green fluorescence emitted by GIF-2264 or GIF-2250-r, suggesting that GIF-2264 or GIF-2250-r are localized to late endosomes / lysosomes.

[0062] Furthermore, the targeting and localization of GIF-2264 or GIF-2250-r to lysosomes were further validated in living cells using LysoBrite (lysosomes, red fluorescence), ER-Tracker (endoplasmic reticulum, blue fluorescence), or MitoMM2 (mitochondria, red fluorescence). The green fluorescence of GIF-2264 or GIF-2250-r overlapped with the red fluorescence of LysoBrite, but not with ER-Tracker or MitoMM2 (Figure 5c), suggesting that these compounds preferentially localize to late endosomes / lysosomes. In other words, the GIF compounds of the present invention are thought to function as lysosomal localized iron ion chelators and lysosomal localized ferroptosis inhibitors.

[0063] These results, taken together, suggest that late endosomes / lysosomes are potential targets for oxytosis and ferroptosis. As shown in the results using the fluorescent probes described above, the accumulation of GIF-2114 (compound of formula (2)) and GIF-2197r (compound of formula (11)) in late lysosomes / endosomes suggests that they are important for protecting HT22 cells from oxytosis / ferroptosis.

[0064] The N,N-dimethylaniline derivatives of the present invention, which possess cytoprotective properties, have been found to exhibit more potent neuroprotective effects through a mechanism different from that of known compounds. GIF-2114 (compound of formula (2)), GIF-2115 (compound of formula (3)), GIF-2196 (compound of formula (12)), and GIF-2197-r (compound of formula (11)) are not antioxidants themselves, but they localize to late endosomes / lysosomes. As a result, it is thought that these four compounds, even at low effective concentrations, can protect cells by regulating ferroptosis and oxyposis through the adjustment of intracellular iron balance and the production of reactive oxygen species. These properties of N,N-dimethylaniline derivatives make them potential candidates for further development in the treatment of oxidative stress-related diseases. Furthermore, the fluorescent probes GIF-2264 or GIF-2250-r are useful tools for imaging late endosomes / lysosomes and extracellular vesicles such as exosomes, as the fluorescence of the compound itself is quenched in culture medium.

Claims

1. A dimethylaniline derivative represented by any of the following formulas (2), (4), (6) to (10), or (13) to (16). 【Chemistry 1】 【Transformation 3】 【Transformation 5】 【Transformation 6】 【Transformation 7】 【Transformation 8】 【Chemistry 9】 【Chemistry 12】 【Chemistry 13】 【Chemistry 14】 【Chemistry 15】

2. An antioxidant comprising the dimethylaniline derivative described in claim 1.

3. A lysosome-localized iron ion chelating agent comprising the dimethylaniline derivative described in claim 1.

4. A lysosomal localized ferroptosis inhibitor comprising the dimethylaniline derivative described in claim 1.

5. A cytoprotective agent comprising the dimethylaniline derivative described in claim 1.

Images