O-alkyl-benzylideneguanidine derivatives and therapeutic use for the treatment of disorders associated with an accumulation of misfolded proteins

Abstract

The present invention relates to a compound of formula (I), or a tautomer and/or a pharmaceutically acceptable salt thereof, ##STR00001## and its uses to treat a disorder associated with protein misfolding stress and in particular with an accumulation of misfolded proteins.

Claims

1. A method for treating a disease associated with protein misfolding stress comprising administering a compound of formula (II): ##STR00089## or a tautomer form thereof, or a pharmaceutically acceptable salt thereof wherein: Hal=F, Cl, Br, I X is either −CR1= or −N=, Y is either −CR2= or −N=, Z is either −CR3= or −N=, W is either −CR4= or −N=, R1 is selected from the group consisting of H, Hal, and alkyl; R2 is selected from the group consisting of H, Hal, alkyl, and C(O)R6; R3 is selected from the group consisting of H, Hal, alkyl and O-alkyl; R4 is H, Cl, F, I or Br; R5is alkyl, cycloalkyl, aralkyl, alkenyl, cycloalkenyl, heterocyclyl, aryl, C(O)-alkyl, and C(O)-aryl, each of which is optionally substituted with one or more R7 groups; R6 is selected from the group consisting of OH, O-alkyl, O-aryl, aralkyl, NH.sub.2, NH-alkyl, N(alkyl).sub.2, NH-aryl, CF.sub.3, alkyl, and alkoxy; and each R7 is independently selected from the group consisting of halogen, OH, CN, COO-alkyl, aralkyl, heterocyclyl, alkyl, SO-alkyl, SO.sub.2-alkyl, SO.sub.2-aryl, COOH, CO-alkyl, CO-aryl, NH.sub.2, NH-alkyl, N(alkyl).sub.2, CF.sub.3, alkyl, and alkoxy; to a patient in the need thereof.

2. The method according to claim 1 wherein Hal is Cl.

3. The method according to claim 1 wherein X is −CR1= and R1 is H or F.

4. The method according to claim 1 wherein Y is −CR2= and R2 is H or F.

5. The method according to claim 1 wherein Z=−CR3= and R3 is H or F.

6. The method according to claim 1 wherein W=−CR4= and R4 is H, Cl or F.

7. The method according to claim 1 wherein R5is alkenyl or alkyl, each of which is optionally substituted with one or more R7 groups selected from the group consisting of halogen, OH, heterocyclyl, S-alkyl, SO-alkyl, SO.sub.2-alkyl, and O-alkyl.

8. The method according to claim 1 wherein the compound is selected from the group consisting of the following: ##STR00090## ##STR00091## ##STR00092## or acceptable salt thereof.

9. The method according to claim 1 wherein the disease is associated with the PPP1R15A pathway.

10. The method according to claim 1 wherein the disease is a tauopathy selected from the group consisting of Alzheimer disease, progressive supranuclear palsy, corticobasal degeneration, frontotemporal lobar degeneration or frontotemporal dementia (FTD) (Pick's disease); synucleinopathies chosen from Parkinson's disease, dementia with Lewy bodies, pure autonomic failure, and multiple system atrophy; polyglutamine and polyalanine diseases selected from the group consisting of Huntington disease, spinobulbar muscular atrophy (or Kennedy disease), dentatorubral-pallidoluysian atrophy, Spinocerebellar ataxia type 1, Spinocerebellar ataxia type 2, Spinocerebellar ataxia type 3 (or Machado-Joseph disease), Spinocerebellar ataxia type 6, Spinocerebellar ataxia type 7 and Spinocerebellar ataxia type 17, oculo-pharyngeal muscular dystrophy, demyelinating disorders like leukodystrophies, Charcot-Marie-Tooth disease and multiple sclerosis, cystic fibrosis, chosen from systemic lupus erythematosus, pancreatitis, sepsis, colitis, ulcerative colitis, Inflammatory Bowel Disease, Systemic Inflammatory Response Syndrome (SIRS), cancers, diabetes, amyotrophic lateral sclerosisseipinopathies, lysosomal storage disorders, amyloidosis diseases, inflammation, metabolic disorders and cardio-vascular disorders chosen from adiposity, hyper-lipidemia, familial hyper-cholesterolemia, obesity, atherosclerosis, hypertension, heart diseases, cardiac ischaemia, stroke, myocardial infraction, trans-aortic constriction, vascular stroke; osteoporosis, nervous system trauma, ischemia, osteoporosis, retinal diseases selected from the group consisting of retinitis pigmentosa, retinal ciliopathies, glaucoma, macular degeneration and aging.

11. The method according to claim 1 wherein the disease associated with protein misfolding stress is associated with an accumulation of misfolded proteins.

Description

BRIEF DESCRIPTION OF THE DRAWING FIGURES

(1) The present invention is further described with reference to the following figures wherein:

(2) FIG. 1 shows dose dependent protection of Hela cells by compound 12 of the invention from ER stress induced by 6 hour exposure to tunicamycin.

(3) FIG. 2 shows dose dependent protection of interferon-gamma injured rat oligodendrocytes by compound 11, compound 12 and compound 17 of the invention.

(4) FIG. 3 shows dose dependent protection of rotenone injured primary mesencephalic rat neurons by compound 5, compound 12 and compound 17 of the invention.

(5) FIG. 4 shows dose dependent protection of amyloid-beta 1-42 injured primary cortical rat neurons by compound 12 of the invention.

(6) FIG. 5 shows the ability of compound 12 and compound 17 at 5 microM and 10 microM respectively to prevent the accumulation of T181P mutated DM20 protein in Human 293T cell.

(7) FIG. 6 shows the ability of compounds 16 to prevent cell death associated with the accumulation of misfold prone Insulin Akita expressed in Min6 cells.

(8) FIG. 7 shows the ability of compound 12, compound 16 and compound 17 at different concentrations to prevent Min6 insulinoma cell death associated with accumulation of misfolded protein induced by 6 hour exposure to tunicamycin.

(9) FIG. 8 shows the ability of compound 11, compound 12, compound 16 and compound 17 at different concentrations to prevent INS1 insulinoma cell death associated with accumulation of misfolded protein induced by 6 hour exposure to tunicamycin.

(10) FIG. 9 shows the ability of compounds 6, 10, 11, 12, 15, 16 and 17 (at 25 microM) to prevent type-I interferon production by mouse embryonic fibroblasts lipofected with poly I:C.

(11) FIG. 10 shows the ability of compound 10 to protect neonatal rat cardiomyocytes against hypoxia-induced apoptosis. The graph shows the percentage of apoptotic cells measured by FACS analysis. Cardiomyocytes were exposed to hypoxia (0.3% O.sub.2) for 36 h in the absence (0 μM) or in the presence of indicated concentrations of Compound 2 (n=3).

(12) The present invention is further described with reference to the following non-limiting examples.

EXAMPLES

1-METHODS & MATERIALS

(13) 1.1- Preparation of the compounds according to the present invention

(14) The reactants and commercials compounds were purchased from Acros Organics, Sigma-Aldrich. The compounds according to the present invention can be prepared according to the following general procedure:

(15) Compounds 1 & 2: preparation of 2-(2-chlorobenzyl)-N′-(3-methylbutoxy)hydrazinecarboximidamide formate salt (compound 1) and 2-(2-chlorobenzyl)-N′-(3-methylbutoxy)hydrazinecarboximidamide (compound 2)

(16) 2-(3-methylbutoxy)-1H-isoindole-1,3(2H)-dione (1-1)

(17) ##STR00020##

(18) Triethylamine (49.58 g) was added drop wise to a stirred solution of N-Hydroxyphthalimide (40 g) and 1-bromo-3-methyl butane (37.4 g) in DMF (600 ml) at room temperature. The reaction mixture was stirred at 70° C. for 18 hours. The reaction mixture was allowed to cool to room temperature. The mixture was concentrated under reduced pressure and the residue thus obtained was suspended in cold water (1000 ml). The resulting suspension was stirred well for some time and the solid was filtered off under reduced pressure. The solid was further washed with demineralized water (200 ml) and hexane (100 ml). The resulting solid was dried under reduced pressure to get a crude material which was purified by column chromatography using silica gel. The desired product eluted at around 2% Methanol in dichloromethane. Evaporation of pure product fractions gave 50.0 g of 2-(3-methylbutoxy)-1H-isoindole-1,3(2H)-dione (Yield: 87.4%). .sup.1H-NMR (DMSO-d6): δ(ppm) 0.93 (d, 6H), 1.57 (q, 2H), 1.82 (m, 1H), 4.16 (t, 2H), 7.86 (s, 4H); LC-MS: m/z=234.25 (M+H).

(19) 1-(amino-oxy)-3-methylbutane hydrochloride (I-2)

(20) ##STR00021##

(21) Hydrazine hydrate (12.8 g) was added drop-wise to a stirred solution of 2-(3-methylbutoxy)-1H-isoindole-1,3(2H)-dione (45 g) in methanol (600 ml) at room temperature. The reaction mixture was stirred at the same temperature for 24 hours. The reaction mixture was filtered off to remove the insoluble by-product and the resulting filtrate was concentrated under reduced pressure to get a crude material which was purified by column chromatography using silica gel. The desired product eluted at around 1% Methanol in dichloromethane. Evaporation of pure product fractions gave the desired intermediate as free base which was converted as hydrochloride salt using 4M HCl in 1,4-dioxane, to get 3.3 g of 1-(aminooxy)-3-methylbutane hydrochloride. .sup.1H-NMR (DMSO-d6): δ(ppm) 0.89 (d, 6H), 1.46 (q, 2H), 1.65 (m, 1H), 4.01 (t, 2H), 10.84 (s, 3H).

(22) N′-(3-methylbutoxy)hydrazinecarboximidamide (1-3)

(23) ##STR00022##

(24) 2N NaOH solution (3.6 ml) was added drop wise to a stirred solution of 1-(amino-oxy)-3-methylbutane hydrochloride (1.2 g) and s-methylisothiosemicarbazide hydro-iodide (2.02 g) in water (3.6 ml) at room temperature and was stirred for 48 hours. Then, the reaction mixtures was concentrated under reduced pressure and the residue was azeotroped with methanol (5 ml). The resulting residue was suspended in ethanol (10 ml) and insoluble inorganic salts were removed by filtration. The filtrate was directly used for the next step without any further processing. N′-(3-methylbutoxy)hydrazinecarboximidamide was confirmed by LCMS analysis. LC-MS: m/z=161.5 (M+H).

(25) 2-(2-chlorobenzyl)-N′-(3-methylbutoxy)hydrazinecarboximidamide formate salt (compound 1)

(26) ##STR00023##

(27) 2-chlorobenzaldehyde (1.81 g) was added drop wise to the filtrate which contain N′-(3-methylbutoxy)hydrazinecarboximidamide at room temperature and was stirred for 2 hours. Then, the reaction mixture was concentrated under reduced pressure and the residue thus obtained was further purified by Prep HPLC using 0.1% HCOOH/water/MeCN to give 0.27 g of 2-(2-chlorobenzyl)-N′-(3-methylbutoxy)hydrazinecarboximidamide as formate salt (Yield: 13.1%) .sup.1H-NMR (DMSO-d6): δ (ppm) 0.88 (d, 6 H), 1.48 (q, 2H), 1.68 (m, 1H), 3.75 (t, 2H), 7.32 (m, 2 H), 7.44 (m, 2 H), 8,10 (m, 1 H), 8.14 (m, 1H), 8.25 (m, 1H), 11.80 (s broad, 2H). LC-MS: m/z=282.88 (M+H).

(28) 2-(2-chlorobenzyl)-N′-(3-methylbutoxy)hydrazinecarboximidamide (compound 2)

(29) ##STR00024##

(30) 2-(2-chlorobenzyl)-N′-(3-methylbutoxy)hydrazinecarboximidamide formate salt (220 mg) was dissolved in water and was basified by saturated NaHCO.sub.3 aqueous solution. The basic aqueous solution was extracted with Dichloromethane and the organic layer was washed with water, dried over sodium sulphate and evaporated under reduced pressure to give 180 mg of 2-(2-chlorobenzyl)-N′-(3-methylbutoxy)hydrazinecarboximidamide as free base (Yield: 95%). .sup.1H-NMR (DMSO-d6): δ (ppm) 0.89 (d, 6H), 1.49 (q, 2H), 1.69 (m, 1H), 3.75 (t, 2H), 5.73 (s broad, 2H), 7.30 (m, 2H), 7.44 (m, 1H), 8,11 (m, 1H), 8.15 (m, 1H), 10.48 (s broad, 1H). LC-MS: m/z=282.82 (M+H).

(31) Compound 3: Preparation of 2-(2-chlorobenzylidene)-N′-[2-(methylsulfonyOethoxy]hydrazinecarboximidamide

(32) 2[2-(methylsulfanyl)ethoxy]-1H-isoindole-1,3(2H)-dione (1-4)

(33) ##STR00025##

(34) 2-chloroethyl methyl sulfide (10.1 g) was added drop-wise to a stirred solution of N-Hydroxyphthalimide (12.5 g), potassium iodide (2.5 g) and potassium carbonate (21.1 g) in DMF (150 ml) at room temperature and was stirred at the 80° C. for 18 hours. The reaction mixture was allowed to cool to room temperature and was dumped in 500 ml of cold water. Then, the solid thus obtained was filtered off under reduced pressure. The resulting solid was dried under reduced pressure to give 9.7 g of 2-[2-(methylsulfanyl)ethoxy]-1 H-isoindole-1,3(2H)-dione (Yield: 52.8%) and was used for the next step without any further processing. .sup.1H-NMR (DMSO-d6): δ (ppm) 2.16 (s, 3 H), 2.84 (t, 2H), 4.29 (t, 2H), 7.87 (s, 4H). LC-MS: m/z=238.4 (M+H).

(35) 2[2-(methylsulfonyl)ethoxy]-1H-isoindole-1,3(2H)-dione (1-5)

(36) ##STR00026##

(37) m-CPBA (11 g) was added portion wise to a stirred solution of 2-[2-(methylsulfanyl)ethoxy]-1H-isoindole-1,3(2H)-dione (9.6 g) in dichloromethane (100 ml) at room temperature and was stirred at room temperature for 6 hours. The crude was concentrated under reduced pressure and the resulting residue was suspended in saturated NaHCO.sub.3 solution (100 ml) and stirred for 30 minutes. The resulting solid was filtered off under reduced pressure and washed with water (50 ml) and was dried under reduced pressure to give 9.0 g of 2[2-(methylsulfonyl)ethoxy]-1H-isoindole-1,3(2H)-dione (Yield: 82.6%) and was used for the next step without any further processing. .sup.1H-NMR (DMSO-d6): δ (ppm) 3.15 (s, 3 H), 3.66 (t, 2H), 4.54 (t, 2H), 7.88 (s, 4 H). LC-MS: m/z=270.3 (M+H).

(38) 1-(aminooxy)-2-(methylsulfonyl)ethane hydrochloride (1-6)

(39) ##STR00027##

(40) 85% methyl hydrazine (2.0 g) was added drop wise to a stirred suspension of 2[2-(methylsulfonyl)ethoxy]-1H-isoindole-1,3(2H)-dione (9.0 g) in dichloromethane (100 ml) at room temperature and was stirred for 6 hours. Then the reaction mixture was filtered off under reduced pressure to remove insoluble by-product. The resulting filtrate was concentrated under reduced pressure at lower temperature. The residue was suspended in 1N HCl (100 ml) and extracted by ethyl acetate (3×250 ml). The resulting aqueous solution containing the desired product was concentrated under reduced pressure to give white solid which was further triturated with diethyl ether and dried under reduced pressure to give 4.0 g of 1-(aminooxy)-2-(methylsulfonyl)ethane hydrochloride (Yield: 68.3%). .sup.1H-NMR (DMSO-d6): δ (ppm) 3.04 (s, 3H), 3.60 (t, 2H), 4.38 (t, 2H), 10.09 (s broad, 2H). LC-MS: m/z=270.3 (M+H).

(41) N′-[2-(methylsulfonyl)ethoxy]hydrazinecarboximidamide (1-7)

(42) ##STR00028##

(43) 2N NaOH solution (4.28 ml) was added drop wise to a stirred solution of 1-(aminooxy)-2-(methylsulfonyl)ethane hydrochloride (1.5 g) and s-methylisothiosemicarbazide hydroiodide (1.99 g) in water (4.5 ml) at room temperature and was stirred for 48 hours. Then, the reaction mixture was concentrated under reduced pressure and the residue was azeotroped with methanol (5 ml). The resulting material was suspended in ethanol (10 ml) and insoluble inorganic salts were removed by filtration. The resulting filtrate which contain N′-[2-(methylsulfonyl)ethoxy]hydrazinecarboximidamide was directly used for the next step without any further processing.

(44) 2-(2-chlorobenzylidene)-N′-[2-(methylsulfonyl)ethoxy]hydrazinecarboximidamide (Compound 3)

(45) ##STR00029##

(46) 2-chlorobenzaldehyde (1.32 g) was added drop wise to the filtrate containing N′-[2-(methylsulfonyl)ethoxy]hydrazinecarboximidamide at room temperature. The mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure and the residue thus obtained was further purified by Prep HPLC using 0.1% NH.sub.3/water/MeCN to give 20 mg of 2-(2-chlorobenzylidene)-N′-[2-(methylsulfonyl)ethoxy]hydrazinecarboximidamide (Yield: 0.7% for 2 steps). .sup.1H-NMR (DMSO-d6): δ (ppm) 3.03 (s, 3 H), 3.45 (m, 2H), 4.12 (m, 2H), 6.11 (s broad, 2H), 7.40 (m, 2H), 7.44 (m, 1 H), 8.15 (m, 1 H), 8.26 (s broad, 1 H), 10.48 (s, 1 H). LC-MS: m/z=318.83 (M+H).

(47) Compound 4: 2-(2-chlorobenzylidene)-N′-(methylsulfony0propoxylhydrazinecarboximidamide

(48) 2[3-(methylsulfanyl)propoxy]-1H-isoindole-1,3(2I-1)-dione (1-8)

(49) ##STR00030##

(50) Diisopropyl azodicarboxylate (77.92 ml) was added drop wise to a stirred solution of N-Hydroxyphthalimide (36.8 g), 3-(methylsulfanyl)-1-propanol (30 g) and triphenylphosphine (37.1 g) in anhydrous THF (600 ml) under nitrogen atmosphere at 0° C. The reaction mixture was stirred at 0° C. for 30 minutes and then it was allowed to warm to room temperature and was stirred for 18 hours. Then, the reaction mixture was concentrated under reduced pressure to get a crude material which was purified by column chromatography using silica gel. The desired product eluted at 4% ethyl acetate in hexane. Evaporation of pure product fractions gave 30 g of 2[3-(methylsulfanyl)propoxy]-1H-isoindole-1,3(2H)-dione (Yield: 42.2%). .sup.1H-NMR (DMSO-d6): δ (ppm) 1.94 (q, 2H), 2.07 (s, 3H), 2.67 (t, 2H), 4.23 (t, 2H), 7.87 (s, 4H). LC-MS: m/z=252.4 (M+H).

(51) 2[3-(methylsulfonyl)propoxy]-1H-isoindole-1,3(2H)-dione (I-9)

(52) ##STR00031##

(53) m-CPBA (61.89 g) was added portion wise to a stirred solution of 2-[3-(methylsulfanyl)propoxy]-1H-isoindole-1,3(2H)-dione (30.0 g) in dichloromethane (550 ml) at room temperature. The mixture was stirred at the room temperature for 5 hours. Then, the reaction mixtures was concentrated under reduced pressure to get a crude material which was suspended in saturated NaHCO.sub.3 solution (250 ml) and stirred well for 30 minutes. The resulting solid was filtered off under reduced pressure and washed with water (100 ml). The solid was dried under reduced pressure to give 22 g of 2[3-(methylsulfonyl)propoxy]-1H-isoindole-1,3(2H)-dione (yield: 65%). .sup.1H-NMR (CDCl3): δ (ppm) 2.32 (m, 2H), 3.00 (s, 3H), 3.50 (t, 2H), 4.39 (t, 2H), 7.83 (m, 4H). LC-MS: m/z=283.9 (M+H).

(54) 1-(aminooxy)-3-(methylsulfonyl)propane hydrochloride (1-10)

(55) ##STR00032##

(56) 85% methyl hydrazine (4.2 g) was added drop wise to a stirred suspension of 2-[3-(methylsulfonyl)propoxy]-1H-isoindole-1,3(2H)-dione (20 g) in dichloromethane (300 ml) at room temperature and was stirred for 6 hours. Then, the solution was filtered off under reduced pressure to remove the insoluble by-product. The resulting filtrate was concentrated under reduced pressure at low temperature. The residue was suspended in 1N HCl (200 ml) and extracted by ethyl acetate (3×500 ml) to remove undesired impurities. The resulting aqueous solution was concentrated under reduced pressure to give a white solid which was further triturated with diethyl ether and dried under reduced pressure to give 8.0 g of 1-(aminooxy)-3-(methylsulfonyl)propane hydrochloride (Yield: 59.8%). .sup.1H-NMR (DMSO-d6): δ (ppm) 2.04 (m, 2H), 3.02 (s, 3H), 3.19 (t, 2H), 4.12 (t, 2H), 11.06 (s broad, 3H).

(57) N′-[3-(methylsulfonyl)propoxy]hydrazinecarboximidamide (I-11)

(58) ##STR00033##

(59) 2N NaOH solution (5.28 ml) was added drop wise to a stirred solution of 1-(aminooxy)-3-(methylsulfonyl)propane hydrochloride (2.0 g) and s-methylisothiosemicarbazide hydroiodide (2.46 g) in water (6.0 ml) at room temperature. The reaction mixture was stirred at the room temperature for 24 hours. Formation of N′-[3-(methylsulfonyl)propoxy]hydrazinecarboximidamide was confirmed by LCMS analysis. Then, the mixture was concentrated under reduced pressure and the residue was azeotroped with methanol (15 ml). The resulting material was suspended in ethanol (15 ml) and the insoluble inorganic salts were removed by filtration. The filtrate was directly used for the next step without any further processing. LC-MS: m/z=210.8 (M+H).

(60) 2-(2-chlorobenzylidene)-N′-[3-(methylsulfonyl)propoxy]hydrazinecarboximidamide (compound 4)

(61) ##STR00034##

(62) 2-chlorobenzaldehyde (1.62 g) was added drop wise to the filtrate containing N′-[3-(methylsulfonyl)propoxy]hydrazinecarboximidamide at room temperature. The resulting reaction mixture was stirred at the same temperature for 2 hours. The crude was concentrated under reduced pressure and the residue thus obtained was further purified by Prep HPLC using 0.1% NH.sub.3/water/MeCN. After purification, the material was stirred in saturated NaHCO.sub.3 solution and the resulting solid was filtered off under reduced pressure and washed with water and dried to give 0.14 g of pure 2-(2-chlorobenzylidene)-N′-[3-(methylsulfonyl)propoxy]hydrazinecarboximidamide (Yield: 4% for 2 steps). .sup.1H-NMR (DMSO-d6): δ (ppm) 2.01 (m, 2H), 2.98 (s, 3H), 3.24 (t, 2H), 3.82 (t, 2H), 5.90 (s, 2H), 7.31 (m, 2H), 7.43 (d, 1H), 8.13 (m, 2H), 10.48 (s, 1H). LC-MS: m/z=333.5 (M+H).

(63) Compound 5: 2-(2-chlorobenzylidene)-N′-(prop-2-en-1-yloxy) hydrazine carboximidamide

(64) N′-(prop-2-en-1-yloxy)hydrazinecarboximidamide (1-12)

(65) ##STR00035##

(66) 2N NaOH solution (6.8 ml) was added drop wise to a stirred solution of 0-Allylhydroxylamine hydrochloride (1.5 g) and s-methylisothiosemicarbazide hydroiodide (3.22 g) in water (4.2 ml) at room temperature. The reaction mixture was stirred at room temperature for 48 hours. Formation of intermediate I-12 N′-(prop-2-en-1-yloxy)hydrazinecarboximidamide was confirmed by LCMS analysis. Then, the mixture was concentrated under reduced pressure and the residue was azeotroped with methanol (5 m1). The resulting material was suspended in ethanol (10 ml) and the insoluble inorganic salts were removed by filtration. The filtrate was directly used for the next step without any further processing. LC-MS: m/z=130.6 (M+H).

(67) 2-(2-chlorobenzylidene)-N′-(prop-2-en-1-yloxy)hydrazinecarboximidamide (Compound 5)

(68) ##STR00036##

(69) 2-chlorobenzaldehyde (1.9 g) was added drop wise to the filtrate containing N′-(prop-2-en-1-yloxy)hydrazinecarboximidamide at room temperature and was stirred for 2 hours. The reaction mixture was concentrated under reduced pressure and the residue thus obtained was further purified by Prep HPLC using 0.1% HCOOH/water/MeCN to give 0.25 g of 2-(2-chlorobenzylidene)-N′-(prop-2-en-1-yloxy)hydrazinecarboximidamide (Yield: 6.1% for 2 steps. .sup.1H-NMR (DMSO-d6): δ (ppm) 3.17 (s, 1H), 4.23 (m, 2H), 5.82 (s broad, 2H), 5.98 (m, 1H), 7.37 (m, 2H), 8.15 (m, 3H). LC-MS: m/z=252.8 (M+H).

(70) Compound 6: 2-(2-chlorobenzylidene)-N′-(2-hydroxyethoxy) hydrazine carboximidamide 2-(2-hydroxyethoxy)-1H-isoindole-1,3(2H)-dione (1-13)

(71) ##STR00037##

(72) 2-Bromotehanol (13.26 ml) was added drop wise to a stirred solution of N-Hydroxyphthalimide (10.0 g) and Sodium acetate (25.14 g) in DMF (50 ml) at room temperature. The resulting reaction mixture was stirred at 80° C. for 1.5 hours. The reaction mixture was allowed to cool to room temperature and was dumped in 500 ml of cold water and the product was extracted by ethyl acetate (2×400 ml). The resulting organic layer were combined and distilled under vacuum. The residue was stirred in cold water and the resulting solid was filtered off under vacuum. The solid was dried under reduced pressure to give 6.0 g of 2-(2-hydroxyethoxy)-1 H-isoindole-1,3(2H)-dione (Yield: 47.3%) which were used for the next step without any further processing. .sup.1H-NMR (DMSO-d6): δ (ppm) 3.70 (q, 2H), 4.18 (t, 2H), 4.83 (t, 1H), 7.87 (s, 4H). LC-MS: m/z=208.34 (M+H).

(73) 2-(aminooxy)ethanol hydrochloride (1-14)

(74) ##STR00038##

(75) 85% methyl hydrazine (1.25 g) was added drop wise to a stirred suspension of 2-(2-hydroxyethoxy)-1H-isoindole-1,3(2H)-dione (6.0 g) in dichloromethane (25 ml) at room temperature and was stirred for 2 hours. Then, the reaction mixture was filtered off under reduced pressure to remove insoluble by-product. The filtrate was concentrated under reduced pressure at lower temperature. The residue was suspended in 2N HCl in Ethylacetate (20 ml) and concentrated under reduced pressure at lower temperature. The resulting solid was triturated with Dichloromethane (2×15 ml) and dried under reduced pressure to give 2.8 g of 2-(aminooxy)ethanol hydrochloride (Yield: 85.5% as mono hydrochloride salt). .sup.1H-NMR (DMSO-d6): δ (ppm) 3.61 (m, 2H), 4.04 (t, 2H), 4.73 (m, 1H), 11.02 (s broad, 2H).

(76) N′-(2-hydroxyethoxy)hydrazinecarboximidamide (1-15)

(77) ##STR00039##

(78) 2N NaOH solution (10.6 ml) was added drop wise to a stirred solution of 2-(aminooxy)ethanol hydrochloride salt (2.4 g) and s-methyl isothiosemicarbazide hydroiodide (4.98 g) in water (8.4 ml) at room temperature and was stirred for 24 hours. Formation of N′-(2-hydroxyethoxy)hydrazine carboximidamide was confirmed by LCMS analysis. The mixtures was concentrated under reduced pressure and the resulting residue was azeotroped with methanol (15 ml). The resulting material was suspended in ethanol (10 ml) and the insoluble inorganic salts were removed by filtration. The filtrate containing N′-(2-hydroxyethoxy)hydrazinecarboximidamide was directly used for the next step without any further processing. LC-MS: m/z=134.6 (M+H)

(79) 2-(2-chlorobenzylidene)-N′-(2-hydroxyethoxy)hydrazinecarboximidamide (Compound 6)

(80) ##STR00040##

(81) 2-chlorobenzaldehyde (3.28 g) was added drop wise to the filtrate containing N′-(2-hydroxyethoxy)hydrazinecarboximidamide at room temperature and was stirred for 2 hours. The reaction mixture was concentrated under reduced pressure and the residue thus obtained was further purified by Prep HPLC using 0.1% NH.sub.3/water/MeCN to give 0.24 g of 2-(2-chlorobenzylidene)-N′-(2-hydroxyethoxy)hydrazinecarboximidamide (Yield: 4.4% for 2 steps). .sup.1H-NMR (DMSO-d6): δ (ppm) 3.68 (m, 2H), 3.97 (m, 2H), 5.82 (s broad, 2H), 5.07 (m, 1H), 7.50 (m, 2H), 7.55 (m, 1H), 8.34 (m, 1H) 8.47 (s, 1H), 8.67 (s, 1H), 11.78 (m, 1H), 12.09 (m, 1H). LC-MS: m/z=256.73 (M+H).

(82) Compound 7: 2-(2-chlorobenzylidene)-N′-(2-chloroethoxy) hydrazine carboximidamide hydrochloride

(83) ##STR00041##

(84) SoCl.sub.2 (0.26 ml) was added drop wise to a stirred solution of 2-(2-chlorobenzylidene)-N′-(2-hydroxyethoxy)hydrazine carboximidamide (0.22 g) in Dichloromethane (10 ml) at 0° C. The reaction mixture was stirred at the room temperature for 24 hours. Then, the reaction mixtures was concentrated under reduced pressure. The resulting residue was triturated with n-pentane (2×5 ml) and dried under reduced pressure to give 0.26 g of 2-(2-chlorobenzylidene)-N′-(2-chloroethoxy)hydrazinecarboximidamide hydrochloride (Yield: 99.5%). LC-MS: m/z=274.8 (M+H).

(85) Compound 8: 2-(2-chlorobenzylidene)-N′-[2-(pyrrolidin-1-yl)ethoxy] hydrazine carboximidamide

(86) ##STR00042##

(87) Pyrrolidine (0.23 g) was added to a stirred solution of 2-(2-chlorobenzylidene)-N′-(2-chloroethoxy)hydrazine carboximidamide hydrochloride (0.27 g), Triethylamine (0.35 g) and Sodium iodide (0.04 g) in THF (10 ml) at room temperature. The resulting mixture was stirred at 50° C. for 24 hours. Then, the reaction mixtures was allowed to cool to room temperature and the crude was dumped in 50 ml of cold water. The product was extracted by ethyl acetate (2×50 ml). Then, organic layer were combined and distilled under vacuum, the residue thus obtained was further purified by Prep HPLC using 0.1% NH.sub.3/water/MeCN to give 14 mg of 2-(2-chlorobenzylidene)-N′-[2-(pyrrolidin-1-Aethoxy]hydrazinecarboximidamide (Yield: 5.3%).). .sup.1H-NMR (MeOD): δ (ppm) 1.91 (m, 4H), 2.75 (m, 4H), 2.88 (t, 2H), 3.97 (t, 2H), 7.32 (m, 2H), 7.41 (m, 1H), 8.07 (m, 1H), 8.32 (s, 1H). LC-MS: m/z=310.33 (M+H).

(88) Compound 10: 2-(2-chlorobenzylidene)-M-ethoxyhydrazinecarboximidamide

(89) N′-(2-ethoxy)hydrazinecarboximidamide (I-16)

(90) ##STR00043##

(91) 1N NaOH solution (5.12 ml) was added drop wise to a stirred solution of ethoxyamine hydrochloride salt (0.5 g) and s-methyl isothiosemicarbazide hydroiodide (1.19 g) in water (5.0 ml) at room temperature and was stirred for 48 hours. Formation of N′-(2-ethoxy)hydrazinecarboximidamide was confirmed by LCMS analysis. The mixtures was concentrated under reduced pressure and the resulting residue was dissolved in ethanol (15 ml). The insoluble solids were removed by filtration. The filtrate was concentrated and N′-(2-ethoxy)hydrazinecarboximidamide was directly used for the next step without any further processing. LC-MS: m/z=118.8 (M+H).

(92) 2-(2-chlorobenzylidene)-N′-ethoxyhydrazinecarboximidamide (compound 10)

(93) ##STR00044##

(94) 2-chlorobenzaldehyde (0.717 g) was added dropwise to N′-(2-ethoxy)hydrazinecarboximidamide in solution in ethanol (10 ml) and sodium acetate (0.42 g) at room temperature and was stirred for 2 hours at 90° C. The reaction mixture was concentrated under reduced pressure and the residue thus obtained was further purified by chromatography to give 21.4 mg of 2-(2-chlorobenzylidene)-N′-(2-ethoxy)hydrazinecarboximidamide (Yield: 1.7% for 2 steps). .sup.1H-NMR (DMSO-d6): 8 (ppm) 1.18 (t, 3H), 3.77 (q, 2H), 5.77 (s broad, 2H), 7.31 (m, 2H), 7.43 (m, 1H), 8.11 (m, 1H), 8.15 (s, 1H), 10.45 (s broad, 1H). LC-MS: m/z=240.9 (M+H).

(95) Compound 11: 2-(2,6-dichlorobenzylidene)-N-ethoxyhydrazinecarboximidamide

(96) ##STR00045##

(97) 2,6-dichlorobenzaldehyde (0.896 g) was added dropwise to 1 equivalent of N′-(2-ethoxy)hydrazinecarboximidamide (1-16) in solution in ethanol (10 ml) and sodium acetate (0.42 g) at room temperature and was stirred for 2 hours at 90° C. The reaction mixture was concentrated under reduced pressure and the residue thus obtained was further purified by chromatography to give 57 mg of 2-(2,6-dichlorobenzylidene)-N′-(2-ethoxy)hydrazinecarboximidamide (Yield: 4.1% for 2 steps). .sup.1H-NMR (DMSO-d6): δ (ppm) 1.77 (t, 3H), 3.78 (q, 2H), 5.48 (s broad, 2H), 7.33 (t, 1H), 7.52 (m, 2H), 8.04 (s, 1H), 8.16 (m, 1H). LC-MS: m/z=277.1 (M+H).

(98) Compound 12: 2-(2-chlorobenzylidene)-N-propoxyhydrazinecarboximidamide

(99) N′-propoxyhydrazinecarboximidamide (I-17)

(100) ##STR00046##

(101) 2N NaOH solution (1.23 ml) was added dropwise to a stirred solution of O-propylhydroxylamine hydrochloride salt (0.28 g) and s-methyl isothiosemicarbazide hydroiodide (0.58 g) in water (2.0 ml) at room temperature and was stirred for 24 hours. Formation of N′-(propoxy)hydrazinecarboximidamide was confirmed by LCMS analysis. The mixtures was concentrated under reduced pressure and the resulting residue was dissolved in ethanol (15 ml). The insoluble solids were removed by filtration. The filtrate was concentrated and N′-(propoxy)hydrazinecarboximidamide was directly used for the next step without any further processing. LC-MS: m/z=132.9 (M+H) 2-(2-chlorobenzylidene)-N-propoxyhydrazinecarboximidamide (compound 12)

(102) ##STR00047##

(103) 2-chlorobenzaldehyde (0.35 g) was added dropwise to N′-(2-propoxy)hydrazinecarboximidamide in solution in ethanol (10 ml) and was stirred for 2 at room temperature. The reaction mixture was concentrated under reduced pressure and the residue thus obtained was further purified by chromatography to give 25 mg of 2-(2-chlorobenzylidene)-N′-(2-propoxy)hydrazinecarboximidamide (Yield: 3.9% for 2 steps). .sup.1H-NMR (DMSO-d6): δ (ppm) 0.88 (t, 3H), 1.58 (m, 2H), 3.66 (t, 2H), 5.75 (s broad, 1H), 7.29 (m, 2H), 7.41 (m, 1H), 8.10 (m, 2H), 10.45 (s broad, 2H). LC-MS: m/z=255.1 (M+H).

(104) Compound 13: 2-(2-chlorobenzylidene)-N-(2-ethoxyethoxy) hydrazinecarboximidamide 2-(2-ethoxyethoxy)-1,3-dimethylidene-2,3-dihydro-1H-isoindole (1-18)

(105) ##STR00048##

(106) The N-hydroxypthalimide (4.0 g) and 1-bromo-2-ethoxyethane (11.25 g) were dissolved in DMF (40.0 ml) and CH.sub.3COONa (10.0 g) was added to the solution at room temperature. The reaction mixture was allowed to stir at 70° C. for 12 hours. The reaction mixture was allowed to cool to room temperature and was and was poured in water and then extracted two times by ethyl acetate. The organic layer was concentrated under reduce pressure and was purified by column chromatography using silica gel. The desired product was eluted with 0-30% ethyl acetate in hexane. Evaporation of pure product fractions gave 4.8 g of 2-(2-ethoxyethoxy)-1,3-dimethylidene-2,3-dihydro-1H-isoindole (1-18) (Yield: 83.3%). .sup.1H-NMR (DMSO-d6): δ (ppm) 0.98 (t, 3H),3.39 (q, 2H), 3.73 (t, 2H), 4.27 (t, 2H), 7.87 (s, 4H). LC-MS: m/z=236.2 (M+H).

(107) 1-(aminooxy)-2-ethoxyethane hydrochloride (1-19)

(108) ##STR00049##

(109) Hydrazine hydrate (1.32 g) was added dropwise to a stirred solution of 2-(2-ethoxyethoxy)-1,3-dimethylidene-2,3-dihydro-1H-isoindole (4.8 g) in methanol (10 ml) at room temperature and was stirred for 30 minutes. Then, the reaction mixture was filtered off under reduced pressure to remove insoluble by-product. The filtrate was concentrated under reduced pressure at lower temperature and triturated ether and insoluble was removed by filtration. Then, to the filtrate, 4N HCl in dioaxane (10.2 ml) was added dropwise and the precipitated salt was collected by filtration and was dried to 2.0 g of 1-(aminooxy)-2-ethoxyethane hydrochloride (Yield: 69.4% as mono hydrochloride salt). .sup.1H-NMR (DMSO-d6): δ (ppm) 1.11 (t, 3H), 3.44 (q, 2H), 3.59 (m, 2H), 4.14 (m, 2H), 11.02 (s broad, 2H). LC-MS: m/z=106.1 (M+H).

(110) N′-(2-ethoxyethoxy)hydrazinecarboximidamide (1-20)

(111) ##STR00050##

(112) 1N NaOH solution (4.23 ml) was added dropwise to a stirred solution of 1-(aminooxy)-2-ethoxyethane hydrochloride salt (0.6 g) and s-methyl isothiosemicarbazide hydroiodide (0.99 g) in water (2.1 ml) at room temperature and was stirred for 48 hours. Formation of N′-(2-ethoxyethoxy)hydrazinecarboximidamide was confirmed by LCMS analysis. The mixtures was concentrated under reduced pressure and the resulting residue was dissolved in ethanol (10 ml). The insoluble solids were removed by filtration. The filtrate was concentrated and N′-(2-ethoxyethoxy)hydrazinecarboximidamide was directly used for the next step without any further processing. LC-MS: m/z=163.0 (M+H).

(113) 2-(2-chlorobenzylidene)-N-(2-ethoxyethoxy)hydrazinecarboximidamide (compound 13)

(114) ##STR00051##

(115) 2-chlorobenzaldehyde (0.59 g) was added dropwise to N′-(2-ethoxyethoxy) hydrazinecarboximidamide in solution in ethanol (5 ml) and was stirred for 2 at room temperature. The reaction mixture was concentrated under reduced pressure and the residue thus obtained was further purified by chromatography to give 19 mg of 2-(2-chlorobenzylidene)-N′-(2-propoxy)hydrazinecarboximidamide (Yield: 1.8% for 2 steps). .sup.1H-NMR (DMSO-d6): δ (ppm) 1.24 (t, 3H), 3.48 (q, 2H), 3.56 (m, 2H), 3.83 (m, 2H), 5.80 (s broad, 1H), 7.43 (m, 1H), 8.12 (m, 1H), 8.17 (s, 1H), 10.50 (s broad, 2H). LC-MS: m/z=285.0 (M+H).

(116) Compound 14: 2-(2-chlorobenzylidene)-N′-[(3-methylbut-2-en-1-yl)oxy]hydrazinecarboximidamide

(117) 2-[(3-methylbut-2-en-1-yl)oxy]-1H-isoindole-1,3(2H)-dione (I-21)

(118) ##STR00052##

(119) Triethylamine (12.13 g) was added dropwise to a stirred solution of N-Hydroxyphthalimide (9.85 g) and 1-bromo-3-methyl butene (9.0 g) in DMF (30 ml) at room temperature. The reaction mixture was stirred at 70° C. for 2 hours. The reaction mixture was allowed to cool to room temperature. The mixture was concentrated under reduced pressure and the residue thus obtained was suspended in cold water. The resulting suspension was stirred well for some time and the solid was filtered off under reduced pressure. The solid was further washed with demineralized water (200 ml) and hexane (100 ml). The resulting solid was dried under reduced pressure to get a crude material which was purified by column chromatography using silica gel to give 9.0 g of —[(3-methylbut-2-en-1-yl)oxy]-1H-isoindole-1,3(2H)-dione (Yield: 64.5%). .sup.1H-NMR (DMSO-d6): δ (ppm) 1.70 (d, 6H), 4.63 (m, 2H), 5.45 (m, 1H), 7.87 (s, 4H). LC-MS: m/z=232.1 (M+H).

(120) 1-(aminooxy)-3-methylbut-2-ene hydrochloride (1-22)

(121) ##STR00053##

(122) Hydrazine hydrate (2.52 g) was added dropwise to a stirred solution of 2-[(3-methylbut-2-en-1-yl)oxy]-1H-isoindole-1,3(2H)-dione (9.0 g) in methanol (120 ml) at room temperature. The reaction mixture was stirred at the same temperature for 30 min. The reaction mixture was filtered off to remove the insoluble by-product and the resulting filtrate was concentrated under reduced pressure to get a crude material which was purified by column chromatography using silica gel. The crude was triturated with ether and insoluble mass was removed by filtration. The filtrate was treated with 4 M HCl in dioxane (19 ml) dropwise and the precipitate was filtered, collected and dried under vacuum to give 2.9 g of 1-(aminooxy)-3-methylbut-2-ene hydrochloride (Yield: 73.6%). .sup.1H-NMR (DMSO-d6): δ (ppm) 1.70 (s, 3H), 1.75 (s, 3H), 1.65 (m, 1H), 4.50 (d, 2H), 5.30 (t, 1H), 10.89 (s, 3H).

(123) N′-[(3-methylbut-2-en-1-yl)oxy]hydrazinecarboximidamide (1-23)

(124) ##STR00054##

(125) 1N NaOH solution (3.63 ml) was added dropwise to a stirred solution of 1-(aminooxy)-3-methylbut-2-ene hydrochloride (0.5 g) and s-methylisothiosemicarbazide hydro-iodide (0.85 g) in water (3 ml) at room temperature and was stirred for 48 hours. Then, the reaction mixtures was concentrated under reduced pressure. The resulting residue was suspended in ethanol (15 ml) and insoluble inorganic salts were removed by filtration. The filtrate was concentrated and directly used for the next step without any further processing. N′-[(3-methylbut-2-en-1-yl)oxy] hydrazinecarboximidamide was confirmed by LCMS analysis. LC-MS: m/z=159.15 (M+H).

(126) 2-(2-chlorobenzylidene)-N′-[(3-methylbut-2-en-1-yl)oxy]hydrazinecarboximidamide (compound 14)

(127) ##STR00055##

(128) 2-chlorobenzaldehyde (0.5 g) was added dropwise to N′-[(3-methylbut-2-en-1-yl)oxy]hydrazinecarboximidamide in solution in ethanol (3 ml) at room temperature and was stirred for 2 hours at 90° C. The reaction mixture was concentrated under reduced pressure and the residue thus obtained was further purified by chromatography to give 139 mg of 2-(2-chlorobenzylidene)-N′-[(3-methylbut-2-en-1-yl)oxy]hydrazinecarboximidamide (Yield: 13.5% for 2 steps). .sup.1H-NMR (DMSO-d6): δ (ppm) 1.64 (s, 3H), 1.71 (s, 3H), 3.17 (s, 1H), 4.25 (d, 2H), 5.39 (t, 1H), 5.75 (s broad, 2H), 7.32 (m, 1H), 7.43 (m, 1H), 8.10 (m, 1H), 8.15 (m, 1H), 8.17 (s broad, 1H). LC-MS: m/z=281.2 (M+H).

(129) Compound 15: 2-(2-chlorobenzylidene)-N-[2-(ethylsulfanyl) ethoxy]hydrazinecarboximidamide

(130) 2-bromoethyl ethyl sulphide (I-24)

(131) ##STR00056##

(132) PBr.sub.3 (10 ml) was added dropwise to 2-(ethylsulfanyl)ethanol in solution in dichloromethane (100 ml) at 0° C. and was stirred for 2 hours. Then the reaction mixture was warmed to room temperature and stirred for 16 hours. The reaction mixture was cooled at 0° C. and 10 ml of water was added. Then reaction mixture was neutralized with saturated Na.sub.2CO.sub.3 solution (˜up to Ph 7) and extracted with dichloromethane (3×250 ml). The organic layers were separated, combined and dried (Na.sub.2SO.sub.4) and concentrated to afford 13.0 g of 2-bromoethyl ethyl sulphide (yield: 72.7%)..sup.1H-NMR (CDCl.sub.3): δ (ppm) 1.30 (t, 3H), 2.62 (q, 2H), 2.97 (m, 2H), 3.50 (m, 2H).

(133) 2[2-(ethylsulfanyl)ethoxy]-1H-isoindole-1,3(2H)-dione (I-25)

(134) ##STR00057##

(135) N-hydroxypthalimide (3.9 g) and 2-bromoethyl ethyl sulphide (12.1 g) were dissolved in DMF (40.0 ml) and CH.sub.3COONa (9.7 g) was added portionwise to the solution at room temperature. The reaction mixture was allowed to stir at 70° C. for 2 hours. The reaction mixture was allowed to cool to room temperature and was and was poured in cold water and then extracted two times by ethyl acetate. The organic layer was concentrated under reduce pressure and was purified by column chromatography using silica gel. To give 6.0 g of 2-[2-(ethylsulfanyl)ethoxy]-1H-isoindole-1,3(2H)-dione (1-25) (Yield: 98%). .sup.1H-NMR (CDCl.sub.3): δ (ppm) 1.29 (t, 3H), 2.63 (q, 2H), 2.94 (t, 2H), 4.36 (t, 2H), 7.77 (m, 2H), 7.86 (m, 2H).

(136) 1-(aminooxy)-2-(ethylsulfanyl)ethane hydrochloride (I-26)

(137) ##STR00058##

(138) Hydrazine hydrate (0.25 g) was added dropwise to a stirred solution of 2-[2-(ethylsulfanyl)ethoxy]-1H-isoindole-1,3(2H)-dione (1.0 g) in methanol (10 ml) at room temperature. The reaction mixture was stirred at the same temperature for 30 min. The reaction mixture was filtered off to remove the insoluble by-product and the resulting filtrate was concentrated under reduced pressure then dissolved in DCM and insoluble removed by filtration. The filtrate was concentrated under reduced pressure then, the crude was triturated with ether and insoluble mass was removed by filtration. The filtrate was treated with 4 M HCl in dioxane (2 ml) dropwise. Then the solvent was removed by evaporation and the the residue was triturated with diethyl ether to provide 454 mg 1-(aminooxy)-2-(ethylsulfanyl)ethane hydrochloride (Yield: 72.5%). .sup.1H-NMR (DMSO-d6): 6 (ppm) 1.18 (s, 3H), 2.53 (m, 2H), 2.79 (t, 2H), 4.16 (t, 2H), 11.14 (s broad, 3H).

(139) N′-[2-(ethylsulfanyl)ethoxy]hydrazinecarboximidamide (1-27)

(140) ##STR00059##

(141) NaOH solution (2.88 ml) was added dropwise to a stirred solution of 1-(aminooxy)-2-(ethylsulfanyl)ethane hydrochloride (0.5 g) and s-methyl isothiosemicarbazide hydroiodide (0.7 g) in water (5 ml) at room temperature and was stirred for 48 hours. The mixtures was concentrated under reduced pressure and the resulting residue was dissolved in ethanol (15 ml). The insoluble solids were removed by filtration. The filtrate was concentrated and N′-[2-(ethylsulfanyl)ethoxy]hydrazinecarboximidamide was directly used for the next step without any further processing.

(142) 2-(2-chlorobenzylidene)-N-[2-(ethylsulfanyl)ethoxy]hydrazinecarboximidamide (compound 15)

(143) ##STR00060##

(144) 2-chlorobenzaldehyde (0.4 g) was added dropwise to N′-[2-(ethylsulfanyl)ethoxy]hydrazinecarboximidamide in solution in ethanol (5 ml) and was stirred for 2 at room temperature. The reaction mixture was concentrated under reduced pressure and the residue thus obtained was further purified by chromatography to give 15 mg of 2-(2-chlorobenzylidene)-N-[2-(ethylsulfanyl)ethoxy]hydrazinecarboximidamide (Yield: 1.5% for 2 steps). .sup.1H-NMR (DMSO-d6): δ (ppm) 1.90 (t, 3H), 2.54 (q, 2H), 2.75 (t, 2H), 3.85 (t, 2H), 5.84 (s broad, 2H), 7.30 (m, 2H), 7.44 (m, 1H), 8.12 (m, 1H), 8.16 (s, 1H), 10.50 (s broad, 1H). LC-MS:

(145) m/z=301.9 (M+H).

(146) Compound 16: 2-[(3-chloropyridin-4-yl)methylidene]-N-ethoxyhydrazinecarboximidamide

(147) ##STR00061##

(148) 3-chloroisonicotinaldehyde (0.72 g) was added dropwise to 1 equivalent of N′-(2-ethoxy)hydrazinecarboximidamide (I-16) in solution in ethanol (5 ml) and sodium acetate (0.42 g) at room temperature and was stirred for 2 hours at 80° C. The reaction mixture was concentrated under reduced pressure and the residue thus obtained was further purified by chromatography to give 184 mg of 2-[(3-chloropyridin-4-yl)methylidene]-N-ethoxyhydrazinecarboximidamide (Yield: 15% for 2 steps). .sup.1H-NMR (DMSO-d6): δ (ppm) 1.19 (t, 3H), 3.79 (q, 2H), 5.96 (s broad, 2H), 8.05 (s, 1H), 8.11 (d, 1H), 8.41 (s, 1H), 10.89 (s broad, 1H). LC-MS: m/z=242.0 (M+H).

(149) Compound 17: 2-(2-chloro-6-fluorobenzylidene)-N-ethoxyhydrazinecarboximidamide

(150) ##STR00062##

(151) 2-chloro-6-flurobenzaldehyde (0.81 g) was added dropwise to 1 equivalent of N′-(2-ethoxy)hydrazinecarboximidamide (I-16) in solution in ethanol (5 ml) and sodium acetate (0.42 g) at room temperature and was stirred for 2 hours at 80° C. The reaction mixture was concentrated under reduced pressure and the residue thus obtained was further purified by chromatography to give 215 mg of 2-(2-chloro-6-fluorobenzylidene)-N-ethoxyhydrazinecarboximidamide (Yield: 17.2% for 2 steps). .sup.1H-NMR (DMSO-d6): δ (ppm) 1.17 (m, 3H), 3.78 (q, 2H), 5.48 (s broad, 2H), 7.30 (m, 3H), 8.01 (s, 1H), 10.54 (s broad, 1H). LC-MS: m/z=258.9 (M+H).

(152) Compound 18: N′-butoxy-2-(2-chlorobenzylidene)hydrazinecarboximidamide

(153) Compound 18 is prepared following the same procedure than compound 12 from 2-chlorobenzaldehyde and N′-(2-butoxy)hydrazinecarboximidamide.

(154) Compound 19: 2-(2-chloro-6-fluorobenzylidene)-N′-propoxyhydrazinecarboximidamide

(155) ##STR00063##

(156) Compound 19 is prepared following the same procedure than compound 17 from 2-chloro-6-flurobenzaldehyde and N′-(2-propoxy)hydrazine carboximidamide (1-17) to give 2-(2-chloro-6-fluorobenzylidene)-N′-propoxyhydrazinecarboximidamide LC-MS: m/z=273.0 (M+H).

(157) Compound 20: 2-(2-chloro-6-fluorobenzylidene)-N′-butoxyhydrazinecarboximidamide

(158) Compound 20 is prepared following the same procedure than compound 17 from 2-chloro-6-flurobenzaldehyde and N′-(2-butoxy)hydrazinecarboximidamide.

(159) Compound 21: 2-(2,6-dichlorobenzylidene)-N-propoxyhydrazinecarboximidamide

(160) ##STR00064##

(161) Compound 21 is prepared following the same procedure than compound 11 from 2,6-dichlorobenzaldehyde and N′-(2-propoxy)hydrazinecarboximidamide (I-17) to give 2-(2-chloro-6-fluorobenzylidene)-N′-propoxyhydrazinecarboximidamide LC-MS: m/z=290.9 (M+H).

(162) Compound 22: 2-(2,6-dichlorobenzylidene)-N-butoxyhydrazinecarboximidamide

(163) Compound 22 is prepared following the same procedure than compound 17 from 2,6-dichlorobenzaldehyde and N′-(2-butoxy)hydrazinecarboximidamide.

(164) Compound 23: 2-[(3-chloropyridin-4-yl)methylidene]-N-propoxyhydrazinecarboximidamide

(165) ##STR00065##

(166) Compound 23 is prepared following the same procedure than compound 16 from 3-chloroisonicotinaldehyde and N′-(2-propoxy)hydrazinecarboximidamide (I-17) to give 2-(2-chloro-6-fluorobenzylidene)-N′-propoxyhydrazinecarboximidamide LC-MS: m/z=255.9 (M+H).

(167) Selected compounds according to the invention are set forth in Table below:

(168) TABLE-US-00001 Compound 1 Compound 2 Compound 3 Compound 4 0 Compound 5 Compound 6 Compound 7 Compound 8 Compound 9 Compound 10 Compound 11 Compound 12 Compound 13 Compound 14 0 Compound 15 Compound 16 Compound 17 Compound 18 Compound 19 Compound 20 Compound 21 Compound 22 Compound 23

(169) In some of the experiments below, the salt of these compounds may be used.

1.2—Mammalian Cell Culture, Constructs and Transfection

(170) HeLa Cells were cultured in Eagle's Minimum Essential Medium (EMEM) supplemented with Glutamine, Sodium Pyruvate, Non-Essential Amino Acids, Penicillin and Streptomycin (Lonza) containing 10% Foetal Bovine Serum (FBS) (Biowest). 293T cells were cultured in Dubelcco's Modified Eagle's Media (DMEM) supplemented with penicillin, streptomycin, glutamine (Lonza) and 10% of fetal bovine serum (FBS) (Biowest).

(171) Min6 cells were cultured in DMEM supplemented with penicillin, streptomycin, glutamine, sodium pyruvate, 50 μM f3-Mercaptoethanol and 15% Foetal Bovine Serum (FBS) (Biowest). INS1 cells were cultured in RPMI supplemented with penicillin, streptomycin, glutamine, sodium pyruvate (Lonza), 50 μM Mercaptoethanol and 10% of fetal bovine serum (FBS) (Biowest). Each cell line was maintained at 37° C. in 5% CO.sub.2 atmosphere.

(172) Human open reading frame (ORF) sequences for PLP1, DM20 and Insulin were obtained from Life Technologies (Invitrogen) (IOH41689, IOH5252 and IOH7334 respectively). Construct cloning into the expression plasmid pDEST26 (Invitrogen) was performed by Gateway® LR Clonase™ II Enzyme Mix (Invitrogen). ORF mutations were carried out using the QuikChange Lightning Site-Directed Mutagenesis Kit (Stratagene) (T181P mutation for PLP1 and DM20 ORFs, Akita (C96Y) for Insulin ORF).

(173) Gene expression into mammalian cells was carried out by nucleofection, using the Amaxa™ 4D-Nucleofector™ System (Lonza) or by transfection using Lipofectamine (Life technologies).

1.3—Cytoprotection from ER Stress

(174) This assay is described in Tsaytler et al. (Science 2011). HeLa Cells were cultured in Eagle's Minimum Essential Medium (EMEM) supplemented with Glutamine, Sodium Pyruvate, Non-Essential Amino Acids, Penicillin and Streptomycin containing 10% Foetal Bovine Serum (FBS), at 37° C. in 5% CO.sub.2 atmosphere. Cells were plated in 96 well plates at a density of 17,000 cells/mL the day before the treatment. ER stress was elicited by addition of 5 μg/mL tunicamycin (Sigma-Aldrich) together with PPP1R15A inhibitors (0.5-10 μM). Media were changed 6h later with fresh media and the cytoprotection was maintained by the addition of PPP1R15A inhibitors (0.5-10 μM). Cell viability was assessed by measuring the reduction of WST-8 into formazan using Cell Counting Kit-8 (Sigma) according to the supplier's recommendation, 48 h or 72h after tunicamycin treatment. Cytoprotection from ER stress is measured in terms of cytoprotective potency effect compared to the reference compound Guanabenz (Tsaytler et al., Science 2011) after ER stress: ‘+’ no cytoprotective effect; ‘+’ lower cytoprotective effect compared to Guanabenz; ‘++’ similar cytoprotective effect compared to Guanabenz; ‘+++’ higher cytoprotective effect compared to Guanabenz.

(175) Table 1 summarizes the results of cytoprotective effect of different compounds of the invention, compared to guanabenz, after the stress induced by a 6 hour exposure of tunicamycin.

1.4—Assessment of Translation Rates in Unstressed Cells

(176) HeLa cells (100,000 cells/ml) were plated in 6-well plates 24 h before each experiment and were either left untreated or treated with compounds (50 μM) for 2.5, 5 and 9 h. Culture medium was replaced by methionine-free DMEM medium (Invitrogen) 30 min before compounds addition. One hour before each time point, 50 μM of Click-iT® AHA (L-azidohomoalanine) (Invitrogen) was added to the culture medium in order to label newly synthesized proteins. At the end of each time point, cells were washed with ice-cold PBS and harvested by Trypsine dissociation (Lonza), then lysed in a 50 mM Tris-HCl buffer containing 1% of SDS (Sigma) and protease and phosphatase inhibitors (Sigma). Protein samples were coupled to alkyne biotin (Invitrogen) using Click-iT® Protein Reaction Buffer Kit (Invitrogen). Samples were denatured at 70° C. for 10 min, resolved on ECL 4-20% precasted gels (GE Healthcare) and transferred to nitrocellulose membranes (GE Healthcare). Alkyne biotin coupled to Click-iT® AHA incorporated to newly synthesized proteins was detected using streptavidin-HRP (Gentex). Revelation was performed by incubation of ECL Prime (GE Healthcare) and read by chemoluminiscence using Fusion Solo 3S (Vilber Lourmat).

1.5—Assessment of Translation Rates in Stressed Cells

(177) Treatments were performed as for measuring translation in unstressed cells, except that Tunicamycin (5 μg/ml) was added together with the compounds.

1.6—Functional GPCR Assay for Adrenergic α2A Receptor (Cellkey Detection Method)

(178) The agonist activity of compounds was evaluated on CHO cells endogenously expressing human alpha2A receptor and was determined by measuring their effects on impedance modulation using the CellKey detection method. Cells were seeded onto 96-well plate at density of 6×10.sup.4 cells/well in HBSS buffer (Invitrogen)+20 mM HEPES (Invitrogen) with 0.1% BSA and are allowed to equilibrate for 60 min at 28° C. before the start of the experiment. Plates were placed onto the system and measurements were made at a temperature of 28° C. Solutions were added simultaneously to all 96 wells using an integrated fluidics system: HBSS (basal control), reference agonist at 100 nM (stimulated control), reference agonist (EC.sub.50 determination) or the test compounds. Impedance measurements are monitored for 10 minutes after ligand addition. The standard reference agonist is epinephrine, which is tested in each experiment at several concentrations to generate a concentration-response curve from which its EC.sub.50 value is calculated.

(179) Dose-response data from test compounds were analysed with Hill software using non-linear regression analysis of the concentration-response curves generated with mean replicate values using Hill equation curve fitting. Results are presented table 1, compounds with EC50>33.3 μM are considered to have no significant alpha-2 adrenergic activity.

1.7—In Vitro Multiple Sclerosis Disease Model: Interferon-Gamma Injured Rat Oligodendrocytes Co-Cultured with Neurons

Culture of Oligodendrocyte Co-Cultured with Neurons

(180) Neurons/OPC were cultured as previously describes by Yang et al. (2005 J Neurosci Methods;149(1) pp50-6) with modifications. Briefly, the full brain (without cerebellum) obtained from 17-day old rat embryos (Wistar, Janvier labs) were removed. The full brains were treated for 20 min at 37° C. with a trypsin-EDTA (Pan Biotech) solution at a final concentration of 0.05% trypsin and 0.02% EDTA. The dissociation was stopped by addition of Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/liter of glucose (Pan Biotech), containing DNAse I grade II (final concentration 0.5 mg/ml; Pan Biotech, Batch: h140508) and 10% fetal calf serum (FCS; Invitrogen, Batch: 41Q7218K). Cells were mechanically dissociated by three forced passages through the tip of a 10-ml pipette. Cells were then centrifuged at 515 g for 10 min at 4° C. The supernatant was discarded, and the pellet was resuspended in a defined culture medium consisting of Neurobasal medium (Invitrogen, Batch: 1636133) with a 2% solution of B27 supplement (Invitrogen, Batch: 1660670), 2 mmol/liter of L-glutamine (Pan Biotech), 2% of PS solution, and, 1% of FCS and 10 ng/ml of platelet derived growth factor (PDGF-AA, Batch: H131205). The cells were seeded at a density of 20 000 cells per well in 96 well plates precoated with PLL (BD corning, Batch: 6614022) and laminine (Sigma, Batch: 083M4034V). The plates were maintained at 37° C. into a humidified incubator, in an atmosphere of air (95%)-CO2 (5%). Half of the medium was changed every 2 days with fresh medium. On days 18, test compounds were pre-incubated 1 hour before interferon-gamma (70 U/ml, 48H, R&D system, Batch: AAL2214081) application.

(181) Test Compounds and Interferon-Gamma Exposure

(182) On day 18 of culture, test compounds (4 concentrations) were solved in culture medium and then pre-incubated with oligodendrocyte co-cultured with neurons for 1 hour before the interferon-gamma (70 U/ml, 48H) application. One hour after test compounds incubation, interferon-gamma was added at 70 U/ml concentration for 48 H still in presence of test compounds. Then, cells were fixed by a cold solution of ethanol (95%, Sigma, Batch: SZBD3080V) and acetic acid (5%, Sigma, Batch: SZBD1760V) for 5 min at −20° C. After permeabilization with 0.1% of saponin (Sigma, Batch: BCBJ8417V), cells were incubated for 2 h with Monoclonal Anti-O4 antibody produced in mouse (Sigma, batch: SLBF5997V) at dilution of 1/1000 in PBS (PAN, Batch: 8410813) containing 1% FCS, 0.1% saponin, for 2 h at room temperature. This antibody are revealed with Alexa Fluor 488 goat anti-mouse IgG (Invitrogen, batch: 1664729) at the dilution 1/400in PBS containing 1% FCS, 0.1% saponin, for 1 h at room temperature.

Analysis of Total Number of O4 Cells

(183) For each condition, 30 pictures per well were taken using ImageXpress (Molecular Device) with 20× magnification. All images were taken with the same conditions. Analysis of total number of O4 cells was performed automatically by using Custom module editor (Molecular Device). Data were expressed in percentage of control conditions (no intoxication, no interferon-gamma=100%) in order to express the interferon-gamma injury. All values were expressed as mean +/−SEM (s.e.mean) (n=6 wells per condition).

(184) 1.8- In vitro Parkinson's Disease Model: Rotenone Injured Primary Mesencephalic Rat Neurons

Culture of Mesencephalic Dopaminergic Neurons

(185) Rat dopaminergic neurons were cultured as described by Schinelli et al., (1988 J. Neurochem 50 pp1900-07) and Visanji et al., (2008 FASEB J. 22(7) pp2488-97). Briefly, the midbrains obtained from 15-day old rat embryos (Janvier Labs, France) were dissected under a microscope. The embryonic midbrains were removed and placed in ice-cold medium of Leibovitz (L15, Pan Biotech, Batch: 9310614) containing 2% of Penicillin-Streptomycin (PS, Pan Biotech, Batch: 1451013) and 1% of bovine serum albumin (BSA, Pan Biotech, Batch: h140603). The ventral portion of the mesencephalic flexure, a region of the developing brain rich in dopaminergic neurons, was used for the cell preparations.

(186) The midbrains were dissociated by trypsinisation for 20 min at 37° C. (Trypsin 0.05% EDTA 0.02%, PanBiotech, Batch: 5890314). The reaction was stopped by the addition of Dulbecco's modified Eagle's medium (DMEM, PanBiotech, Batch: 1300714) containing DNAase I grade II (0.1 mg/ml, PanBiotech, Batch: H140508) and 10% of foetal calf serum (FCS, Gibco, Batch: 41Q7218K). Cells were then mechanically dissociated by 3 passages through a 10 ml pipette. Cells were then centrifuged at 180×g for 10 min at +4° C. on a layer of BSA (3.5%) in L15 medium. The supernatant was discarded and the cell pellets were re-suspended in a defined culture medium consisting of Neurobasal (Invitrogen, Batch: 1636133) supplemented with B27 (2%, Invitrogen, Batch: 1660670), L-glutamine (2 mM, PanBiotech, Batch: 8150713) and 2% of PS solution and 10 ng/ml of Brain-derived neurotrophic factor (BDNF, PanBiotech, Batch: H140108) and 1 ng/ml of Glial-Derived Neurotrophic Factor (GDNF, Pan Biotech, Batch: H130917). Viable cells were counted in a Neubauer cytometer using the trypan blue exclusion test. The cells were seeded at a density of 40 000 cells/well in 96 well-plates pre-coated with poly-L-lysine (Corning Biocoat, Batch: 6614022) and maintained in a humidified incubator at 37° C. in 5% CO.sub.2/95% air atmosphere. Half of the medium was changed every 2 days with fresh medium.

(187) On day 6 of culture, the medium was removed and fresh medium was added, without or with rotenone (Sigma, Batch: 021M2227V) at 10 nM diluted in control medium, 3 wells per condition were assessed. Test compounds were solved in culture medium and then pre-incubated with mesencephalic neurons for 1 hour before the rotenone application.

(188) After 24 hours of intoxication, cells were fixed by a solution of 4% paraformaldehyde (Sigma, batch SLBF7274V) in PBS (Pan Biotech, Batch: 4831114), pH=7.3 for 20 min at room temperature. The cells were washed again twice in PBS, and then were permeabilized and non-specific sites were blocked with a solution of PBS containing 0.1% of saponin (Sigma, batch: BCBJ8417V) and 1% FCS for 15 min at room temperature. Then, cells were incubated with Monoclonal Anti-Tyrosine Hydroxylase antibody produced in mouse (TH, Sigma, batch: 101M4796) at dilution of 1/10000 in PBS containing 1% FCS, 0.1% saponin, for 2 h at room temperature. This antibody was revealed with Alexa Fluor 488 goat anti-mouse IgG (Molecular Probes, batch: 1531668) at the dilution 1/800 in PBS containing 1% FCS, 0.1% saponin, for 1 h at room temperature.

Analysis of Total Number of TH Positive Neurons

(189) The immunolabeled cultures were automatically examined with ImageXpress (Molecular device USA). For each condition, 20 automatically fields per well (representing ˜80% of the total surface of the well) from 3 wells were analyzed. The total number of TH neurons was automatically analyzed using Custom module editor (Molecular Devices, USA). Data were expressed in percentage of control conditions (no intoxication, no rotenone=100%) in order to express the rotenone injury. All values were expressed as mean +/−SEM (s.e. mean) of the 1 culture (n=3 wells per condition per culture).

(190) 1.9-In Vitro Alzheimer Disease Model: Amyloid-Beta 1-42 Injured Primary Cortical Rat Neurons.

Culture of Rat Cortical Neurons

(191) Rat cortical neurons were cultured as described by Singer et al., (1999 J. Neuroscience 19 pp2455-63) and Callizot et al., (2013 J.Neurosci. Res. 91 pp706-16). Pregnant females (Wistar; Janvier Labs) at 15 days of gestation were killed by cervical dislocation. Fetuses were collected and immediately placed in ice-cold L15 Leibovitz medium (Pan Biotech, Batch: 9310614) with a 2% penicillin (10,000 U/mI) and streptomycin (10 mg/ml) solution (PS; Pan Biotech, Batch: 1451013) and 1% bovine serum albumin (BSA; Pan Biotech, Batch: h140603). Cortex was treated for 20 min at 37° C. with a trypsin-EDTA (Pan Biotech, Batch: 5890314) solution at a final concentration of 0.05% trypsin and 0.02% EDTA. The dissociation was stopped by addition of Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/liter of glucose (Pan Biotech, batch: 1300714), containing DNAse I grade II (final concentration 0.5 mg/ml; Pan Biotech, Batch: h140508) and 10% fetal calf serum (FCS; Invitrogen, Batch: 41Q7218K). Cells were mechanically dissociated by three forced passages through the tip of a 10-ml pipette. Cells were then centrifuged at 515 g for 10 min at 4° C. The supernatant was discarded, and the pellet was resuspended in a defined culture medium consisting of Neurobasal medium (Invitrogen, Batch: 1636133) with a 2% solution of B27 supplement (Invitrogen, Batch: 1660670), 2 mmol/liter of L-glutamine (Pan Biotech, Batch: 8150713), 2% of PS solution, and 10 ng/ml of brain-derived neurotrophic factor (BDNF; Pan Biotech, Batch: H140108). Viable cells were counted in a Neubauer cytometer, using the trypan blue exclusion test. The cells were seeded at a density of 30,000 per well in 96-well plates precoated with poly-L-lysine (Corning Biocoat, Batch: 6614022) and were cultured at 37° C. in an air (95%)-0O.sub.2 (5%) incubator. The medium was changed every 2 days. The cortical neurons were intoxicated with A-beta solutions (see below) after 11 days of culture.

Test Compounds and Amyloid-Beta 1-42 Exposure

(192) The Amyloid-beta1-42 preparation was done following the procedure described by Callizot et al., 2013. Briefly, Amyloid-beta 1-42 peptide (Bachem, Batch: 1014012) was dissolved in the defined culture medium mentioned above, devoid of serum, at an initial concentration of 40 μmol/liter. This solution was agitated for 3 days at 37° C. in the dark and immediately used after being properly diluted in culture medium to the concentrations used. Test compounds were solved in culture medium and then pre-incubated with primary cortical neurons for 1 hour before the Amyloid-beta 1-42 application. Amyloid-beta 1-42 preparation was added to a final concentration of 20 μM (including to ˜2 μM of toxic oligomers measured by WB) diluted in control medium in presence of drugs. After 24 hours of intoxication, cells were fixed by a cold solution of ethanol (95%, Sigma, Batch: SZBD3080V) and acetic acid (5%, Sigma, Batch: SZBD1760V) for 5 min at −20° C. After permeabilization with 0.1% of saponin (Sigma, Batch: BCBJ8417V), cells were incubated for 2 h with mouse monoclonal antibody anti microtubule-associated-protein 2 (MAP-2; Sigma, Batch: 063M4802) at dilution of 1/400 in PBS (Pan biotech, Batch: 4831114) containing 1% foetal calf serum (Invitrogen, Batch: 41Q7218K) and 0.1% of saponin. This antibody was revealed with Alexa Fluor 488 goat anti-mouse IgG

(193) (Molecular probe, Batch: 1572559) at the dilution of 1/400 in PBS containing 1% foetal calf serum and 0.1% of saponin for 1 H at room temperature.

Analysis of Total Number of Neurons

(194) The immunolabeled cultures were automatically examined with ImageXpress (Molecular device USA) at ×20 magnification. For each condition, 30 automatically fields per well (representing ˜80% of the total surface of the well) from 3 wells were analyzed. The total number of neurons was automatically analyzed using Custom module editor (Molecular Devices, USA). Data were expressed in percentage of control conditions (no intoxication, no Amyloid-beta 1-42=100%) in order to express the A-beta 1-42 injury. All values were expressed as mean +/−SEM (s.e.mean) (n=3 wells per condition per culture).

(195) 1.10- In Vitro Model of Leukodystrophy (PMD): Overexpression of Mutated PLP1 and DM20 In Human Cell Line

(196) One day before transfection, 293T cells were plated at 300,000 cells/mL. 293T cells were transfected with PLP1 and DM20 mutant constructs using Lipofectamine 2000 according to manufacturer's procedure. After transfection, cells were treated with molecules or left untreated.

(197) As a control, cells were transfected with native forms of the proteins. 48h later, cellular lysates were harvested. Protein accumulation was assessed by western-blot.

(198) 1.11-In Vitro Model of Type 2 Diabetes: Min6 And INS1 Cell Lines Cytoprotection from ER Stress

(199) Cells were plated in 96 well plates at a density of 0.5.10.sup.6 cells/mL for Min6 cell line, 0,4.10.sup.6 cells/mL for INS1 cell line the day before the treatment. ER stress was elicited by addition of 2.5 μg/mL tunicamycin (Sigma Aldrich) together with phosphatases inhibitors. Media were changed 6h later with fresh media and the cytoprotection was maintained by the addition of phosphatases inhibitors. Cell viability was assessed by measuring the reduction of WST-8 into formazan using Cell Counting Kit-8 (Sigma) according to the supplier's recommendation, 72h after tunicamycin treatment.

Protection Against Accumulation of Misfold Prone Insulin.SUP.Akita

(200) Min6 cells were nucleofected with Insulin.sup.Akita mutant constructs and seeded in 96 well-plates at 300,000 cells/mL and 24h later, cells were treated with molecules or left untreated. As a control, cells were nucleofected with non-relevant plasmid. 6 days later, a selective agent was added (G418). Cell viability was assessed by measuring the reduction of WST-8 into formazan using Cell Counting Kit-8 (Sigma) according to the supplier's recommendation, 9 days after treatment.

1.12-In Vitro Inflammation/Infection Disease Model: Poly I:C Induced Mouse Embryonic Fibroblasts

Experimental Protocols

(201) Mouse Embryonic Fibroblasts (MEFs) were lipofected with poly I:C and treated with two concentrations of compounds of the invention (25 μM) for 6h. After 6h of culture, elF2alpha-phosphorylation (elF2a-P) and PPP1R15A (GADD34) expression was monitored by western blotting, while type-1 Interferon(IFN)-beta production was quantified in culture supernatants by ELISA. Control (nt) and poly I:C/DMSO are respectively negative and positive controls. Poly I:C (polyinosinic:polycytidylic acid or polyinosinic-polycytidylic acid sodium salt) is an immunostimulant used to simulate viral infections. Poly I:C which is structurally similar to double-stranded RNA, is known to interact with toll-like receptor 3 which is expressed in the intracellular compartments of B-cells and dendritic cells. Guanabenz (25 μM) was used as reference inhibitory compound.

Cell Culture

(202) MEFs were cultured in DMEM, 10% FCS (HyClone, Perbio), 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine, 1× MEM non-essential amino acids and 50 μM 2-mercaptoethanol. MEFS were treated for the indicated time with 10 μg/ml poly I:C (InvivoGen) in combination with lipofectamine 2000 (Invitrogen).

Immunoblotting

(203) Cells were lysed in 1% Triton X-100, 50 mM Hepes, 10 mM NaCl, 2.5 mM MgCl.sub.2, 2 mM EDTA, 10% glycerol, supplemented with Complete Mini Protease Inhibitor Cocktail Tablets (Roche). Protein quantification was performed using the BCA Protein Assay (Pierce). 25-50 μg of Triton X-100-soluble material was loaded on 2%-12% gradient or 8% SDS-PAGE before immunoblotting and chemi-luminescence detection (SuperSignal West Pico Chemi-luminescent Substrate, Pierce). Rabbit polyclonal antibodies recognizing GADD34 (C-19) were from Santa Cruz Biotechnology and anti-elF2alpha[pS.sup.52] were from Invitrogen.

Elisa

(204) IFN-beta quantification in culture supernatant was performed using the Mouse Interferon Beta ELISA kit (PBL Interferon Source) according to manufacturer instructions.

1.13—Hypoxia-Induced Apoptosis in Cultured Neonatal Rat Cardiomyocytes

Cell Culture

(205) Primary cultures of neonatal rat cardiomyocytes were obtained from the ventricles of 1-day-old Sprague Dawley rats (Janvier, France). The rats were euthanized and their hearts excised. Hearts cut into small pieces (1-2 mm.sup.3) and enzymatically digested using the Neonatal Heart Dissociation Kit rat and the gentleMACS™ Dissociator (MiltenyiBiotec, Germany). After dissociation, the homogenates were filtered (70 μm) to obtain a single-cell suspension. Isolated cells were collected by centrifugation and resuspended in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% horse serum (HS), 5% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cultures were enriched with myocytes by pre-plating for 90 min to deplete the population of non-myocytes. Non-attached cells were plated onto 6- or 96-well plates at an appropriate cell density. The cells were cultured at 37° C. in 95% air/5% CO.sub.2 for 24 h. Then the culture medium was exchanged with fresh DMEM containing 1% FBS and different concentrations of test compound thirty minutes before incubation in a normal or a hypoxic (N.sub.2/CO.sub.2, 95%/5%; 0.3% O.sub.2) culture chamber.

Treatment with Test Compound

(206) Purified neonatal rat cardiomyocytes were seeded in a 96-well plate at 10.sup.6 cells/2 mL for flow cytometry experiments.

(207) After 24 hours, the cardiomyocytes were treated with different concentrations of test compound in culture medium with 0.1% DMSO. The positive controls cells were treated with culture medium (0.1% DMSO). Thirty minutes after starting the treatments, the cells were incubated in the hypoxic culture chamber (N.sub.2/CO.sub.2, 95%/5%; final measured O.sub.2: 0.3%) for 36 hours. The negative controls cells were left in normoxic conditions at 37° C. with culture medium (1% FBS, 0.1% DMSO) for the same time periods.

Apoptotic Cell Measurement

(208) At the end of the treatment period, flow cytometry were performed to measure the amount of apoptotic cells. The Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit from Miltenyi was used. Cells were washed twice with PBS and re-suspended in binding buffer. FITC-Annexin V and propidium iodide were added according to the manufacturer's protocol. The mixture was incubated for 15 min in the dark at room temperature, and cellular fluorescence was then measured by FACS scan flow cytometry.

2—RESULTS

2.1 —Cytoprotection & Compound Selectivity

(209) The results of the different assays ran with selected compounds of the invention are shown below in Table 1.

(210) As example, FIG. 1 represents the cytoprotective effect of compound 12 after the stress induced by an exposure of tunicamycin.

(211) TABLE-US-00002 TABLE 1 Cytoprotection from Functional Compound ER stress compared adrenergic alpha2 N.sup.o to guanabenz receptor assay 1 + 2 + 3 + 4 ++ 5 + 6 ++ EC50 > 33.3 μM 7 + 8 + 9 + 10 ++ EC50 > 33.3 μM 11 +++ EC50 > 0.7 μM  12 +++ EC50 > 33.3 μM 13 ++ EC50 > 33.3 μM 14 ++ 15 ++ EC50 > 33.3 μM 16 + EC50 > 33.3 μM 17 +++ EC50 > 33.3 μM 19 +++ 21 ++ 23 ++

2.2—Multiple Sclerosis

(212) FIG. 2 shows dose dependent protection of interferon-gamma injured rat oligodendrocytes by compounds 11, 12 and 17 of the invention.

(213) These data show that the compounds of this invention are promising effective treatment of Multiple Sclerosis.

2.3—Parkinson's Disease (PD)

(214) FIG. 3 shows dose dependent protection of rotenone injured primary mesencephalic rat neurons by compounds 5, 11 and 12 of the invention.

(215) These data show that the compounds of this invention are promising effective treatment of synucleopathies, and more specifically Parkinson's disease.

2.4—Alzheimer Disease (AD) & Amyloidosis

(216) FIG. 4 shows dose dependent protection of amyloid-beta 1-42 injured primary cortical rat neurons by compound 12 of the invention.

(217) These data show that the compounds of this invention are promising effective treatment of Amyloidosis and more specifically Alzheimer disease.

(218) 2.5—Leukodystrophy: Pelizaeus-Merzbacher Disease (PMD),

(219) T181P and L223P mutations in PLP1 and DM20 proteins have been described to cause a severe phenotype of Pelizaeus-Merzbacher disease (Strautnieks et al. 1992, Am. J. Hum. Genet. 51 (4): 871-878; Gow and Lazzarini, 1996 Nat Genet. 13(4):422-8).

(220) The Compound 12 and 17 of the invention (5 microM) is able to prevent the accumulation of T181P mutated DM20 protein expressed in Human 293T cell (FIG. 5).

(221) These data show that the compounds of this invention, specifically compounds 12 and 17, are promising effective treatment of demyelinating disorders like leukodystrophies, more specifically PMD.

(222) 2.6—Type 2 Diabetes

(223) FIG. 6 represents the results of over expression of pre-pro-insulin bearing Akita mutation in Min6 cells with compound 16 of the invention.

(224) The compounds 12, 16 and 17 at different concentrations prevent Min6 insulinoma cell death associated with accumulation of misfolded protein induced by 6 hour exposure to tunicamycin (FIG. 7)

(225) The compounds 11, 12, 16 and 17 at different concentrations prevent INS1 insulinoma cell death associated with accumulation of misfolded protein induced by 6 hour exposure to tunicamycin (FIG. 8).

(226) These data show that the compounds of the invention are promising effective treatment of pre-diabetes and diabetes, preferably type 2 pre-diabetes and type 2 diabetes.

2.7- Infection-Related or Non-Infectious Inflammatory Conditions

(227) Normal response of MEFs to poly I:C is characterized by PPP1R15A expression, increase in elF2alpha-P (variable in time and related to the levels PPP1R15A expression) mediated by PKR activation and type-I IFN production (range 500 to 700 pg/ml). Knock out PPP1R15A MEFs are unable to produce this cytokine in response to poly I:C.

(228) The potency of compounds of the invention to inhibit PPP1R15A was evaluated by measuring the increase of elF2alpha phosphorylation, the decrease of PPP1R15A expression due to its own pharmacological inhibition resulting in general protein synthesis inhibition and type-I IFN production.

(229) The evaluated compounds of the invention were found efficient at 25 μM to increase elF2alpha phosphorylation, to decrease of PPP1R15A expression and to prevent type-I IFN production. As example, FIG. 9 shows the ability of compounds 6, 10, 11, 12, 15, 16 and 17 (at 25 microM) to prevent type-I IFN production by mouse embryonic fibroblasts lipofected with poly I:C.

(230) These data show that the compounds of this invention are promising effective treatment of infection-related or non-infectious inflammatory conditions.

2.8-Cardiac Ischemia

(231) Compound 10 of the invention protects cultured neonatal rat cardiomyocytes from hypoxia-induced apoptosis (FIG. 10). These data show that the compounds of this invention are promising effective treatment of ischemia, specifically cardiac ischemia.

(232) Various modifications and variations of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the relevant fields are intended to be covered by the present invention.