NOVEL 1,4-BIS(3-AMINOPROPYL)PIPERAZINE DERIVATIVE AND ITS USE

Abstract

The present invention relates to the compound having Formula (I) or pharmaceutically acceptable salts or solvates thereof, and its use in treating and/or preventing a Tauopathy.

##STR00001##

Claims

1-7. (canceled)

8. A method of treating and/or preventing a disease selected from Amyotrophic lateral sclerosis and parkinsonism-dementia complex, Chronic traumatic encephalopathy, cerebral amyloid angiopathy, Dementia pugilistica, Familial British Dementia, Familial Danish Dementia, Gerstmann-Straussler-Scheinker disease, Guadeloupean parkinsonism, Hallervorden-Spatz disease, inclusion body myositis, multiple system atrophy, Steinert Myotonic dystrophy, Myotonic dystrophy type II, Neurodegeneration with brain iron accumulation, Niemann-Pick disease, Non-Guamanian motor neuron disease with neurofibrillary tangles, Postencephalitic parkinsonism, Subacute sclerosing panencephalitis, multi-infarct dementia, chronic traumatic encephalopathy (CTE), traumatic brain injury (TBI), comprising administering to a patient in need thereof a pharmaceutically effective amount of a compound of Formula I ##STR00005## or a pharmaceutically acceptable salt thereof.

9. A method according to claim 8, wherein the disease is Steinert Myotonic dystrophy and Myotonic dystrophy type II.

10. A method according to claim 8, wherein the disease is Steinert Myotonic dystrophy.

11. A method according to claim 8, wherein the compound of Formula I is administered as a composition of the compound or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant.

12. A method for decreasing pathological Tau protein phosphorylation in a patient suffering from Amyotrophic lateral sclerosis and parkinsonism-dementia complex, Chronic traumatic encephalopathy, cerebral amyloid angiopathy, Dementia pugilistica, Familial British Dementia, Familial Danish Dementia, Gerstmann-Straussler-Scheinker disease, Guadeloupean parkinsonism, Hallervorden-Spatz disease, inclusion body myositis, multiple system atrophy, Steinert Myotonic dystrophy, Myotonic dystrophy type II, Neurodegeneration with brain iron accumulation, Niemann-Pick disease, Non-Guamanian motor neuron disease with neurofibrillary tangles, Postencephalitic parkinsonism, Subacute sclerosing panencephalitis, multi-infarct dementia, chronic traumatic encephalopathy (CTE) or traumatic brain injury (TBI), in need thereof, comprising administering to said patient an effective amount of a compound of Formula I ##STR00006## or a pharmaceutically acceptable salt thereof.

13. A method according to claim 12, wherein the disease is Steinert Myotonic dystrophy and Myotonic dystrophy type II.

14. A method according to claim 12, wherein the disease is Steinert Myotonic dystrophy.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0058] FIG. 1: Determination by ID gel electrophoresis of the expression of the Tau protein by using Pan-Tau antibodies (Tau-Nter, Tau-Cter) and of the phosphorylation of the Tau protein by using phospho-dependent Tau antibodies (S199P, S396) and non-phosphorylated epitote comprised between 198-205 (Tau-1), in non-induced conditions (NI), induced but non-treated conditions (NT) and with administration of Compound I.HQ.

[0059] FIG. 2: Determination by 2D gel electrophoresis of the expression of the Tau protein by using Pan-Tau antibodies (Tau-Nter, Tau-Cter) and of the phosphorylation of the Tau protein by using phospho-dependent Tau antibody (S396), with an induced but non-treated condition (Ctrl) and with administration of Compound I.HCl.

[0060] FIG. 3: Increase Tau C-terminal catabolites and reduce phosphorylation at serine 199. The 2D pattern of Tau proteins from the brain of WT animals treated with 0.5 mg/Kg or 1 mg/Kg was revealed a pan-Tau antibody (Tau-Cter) and a phospho-dependent antibody against the phosphorylated serine 199.

[0061] FIG. 4: Morris water maze evaluation of the spatial memory of Wild-type C57BL/6J animal (WT) and Thy-Tau22 mice, treated or not with 0.5 mg/Kg of Compound I-HQ.

[0062] A: Representation of the distance to reach the platform (path length) in function of time (days).

[0063] B: Representation of the percent of time spent in the target quadrant (T) (where the platform is positioned during the learning phase) versus the others (O).

[0064] FIG. 5: Immunohistochemical measurement of tau pathology in Thy-Tau22 mice untreated (0 mg/kg) or treated with Compound I-HQ (0.5 mg/kg Compound I-HQ).

[0065] FIG. 6: Biochemical analysis of Tau expression, catabolites and phosphorylation by ID SDS-PAGE and Western-blot.

CHEMISTRY EXAMPLES

[0066] The following abbreviations are used throughout the present application: C.: Celsius degrees, BINAP: 2,2-bis(diphenylphosphino)-1,1-binaphtyl, dba: dibenzylideneacetone, DCE: dichloroethane, DCM: dichloromethane, : NMR chemical shifts expressed in ppm, eq: equivalent(s), Et: ethyl, g: gram(s), HPLC: high performance liquid chromatography, L: liter(s), LCMS: HPLC coupled to a mass spectrometer, Me: methyl, mg: milligram(s), min: minute(s), mL: milliliter(s), mol: mole(s), mmol: millimole(s), o: micromole(s), MS: Mass Spectrometry, NMR: nuclear magnetic resonance, ppm: party per million, RT: room temperature (ca 15-25 C.), Xantphos: 4,5-Bis(diphenylphosphino)-9,9-dimethylxanthene, rt: retention time.

[0067] All reported temperatures are expressed in degrees Celsius ( C.); all reactions were carried out at room temperature (RT) unless otherwise stated.

[0068] Material and Methods

[0069] Chemicals and solvents were obtained from commercial sources, and used without further purification unless otherwise precised. Reactions were monitored by TLC performed on Macherey-Nagel Alugram Sil 60/UV.sub.254 sheets (thickness 0.2 mm). Purification of products was carried out by either column chromatography using Macherey-Nagel silica gel (230-400 mesh).

[0070] NMR spectra were recorded on a Bruker DRX 300 spectrometer (operating at 300 MHz for .sup.1H and 75 MHz for .sup.13C). Chemical shifts are expressed in ppm relative to either tetramethylsilane (TMS) or to residual proton signal in deuterated solvents. Chemical shifts are reported as position (6 in ppm), multiplicity (s=singlet, d=doublet, t=triplet, sept=septuplet, br=broad and m=massif), coupling constant (J in Hz), relative integral and assignment. The attributions of protons and carbons were achieved by analysis of ID and 2D experiments (.sup.1H, .sup.13C).

[0071] Mass spectra were recorded on a Varian triple quadrupole 1200 W mass spectrometer equipped with a non-polar C18 TSK-gel Super ODS (4.650 mm) column, using electro spray ionization and a UV detector (diode array).

[0072] The compound of formula I can be prepared according to the three methods set out in Scheme 1 below.

##STR00004##

Example 1: synthesis of tert-Butyl N-[3-(4-{3-[(1,2,3,4-tetrahydroacridin-9-yl)amino]propyl}piperazin-1-yl)propyl] carbamate (1.2)

[0073] To an oven-dried flask and under nitrogen atmosphere, were placed 9-chloro-1,2,3,4-tetrahydroacridine (0.50 g, 2.3 mmol), Cs.sub.2C0 3 (1.05 g, 3.22 mmol) in dioxane (7.45 mL). The mixture was deoxygenated by passing a stream of nitrogen through it. Xantphos (0.20 g, 0.35 mmol) and Pd.sub.2(dba).sub.3 (0.11 g, 0.12 mmol) were added. Then tert-butyl N-{3-[4-(3-aminopropyl)piperazin-1-yl]propyl}carbamate 1.1 prepared according to J. Med. Chem. 2003, 46, 542-557 (0.83 mg, 2.76 mmol) dissolved in dioxane (7.45 mL) was added to the mixture. The reaction mixture was stirred for 24 hours. The solution was filtered through a celite pad and evaporated. The residue was purified by flash chromatography (eluent DCM/MeOH 0 to 10% then DCM/MeOH sat NH3 0 to 4%. The title compound (1.00 g, 90% yield) was obtained as yellow oil.

[0074] H NMR (300 MHz), (ppm, CDCl.sub.3): 7.95 (dd, .sup.3J=1.1 Hz, .sup.3J=8.5 Hz, 1H); 7.82 (dd, .sup.3J=0.9 Hz, .sup.3J=8.6 Hz, 1H); 7.45 (ddd, .sup.4J=1.2 Hz, .sup.3J=6.8 Hz, .sup.3J=8.2 Hz, 1H); 7.24 (ddd, .sup.4J=1.2 Hz, .sup.3J=6.8 Hz, .sup.3J=8.3 Hz, 1H); 5.53 (br t, 3=5.2 Hz, 1H); 5.06 (br t, 31=5.4 Hz, 1H); 3.48 (m, 2H); 3.12 (m, 2H); 2.98 (t, .sup.3J=6.0 Hz, 2H); 2.66 (t, .sup.3J=6.0 Hz, 2H); 2.55-2.32 (M, 12H); 1.85-1.72 (M, 6H); 1.59 (m, 2H), 1.35 (s, 9H).

[0075] .sup.13C NMR (75 MHz), (ppm, CDCl.sub.3): 158.4; 156.0; 151.0; 147.5; 128.7; 128.0; 123.3; 123.0; 120.3; 115.9; 78.7; 57.0; 53.2; 48.9; 39.8; 34.0; 28.4; 27.2; 26.4; 25.7; 22.9.

[0076] LC-MS (ESI) mlz Calculated: 482.3, Found: 482.4 [M+H].sup.+, 382.2 [M+H-Boc].sup.+; rt: 1.8 min

Example 2: N-{3-[4-(3-Aminopropyl)piperazin-1-yl]propyl}-1,2,3,4-tetrahydroacridin-9-amine (1.3)

[0077] tert-Butyl N-[3-(4-{3-[(1,2,3,4-tetrahydroacridin-9-yl)amino]propyl}piperazin-1-yl)propyl] carbamate 1.2 (90 mg, 0.19 mmol) was dissolved in dioxane saturated with HCl (5 mL). The reaction mixture was stirred for 15 hours. The solvent was evaporated. The residue was dissolved in MeOH(NH.sub.3) and stirred for 2 hours. The solvent was evaporated. The residue was dissolved in 15 mL of DCM and washed with saturated NaHCO.sub.3 solution (310 mL). The organic layer was dried over MgSO.sub.4, filtered and evaporated. The residue was purified by column chromatography (eluent DCM/MeOH(NH.sub.3) 9.4:0.6 (v/v)). The title compound (70 mg, 98% yield) was obtained as yellow oil.

[0078] H NMR (300 MHz), (ppm, CDCl.sub.3): 8.01 (dd, .sup.4J=0.9 Hz, .sup.3J=8.5 Hz, 1H); 7.87 (dd, .sup.4J=0.9 Hz, .sup.3J=8.5 Hz, 1H); 7.51 (ddd, .sup.4J=1.4 Hz, .sup.3J=6.8 Hz, .sup.3J=8.3 Hz, 1H); 7.30 (ddd, V=1.3 Hz, .sup.3J=6.8 Hz, .sup.3J=8.3 Hz, 1H); 5.13 (br s, 1H); 3.55 (t, 3=6.0 Hz, 2H); 3.04 (t, .sup.3J=6.4 Hz, 2H); 2.71 (M, 4H); 2.65-2.37 (M, 12H); 1.91-1.79 (M, 8H); 1.64 (m, 2H).

[0079] .sup.13C NMR (75 MHz), (ppm, CDCl.sub.3): 158.4; 151.1; 147.5; 128.7; 128.1; 123.4; 123.0; 120.3; 115.9; 57.0; 53.4; 49.0; 40.8; 34.0; 30.5; 27.2; 25.7; 22.9.

[0080] LC-MS (ESI) mlz Calculated: 382.3, Found: 382.2 [M+H].sup.+; rt: 1.3 min;

Example 3: N-[3-(4-{3-[(2-Methylpropyl)amino]propyl}piperazin-1-yl)propyl]-1,2,3,4-tetrahydroacridin-9-amine (1.4)

[0081] To a stirred solution of N-{3-[4-(3-aminopropyl)piperazin-1-yl]propyl}-1,2,3,4-tetrahydroacridin-9-amine 1.3 (100 mg, 0.26 mmol) in toluene (5 mL) was added isobutyraldehyde (0.04 mL, 0.4 mmol). The reaction mixture was refluxed with a Dean-Stark apparatus for 1 hour (until no more drops of water appeared). The toluene was evaporated and the residue was dissolved in DCE (5 mL). NaBH 4 (15 mg, 0.4 mmol) was added and the mixture was stirred for 15 hours. 10 mL of saturated NaHCO 3 solution was added. The mixture was stirred for 1 hour. 5 mL of DCM was added and the layers were separated. The organic layer was washed with saturated NaHCO 3 solution (210 mL). The organic layer was dried over MgS0.sub.4, filtered and evaporated. The compound was used without purification for the next step.

[0082] H NMR (300 MHz), (ppm, CDCl.sub.3): 8.03 (dd, .sup.4J=1.0 Hz, .sup.3J=8.5 Hz, 1H); 7.90 (dd, .sup.4J=0.9 Hz, .sup.3J=8.5 Hz, 1H); 7.54 (ddd, .sup.4J=1.3 Hz, .sup.3J=6.8 Hz, .sup.3J=8.3 Hz, 1H); 7.33 (ddd, .sup.4J=1.3 Hz, .sup.3J=6.8 Hz, .sup.3J=8.3 Hz, 1H); 5.14 (br s, 1H); 3.57 (m, 2H); 3.05 (t, .sup.3J=6.2 Hz, 2H); 2.74 (t, .sup.3J=5.5 Hz, 2H); 2.65 (t, .sup.3J=6.9 Hz, 2H); 2.56-2.40 (M, 14H); 1.94-1.66 (M, 11H); 0.92 (d, .sup.3J=6.6 Hz, 6H).

[0083] .sup.13C NMR (75 MHz), (ppm, CDCl.sub.3): 158.5; 151.1; 147.6; 128.8; 128.1; 123.4; 123.0; 120.3; 116.0; 58.2; 57.4; 53.5; 49.0; 34.1; 28.5; 27.2; 25.7; 23.0; 20.7.

[0084] LC-MS (ESI) mlz Calculated: 438.3, Found: 438.3 [M+H].sup.+; rt: 1.5 min.

Example 4: Isobutyraldehyde-</7: 2-(.SUP.2.H.SUB.3.)methyl(2,3,3,3-.SUP.2.H.SUB.4.)propanal

N-Methoxy-2-(.SUP.2.Hg)methyl-N-methyl(.SUP.2.H.SUB.4.)propanamide

[0085] To a stirred solution of 2-(2H.sub.3)methyl(.sup.2H.sub.4)propanoic acid (93 L, 1 mmol) in DCM (3.8 mL) was added N-methylmorpholine (0.715 mL, 6.5 mmol), EDC 1HC1 (249 mg, 1.3 mmol), HOBt (175 mg, 1.3 mmol) and N,O-dimethylhydroxylamine hydrochloride (204 mg, 2.1 mmol). The mixture was stirred for 15 hours. 5 mL of DCM were added to the reaction mixture and the organic layer was washed with saturated NaHCO 3 solution (25 mL), HCl 1M solution (25 mL) and brine (15 mL).

[0086] The organic layer was used without evaporation or purification for the next step.

[0087] H NMR (300 MHz), (ppm, CDCl.sub.3): 3.70 (s, 3H, H.sub.b); 3.19 (s, 3H, H.sub.a).

[0088] .sup.13C NMR (75 MHz), (ppm, CDCl.sub.3): 178.3; 61.4; 32.3; 29.1; 18.3.

[0089] LC-MS (ESI) mlz Calculated: 139.2, Found: 139.8 [M+H]+

Isobutyraldehvde-<j7: 2-(.SUP.2.H.SUB.3.)methyl(2,3,3,3-.SUP.2.H.SUB.4.)propanal

[0090] To a solution of N-Methoxy-2-(.sup.2H.sub.3)methyl-N-methyl(.sup.2H.sub.4)propanamide in DCM (5 mL, 1 mmol) was added LiAlH.sub.4 (0.9 mL, 0.9 mmol). The mixture was stirred for 1 hour. Saturated KHS0.sub.4 solution (4.2 mL) was added dropwise. 5 mL of DCM were added and the organic layer was washed with saturated NaHCO 3 solution (25 mL), HCl 1M solution (25 mL) and brine (15 mL). The organic layer was dried, filtered and used as it is.

Example 5: N-[3-(4-{3-[Bis(2-methylpropyl)amino]propyl}piperazin-1-yl)propyl]-1,2,3,4-tetrahydroacridin-9-amine (I)

[0091] First Protocol (iv)

[0092] To a stirred solution of N-[3-(4-{3-[(2-methylpropyl)amino]propyl}piperazin-1-yl)propyl]-1,2,3,4-tetrahydroacridin-9-amine 1.4 (230 mg, 0.52 mmol) in toluene (10 mL) was added isobutyraldehyde (0.07 mL, 0.8 mmol). The reaction mixture was heated with a Dean-Stark apparatus for 1 hour (until no more drops of water appeared). The toluene was evaporated and the residue was dissolved in DCE (10 mL). NaBH.sub.4 (30 mg, 0.8 mmol) was added and the mixture was stirred for 15 hours. 15 mL of saturated NaHCO 3 solution was added. The mixture was stirred for 1 hour. 10 mL of DCM was added and the layers were separated. The organic layer was washed with saturated NaHCO 3 solution (215 mL). The organic layer was dried over MgSO 4, filtrated and evaporated. The residue was purified by column chromatography on alumina (eluent DCM/MeOH(NH.sub.3) 9.8:0.2 (v/v)).

[0093] The title compound (30 mg, 12% yield) was obtained as colorless oil.

[0094] Second Protocol (v)

[0095] To the organic layer containing isobutyralehyde-<i7 (Example 4) was added N-{3-[4-(3-aminopropyl)piperazin-1-yl]propyl}-1,2,3,4-tetrahydroacridin-9-amine 1.3 (80 mg, 0.21 mmol). The mixture was stirred for 1 hour and STAB (190 mg, 0.899 mmol) was added. The mixture was stirred for 15 hours. 5 mL of saturated NaHCO 3 solution was added and the mixture was stirred for 1 hour. The layers were separated and the organic layer was washed with saturated NaHCO 3 solution (25 mL). The organic layer was dried, filtrated and evaporated. The residue was purified by flash chromatography (DCM/MeOH(NH.sub.3), 10:0 to 9.5:0.5 (v/v)).

[0096] The title compound (36 mg, 34% yield) was obtained as colorless oil.

[0097] Third Protocol (vii)

[0098] In a oven-dried flask and under a nitrogen atmosphere, were placed 9-chloro-1,2,3,4-tetrahydroacridine (257 mg, 1.12 mmol), (+/) BINAP (167 mg, 0.15eq), Cs.sub.2CO 3 (546 mg, 1.4eq), Pd.sub.2(dba).sub.3 (162 mg, 0.15eq) and 3 mL of dry 1,4-dioxane. N-[3-[4-(3-aminopropyl)piperazin-1-yl]propyl]-N-isobutyl-2-methyl-propan-1-amine 1.5 (349 mg, 2.07 mmol) prepared according to Ryckebusch A et al. J. Med. Chem. 2003, 46, 542-557 in dry dioxane (3 mL) was added and the mixture was stirred at 90 C. for 12 h. The solution was filtered through a celite pad and evaporated. The residue was purified by flash chromatography on silica gel (CH.sub.2Cl.sub.2/MeOH//95/5).

[0099] The title compound (419 mg, 12% yield) was obtained as colorless oil.

Characterization of N-r3-r4-(3-aminopropyl)piperazin-1-yllpropyll-N-isobutyl-2-methyl-propan-1-amine 1.5

[0100] .sup.1H NMR (300 MHz), (ppm, CD.sub.3OD): 3.05 (t, .sup.3J=1.2 Hz, 2H); 3.00-2.60 (M, 18H,); 2.10-1.80 (M, 6H); 1.02 (d, .sup.3J=6.5 Hz, 12H).

[0101] .sup.13C NMR (75 MHz), (ppm, CD.sub.3OD): 61.1; 53.8; 53.5; 50.7; 37.6; 24.2; 22.5; 21.3; 19.5.

[0102] LC-MS (ESI) m/z calculated: 313.3, found: 313.2 [M+H].sup.+; rt: 1.15 min

Characterization of N43-(4-134Bis(2-methylpropyl)aininolpropyl}piperazin-1-yl)propyll-1,2,3,4-tetrahydroacridin-9-amine I

.SUP.1.H-Compound

[0103] H NMR (300 MHz), (ppm, CDCl.sub.3): 8.45 (d, .sup.3J=8.6 Hz, 1H); 7.8-7.9 (M, 2H); 7.64 (ddd, .sup.3J=8.6 Hz, .sup.3J=6.7 Hz, .sup.4J=1.1 Hz, 1H); 4.14 (t, .sup.3J=6.6 Hz, 2H); 3.6-4.1 (M, 8H); 3.35-3.55 (M, 6H); 3.11 (d, .sup.3J=6.8 Hz, 4H); 3.0-3.1 (M, 2H); 2.79 (m, 2H); 2.5-2.3 (M, 4H); 2.20 (sept, .sup.3J=6.6 Hz, 2H); 1.9-2.0 (M, 4H); 1.09 (d, .sup.3J=6.5 Hz, 12H).

[0104] .sup.13C NMR (75 MHz), (ppm, CDCl.sub.3): 156.5; 150.9; 138.2; 132.8; 125.5; 124.9; 118.8; 115.9; 112.2; 61.1; 51.2; 44.3; 28.1; 24.7; 24.0; 21.6; 20.4; 19.5; 19.4; 18.1.

[0105] LC-MS (ESI) m/z Calculated: 494.4, Found: 494.4 [M+H].sup.+; rt: 1.4 min;

<W4-Compound

[0106] H NMR (300 MHz), (ppm, CDCl.sub.3): 8.04 (dd, .sup.4J=0.9 Hz, .sup.3J=8.6 Hz, 1H, H.sub.5); 7.90 (dd, .sup.4J=0.7 Hz, .sup.3J=7.7 Hz, 1H, .sup.34); 7.54 (ddd, .sup.4J=1.3 Hz, .sup.3J=6.8 Hz, .sup.3J=8.2 Hz, 1H, H.sub.7); 7.32 (ddd, .sup.4J=1.2 Hz, .sup.3J=6.8 Hz, .sup.3J=8.3 Hz, 1H, H.sub.6); 5.27 (br s, 1H, NH); 3.59 (m, 2H, H.sub.d); 3.07 (t, .sup.3J=5.4 Hz, 2H, H.sub.4); 2.73 (t, .sup.3J=5.8 Hz, 2H, H.sub.1); 2.57-2.40 (M, 14H, H.sub.a, , H.sub.b, , H.sub.d); 2.04 (s, 4H, ); 1.96-1.82 (M, 6H, H.sub.2, H.sub.3, H.sub.c); 0.92 (m, 2H, ).

[0107] .sup.13C NMR (75 MHz), (ppm, CDCl.sub.3): 158.3; 151.2; 147.3; 128.6; 128.2; 123.4; 123.1; 120.2; 115.8; 63.7; 57.4; 53.5; 49.2; 33.9; 27.2; 25.8; 22.9; 20.4; 19.4.

[0108] LC-MS (ESI) m/z Calculated: 508.5, Found: 508.4 [M+H].sup.+; rt: 1.5 min;

Example 6: N-[3-(4-{3-[Bis(2-methylpropyl)amino]propyl}piperazin-1-yl)propyl]-1,2,3,4-tetrahydroacridin-9-amine hydrochloride

[0109] The hydrochloride of the 1 compound of Example 5 obtained according to the third protocol of Example 5 was formed after treatment with MeOH/HCl. Compound of example 5 was solubilized in MeOH, treated with HCl 1M until pH=1 and then lyophilized.

Biological Examples

[0110] Materials and Methods

[0111] Antibodies

[0112] Pan-Tau and phospho-dependent Tau antibodies included Tau-Nter (M19G, 1/10 000, Sergeant et al., 1999), Tau-Cter (1/10 000, Sergeant et al., 1999, Le Freche et al., 2012), Tau-1 (1/5000, Merck Millipore), S199P (directed against the phospho-serine 199, 1/4000, Sergeant et al., 1999), Tau-Phospho S396 (directed against the phosphor-serine 396, 1/10000, Lifetechnologies), Tau-Phospho 404 (1/10 000, Lifetechnologies), 422 (clone 2H9, a homemade monoclonal antibody developed against a phospho-peptide containing the phosphorylated serine 422 residue of Tau, numbering according to the longest human brain Tau-441 isoform), AT8 (1/1000, Thermo Scientific), AT270 (1/2000, Thermo Scientific), AT100 (1/1000, Thermo Scientific). Neuron specific enolase (1/10 000), -actin (1/5000) and -tubulin (1/10 000) were from Sigma-Aldrich.

[0113] Cell Culture and Transfection

[0114] Human neuroblastoma cell line SKNSH-SY5Y cells expressing the human Tau isoform 412 (with the exon 2 and 10 encoding sequences) (or SY5Y-Tau46) was as previously described in Bretteville et al., 2009.

[0115] Drug Treatment

[0116] Neuroblastoma SKNSH-SY5Y cells expressing the human Tau isoform 412 (with the exon 2 and 10 encoding sequences) were treated with 10 M of the hydrochloride salt of N-[3-(4-{3-[Bis(2-methylpropyl)amino]propyl}piperazin-1-yl)propyl]-1,2,3,4-tetrahydroacridin-9-amine (Example 6, hereinafter named Compound I.HQ) which was diluted in the culture medium as described in Bretteville et al., 2009.

[0117] Animal Treatment

[0118] Wild-type C57BL/6J animals of 3 months were purchased at Charles Rivers Laboratories (France) and were allow to acclimate in the animal facility for at least one week before any treatment. Thy-Tau22 transgenic colonies (C57BL/6J genetic background) were obtained by crossing heterozygous Thy-Tau22 males with C57BL/6 WT females. Thy-Tau22 mouse transgenic line exhibits progressive neuron-specific Alzheimer Disease-like Tau pathology devoid of motor deficits. In this model, a progressive development of NFT is observed in the hippocampus and amygdala, which parallels behavioral impairments (Schindowski et al., 2006). Furthermorever, in the hippocampus, hyper- and abnormally phosphorylated Tau species accumulate within the somato-dendritic area (Schindowski et al., 2006).

[0119] All animals were housed in a pathogen-free facility at 5 to 6 animals per cage (Techniplast Cages 1284L). They had ad libitum access to food and water in a 12/12-hour light-dark cycle and maintained in constant temperature of 22 C.

[0120] For animal treatment, animals were randomly distributed and the compound of Formula I was given in the drinking water at final concentration of 0.5 or 1 mg/kg. Drinking bottles were changed once per week, volume consumption was measured along the treatment period. Food consumption and body weight were assessed. In wild-type animals the pilot study of drug treatment was performed for one month to establish the innocuousness of the treatment with the compound of Formula I. This was based on the physiological, social and behavioural assessments. Thy-Tau22 males were treated for 4 months starting at 3 month old. All protocols were approved by the local ethics committee (n 342012, CEEA).

[0121] Spatial Memory Assessment Using Morris Water Maze

[0122] Before any behavioral test, exploratory and locomotion of treated and untreated animal were evaluated in a Open field (OF) 25 cm25 cm arena. Four acquisitions in joined arena were performed simultaneously. Each mouse was placed in the arena and allowed to explore for at least 5 min. Parameters including distance, speed, velocity were acquired by video recording using the EthoVision video tracking equipment and software (Noldus, Information Technology, Paris, France) in a dedicated room. The anxiety, which could interfere with memory test, was evaluated by the elevated plus maze (EPM). Mice were placed in the centre of a plus-shaped maze consisting of two 10 cm wide open arms and two enclosed arms elevated at 50 cm from the floor. Locomotion, distance, speed and velocity were measured as well as the number of arm entries, time spent in the open versus closed arms, percentage of open arm entries and time per minute spent in the open arms on a total of 5 min test.

[0123] Morris Water Maze

[0124] Spatial learning and memory abilities were assessed in a standard hidden-platform acquisition and retention version of the Morris water maze task (Van der Jeugd et al., 2013). A 90-cm circular pool was filled with water opacified with nontoxic white paint and kept at 21 C. A 10-cm round rescue platform was hidden 1 cm beneath the surface of the water at a fixed position. Four positions around the edge of the tank were arbitrarily designated in order to divide it into four cardinal points (North, East, West and South) delineating four quadrants. This target quadrant contains the rescue platform, surrounded by to adjacent quadrant and a opposite quadrant. In the acquisition trial, each mouse was given four swimming trials per day with at least 10 min of intertrial interval, for four consecutive training days. The start position was pseudo-randomized across trials. Mice that failed to find the hidden platform within 2 min were manually positioned on the platform. They were allowed to remain on it for 15 s before putting them back to their cages. The time required to find the hidden rescue platform (escape latency) was used as a spatial learning index and was recorded using the Ethovision XT video tracking system (Noldus France). Swimming speed and total distance were also measured. 72 h after the acquisition phase, the hidden platform was removed and spatial memory was evaluated in a 60 s probe trial. The proportion of time spent in the target quadrant vs. the other quadrants was considered as a spatial memory index.

[0125] Mouse Brain Samples

[0126] After behavioral assessment, blood was collected in 1.5 mL polypropylene tubes (Eppendorf, France), mice were sacrificed by cervical dislocation to avoid any modifications instrumental to anesthesia (Lefreche et al. 2012). Hippocampi and cortices were dissected and snap frozen in 1.5 mL tubes (Eppendorf). Tissue was homogenized in Tris-HCl buffer (10 mM pH 7.4) containing 320 mM sucrose, protease inhibitors (Complete mini EDTA-free, Roche) sonicated and spun at 12,000g for 10 min at 4 C. The supernatant was recovered, protein concentration was achieved using the BCA Protein quantification assay kit (Pierce) according to the manufacturer instruction. The supernatant was further processed for biochemical analyses or kept at 80 C. For ID or 2D gels, the same lysis buffer was used. Thus, equal amount protein in Tris buffer were added with a solution containing 7M urea, 2M thiouree with 2% SDS and sonicated at 60 Hz 30 pulses.

[0127] ID SDS-PAGE and Western-Blotting

[0128] For the western-blot analysis of Tau proteins, SDS-PAGE was performed according to the manufacturer's instructions using Bis-Tris 4-12% gradient acrylamide gel (NuPage Bis-Tris PreCast 12 wells, Life Technologies). The apparent molecular weights were calibrated using the molecular weight markers (Novex and Magic Marks, Life Technologies). Electrophoresis was performed under a continuous tension of lOOV/per gel for 1 h. Proteins were reversibly stained with Ponceau Red to check the quality of the efficacy of protein transfer. For western-blotting, the membranes were blocked in 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% Tween-20 (v/v) (TBS-T) and 5% (w/v) of skimmed milk (TBS-M) for 30 min and they were incubated overnight at 4 C. the primary antibody. The nitrocellulose membrane is rinsed three times by gentle shaking in TBS-T during 10 min each. The membrane was then incubated during 1 h at room temperature with secondary antibody (Peroxydase Labeled Anti-Rabbit IgG (H+L chains), Vector Laboratories). The immunoreactive complexes were revealed using the ECL Western Blotting kit and image acquisition was performed with the LAS-3000 Luminescence Image Analyser (FujiFilm Lifesciences) or exposed to Amersham Hyperfilm ECL (G&E healthcare). Digitized images were processed with ImageJ Software (NIH).

[0129] Two-Dimensional Gel Electrophoresis

[0130] Two-dimensional gel electrophoresis was performed according to the recently published procedure (Fernandez-Gomez et al., 2014). Briefly, cells were rinsed once with PBS and lysed in UTS buffer (7M urea, 2M thiourea, 2% SDS) and sonicated at 60 Hz 30 pulses. Lysate was precipitated with methanol/chloroform. 50 g of proteins were precipitate using the chloroform/methanol procedure and the pellet was dissolved in 2D buffer [urea 7M, thiourea 2M, 4% CHAPS, and 0.6% Pharmalytes 3-10 (G&E Healthcare)]. Immobilized pH gradient strip 3-11 Immobiline DryStrip of 11 cm (G&E Healthcare) were rehydrated with 200 g containing a final amount of 50 g of protein and isoelectrofocusing was achieved with an IPGPhor III (G&E Healthcare) according to the manufacturer's instructions. The strips were layered onto a 4-12% Bis-Tris Poly-acrylamide Gel XT Precast Criterion (BioRad) and run with a constant tension of 100V as described for ID SDS-PAGE. The Western blot was performed as above described for ID SDS-PAGE and Western-blotting.

[0131] Quantification of Tau in Mouse Plasma

[0132] hTau dosage was achieved using the total Tau and phospho-181 hTau Innotest ELISA kits (Fujirebio Europe) according to the manufacturer's instructions. hTau or phospho-Tau dosage was performed using 25 or 75 L of plasma.

[0133] Immunofluorescence

[0134] Neuroblastoma SY5Y-APPWT cells were plated in cell culture chamber slides (Labtek) and allowed to grow in DMEM supplemented with 10% Fetal calf serum. Treatment with compounds were performed following the same protocol described in the drug treatment paragraph. After 48 h, cells were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (PBS) for 15 min at room temperature. Cells were washed 3 times with ice cold PBS and blocked with PBS supplemented with 1% BSA. Antibodies were incubated overnight at 4 C. in PBS-BSA, rinsed 3-times with ice cold-PBS and incubated with a secondary FTIC or Rhodamine-conjugated secondary antibody (Lifesciences Technologies). The coverslips were mounted onto slides with Vectashield with DAPI mounting medium (Vector laboratories). Images were acquired with a Zeiss Apotome microscope or Leica confocal microscope. All data were analyzed using Photoshop Element 6 Software (Adobe) without any modification of raw images.

[0135] Immunohistochemistry

[0136] Serial coronal sections (n=3) of the same brain coordinates (George Paxinos et al.) were incubated with the phospho-dependent AT100 or AT8 antibodies. They respectively recognized phospho-epitope of tau at residues Thr 212-Ser 214 and Ser 202-Thr 205. Quantification of labeled neurons was achieved using the NIH-Image J software and expressed in arbitrary units (A.U).

[0137] Statistical Analyses

[0138] Results were reported as means SEM. Differences between mean values were evaluated using the Student's t-test, one Way-ANOVA followed by a post-hoc Fisher's LSD test using GraphPad Prism 6 Software. P values<0.05 were considered significant.

[0139] Results

[0140] Modulatory Effect on Tau Phosphorylation and Tau Degradation

[0141] The effect of the compound of Formula I on Tau phosphorylation was analyzed in vitro and in vivo. Neuroblastoma SKNSH-SY5Y cells expressing the human Tau isoform 412 (with the exon 2 and 10 encoding sequences) were treated with 10 M of the compound of Formula I.

[0142] In Vitro Experiments

[0143] Expression and total phosphorylation was assessed by ID and 2D gel electrophoresis using phospho-dependent and pan-Tau antibodies (FIGS. 1 and 2). The phospho-dependant Tau antibody S199P is directed against the phosphorylated serine 199 residue (numbering according to the longest human Tau isoform) and is an early marker of Tau hyperphosphorylation during neurofibrillary degeneration (Maurage et al., 2003). The S396 antibody is directed to an epitope comprising the phosphorylated serine 396 of Tau (numbering according to the longest brain tau isoform of 441 amino acids). Tau expression is induced by tetracycline treatment and cells were treated afterwards with the compound of Formula I. Change of Tau expression or phosphorylation is compared to the induced but non-treated condition.

[0144] No significant change of Tau expression was observed with the compound of Formula I although Tau phosphorylation at serines 199 and 396 was reduced in cells treated with the compound of Formula I (FIG. 1).

[0145] These results were corroborated using 2D gel electrophoresis. In control conditions, isovariants of Tau were resolved between pi 6.5 and 10.5. Following treatment with the compound of Formula I, a reduction of the isovariants at the neutral pi was observed with Tau396 phospho-dependent antibody (S396) as well as with both N- and C-terminal directed pan-Tau antibodies (FIG. 2).

[0146] In Vivo Experiments

[0147] To further determine the effect of the compound of Formula I on Tau metabolism, wild-type C57/1BL6 animals were treated with 0.5 and 1 mg/Kg of the compound of Formula I for 1 month and endogenous MAPT expression was assessed by 2D gel electrophoresis. Tau protein isovariants consist of three series ranging from pi 10 to 6. The major and most basic isovariants correspond to the mouse Tau isoform comprising the sole exon 10 encoding sequence. The two other series of isoforms of molecular weight of 64 and 69 kDa are isovariants of Tau isoforms which, in addition to the exon 10, contain exon 2 or exon 2+3 encoding sequences, respectively.

[0148] As shown at FIG. 3 with the Tau-Cter antibody, the distribution of Tau isovariants did not significantly change in treated animals although low molecular weight Tau catabolic products (indicated by arrows on FIG. 3) increased in number and intensity at 0.5 mg/Kg and even more significantly at 1 mg/Kg of the compound of Formula I.

[0149] Interestingly when the antibody SI99 is used, 5 isovariants of Tau are detected in the non-treated condition and the number of isovariants stained diminished to 3 and 2 isovariants at both dosages (0.5 mg/Kg and 1 mg/Kg) of the compound of Formula I, respectively. The surface of the spots was also reduced suggesting a lower abundance of the phospho-site after treatment, although the overall 2D profile of the full-length Tau isoforms is preserved after treatment.

[0150] Altogether, these results suggest that the compound of Formula I modulates the endogenous Tau metabolism, in particular by reducing Tau phosphorylation and by increasing Tau proteolysis.

[0151] Preserved Spatial Memory and Reduced Tau Pathology in Thy-Tau22 Transgenic Animals

[0152] The hippocampal development of NFT in Thy-Tau22 is correlated to an impairment of the spatial memory (Laurent et al., 2015). The spatial memory can be preserved by voluntary exercise (Belarbi et al, 2011) or immunotherapy (Troquier et al., 2012) thus making Thy-Tau22 a good model to test beneficial or detrimental effect of drugs. Thy-Tau22 mice were treated for 4 months with 0.5 mg/kg of Compound I.HCl and the spatial memory was assessed using the morris water maze.

[0153] As shown at FIG. 4A, the learning phase is not significantly different between the animal groups in the upper panel. After 5 days of learning, the distance to reach the platform is reduced showing that mice have successfully learnt. In the probe test, performed after 72 hours following the last learning day, the Compound of Formula I had no significant effect whereas in Thy-Tau22 mice the spatial memory was preserved.

[0154] In wild-type animals, the animal had a significant preference for the target quadrants than the three other quadrants independently of the treatment (FIG. 4B). Thus, the Compound of Formula I did not impact the cognitive performance of wild-type animals.

[0155] Thy-Tau22 animals spent equal % of time in the target quadrant (where the platform is positioned during the learning phase) versus the others. The lack of distinction is interpreted as a loss of spatial memory. In Thy-Tau22 treated animal, mice spent more time in the target quadrant and therefore showed a preserved spatial memory. The ANOVA statistical analysis showed that a treatment with the compound of Formula I protected or preserved the Thy-Tau22 mice from developing spatial memory impairments.

[0156] The burden of NFT in the hippocampal CA1 was investigated by immunohistochemistry and quantification. As mentioned above, AT100 recognizes pathological sites of Tau (residues Thr 212-Ser 214), and AT8 is used to detect the presence of phosphorylated tau (residues Ser 202-Thr 205). As shown at FIG. 5, the average surface of staining measured after AT100 staining was significantly reduced in Compound I.HCl treated animal whereas no significant differences were observed after AT8 immuno staining. AT100 recognized a pathological Tau epitope suggesting that Compound I.HQ impacts on the development of tau pathology but not necessarily on phosphorylated Tau proteins labeled by AT8.

[0157] To further assess this hypothesis, Tau expression and phosphorylation was further investigated by western-blotting (FIG. 6).

[0158] Brain proteins (of hippocampus) from untreated and Compound I.HCl treated thy-tau22 animals were resolved by ID SDS-PAGE and Tau proteins were labeled with Pan-Tau antibodies (N-ter, C-ter). Catabolites detected with these antibodies are indicated as f-Nter and f-Cter. Phosphorylated variants of Tau were stained with the pSerl99 (against phosphorylated serine 199), pSer262 (against the phosphorylated serine 262), pSer396 (against the phosphorylated serine 396), pSer404 (against the phosphorylated serine 404), pSer422 (against the phosphorylated serine 422) and Tau-1 (against unphosphorylated epitope comprised between amino acid residues 198-205). The quantification of each phospho-epitote is expressed as the ratio of phospho-epitope upon the Total hTau signal determined by the N-ter staining. This ratio is given as the % of variation to the control condition (untreated Thy-Tau22 animals) whose value is arbitrary given the value of 100%.

[0159] A significant reduction of Tau phosphorylation at phospho-sites 396 and 404 was observed whereas other phospho-sites were not significantly modified (FIG. 6). More interestingly, Tau C-terminal catabolites products at 43 kDa were significantly increased whereas the N-terminal catabolites were significantly decreased (FIG. 6). These results suggest that a treatment with the Compound I-HCl reduces the burden of Tau pathology by reducing the phosphorylation of Tau and increasing the catabolism of Tau which is sufficient to preserve the Thy-Tau22 animal model of neurofibrillary degeneration from development of spatial memory impairment.

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