Benzylideneguanidine derivatives and therapeutic use for the treatment of protein misfolding diseases

Abstract

The present invention relates to a compound of formula (I), or a tautomer and/or a pharmaceutically acceptable salt thereof, wherein: R.sub.1 is alkyl, Cl, F or Br; R.sub.2 is H or F; R.sub.3 is selected from H and alkyl; R.sub.4 is selected from H and C(O)R.sub.6; R.sub.5 is H; or R.sub.4 and R.sub.5 are linked to form a heterocyclic group which is optionally substituted with one or more R.sub.10 groups; R.sub.6 is selected from R.sub.7, OR.sub.7 and NR.sub.8R.sub.9; R.sub.7, R.sub.8 and R.sub.9 are each independently selected from alkyl, cycloalkyl, aralkyl, cycloalkenyl, heterocyclyl and aryl, each of which is optionally substituted with one or more R.sub.10 groups; each R.sub.10 is independently selected from halogen, OH, CN, NO.sub.2, COO-alkyl, aralkyl, 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; X and Z are each independently CR.sub.11, and Y is selected from CR.sub.11 and N; and R.sub.11 is H or F; for use in treating a disorder associated with protein misfolding stress and in particular associated with accumulation of misfolded proteins.

Claims

1. A compound of formula (II), or a pharmaceutically acceptable salt thereof, ##STR00025## or a tautomeric form thereof wherein: R.sub.1 is alkyl, Cl, F or Br; R.sub.2 is H or F; R.sub.3 is selected from H and alkyl; R.sub.4 is selected from H and C(O)R.sub.6; R.sub.5 is H; or R.sub.4 and R.sub.5 are linked to form a heterocyclic group which is optionally substituted with one or more R.sub.10 groups; R.sub.6 is selected from R.sub.7, OR.sub.7 and NR.sub.8R.sub.9; R.sub.7, R.sub.8 and R.sub.9 are each independently selected from alkyl, cycloalkyl, aralkyl, cycloalkenyl, heterocyclic, aryl and heteroaryl, each of which is optionally substituted with one or more R.sub.10 groups; each R.sub.10 is independently selected from halogen, OH, CN, NO.sub.2, COO-alkyl, aralkyl, 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; and each R.sub.11 is H or F.

2. A compound of formula (III), or a pharmaceutically acceptable salt thereof, ##STR00026## or a tautomeric form thereof wherein: R.sub.1 is alkyl, Cl, F or Br; R.sub.2 is H or F; R.sub.3 is selected from H and alkyl; R.sub.4 is C(O)R.sub.6; R.sub.5 is H; or R.sub.4 and R.sub.5 are linked to form a heterocyclic group which is optionally substituted with one or more R.sub.10 groups; R.sub.6 is selected from R.sub.7, OR.sub.7 and NR.sub.8R.sub.9; R.sub.7, R.sub.8 and R.sub.9 are each independently selected from alkyl, cycloalkyl, aralkyl, cycloalkenyl, heterocyclic, aryl and heteroaryl, each of which is optionally substituted with one or more R.sub.10 groups; each R.sub.10 is independently selected from halogen, OH, CN, NO.sub.2, COO-alkyl, aralkyl, 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; Y is selected from CR.sub.11 and N; and each R.sub.11 is H or F with the exception of the following compound: R.sub.1Cl, R.sub.2H, each R.sub.11 is H, Y is CH, R.sub.3 is H and R.sub.4 and R.sub.5 are linked together to form the following cycle: ##STR00027##

3. A compound according to claim 2 of the formula (IIIa), or a pharmaceutically acceptable salt thereof, ##STR00028## wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.10 are as defined in claim 2.

4. A compound of formula (IV), or a pharmaceutically acceptable salt thereof, ##STR00029## or a tautomeric form thereof wherein: R.sub.1 is alkyl or Br; R.sub.2 is H; R.sub.3 is selected from H and alkyl; R.sub.4 is selected from H and C(O)R.sub.6; R.sub.5 is H; or R.sub.4 and R.sub.5 are linked to form a heterocyclic group which is optionally substituted with one or more R.sub.10 groups; R.sub.6 is selected from OR.sub.7 and NR.sub.8R.sub.9; R.sub.6 is selected from R.sub.7, ORS and NR.sub.8R.sub.9; R.sub.7, R.sub.8 and R.sub.9 are each independently selected from alkyl, cycloalkyl, aralkyl, cycloalkenyl, heterocyclyl and aryl, each of which is optionally substituted with one or more R.sub.10 groups; each R.sub.10 is independently selected from halogen, OH, CN, NO.sub.2, COO-alkyl, aralkyl, 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; Y is C.sub.11; R.sub.11 is H or F, with the exception of the following compound: R.sub.1 is Br, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are H, Y is CH.

5. A compound selected from, ##STR00030## and pharmaceutically acceptable salts and tautomeric forms thereof.

6. A process of preparation of a compound according to claim 1 comprising the following step: ##STR00031## wherein R.sub.1, R.sub.2, X, Y or Z are defined as in claim 1.

7. A pharmaceutical composition comprising a compound according to claim 1, 2, 4 or 5 admixed with a pharmaceutically acceptable diluent, excipient or carrier.

8. A combination comprising a compound according to claim 1, and a second active agent.

9. A process of preparation of a compound according to claim 2 comprising the following step: ##STR00032## wherein R.sub.1, R.sub.2, X, Y or Z are defined as in claim 2.

10. A pharmaceutical composition comprising a compound according to claim 2 admixed with a pharmaceutically acceptable diluent, excipient or carrier.

11. A combination comprising a compound according to claim 2 and a second active agent.

12. A process of preparation of a compound according to claim 4 comprising the following step: ##STR00033## wherein R.sub.1, R.sub.2, X, Y or Z are defined as in claim 4.

13. A pharmaceutical composition comprising a compound according to claim 4 admixed with a pharmaceutically acceptable diluent, excipient or carrier.

14. A combination comprising a compound according to claim 4 and a second active agent.

15. A process of preparation of a compound according to claim 5 comprising the following step: ##STR00034## wherein R.sub.1, R.sub.2, X, Y or Z are defined as in claim 5.

16. A pharmaceutical composition comprising a compound according to claim 5 admixed with a pharmaceutically acceptable diluent, excipient or carrier.

17. A combination comprising a compound according to claim 5 and a second active agent.

18. A compound of the formula: ##STR00035## or a pharmaceutically acceptable salt thereof.

19. A process of preparation of a compound according to claim 18 comprising reacting 2-chloro-4-fluorobenzaldehyde with hydrazinecarboximidamide in a solvent.

20. A process according to claim 19, wherein the solvent is ethanol.

21. A pharmaceutical composition comprising a compound according to claim 18, 2, 4 or 5 admixed with a pharmaceutically acceptable diluent, excipient or carrier.

22. A combination comprising a compound according to claim 18, and a second active agent.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(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 of the formula (I), Example 1 of the invention, from ER stress induced by 6 hour exposure to tunicamycin. See description test 1.

(3) FIG. 2 shows that Compound of the formula (I), Example 1 of the invention, postpones translation recovery in stressed cells. More specifically, FIG. 2 shows that translation is attenuated 2 h following Tunicamycin addition. Translation recovery is noticeable in cells treated with tunicamycin only. Example 1 of the invention prolongs translation attenuation in tunicamycin treated cells. See description test 3.

(4) FIG. 3 shows Compound of the formula (I), Example 1 of the invention, unlike Guanabenz, has a no activity for adrenergic 2A receptor as measured by a functional assay for the adrenergic 2A receptor. See description test 5.

(5) FIG. 4 shows that a Compound of the formula (I), Example 1 of the invention prevents ER-retention of P0S63del, the mutant protein associated with Charcot Marie Tooth 1B. Y axis: number of cells. UT: untreated.

(6) FIG. 5 shows that a Compound of the formula (I), Example 1 of the invention reduces accumulation of two unrelated disease-causing, misfolded proteins: mutant huntingtin amino-terminal fragment (Htt48Q) associated with Huntington's disease and SOD1 mutant (A4V), associated with amyotrophic lateral sclerosis. Y axis: percentage accumulation of protein, relative to untreated cells. UT: untreated.

(7) FIG. 6 shows that a Compound of the formula (I), Example 1 of the invention reduces accumulation of rhodopsin mutant P23H associated with retinitis pigmentosa. Y axis: number of cells. UT: untreated.

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

EXAMPLES

(9) Methods & Materials

(10) Example 1 was purchased from Chemdiv ref: 1683-6588

(11) Example 2 was purchased from Chembridge ref: 5173161

(12) Example 4 was purchased from Enamine ref: Z49562642

(13) Example 6 was purchased from Chemdiv ref: 1683-6502

(14) Preparation of the Compounds According to the Present Invention

(15) 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:

(16) General Procedure A:

(17) ##STR00011##

(18) To a solution of benzaldehyde (1 eq.) in ethanol (300 ml) was sequentially added Aminoguanidine hydrochloride (1 eq.) and sodium acetate (1 eq.) at 25 C. The resulting reaction mixture was heated at 80 C. for next 6 hours. Reaction completion was monitored on TLC using dichloromethane/methanol (8/2) as mobile phase. After completion of reaction, the reaction mixture was allowed to cool down to 25 C. and dumped in the saturated solution of NaHCO.sub.3 (700 ml). The resulting precipitate were filtered off under vacuum and washed with water (100 ml). The resulting solid material was titurated with diethylether (225 ml) and dried under vacuum to provide the desired substituted aminoguanidine derivative.

(19) The following compounds were prepared according general procedure A:

Example 1: 1-[(E)-[(2-chlorophenyl)methylidene]amino]-guanidine

(20) Prepared following general procedure A from 2-chlorobenzaldehyde. .sup.1H-NMR (DMSO-d.sub.6): (ppm) 5.61 (s, 2H); 6.06 (s, 2H); 7.22-7.32 (m, 2H); 7.40 (dd, 1H); 8.15 (dd, 1H); 8.28 (s, 1H); MS (ESI+): m/z=197.4 [M+H].sup.+

Example 3: 1-[(E)-[(2-fluorophenyl)methylidene]amino]-guanidine

(21) Prepared following general procedure A from 2-fluorobenzaldehyde.

Example 7: 1-[(E)-[(2-chloro-4-fluorophenyl)methylidene]amino]guanidine

(22) Prepared following general procedure A from 2-chloro-4-fluorobenzaldehyde in 67% yield. .sup.1H-NMR (DMSO-d.sub.6): (ppm) 5.80 (brs, 2H); 5.84 (brs, 2H); 7.19-7.34 (m, 4H); 8.16 (s, 1H); MS (ESI+): m/z=215.1 [M+H].sup.+

Example 13: 1-[(E)-[(3-chloropyridin-4-yl)methylidene]amino]guanidine

(23) Prepared following general procedure A from 3-chloroisonicotinaldehyde in 50% yield. .sup.1H-NMR (DMSO-d.sub.6): (ppm) 6.01 (brs, 2H); 6.33 (brs, 2H); 8.10 (d, 1H); 8.14 (s, 1H); 8.37 (dd, 1H); 8.52 (s, 1H); MS (ESI+): m/z=198.4 [M+H].sup.+

Example 15: 1-[(E)-[(2-chloro-6-fluorophenyl)methylidene]amino]guanidine

(24) Prepared following general procedure A from 2-chloronicotinaldehyde in 56% yield. .sup.1H-NMR (DMSO-d.sub.6): (ppm) 5.84 (brs, 2H); 5.88 (brs, 2H); 7.18-7.35 (m, 3H); 8.16 (s, 1H); MS (ESI+): m/z=215.4 [M+H].sup.+.

Intermediate 1: 3-chloro-5-fluoroisonicotinaldehyde

(25) To a stirred solution of N,N-Diisopropylamine (0.864 g, 0.006690 mol) in THF (6 ml) was added n-buLi (1.6M in hexane) (7.6 ml, 0.012164 mol) dropwise over a period of 15 minutes at 78 C. The resulting reaction mixture was stirred at 78 C. for 15 minutes and then it was allowed to warm at 0 C. whereby it was further stirred for 1 hour. The resulting reaction mixture was again cooled at 78 C. and a solution of 3-chloro-5-fluoropyridine (0.8 g, 0.006082 mol) in THF (6 ml) was added dropwise over period of 10 minutes. The resulting reaction mixture was stirred at 78 C. for 1 hour, thereafter methyl formate (0.73 g, 0.012164 mol) was added dropwise at 78 C. The resulting reaction mixture was further stirred at 78 C. for 1 more hour. The reaction was monitored on TLC using Hexane:ethylaceate (5:5) as mobile phase. After completion of reaction, the reaction mixture was dumped in saturated solution of NH.sub.4Cl (50 ml) and extracted with Ethyl acetate (425 ml). The combined organic extract was washed with demineralised water (50 ml), brine (25 ml), dried over sodium sulphate and concentrated under vacuo. Distillation of the organic layer provided the desired aldehyde (0.6 g, 61.85% yield) in crude form. This crude compound was directly used for the next step without any further treatment.

Example 16: 1-[(E)-[(3-chloro-5-fluoropyridin-4-yl)methylidene]amino]guanidine

(26) Prepared following general procedure A from 3-chloro-5-fluoroisonicotinaldehyde in 14% yield. .sup.1H-NMR (DMSO-d.sub.6): (ppm) 5.95-6.30 (m, 4H); 8.10 (s, 1H); 8.46-8.52 (m, 2H); MS (ESI+): m/z=216.0 [M+H].sup.+.

Example 8: N{N-[(E)-[(2-chlorophenyl)methylidene]amino]carbamimidoyl}acetamide

(27) ##STR00012##

(28) To a solution of 1-[(E)-[(2-chlorophenyl)methylidene]amino]-guanidine (0.50 g, 0.002543 mol) in DMSO (10 ml) was added acetic anhydride (0.26 g, 0.002543 mol) at 25 C. The resulting reaction mixture was stirred at 25 C. for next 15 hours. Reaction completion was monitored on TLC using dichloromethane/Methanol (9.5/0.5) as mobile phase. After completion of reaction the reaction mixture was dumped in the water (100 ml) and extracted with ethyl acetate (2150 ml). The combined organic extract was washed with brine (100 ml), dried over sodium sulphate, filtered and concentrated in vacuo. The resulting crude material was further purified by flash chromatography using dichloromethane:methanol as mobile phase whereby the desired product eluted at around 1.0% methanol in dichloromethane. Distillation of the pure product fractions provided N{N-[(E)-[(2-chlorophenyl)methylidene]amino]carbamimidoyl}acetamide (0.080 g, 13% yield). .sup.1H-NMR (DMSO-d.sub.6): (ppm) 2.97 (s, 3H); 7.25-7.41 (m, 3H); 7.42-7.53 (m, 1H); 7.79 (brs, 1H); 8.22-8.29 (m, 1H); 8.48 (s, 1H); 10.58 (brs, 1H); MS (ESI+): m/z=239.2 [M+H].sup.+.

Example 9: methyl N{N-[(E)-[(2-chlorophenyl)methylidene]amino]carbamimidoyl}carbamate

(29) ##STR00013##

(30) To a suspension of 1-[(E)-[(2-chlorophenyl)methylidene]amino]-guanidine (0.15 g, 0.000762 mol) in dichloromethane (5 ml) was added triethylamine (0.32 ml, 0.002288 mol) at 25 C. The resulting reaction mixture was cooled to 0 C. using ice/salt bath; thereafter methylchloroformate (0.09 ml, 0.001144 mol) was added in to the reaction mixture at 0 C. The resulting reaction mixture was stirred at room temperature for 15 hours. Reaction completion was monitored on TLC using dichloromethane/methanol (9/1) as mobile phase. After completion of reaction, the reaction mixture was dumped in saturated solution of NaHCO.sub.3 (20 ml) and extracted with dichloromethane (325 ml). The combined organic extract was washed with D.M. water (20 ml), brine (20 ml), dried over sodium sulphate, filtered and concentrated in vacuo. The resulting crude material was further purified by flash column chromatography using dichloromethane:methanol as mobile phase whereby the desired product eluted at around 1.0% methanol in dichloromethane. Distillation of the pure product fractions provided methyl N{N-[(E)-[(2-chlorophenyl)methylidene]amino]carbamimidoyl}carbamate (0.065 g, 37% yield). .sup.1H-NMR (DMSO-d.sub.6): (ppm) 3.60 (s, 3H); 7.34-7.43 (m, 2H); 7.45-7.52 (m, 1H); 7.67 (brs, 1H); 7.92 (brs, 1H); 8.22-8.30 (m, 1H); 8.44 (s, 1H); 11.02 (brs, 1H); MS (ESI+): m/z=255.4 [M+H].

(31) Selected compounds according to the invention are set forth in Table 1 below:

(32) TABLE-US-00001 Compound Number Structure Chemical Name Example 1 1-[(E)-[(2- chlorophenyl)methylidene]amino]- guanidine Example 2 1-[(E)-[(2- bromophenyl)methylidene]amino]- guanidine Example 3 1-[(E)-[(2- fluorophenyl)methylidene]amino]- guanidine Example 4 1-[(E)-[(2- methylphenyl)methylidene]amino] guanidine Example 6 2-chlorobenzaldehyde (6-methyl-5-oxo- 4,5-dihydro-1,2,4-triazin-3-yl)hydrazone Example 7 1-[(E)-[(2-chloro-4- fluorophenyl)methylidene]amino]guanidine Example 8 0 N-{N-[(E)-[(2- chlorophenyl)methylidene]amino] carbamimidoyl}acetamide Example 9 methyl N-{N-[(E)-[(2- chlorophenyl)methylidene]amino] carbamimidoyl}carbamate Example 13 1-[(E)-[(3-chloropyridin-4- yl)methylidene]amino]guanidine Example 15 1-[(E)-[(2-chloro-6- fluorophenyl)methylidene]amino]guanidine Example 16 1-[(E)-[(3-chloro-5-fluoropyridin-4- yl)methylidene]amino]guanidine

(33) In some of the experiments below, the salt of these compounds may be used; for example, the acetate salt of example 1 formed with acetic acid may be used.

(34) Cytoprotection from ER Stress (Test 1)

(35) HeLa cells were cultured in Dulbecco's Modified Eagle's Media (DMEM) supplemented with penicillin, streptomycin, containing 5% fetal bovine serum (FBS), at 37 C. in 5% CO.sub.2 atmosphere. Cells were plated in 24 well plates at a density of 15,000 cells/ml 24 hours prior treatment. ER stress was elicited by addition of fresh media containing 2.5 g/ml tunicamycin (Sigma-Aldrich) together with eIF2 phosphatases inhibitors (0.2-5 M). Media were changed 6 h later with fresh media containing phosphatase inhibitors (0.2-5 M). Inhibitors were dissolved in DMSO (50 mM) and DMSO was used as a mock treatment. Cell viability was assessed by measuring the reduction of WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium]into formazan using Cell viability Counting Kit-8 (Dojindo) according to the supplier's recommendation, 48 h after tunicamycin treatment. Cytoprotection from ER stress is measured in terms of the percentage increase in viable cells (relative to control) after ER stress. The result for Example 1 of the invention is shown in FIG. 1.

(36) Assessment of Translation Rates in Unstressed Cells (Test 2)

(37) HeLa cells (80,000 cells/ml) were plated in 12-well plates 24 h before each experiment and either untreated or treated with compounds (50 M) for 0.5, 1, 2.5, 5 and 7.5 h. At the end of each time point, 30.6 Ci/ml .sup.35S-methionine (EasyTag, PerkinElmer) was added to the culture medium for 10 min at 37 C. Following labelling, cells were washed with ice-cold PBS and lysed in 75 l Laemmli Buffer. Lysates were sonicated, boiled at 95 C. for 5 min and resolved on NuPAGE 4-12% gradient gels. Gels were then stained with Coomassie Brilliant Blue R-250 and analyzed by phosphorimaging.

(38) Assessment of Translation Rates in Stressed Cells (Test 3)

(39) Treatments were performed as for measuring translation in unstressed cells, except that Tunicamycin (2.5 g/ml) was added together with the compounds. The result for Example 1 of the invention is shown in FIG. 3.

(40) Immunoprecipitations (Test 4)

(41) HeLa cells (80,000 cells/ml) were plated the day before the indicated treatments, transfected with GFP-PPP1R15A or FLAG-PPP1R15B expression plasmids using Lipofectamine 2000 (Invitrogen) according to manufacturer's procedure. Two days following transfection, cells were treated for 6 h with compounds (50 M) and then washed in PBS and lysed in IP buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.2% Triton X-100, 10% glycerol, and EDTA-free protease inhibitor cocktail). Lysates were clarified by centrifugation at 15,000 g for 15 min at 4 C. and pre-cleared on protein G beads for 1 hour at 4 C. Proteins were immunoprecipitated with 1.5 l GFP antibody (JL-8, Clontech, 632380), bound to 20 l of protein-G-sepharose beads (GE Healthcare, 17-0618-01). The beads were then washed 3 times with cold IP buffer and boiled in 50 l Laemmli Buffer (25 mM Tris-HCl pH 6.8, 1% SDS, 25 mM DTT, 7.5% Glycerol, 0.05% Bromophenol blue). The immunoprecipitated protein complexes (17 l) were separated on 4-12% NuPAGE gradient gels (Invitrogen), transferred to Optitran BA-S 83 reinforced Nitrocellulose membrane and revealed with GFP and PP1 antibodies (sc-7482, Santa Cruz).

(42) Functional Aequorin Assay for Adrenergic 2A Receptor (Test 5)

(43) CHO-K1 cells coexpressing mitochondrial apoaequorin, G16 and recombinant human Adrenergic 2A receptor grown to mid-log phase in culture media without antibiotics were detached with PBS-EDTA, centrifuged and resuspended in DMEM/HAM's F12 with HEPES, without phenol red+0.1% BSA protease free buffer at a concentration of 110.sup.6 cells/ml. Cells were incubated at room temperature for at least 4 h with coelenterazine h. On each day of the test, reference agonist (UK14304) was tested to evaluate the performance of the assay and determine EC.sub.50. Then, 50 l of cell suspension was mixed with 50 l of test agonist in a 96-well plate. The resulting emission of light was recorded using Hamamatsu Functional Drug Screening System 6000 luminometer. To standardize the emission of recorded light (determination of the 100% signal) across plate and across different experiments, some of the wells contained 100 M digitonin or a saturating concentration of AT (20 M). Dose-response data from test compounds were analysed with XLfit (IDBS) software using nonlinear regression applied to a sigmoidal dose-response model. The result for Example 1 of the invention is shown in FIG. 4. Advantageously, in contrast to Guanabenz, Example 1 is not considered to be a potent alpha-2 agonist. This loss in alpha-2 adrenergic activity renders the compound therapeutically useful in the treatment of the disorders claimed herein. The absence of alpha-2 adrenergic activity means that the compound can be administered at a dosage suitable to treat the disorders claimed herein, but without any significant effect on blood pressure, thereby avoiding the need to co-administer with a known alpha-2 adrenergic antagonist (an alpha blocker).

(44) Selectivity Assessment

(45) Selectivity was inferred from the results of test 1, 2, 3, and 5:

(46) A selective inhibitor of PPP1R15A should, protect cells from ER stress (Test 1), does not inhibit translation in non-stressed cells (Test 2), prolongs translation attenuation after Tunicamycin (Test 3), and selectively dissociates PPP1R15A-PP1 holophosphatase but not PPP1R15b-PP1 holophosphatase (Test 4).

(47) Results

(48) The results of Tests 1 to 4 for selected compounds of the invention are shown below in Table 1.

(49) TABLE-US-00002 TABLE 1 Test 1 Test 2 Selectivity Survival Translation Test 3 Test 4 assessment following inhibition in Translation Dissociation Selectivity towards ER stress non-stressed attenuation PPP1R15A/PP1 or PPP1R15A or Ex (increase %)) cells after Tm PPP1R15B/PP1.sup.b PPP1R15B.sup.a 1 180 NO prolonged Dissociation Selectively inhibits PPP1R15A/PP1 BUT PPP1R15A, NOT NOT PPP1R15B/PP1.sup.b PPP1R15B 2 40 YES prolonged Inhibits both PPP1R15A and PPP1R15B 3 80 YES prolonged Preferentially inhibits PPP1R15A 4 100 YES prolonged Inhibits both PPP1R15A and PPP1R15B 6 120 NO prolonged Selectively inhibit PPP1R15A, not B 7 160 YES prolonged Dissociation Inhibits both PPP1R15A/PP1 AND PPP1R15A and PPP1R15B/PP1.sup.b PPP1R15B 8 100 YES prolonged Inhibits both PPP1R15A and PPP1R15B 9 20 13 60-80 15 160 NO prolonged Potentially selective 16 140 YES Potentially non selective .sup.ainferred from translation inhibition in stressed/non-stressed cells .sup.bconfirms selectivity towards PPP1R15A-PP1 or lack thereof.
Cell-Based Assays:
Material and Methods

(50) Cell Culture and Reagents 293T cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and transfected in 6- or 12-well plates by using the calcium phosphate method leading usually to 70% transfection efficiency. Routinely, 45,000 cells/ml were plated before transfection as described in (Rousseau et al. 2009). Myelin P0S63del-DSred construct was described in (Pennuto et al. 2008), the Huntingtin construct was described in (Rousseau et al. 2009), the SOD1A4V construct is described in (Mnch et al. 2011) and the P23H construct is described in (Mendes and Cheetham 2008).

(51) Fluorescence Microscopy

(52) Transfected cells were fixed with 4% paraformaldehyde and labeled with indicated antibodies. Micrographs were taken at 100 magnification on a Leica TCS SP2AOBS confocal microscope or Leica DMRB Fluorescence microscope.

(53) Immunoblotting

(54) Routinely, 70% confluent cells from a well of a 12-well plate were lysed in 140 l of boiling Laemmli buffer (25 mM Tris-HCl, pH 6.8, 1% SDS, 25 mM dithiothreitol, 7.5% glycerol, 0.05% bromphenol blue) for immunoblot analysis. 18 l of protein extracts were loaded on 2-12% NuPAGE gels and transferred to Optitran BA-S 83 reinforced nitrocellulose membrane (Whatman and Schleicher & Schuell). Equal loading of protein extracts analyzed by immunoblot was controlled by Ponceau Red staining and vimentin (data not shown). Membranes were saturated in 5% dried skimmed milk in phosphate-buffered saline and probed with Htt 2b4 antibody or HA antibody to reveal HA-tagged SOD1. The appropriate secondary antibody coupled to peroxidase was revealed using the SuperSignal West Pico Chemiluminescent kit (Pierce). Chemiluminescent images were acquired using the Chemi-Smart 5000 (Vilber-Lourmat) allowing quantitative detection of chemilumi-nescence. Signals of interest were quantified using ImageJ.

(55) Assay for Charcot Marie Tooth 1B (Test 6)

(56) Deletion of serine 63 from P0 (P0S63del) causes Charcot-Marie-Tooth 1B neuropathy in humans and a similar demyelinating neuropathy in transgenic mice. The mutant protein misfolds and accumulates in the ER, induces the UPR and fails to be incorporated into myelin (D'Antonio et al., 2009, J Neurosci Res, 87, 3241-9). 293T cells were transfected with labeled P0S63 del-P0S63del-DSredand analyzed by confocal microscopy, 48 h post-transfection in the presence or absence of compound of formula (I). In accordance with the methodology described in (Pennuto et al. 2008), cells with ER-retained P0S63del-DSred were scored. FIG. 4 shows that in untreated cells, P0S63del accumulates in the ER but Example 1 prevents this accumulation. Since accumulation of misfolded P0 causes CMT-1B and having shown that Example 1 reduces accumulation of the disease-causing protein, the compound of formula (I) should be useful to treat CMT-1B as well as other forms of CMT where the disease causing-protein is misfolded and retained in the ER.

(57) Assay for Huntington's Disease and Amyotrophic Lateral Sclerosis (Test 7)

(58) We tested for accumulation of mutant huntingtin amino-terminal fragment (Htt48Q) associated with Huntington's disease and SOD1 mutant (A4V), associated with amyotrophic lateral sclerosis.

(59) We used a method previously described in WO/2008/041133. 293T cells were transfected with plasmids encoding for Htt48 or SOD1.sup.A4V and treated with Example 1 in DMSO or DMSO alone 4 h post-transfection. SDS lysates collected 48 h post-transfection were analyzed on a NuPAGE followed by immunoblot with Huntingtin antibody (2B4) or HA (SOD1). FIG. 5 shows the quantifications of the signal on immunoblots, normalized to untreated cells. Example 1 reduces accumulation of both proteins. Having shown that Example 1 reduces accumulation of the proteins causing Huntington's disease and Amyotrophic lateral sclerosis, the compound of formula (I) should be useful to treat such diseases as well as other neurodegenerative diseases caused by accumulation of misfolded proteins.

(60) Assay for Rhodopsin P23H Aggregation (Test 8)

(61) We tested for aggregation of rhodopsin associated for retinitis pigmentosa as described (Mendes & Cheetham 2008).

(62) 293T cells were transfected with plasmid encoding the P23H mutant of rhodopsin and treated with Example 1 in DMSO or DMSO alone 4 h post-transfection. Cells were analyzed by microscopy. FIG. 6 shows the cells with or without aggregates. Example 1 reduces aggregates. Since accumulation of misfolded rhodopsin causes RP and having shown that Example 1 reduces accumulation of the disease-causing protein, the compound of formula (I) should be useful to treat Retinitis Pigmentosa.

(63) Examples 1 and 6 are confirmed selective inhibitors of PPP1R15A.

(64) Examples 2, 4, 7, 8 inhibit both PPP1R15A and B.

(65) 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.