Fluorescent synthetic retinoids

Abstract

There are described novel compounds of formula I: which R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9 and R.sup.10 are each as herein defined. ##STR00001##

Claims

1. A compound of formula I: ##STR00027## in which R.sup.1 is hydrogen, alkyl C1-10 or acyl; R.sup.2, R.sup.3, R.sup.4 and R.sup.5, which may be the same or different, are each hydrogen or alkyl C1-4, or together one pair of R.sup.2 and R.sup.4 or R.sup.3 and R.sup.5 represent a bond; R.sup.6 and R.sup.7, which may be the same or different, are each hydrogen, alkyl C1-4 or together one pair of R.sup.4 and R.sup.6 or R.sup.5 and R.sup.7 represent a bond, or R.sup.6 and R.sup.7 together form a group: CR.sup.11R.sup.12; provided that the pair of R.sup.4 and R.sup.6 or R.sup.5 and R.sup.7 does not represent a bond if a pair from R.sup.2, R.sup.3, R.sup.4 and R.sup.5 represents a bond; R.sup.8 and R.sup.9, which may be the same or different, are each hydrogen, alkyl C1-10, aryl, aralkyl, halogen, trifluoroalkyl, cyano, nitro, NR.sup.aR.sup.b, OR.sup.a, C(O)R.sup.a, C(O)OR.sup.a, OC(O)R.sup.a, S(O)R.sup.aR.sup.b, and C(O)NR.sup.aR.sup.b; R.sup.11 and R.sup.12, which may be the same or different, are each hydrogen or alkyl C1-10; and R.sup.a and R.sup.b, which may be the same or different, are each hydrogen or alkyl C1-10; R.sup.10 is a group II, III, IV, V, VI, VII, VIII, IX, or XI: ##STR00028## ##STR00029## in which R.sup.13 is hydrogen or alkyl C1-10; with the proviso that when R.sup.10 is a group II or a group V, one pair of R.sup.2 and R.sup.4, R.sup.3 and R.sup.5, R.sup.4 and R.sup.6 or R.sup.5 and R.sup.7 represent a bond; and isomers thereof; in free or in salt form.

2. The compound according to claim 1 in which R.sup.10 is a group II, as defined in claim 1.

3. The compound according to claim 1 in which R.sup.10 is a group III, as defined in claim 1.

4. The compound according to claim 1 in which R.sup.10 is a group IV, as defined in claim 1.

5. The compound according to claim 1 in which R.sup.10 is a group V, as defined in claim 1.

6. The compound according to claim 1 in which R.sup.10 is a group VI, as defined in claim 1.

7. The compound according to claim 1 in which R.sup.10 is a group VII, as defined in claim 1.

8. The compound according to claim 1 in which R.sup.10 is a group VIII, as defined in claim 1.

9. The compound according to claim 1 in which R.sup.10 is a group IX, as defined in claim 1.

10. The compound according to claim 1 in which R.sup.10 is a group XI, as defined in claim 1.

11. The compound according to claim 1 in which the moiety CO.sub.2R.sup.13 is positioned in a para relationship.

12. The compound according to claim 1 in which the moiety CO.sub.2R.sup.13 is positioned in a meta relationship.

13. The compound according to claim 1, which is selected from the group consisting of: 6-(1,4,4-trimethyl-1,2,3,4-tetrahydroquinolin-6-yl)-naphthalene-2-carboxylic acid methyl ester (11); 3-[4-(1,4,4-trimethyl-1,2,3,4-tetrahydroquinolin-6-yl)-phenyl]-acrylic acid methyl ester (13); and 4-2-[2,4,4-trimethyl-1-(propan-2-yl)-1,4-dihydroquinolin-6-yl]ethynylbenzoic acid, (17); and isomers thereof; in free or in salt form.

Description

(1) The present invention will now be described by way of example only with reference to the accompanying figures in which:

(2) FIG. 1 illustrates normalised excitation spectra of EC23 in a range of solvents;

(3) FIG. 2 illustrates normalised emission spectra of EC23 in a range of solvents, with excitation at 300 nm;

(4) FIG. 3 illustrates normalised excitation spectra of compound 9 of Example 3 in a range of solvents;

(5) FIG. 4 illustrates normalised emission spectra of compound 9 of Example 3 in a range of solvents, with excitation in the range of 275-300 nm;

(6) FIG. 5 illustrates the normalised excitation spectrum of compound 17 of Example 6 in chloroform;

(7) FIG. 6 illustrates the normalised emission spectrum of compound 17 of Example 6 in chloroform, with excitation at 378 nm;

(8) FIG. 7 illustrates a .sup.1H NMR spectrum of compound 9 of Example 3 after storage at ambient temperature in the absence of light;

(9) FIG. 8 illustrates a .sup.1H NMR spectrum of compound 9 of Example 3 after treatment with typical laboratory light for 72 hours at ambient temperature;

(10) FIG. 9 illustrates compound 9 of Example 3 activity in stem cells compared to ATRA, EC23 and DMSO-Nestin staining;

(11) FIG. 10 illustrates compound 9 of Example 3 activity in stem cells compared to ATRA, EC23 and DMSO-CK8 staining;

(12) FIG. 11 illustrates compound 9 of Example 3 activity in stem cells compared to ATRA, EC23 and DMSO-TUJ-1 staining;

(13) FIG. 12 illustrates compound 9 of Example 3 activity in stem cells compared to ATRA, EC23 and DMSO-Oct 4 staining;

(14) FIG. 13 illustrates compound 9 of Example 3 activity in stem cells compared to ATRA, EC23 and DMSO-Sox 2 staining;

(15) FIG. 14 illustrates flow cytometry evaluation of compound 9 of Example 3 compared to ATRA, EC23 and DMSO, the expression of stem cell marker SSEA-3 is measured;

(16) FIG. 15 illustrates flow cytometry evaluation of compound 9 of Example 3 compared to ATRA, EC23 and DMSO, the expression of stem cell marker TRA160 is measured;

(17) FIG. 16 illustrates flow cytometry evaluation of compound 9 of Example 3 compared to ATRA, EC23 and DMSO, the expression of stem cell marker A2B5 is measured;

(18) FIG. 17 illustrates phase contrast images of cell populations treated with compound 9 of Example 3, ATRA, EC23 and DMSO;

(19) FIG. 18 illustrates MTT cell viability analysis of compound 9 of Example 3 with comparison to ATRA, EC23 and DMSO at a treatment concentration of 1 M;

(20) FIG. 19 illustrates MTT cell viability analysis of compound 9 of Example 3 with comparison to ATRA, EC23 and DMSO at a treatment concentration of 10 M;

(21) FIG. 20 illustrates TERA-2 stem cells treated with compound 9 over a range of concentrations, imaged using confocal microscopy after 7 days;

(22) FIG. 21 illustrates SHSY5Y cells (neuroblastoma) treated with compound 9 of Example 3 (10 M), and imaged using a confocal microscope after 8 hours;

(23) FIG. 22 Fibroblast cells treated with compound 9 of Example 3 (10 M), and imaged using a confocal microscope after 24 hours;

(24) FIG. 23 illustrates TERA-2 stem cells treated with compound 9 of Example 3 (10 M) for 7 days, fixed with 4% paraformaldehyde, and imaged using a confocal microscope;

(25) FIG. 24 illustrates HaCat keratinocyte skin cells treated with compound 9 of Example 3 (10 M) for 5 days;

(26) FIG. 25 illustrates HaCat keratinocyte skin cells treated with compound 9 of Example 3 (10 M) for 5 days, and stained with Involucrin and K14;

(27) FIG. 26 illustrates HaCat keratinocyte skin cells treated with compound 17 of Example 6 (10 M) for 5 days;

(28) FIG. 27 illustrates HaCat keratinocyte skin cells treated with compound 17 of Example 6 (10 M) for 5 days, and stained with Involucrin and K14; and

(29) FIG. 28 illustrates the Raman spectrum of compound 9 of Example 3. A high intensity acetylene band is observed at 2190 cm.sup.1, this lies in the cellular silent region (1800-2800 cm.sup.1), wherein signals of biological origin, such as amide bonds, are not observed.

(30) In the figures, any reference to DC271 is a reference to compound 9 of Example 3.

(31) The following abbreviations are used in the Examples and other parts of the description:

(32) ATRA: All Trans-Retinoic Acid

(33) B.sub.2pin.sub.2: bis(pinacolato)diboron

(34) DCM: dichloromethane

(35) DMF: N,N-dimethylformamide

(36) DMSO: dimethylsulfoxide

(37) dppf: 1,1-ferrocenediyl-bis(diphenylphosphine)

(38) EDTA: ethylenediaminetetraacetic acid

(39) EtOAc: ethyl acetate

(40) GCMS: gas chromatography-mass spectrometry

(41) h: hour(s)

(42) KOAc: potassium acetate

(43) RT: room temperature

(44) THF: tetrahydrofuran

GENERAL EXPERIMENTAL

(45) Reagents were purchased from Sigma-Aldrich, Acros Organics, Alfa-Aesar and Fluorochem and used without further purification unless otherwise stated. Solvents were used as supplied, and dried before use with appropriate drying agents if stated. Reactions were monitored in situ by TLC, or NMR spectroscopy. Thin layer chromatography (TLC) was conducted using Merck Millipore silica gel 60G F254 25 glassplates with visualisation by UV lamp. Flash column chromatography was performed using SiO.sub.2 from Sigma-Aldrich (230-400 mesh, 40-63 am, 60 ) and monitored using TLC. NMR spectra were recorded on Varian VNMRS-700, Varian VNMRS-600, Bruker Avance-400 or Varian Mercury-400 spectrometers operating at ambient probe temperature unless otherwise stated. NMR spectra were recorded in CDCl.sub.3 or DMSO-d.sub.6 purchased from Goss Scientific. NMR peaks are reported as singlet (s), doublet (d), triplet (t), quartet (q), broad (br), heptet (hept), combinations thereof, or as a multiplet (m). ES-MS was performed by the Durham University departmental service using a TQD (Waters UK) mass spectrometer and Acquity UPLC (Waters Ltd, UK), and accurate mass measurements were obtained using a QTOF Premier mass spectrometer and an Acquity UPLC (Waters Ltd, UK). GCMS was performed by the Durham University departmental service using a Shimadzu QP2010-Ultra. IR spectra were recorded on a Perkin Elmer FT-IR spectrometer. Melting points were obtained using a Gallenkamp melting point apparatus. Elemental analyses were obtained by the Durham University departmental service using an Exeter Analytical CE-440 analyzer.

Synthetic Procedures

Example 1

6-Iodo-4,4-dimethyl-1-(propan-2-yl)-1,2,3,4-tetrahydroquinoline (4)

(46) ##STR00015##

1(a) N-(4-iodophenyl)-3-methylbut-2-enamide, (1)

(47) To a solution of 4-iodoaniline, (25.0 g, 114.0 mmol) in DCM (400 mL) was added 3,3-dimethylacroloyl chloride (13.36 mL, 120.0 mmol) and the resultant white suspension was stirred for 0.5 h, after which pyridine (9.70 mL, 120 mmol) was added and the solution stirred at RT for 16 h. The solution was quenched with H.sub.2O, diluted with DCM, washed with sat. NH.sub.4Cl, H.sub.2O and brine, dried (MgSO.sub.4) and evaporated to give a crude light brown solid (33 g). This was recrystallised from EtOH to give 1 as a white crystalline solid (31.8 g, 93%): .sup.1H NMR (700 MHz, CDCl.sub.3) 1.91 (s, 3H), 2.22 (s, 3H), 5.68 (s, 1H), 7.01 (s, 1H), 7.33 (m, 2H), 7.60 (d, J=8.8 Hz, 2H); .sup.13C NMR (101 MHz, CDCl.sub.3) 20.2, 27.7, 87.2, 118.5, 121.8, 138.0, 138.2, 154.5, 165.2; IR (neat) .sub.max/cm.sup.1 3294m, 3094, 2964w, 2890w, 1666m, 1586m, 1430m, 821s, 650m; MS (ES): m/z=302.0 [M+H].sup.+; HRMS (ES) calcd. for C.sub.11H.sub.13NOI [M+H].sup.+: 302.0042. found: 302.0050. Found: C, 43.87; H, 4.02; N 4.64. Calc. for C.sub.11H.sub.12NOI: C, 43.88; H, 4.02; N 4.65%; m.p.=136-138 C.

1(b) 6-Iodo-4,4-dimethyl-1,2,3,4-tetrahydroquinolin-2-one, (2)

(48) AlCl.sub.3 (7.66 g, 57.5 mmol) was added to anhydrous DCM (150 mL) and the resultant slurry stirred for 0.5 h. To this was added 1 (11.5 g, 38.3 mmol) and the solution stirred vigorously for 2.5 h at RT. The reaction was quenched slowly with H.sub.2O, diluted with DCM, washed with 5% NaOH until the solution turned off-white, then further washed with H.sub.2O and brine, dried (MgSO.sub.4) and evaporated to give a crude yellow solid. This was recrystallised from EtOH to give 2 as a white crystalline solid (10.2 g, 88%): .sup.1H NMR (700 MHz, CDCl.sub.3) 1.32 (s, 6H), 2.47 (s, 2H), 6.62 (d, J=8.3 Hz, 1H), 7.47 (dd, J=8.3, 1.9 Hz, 1H), 7.56 (d, J=1.8 Hz, 1H), 9.20 (s, 1H); .sup.13C NMR (176 MHz, CDCl.sub.3) 27.7, 34.2, 45.2, 86.8, 118.1, 133.7, 135.1, 135.9, 136.6, 171.3; IR (neat) .sub.max/cm.sup.1 3164m, 3102, 3040w, 2953m, 1671s, 1596m, 1484m, 817s; MS (ES): m/z=302.0 [M+H].sup.+; HRMS (ES) calcd. for C.sub.11H.sub.13NOI [M+H].sup.+: 302.0042. found: 302.0042. Found: C, 43.91; H, 4.02; N 4.63. Calc. for C.sub.11H.sub.12INO: C, 43.87; H, 4.02; N 4.65%; m.p.=199-202 C.

1(c) 6-Iodo-4,4-dimethyl-1-(propan-2-yl)-1,2,3,4-tetrahydroquinolin-2-one, (3)

(49) To a solution of 2 (7.05 g, 23.4 mmol) in anhydrous DMF (200 mL) was added crushed KOH (4.08 g, 70.2 mmol) and the resultant slurry stirred for 1 h at 50 C. To this was added 2-iodopropane (7.00 mL, 70.2 mmol) and the solution stirred at 50 C. for 40 h. The reaction was quenched with H.sub.2O, diluted with EtOAc, washed with sat. NH.sub.4Cl, H.sub.2O and brine, dried (MgSO.sub.4) and evaporated to give a crude clear oil (7.2 g). This was purified by SiO.sub.2 chromatography (hexane:EtOAc, 9:1, with 1% Et.sub.3N, as eluent) to give 3 as a colourless oil (3.93 g, 49%): .sup.1H NMR (700 MHz, CDCl.sub.3) 1.25 (s, 6H), 1.49 (s, 3H), 1.50 (s, 3H), 2.38 (s, 2H), 4.66 (hept, J=7.0 Hz, 1H), 6.87 (d, J=8.6 Hz, 1H), 7.50 (dd, J=8.6, 2.1 Hz, 1H), 7.52 (d, J=2.1 Hz, 1H); .sup.13C NMR (176 MHz, CDCl.sub.3) 20.3, 26.8, 33.1, 47.2, 48.8, 86.9, 118.9, 133.4, 135.7, 138.9, 139.1, 169.5; IR (neat) .sub.max/cm.sup.1 2961m, 2934w, 2870w, 1667s, 1582m, 1482m, 809s; MS (ES): m/z=344.0 [M+H].sup.+; HRMS (ES) calcd. for C.sub.11H.sub.13NOI [M+H].sup.+: 344.0511. found: 344.0512.

1(d) 6-Iodo-4,4-dimethyl-1-(propan-2-yl)-1,2,3,4-tetrahydroquinoline, (4)

(50) To a solution of 3 (1.25 g, 3.63 mmol) in anhydrous toluene (15 mL) at 0 C. was added borane dimethyl sulfide complex (2.0 M in THF, 1.91 mL, 3.81 mmol) dropwise and the resultant solution stirred at reflux for 16 h. The solution was cooled to RT, and 10% aq. Na.sub.2CO.sub.3 (25 ml) was added and the solution stirred for 0.5 h, diluted with EtOAc, washed with H.sub.2O and brine, dried (MgSO.sub.4) and evaporated to give a crude colourless oil (1.12 g). This was purified by SiO.sub.2 chromatography (hexane:EtOAc, 9:1, with 1% Et.sub.3N, as eluent) to give 4 as a colourless oil (1.08 g, 90%): .sup.1H NMR (700 MHz, CDCl.sub.3) 1.19 (s, 3H), 1.19 (s, 3H), 1.24 (s, 6H), 1.65-1.67 (m, 2H), 3.14-3.17 (m, 2H), 4.06 (hept, J=6.6 Hz, 1H), 6.46 (d, J=8.8 Hz, 1H), 7.28 (dd, J=8.9, 2.1 Hz, 1H), 7.39 (d, J=2.2 Hz, 1H); .sup.13C NMR (176 MHz, CDCl.sub.3) 18.9, 30.3, 32.4, 36.6, 36.8, 47.3, 76.1, 113.4, 134.5, 134.8, 135.6, 144.0; IR (neat) .sub.max/cm.sup.1 2957m, 2927w, 2863w, 1580m, 1489m, 792s, 684w; MS (ES): m/z=330.1 [M+H].sup.+; HRMS (ES) calcd. for C.sub.11H.sub.13NOI [M+H].sup.+: 330.0719. found: 330.0717.

Example 2

Methyl 4-ethynylbenzoate (7)

(51) ##STR00016##

2(a) Methyl 4-iodobenzoate (5)

(52) 4-Iodobenzoic acid (25 g, 100.8 mmol) was suspended in MeOH (250 mL), and conc. H.sub.2SO.sub.4 (5 mL) was added and the resultant solution was stirred at reflux overnight. The clear solution was then cooled slowly to RT, and then to 0 C. The resultant solid was filtered, washed with cold MeOH and dried to give 5 as a colourless crystalline solid (23.7 g, 90%): .sup.1H NMR (600 MHz, CDCl.sub.3) 3.90 (s, 3H), 7.73 (d, J=8.6 Hz, 2H), 7.79 (d, J=8.6 Hz, 2H); .sup.13C NMR (176 MHz, CDCl.sub.3) 52.5, 100.9, 129.8, 131.2, 137.9, 166.7; IR (neat) .sub.max/cm.sup.1 3040w, 2996w, 2946w, 1709s, 1596m, 1436m, 1269s, 1114s, 843s, 683m; MS (GC): m/z=261.9 [M].sup.+. Found: C, 36.54; H, 2.71. Calc. for C8H7IO2: C, 36.67; H, 2.69%.

2(b) Methyl 4-((trimethylsilyl)ethynyl)benzoate (6)

(53) An oven-dried 500 mL Schlenk flask was evacuated under reduced pressure and refilled with Ar, before Pd(PPh.sub.3).sub.2Cl.sub.2 (1.18 g, 1.68 mmol), CuI (1.68 g, 1.68 mmol) and 5 (22.0 g, 83.98 mmol) were added and the flask sealed with a septum. Triethylamine (200 mL) and trimethylsilylacetylene (13.94 mL, 100.8 mmol) were added and the flask evacuated/filled with Ar again (3). The mixture was stirred at RT overnight. The solution was diluted with hexane, passed through Celite/SiO.sub.2 under vacuum, and evaporated to give 6 as an off-white solid (19.8 g). This was carried to the next step without purification: MS (GC): m/z=232.1 [M].sup.+. Found: C, 66.90; H, 6.88. Calc. for C13H16O2Si: C, 67.2; H, 6.94%.

2(c) Methyl 4-ethynylbenzoate (7)

(54) To a MeOH:DCM solution (2:1, 300 mL) was added 6 (18.5 g, 79.5 mmol) and K.sub.2CO.sub.3 (22.0 g, 159 mmol). The mixture was stirred under Ar for 6 h. The solution was then evaporated to volume, diluted with hexane, passed through Celite and evaporated to give a light brown solid, which was purified by sublimation under reduced pressure to give 7 as a white solid (11.1 g, 83% over two steps): .sup.1H NMR (600 MHz, CDCl.sub.3) 3.23 (s, 1H), 3.91 (s, 3H), 7.54 (d, J=8.4 Hz, 2H,), 7.98 (d, J=8.6 Hz, 2H); .sup.13C NMR (176 MHz, CDCl.sub.3) 52.5, 80.2, 83.0, 126.9, 129.6, 130.3, 132.3, 166.6; IR (neat) .sub.max/cm.sup.1 3035w, 3006w, 2950w, 2103w, 1699s, 1605m, 1433m, 1277s, 1107s, 859s; MS (GC): m/z=160.1 [M].sup.+. Found: C, 74.62; H, 5.01. Calc. for C10H8O2: C, 74.99; H, 5.03%.

Example 3

4-2-[4,4-Dimethyl-1-(propan-2-yl)-1,2,3,4-tetrahydroquinolin-6-yl]ethynylbenzoic acid (9)

(55) ##STR00017##

3(a) 4-2-[4,4-Dimethyl-1-(propan-2-yl)-1,2,3,4-tetrahydroquinolin-6-yl]ethynylbenzoate, (8)

(56) An oven-dried Schlenk flask was evacuated under reduced pressure and refilled with Ar, before Pd(PPh.sub.3).sub.2Cl.sub.2 (0.0744 g, 0.106 mmol), CuI (0.0202 g, 0.106 mmol) and 7 (0.219 g, 1.37 mmol) were added and the flask sealed with a septum. A solution of 4 (0.349 g, 1.06 mmol) in triethylamine (6 mL) was added and the flask evacuated/filled with Ar again (3). The mixture was stirred at RT for 72 h. The mixture was diluted with Et.sub.2O, passed through Celite/SiO.sub.2 under vacuum, and evaporated to give a crude orange solid (0.47 g). This was purified by SiO.sub.2 chromatography (hexane:EtOAc, 8:2, with 1% Et.sub.3N, as eluent) to give 8 as an orange solid (0.105 g, 27%): .sup.1H NMR (700 MHz, CDCl.sub.3) 1.21/1.23 (s, 6H), 1.28 (s, 6H,), 1.66-1.71 (m, 2H), 3.19-3.24 (m, 2H), 3.92 (s, 3H), 4.15 (hept, J=6.6 Hz, 1H), 6.64 (d, J=8.7 Hz, 1H), 7.24-7.25 (m, 1H), 7.36 (d, J=1.8 Hz, 1H), 7.54 (d, J=8.3 Hz, 2H), 7.98 (d, J=8.3 Hz, 2H); .sup.13C NMR (151 MHz, CDCl.sub.3) 19.1, 30.1, 32.2, 36.7, 36.8, 47.4, 52.3, 86.8, 95.2, 108.0, 110.6, 128.5, 129.5, 129.6, 131.1, 131.2, 131.7, 145.0, 167.0; MS (ES):m/z=362.2 [M+H].sup.+; HRMS (ES) calcd. for C24H28NO2 [M+H].sup.+: 362.2120. found: 362.2114.

3(b) 4-2-[4,4-Dimethyl-1-(propan-2-yl)-1,2,3,4-tetrahydroquinolin-6-yl]ethynylbenzoic acid (9)

(57) ##STR00018##

(58) An oven-dried Schlenk flask was evacuated under reduced pressure and refilled with Ar, before Pd(PPh.sub.3).sub.2Cl.sub.2 (0.253 g, 0.36 mmol), CuI (0.0686 g, 0.36 mmol) and 7 (0.634 g, 3.96 mmol) were added and the flask sealed with a septum. A solution of 4 (1.185 g, 1.06 mmol) in triethylamine (30 mL) was degassed by sonication under vacuum, and backfilling with Ar (3). This solution was then added to the Schlenk flask, degassed under vacuum and backfilled with Ar once more, and the resultant mixture stirred at RT for 72 h. The reaction mixture was then evaporated to dryness, and eluted through a thin Celite/SiO.sub.2 plug with hexane, and then hexane:EtOAc (9:1). The organics were then washed with sat. NH.sub.4Cl, 3% aq. EDTA, H.sub.2O and brine, dried (MgSO.sub.4) and evaporated to give an orange solid (1.38 g). This was dissolved in THF (30 mL) and aq. 20% NaOH (3 mL) was added. The resultant solution was stirred at reflux for 40 h, whereupon the mixture was cooled and H.sub.2O added. The solution was neutralised with 5% HCl, diluted with EtOAc, washed with sat. NaHCO.sub.3, H.sub.2O and brine, dried (MgSO.sub.4) and evaporated to give a crude yellow solid (1.0 g). This was recrystallised twice by solvent layering (DCM/hexane) to give 17 as bright yellow needles (0.73 g, 58% over two steps): .sup.1H NMR (700 MHz; (CD.sub.3).sub.2SO) 1.16 (s, 3H), 1.17 (s, 3H), 1.22 (s, 6H), 1.60-1.64 (m, 2H), 3.17-3.21 (m, 2H), 4.15 (hept, J=7.0 Hz), 6.70 (d, J=9.3 Hz, 1H), 7.19 (dd, J=8.6, 2.1 Hz, 1H), 7.30 (d, J=2.1 Hz, 1H), 7.56 (d, J=8.5 Hz, 2H), 7.92 (d, J=8.6 Hz, 2H), 13.02 (s, 1H); .sup.13C NMR (700 MHz, (CD.sub.3).sub.2SO) 18.6, 29.7, 31.6, 35.8, 36.1, 46.7, 86.5, 94.9, 106.5, 109.5, 110.5, 128.9, 129.4, 130.7, 130.7, 131.2, 144.7, 166.8; MS (ES):m/z=348.2 [M+H].sup.+; HRMS (ES) calcd. for C.sub.23H.sub.26NO.sub.2 [M+H].sup.+: 348.1964. found: 348.1965.

Example 4

3-[4-(1,4,4-Trimethyl-1,2,3,4-tetrahydroquinolin-6-yl)-phenyl]-acrylic acid methyl ester (13)

(59) ##STR00019##

4(a) Methyl-(3-methyl-but-2-enyl)-phenyl-amine

(60) ##STR00020##
In a 500 mL round bottomed flask a solution of N-methylanaline (3.24 g, 30.32 mmol), 1-bromo-3-methyl-but-2-ene (5.0 g, 33.56 mmol) and K.sub.2CO.sub.3 (4.63 g, 33.56 mmol) in 160 ml MeCN was heated at 85 C. for 18 h at which time analysis via in situ ES.sup.+-MS showed the reaction to be complete. The mixture was diluted with Et.sub.2O (100 mL) and washed with H.sub.2O (3100 mL). The organic layer was dried with MgSO.sub.4, filtered and evaporated in vacuo to give a crude oil which was filtered through a silica pad, eluting with hexane. The solvent was removed in vacuo to give the title compound as a clear oil (3.82 g, 72%); m/z (ES.sup.+-MS) 176 (MH.sup.+); .sup.1H NMR (499.76 MHz, CDCl.sub.3) 7.28 (2H, d, J=7.0 Hz), 6.79 (2H, d, J=7.0 Hz), 6.75 (1H, tr, J=7.0 Hz), 5.25 (1H, tr, J=6.0 Hz), 3.93 (2H d, J=6.0 Hz), 2.93 (3H, s) 1.76 (6H, s); .sup.13C{.sup.1H} NMR (100.61 MHz, CDCl.sub.3) 149.86, 134.54, 129.08, 120.91, 116.42, 112.97, 50.53, 37.91, 25.70. 17.92; HRMS calcd for C.sub.12H.sub.18N ([M+H].sup.+) 176.14338. found 176.14336.

4(b) 1,4,4-Trimethyl-1,2,3,4-tetrahydroquinoline

(61) In a 500 mL round bottomed flask a mixture of methyl-(3-methyl-but-2-enyl)-phenyl-amine (18.0 g, 102.86 mmol) and polyphosphoric acid (75 mL) was heated at 120 C. for 18 h, at which time analysis of purified aliquot of the mixture via .sup.1H NMR spectroscopy showed the reaction to be complete. The mixture was diluted by the slow addition of H.sub.2O (100 mL) over 5 minutes. The solution was cautiously basified via the addition of aqueous KOH and then extracted with Et.sub.2O (1 L). The organic layer was washed with H.sub.2O (3200 mL), dried with MgSO.sub.4, filtered and the solvent removed in vacuo to give a crude oil which was filtered through a silica pad, eluting with hexane. The solvent was removed in vacuo to give the title compound as a clear oil (14.93 g, 83%); m/z (EI-MS) 175 (50%, M.sup.+), 160 (60%, M.sup.+-Me); .sup.1H NMR (499.76 MHz, CDCl.sub.3) 7.23 (1H, dd, J=7.5, 1.5 Hz), 7.11 (1H, triplet of doublets, J=7.5, 1.5 Hz), 6.63 (1H, triplet of doublets, J=7.5, 1.5 Hz), 6.62 (1H, d, J=7.5 Hz), 3.25 (2H, tr, J=6.0 Hz), 2.92 (3H, s), 1.80 (2H, tr, J=6.0 Hz); .sup.13C{.sup.1H} NMR (125.67 MHz, CDCl.sub.3) 145.74, 131.61, 126.94, 126.02, 116.25, 111.09, 47.88, 39.50, 37.50, 32.19, 31.21, HRMS calcd for C.sub.12H.sub.18N ([M+H].sup.+) 176.14338. found 176.14332.

4(c) 6-Iodo-1,4,4-trimethyl-1,2,3,4-tetrahydroquinoline

(62) To a solution of 1,4,4-trimethyl-1,2,3,4-tetrahydro-quinoline (2.10 g, 12.0 mmol) and iodine (3.05 g, 12.0 mmol) in DCM (100 mL) was added red HgO (2.59 g, 12.0 mmol). The reaction was stirred at room temperature until analysis via .sup.1H NMR showed the reaction to be complete (2 h). The mixture was filtered, washed with dilute aqueous Na.sub.2S.sub.2O.sub.3 (100 mL) and H.sub.2O (100 mL). The organic layer was dried with MgSO.sub.4 and the solvent removed in vacuo. The residue was filtered through an alumina plug, eluting with DCM and the solvent removed in vacuo to give the title compound as a pale yellow oil (2.50 g, 69%); m/z (EI-MS) 301 (100%, M.sup.+), 286 (80%, M.sup.+-Me); .sup.1H NMR (499.67 MHz, CDCl.sub.3) 7.40 (1H, d, J=2.0 Hz), 7.32 (1H, dd, J=8.5, 2.0 Hz), 6.35 (1H, d, J=8.5 Hz), 3.24 (2H, tr, J=6.0 Hz), 2.89 (3H, s), 1.74 (2H, tr, J=6.0 Hz) 1.27 (6H, s); .sup.13C{.sup.1H} NMR (125.67 MHz, CDCl.sub.3) 144.92, 135.49, 134.34, 127.22, 126.52, 113.35, 47.58, 39.30, 36.87, 32.29, 30.79; HRMS calcd for C.sub.12H.sub.17NI([M+H].sup.+) 302.04003. found 302.04008.

4(d) 1,4,4-Trimethyl-6-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1,2,3,4-tetrahydroquinoline (12)

(63) In a dry, N.sub.2 filled glovebox, Pd(dppf)Cl.sub.2 (0.126 g, 0.15 mmol), 6-iodo-1,4,4-trimethyl-1,2,3,4-tetrahydro-quinoline (0.93 g, 3.09 mmol), B.sub.2pin.sub.2 (0.78 g, 3.09 mmol) and KOAc (0.61 g, 6.18 mmol) were mixed in a thick walled glass tube fitted with a Young's tap. Degassed DMSO (10 mL) was added and the mixture heated at 80 C. for 18 h, at which time GCMS analysis showed the reaction to be complete. The mixture was diluted with Et.sub.2O (100 mL) and washed with H.sub.2O (3100 mL). The organic layer was dried with MgSO.sub.4, filtered and the solvent removed in vacuo to give a residue which was filtered through a silica pad, eluting with 1:1 DCM/hexane. Removal of the solvent in vacuo gave a crude product that was recrystallised from MeOH at 20 C. to give 12 as white needles (0.66 g 70%); mp 140-141 C.; m/z (EI-MS) 301 (100%, M.sup.+), 286 (100%, M.sup.+-Me); .sup.1H NMR (699.73 MHz, CDCl.sub.3) 7.63 (1H, s) 7.55 (1H, d, J=8.0 Hz), 6.56 (1H, d, J=8.0 Hz), 3.29 (2H, tr, J=6.0 Hz), 2.94 (3H, s), 1.75 (2H, tr, J=6.0 Hz), 1.33 (12H, s), 1.31 (6H, s); .sup.13C{.sup.1H} NMR (175.73 MHz, CDCl.sub.3):

(64) 147.8, 134.4, 132.3, 130.3, 110.1, 83.2, 47.7, 39.2, 37.2, 32.1, 30.7, 25.0, the resonance of the carbon attached to boron was not observed; .sup.11B{.sup.1H} NMR (128.38 MHz, CDCl.sub.3) 31.01; elemental analysis calcd. (%) for C.sub.18H.sub.28BNO.sub.2: C 71.77, H 9.37, N 4.65. found: C 71.79, H 9.27, N 4.60.

4(e) 3-[4-(1,4,4-Trimethyl-1,2,3,4-tetrahydroquinolin-6-yl)-phenyl]-acrylic acid methyl ester (13)

(65) ##STR00021##

(66) In a dry, N.sub.2 filled glovebox, Pd(dppf)Cl.sub.2 (25 mg, 0.03 mmol), 1,4,4-trimethyl-6-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1,2,3,4-tetrahydroquinoline (0.49 g, 1.55 mmol), 3-(4-bromo-phenyl)-acrylic acid methyl ester (0.37 g, 0.83 mmol) and K.sub.3PO.sub.4.2H.sub.2O (0.77 g, 3.10 mmol) were mixed in a thick walled glass tube fitted with a Young's tap. Degassed .sup.iPrOH (10 mL) and H.sub.2O (1 mL) were added and the mixture heated at 80 C. for 18 h, at which time GCMS analysis showed the reaction to be complete. The solvent was removed in vacuo and the residue dissolve in DCM (100 mL) and washed with H.sub.2O (320 mL). The organic layer was dried with MgSO.sub.4, filtered and the solvent removed in vacuo to give a residue which was filtered through a silica pad, eluting with DCM. Removal of the solvent in vacuo gave a yellow solid which was recrystallised from MeOH at 20 C. to give yellow white needles of 13 (0.32 g, 62%); mp 121-123 C.; UV-vis (CHCl.sub.3) .sub.max () 380 nm (23900 L mol.sup.1 cm.sup.1); .sub.em (CHCl.sub.3) 536 nm; m/z (ES.sup.+-MS) 336 ([MH].sup.+); .sup.1H NMR (499.77 MHz, CDCl.sub.3) 7.73 (1H, d, J=16.0 Hz), 7.58 (2H, d, J=8.5 Hz), 7.56 (2H, d, J=8.5 Hz), 7.48 (1H, s), 7.37 (1H, d, J=8.0 Hz), 6.66 (1H, d, J=8.0 Hz), 6.45 (1H, d, J=16.0 Hz), 3.83 (3H, s), 3.30 (2H, tr, J=5.5 Hz), 2.97 (3H, s), 1.81 (2H, tr, J=5.5 Hz), 1.35 (6H, s); .sup.13C{.sup.1H} NMR (125.67 MHz, CDCl.sub.3) 167.87. 145.49, 144.98, 143.89, 131.85 (2 peaks overlapped), 12.71, 127.45, 126.49, 125.63, 124.56, 116.56, 111.28, 51.79, 47.75, 39.40, 37.24, 32.34, 30.97; HRMS calcd for C.sub.22H.sub.26NO.sub.2 ([MH].sup.+) 336.19581. found 336.19577.

Example 5

6-(1,4,4-Trimethyl-1,2,3,4-tetrahydroquinolin-6-yl)-naphthalene-2-carboxylic acid methyl ester (11)

(67) ##STR00022##

5(a) 6-(5,5-Dimethyl-[1,3,2]dioxaborinan-2-yl)-1,4,4-trimethyl-1,2,3,4-tetrahydroquinoline (10)

(68) In a dry, N.sub.2 filled glovebox, Pd(dppf)Cl.sub.2 (0.135 g, 0.17 mmol), 6-iodo-1,4,4-trimethyl-1,2,3,4-tetrahydro-quinoline (1.0 g, 3.32 mmol), B.sub.2pin.sub.2 (0.75 g, 3.32 mmol) and KOAc (0.65 g, 6.64 mmol) were mixed in a thick walled glass tube fitted with a Young's tap. Degassed DMSO (10 mL) was added and the mixture heated at 80 C. for 18 h, at which time GCMS analysis showed the reaction to be complete. The mixture was diluted with Et.sub.2O (100 mL) and washed with H.sub.2O (3100 mL). The organic layer was dried with MgSO.sub.4, filtered and the solvent removed in vacuo to give a residue which was filtered through a silica pad, eluting with 1:1 DCM/hexane. Removal of the solvent in vacuo gave a crude product that was recrystallised from MeOH at 20 C. to give white needles of 10 (0.80 g, 88%); mp 151-153 C.; m/z (EI-MS) 287 (90%, M.sup.+), 272 (100%, M.sup.+-Me); .sup.1H NMR (499.77 MHz, CDCl.sub.3) 7.64 (1H, d, J=1.5 Hz), 7.54 (1H, dd, J=8.5, 1.5 Hz), 7.27 (1H, s), 6.57 (1H, d, J=8.5 Hz), 3.75 (4H, s), 3.28 (2H, tr, J=6.0 Hz), 2.94 (3H, s), 1.76 (2H, tr, J=6.0 Hz), 1.32 (6H, s), 1.02 (6H, s); .sup.13C{.sup.1H} NMR (125.67 MHz, CDCl.sub.3) 147.49, 133.29, 131.42, 130.19, 110.09, 72.33, 47.75, 39.24, 37.29, 32.05, 32.03, 30.83, 20.12, the resonance of the carbon attached to boron was not observed; .sup.11B{.sup.1H} NMR (128.38 MHz, CDCl.sub.3) 27.02; elemental analysis calcd. (%) for C.sub.17H.sub.26BNO.sub.2: C 71.09, H 9.12, N 4.88. found: C 71.00, H 9.12, N 4.81.

5(b) 6-(1,4,4-Trimethyl-1,2,3,4-tetrahydroquinolin-6-yl)-naphthalene-2-carboxylic acid methyl ester, (11)

(69) ##STR00023##
In a dry, N.sub.2 filled glovebox, Pd(dppf)Cl.sub.2 (13 mg, 0.02 mmol), 6-(5,5-dimethyl-[1,3,2]dioxaborinan-2-yl)-1,4,4-trimethyl-1,2,3,4-tetrahydro-quinoline (0.25 g, 0.87 mmol), 6-bromo-naphthalene-2-carboxylic acid methyl ester (0.22 g, 0.83 mmol) and K.sub.3PO.sub.4.2H.sub.2O (0.43 g, 1.74 mmol) were mixed in a thick walled glass tube fitted with a Young's tap. Degassed DMSO (15 mL) and H.sub.2O (3 mL) were added and the mixture heated at 80 C. for 18 h, at which time GCMS analysis showed the reaction to be complete. The mixture was diluted with Et.sub.2O (100 mL) and washed with H.sub.2O (3100 mL). The organic layer was dried with MgSO.sub.4, filtered and the solvent removed in vacuo to give a residue which was filtered through a silica pad, eluting with DCM. Removal of the solvent in vacuo gave a yellow solid which was recrystallised from MeOH at 20 C. to give yellow white needles of 11 (0.28 g, 94%); mp 166-167 C.; UV-vis (CHCl.sub.3) .sub.max (E) 243 nm (53200 L mol.sup.1 cm.sup.1); .sub.em (CHCl.sub.3) 494 nm; m/z (EI-MS) 359 (100%, M.sup.+), 344 (60%, M+Me); .sup.1H NMR (699.73 MHz, CDCl.sub.3) 8.60 (1H, s), 8.06 (1H, dd, J=8.5, 1.5 Hz), 7.99 (1H, s), 1.97 (1H, d, J=8.5 Hz), 7.90 (1H, d, J=8.0 Hz), 7.81 (1H, dd, J=8.5, 1.5 Hz), 7.60 (1H, d, J=2.0 Hz), 7.49 (1H, dd, J=8.5, 2.0 Hz), 6.71 (1H, d, J=8.5 Hz), 3.99 (3H, s), 3.32 (2H, tr, J=6.0 Hz), 2.99 (3H, s), 1.84 (2H, tr, J=6.0 Hz), 1.39 (6H, s); .sup.13C{.sup.1H} NMR (175.73 MHz, CDCl.sub.3) 167.55, 145.49, 141.72, 136.29, 131.99, 131.08, 129.74, 128.74, 128.18, 126.62, 126.31, 126.05, 125.62, 124.97, 123.74, 111.41, 52.29, 47.79, 39.43, 37.31, 32.40, 31.03; HRMS calcd for C.sub.24H.sub.25NO.sub.2 (M.sup.+) 359.18798. found 359.18789.

Example 6

4-2-[2,4,4-Trimethyl-1-(propan-2-yl)-1,4-dihydroquinolin-6-yl]ethynylbenzoic acid, (17)

(70) ##STR00024##

6(a) 6-Iodo-2,4,4-trimethyl-1-(propan-2-yl)-1,4-dihydroquinoline, (15)

(71) ##STR00025##

(72) To a solution of 3 (1.17 g, 3.42 mmol) in anhydrous THF (50 mL) was added MeMgBr (3.0 M in Et.sub.2O, 2.28 mL, 6.84 mmol) and the resultant solution stirred at reflux for 16 h. The solution was cooled, quenched with 20% HCl (1.14 mL) and H.sub.2O, diluted with EtOAc, washed with H.sub.2O and brine, dried (MgSO.sub.4) and evaporated to give a crude colourless oil (0.95 g). This was immediately purified by SiO.sub.2 chromatography (hexane:EtOAc, 97.5:2.5, with 1% Et.sub.3N, as eluent) to give 15 as a pink oil (0.35 g, 30%) which was immediately used in the next reaction: .sup.1H NMR (400 MHz, CDCl.sub.3) 1.20 (s, 6H), 1.45 (s, 3H), 1.46 (s, 3H), 1.98 (d, J=0.9 Hz, 3H), 4.16 (hept, J=7.1 Hz, 1H), 4.50 (q, J=1.2 Hz, 1H), 6.73 (d, J=8.7 Hz, 1H,), 7.34 (dd, J=8.7, 2.2 Hz, 1H), 7.42 (d, J=2.1 Hz, 1H).

6(b) 4-2-[2,4,4-Trimethyl-1-(propan-2-yl)-1,4-dihydroquinolin-6-yl]ethynyl benzoic acid, (17)

(73) ##STR00026##

(74) Pd(PPh.sub.3).sub.2Cl.sub.2 (0.073 g, 0.104 mmol), CuI (0.0198 g, 0.104 mmol) and 7 (0.176 g, 1.10 mmol) were added to a Schlenk flask under Ar. The flask was evacuated and refilled with Ar. 15 (0.356 g, 1.04 mmol), dissolved in triethylamine (12 mL), was added and the flask evacuated/filled with Ar again (3). The mixture was stirred at RT for 72 h. The solution was diluted with Et.sub.2O, passed through Celite/SiO.sub.2 under vacuum, and evaporated to give a crude green solid (0.4 g). This was purified by SiO.sub.2 chromatography (hexane:EtOAc, 8:2, with 1% Et.sub.3N, as eluent) to give 16 (scheme IV) as a pale green solid (0.12 g, 30%). 16 (0.073 g, 0.195 mmol) was then dissolved in THF (10 mL), and to this was added aq. 20% NaOH (2 mL). The resultant solution was stirred at reflux for 40 h, whereupon the mixture was cooled and H.sub.2O and Et.sub.2O added. The solution was acidified to pH 7 with 5% HCl, diluted with Et.sub.2O, washed with brine, dried (MgSO.sub.4) and evaporated to give 17 as a yellow solid (0.070 g, 99%): .sup.1H NMR (400 MHz; CDCl.sub.3) 1.24 (s, 6H), 1.47 (s, 3H), 1.49 (s, 3H), 2.01 (d, J=0.9 Hz, 3H), 4.23 (hept, J=7.2 Hz, 1H), 4.53 (d, J=1.1 Hz, 1H), 6.93 (d, J=8.6 Hz, 1H), 7.27-7.29 (m, 1H), 7.37 (d, J=2.0 Hz, 1H), 7.54 (d, J=8.3 Hz, 2H), 8.03 (d, J=8.4 Hz, 2H).

Example 7

Initial Fluorescence Characterisation of Compound 9 of Example 3 and Compound 17 of Example 6

(75) Absorption and emission spectra of 9 were obtained in a variety of solvents (FIG. 3 and FIG. 4). Comparison of 9 with EC23 (FIG. 1 and FIG. 2) shows significant increases in the maximal absorption and emission wavelengths. The fluorescence from 9 was easily detected at concentrations as low as 1 nM, and solvent-dependent effects were observed, with high intensity fluorescence detected in non polar solvents, while significant fluorescence quenching was observed in water, in particular. The fluorescence emission wavelength is also highly dependent on solvent polarity, with a significant red shift occurring in polar solvents when compared to non polar solvents. This initial characterisation indicated that when applied to cells, the fluorescence of 9 could be expected to be discernable in discrete cellular locations, depending on the local polarity.

(76) Compound 17 exhibits a similar emission profile to compound 9 (FIG. 6), but also exhibits a longer maximal absorption wavelength (FIG. 5). This absorption band peaks at around 379 nm, and trails into the indigo/blue (440 nm). This longer wavelength absorption band indicates that compound 17 will be more effectively excited than compound 9 with the 405 nm excitation source that is typical on fluorescence microscopes.

(77) Light Stability of Compound 9 of Example 3

(78) A .sup.1H NMR spectrum of compound 9 in DMSO-d.sub.6 was recorded after storage at ambient temperature in the absence of light (FIG. 7). The same sample of compound 9 was then exposed to standard laboratory light at a distance of 30 cm for 72 hours, and the .sup.1H NMR spectrum recorded (FIG. 8). Compound 9 is stable towards typical laboratory lighting over this time period, although a small proportion converts to a structurally similar enamine form. Compound 9 remains stable until around 16 day's exposure, where some indication of degradation becomes apparent. More significant degradation is observed after 22 day's exposure, although compound 9 still represents the major constituent of the sample (>60%).

(79) Biological Evaluation of Compound 9 and Compound 17

(80) Defining properties of retinoids are their ability to induce differentiation of specific cell types and to induce the expression of genes which are directly responsive to retinoic acid by being linked to DNA of defined sequences (retinoic acid response elements, RAREs) which binds ligand-activated retinoic acid receptors (RARs), thus enabling recruitment of the gene transcription machinery to the gene regulatory sequences (promoter) necessary for messenger RNA transcripts of the gene to be expressed.

(81) To show that the fluorescent retinoids exhibit retinoid activity, TERA-2 cells (an embryonal carcinoma cell line) were treated with 1 and 10 M ATRA, EC23 and compound 9, and the resultant samples stained with a variety of immunocytochemical stains. FIG. 9 shows the result of the treatment of TERA-2 cells with 1 and 10 M ATRA, EC23 and compound 9, and with the vehicle solvent, DMSO, on the presence of nestin, an intermediate filament that is typically expressed in neural stem cells. All conditions were positive for nestin with staining possibly to a lesser extent in 10 M EC23 and compound 9 samples.

(82) FIG. 10 shows the result of the treatment of TERA-2 cells with 1 and 10 M ATRA, EC23 and compound 9, and with the vehicle solvent, DMSO, on the presence of cytokeratin 8 (CK8), a marker of non-neural differentiation. The staining appears less intense in 10 M samples of both ATRA and EC23, as is typical with a reduction in non-neural differentiation, but slightly brighter with compound 9 when compared with 1 M samples. DMSO treatment shows very bright staining for CK8.

(83) FIG. 11 shows the result of the treatment of TERA-2 cells with 1 and 10 M ATRA, EC23 and compound 9, and with the vehicle solvent, DMSO, on the presence of TUJ-1, a pan neuronal marker. Samples treated with ATRA, EC23 and compound 9 show significant staining for TUJ-1, with increased staining evident with 10 M treatment. DMSO treated cells show only limited TUJ-1 staining.

(84) FIG. 12 shows the result of the treatment of TERA-2 cells with 1 and 10 M ATRA, EC23 and compound 9, and with the vehicle solvent, DMSO, on the presence of Oct 4, a transcription factor that is a marker of pluripotency. DMSO treated cells show obvious positive staining for Oct 4, and staining is also evident in 1 M ATRA treatment. All other conditions do not exhibit staining for Oct 4, indicating that EC23 and compound 9 readily downregulate markers of pluripotency through the promotion of differentiation.

(85) FIG. 13 shows the result of the treatment of TERA-2 cells with 1 and 10 M ATRA, EC23 and compound 9, and with the vehicle solvent, DMSO, on the presence of Sox 2, a transcription factor that is a marker of pluripotency. DMSO treated cells show obvious positive staining for Sox 2, with significantly reduced staining in cells treated with ATRA, EC23 and compound 9. This observations suggests that ATRA, EC23 and compound 9 readily downregulate markers of pluripotency through the promotion of differentiation.

(86) FIG. 14 shows flow cytometric analysis of TERA-2 cells treated with ATRA, EC23 and compound 9, and DMSO. The expression of stem cell marker SSEA-3 is measured, which is generally reduced when cells are treated with retinoids. SSEA-3 flow cytometry shows that expression of SSEA-3 is significantly decreased in retinoid treated cells compared to DMSO treated cells. Compound 9 treated cells showed higher levels of SSEA-3 than ATRA and EC23 at both 1 and 10 M treatments.

(87) FIG. 15 shows flow cytometric analysis of TERA-2 cells treated with ATRA, EC23 and compound 9, and DMSO. The expression of stem cell marker TRA160 is measured, which is generally reduced when cells are treated with retinoids. TRA160 flow cytometry shows that expression of TRA160 is significantly decreased in retinoid treated cells compared to DMSO treated cells. Compound 9 treated cells showed slightly higher levels of TRA160 than ATRA and EC23 at both 1 and 10 M treatments.

(88) FIG. 16 shows flow cytometric analysis of TERA-2 cells treated with ATRA, EC23 and compound 9 and DMSO. The expression of early neuronal marker A2B5 is measured, which is generally increased when cells are treated with retinoids. A2B5 flow cytometry shows that expression of A2B5 is significantly increased in retinoid treated cells compared to DMSO treated cells. ATRA treated cells express higher levels of A2B5 followed by EC23 and compound 9.

(89) FIG. 17 shows phase contrast images of cell populations that have been treated with ATRA, EC23 and compound 9, and DMSO. In cell populations treated with DMSO, the cells are small, and densely packed together. In contrast, cell populations treated with ATRA, EC23 and compound 9 are less dense, and cells are much more spread out.

(90) FIG. 18 and FIG. 19 shows an MTT cell viability analysis of 1 and 10 M treatments of ATRA, EC23 and compound 9, and DMSO. All treatments exhibit comparable viability to DMSO, suggesting cells treated with retinoids do not experience significant toxic effects.

(91) FIG. 20 shows TERA-2 cells treated with compound 9 at 10, 1, 0.1, 0.01 M concentrations, and imaged using a confocal fluorescence microscope after 7 days. Even at the lowest treatment concentration, the fluorescence of compound 9 is visible, with 0.1-10 M treatments easily imaged. Compound 9 is mainly localised around the nuclear envelope, and appears also to be localised around other cellular structures.

(92) FIGS. 21, 22 and 23 respectively show SHSY5Y cells (neuroblastoma) and fibroblast cells and TERA-2 cells treated with 10 M compound 9, and imaged using a confocal fluorescence microscope after 8 hours (SHSY5Y) and 24 hours (fibroblasts) and 7 days (TERA-2). Compound 9 is again clearly visible with obvious localisation around the nuclear envelope.

(93) FIG. 24 shows HaCat keratinocyte skin cells that were treated with 10 M compound 9 for 5 days, fixed and then imaged with a confocal fluorescence microscope.

(94) FIG. 25 shows HaCat keratinocyte skin cells treated with compound 9 (10 M) for 5 days. The fixed coverslips were then stained with Involucrin (green) and K14 (red) and imaged using a confocal microscope. The fluorescence of compound 9 is coloured in blue. Involucrin selectively stains Cellular Retinoic Acid Binding Protein (CRABP), which transports retinoids in and around the nucleus. K14 is a prototypic marker of dividing basal keratinocytes and helps in the maintenance of epidermal cell shape.

(95) FIG. 27 shows HaCat keratinocyte skin cells treated with compound 17 (10 M) for 5 days. The fixed coverslips were then stained with Involucrin (green) and K14 (red) and imaged using a confocal microscope. The fluorescence of compound 17 is coloured in blue. Involucrin selectively stains Cellular Retinoic Acid Binding Protein (CRABP), which transports retinoids in and around the nucleus. K14 is a prototypic marker of dividing basal keratinocytes and helps in the maintenance of epidermal cell shape. As in FIG. 26, the fluorescence from compound 17 is significantly more intense than that exhibited by compound 9 under identical conditions (FIG. 25).

(96) FIG. 28 shows the Raman spectrum of compound 9. A high intensity acetylene band is observed at 2190 cm.sup.1. This lies in the cellular silent region (1800-2800 cm.sup.1), wherein signals of biological origin, such as amide bonds, are not observed. This spectral separation allows Raman bands in the cellular silent region to be more easily detected when imaging or analysing cellular samples using Raman microscopy/spectroscopy.