Photoresponsive Nutlin Derivatives and Uses Thereof

Abstract

The invention relates to the field of medicine and medicinal chemistry, more in particular to the design, manufacture and use of anti-cancer drugs that can be activated by an external stimulus that can be applied in a spatiotemporal fashion. Provided herein is a compound having the chemical structure

##STR00001##

or a pharmaceutically acceptable salt thereof.

Claims

1. A compound having the chemical structure ##STR00017## wherein V is —H or —F; W is —F, —Cl or —Br; X is selected from the group consisting of H, F, Cl, Br, I, cyano, nitro, ethynyl, cyclopropyl, methyl, ethyl, isopropyl, vinyl and methoxy; Y is one to four group(s) independently selected from the group consisting of —H, —F, —Cl, —Br, —I, —CN, —OH, nitro, lower alkyl, cycloalkyl, lower alkoxy, lower alkenyl, cycloalkenyl and lower alkynyl; Z is Cl, F, Br, I or lower alkoxy; R.sub.1 is —H or —CH.sub.3 R.sub.2 is O, S or C(CN).sub.2 or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, wherein X is selected from the group consisting of F, Cl, Br and I, preferably wherein X is —Cl or —F.

3. The compound according to claim 1, wherein Y is selected from —H, —F and —Cl.

4. The compound according to claim 1, wherein Z is Cl or —OCH.sub.3.

5. The compound according to claim 1, wherein W is —Cl, and/or wherein V is —F.

6. The compound according to claim 1, wherein R.sub.1 is —CH.sub.3.

7. The compound according to claim 1, wherein R.sub.2 is O.

8. A compound of the formula ##STR00018## or a pharmaceutically acceptable salt thereof.

9. A pharmaceutical composition comprising a compound according to claim 1 and a pharmaceutically acceptable carrier, vehicle or diluent.

10. The compound according to claim 1 for use as a photo-activatable inhibitor of the interaction between MDM2 and p53.

11. The compound according to claim 1 for use in a method of treating in a subject a disorder mediated by a p53-MDM2 interaction.

12. The compound for use according to claim 11, wherein said disorder is cancer, in particular a solid tumor, more particularly a breast, colon, lung or prostate tumor.

13. The compound for use according to claim 11, wherein said method comprises administering said compound to a subject in need thereof, followed by selectively illuminating with light, preferably λ≥400 nm, at a predetermined time and/or location of the body of said subject, thereby converting said compound into a biologically active agent.

14. The compound for use according to claim 13, comprising the use of an optical probe to illuminate a location within the body of said subject.

15. The compound for use according to claim 10, wherein said compound is of the formula ##STR00019## or a pharmaceutically acceptable salt thereof.

16. A method for treating a disorder mediated by a p53-MDM2 interaction in a subject, comprising administering to the subject a therapeutically effective amount of a compound according to claim 1.

17. The method according to claim 16, wherein said disorder is cancer, in particular a solid tumor, more particularly a breast, colon, lung or prostate tumor.

18. The method according to claim 16, wherein said method comprises administering said compound to a subject in need thereof, followed by selectively illuminating with light, preferably λ≥400 nm, at a predetermined time and/or location of the body of said subject, thereby converting said compound into a biologically active agent.

19. The method according to claim 18, comprising the use of an optical probe to illuminate a location within the body of said subject.

20. The method according to claim 16, wherein said compound is of the formula ##STR00020## or a pharmaceutically acceptable salt thereof.

Description

LEGEND TO THE FIGURES

[0047] FIG. 1: Strategy towards photocleavable nutlin derivatives. a) Idasanutlin, a potent MDM2 inhibitor allowing the stabilization of p53 levels in tumor cells. b) Molecular docking showcases the possible interaction with Lys90 as a potential site to alter the activity (PDB: 4JRG)..sup.29 c) Irradiation of PPG-idasanutlin led to the formation of idasanutlin and PPG(6) as the sole products. d) Absorption spectra of PPG-idasanutlin, idasanutlin and PPG (6) in buffer. (TRIS, BIS-TRIS, MES, NaOAc, 25 mM each, pH=7.0) at 20 μM concentration. e) UV-vis spectra of PPG-idasanutlin upon exposure to 400 nm light showing a clean photochemical conversion (isosbestic point at 350 nm) to the desired products.

[0048] FIG. 2: A schematic representation of the principle behind phototriggered p53 stabilization. The photonutlin compound (PPG-idasanutlin) is not able to inhibit the MDM2-p53 protein-protein interaction, which results in p53 ubiquitylation and degradation. Irradiation with 400 nm light releases the active inhibitor idasanutlin which prevents MDM2-p53 binding and as a consequence increases the p53 level, leading to senescence or cell death.

[0049] FIG. 3: Functional p53 induction upon λ=400 nm irradiation in PPG-idasanutlin treated cells, a) RPE-1 cells were treated with indicated compounds (all 10 μM final) and fixed 4 h after 5 min (−/+400 nm) irradiation. Anti-p53 staining indicates p53 protein expression in the nucleus. DNA stained by DAPI and actin staining shows the cytoskeleton of the cell. b) Quantification of the mean p53 intensity per nucleus in cells treated as in (a). Error bars represent mean +sd, ****P<0.0001 (unpaired t-test), Dots represent individual cells, n>125 cells per condition combined from 2 independent experiments. c) Representative western blot showing p53 protein levels in cells 4 h after addition of DMSO or PPG-idasanutlin and irradiation for indicated time periods. Hsp90 is used as a loading control. d) Selective outgrowth disadvantage in RPE-1 cells 6 days after PPG-idasanutlin treatment +400 nm irradiation for 5 min. e) Representative Western blot showing p53 protein levels in three cell lines (U2OS, RKO, BJhTert) 4 h after indicated treatments. f) Selective outgrowth inhibition in indicated cell lines 6 days after PPG-idasanutlin treatment +400 nm irradiation for 5 min. In all the experiments <1% DMSO was used.

[0050] FIG. 4: Cell outgrowth experiments showing that PPG 6 (the photoproduct formed upon photocleavage of photonutlin) does not perturb cellular outgrowth.

[0051] FIG. 5: Spatiotemporal control of PPG-idasanutlin. a) Schematic representation of microwell set-up for laser irradiation of individual RPE-1 cells to activate PPG-idasanutlin. Laser target area (represented. by red circle) for single pulse (0.1 sec irradiation at 5 um interspaced position) indicated with scale. Individual irradiated cells followed by measuring nuclear p53-venus levels (fluorescence) every 15 min for 3 h after laser irradiation. Approximately 200 cells in each microwell. b) Percentage of cells that divide within 8 h after indicated treatments. Mean±sem of three independent experiments. Error bars indicate 95% confidence intervals. c, d) p53-venus fluorescent signal in individual RPE-1 cells tracked over time after indicated treatments as represented in (a). Line-graphs represent mean of individual cells. n>42 cells per condition pooled from three independent experiments. ***p<0.005, ****p<0.0001 significance in 2-way anova interaction score.

EXPERIMENTAL SECTION

[0052] Materials and Methods

[0053] General. All chemicals for synthesis were Obtained from commercial sources and used as received unless stated otherwise.

[0054] Thin Layer Chromatography (TLC) was performed using commercial Kieselgel 60, F254 silica gel plates with fluorescence-indicator UV.sub.254 (Merck, TLC silica gel 60 F.sub.254). For detection of components, UV light at λ=254 nm or λ=365 nm was used. Alternatively, oxidative staining using aqueous basic potassium permanganate solution (KMnO.sub.4) or aqueous acidic cerium phosphomolybdic acid solution (Seebach's stain) was used. Flash chromatography was performed on silica gel (Silicycle Siliaflash P60, 40-63 mm, 230-400 mesh). Drying of solutions was performed with MgSO.sub.4 and volatiles were removed with a rotary evaporator. Nuclear Magnetic Resonance spectra were measured with an Agilent Technologies 400-MR (400/54 Premium Shielded) spectrometer (400 MHz). All spectra were measured at room temperature (22-24° C.). Chemical shifts for .sup.1H- and .sup.13C-NMR measurements were determined relative to the residual solvent peaks in ppm (δ.sub.H 7.26 for CHCl.sub.3, 2.50 for DMSO and 2.05 ppm for Acetone, δ.sub.C 77.16 for CHCl.sub.3 and 39.52 for DMSO). The following abbreviations are used to indicate signal multiplicity: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; brs, broad signal. All .sup.13C-NMR spectra are .sup.1H-broadband decoupled. High-resolution mass spectrometric measurements were performed on a Thermo scientific LTQ Orbitrap XL with ESI ionization. For spectroscopic measurements, solutions in Uvasol® grade solvents were measured in a 10 mm quartz cuvette. UV-Vis absorption spectra were recorded on an Agilent 8453 UV-Visible absorption Spectrophotometer.

[0055] Cell Culture

[0056] hTert-immortalized retinal pigment epithelium (RPE-1) cells (ATCC) and hTert-immortalized BJ cells were maintained in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12, Gibco) supplemented with ultraglutamine, penicillin/streptomycin and 10% fetal calf serum. RPE-1 cells with a fluorescently tagged version of p53 (p53-venus) were a kind gift from the Lahav lab..sup.3 RKO colon carcinoma cells were maintained in Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with ultraglutamine, penicillin/streptomycin and 10% fetal calf serum. U2OS cells were maintained in Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with ultraglutamine, penicillin/streptomycin and 6% fetal calf serum.

[0057] Immunofluorescent Staining and Live Cell Imaging

[0058] RPE-1 cells were plated on coverslips at equal density and treated with indicated drugs before −/+irradiation (400 nm for 5 min). For immune fluorescent staining, cells were fixed 4 h after irradiation by wash out of the medium, single wash in phosphate-buffered saline (1× PBS) and incubation in 3.7% formaldehyde for 5 min. Next, fixed cells were permeabilized with 0.2% TritonX in 1× PBS for 5 min before blocking in 3% fetal bovine serum (BSA) in 1× PBS supplemented with 0.1% Tween (PBST) for 1 h (all at room temperature (RT)). Cells were incubated overnight at 4° C. with primary antibody in PBST with 3% BSA, washed three times with PBST, and incubated with secondary antibody and DAPI in PBST with 3% BSA for 2 h at RT. After a final three time wash with PBST coverslips were mounted on microscopic analysis slides using ProLong Gold antifade reagent (Thermo Fisher). The following antibodies were used: anti-p53 (sc-126, Santa Cruz, 1/1000), phalloidin (A12380, Molecular Probes, 1/1000) and goat anti-mouse/Alexa. 488 (A11029, Molecular Probes, 1/1000).

[0059] For live cell imaging, cells were grown in picovitro microwells covered by a silicon membrane.sup.4 in Leibovitz's L-15 (Gibco) CO.sub.2-independent medium supplemented with ultraglutamine, penicillin/streptomycin and 10% fetal calf serum. Images for both fixed slides and live cell imaging were obtained using a Delta Vision Elite (applied precision) equipped with a 60× 1.6 NA or 10× 0.75 NA lens (Olympus) and cooled CoolSnap CCD camera. Directed laser irradiation was performed using a brief (0.1 s) pulse of a 405 nm laser on the Delta Vision Elite microscope equipped with a X4 laser module (Applied Precision). Image analysis was done using ImageJ software. Automated single cell analysis from live cell imaging was done as described before..sup.5

[0060] Western Blot

[0061] RPE-1 cells were plated at equal density in 6-well plates followed by indicated treatments −/+irradiation (400 nm for 5 min) 24 h later. Cells were fixed and collected 4 h after treatment by wash out of the medium, single wash in 1× PBS followed by addition of laemmli buffer (4% SDS, 20% glycerol and 0.125 M Tris HCl). Equal amounts of proteins were separated by SDS-PAGE electrophoresis followed by semi-dry transfer to a nitrocellulose membrane. Membranes were blocked in 5% milk in PBST for 1 h at RT before overnight incubation with primary antibody in PBST with 3% BSA at 4° C. Membranes were washed 3 times with PBST followed by incubation with secondary antibody in PBST with 5% milk for 2 h at RT. Antibody staining was visualized using ECL (GE Healthcare). The following primary antibodies were used: anti-p53 (sc-126, Santa Cruz, 1/1000), anti-Cdk4 (C-22) (se-260, Santa Cruz, 1/1000). Peroxidase-conjugated-goat anti-mouse (P0447 DAKO, 1/1000) and goat anti-rabbit (P448 DAKO, 1/1000) were used as secondary antibodies.

[0062] Clonogenic Outgrowth

[0063] Cells were plated at equal amounts (1000 cells per well) in a 24-wells plate followed by indicated treatments −/+irradiation (400 nm for 5 min) 24 h later. Cells were cultured under normal cell culture conditions for 6 days to allow colony outgrowth. Plates were fixed using 99.8% ice-cold methanol (Honeywell) for 10 min at RT. After a 1 time wash in H.sub.2O, cells were incubated in 0.2% crystal violet (Sigma) in H.sub.2O for at least 3 h at RT to stain cellular outgrowth.

Example 1: Synthesis of PPG-idasanutlin

[0064] Scheme 1 depicts the overall synthesis of PPG-idasanutlin.

##STR00006## ##STR00007## ##STR00008##

[0065] Example 1A: Synthesis of methyl 4-(2-bromoacetamido)-3-methoxybenzoate (1)

##STR00009##

[0066] To a solution of methyl 4-amino-3-methoxybenzoate (500 mg, 2.76 mmol) in DCM (18 mL) at 0° C. was added Et.sub.3N (768 μL, 5.52 mmol) under N.sub.2 atmosphere. Subsequently, 2-bromoacetyl-bromide (610 mg, 263 μL, 3.03 mmol) was slowly added and the reaction mixture was stirred for 45 min at 0° C. Subsequently, 1M aq. HCl (10 mL) was added and the solution was extracted with DCM (2×20 mL). The organic layers were washed with sat. aq. NaHCO.sub.3 (20 mL) and brine (20 mL) and dried (MgSO.sub.4). Evaporation of the volatiles in vacuo and recrystallization from EtOH and MeCN yielded the pure product (654 mg, 78%).

[0067] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.95 (s, 1H), 8.41 (d, J=8.5 Hz, 1H), 7.70 (dd, J=8.5, 1.8 Hz, 1H), 7.57 (d, J=1.8 Hz, 1H), 4.04 (s, 2H), 3.98 (s, 3H), 3.91 (s, 3H).

[0068] .sup.13C NMR (100 MHz, CDCl.sub.3): δ 166.6, 163.5, 147.7, 131.0, 125.9, 123.2, 118.4, 110.8, 56.1, 52.1, 29.5.

Example 1B: Synthesis of methyl 4-(2-aminoacetamido)-3-methoxybenzoate (2)

[0069] ##STR00010##

[0070] To an aqueous solution of NH.sub.3 (20 mL) was slowly (dropwise) added 1 (1.40 g, 4.63 mmol) in EtOH (45 mL) in 30 min. Subsequently, the reaction was stirred for 5 h at RT and extracted with DCM (3×50 mL). The organic layers were washed with brine (3×50 mL) and dried (MgSO.sub.4). Evaporation of the volatiles in vacuo yielded the pure product (945 mg, 86%).

[0071] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 10.00 (s, 1H), 8.52 (d, J=8.5 Hz, 1H), 7.69 (dd, J=8.4, 1.8 Hz, 1H), 7.56 (d, J=1.8 Hz, 1H), 3.96 (s, 3H), 3.90 (s, 3H), 3.52 (s, 2H). Data in accordance with literature.

[0072] .sup.13C NMR (100 MHz, CDCl.sub.3): δ 171.2, 166.7, 147.7, 131.6, 124.9, 123.3, 118.3, 110.7, 55.9, 52.0, 45.6.

Example 1C: Synthesis of methyl (E)-4-(2-((3,3-dimethylbutylidene)amino)acetamido)-3-methoxybenzoate (3)

[0073] ##STR00011##

[0074] To a solution 2 (300 mg, 1.26 mmol) in dry DCM (10 mL) under N2 atmosphere was added 3,3-dimethylbutanal (174 mL, 1.38 mmol) and MgSO4 (227 mg, 1.89 mmol) and the resulting suspension was stirred for 16 h at RT. Subsequently, the suspension was filtered and the residue washed with DCM (10 mL). Evaporation of the filtrate yielded the crude product (302 mg, 67%) as a yellow oil which was used in the next step without further purification.

[0075] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 9.45 (s, 1H), 8.52 (d, J=8.5 Hz, 1H), 7.84 (tt, J=5.6, 1.5 Hz, 1H), 7.69 (dd, J=8.4, 1.8 Hz, 1H), 7.55 (s, 1H), 4.21 (s, 2H), 3.93 (s, 3H), 3.90 (s, 3H), 2.27 (d, J=5.7 Hz, 2H), 1.03 (s, 9H).

Example 1D: Synthesis of (Z)-3-(3-chloro-2-fluorophenyl)-2-(4-chloro-2-fluorophenyl)acrylonitrile (4)

[0076] ##STR00012##

[0077] To a solution of 3-chloro-2-fluorobenzaldehyde (468 mg, 2.95 mmol) and 2-(4-chloro-2-fluorophenyl)acetonitrile (500 mg, 2.95 mmol) in EtOH (10 mL) and H.sub.2O (40 μL) was added NaOEt (10.0 mg, 0.15 mmol) and subsequently the reaction mixture was stirred for 3 h at RT. The resulting suspension was filtered and the precipitate was washed with EtOH. Evaporation in vacuo yielded the crude product which was dissolved in DCM (10 mL) and washed with brine (3×20 mL) and dried (MgSO4). Evaporation of the volatiles yielded the pure product (650 mg, 71%) as a white solid.

[0078] .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 7.97 (td, J=6.8, 1.4 Hz, 1H), 7.88 (s, 1H), 7.77 (ddd, J=8.6, 7.4, 1.6 Hz, 1H), 7.72 (t, J=8.5 Hz, 1H), 7.66 (dd, J=11.0, 2.1 Hz, 1H), 7.47 (ddd, J=8.4, 2.1, 0.8 Hz, 1H), 7.42 (td, J=8.0, 1.1 Hz, 1H),

[0079] .sup.19F NMR (376 MHz, DMSO-d.sub.6): δ −111.41 (dd, J=11.1, 8.6 Hz), −115.42 (t, J=7.0 Hz).

Example 1E: Synthesis of 7-(diethylamino)-2-oxo-2H-chromene-4-carbaldehyde (5)

[0080] ##STR00013##

[0081] A solution of 7-(diethylamino)-4-methyl-coumarin (500 mg, 2.16 mmol) and SeO.sub.2 (480 mg, 4.32 mmol) in p-xylene (20 mL) was heated to 150° C. for 16 h under N2 atmosphere in the dark. Subsequently, the solution was filtered while hot and concentrated in vacuo. Purification by column chromatography (DCM) yielded the pure product (223 mg, 42%) as an orange viscous oil.

[0082] .sup.1H NMR, (400 MHz, CDCl.sub.3): δ 10.03 (s, 1H), 8.31 (d, J=9.2 Hz, 1H), 6.63 (dd, J=9.2, 2.6 Hz, 1H), 6.53 (d, J=2.6 Hz, 1H), 6.45 (s, 1H), 3.43 (q, J=7.1 Hz, 4H), 1.22 (t, J=7.1 Hz, 6H).

Example 1F: Synthesis of 7-(diethylamino)-4-(1-hydroxyethyl)-2H-chromen-2-one (6)

[0083] ##STR00014##

[0084] To a solution of 5 (220 mg, 0.89 mmol) in dry THF (8 mL) under N2 atmosphere at −78° C. was slowly added MeMgBr in THF (3M, 534 μL, 1.60 mmol) and the reaction mixture was stirred for 2.5 h at −78° C. in the dark. Subsequently, sat. aq. NH.sub.4Cl was added (10 mL) and the mixture was allowed to warm to RT. The organic layer was separated and the aqueous layer extracted with EtOAc (2×10 mL). The combined organic layers were washed with brine (20 mL), dried and evaporated in vacuo to yield the crude product. Column chromatography (pentane:acetone, 3:1) yielded the pure product (125 mg, 54%) as an orange solid.

[0085] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.42 (d, J=9.0 Hz, 1H), 6.57 (dd, J=9.0, 2.7 Hz, 1H), 6.52 (d, J=2.6 Hz, 1H), 6.27 (d, J=0.9 Hz, 1H), 5.22-5.09 (m, 1H), 3.41 (q, J=7.1 Hz, 4H), 1.57 (d, J=4.7 Hz, 3H), 1.21 (t, J=7.1 Hz, 6H).

Example 1G: Synthesis of Idasanutlin (4-((2R,3S,4R,5S)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4-cyano-5-neopentylpyrrolidine-2-carboxamido)-3-methoxybenzoic acid (7))

[0086] ##STR00015##

[0087] A solution of CuOAc (0.56 mg, 4.58 μmol) and R-BINAP (3.0 mg, 4.81 μmol) in THF (5 mL) was slowly added to a suspension of 3 (302 mg, 0.95 mmol) and 4 (279 mg, 0.90 mmol) in THF (5 ml) under N.sub.2 atmosphere at RT. Subsequently, Et.sub.3N (123 mL, 0.88 mmol) was added and the resulting mixture was stirred for 5 h at RT. Next, THF (10 mL) was added and the resulting solution was washed twice with aq. NH.sub.4OAc (10 mL, 10% w/w) and brine (10 mL). Subsequently, the organic layers were evaporated and the crude product was dissolved in THF (7 mL) and EtOH (3 mL). 2.5M aq. NaOH (1 mL) was added and the mixture was stirred for 18 h at RT. The solution was acidified with AcOH to pH=6.0 and the volatiles were partially evaporated (2 mL). After addition of H.sub.2O (10 mL) the precipitate was filtered to give the crude product (493 mg, 89%) as an off-white solid. Subsequent enantio-enrichment and purification was performed by crystallization. The crude product (493 mg) was dissolved in THF (6 mL) and heated to 65° C. Subsequently EtOAc (2 mL) was added and the resulting solution was heated for 15 min after which it was cooled to RT and filtered. The residue was washed with EtOAc (5 mL) and the filtrate evaporated in vacuo. The crude product was dissolved in MeCN (7 mL) and heated to 80° C. after which it was slowly cooled to 10° C. The precipitate was filtered yielding the pure product (118 mg, 21%) as a white solid.

[0088] .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 12.85 (s, 1H), 10.45 (s, 1H), 8.35 (d, J=8.8 Hz, 1H), 7.71 (t, J=7.3 Hz, 1H), 7.62-7.50 (m, 4H), 7.44-7.28 (m, 3H), 4.66-4.52 (m, 2H), 4.37 (s, 1H), 3.91-3.85 (m, 4H), 1.63 (dd, J=14.2, 9.9 Hz, 1H), 1.25 (d, J=14.2 Hz, 1H), 0.96 (s, 9H).

[0089] HR-MS (ESI, [M+H].sup.+): Calcd. for C.sub.31H.sub.31Cl.sub.2F.sub.2N.sub.3O.sub.4: 616.1576; Found: 616.1575

Example 1H: Synthesis of PPG-idasanutlin (1-(7-(diethylamino)-2-oxo-2H-chromen-4-yl)ethyl 4-((2R,3S,4R,5S)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4-cyano-5-neopentylpyrrolidine-2-carboxamido)-3-methoxybenzoate ((8))

[0090] ##STR00016##

[0091] To a solution of 7 (100 mg, 0.16 mmol) and 6 (47 mg, 0.18 mmol) in dry DCM (3 mL) under N.sub.2 atmosphere was added EDC.HCl (37 mg, 0.19 mmol) and DMAP (5 mg, 0.04 mmol) at 0° C. Subsequently, the reaction mixture was allowed to warm to RT and stirred for 16 h. Subsequently, DCM (10 mL) was added and the resulting solution was washed with 0.5M aq. HCl (3×10 mL), sat. aq. NaHCO3 (2×10 mL) and brine (10 mL) and dried (MgSO4). All the volatiles were evaporated to yield the crude product (135 mg). Column chromatography (pentane:ethyl acetate, 3:1) yielded the pure product (55 mg, 40%) as a bright yellow solid.

[0092] .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 10.51 (s, 1H) 8.42 (d, J=7.0 Hz, 1H), 7.74-7.66 (m, 3H), 7.62 (s, 1H), 7.59-7.49 (m, 2H), 7.42-7.31 (m, 3H), 6.72 (d, J=9.3 Hz, 1H), 6.54 (s, 1H), 6.24 (q, J=6.6 Hz, 1H), 6.02 (s, 1H), 4.64-4.55 (m, 2H), 4.44-4.36 (m, 1H), 3.99-3.90 (m, 4H), 3.42 (q, J=6.7 Hz, 4H), 1.65 (d, J=6.8 Hz, 3H), 1.63-1.60 (m, 1H), 1.25 (d, J=14.3 Hz, 1H), 1.11 (t, J=6.9 Hz, 6H), 0.96 (s, 9H).

[0093] .sup.13C NMR (151 MHz, DMSO-d.sub.6): δ 171.8, 164.7, 161.3, 160.8, 159.1, 156.8, 156.7, 156.6, 155.2, 150.9, 148.1, 135.2, 132.1, 131.4, 130.5, 129.1, 126.3, 126.1, 125.7, 124.5, 123.5, 120.0, 119.6, 118.1, 117.7, 111.5, 109.4, 105.3, 103,8, 97.5, 68.8, 65.1, 63,7, 56.3, 50.6, 44,4, 44.3, 30.5, 29,9, 21.2, 12.7.

[0094] .sup.19F NMR (376 MHz, DMSO-d.sub.6): δ −108.31 (dd, J=12.2, 8.8 Hz), −120.95. HR-MS (ESI, [M+H]+): Calcd. for C.sub.46H.sub.47Cl.sub.2F.sub.2N.sub.4O.sub.6: 859.2835; Found: 859.2841

Example 2: Photochemical Behavior of Photonutlin

[0095] Following the synthesis of PPG-idasanutlin as described in Example 1, its photochemical behavior under physiological conditions was investigated, UV-vis spectroscopy and UPLC-MS measurements were performed in aqueous buffer at pH=7.0. Upon photodeprotection with λ=400 nm light, solely the formation of idasanutlin and hydroxycoumarin was observed. See FIG. 2. The rate of photocleavage proved to be high, allowing the major photorelease of idasanutlin within 5 min of irradiation, with a 0.1% quantum yield. Moreover, no significant spontaneous hydrolysis of PPG-idasanutlin for >24 h in buffer at room temperature was observed (data not shown). Together, these findings demonstrate the application of PPG-idasanutlin under physiological assay conditions using short irradiation times with biocompatible visible (>400 nm) light.

Example 3: Biological Activity of Photonutlin

[0096] Next, the biological activity of PPG-idasanutlin was investigated. Initial studies aimed at confirming a difference in p53 activation upon λ=400 nm light exposure after addition of the protected idasanutlin derivative (PPG-idasanutlin). To that end, non-transformed, p53-proficient retinal pigment epithelial cells (RPE-1) were treated with either DMSO (control), nutlin-3, idasanutlin or PPG-idasanutlin, followed by −/+irradiation with 400 nm light (FIG. 3). Immunofluorescent staining revealed a significant increase in nuclear p53 protein levels in cells 4 h after addition of nutlin-3 or idasanutlin, regardless of the irradiation with 400 nm light.

[0097] Importantly, treatment with PPG-idasanutlin only resulted in a significant increase in p53 protein level when these cells were irradiated with 400 nm light (photorelease of idasanutlin, see FIG. 3.a,b). To examine the level of control over the dose-response of idasanutlin (employing PPG-idasanutlin), p53 protein levels in RPE-1 cells were determined by immunostaining after both increasing duration of 400 nm light irradiation and varying doses of PPG-idasanutlin (FIG. 3.c). The clear dose-response dependent accumulation of p53 protein shows the highly effective light controllable dose responsiveness of the biological effect using PPG-idasanutlin (FIG. 3.c).

Example 4: Growth Inhibition by Photonutlin

[0098] Subsequently, the functional ability of PPG-idasanutlin to photocontrol growth of rapidly dividing cells was investigated. Colony outgrowth of RPE-1 cells treated with PPG-idasanutlin was selectively blocked after irradiation with 400 nm, while irradiation did not perturb outgrowth of DMSO treated cells (FIG. 3.d).This showcases the use of 400 nm light in living systems as a valid approach to photocontrol biological function. It should be emphasized that in the outgrowth experiment seen in FIG. 3.d, treatment of cells with PPG-idasanutlin without 400 nm irradiation did not show any growth inhibition, confirming the lack of inherent activity of the protected idasanutlin. Hence, PPG-idasanutlin has no functional effect on p53 stabilization nor compromises cellular outgrowth..sup.33

[0099] To verify whether (re-)activation of p53 by our light controllable PPG-idasanutlin is more generally applicable and not dependent on the non-transformed RPE-1 cells used in these experiments, additional non-transformed (BJ-hTert) and tumor (RKO (colon carcinoma), U2OS (osteosarcoma)) cell lines were included for follow-up analysis. Selective stabilization of p53, after treatment with photonutlin and light irradiation, was observed in all cell lines tested (FIG. 3.e). The light-controlled p53 activation invariably led. to a dramatic reduction in cellular outgrowth (FIG. 3.f) proving the possibility to control tumor cellular growth using photonutlin and light.

[0100] Furthermore, it could be demonstrated that the PPG photoproduct formed after photocleavage does not perturb cellular outgrowth (FIG. 4).

Example 5: Spatiotemporal Control of p53 Stabilization by Photonutlin

[0101] To demonstrate the spatiotemporal control of the designed system we sought to investigate the selective enhancement of p53 levels in individual RPE-1 cells within a cell population using light irradiation. Using RPE-1 cells that stably expressed a venus-tagged version of p53 (p53-venus), p53 protein accumulation could be tracked with high time-resolution in individual cells by live-cell microscopy. RPE p53-venus cells were grown in 620 μm wide microwells and a 405 nm laser was used to irradiate individual cells in the colony with a single 0.1 second pulse at 5 μm inter spaced positions to acquire micrometer precision (FIG. 5.a). To determine the functionality of the high spatiotemporal control obtained in this set-up, cell cycle progression was monitored in single cells following laser activation of PPG-idasanutlin (photorelease of idasanutlin). Functional p53 activation will halt cell division, causing fewer cells to pass through mitosis..sup.9 Indeed, the percentage of cells that divide within 8 h after the indicated treatment strongly drops in cells that were irradiated after treatment with photonutlin (FIG. 5.b). This shows that a specific cellular fate can be induced at single-cell resolution by laser irradiation as presented in FIG. 5.a.

[0102] Quantification of the nuclear p53-venus signal at 15 min intervals in single cells treated with PPG-idasanutlin revealed the selective stabilization of p53 protein following irradiation with the 405 nm laser (FIG. 5.c). A significantly lesser extent of p53 stabilization was detectable in neighbouring cells that were not irradiated by the 405 nm laser (FIG. 5.c). The limited stabilization of the non-irradiated neighbouring cells is most likely explained by diffusion of activated photonutlin within the excess of liquid cell culture medium in this 2D cell culture set-up. In contrast, p53 stabilization was completely absent in non-irradiated cells from adjacent wells at micrometer distance, where diffusion could not take place. In addition, p53 levels did not increase due to laser-induced damage to the cells, since p53 levels were unaltered in cells following an identical irradiation protocol in absence of PPG-idasanutlin (FIG. 5.d).

[0103] Together these results show the selective activation of PPG-idasanutlin resulting in the release of idasanutlin, using an extremely short (0.1 sec) pulse of 405 nm laser irradiation at micrometer, single-cell resolution, which offers promising opportunities for applications of photonutlins in a 3D setting like (tumor) tissue.

REFERENCES

[0104] (1) Stewart, B.; Wild, C. World Cancer Report 2014 http://publications.iarc.fr/Non-Series-Publications/World-Cancer-Reports/World-Cancer-Report-2014 (accessed Jun. 16, 2017). [0105] (2), Moslehi, J. J. Cardiovascular Toxic Effects of Targeted Cancer Therapies. N. Engl. J. Med. 2016, 375, 1457-1467. [0106] (3) Bild, A. H.; Yao, G.; Chang, J. T.; Wang, Q.; Potti, A. Chasse, D.; Joshi, M.-B.; Harpole, D.; Lancaster, J. M.; Berchuck, A.; Olson Jr, J. A.; Marks, J. R.; Dressman, H. K.; West, M.; Nevins, J. R. Oncogenic Pathway Signatures in Human Cancers as a Guide to Targeted Therapies. Nature 2006, 439, 353-357. [0107] (4) van't Veer, L. J.; Bernards, R. Enabling Personalized Cancer Medicine through Analysis of Gene-Expression Patterns. Nature 2008, 452, 564-570. [0108] (5) Vogelstein, B.; Kinzler, K. W. P53 Function and Dysfunction. Cell 1992, 70, 523-526. [0109] (6) Kastenhuber, E. R.; Lowe, S. W. Putting P53 in Context. Cell 2017, 170, 1062-1078. [0110] (7) Lakoma, A.; Barbieri, E.; Agarwal, S.; Jackson, J.; Chen, Z.; Kim, Y.; Mcvay, M.; Shohet, J.; Kim, E. The MDM2 Small-Molecule Inhibitor RG7388 Leads to Potent Tumor Inhibition in P53 Wild-Type Neuroblastoma. Cell Death Discov. 2015, 1, 15026 [0111] (8) Brown, C. J.; Lain, S.; Verma, C. S.; Fersht, A. R.; Lane, D. P. Awakening Guardian Angels: Drugging the P53 Pathway. Nat. Rev. Cancer 2009, 9, 862-873. [0112] (9) Krenning, L.; Feringa, F. M.; Shaltiel, I. A.; Van Den Berg, J.; Medema, R. H. Transient Activation of P53 in G2 Phase Is Sufficient to Induce Senescence. Mol. Cell 2014, 55, 59-72. [0113] (10) Burgess, A.; Chia, K. M.; Haupt, S.; Thomas, D.; Haupt, Y.; Lim, E. Clinical Overview of MDM2/X-Targeted Therapies. Front. Oncol. 2016, 6,7. [0114] (11) Haupt, Y.; Maya, R,; Kazaz, A.; Oren, M. Mdm2 Promotes the Rapid Degradation of P53. Nature 1997, 387, 296-299. [0115] (12) Honda, R.; Tanaka, H.; Yasuda, H. Oncoprotein MDM2 Is a Ubiquitin Ligase E3 for Tumor Suppressor P53. FEBS Lett. 1997, 420, 25-27. [0116] (13) Kubbutat, M. H. G.; Jones, S. N.; Vousden, K. H. Regulation of P53 Stability by Mdm2. Nature 1997, 387, 299-303. [0117] (14) Vassilev, L. T.; Vu, B. T.; Graves, B.; Carvajal, D.; Podlaski, F.; Filipovic, Z.; Kong, N.; Kammlott, U.; Lukacs, C.; Klein, C.; Fotouhi, N.; Liu, E. A, In Vivo Activation of the P53 Pathway by Small-Molecule Antagonists of MDM2. Science 2004, 303, 844-848. [0118] (15) Harris, S. L.; Levine, A. J. The P53 Pathway: Positive and Negative Feedback Loops. Oncogene 2005, 24, 2899-2908. [0119] (16) Ray-Coquard, I.; Blay, J.; Italiano, A.; Le Cesne, A.; Penel, N.; Zhi, J.; Heil, F.; Rueger, R.; Graves, B.; Ding, M.; Geho, D.; Middleton, S. A.; Vassilev, L. T.; Nichols, G. L.; Bid, B. N. Effect of the MDM2 Antagonist RG7112 on the P53 Pathway in Patients with MDM2-Amplified, Well-Differentiated or Dedifferentiated Liposarcoma: An Exploratory Proof-of-Mechanism Study. Lancet Oncol. 2012, 13, 1133-1140. [0120] (17) Lerch, M. M.; Hansen, M. J.; van Dam, G. M.; Szymanski, Feringa, B. L. Emerging Targets in Photopharmacology. Angew. Chem., Int. Ed. 2016, 55, 10978-10999. [0121] (18) Broichhagen, J.; Frank, J. A.; Trauner, D. A Roadmap to Success in Photopharmacology. Acc. Chem. Res. 2015, 48, 1947-1960. [0122] (19) Velema, W. A.; van der Berg, J. P.; Szymanski, W.; Driessen, A. J. M.; Feringa, B. L. Orthogonal Control of Antibacterial Activity with Light. ACS Chem. Biol. 2014, 9, 1969-1974. [0123] (20) Stanton-Humphreys, M. N.; Taylor, R. D. T.; McDougall, C.; Hart, M. L.; Brown, C. T. a; Emptage, N. J.; Conway, S. J. Wavelength-Orthogonal Photolysis of Neurotransmitters in Vitro. Chem. Commun. 2012, 48, 657-659. [0124] (21) Gandioso, A.; Cano, M.; Massaguer, A.; Marchan, V. A Green Light-Triggerable RGD Peptide for Photocontrolled Targeted Drug Delivery: Synthesis and Photolysis Studies. J. Org. Chem. 2016, 81, 11556-11564. [0125] (22) Klán, P.; Šolomek, T.; Bochet, C. G.; Blanc, A.; Givens, R.; Rubina, M.; Popik, V.; Kostikov, A.; Wirz, J. Photoremovable Protecting Groups in Chemistry and Biology: Reaction Mechanisms and Efficacy. Chem. Rev. 2013, 113, 119-191. [0126] (23) Hansen, M. J.; Velema, W. A.; Lerch, M. M.; Szymanski, W.; Feringa, B. L. Wavelength-Selective Cleavage of Photoprotecting Groups: Strategies and Applications in Dynamic Systems. Chem. Soc. Rev. 2015, 44, 3358-3377. [0127] (24) Weis, S.; Shafiq, Z.; Gropeanu, R. A.; del Campo, A. Ethyl Substituted Coumarin-4-Yl Derivatives as Photoremovable Protecting Groups for Amino Acids with Unproved Stability for SPPS. J. Photochem. Photobiol. A Chem. 2012, 241, 52-57. [0128] (25) Gandioso, A.; Contreras, S.; Melnyk, I.; Oliva, J.; Nonell, S.; Velasco, D.; García-Amorós, J.; Marchán, V. Development of Green/Red-Absorbing Chromophores Based on a Coumarin Scaffold That Are Useful as Caging Groups. J. Org. Chem. 2017, 82, 5398-5408. [0129] (26) Shu, L.; Gu, C.; Fishlock, D.; Li, Z. Practical Synthesis of MDM2 Antagonist RG7388. Part 1; A Cu(II)-Catalyzed Asymmetric [3+2] Cycloaddition. Org. Process Res. Dev. 2016, 20, 2050-2056. [0130] (27) Rimmler, G.; Alker, A.; Bosco, M.; Diodone, R.; Fishlock, D.; Hildbrand, S.; Kuhn, B.; Moessner, C.; Peters, C.; Rege, P. D.; Schantz, M. Practical Synthesis of MDM2 Antagonist RG7388, Part 2: Development of the Cu(I) Catalyzed [3+2] Asymmetric Cycloaddition Process for the Manufacture of Idasanutlin. Org. Process Res. Dev. 2016, 20, 2057-2066. [0131] (28) Tsakos, M.; Schaffert, E. S.; Clement, L. L.; Villadsen, N. L.; Poulsen, T. B. Ester Coupling Reactions—an Enduring Challenge in the Chemical Synthesis of Bioactive Natural Products. Nat. Prod. Rep. 2015, 32, 605-632. [0132] (29) Ding, Q.; Zhang, Z.; Liu, J.-J.; Jiang, N.; Zhang, J.; Ross, T. M.; Chu, X.-J.; Bartkovitz, D.; Podlaski, F.; Janson, C.; Tovar, C.; Filipovic, Z. M.; Higgins, B.; Glenn, K.; Packman, K.; Vassilev, L. T.; Graves, B. Discovery of RG7388, a Potent and Selective P53-MDM2 Inhibitor in Clinical Development. J. Med. Chem. 2013, 56, 5979-5983.