SYNTHETIC RETINOIDS FOR USE IN RAR ACTIVATION

Abstract

The present invention relates to compounds of formula I: in which A.sup.1-A.sup.7 and R.sup.1 to R.sup.5 are defined herein, for use in the treatment of a condition or disease which is alleviated by the activation of retinoic acid receptors (RAR). The invention also relates to pharmaceutical compounds comprising such compounds, and related methods of treatment. In an aspect, the invention relates to a method of screening compounds for therapeutic potential in the treatment of a condition or disease which is alleviated by the activation of retinoic acid receptors (RAR). Aspects of the invention relate to novel compounds of formula I in which at least one of A.sup.1 to A.sup.3 is or at least one of A.sup.4 is CR.sup.12 or A.sup.5 is CR.sup.13 in which R.sup.12/R.sup.13 is halogen.

Claims

1. A compound of formula I: ##STR00028## in which: A.sup.1 is N or CR.sup.6; A.sup.2 is N or CR.sup.7; A.sup.3 is N or CR.sup.8; R.sup.6 and R.sup.8, are each independently hydrogen, C.sub.1-C.sub.10 alkyl, F, Br or Cl; R.sup.7 is independently hydrogen, C.sub.1-C.sub.10 alkyl, F, Br, Cl or —OCR.sup.9 in which R.sup.9 is H or C.sub.1-C.sub.6 alkyl; R.sup.1 to R.sup.4 are each independently alkyl C.sub.1-C.sub.10 alkyl, or R.sup.1 and R.sup.2 and/or R.sup.3 and R.sup.4 join to form a 3-membered ring; A.sup.4 is N or CR.sup.12; A.sup.5 is N or CR.sup.13; A.sup.6 is N or CR.sup.14; A.sup.7 is N or CR.sup.15; each R.sup.12 to R.sup.15 is independently H, halogen or haloalkyl C.sub.1-C.sub.10 , and R.sup.5 is —C(═O)R.sup.16 or —C(═O)OR.sup.16 in which R.sup.16 is H or C.sub.1-10 alkyl; with the proviso that at least one of A.sup.1 to A.sup.7 is N or at least one of R.sup.12 to R.sup.15 is F, Cl or Br; and isomers thereof; in free or in salt form; for use in the treatment of a condition or disease which is alleviated by the activation of retinoic acid receptors (RAR).

2. A compound as claimed in claim 1, wherein at least one of A.sup.1 to A.sup.3 is N.

3. A compound as claimed in claim 1, wherein A.sup.1 and A.sup.3 are N.

4. A compound as claimed in claim 1, wherein A.sup.2 is CR.sup.7.

5. A compound as claimed in claim 4, where R.sup.7 is hydrogen.

6. A compound as claimed in claim 1, wherein R.sup.5 is —COOH.

7. A compound as claimed in claim 1, wherein at least one of A.sup.4, A.sup.5 or A.sup.6 is CF.

8. A compound according to claim 1, which is selected from: ##STR00029## ##STR00030## ##STR00031## ##STR00032## ##STR00033##

9. A compound according to claim 8, which is selected from: ##STR00034##

10. A compound as claimed in claim 1, wherein the disease or condition which is alleviated by the activation of RAR is selected from Amyotrophic lateral sclerosis (ALS), Parkinson's disease, multiple sclerosis (MS), Alzheimer's disease, early-stage Alzheimer's disease, intermediate-stage Alzheimer's disease, late-stage Alzheimer's disease, cognitive disorders, memory impairment, memory deficit, senile dementia, cognitive impairment, mild cognitive impairment, stroke, traumatic brain injury, epilepsy and spinal cord injury.

11. A compound of formula I as defined in claim 1 in the manufacture of a medicament for use in the treatment of a disease or condition which is alleviated by the activation of RAR.

12. A method of treatment of a patient with a disease or condition which is alleviated by the activation of RAR, the method comprising administering to a patient a therapeutically effective amount of a compound of formula I, wherein formula I is as defined in claim 1.

13. A pharmaceutical composition comprising a compound of formula I as defined in claim 1, optionally in conjunction with one or more pharmaceutically acceptable excipients, diluents or carriers, for use in the treatment of a disease or condition which is alleviated by the activation of RAR.

14. A compound of formula I: ##STR00035## in which: A.sup.1 is N or CR.sup.6; A.sup.2 is N or CR.sup.7; A.sup.3 is N or CR.sup.8; R.sup.6 and R.sup.8, are each independently hydrogen, C.sub.1-C.sub.10 alkyl, F, Br or Cl; R.sup.7 is independently hydrogen, C.sub.1-C.sub.10 alkyl, F, Br, Cl or —OCR.sup.9 in which R.sup.9 is H or C.sub.1-C.sub.6 alkyl; R.sup.1 to R.sup.4 are each independently alkyl C.sub.1-C.sub.10 alkyl, or R.sup.1 and R.sup.2 and/or R.sup.3 and R.sup.4 join to form a 3-membered ring; A.sup.4 is N or CR.sup.12; A.sup.5 is N or CR.sup.13; A.sup.6 is N or CR.sup.14; A.sup.7 is N or CR.sup.15; each R.sup.12 to R.sup.15 is independently H, halogen or haloalkyl C.sub.1-C.sub.10, and R.sup.5 is —C(═O)R.sup.16, or —C(═O)OR.sup.16 in which R.sup.16 is H or C.sub.1-10 alkyl; with the proviso that at least one of A.sup.1 to A.sup.3 is N or at least one of A.sup.4 is CR.sup.12 or A.sup.5 is CR.sup.13 in which R.sup.12/R.sup.13 is halogen; and isomers thereof; in free or in salt form.

15. A compound as claimed in claim 14 in which at least one of A.sup.1 to A.sup.3 is N or at least one of A.sup.4 is CR.sup.12 or A.sup.5 is CR.sup.13 in which R.sup.12/R.sup.13 is F.

16. A compound as claimed in claim 14 in which at least one of A.sup.1 to A.sup.3 is N.

17. A compound as claimed in claim 14 in which A.sup.1and A.sup.3 are both N.

18. A method of screening compounds for therapeutic potential in the treatment of conditions or diseases which are alleviated by the activation of RAR, the method comprising: performing an assay to determine the efficacy (E.sub.max) of a compound in activating a RAR as an indicator of genomic activity; performing an assay to determine the efficacy (E.sub.max) of a compound as an indicator of non-genomic activity; for each assay, comparing the E.sub.max to a baseline value; and selecting those compounds which have an E.sub.max above the baseline value in both assays for further investigation.

19. A method as claimed in claim 18, wherein the assay to determine the efficacy (E.sub.max) of a compound as an indicator of non-genomic activity is a kinase phosphorylation assay.

20. A method as claimed in claim 19, wherein the kinase phosphorylation assay is an ERK1/2 phosphorylation assay.

Description

EXAMPLES

[0093] The invention will now be described by way of example only with reference to the accompanying figures, in which:

[0094] FIG. 1 illustrates the synthesis of coupling partners;

[0095] FIG. 2 illustrates the synthesis of exemplary retinoid compounds DC526, DC528, DC641 and DC645;

[0096] FIG. 3 illustrates the genomic activity of retinoids as indicated by use of the X-Gal assay;

[0097] FIG. 4 illustrates the non-genomic activity of retinoids as indicated by their ability to induce phosphorylation of ERK1/2;

[0098] FIG. 5 illustrates average neurite length of differentiated SH-SY5Y cells following treatment with retinoids;

[0099] FIG. 6 shows the relationship between the ability of retinoids to induce both genomic activity [(transcriptional activity: E.sub.max(efficacy)] and non-genomic activity [ERK1/2 activity:E.sub.max(efficacy)] and the induction of neurite outgrowth (fold increase at 10 μM), with the highlighted retinoids showing both activities;

[0100] FIG. 7 shows data relating to the transcriptional regulation of Alzheimer's disease-related genes in rat mixed primary neuronal/glial cultures following treatment with retinoids;

[0101] FIG. 8 compares the activity of exemplary retinoid DC645 with that of ATRA and EC23.

[0102] FIG. 8(a) compares the genomic activity of the retinoids by means of the X-Gal assay; FIG. 8(b) compares the non-genomic activity of the retinoids by measuring their ability to induce ERK 1/2 phosphorylation; FIG. 8(c) compares the ability of the compounds to induce neurite outgrowth in SH-SY5Y cells; and FIG. 8(d) shows the regulation of Alzheimer's disease-related genes by retinoid compounds.

EXAMPLE 1: SYNTHESIS OF EXEMPLARY COMPOUNDS OF FORMULA I

1.1 Synthesis of Coupling Partners

1.1.1. Synthesis of 6-Iodo-1,1,4,4-tetramethyl-1,2,3,4-tetrahydronaphthalene, 1

[0103] The synthesis of 6-Iodo-1,1,4,4-tetramethyl-1,2,3,4-tetrahydronaphthalene (1) is illustrated in FIG. 1(a). 1,1,4,4-Tetramethyl-1,2,3,4-tetrahydronaphthalene (11.46 g, 60.9 mmol), I.sub.2 (7.77 g, 30.6 mmol) and H.sub.5IO.sub.6 (3.49 g, 15.3 mmol) were added to a mixture of gl. ethanoic acid/acetic acid (AcOH) (250 mL), H.sub.2O (25 mL), and H.sub.2SO.sub.4 (13 mL), and the resultant solution was stirred at 70° C. for 6 hours. The solution was cooled, and extracted with ethyl ethanoate/ethyl acetate (EtOAc). The organics were washed with sat. Na.sub.2S.sub.2O.sub.3, H.sub.2O and brine, dried (MgSO.sub.4) and evaporated to give a crude orange oil (17 g). This was purified by dry column vacuum chromatography (eluting with heptane) to give compound 1 as a white solid (16.3 g, 85%): 1H NMR (700 MHz, CDCl3) δ 1.25 (s, 6H), 1.26 (s, 6H), 1.66 (s, 4H), 7.04 (d, J=8.4 Hz, 1H), 7.43 (dd, J=8.4, 1.9 Hz, 1H), 7.60 (d, J=1.9 Hz, 1H); 13C NMR (176 MHz, CDCl3) δ 31.9, 32.0, 34.4, 34.6, 35.0, 35.1, 91.3, 128.9, 134.8, 135.8, 144.8, 147.9; all other data matched the literature (V. B. Christie et al, Org. Biomol. Chem., 2008, 6, 3497-3507).

1.1.2 Synthesis of 6-Ethynyl-1,1,4,4-tetramethyl-1,2,3,4-tetrahydronaphthalene, 3

[0104] The synthesis of 6-ethynyl-1,1,4,4-tetramethyl-1,2,3,4-tetrahydronaphthalene (3) is illustrated in FIG. 1(b). Triethylamine (Et.sub.3N) (150 mL) was degassed by sparging with N.sub.2 for 1 h. Compound 1 (10.0 g, 31.8 mmol), Pd(PPh.sub.3).sub.2Cl.sub.2 (0.223 g, 0.32 mmol), CuI (0.061 g, 0.32 mmol) and trimethylsilylacetylene (5.29 mL, 38.2 mmol) were then added under N.sub.2 and the resultant slurry was stirred at RT for 20 h. The mixture was diluted with heptane and passed through Celite/SiO.sub.2 (eluting with heptane), and the resultant solution was evaporated to give a crude brown oil (10.36 g). This was purified by SiO.sub.2 chromatography (100% heptane) to give compound 2 as a yellow oil (9.99 g, >100%). Compound 2 (9.99 g, 35.1 mmol) was dissolved in methanol (MeOH)/methyl tert-butyl ether (MTBE) (1:1, 160 mL). A solution of sodium hydroxide (NaOH) (0.96 g, 24.0 mmol) in H.sub.2O (15 mL) was then added and the resultant solution was stirred at RT for 16 h. The solution was diluted with MTBE, washed with H.sub.2O and brine, dried (MgSO.sub.4) and evaporated to give a crude yellow oil (6.6 g). This was purified by SiO.sub.2 chromatography (100% heptane) to give compound 3 as a colourless oil that slowly solidified (6.06 g, 90% over two steps): all data matched the literature (V. B. Christie et al, Org. Biomol. Chem., 2008, 6, 3497-3507).

1.1.3 Synthesis of 4-bromo-3-fluorobenzoate, 4

[0105] The synthesis of 4-bromo-3-fluorobenzoate (4) is illustrated in FIG. 1(c). 4-Bromo-2-fluorobenzoic acid (25.0 g, 114.2 mmol) was suspended in MeOH (250 mL), whereupon conc. H.sub.2SO.sub.4(4 mL) was added and the resultant solution was stirred at reflux overnight. The clear solution was then cooled, and H.sub.2O (100 mL) was added, whereupon a white precipitate formed. This was filtered, washed with H.sub.2O and dried to give a crude white solid. This was recrystallised from heptane to give compound 4 as a colourless crystalline solid (21.34 g, 80%): .sup.1H NMR (600 MHz, CDCl.sub.3) δ 3.91 (s, 3H), 7.31-7.36 (m, 2H), 7.80 (t, J=8.0 Hz, 1H); .sup.13C NMR (151 MHz, CDCl.sub.3) δ 52.4, 117.6 (d, J=9.9 Hz), 120.6 (d, J=25.6 Hz), 127.5 (d, J=3.9 Hz), 127.9 (d, J=9.6 Hz), 133.1, 161.56 (d, J=264.9 Hz), 164.1 (d, J=3.9 Hz); .sup.19F NMR (376 MHz, CDCl.sub.3) δ −106.6; IR (ATR) ν.sub.max/cm.sup.−1 3104w, 3086w, 2961w, 1712s, 1599s, 1571m, 1403m, 1215s, 882s; MS (ASAP): m/z=233.0 [M+H].sup.+; HRMS (ASAP) calcd. for C.sub.3H.sub.7O.sub.2BrF [M+H].sup.+: 232.9613, found: 232.9621 (Zimmerman et al., J. Med. Chem. 2014, 57:2334-2356).

1.1.4 Synthesis of Methyl 4-ethynyl-3-fluorobenzoate, 6

[0106] The synthesis of Methyl 4-ethynyl-3-fluorobenzoate (6) is illustrated in FIG. 1(d). Et.sub.3N (120 mL) was degassed by sparging with N.sub.2 for 1 hour. Compound 4 (5.0 g, 21.45 mmol), trimethylsilylacetylene (3.56 mL, 25.74 mmol), Pd(PPh.sub.3).sub.2Cl.sub.2 (301 mg, 0.429 mmol) and CuI (82 mg, 0.429 mmol) were then added under N.sub.2 and the resultant suspension was stirred at room temperature for 16 hours. The suspension was diluted with heptane and passed through Celite®/SiO.sub.2 and the extracts were evaporated to a give a crude oil (6.44 g). This was purified by dry column vacuum chromatography (100% heptane, to 9:1, heptane/EtOAc), and the isolated product further purified by Kugelrohr distillation (150° C., 7.4 Torr) to give compound 5 as a yellow oil (5.58 g, >100%) which was carried directly to the next step: .sup.1H NMR (400 MHz, CDCl.sub.3) δ 0.27 (s, 9H), 3.92 (s, 3H), 7.50 (dd, J=8.0, 6.8 Hz, 1H), 7.71 (dd, J=9.6, 1.5 Hz, 1H), 7.75 (dd, J=8.0, 1.6 Hz, 1H). To a MeOH:MTBE solution (5:50, 55 mL) was added compound 5 (5.58 g, 22.3 mmol) and K.sub.2CO.sub.3 (6.16 g, 44.6 mmol), and the resultant mixture was stirred under N.sub.2 for 6 hours at room temperature. The solution was then diluted with EtOAc, washed with sat. NH.sub.4Cl, H.sub.2O and brine, dried (MgSO.sub.4) and evaporated to give a crude solid (3.6 g). This was purified by dry column vacuum chromatography (100% heptane, to 8:2, heptane/EtOAc) to give compound 6 as a white solid (3.07 g, 80% over two steps): .sup.1H NMR (600 MHz, CDCl.sub.3) δ 3.45 (s, 1H), 3.92 (s, 3H), 7.53 (dd, J=8.0, 6.8 Hz, 1H), 7.72 (dd, J=9.6, 1.6 Hz, 1H), 7.77 (dd, J=8.0, 1.6 Hz, 1H); .sup.13C NMR (151 MHz, CDCl.sub.3) δ 52.5, 76.3, 85.1 (d, J=3.3 Hz), 115.3 (d, J=16.0 Hz), 116.5 (d, J=22.9 Hz), 124.9 (d, J=3.7 Hz), 132.2 (d, J=7.4 Hz), 133.9 (d, J=1.3 Hz), 162.9 (d, J=253.5 Hz), 165.3 (d, J=2.7 Hz); .sup.19F NMR (376 MHz, CDCl.sub.3) δ −109.3; IR (ATR) νmax/cm.sup.−1 3238m, 3090w, 2967w, 2111w, 1710s, 1564m, 1501m, 1440m, 1308s, 1212s, 766s; MS (ASAP) m/z=179.0 [M+H].sup.+; HRMS (ASAP) calcd. for C.sub.10H.sub.8O.sub.2F [M+H].sup.+: 179.0508, found 179.0495.

1.1.5 Synthesis of Methyl 5-bromopyridine-2-carboxylate, 8

[0107] The synthesis of methyl 5-bromopyridine-2-carboxylate (8) is illustrated in FIG. 1(e). 5-Bromopyridine-2-carboxylic acid (20.0 g, 99.0 mmol) was suspended in MeOH (150 ml), whereupon conc. H.sub.2SO.sub.4 (5 mL) was carefully added and the resultant solution was stirred at reflux for 6 h. The solution was cooled, diluted with EtOAc, washed with H.sub.2O and brine (50 mL), dried (MgSO.sub.4) and evaporated to give a crude colourless solid. This was distilled in vacuo using a Kugelrohr (200° C., 7.4 Torr), and the resultant white solid was further recrystallised from heptane/MeOH (10:1) to give compound 8 as a white solid (17.21 g, 80%): .sup.1H NMR (700 MHz, CDCl.sub.3) δ 3.90 (s, 4H), 7.86 7.93 (m, 3H), 8.68 (d, J=2.0 Hz, 1H); .sup.13C NMR (176 MHz, CDCl.sub.3) δ 52.8, 124.8, 126.0, 139.5, 146.0, 150.7, 164.7; ν.sub.max/cm.sup.−1 3059w, 3008w, 2957w, 1710s, 1571w, 1558w, 1436m, 1305s, 1131s, 696s; MS (ASAP): m/z=216.0 [M+H].sup.+; HRMS (ASAP) calcd. for C.sub.10H.sub.9NOI [M+H].sup.+: 215.9660, found: 215.9664 (Tung et al., Eur. J. Med. Chem. 2017, 126, 1011-1020).

1.1.6 Synthesis of 2,2,5,5-Tetramethylhexanedioic acid, 9

[0108] The synthesis of 2,2,5,5-Tetramethylhexanedioic acid (9) is illustrated in FIG. 1(f). To a 2L three-neck flask equipped with mechanical stirrer was added H.sub.2O (600 mL), conc. H.sub.2SO.sub.4 (7.5 mL), then pivalic acid (51.0 g, 500 mmol), and the resultant slurry was cooled to 0° C. Over 15 mins, H.sub.2O.sub.2(30%, 43 mL) and a solution of FeSO.sub.4.7H.sub.2O (139.0 g, 500 mmol) in H.sub.2O (288 mL) and conc. H.sub.2SO.sub.4 (27.5 mL) were added dropwise, with vigorous stirring. After completion of the addition, the suspension was stirred for a further 15 mins, before the solution was concentrated to ca. 250 mL. The precipitated solids were filtered, then dried further under vacuum using a rotary evaporator to give a crude residue. This was recrystallised from AcOH to give 9 as a colourless crystalline solid (3.39 g, 3%): .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 1.06 (s, 12H), 1.37 (s, 4H), 12.05 (s, 2H).

1.1.7 Synthesis of 1,6-Diethyl 2,2,5,5-tetramethylhexanedioate, 10

[0109] The synthesis of 1,6-Diethyl 2,2,5,5-tetramethylhexanedioate (10) is illustrated in FIG. 1(g). To a solution of 9 (3.39 g, 16.76 mmol) in EtOH (40 mL) was added conc. H.sub.2SO.sub.4 (2 mL), and the resultant suspension was stirred at reflux for 16 h. The solution was cooled, and the solvent evaporated to a give crude residue which was dissolved in EtOAc. The organics were washed with sat. NaHCO.sub.3, H.sub.2O and brine, dried (MgSO.sub.4) and evaporated to give a crude oil (3.8 g). This was purified by SiO.sub.2 chromatography (95:5, heptane/EtOAc) to give 10 as a colourless oil (3.41 g, 79%): .sup.1H NMR (700 MHz, CDCl.sub.3) δ 1.14 (s, 11H), 1.24 (t, J=7.1 Hz, 6H), 4.11 (q, J=7.1 Hz, 4H); .sup.13C NMR (176 MHz, CDCl.sub.3) δ 14.2, 25.0, 35.5, 41.8, 60.2, 177.7; IR (ATR) ν.sub.max/cm.sup.−1 2978m, 2934w, 2880w, 1726s, 1475m, 1308m, 1176s, 1110m, 771w; MS(ES): m/z=259.5 [M+H].sup.+.

1.1.8 Synthesis of Trimethyl({3,3,6,6-tetramethyl-2-[(trimethylsilyl)oxy]cyclohex-1-en-1-yl}oxy)silane, 11

[0110] The synthesis of Trimethyl({3,3,6,6-tetramethyl-2-[(trimethylsilypoxy]cyclohex-1-en-1-yl}oxy)silane, 11 is illustrated in FIG. 1(h). To anhydrous toluene (50 mL) was added sodium (1.20 g, 52.1 mmol) under N.sub.2, and the resultant mixture was heated to reflux until the sodium melted. The flask was then removed from the heat, and then 10 (2.69 g, 10.41 mmol) and chlorotrimethylsilane (6.72 mL, 53.0 mmol) were added and the resultant suspension was then stirred at reflux overnight. The purple suspension was then cooled, and filtered under a flow of N.sub.2, washing with toluene, then tetrahydrofuran (THF). The filtrate was then evaporated to give a crude light yellow oil (3.2 g), which was purified by Kugelrohr distillation (120° C., 3.6 Torr) to give 11 as a clear oil (2.64 g, 81%): .sup.1H NMR (400 MHz, CDCl.sub.3) δ 0.19 (s, 18H), 1.03 (s, 12H), 1.44 (s, 4H). All other data matched the literature (Kikuchi et al. J. Med. Chem. 2000; 43: pp.409-419).

1.1.9 Synthesis of 3,3,6,6-tetramethylcyclohexane-1,2-dione, 12

[0111] The synthesis of 3,3,6,6-tetramethylcyclohexane-1,2-dione is illustrated in FIG. 1(i). To a solution of 11 (2.6 g, 8.2 mmol) in dichloromethane (DCM) was added bromine (0.42 mL, 8.2 mmol) dropwise over 5 mins. The resultant yellow solution was stirred at RT for 1 h, before being diluted with DCM, and treated with sat. Na.sub.2S.sub.2O.sub.3, then washed with H.sub.2O, dried (MgSO.sub.4) and evaporated to give a crude yellow solid (1.5 g). This was purified by recrystallisation from heptane to give 12 as a yellow crystalline solid (1.07 g, 78%): .sup.1H NMR (700 MHz, CDCl.sub.3) δ 1.14 (s, 4H), 1.85 (s, 12H); .sup.13C NMR (176 MHz, CDCl.sub.3) δ 22.9, 34.7, 48.6, 207.3; IR (ATR) ν.sub.max/cm.sup.−1 2973m, 2940w, 2870w, 1706s, 1599w, 1459m, 1372m, 1102m, 931m; MS(ES): m/z=169.3 [m+H].sup.+. All other data matched the literature. (Kikuchi et al. J. Med. Chem. 2000; 43: pp. 409-419).

1.1.10 Synthesis of methyl 5,5,8,8-tetramethyl-5,6,7,8-tetrahydroquinoxaline-2-carboxylate, 13

[0112] The synthesis of methyl 5,5,8,8-tetramethyl-5,6,7,8-tetrahydroquinoxaline-2-carboxylate, 13 is illustrated in FIG. 1(j). 12 (0.80 g, 4.76 mmol) and DL-2,3-diaminopropionic acid hydrochloride (0.67 g, 4.76 mmol) were combined in MeOH (30 mL). NaOH (0.76 g, 19.04 mmol) was added and the resultant mixture was stirred at reflux for 24 h. The solution was then cooled to 0° C., H.sub.2SO.sub.4 carefully added, and the solution was stirred at reflux for a further 6 h. The solution was cooled, and the solvent evaporated to give a crude residue which was dissolved with EtOAc, washed with sat. NaHCO.sub.3, H.sub.2O and brine, dried (MgSO.sub.4) and evaporated to give a crude yellow oil (0.9 g). This was purified by SiO.sub.2 chromatography (95:5, heptane/EtOAc) to give 13 as a colourless oil (0.633 g, 54%): .sup.1H NMR (400 MHz, CDCl.sub.3) δ 1.33 & 1.36 (s, 12H), 1.81 (s, 4H), 3.98 (s, 3H), 9.00 (s, 1H). All other data matched the literature (Kikuchi et al. J. Med. Chem. 2000; 43: pp. 409-419).

[0113] 1.1.11 Synthesis of (5,5,8,8-Tetramethyl-5,6,7,8-tetrahydroquinoxalin-2-yl)methanol, 14

[0114] The synthesis of (5,5,8,8-Tetramethyl-5,6,7,8-tetrahydroquinoxalin-2-yl)methanol, 14 is illustrated in FIG. 1(k). To a solution of 13 (5.09 g, 20.5 mmol) in THF (80 mL) was added NaBH.sub.4 (2.33 g, 61.5 mmol). The solution was then heated to reflux, whereupon MeOH (16 mL) was slowly added over 1 h. The resultant solution was then stirred at reflux overnight. The solution was cooled, quenched with 1 M HCl, and the solvent then evaporated. The residue was dissolved in DCM, washed with water, dried (MgSO.sub.4) and evaporated to give a crude yellow oil (4 g). This was purified by SiO.sub.2 chromatography (8:2, heptane/EtOAc, as eluent) to give 14 as a colourless oil (3.96 g, 88%): .sup.1H NMR (400 MHz, CDCl.sub.3) δ 1.33 & 1.36 (s, 12H), 1.80 (s, 4H), 3.50 (br, 1H), 4.75 (s, 2H), 8.32 (s, 1H); all other data matched the literature (Kikuchi et al. J. Med. Chem. 2000; 43: pp. 409-419).

1.1.12 Synthesis of 5,5,8,8-Tetramethyl-5,6,7,8-tetrahydroquinoxaline-2-carbaldehyde, 15

[0115] The synthesis of 5,5,8,8-Tetramethyl-5,6,7,8-tetrahydroquinoxaline-2-carbaldehyde, 15 is illustrated in FIG. 1(l). Oxalyl chloride (2.28 mL, 26.96 mmol) was added to anhydrous DCM (100 mL) under N.sub.2. The resultant solution was cooled to −78° C., whereupon DMSO (3.83 mL, 53.92 mmol) was added dropwise so as to maintain the temperature below −60° C. The solution was stirred for 15 mins before 14, as a solution in anhydrous DCM (3.96 g, 17.97 mmol, in 20 mL) was added dropwise so as to main the temperature below −60° C. The solution was stirred for a further 15 mins before Et.sub.3N (18.03 mL, 129.38 mmol) was added. The solution was then stirred for 10 mins, before being allowed to reach room temperature (RT) over 30 mins. H.sub.2O was added, and the resultant mixture was diluted with DCM, washed with H.sub.2O, dried (MgSO.sub.4) and evaporated to give a crude oil (4 g). This was purified by dry column vacuum chromatography (DCVC) (heptane to 9:1, heptane/EtOAc) to give 15 as a colourless oil that slowly crystallises (3.39 g, 86%): .sup.1H NMR (700 MHz, CDCl.sub.3) δ 1.35 & 1.37 (s, 12H), 1.83 (s, 4H), 8.90 (s, 1H), 10.08 (s, 1H); .sup.13C NMR (176 MHz, CDCl.sub.3) δ 29.7, 29.7, 33.8, 33.8, 37.4, 37.9, 139.8, 144.2, 159.0, 163.7, 193.4; IR (ATR) ν.sub.max/cm.sup.−1 2979m, 2964m, 2928m, 2862m, 2823w, 1707s, 1553m, 1457m, 1126s, 1078s, 737s; MS(ES): m/z =219.3 [M+H].sup.+; HRMS (ES) calcd. for C.sub.13H.sub.19ON.sub.2 [M+H].sup.+: 216.1497, found 216.1503.

1.1.13 Synthesis of Dimethyl 1-diazo-2-oxopropylphosphonate, 16

[0116] The synthesis of Dimethyl 1-diazo-2-oxopropylphosphonate, 16 is illustrated in FIG. 1(m). To a solution of dimethyl 2-oxopropylphosphonate (4.49 mL, 32.5 mmol) in anhydrous toluene (30 mL) at 0° C. was added NaH (60% dispersion in mineral oil, 1.20 g, 30.00 mmol) portionwise with vigorous stirring. After gas evolution had ceased 4-acetamidobenzenesulfonyl azide (7.21 g, 30.0 mmol), as a solution in anhydrous THF (10 mL), was added dropwise. The resultant suspension was stirred at RT for 16 h, whereupon heptane was added, and the suspension was filtered through Celite®, and rinsed with MTBE. The organic extracts were then evaporated to give a crude oil (5 g), which was purified by SiO.sub.2chromatography (1:1, heptane/EtOAc) to give dimethyl 1-diazo-2-oxopropylphosphonate as a light yellow oil (3.14 g, 54%): .sup.1H NMR (400 MHz, CDCl.sub.3) δ 2.24 (s, 3H), 3.82 (d, J=11.9 Hz, 6H), all other data matched the literature (Pietruszka et al. Synthesis, 2006, pp. 4266-4268).

1.1.14 2-Ethynyl-5,5,8,8-tetramethyl-5,6,7,8-tetrahydroquinoxaline, 17

[0117] The synthesis of 2-Ethynyl-5,5,8,8-tetramethyl-5,6,7,8-tetrahydroquinoxaline, 17 is illustrated in FIG. 1(n). To a solution of 15 (1.18 g, 5.40 mmol) in anhydrous MeOH (50 mL) under N.sub.2 was added K.sub.2CO.sub.3 (1.49 g, 10.80 mmol) and dimethyl-1-diazo-2-oxopropylphosphonate (0.98 mL, 6.48 mmol), and the resultant suspension was stirred at RT for 16 h. The solution was diluted with EtOAc, washed with 5% NaHCO.sub.3, H.sub.2O and brine, dried (MgSO.sub.4) and evaporated to give a crude orange oil (0.4 g). This was purified by SiO.sub.2 chromatography (95:5, heptane/EtOAc, as eluent) to give 17 as a colourless oil that slowly crystallised (0.84 g, 73%): .sup.1H NMR (700 MHz, CDCl.sub.3) δ 1.30 & 1.31 (s, 12H), 1.77 (s, 4H), 3.22 (s, 1H), 8.45 (s, 1H); .sup.13C NMR (176 MHz, CDCl.sub.3) δ 29.6, 29.7, 33.8, 34.0, 37.2, 37.3, 79.0, 81.0, 135.4, 144.5, 158.1, 158.6; IR (ATR) ν.sub.max/cm.sup.−1 3279s, 2986w, 2945m, 2917m, 2863w, 2110w, 1519w, 1470m, 1459m,1274m, 1078s, 674s; MS(ES): m/z=215.3 [M+H].sup.+; HRMS (ES) calcd. for C.sub.14H.sub.19N.sub.2 [M+H].sup.+: 215.1548, found 215.1548.

1.2 Synthesis of 3-Fluoro-4-[2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)ethynyl]benzoic acid, DC526

[0118] The synthesis of exemplary compound DC526 is illustrated in FIG. 2a.

[0119] Et.sub.3N (80 mL) was degassed by sparging with N.sub.2 for 1 hour. Compound 1 (0.80 g, 2.55 mmol), compound 6 (0.54 g, 3.05 mmol), Pd(PPh.sub.3).sub.2Cl.sub.2 (179 mg, 0.255 mmol) and CuI (49 mg, 0.255 mmol) were then added under N.sub.2 and the resultant suspension was stirred at room temperature for 72 hours. The suspension was diluted with MTBE and passed through Celite®/SiO.sub.2 and the extracts were evaporated to a give a crude solid (1.1 g). This was purified by dry column vacuum chromatography (100% heptane, to 95:5, heptane/EtOAc) to give compound 7 as a colourless oil which slowly crystallised (0.82 g, 88%), which was carried directly to the next step. Compound 7 (0.80 g, 2.20 mmol) was dissolved in THF (40 mL), 20% NaOH (3 mL) added, and the resultant solution was stirred at reflux for 16 hours. The mixture was cooled, acidified to pH 1 with 5% HCl, extracted with EtOAc, washed with H.sub.2O and brine, dried (MgSO.sub.4) and evaporated to give a crude white solid which was recrystallised from Acetonitrile (MeCN) to give DC526 as a colourless crystalline solid (0.60 g, 77%): .sup.1H NMR (600 MHz, DMSO-d.sub.6) 1.24 & 1.26 (s, 12H), 1.64 (s, 4H), 7.32 (dd, J=8.1, 1.8 Hz, 1H), 7.40 (d, J=8.2 Hz, 1H), 7.53 (d, J=1.8 Hz, 1H), 7.70 7.78 (m, 2H), 7.80 (dd, J=7.9, 1.6 Hz, 1H), 13.44 (br, 1H); .sup.13C NMR (151 MHz, DMSO-d.sub.6) δ 31.3, 31.4, 33.9, 34.1, 34.2, 34.3, 81.0, 97.4 (d, J=3.1 Hz), 115.2, 115.3 (d, J=15.8 Hz), 116.0 (d, J=22.3 Hz), 118.5, 125.4 (d, J=3.4 Hz), 127.1, 128.7, 129.6, 132.7 (d, J=7.1 Hz), 133.6, 145.2, 146.5, 161.4 (d, J=250.3 Hz), 165.7 (d, J=2.5 Hz); .sup.19F NMR (376 MHz, DMSO-d.sub.6) δ −110.0; IR (ATR) ν.sub.max/cm.sup.−1 2967m, 2928m, 2857m, 2210w, 1686s, 1617m, 1566m, 1421m, 1307m, 1218m, 834s, 764m; MS(ASAP): m/z=351.2 [M+H].sup.+; HRMS (ASAP) calcd. for C.sub.23H.sub.24O.sub.2F [M+H].sup.+: 351.1760, found 351.1766.

1.3 Synthesis of 5-[2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)ethynyl]pyridine-2-carboxylic acid, DC528

[0120] The synthesis of exemplary compound DC528 is illustrated in FIG. 2b Et.sub.3N/THF (1:1, 120 mL) was degassed by sparging with N.sub.2 for 1 h. Pd(PPh.sub.3).sub.2Cl.sub.2 (0.265 g, 0.38 mmol), Cul (0.072 g, 0.38 mmol), compound 3 (0.8 g, 4.80 mmol) and compound 8 (0.98 g, 4.52 mmol) were added under N.sub.2 and the resultant solution was stirred at 50° C. for 40 h. The solution was diluted with heptane, and eluted through a Celite®/SiO.sub.2 plug with heptane. The organics were then washed with sat. NH.sub.4Cl, H.sub.2O and brine, dried (MgSO.sub.4) and evaporated to give a crude brown solid (1.9 g). This was purified by dry column vacuum chromatography (100% heptane to 8:2, heptane/EtOAc), to give compound 9 as a white solid (0.36 g, 27%), which was carried directly to the next step: .sup.1H NMR (400 MHz, CDCl.sub.3) δ 1.29 (d, J=7.9 Hz, 12H), 1.69 (s, 4H), 4.02 (s, 3H), 7.31 (d, J=1.1 Hz, 2H), 7.51 (s, 1H), 7.94 (dd, J=8.1, 2.1 Hz, 1H), 8.12 (dd, J=8.1, 0.9 Hz, 1H), 8.85 (dd, J=2.1, 0.9 Hz, 1H). Compound 9 (0.33 g, 0.95 mmol) was dissolved in THF (30 mL), 20% NaOH (3 mL) added, and the resultant solution was stirred at reflux for 16 h. The mixture was cooled, acidified to pH 1 with 5% HCl, extracted with EtOAc, washed with H.sub.2O and brine, dried (MgSO.sub.4) and evaporated to give a crude white solid. This was recrystallised from MeCN to give DC528 as a colourless crystalline solid (0.30 g, 96%): .sup.1H NMR (700 MHz, DMSO-d.sub.6) δ 1.25 & 1.27 (s, 12H), 1.65 (s, 4H), 7.35 (dd, J=8.1, 1.8 Hz, 1H), 7.41 (d, J=8.1 Hz, 1H), 7.57 (d, J=1.8 Hz, 1H), 8.06 (dd, J=8.1, 0.9 Hz, 1H), 8.12 (dd, J=8.1, 2.1 Hz, 1H), 8.85 (dd, J=2.1, 0.9 Hz, 1H), 13.35 (s, 1H); .sup.13C NMR (176 MHz, DMSO-d.sub.6) δ 31.3, 31.4, 33.9, 34.1, 34.2, 34.3, 39.5, 84.8, 95.4, 118.4, 122.7, 124.3, 127.1, 128.7, 129.8, 139.5, 145.2, 146.4, 146.9, 151.3, 165.6; IR (ATR) ν.sub.max/cm.sup.−1 3283br, 2955m, 2920m, 2856m, 2208m, 1752s, 1586m, 1336s, 1247m, 1017m, 833m; MS(ES): m/z=334.2 [M+H].sup.+; HRMS (ES) calcd. for C.sub.22H.sub.24NO.sub.2 [M+H].sup.+: 334.1807, found 334.1808.

1.4 Synthesis of Methyl 4-[2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydroquinoxalin-2-yl)ethynyl]benzoate, DC641

[0121] The synthesis of exemplary compound DC641 is illustrated in FIG. 2c. Et.sub.3N (20 mL) was degassed by sparging with Ar for 1 h. Methyl 4-iodobenzoate (0.31 g, 1.20 mmol), DC640 (0.30 g, 1.40 mmol), Pd(PPh.sub.3).sub.2Cl.sub.2 (83 mg, 0.12 mmol) and CuI (22 mg, 0.12 mmol) were then added under Ar and the resultant suspension was stirred at RT for 16 h. The suspension was diluted with MTBE and passed through Celite®/SiO.sub.2and the extracts were evaporated to a give a crude solid (0.5 g). This was purified by dry column vacuum chromatography (100% heptane to 85:15, heptane/EtOAc) to give an off-white solid which was subsequently recrystallised from MeOH to give DC641 as a colourless crystalline solid (0.29 g, 71%), which was carried directly to the next step: .sup.1H NMR (400 MHz, CDCl.sub.3) δ 1.33 & 1.35 (s, 12H), 1.81 (s, 4H), 3.94 (s, 3H), 7.64 7.71 (m, 2H), 8.01-8.08 (m, 2H), 8.52 (s, 1H).

[0122] 1.5 Synthesis of 4-[2-(5,5,8,8-Tetramethyl-5,6,7,8-tetrahydroquinoxalin-2-yl)ethynyl]benzoic acid, DC645

[0123] The synthesis of exemplary compound DC645 is illustrated in FIG. 2d. DC641 (0.28 g, 0.8 mmol) was dissolved in THF (20 mL), 20% NaOH (2mL) added, and the resultant solution was stirred at reflux for 16 h. The mixture was cooled, acidified to pH 1 with 5% HCl, extracted with EtOAc, washed with H.sub.2O and brine, dried (MgSO.sub.4) and evaporated to give a crude white solid which was recrystallised from MeCN to give DC645 as a colourless crystalline solid (0.24 g, 89%): .sup.1H NMR (700 MHz, DMSO-d.sub.6) δ 1.29 (s, 12H), 1.78 (s, 4H), 7.73 7.79 (m, 2H), 7.98 8.02 (m, 2H), 8.68 (s, 1H), 13.24 (br, 1H); .sup.13C NMR (176 MHz, DMSO-d.sub.6) δ 29.4, 29.4, 33.1, 33.2, 37.0, 37.1, 88.9, 89.8, 125.2, 129.6, 131.3, 131.9, 135.2, 144.4, 157.6, 158.0, 166.5; IR (ATR) ν.sub.max/cm.sup.−1 2961w, 2925w, 2958w, 2223w, 1683s, 1606m, 1558w, 1428m, 1282s, 862s, 769m; MS(ASAP): m/z=334.2 [M].sup.+; HRMS (ASAP) calcd. for C.sub.21H.sub.22N.sub.2O.sub.2 [M].sup.+: 334.1681, found 334.1686.

EXAMPLE 2: BIOLOGICAL EVALUATION

2.1 Genomic Activity of Synthetic Retinoids

[0124] The genomic activity of synthetic retinoids was evaluated by determining their efficiency of inducing transcription using the X-Gal Assay. The X-Gal Assay utilizes Sil-15 reporter cells in which the transcription of the LacZ gene is under control of a promoter linked to a retinoic acid response element (RARE). After treatment of the reporter cells with retinoids, the ability of the compounds to induce transcription can be attained by monitoring and quantifying the activity of β-galactosidase produced by the reporter cells.

[0125] Sil-15 cells were plated at 100,000 cells per well in 96-well plates coated in 0.1% gelatin. The following day, serial dilutions of retinoid ligands, prepared in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% foetal calf serum (FCS) were added at concentrations from 10.sup.−6 M to 10.sup.−14 M and the plates were incubated overnight. All concentrations for the ATRA standard curve and the other retinoid ligands were tested in triplicate.

[0126] The next day, the assay plates were washed twice with phosphate buffered saline (PBS), fixed with 100 μl per well of 1% glutaraldehyde and 1 mM MgCl.sub.2 in PBS for 15 minutes and washed twice with PBS. β-galactosidase activity was detected by the addition of 100 μl of freshly-prepared 0.2% X-Gal in 1 mM MgCl.sub.2, 3.3 mM potassium ferricyanide and 3.3 mM potassium ferrocyanide in PBS per well. Plates were incubated for 6 hours at 37° C. in 5% CO.sub.2 and colour change was detected by reading the plates at 650 nm on an Emax Precision Microplate Reader (Molecular Devices).

[0127] The resulting data, as shown in Table 1 and illustrated in FIG. 3, demonstrates that several of the synthetic retinoids displayed greater genomic activity than that of the endogenous ligand (ATRA) as demonstrated by increased ability to induce transcription in the reporter cells. Notably, DC527, DC540, DC525, DC528, DC526 and DC645 all demonstrated increased genomic activity and potency when compared to retinoic acid (ATRA).

2.2 Non-Genomic Activity of Synthetic Retinoids

[0128] The non-genomic activity of synthetic retinoids was evaluated by measuring their ability to induce phosphorylation of ERK1/2 using the AlphaLISA® SureFire® Ultra ERK1/2 kit.

[0129] In this assay, SH-SY5Y cells (100,000 cells/well) were plated in 96-well plates and serum-starved in DMEM for 24 hours. Retinoids were tested at concentrations from 10-5 M to 10-11 M and at a final concentration of 0.1% DMSO in the medium. The assay was carried out on the SH-SY5Y cells in serum-free DMEM, cells were stimulated by retinoid for 30 minutes at 37° C.

[0130] At the end of the assay, the medium was removed, and cells were lysed in 50 μl of freshly prepared 1× lysis buffer (supplied in the kit). The 96-well plate was agitated on an orbital shaker (SO1, Stuart Scientific) at approximately 350 rpm for 10 minutes at room temperature.

[0131] In the meantime, the activation buffer was diluted 25-fold in the reaction buffers. Under green light in a dark room, the acceptor beads were diluted 50-fold in the freshly prepared reaction mix while the donor beads were diluted 50-fold in dilution buffer to obtain two final reaction mixtures.

[0132] 10 μl of cell lysate was then transferred per well into 384-well white Proxiplates plates (PerkinElmer) and 5 μl of each prepared acceptor and donor reaction mixture was added under green light in a dark room. Plates were wrapped with aluminium foil and incubated at room temperature for at least 3 hours. Readings were taken using an Envision system (PerkinElmer Life Sciences) using the AlphaScreen® settings.

[0133] The results, shown in Table 1 and illustrated in FIG. 4 demonstrate the ability of retinoids to induce Erk 1/2 phosphorylation in SH-SY5Y cells. Several retinoids were identified having both higher potency and efficacy than retinoic acid such as DC527, DC540, DC528, DC526 and DC645, with DC525 identified as having higher potency.

2.3 Induction of Neurite Outgrowth

[0134] SY-SY5Y cells were plated at 10,000 cells/well in 12-well plates containing acid-treated/poly-L-lysine-coated coverslips. After 24 hours, each retinoid was added to the medium at a concentration of 10 nM and the plates were incubated for 5 days. After retinoid treatment, SH-SY5Y cells on coverslips were washed twice in PBS, fixed in 4% paraformaldehyde (PFA) for 20 minutes at room temperature, and washed twice with PBS.

[0135] For immunocytochemical staining of neurites, cells on coverslips were washed three times in PBS, and incubated in blocking solution (10% donkey serum and 0.1% triton X-100 in PBS) for 1 hour at room temperature. Cells were then labelled by incubation overnight at 4° C. with β-III tubulin primary antibody (Sigma-Aldrich) diluted 1:1000 in blocking buffer. Following incubation overnight, cells were washed three times with PBS containing 0.1% Triton X-100 solution (PBST) before incubation with anti-mouse monoclonal secondary antibody (1:300 in PBST; Jackson Immunoresearch) for 2 hours at room temperature. Finally, after three washes in PBST and a final wash in PBS, the coverslips were mounted on slides and stored at 4° C.

[0136] ImageJ software with the NeuronJ plugin was used to quantify neurite outgrowth on stained cells. For each experiment, 10 different randomly selected images were taken from each cover slip using a Nikon Eclipse E400 fluorescence microscope. Each image was converted into an 8-bit image (necessary for the NeuronJ plugin) and optimised with the brightness and contrast tool in GIMP (GNU Image Manipulation Program). For each image, individual traces were drawn for each clearly-identifiable neurite using the tracing tool in the NeuronJ plugin. Neurite length was measured in pixels and transformed into the corresponding length in μm depending on the magnification used. The average neurite length for each image was calculated by dividing total neurite length by the total number of neurites per image. Ten images per cover slip were measured and the mean calculated for the coverslip overall. Coverslips were in triplicate for each retinoid and concentration.

[0137] Data shown in FIG. 5 demonstrates that several of the retinoids showed the ability to induce neurite outgrowth in SY-SY5Y cells. Furthermore, cells treated with several of these compounds (DC527, DC525, DC528, DC526 and DC645) showed a significantly larger average neurite length than cells treated with ATRA.

[0138] Table 1 below summarises the genomic (transcriptional activity) and non-genomic activity (p-ERK ½ activity) for the retinoid compounds tested, along with increase in neurite outgrowth, and these results were plotted in FIG. 5. From this figure it is evident that the compounds which showed high efficacy in both the genomic and non-genomic assays demonstrated increased neurite outgrowth induction. This indicates that “dual-potent” compounds, i.e. those which induce both genomic and non-genomic activities, are likely to prove more efficacious in clinical use, allowing this dual assay method to act as a potentially powerful screening tool for new therapeutic compounds.

TABLE-US-00001 TABLE 1 Genomic activity, non-genomic activity and neurite outgrowth Genomic Activity (X-Gal Non-genomic activity (ERK½ Neurite RA Based Reporter Assay) Phosphorylation Assay) Outgrowth Potency (EC.sub.50) nM Efficacy (E.sub.max) Potency (EC.sub.50) nM Efficacy (E.sub.max) Fold increase Compound (95% Cl) (95% Cl) (95% Cl) (95% Cl) (10 nM) ATRA  1.044 170.1  33.07 48.55 1.4 (83.96 to 1.291) (164.4 to 175.7) (23.76 to 44.85) (46.72 to 50.42) EC23  0.1279 169.18  31.75 91.93 2.2 (0.08006 to 0.2033) (159.1 to 180.7) (20.21 to 49.83) (86.3 to 100.29) DC527  0.003475 209.8  30.55 92.51 1.9 (0.002472 to 0.004869) (201.4 to 218.3) (19.47 to 48.53) (85.7 to 99.62) DC540  0.01504 185.3 159.4 79.73 1.7 (0.01203 to 0.01888) (180.4 to 190.2) (117.7 to 215.5) (75.14 to 84.46) DC525  0.2103 190  2.491 42.44 1.9 (0.1505 to 0.2952) (181.9 to 198.3) (1.966 to 3.152) (40.92 to 43.98) DC528  0.146 206.5  14.22 69.15 2.2 (0.06294 to 0.3296) (187.3 to 226.2) (9.415 to 21.28) (65.02 to 73.35) DC526  0.05807 149.1  26.35 50.22 2.1 (0.02068 to 0.1292) (134.2 to 164) (19.51 to 35.49) (47.4 to 53.09) DC564  5.31 177.1  18.07 44.46 1.3 (3.966 to 7.03) (168.1 to 186.3) (9.765 to 35.5) (40.82 to 48.35) DC559  1.379 139.8  15.66 40.68 1.4 (81.11 to 2.297) (127.8 to 151.9) (12.25 to 20.1) (39.24 to 42.15) DC567  13.57  97.21  18.07 44.46 1.6 (8.721 to 21.17) (89.5 to 105.3) (9.765 to 35.5) (40.82 to 48.35) DC712  0.3818 179.7  6.349 94 1.2 (0.3011 to 0.4824) (173.1 to 186.3) (46.62 to 84.46) (89.63 to 98.43) DC657  0.1463 154.9  3.496 72.45 1.4 (0.08618 to 0.2479) (144.4 to 165.5) (2.147 to 5.656) (68.39 to 76.62) DC667  0.1336 169.8  4.849 44.3 1.4 (0.09097 to 0.1948) (160.5 to 179.2) (2.969 to 7.672) (41.6 to 47.05) DC707  0.8256 192.2  4.323 44.87 1.2 (0.5211 to 1.266) (179.6 to 205) (2.302 to 7.727) (41.83 to 47.98) DC645  0.06965 185.9  8.538 91.57 2.3 (0.05443 to 0.08835) (180.2 to 191.6) (6.708 to 10.75) (88.37 to 94.79) DC529  3.891 174  30.37 28.85 1.3 (2.935 to 5.117) (165.8 to 182.3) (21.26 to 43.1) (27.3 to 30.44) DC706 603.2 265.9  90.8 36.87 1.3 (409.4 to 1022) (228.8 to 339.2) (58.85 to 138.4) (34.15 to 39.68) DC661  0.2701 171.4  2.004 53.91 1.6 (0.2064 to 0.3532) (165.3 to 177.5) (1.39 to 2.93) (51.5 to 56.36) DC646  3.159 176.3 143.5 77.63 1.7 (2.385 to 4.169) (167.8 to 184.9) (117.8 to 174.9) (74.75 to 80.58) DC656  0.4274 177.2 501.6 56.77 1.6 (0.335 to 0.5427) (170.6 to 183.9) (260.3 to 854.2) (50.45 to 63.47)

2.4 Regulation of Alzheimer's Disease Related Genes

[0139] To examine the influence retinoids may have on the expression of genes involved in Alzheimer's disease, the expression of a group of Alzheimer's-related genes in rat mixed primary neuronal/glial cultures was assessed following retinoid treatment.

[0140] Approximately 300,000 rat neuronal/glial cells were treated first with 1 μg/ml lipopolysaccharide (Sigma-Aldrich) for 6 hours to induce inflammation. Following the induction of inflammation, cells were treated with 10 nM retinoids for 24 hours. The retinoids which proved most potent in previous assays were selected for use in these experiments. Following treatment, RNA was extracted from the treated cells for qPCR analysis.

[0141] Total RNA was extracted using a Qiagen RNeasy mini kit (CAT # 74104, Qiagen) according to the manufacturer's protocol. Briefly, cell and brain tissue samples were homogenized in 350 μl and 1200 μl of RLT buffer mixed with β-ME (1 ml buffer: 10 μl β-ME ratio) respectively. Then, samples were centrifuged for 3 min at 13,000 rpm. The supernatant was first mixed with 70% ethanol at a 1:1 volume ratio (CAT # E7023, Sigma-Aldrich) before transferring it to a spin column placed in a collection tube. The samples were centrifuged for 1 min at 10,000 rpm to bind RNA to the membrane of the spin column.

[0142] For the on-column DNase digestion, the samples in the columns were washed with 350 μl RW1 buffer and centrifuged at 10,000 rpm for 1 min. Then, 80 μl of the DNase mixture (10 μl DNase enzyme with 70 μl RDD buffer; CAT # 79254, Qiagen) was added above each sample. Samples were incubated at room temperature for 15 min before adding 350 μl RW1 buffer. Samples were centrifuged at 10,000 rpm for 1 min.

[0143] Afterwards, 500 μl of RPE buffer (mixed with 100% ethanol at 1:4 ratio) was added above the samples. Samples were centrifuged for 1 min at 10,000 rpm (the step was repeated 2 times). The spin columns were centrifuged again at 13,000 rpm for 1 min to dry the membranes. The spin columns were removed from the collection tubes and placed inside another 1.5 ml RNase free collection tubes. The RNA was eluted by adding 30 μl of RNase free water directly to the spin column membrane. The samples were incubated 5 min at room temperature before spinning them for 1 min at 10,000 rpm.

[0144] RNA concentration was measured using a NanoDrop™ 2000c spectrophotometer (Thermo Fisher Scientific). The instrument was calibrated using the RNase free water used to elute the RNA as a blank and 2 μl was used of each RNA sample. RNA samples were stored in a −70° C. freezer to minimize RNA degradation.

[0145] qPCR reactions were performed using PerfeCTa SYBR Green SuperMix (CAT # 733-1246, VWR). 10 μl of reaction mix was added to each well of a 384-well plate (CAT # 04729749001, Roche) in triplicate. Each reaction contained 2 μl of 4 times diluted cDNA template, 5 μl 2×SYBR green mix and 250 nM primers.

[0146] Primers were designed using Primer-BLAST with melting temperatures of about 60° C. Before using them in qPCR, the primers were checked for specificity by PCR and the PCR products were sent for sequencing.

[0147] Standard curves (made using 5-fold dilutions of the stock cDNA) and blank controls were run for all sets of primers tested in qPCR. The plates were then sealed and centrifuged briefly to ensure all the reagents were at the base of the wells. The plates were run on a Roche LightCycler 480 that was programmed to hold the plate at 95° C. for 5 min. The qPCR then ran for 45 cycles of 95° C. for 15 seconds (s), 60° C. for 15 s and 72° C. for 15 s. Afterwards, the melting curve was obtained by running the plate at 95° C. for 5 s followed by 58° C. for 1 min.

[0148] Results were analysed using the delta delta CT method within the LightCycler 480 1.5 software. The expression of genes of interest was normalized to the appropriate reference gene according to each experiment.

[0149] The data shown in FIG. 7 demonstrates that DC645, DC528 and DC526 downregulated inflammation genes (Ccl5, TNFα and Nos2) and upregulated neuroprotection (Abca1, Abcg1, Igf1 and Igf2) and non-amyloidogenic pathway genes (Nep, Ide and Adam10) taking amyloid precursor down the pathway that does not generate toxic amyloid fragments.

EXAMPLE 3: MDCK-MDR1 PERMEABILITY ASSAY

[0150] This assay is used to measure the permeability of test compound in the apical to basolateral (A-B) and basolateral to apical (B-A) direction across MDCK-MDR1 cells and to determine the efflux ratio (ER), which shows whether the compound undergoes active efflux. Thus, this assay is a valuable in vitro surrogate for blood-brain permeability and CNS exposure

3.1 Experimental Procedure

[0151] MDCK-MDR1 cells obtained from the NIH (Rockville, Md., USA) are used between passage numbers 6-30. Cells are seeded onto Millipore Multiscreen Transwell plates at 3.4×10.sup.5cells/cm.sup.2. The cells are cultured in DMEM and media is changed on day 3. On day 4 the permeability study is performed. Cell culture and assay incubations are carried out at 37° C. in an atmosphere of 5% CO.sub.2 with a relative humidity of 95%. On the day of the assay, the monolayers are prepared by rinsing both apical and basolateral surfaces twice with Hanks Balanced Salt Solution (HBSS) at the desired pH warmed to 37° C. Cells are then incubated with HBSS at the desired pH in both apical and basolateral compartments for 40 min to stabilise physiological parameters. The dosing solutions are prepared by diluting test compound with assay buffer to give a final test compound concentration of 10 μM. Analytical standards are prepared from test compound DMSO dilutions and transferred to buffer, maintaining a 1% v/v DMSO concentration; buffer is composed of supplemented HBSS pH 7.4. For assessment of A-B permeability, HBSS is removed from the apical compartment and replaced with test compound dosing solution. The apical compartment insert is then placed into a companion plate containing fresh buffer (containing 1% v/v DMSO). For assessment of B-A permeability, HBSS is removed from the companion plate and replaced with test compound dosing solution. Fresh buffer (containing 1% v/v DMSO) is added to the apical compartment insert, which is then placed into the companion plate. At 60 min the apical compartment inserts and the companion plates are separated and apical and basolateral samples diluted for analysis. Test compound permeability is assessed in duplicate. Compounds of known permeability characteristics are run as controls on each assay plate. Test and control compounds are quantified by LC-MS/MS cassette analysis using a 7-point calibration with appropriate dilution of the samples. The starting concentration (C.sub.0) is determined from the dosing solution and the experimental recovery calculated from C.sub.0 and both apical and basolateral compartment concentrations.

3.2 Data Analysis

[0152] The permeability coefficient (P.sub.app) for each compound is calculated from the following equation:

[00001]$P_{app}=\left(\frac{dQ/dt}{C_0\times A}\right)$

[0153] Where dQ/dt is the rate of permeation of the drug across the cells, C.sub.0 is the donor compartment concentration at time zero and A is the area of the cell monolayer. C.sub.0 is obtained from analysis of the dosing solution. An efflux ratio (ER) is calculated from mean A-B and B-A data. This is derived from:

[00002]$\mathrm{ER}=\frac{P_{app\left(B-A\right)}}{P_{app\left(A-B\right)}}$

3.3. Biological Study

[0154] The membrane permeability of a number of compounds of Formula I were measured in the MDCK-MDR1 assay described previously. The permeability (P.sub.app, nm/s) and efflux ratio (ER) for compounds was determined and compared with those for the closely-related reference compound EC23. The results are summarised in Table 2:

TABLE-US-00002 TABLE 2 Permeability and efflux ratio of compounds of formula I, versus reference compound EC23. Papp Compound/Structure (nm/s) ER EC23 [00020]   0 — DC526 [00021]   0 — DC528 [00022]   0 — DC645 [00023] 349 0.6

[0155] The results shown in Table 2 demonstrate that some compounds of Formula (I) can show considerable improvements in permeability and lack of efflux in the MDCK-MDR1 assay and, thus, can be expected to show considerable improvements in blood brain barrier penetration and CNS exposure. As treatment of CNS disorders, including ALS and Alzheimer's disease, requires exposure of CNS targets to sufficiently-high concentrations of therapeutic agent, it is clear that optimal CNS exposure is an essential characteristic in a drug for the treatment of these disorders. Thus, investigated compounds of Formula (I) show significantly improved drug-like characteristics compared to the literature compound EC23. This improvement in CNS exposure represents a surprising and unexpected discovery.

EXAMPLE 4: TURBIDIMETRIC AQUEOUS SOLUBILITY

Example 4.1 Experimental Procedure

[0156] Test compound (10 mM in DMSO) is serially diluted to give solutions of 0.1, 0.3, 1 and 3 mM in DMSO. Each test compound concentration is then further diluted 1 in 100 in buffer (0.01 M phosphate buffered saline pH 7.4) so that the final DMSO concentration was 1% and the final test compound concentrations were 1, 3, 10, 30 and 100 μM. The experiment was performed at 37° C. and each concentration sample was incubated in 7 replicate wells. The plates were incubated for 2 hr at 37° C. before the absorbance was measured at 620 nm. The solubility of the sample was estimated from the concentration of test compound that produced an increase in absorbance above vehicle control (i.e., 1% DMSO in buffer).

4.2 Biological Study

[0157] The solubility of a number of compounds of the Formula (I) were measured in the turbidimetric aqueous solubility assay described previously. The solubility for compounds was determined and compared with those for the closely-related reference compound EC23. The results are summarised in Table 3:

TABLE-US-00003 TABLE 3 Solubility of compounds of formula I, versus reference compound EC23. Compound/Structure μM EC23 [00024]    6.5 DC526 [00025]    20 DC528 [00026]    20 DC645 [00027] >100

[0158] The results shown in the above table demonstrate that compounds of Formula I can show considerable improvements in solubility and, thus, can also be expected to show considerable improvements in a number of biological properties, including absorption and target exposure. Again, this represents a surprising and unexpected discovery.

[0159] It will be understood that where, throughout the description, compounds are described for use in the treatment of a condition or disease which is alleviated by the activation of retinoic acid receptors (RAR), that this represents a disclosure of these compounds per se.

[0160] All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. Each feature disclosed in this specification (including any accompanying claims, abstract and drawings) may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features. The invention is not restricted to the details of the foregoing embodiment(s). The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0161] With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity.

[0162] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations.

[0163] However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and/or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations).

[0164] It will be appreciated that various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modifications may be made without departing from the scope of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, with the true scope being defined by the following claims.