Synthetic retinoids (in cell modulation)

Abstract

There are described novel compounds of formula I: (I) in which, in which A.sup.1, A.sup.2, A.sup.3, A.sup.4, R.sup.1 and R.sup.2 are each as herein defined, for use in the treatment or alleviation of an RAR mediated condition; and methods related thereto.

Claims

1. A compound of formula I: ##STR00064## in which A.sup.1 is N or CR.sup.3; A.sup.2 is N or CR.sup.4; A.sup.3 is N or CR.sup.5; R.sup.3, R.sup.4 and R.sup.5, which may be the same or different, are each hydrogen, alkyl.sub.C1-10, alkene.sub.C2-12, aryl, aralkyl, halogen, trifluoroalkyl, cyano, nitro, —NR.sup.aR.sup.b, —OR.sup.a, glycol, —C(O)R.sup.a, —C(O)OR.sup.a, —OC(O)R.sup.a, —S(O)R.sup.aR.sup.b, —C(O)NR.sup.aR.sup.b or a solubilising group; R.sup.7 is hydrogen, propynyl, —(CH.sub.2).sub.nC≡CH, —(CH.sub.2).sub.nSH, —(CH.sub.2).sub.nSO.sub.2F or —(CH.sub.2).sub.nC═CH.sub.2, alkyl.sub.C1-10, said alkyl being optionally substituted by aryl or heteroaryl; R.sup.8, R.sup.9, R.sup.10 and R.sup.11, which may be the same or different, are each hydrogen or alkyl.sub.C1-4, aryl, halogen, trifluoroalkyl, —OR.sup.c or glycol, or together one pair of R.sup.8 and R.sup.10 or R.sup.9 and R.sup.11 represent a bond; R.sup.12 and R.sup.13, which may be the same or different, are each hydrogen, alkyl.sub.C1-4 or together one pair of R.sup.10 and R.sup.12 or R.sup.11 and R.sup.13 represent a bond, or R.sup.12 and R.sup.13 together form a group:
═CR.sup.14R.sup.15 provided that the pair of R.sup.10 and R.sup.12 or R.sup.11 and R.sup.13 does not represent a bond if a pair from R.sup.8, R.sup.9, R.sup.10 and R.sup.11 represents a bond; R.sup.14 and R.sup.15, which may be the same or different, are each hydrogen or alkyl.sub.C1-10; and R.sup.a, R.sup.b and R.sup.c, which may be the same or different, are each hydrogen or alkyl.sub.C1-10; n is an integer from 1 to 6; R.sup.2 is a group III: ##STR00065## in which X.sup.a is —C≡C—, —CH═CH— or —N═CH—; X.sup.b is —C≡C— or is absent; A.sup.5 is N or CR.sup.17; A.sup.6 is N or CR.sup.18; A.sup.7 is N or CR.sup.19; A.sup.8 is N or CR.sup.20; R.sup.17, R.sup.18, R.sup.19 and R.sup.20, which may be the same or different, are each hydrogen, alkyl.sub.C1-10, alkene.sub.C2-12, aryl, aralkyl, halogen, trifluoroalkyl, cyano, nitro, —NR.sup.dR.sup.e, —OR.sup.d, glycol, —C(O)R.sup.d, —C(O)OR.sup.d, —OC(O)R.sup.d, —S(O)R.sup.dR.sup.e, —C(O)NR.sup.dR.sup.e or a solubilising group; R.sup.16 is —CR.sup.21═CR.sup.22Y, —C≡C—R.sup.23 or together with R.sup.18 forms a ring IV: ##STR00066## A.sup.9 is N or CR.sup.24; A.sup.10 is N or CR.sup.25; A.sup.11 is N or CR.sup.26; R.sup.23 is a group V: ##STR00067## in which A.sup.12 is N or CR.sup.27; A.sup.13 is N or CR.sup.28; A.sup.14 is N or CR.sup.29; A.sup.15 is N or CR.sup.30; R.sup.21 and R.sup.22, which may be the same or different, are each hydrogen, alkyl.sub.C1-10, alkene.sub.C2-12, aryl, halogen or trifluoroalkyl; R.sup.24, R.sup.25, R.sup.26, R.sup.27, R.sup.28, R.sup.29 and R.sup.30, which may be the same or different, are each hydrogen, alkyl.sub.C1-10, alkene.sub.C2-12, aryl, halogen, trifluoroalkyl, —OR.sup.f, glycol or a solubilising group; R.sup.d, R.sup.e and R.sup.f, which may be the same or different, are each hydrogen or alkyl.sub.C1-10; Y is —CO.sub.2R.sup.31, —COH, —CO.sub.2CH.sub.2C≡CH, —CN, —SF.sub.5, —SO.sub.3H, —SO.sub.2NH.sub.2, —SO.sub.2CF.sub.3, —CF.sub.3, —CO.sub.2(CH.sub.2).sub.mSH, —CO.sub.2(CH.sub.2).sub.mSO.sub.2F, —CO.sub.2(CH.sub.2).sub.mCH═CH.sub.2, —C═NR.sup.32 or —C═N.sup.+R.sup.33R.sup.34; R.sup.31 is hydrogen, alkyl.sub.C1-10, alkene.sub.C2-12, aryl or a photocleavable group; R.sup.32, R.sup.33 and R.sup.34, which may be the same or different, are each hydrogen, alkyl.sub.C1-10, alkene.sub.C2-12 or aryl; m is an integer from 1 to 9; with the proviso that when R.sup.1 together with R.sup.6 forms a ring II in which R.sup.7 is hydrogen or alkyl C.sub.1-10, X.sub.a is —C≡C— and X.sub.b is absent, then Y is not —CO.sub.2R.sup.13; and isomers thereof; in free or in salt form.

2. The compound according to claim 1 wherein A.sup.1 is CR.sup.3, A.sup.2 is CR.sup.4, A.sup.3 is CR.sup.5; and R.sup.2, R.sup.3, R.sup.4, and R.sup.5 are each as defined in claim 1.

3. The compound according to claim 1 wherein R.sup.2 is a group III: ##STR00068## wherein X.sup.a is —C≡C— and X.sup.b is —C≡C—; and A.sup.5, A.sup.6, A.sup.7, A.sup.8 and R.sup.16 are each as defined in claim 1.

4. The compound according to claim 3 wherein X.sup.b is absent and X.sup.a is selected from a group consisting of: —C≡C—, —CH═CH—, and —N═CH—.

5. The compound according to claim 3 wherein A.sup.5 is CR.sup.17, A.sup.6 is CR.sup.18, A.sup.7 is CR.sup.19 and A.sup.8 is CR.sup.20; and X.sup.a, X.sup.b, R.sup.16, R.sup.17, R.sup.18, R.sup.19 and R.sup.20 are each as defined in claim 1.

6. The compound according to claim 3 wherein A.sup.5 is CR.sup.17, A.sup.6 is CR.sup.18, A.sup.7 is CR.sup.19 and A.sup.8 is CR.sup.20; and R.sup.17, R.sup.19 and R.sup.20 are each as defined in claim 1; R.sup.16 together with R.sup.18 forms a ring IV: ##STR00069## wherein A.sup.9, A.sup.10, A.sup.11 and Y are each as defined in claim 1.

7. The compound according to claim 3 wherein R.sup.2 is a group III; ##STR00070## and R.sup.16 is —C≡C—R.sup.23 wherein R.sup.23 is a group V: ##STR00071## in which A.sup.12 is CR.sup.27, A.sup.13 is CR.sup.28, A.sup.14 is CR.sup.29 and A.sup.15 is CR.sup.30; and R.sup.27, R.sup.28, R.sup.29, R.sup.30 and Y are each as defined in claim 1.

8. The compound according to claim 7 wherein R.sup.16 is —C≡C—R.sup.23, R.sup.23 is a group V and Y is —CO.sub.2R.sup.31, —COH, —CO.sub.2CH.sub.2C≡CH, —CN, —SF.sub.5, —SO.sub.3H, —SO.sub.2NH.sub.2, —SO.sub.2CF.sub.3, in which R.sup.31 is hydrogen, alkyl.sub.C1-10, alkene.sub.C2-12, aryl or a photocleavable group.

9. The compound according to claim 7 wherein Y is —CO.sub.2R.sup.31 in which R.sup.31 is hydrogen, alkyl.sub.C1-10, alkene.sub.C2-12, aryl or a photocleavable group.

10. The compound according to claim 1 wherein R.sup.7 is alkyl C1-10.

11. The compound according to claim 1 wherein R.sup.8, R.sup.9, R.sup.10 and R.sup.11 are each hydrogen.

12. The compound according to claim 1 wherein R.sup.8 and R.sup.10 or R.sup.9 and R.sup.11 represent a bond.

13. The compound according to claim 1 wherein R.sup.12 and R.sup.13 are the same or different; R.sup.12 and R.sup.13 may each represent alkyl C1-4.

14. The compound according to claim 1 wherein R.sup.2 is selected from a group consisting of group VI: ##STR00072## wherein R.sup.31 is as defined in claim 1.

15. The compound of formula I according to claim 1 that is selected from the group consisting of: (2E)-3-(4-2-[4,4-dimethyl-1-(propyn-2-yl)-1,2,3,4-tetrahydroquinolin-6-yl] ethynylphenyl)prop-2-enoic acid; and (2E)-3-(4-2-[4,4-dimethyl-1-(propyn-2-yl)-1,2,3,4-tetrahydroquinolin-6-yl] ethynylphenyl)prop-2-enoic acid methyl ester; and isomers thereof; in free or in salt form.

16. A composition comprising the compound according to claim 1 in combination with one or more pharmaceutically acceptable excipients; said composition being for use in the generation of reactive oxygen species when said compound is activated by light.

17. The composition according to claim 16 for use in the treatment of one or more of a cancer selected from head and neck tumours, breast cancer, gynaecological tumours, brain tumours, colorectal cancer, prostate cancer, mesothelioma, and pancreatic cancer, or a disease caused by a pathogenic organism selected from bacteria, viruses, fungi, parasites, protozoa and toxins, as well as cells and tissues infected or infiltrated therewith.

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 is a Jablonski diagram showing the formation of singlet oxygen;

(3) FIG. 2 illustrates the biological effects of ROS generation;

(4) FIG. 3 illustrates DC324 localised to one side of the nucleus in large patches of cells;

(5) FIG. 4 illustrates DC324 co-imaged with BODIPY® Golgi stain;

(6) FIG. 5 illustrates that DC324 was the most rapid inducer of cell death;

(7) FIG. 6 illustrates that cell death observations were consistent across different cell lines;

(8) FIG. 7 illustrates that the majority of DMSO treated cells appeared healthy after 47 exposures to 405 nm laser, at 50% strength;

(9) FIG. 8 illustrates that the majority of ATRA treated cells appeared healthy after 47 exposures to 405 nm laser, at 50% strength;

(10) FIG. 9 illustrates that membrane blebbing was induced in DC324 treated cells by 405 nm laser, at 50% strength;

(11) FIG. 10 illustrates DC324 treated cells putatively producing apoptotic bodies;

(12) FIG. 11 illustrates DC324 fluorescence from treated HaCaT cells, imaged with 405 nm laser light, at 50% strength;

(13) FIG. 12 illustrates the dose-dependent response of HaCaT cells treated with DC324 and DC473 and 10 seconds of UV;

(14) FIG. 13 illustrates control treatments stained for superoxide after exposure to UV;

(15) FIG. 14 illustrates active compounds stained for superoxide after exposure to DAPI;

(16) FIG. 15 illustrates inactive DC324 compound treated cells stained for superoxide after UV exposure;

(17) FIG. 16 illustrates DC473 compound treated cells after 2 hours and 24 hours UV exposure;

(18) FIG. 17 illustrates a comparison of DC324 and DC473 compound treated cells;

(19) FIG. 18 illustrates the combined normalised absorbance spectra of DC324, DC473 and DC474 in CHCl.sub.3 (10 μM);

(20) FIG. 19 illustrates the combined normalised emission spectra of DC324, DC473 and DC474 in CHCl.sub.3 (100 nM) with excitation at their respective maximal absorption wavelengths;

(21) FIG. 20 illustrates the combined normalised absorbance spectra of DC473, DC474 and click-conjugate compound 16 in CHCl.sub.3 (10 μM); and

(22) FIG. 21 illustrates the combined normalised emission spectra of DC473, DC474, and click-conjugate compound 16 in CHCl.sub.3 (100 nM) with excitation at their respective maximal absorption wavelengths.

(23) In the figures, any reference to DC271 is a reference to compound 9 of Example 3 of WO 2016/055800. Reference to DC324 is a reference to compound 9 of Example 1 herein; and reference to DC473 is a reference to compound 14 of Example 2.5 herein; reference to DC474 is a reference to compound 15 of Example 2.6 herein.

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

(25) ATRA: All Trans-Retinoic Acid

(26) DCM: dichloromethane

(27) DMF: N,N-dimethylformamide

(28) DMSO: dimethylsulfoxideEDTA: ethylenediaminetetraacetic acid

(29) EtOAc: ethyl acetate

(30) GCMS: gas chromatography-mass spectrometry

(31) h: hour(s)

(32) KOAc: potassium acetate

(33) RT: room temperature

(34) THF: tetrahydrofuran

GENERAL EXPERIMENTAL

(35) 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 μm, 60 Å) 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.

(36) Synthetic Procedures

Example 1

(2E)-3-(4-2-[4,4-Dimethyl-1-(propan-2-yl)-1,2,3,4-tetrahydroquinolin-6-yl]ethynylphenyl)prop-2-enoic Acid (9)

1.1 N-(4-Iodophenyl)-3-methylbut-2-enamide (1)

(37) ##STR00048##

(38) To a solution of 4-iodoaniline (25.0 g, 114.0 mmol) in DCM (400 mL) was added 3,3-dimethylacryloyl 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 diluted with DCM and H.sub.2O, 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) which was recrystallised from EtOH to give compound 1 as a white crystalline solid (31.8 g, 93%): m.p.=136-138° C.; .sup.1H NMR (700 MHz, CDCl.sub.3) δ 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) δ 20.2, 27.6, 87.2, 118.7, 122.0, 137.8, 138.2, 154.1, 165.5; IR (neat) ν.sub.max/cm.sup.−1 3294 m, 3094, 2964 w, 2890 w, 1666 m, 1586 m, 1430 m, 821 s, 650 m; 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%.

1.2 6-Iodo-4,4-dimethyl-1,2,3,4-tetrahydroquinolin-2-one (2)

(39) ##STR00049##

(40) Compound 1 (11.5 g, 38.3 mmol) and AlCl.sub.3 (7.66 g, 57.5 mmol) were added to anhydrous DCM (150 mL) under Ar and the resultant solution stirred vigorously for 2.5 h at RT. The reaction was cooled to 0° C., quenched slowly with H.sub.2O, diluted with DCM, stirred with 5% NaOH (w/v) 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 (12.0 g). This was recrystallised from EtOH to give compound 2 as a white crystalline solid (10.2 g, 88%): m.p.=199-202 OC; .sup.1H NMR (700 MHz, CDCl.sub.3) δ 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) δ 27.7, 34.2, 45.2, 86.8, 118.1, 133.7, 135.1, 135.9, 136.6, 171.3; IR (neat) ν.sub.max/cm.sup.−1 3164 m, 3102, 3040 w, 2953 m, 1671 s, 1596 m, 1484 m, 817 s; 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.12NOI: C, 43.88; H, 4.02; N, 4.65%.

1.3 6-Iodo-4,4-dimethyl-1-(propan-2-yl)-1,2,3,4-tetrahydroquinolin-2-one (3)

(41) ##STR00050##

(42) To a solution of compound 2 (25.9 g, 85.9 mmol) in anhydrous DMF (200 mL) was added crushed KOH (14.5 g, 257 mmol) and the resultant slurry stirred for 1 h at 50° C. under Ar. To this was added 2-iodopropane (25.6 mL, 257 mmol) and the solution stirred at 50° C. for 40 h under Ar. 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 (29.0 g). This was purified by SiO.sub.2 chromatography (hexane:EtOAc, 9:1, with 1% Et.sub.3N, as eluent) to give compound 3 as a colourless oil (14.8 g, 50%): R.sub.f 0.51 (hexane:EtOAc, 8:2, with 1% Et.sub.3N); .sup.1H NMR (400 MHz, CDCl.sub.3) δ 1.25 (s, 6H), 1.50 (d, J=7.0 Hz, 6H), 2.38 (s, 2H), 4.66 (sept, 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 (101 MHz, CDCl.sub.3) δ 20.3, 26.8, 33.1, 47.2, 48.8, 86.9, 119.0, 133.4, 135.9, 139.1, 139.3, 169.8; IR (neat) ν.sub.max/cm.sup.−1 2961 m, 2934 w, 2870 w, 1667 s, 1582 m, 1482 m, 809 s; MS (ES): m/z=344.0 [M+H].sup.+; HRMS (ES) calcd. for C.sub.14H.sub.19NOI [M+H].sup.+: 344.0511, found: 344.0512. Found: C, 49.21; H, 5.29; N, 4.08. Calc. for C.sub.14H.sub.18NOI: C, 48.99; H, 5.29; N, 4.08%.

1.4 6-Iodo-4,4-dimethyl-1-(propan-2-yl)-1,2,3,4-tetrahydroquinoline (4)

(43) ##STR00051##

(44) To a solution of compound 3 (1.25 g, 3.63 mmol) in anhydrous toluene (15 mL) 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 under Ar. The solution was cooled to RT, 10% aq. Na.sub.2CO.sub.3 (25 ml) added and then stirred for 0.5 h. The resultant solution was 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 compound 4 as a colourless oil (1.08 g, 90%): .sup.1H NMR (700 MHz, CDCl.sub.3) δ 1.19 (d, J=6.6 Hz, 6H), 1.24 (s, 6H), 1.65-1.67 (m, 2H), 3.14-3.17 (m, 2H), 4.06 (sept, 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) δ 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) ν.sub.max/cm.sup.−1 2957 m, 2927 w, 2863 w, 1580 m, 1489 m, 792 s, 684 w; MS (ES): m/z=330.1 [M+H].sup.+; HRMS (ES) calcd. for C.sub.14H.sub.21NI [M+H].sup.+: 330.0719, found: 330.0717.

1.5 4-Iodobenzenediazonium tetrafluoroborate (5)

(45) ##STR00052##

(46) 4-Iodoaniline (10.95 g, 50 mmol) was added to tetrafluoroboric acid solution (48% in H.sub.2O, 25 mL), and the suspension was cooled to 0° C. before a solution of NaNO.sub.2 (3.79 g, 55 mmol) in H.sub.2O (13.73 mL) was added dropwise with vigorous stirring so as to maintain the internal temperature below 5° C. After addition the suspension was further stirred for 1 h at 0° C., before the precipitated solid was isolated by filtration, washed with cold MeOH and dried to give a crude brown solid. This was dissolved in a minimal amount of acetone (around 55 mL), and to which Et.sub.2O was slowly added to precipitate a yellow solid. This was filtered, washed with cold Et.sub.2O and dried to give compound 5 as a pale yellow solid (13.13 g, 83%): .sup.1H NMR (700 MHz, (CD.sub.3).sub.2SO) δ 8.35 (d, J=9.0 Hz, 2H), 8.43 (d, J=9.0 Hz, 2H); .sup.13C NMR (151 MHz, (CD.sub.3).sub.2SO) δ 113.6, 115.1, 132.8, 140.2; IR (neat) ν.sub.max/cm.sup.−1 3090 w, 2282 s, 1548 m, 1461 w, 824 s, 523 m. Found: C, 22.83; H, 1.30; N, 8.83, Calc. for C.sub.6H.sub.4BF.sub.4IN.sub.2: C, 22.67; H, 1.27; N, 8.81%.

1.6 Methyl (2E)-3-(4-iodophenyl)prop-2-enoate (6)

(47) ##STR00053##

(48) Pd(OAc).sub.2 (0.138 g, 0.61 mmol), CaCO.sub.3 (2.40 g, 24.0 mmol) and compound 5 (5.54 g, 17.4 mmol) were suspended in MeOH (60 mL). Methyl acrylate (2.16 mL, 24.0 mmol) was added, and the suspension was stirred vigorously for 1.5 h. The solution was diluted with DCM, filtered through Celite and evaporated to give a crude light brown solid (6.3 g). This was purified by SiO.sub.2 chromatography (hexane:DCM, 1:1, as eluent) to give compound 6 as a white solid (3.74 g, 75%): .sup.1H NMR (600 MHz, CDCl.sub.3) δ 3.81 (s, 3H), 6.44 (d, J=16.0 Hz, 1H), 7.24 (d, J=8.4 Hz, 2H), 7.60 (d, J=16.0 Hz, 1H), 7.73 (d, J=8.4 Hz, 2H); .sup.13C NMR (151 MHz, CDCl.sub.3) δ 52.0, 96.7, 118.8, 129.7, 134.1, 138.3, 143.8, 167.3; IR (neat) ν.sub.max/cm.sup.−1 3080 w, 3000 w, 2850 w, 1708 s, 1636 m, 1580 m, 1483 m, 815 s, 493 m; MS (ES): m/z=288.9 [M+H].sup.+; HRMS (ES) calcd. for C.sub.10H.sub.10IO.sub.2 [M+H].sup.+: 288.9726, found: 288.9733. Found: C, 41.86; H, 3.14. Calc. for C.sub.10H.sub.9IO.sub.2: C, 41.96; H, 3.15%.

1.7 Methyl (2E)-3-4-[2-(trimethylsilyl)ethynyl]phenylprop-2-enoate (7)

(49) ##STR00054##

(50) Triethylamine (80 mL) was added to an oven-dried Schlenk flask, and was then degassed via sonication under vacuum, followed by refilling with Ar (×5). Pd(PPh.sub.3).sub.2Cl.sub.2 (0.217 g, 0.31 mmol), CuI (0.06 g, 0.31 mmol) and compound 6 (3.57 g, 12.38 mmol) and trimethylsilylacetylene (1.76 mL, 12.44 mmol) were then added and the mixture was stirred at RT overnight. The solution was diluted with Et.sub.2O, passed through Celite/SiO.sub.2 under vacuum, and evaporated to give a light brown solid (4.5 g). This was purified by SiO.sub.2 chromatography (hexane:EtOAc, 9:1, as eluent) to give compound 7 as a white solid (2.65 g, 83%): .sup.1H NMR (400 MHz, CDCl.sub.3) δ 0.25 (s, 9H) 3.80 (s, 3H), 6.42 (d, J=16.0 Hz, 1H), 7.40-7.50 (m, 4H), 7.64 (d, J=16.0 Hz, 1H).

1.8 Methyl (2E)-3-(4-ethynylphenyl)prop-2-enoate (8)

(51) ##STR00055##

(52) Compound 7 (2.21 g, 8.55 mmol) was dissolved in THF (25 mL), and cooled to −20° C. Tetrabutylammonium fluoride (1.0 M in THF, 8.98 mL, 8.98 mmol) was then added dropwise and the resultant solution stirred at −20° C. for 1 h, after which H.sup.2O was added, and the solution extracted with EtOAc (3×). The organics were washed with brine, dried (MgSO.sub.4) and evaporated to give a crude brown solid. This was purified by SiO.sub.2 chromatography (hexane:EtOAc, 9:1, as eluent) to give compound 8 as a white solid (1.52 g, 95%): m.p.=93-95° C.; .sup.1H NMR (600 MHz; CDCl.sub.3) δ 3.18 (s, 1H), 3.81 (s, 3H), 6.44 (d, J=16.0 Hz, 1H), 7.46-7.51 (m, 4H), 7.66 (d, J=16.0 Hz, 1H); .sup.13C NMR (151 MHz; CDCl.sub.3) δ 52.0, 79.4, 83.3, 119.1, 124.2, 128.1, 132.8, 134.9, 143.9, 167.4; IR (neat) ν.sub.max/cm.sup.−1 3260 m, 2996 w, 2946 w, 2108 w, 1700 s, 1634 m, 1554 m, 1431 m, 1206 s, 831 s; MS (EI): m/z=186.1 [M].sup.+. Found: C, 77.40; H, 5.37. Calc. for C.sub.12H.sub.10O.sub.2: C, 77.40; H, 5.41%.

1.9 (2E)-3-(4-2-[4,4-Dimethyl-1-(propan-2-yl)-1,2,3,4-tetrahydroquinolin-6-yl]ethynylphenyl)prop-2-enoic Acid (9) (DC324)

(53) ##STR00056##

(54) Compound 4 (0.61 g, 1.85 mmol) was dissolved in triethylamine (12 mL), and the resultant solution was degassed by sonication under vacuum, before the atmosphere was replaced with Ar (5×). Pd(PPh.sub.3).sub.2Cl.sub.2 (0.13 g, 0.185 mmol), CuI (0.0352 g, 0.185 mmol) and compound 8 (0.362 g, 1.94 mmol) were then added under Ar. The resultant suspension was stirred at RT for 72 h. The suspension was diluted with hexane and passed through a thin Celite/SiO.sub.2 plug (eluting with hexane, then hexane:EtOAc (8:2)). The extracts were washed with sat. NH.sub.4Cl (3×), brine, dried (MgSO.sub.4) and evaporated to give the coupling product as an orange solid (0.7 g). This was dissolved in THF (20 mL), 20% NaOH (2 mL) added, and the resultant solution was stirred under reflux for 40 h. The mixture was cooled, acidified to pH 1 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 which was recrystallised from MeOH to give compound 9 as an orange crystalline solid (0.46 g, 67% over two steps): .sup.1H NMR (700 MHz; (CD.sub.3).sub.2SO) δ 1.16 (d, J=6.6 Hz, 6H)), 1.22 (s, 6H), 1.60-1.65 (m, 2H), 3.16-3.21 (m, 2H), 4.14 (hept, J=6.6 Hz, 1H), 6.54 (d, J=16.0 Hz, 1H), 6.69 (d, J=9.4 Hz, 1H), 7.17 (dd, J=8.6, 2.1 Hz, 1H), 7.29 (d, J=2.2 Hz, 1H), 7.46-7.51 (m, 2H), 7.58 (d, J=16.0 Hz, 1H), 7.66-7.72 (m, 2H), 12.41 (s, 1H); .sup.13C NMR (176 MHz; (CD.sub.3).sub.2SO) δ 18.6, 29.7, 31.6, 35.9, 36.1, 46.6, 86.9, 94.0, 106.8, 110.5, 119.5, 125.2, 128.4, 128.8, 130.6, 131.1, 131.2, 133.3, 143.0, 144.5, 167.5; MS(ES): m/z=374.2 [M+H].sup.+; HRMS (ES) calcd. for C.sub.25H.sub.28NO.sub.2 [M+H].sup.+: 374.2120, found 374.2118.

Example 2

(2E)-3-(4-{2-[4,4-Dimethyl-1-(prop-2-yn-1-yl)-1,2,3,4-tetrahydroquinolin-6-yl]ethynyl}phenyl)prop-2-enoic Acid (15) (DC474)

2.1 (3-Bromoprop-1-yn-1-yl)trimethylsilane (10)

(55) ##STR00057##

(56) A solution of propargyl bromide (80% in toluene, 6.69 mL, 60.0 mmol) in THF (75 mL) was cooled to −78° C. under Ar. Lithium bis(trimethylsilyl)amide (10.37 g, 62.0 mmol) was added under Ar, and the solution then stirred for 0.5 h. Chlorotrimethylsilane (10.15 mL, 80.0 mmol) was then added dropwise, and the solution stirred for 0.5 h, whereupon sat. NH.sub.4Cl (30 mL) was added, and the solution then warmed to RT. The solution was diluted with EtOAc, washed with brine, dried (MgSO.sub.4) and evaporated to give a crude oil. This was purified by SiO.sub.2 chromatography (hexane as eluent), and then further purified by Kugelrohr distillation (70-90° C., ambient pressure) to give compound 10 as a clear oil (8.09 g, 70%): .sup.1H NMR (400 MHz; CDCl.sub.3) δ 0.18 (s, 9H), 3.91 (s, 2H); .sup.13C NMR (101 MHz; CDCl.sub.3) δ −0.1, 14.9, 92.5, 100.2; IR (neat) ν.sub.max/cm.sup.−1 2960 w, 2906 w, 2180 w, 1251 m, 1204 m, 1038 m, 837 s; MS(EI): m/z=174.9 [M-CH.sub.3].sup.+.

2.2 6-Iodo-4,4-dimethyl-1,2,3,4-tetrahydroquinoline (11)

(57) ##STR00058##

(58) To a solution of compound 2 (4.00 g, 13.28 mmol) in anhydrous toluene (30 mL) was added borane dimethyl sulfide complex (2.0 M in THF, 8.30 mL, 16.6 mmol) dropwise and the resultant solution stirred under reflux for 16 h. The solution was cooled to RT, 10% aq. Na.sub.2CO.sub.3 (25 ml) added and the solution stirred for 0.5 h. The solution was then diluted with EtOAc, washed with brine, dried (MgSO.sub.4) and evaporated to give a crude red oil. This was purified by SiO.sub.2 chromatography (hexane:EtOAc, 9:1, with 1% Et.sub.3N, as eluent) to give compound 11 as a colourless oil (3.36 g, 88%): .sup.1H NMR (700 MHz; CDCl.sub.3) δ 1.27 (s, 6H), 1.68-1.71 (m, 2H), 3.28-3.32 (m, 2H), 3.93 (br, 1H), 6.24 (d, J=8.4 Hz, 1H), 7.19 (dd, J=8.4, 2.0 Hz, 1H), 7.41 (d, J=2.1 Hz, 1H); .sup.13C NMR (176 MHz; CDCl.sub.3) δ 30.9, 32.0, 36.8, 38.4, 77.7, 116.5, 133.1, 135.1, 135.3, 143.4; IR (neat) ν.sub.max/cm.sup.−1 3400 br, 2956 w, 2927 w, 2862 w, 1589 m, 1524 m, 1492 s, 1352 m, 1282 s, 804 s; MS(ES): m/z=288.0 [M+H].sup.+; HRMS (ES) calcd. for C.sub.11H.sub.15NI [M+H].sup.+: 288.0246, found 288.0242.

2.3 6-Iodo-4,4-dimethyl-1-[3-(trimethylsilyl)prop-2-yn-1-yl]-1,2,3,4-tetrahydroquinoline (12)

(59) ##STR00059##

(60) K.sub.2CO.sub.3 (1.39 g, 10.08 mmol) was added to a solution of compound 11 (2.07 g, 7.20 mmol) in anhydrous DMF (25 mL) under Ar and the resultant slurry was stirred for 1 h. Compound 10 (1.65 mL, 10.08 mmol) was added, and the solution was stirred at RT for 72 h. The solution was diluted with H.sub.2O, and extracted with EtOAc (3×). The organics were washed with sat. NH.sub.4Cl, H.sub.2O and brine, dried (MgSO.sub.4) and evaporated to give a crude yellow oil. This was purified by SiO.sub.2 chromatography (hexane:EtOAc, 96:4, with 1% Et.sub.3N as eluent) to give compound 12 as a light yellow oil (2.71 g, 95%): .sup.1H NMR (700 MHz; CDCl.sub.3) δ 0.13 (s, 9H), 1.26 (s, 6H), 1.74-1.78 (m, 2H), 3.27-3.31 (m, 2H), 3.99 (s, 2H), 6.49 (d, J=8.7 Hz, 1H), 7.33 (dd, J=8.7, 2.2 Hz, 1H), 7.42 (d, J=2.2 Hz, 1H); .sup.13C NMR (176 MHz, CDCl.sub.3) δ 0.2, 30.7, 32.4, 36.9, 42.0, 45.6, 78.8, 89.0, 101.4, 114.7, 134.6, 135.5, 135.5, 143.3; IR (neat) ν.sub.max/cm.sup.−12958 w, 2925 w, 2856 w, 2169 w, 1584 m, 1491 m, 1332 m, 1248 m, 838 s; MS(ES): m/z=288.0 [M+H].sup.+; HRMS (ES) calcd. for C.sub.17H.sub.25SiNI [M+H].sup.+: 398.0801, found 398.0797.

2.4 Methyl (2E)-3-[4-(2-{4,4-dimethyl-1-[3-(trimethylsilyl)prop-2-yn-1-yl]-1,2,3,4-tetrahydroquinolin-6-yl})ethynyl)phenyl]prop-2-enoate (13)

(61) ##STR00060##

(62) Compound 12 (1.61 g, 4.05 mmol) was dissolved in triethylamine (35 mL), and the resultant solution was degassed by sonication under vacuum, before the atmosphere was replaced with Ar (5×). Pd(PPh.sub.3).sub.2Cl.sub.2 (0.28 g, 0.405 mmol), CuI (0.077 g, 0.405 mmol) and compound 8 (0.79 g, 4.25 mmol) were then added under Ar. The resultant suspension was stirred at RT for 72 h. The suspension was diluted with hexane and passed through a thin Celite/SiO.sub.2 plug (eluting with hexane, then Et.sub.2O). The extracts were washed with sat. NH.sub.4Cl (3×), brine, dried (MgSO.sub.4) and evaporated to give the coupling a crude yellow oil. This was purified by SiO.sub.2 chromatography (hexane:EtOAc, 9:1, with 1% Et.sub.3N as eluent) to give compound 13 as a thick yellow oil (1.25 g, 68%): .sup.1H NMR (700 MHz, CDCl.sub.3) δ 0.12 (s, 9H), 1.30 (s, 6H), 1.77-1.80 (m, 2H), 3.34-3.37 (m, 2H), 3.81 (s, 3H), 4.05 (s, 2H), 6.43 (d, J=16.0 Hz, 1H), 6.68 (d, J=8.6 Hz, 1H), 7.27 (dd, J=8.5, 2.0 Hz, 1H), 7.38 (d, J=2.0 Hz, 1H), 7.45-7.52 (m, 4H), 7.67 (d, J=16.0 Hz, 1H); .sup.13C NMR (176 MHz, CDCl.sub.3) δ 0.2, 30.5, 32.3, 36.9, 42.0, 45.7, 51.9, 87.3, 89.0, 93.8, 101.3, 110.5, 112.2, 118.0, 126.5, 128.2, 129.6, 130.8, 131.8, 132.5, 133.4, 144.1, 144.4, 167.6; IR (neat) ν.sub.max/cm.sup.−1 3042 w, 2957 w, 2927 w, 2858 w, 2195 w, 1718 s, 1634 m, 1595 s, 1515 s, 1324 s, 1170 s, 842 s; MS(ES): m/z=456.2 [M+H].sup.+; HRMS (ES) calcd. for C.sub.26H.sub.26NO.sub.2 [M+H].sup.+: 456.2359, found 456.2345.

2.5 Methyl (2E)-3-(4-2-[4,4-dimethyl-1-(prop-2-yn-1-yl)-1,2,3,4-tetrahydroquinolin-6-yl]ethynyl)phenyl)prop-2-enoate (14) (DC473)

(63) ##STR00061##

(64) Compound 13 (1.20 g, 2.64 mmol) was dissolved in THF (30 mL), and cooled to −20° C. Tetrabutylammonium fluoride (1.0 M in THF, 2.90 mL, 2.90 mmol) was then added dropwise and the resultant solution stirred at −20° C. for 1 h, after which H.sub.2O was added, and the solution extracted with EtOAc (3×). The organics were washed with brine, dried (MgSO.sub.4) and evaporated to give a crude solid. This was purified by SiO.sub.2 chromatography (hexane:EtOAc, 8:2, with 1% Et.sub.3N as eluent) to give compound 14 as a yellow oil that slowly crystallises to give an orange solid (0.83 g, 82%): m.p.=101-102° C.; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 1.30 (s, 6H), 1.76-1.83 (m, 2H), 2.16-2.20 (m, 1H), 3.33-3.39 (m, 2H), 3.81 (s, 3H), 4.05 (d, J=2.4 Hz, 2H), 6.43 (d, J=16.0 Hz, 1H), 6.68 (d, J=8.6 Hz, 1H), 7.28 (dd, J=8.5, 1.9 Hz, 1H), 7.39 (d, J=1.9 Hz, 1H), 7.43-7.54 (m, 4H), 7.67 (d, J=16.0 Hz, 1H); .sup.13C NMR (101 MHz, CDCl.sub.3) δ 30.7, 32.3, 36.9, 41.0, 45.9, 51.9, 72.0, 79.3, 87.3, 93.7, 110.9, 111.9, 118.0, 126.5, 128.2, 129.8, 130.8, 131.9, 132.6, 133.5, 143.9, 144.4, 167.6; IR (neat) ν.sub.max/cm.sup.−13288 w, 2954 w, 2927 w, 2861 w, 2194 m, 1716 s, 1633 m, 1595 s, 1515 s, 1496 m, 1324 s, 1170 s, 830 s; MS(ES): m/z=384.4 [M+H].sup.+; HRMS (ES) calcd. for C.sub.26H.sub.26NO.sub.2 [M+H].sup.+: 384.1964, found 384.1963.

2.6 (2E)-3-(4-{2-[4,4-Dimethyl-1-(prop-2-yn-1-yl)-1,2,3,4-tetrahydroquinolin-6-yl]ethynyl}phenyl)prop-2-enoic Acid (15) (DC474)

(65) ##STR00062##

(66) Compound 14 (0.824 g, 2.15 mmol) was dissolved in THF (25 mL), 20% NaOH (2.5 mL) added, and the resultant solution was stirred under reflux for 40 h. The mixture was cooled, acidified to pH 1 with 5% HCl, diluted with EtOAc, washed with sat. NH.sub.4Cl, H.sub.2O and brine, dried (MgSO.sub.4) and evaporated to give a crude yellow solid which was recrystallised from MeCN to give compound 15 as an orange crystalline solid (0.50 g, 63%): m.p.=193-195 OC (decomposition); .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO) δ 1.24 (s, 6H), 1.69-1.75 (m, 2H), 3.12 (t, J=2.3 Hz, 1H), 3.27-3.32 (m, 2H), 4.14 (d, J=2.3 Hz, 2H), 6.55 (d, J=16.0 Hz, 1H), 6.74 (d, J=8.7 Hz, 1H), 7.23 (dd, J=8.5, 2.0 Hz, 1H), 7.35 (d, J=2.1 Hz, 1H), 7.49-7.53 (m, 2H), 7.59 (d, J=16.0 Hz, 1H), 7.68-7.72 (m, 2H), 12.44 (s, 1H); .sup.13C NMR (101 MHz, (CD.sub.3).sub.2SO) δ 30.3, 31.6, 36.0, 44.8, 74.3, 79.8, 87.1, 93.4, 109.2, 112.2, 119.7, 124.9, 128.4, 129.1, 130.2, 131.2, 132.1, 133.5, 143.0, 143.9, 167.5; IR (neat) ν.sub.max/cm.sup.−1 3278 w, 2962 w, 2920 w, 2847 w, 2196 w, 1684.9, 1623n, 1515n, 1217 s, 836 w; MS(ES): m/z=370.8 [M+H].sup.+; HRMS (ES) calcd. for C.sub.25H.sub.24NO.sub.2 [M+H].sup.+: 370.1807, found 370.1804.

Example 3

3.1 Example Click Conjugation of Compound 15 (DC474) with Benzyl Azide to Give Methyl (2E)-3-[4-(2-{1-[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]-4,4-dimethyl-1,2,3,4-tetrahydroquinolin-6-yl}ethynyl)phenyl]prop-2-enoate (16)

(67) ##STR00063##

(68) Compound 15 (75 mg, 0.203 mmol) and benzyl azide (0.028 mL, 0.223 mmol) were suspended in H.sub.2O/BuOH (1:1, 1 mL), before sodium ascorbate (1M in H.sub.2O, 0.020 mL, 0.02 mmol) and CuSO.sub.4.5H.sub.2O (5 mg, 0.02 mmol) were added. The suspension was stirred vigorously for 16 h, before being diluted with cold H.sub.2O, and the precipitated solid was filtered and dried to give compound 16 as a yellow solid (59 mg, 57%). .sup.1H NMR indicated 87% conversion to compound 16.

Example 4

(69) 4.1 Cell Culture and Media

(70) Immortalised HaCaT cell lines were maintained in Dulbecco's Modified Eagle's Medium (5 mL) containing 10% Foetal Bovine Serum and 1% Penicillin Streptomycin antibiotic.

(71) 4.2 Fixed Cell Imaging

(72) HaCaT human keratinocyte cells were seeded onto acid washed glass coverslips and treated, with a single dose, of either a 1 μM or a 10 μM solution of the compound. They were left in this compound containing media for 2 to 72 h before live-staining with Mitotracker red or BODIPY® TR Ceramide complexed to BSA. The cells were then fixed with 4% PFA, and mounted onto glass slides with Mowiol®. Images were taken on a Zeiss LSM 880 microscope. Note: these experiments were also carried out using a mouse embryonic fibroblast cell line, J2. However, some background fluorescence was present in J2 s at the same wavelengths as the compound. Therefore, HaCaTs were favoured for imaging.

(73) Treatment with Compounds

(74) An acid washed glass coverslip was placed in the bottom of each well of a 24 well plate. In each well, HaCaT cells (12,000 approx.) were seeded in serum containing media (1 mL) and allowed 24 h to settle before being treated. The media was replaced with media containing the compound/DMSO control (1 mL). For the 10 μM wells, compound (10 μL, 1 mM) was added to serum containing media (9990 μL). For the 1 μM wells, compound (1 μL, 1 mM) was added to serum containing media (9999 μL). The cells were stained and fixed 72 h or 48 h after being treated with the compound. The appropriate vehicle only experimental controls were included.

(75) MitoTracker® red staining: The cells were incubated for 30 min with MitoTracker® Red diluted in serum containing media (1 mL, 0.1 μM). They were then rinsed twice with PBS before incubation with PFA (0.5 mL, 4%), for 5 min. Cells were rinsed in PBS before mounting onto glass slides with Mowiol®.

(76) BODIPY® TR Ceramide complexed to BSA staining: The cells were incubated for 30 min at 4° C. with BODIPY® TR Ceramide complexed to BSA diluted in PBS (1 mL, 5 μM). They were then rinsed twice with cold PBS before incubation in fresh PBS at 37° C. for a further 30 min. The cells were fixed by incubation with PFA (0.5 mL, 4%), for 5 min. Cells were rinsed in PBS before mounting onto glass slides with Mowiol®.

(77) Imaging: Images taken using the Zeiss LSM 880 AxioObserver confocal microscope, with the Plan-Apochromat 63x/1.4 Oil DIC M27 objective lens. Acquisition settings can be found in table 1.

(78) TABLE-US-00001 TABLE 1 Acquisition Settings Green Channel (Compound) Red Channel (Stain) Detection wavelength/nm 431-560 600-735 Excitation wavelength/nm 405 594 Emission wavelength/nm 460 668

(79) TABLE-US-00002 TABLE 1 Absorption and fluorescence emission maxima of dyes, in nm, determined in methanol BODIPY ® TR Ceramide complexed to BSA MitoTracker ® Red Absorbance wavelength/nm 589 max 579 max 300-640 range 300-630 range Emission wavelength/nm 617 max 599 max 575-724 range 560-700 range

(80) TABLE-US-00003 TABLE 3 Absorbance and emission spectra data DC473 DC474 DC324 (In chloroform, (In chloroform, (In chloroform, excitation excitation excitation at 400 nm) at 380 nm) at 300 nm) Absorbance 400 max 380 max 380 max wavelength/ 275-475 range 275-450 range 275-450 range nm Emission 560 max 520 max 540 max wavelength/ 450-700 range 440-680 range 450-700 range nm

Example 5

(81) Living Cell Imaging

(82) 5.1 Cell Death on Zeiss Live Cell Observer

(83) One day prior to imaging, HaCaT or J2 cells (20,000 approx.) were seeded into each well of a 6 well or 24 well plastic plate. Cells were allowed to incubate overnight in serum containing media. One to four hours before imaging the normal media was replaced with compound containing media, 1 mL per well. The cells were imaged in a 37° C. heated chamber and supplied with 5% CO.sub.2. Zeiss Live Cell Observer microscope: OSRAM 1×HBO 103 W/2 100 Watt Mercury Bulb, DAPI Excitation filter=335-385 nm, DAPI Emission filter=420-470 nm. Note: Cells were imaged in DMEM media which contains phenol red.

(84) 5.2 Cell Death on Zeiss 880 Confocal

(85) One day prior to imaging, HaCaT cells (4000 approx.) were seeded into each well of an 8 well glass slide. Cells were allowed to incubate overnight in serum containing media. One hour before imaging the normal media was replaced with compound containing media, 1 mL per well. The cells were imaged in a 37° C. heated chamber using the Zeiss LSM 880 with Airyscan confocal laser scanning microscope. Note: Cells were imaged in DMEM media which contains phenol red.

(86) 5.3 ROS Staining

(87) One day prior to imaging, HaCaT cells (20,000 approx) were seeded into each well of a 24 well plastic plate. Cells were allowed to incubate overnight in serum containing media. Three hours prior to imaging, the media in each well was replaced with fresh serum containing media which included a 1:500 dilution of Superoxide Detection Reagent, CellRox. Note: A 1:2500 dilution was originally tried and was less effective. The cells were incubated with the ROS stain at 37° C. for one hour. An hour and a half before imaging, the media in the negative control well was replaced with N-acetyl-L-cysteine diluted in serum containing media (1 mL, 10 mM). One hour before imaging the media in all but one of the other wells was replaced with compound containing media, 1 mL per well. Thirty minutes prior to imaging, the media in the positive control well was replaced with pyocyanine diluted in serum containing media (1 mL, 500 μM). Pyocyanine induces ROS within 20-30 minutes. The cells were imaged in a heated chamber and supplied with 5% CO.sub.2. The cells were imaged in a 37° C. heated chamber using the Zeiss LSM 880 with Airyscan confocal laser scanning microscope. Cells were irradiated with 405 nm laser to activate compounds and ROS dye was excited with 633 nm laser Note: Cells were imaged in DMEM media which contains phenol red.

(88) 6. Results

(89) 6.1 Initial Screening of Compounds

(90) Following exposure to UV light, no membrane blebbing was observed in ATRA, EC23 and DMSO treated cells. DC324 and DC473 induced membrane blebbing and cell death following exposure to UV light.

(91) 6.2 DC324

(92) DC324 was not observed filling the nucleus.

(93) Referring to FIG. 3: DC324 localised to one side of the nucleus in large patches of cells. DC324 emits above zero fluorescence in the 405-640 range. The red channel was recorded 600-735 nm. This image shows signs of DC324 emission in the red channel.

(94) Referring to FIG. 4: DC324 co-imaged with BODIPY® Golgi stain. Some regions show overlap between the Golgi and DC324. DC324 appears to be entering the nucleus in one of these cells. Cells fixed after 72 hours in 1 μM DC324.

(95) 6.3 Cell Death

(96) 6.3.1 UV Light

(97) Referring to FIG. 5: DC324 was the most rapid inducer of cell death. A single 10 s treatment with UV light was sufficient to induce necrosis in 10 μM DC324 treated cells, within two hours using a 10× objective lens and at Phase 0. Part C shows healthy cells from the same well, in an area away from the site of UV exposure. The localised effect of the UV light indicates that the cell death is not due to heat transfer.

(98) Referring to FIG. 6: Cell death observations were consistent across different cell lines. DC324 treated J2 cells, a mouse fibroblast cell line, displayed membrane blebbing within two hours following a single UV treatment. UV untreated cells appear healthy after 26 hours in the compound in the same well as the treated area. This suggests that the cause of death is localised, not due to heat transfer, and due to the combined effect of the UV light and the DC324. A 10× objective lens at Phase 0 was used.

(99) 6.3.2 405 nm Light

(100) Images of 10 μM compound treated cells were taken every twenty minutes and at each time point a treatment of 405 nm light at 50% laser strength was applied. Cell death was not as rapid under the 405 nm laser light as under the UV light, only DC324 was significantly different to the DMSO control.

(101) Referring to FIG. 7: The majority of DMSO treated cells appeared healthy after 47 exposures to 405 nm laser, at 50% strength. No membrane blebbing was observed.

(102) Referring to FIG. 8: The majority of ATRA treated cells appeared healthy after 47 exposures to 405 nm laser, at 50% strength. No membrane blebbing was observed.

(103) Referring to FIG. 9: Membrane blebbing was induced in DC324 treated cells by 405 nm laser, at 50% strength. The first bleb appeared after 21 exposures, however, the majority of cells presented membrane blebbing between 30 and 40 exposures. The cell death induced by 405 nm light appeared more characteristic of apoptosis than the cell death caused by UV light. Cells appeared to shrink rather than expand, and apoptotic bodies are visible.

(104) Referring to FIG. 10: DC324 treated cells putatively producing apoptotic bodies. This is an image from the same experiment discussed previously. It was taken at the 37.sup.th exposure to 405 nm light, 740 minutes from the first exposure, and 770 minutes after the addition of DC324. A number of the cells appear to be releasing small spheres from all edges.

(105) Referring to FIG. 11: DC324 fluorescence from treated HaCaT cells, imaged with 405 nm laser light, at 50% strength. Initially, DC324 is brightest around the edges of the cells. DC324 is then concentrated to the centre of the cells and is also visible in the membrane blebs.

(106) 6.4 Dose Response

(107) The speed of death was tested at a number of concentrations in order to determine an estimate for the EC.sub.50 (half maximal effective concentration) value. Morphological changes were recorded using time lapse imaging at 5 minute intervals. The time taken between 10 s of UV light treatment and the first sign of damage to the cell membranes was recorded. Each cell in the field of view was treated as an individual data point, before taking an average of the time for each concentration.

(108) Referring to FIG. 12: The dose-dependent response of HaCaT cells treated with DC324 and DC473 and 10 seconds of UV. UV exposure was administered through the DAPI filter and a 20× objective lens, phase 0. EC.sub.50 values for both compounds were determined based on the average viable fraction of the cellular population 24 hours after the initial irradiation event of 0.20 (±0.007) μM and 0.18 (±0.006) μM for DC324 and DC473, respectively.

(109) 6.5 ROS Staining

(110) Referring to FIG. 13: Control treatments stained for superoxide after exposure to UV. Staining for superoxide production under the rhodamine filter. Fluorescence in the negative control indicates possible background fluorescence from HaCaT cells. One cell in the negative control is induced to produce increased fluorescence post-UV imaging. This could be due to an increase in superoxide production in an apoptotic cell, or merely due to the change in shape of the cell as it dies. The positive control treated cells were visibly rounded and dying when imaged under phase. Therefore, the reactive oxygen species had already been produced before the imaging began. However, the superoxide stain was brighter for the positive control, indicating that superoxide was still present. Several smaller patches of the DMSO treated cells showed superoxide production prior to exposure to UV, these cells were most affected by the DAPI treatment. Compared with the initial picture taken 60 seconds after UV treatment, the image taken 260 seconds after UV treatment shows that there is an increase in fluorescence in a defined circle in the centre of these cells.

(111) Referring to FIG. 14: Active compounds stained for superoxide after exposure to DAPI. As seen with the DMSO control, the ATRA treated cells which already had some evidence of superoxide production before UV treatment produced an increased level of fluorescence post exposure to UV. As with the DMSO control, this fluorescence formed a defined circle in the centre of the cell, 260 seconds after exposure to UV. For all treatments, healthy normal cells produced a brighter fluorescence in small circles in the cell.

(112) Referring to FIG. 15: DC324 compound treated cells stained for superoxide after UV exposure. The intensity of the small bright circles in the centre of the cells increased 60 seconds after UV treatment, compared with before exposure. This indicates superoxide production. This initial increase in brightness faded as the ROS dye photo-bleaches. Imaging the ROS dye under the rhodamine filter induced photo-bleaching much more rapidly with the DC324 treated cells than with the other compounds.

(113) In order to gain more insight into the mode of action leading to cell death, we examined the photo-initiated ROS production using the redox reactive dye, CellRox, which fluoresces in response to oxidation by reactive oxygen species. DC324-treated cells stimulated with 405 nm light exhibited a strong CellRox fluorescence signal after irradiation, particularly in intracellular organelles. CellRox fluorescence was quantified in the cell before and after irradiation; a steady increase in the production of ROS-stimulated relative fluorescence was observed immediately following irradiation in DC324-treated cells, but not cells treated with EC23, a synthetic retinoid analogue of DC324 with ATRA-like biochemical properties, which acted as a negative control, to demonstrate that co-treatment with any retinoid or near ATRA-like analogue and light does not kill cells.

(114) Referring to FIG. 16 DC473 compound treated cells after 2 hours and 24 hours UV exposure.

(115) Referring to FIG. 17 a comparison is made of DC324 and DC473 compound treated cells.

(116) Referring to FIG. 18 a comparison is made between the absorption spectra of DC324, DC473 and DC474 in CHCl.sub.3.

(117) Referring to FIG. 19 a comparison is made between the emission spectra of DC324, DC473 and DC474 in CHCl.sub.3.

(118) Referring to FIG. 20 a comparison is made between the absorption spectra of DC473, DC474 and click-conjugate compound 16 in CHCl.sub.3.

(119) Referring to FIG. 21 a comparison is made between the emission spectra of DC473, DC474 and click-conjugate compound 16 in CHCl.sub.3.

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