PHENAZINE DERIVATIVE AND USE THEREOF FOR THE TREATMENT OF CANCER

Abstract

A compound of formula (I),

##STR00001##

wherein R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are selected from a saturated or unsaturated, branched or unbranched, cyclic or non-cyclic alkyl, or an amide, or a functional group, or a salt or a solvate thereof, or a protonated form thereof, and to the use thereof for the treatment of cancer.

Claims

1.-10. (canceled)

11. A compound of the following formula I: ##STR00049## where, independently of each other, R1 and R2 are: either a linear or branched, saturated or unsaturated, cyclic or non-cyclic C1-C18 alkyl, optionally substituted by one or more groups chosen from a hydroxyl group, an amino group, an aminoalkyl group, a C1-C5 alkoxy group, a C1-C5 alkyl, a peptide, a pyridine group, a phosphine group, a thiol, a C2 alkene, a C2 alkyne group and a halogen, or a benzyl radical optionally substituted by one or more radicals chosen from a hydroxyl group, an amino group, an aminoalkyl group, a C1-C5 alkoxy group, a C1-C5 alkyl group, and a halogen, or (hetero)aryl groups, optionally substituted by one or more groups chosen from a hydroxyl group, an amino group, an aminoalkyl group, a C1-C5 alkoxy group, a C1-C5 alkyl, a peptide, a pyridine group, a phosphine group, a thiol, a C2 alkene, a C2 alkyne group and a halogen, or a benzyl radical optionally substituted by one or more radicals chosen from a hydroxyl group, an amino group, an aminoalkyl group, a C1-C5 alkoxy group, a C1-C5 alkyl group, and a halogen, and R2 may in particular be a hydrogen atom, and independently of each other, R3 and R4 are H or R1 or R2, or R3 and R4 are either or both a carbonyl functional group forming amine functions including peptides or not, or a salt or solvate thereof, or a protonated form thereof.

12. The compound according to claim 11, wherein R1 and R2, independently of one another, are linear or branched, saturated or unsaturated, cyclic or non-cyclic C4-C10 alkyls.

13. The compound according to claim 11, wherein the protonated form of the compound of formula 1 is chosen from the following compounds: the compound of formula Ia: ##STR00050## the compound of formula Ib: ##STR00051## and the compound of formula Ic: ##STR00052## where X represents Cl, Br, OH, F, I, BF.sub.4 or PF.sub.6 or trifluoromethanesulfate (Otf).

14. The compound according to claim 11, said compound having the following formula II or III: ##STR00053## where X represents Cl, Br, OH, F or I.

15. A pharmaceutical composition comprising, as active substance, a compound according to claim 11, in association with a pharmaceutically acceptable vehicle.

16. A method for treating a pathology by photodynamic therapy comprising administering an effective amount of a compound according to claim 11 to a patient in a need thereof.

17. The method according to claim 16, for treating tumors.

18. A method for diagnosing cancer, comprising administering to an individual liable to be afflicted by a cancer a compound according to claim 11, and detecting one or two photon fluorescence.

19. A method for visualizing living cells, cytoplasmic or tissues, by fluorescence microscopy, comprising: bringing the living cells, the cytoplasmic organelles or the tissues, into contact with a compound according to claim 11, at a concentration of 2 to 500 nmol.Math.L.sup.−1, to obtain marked living cells, cytoplasmic organelles or tissues, exposing marked living cells, cytoplasmic organelles or tissues to a light beam having a wavelength varying from 450 to 850 nm, and detecting by appropriate one- or two-photon fluorescence detection means of the fluorescent emitted by the marked living cells, cytoplasmic organelles or tissues.

20. A method for eradicating a cell comprising: contacting the cell with a compound according to claim 11, in order to obtain a contacted cell, exposing the contacted cell with a light source emitting one or two photons, wherein the compound being used at a concentration varying from 1 to 1000 nmol.Math.L.sup.−1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0112] FIG. 1 1H NMR spectra of I in MeCN-d3: without added NaOD (A), with added NaOD (B) (the window between δ=5.7 ppm and 0 ppm was omitted for reasons of clarity): The table indicates the protonated form and the non-protonated form in the presence of NaOD. The x-axis represents values in ppm.

[0113] FIG. 2 shows the absorption spectrum ε (M.sup.−1cm.sup.−1) as a function of the wavelength λ (in nm) of the compound shown.

[0114] FIG. 3 shows the absorption spectrum ε (M.sup.−1cm.sup.−1) as a function of the wavelength λ (in nm) of the compound shown.

[0115] FIG. 4 shows the absorption spectrum ε (M.sup.−1cm.sup.−1) as a function of the wavelength λ (in nm) of the compound shown.

[0116] FIG. 5 shows the absorption spectrum ε (M.sup.−1cm.sup.−1) as a function of the wavelength λ (in nm) of the compound shown.

[0117] FIG. 6 shows the absorption spectrum (A) as a function of the wavelength λ (in nm) and the emission spectrum (B) as a function of the wavelength λ (in nm) of the following compound in acetonitrile:

##STR00016##

[0118] FIG. 7 shows the emission spectrum as a function of the wavelength A (in nm) of the following compound in acetonitrile:

##STR00017##

[0119] in the presence of 0 DBU equivalents (a), 0.1 DBU equivalents (b), 0.2 DBU equivalents (c), 0.4 DBU equivalents (d), 0.6 DBU equivalents (e), 0.8 DBU equivalents (f), 1.0 DBU equivalents (g), 1.2 DBU equivalents (h), 1.4 DBU equivalents (i), 1.6 DBU equivalents (j), 1.8 DBU equivalents (k), 2.5 DBU equivalents (l).

[0120] FIG. 8 shows the capture of the compound of formula

##STR00018##

[0121] by human breast cancer cells (MCF-7). The MCF-7 cells were incubated for 16 h with the compound at concentrations of 0 μM (B), 0.1 μM (E) or 0.5 μM (H). The nuclei were stained with Hoechst 33342 (A, D and G). Single-photon fluorescence imaging was performed on live cells at the excitation wavelength of 514 nm with a Carl Zeiss microscope. Images C, F, and I represent the superposition of the signals of images A+B, D+E and G+H, respectively.

[0122] FIG. 9 shows the capture of the compound of formula

##STR00019##

[0123] by human breast cancer cells (MCF-7). The MCF-7 cells were incubated for 16 h with the compound at concentrations of 0 μM (B), 0.5 μM at 790 nm (E) or 0.5 μM at 810 nm (H). The nuclei were stained with Hoechst 33342 (A, D and G). Single-photon fluorescence imaging was performed on live cells at the excitation wavelengths of 790 or 810 nm with a Carl Zeiss microscope. Images C, F, and I represent the superposition of the signals of images A+B, D+E and G+H, respectively.

[0124] FIG. 10 shows the incorporation kinetics of the compound depicted in FIG. 8 using a CLARIOstar plate reader to quantify internalization. Data are percent internalization (remaining fluorescence/total fluorescence) as a function of time in hours. Data are means of three experiments±standard deviation.

[0125] FIG. 11 shows the survival of MCF7 cells incubated for 5 hours with the compound described in FIG. 9 at a concentration of 0.5 μM, without irradiation (A2), after irradiation at 790 nm (B2) or 810 nm (C2). As a control, untreated MCF7 cells without irradiation (A1), after irradiation at 790 nm (B1) or 810 nm (C1) are presented. The y-axis shows the percentage of live MCF-7 cells (numbered values are indicated above each bar). Data are means of three experiments±standard deviation.

[0126] FIG. 12 is a bar graph showing the effectiveness of single-photon photodynamic therapy at 540 nm. MCF-7 cells were incubated with the compound for 5 h at doses of 0 nM (A), 1 nM (B), 10 nM (C) or 100 nM (D) and irradiated (black columns) or not (gray columns) at 530 nm for 20 minutes. Two days later, the cells were subjected to a colorimetric cell viability assay (MTT). The experiment was carried out 3 times. The y-axis shows the percentage of live MCF-7 cells.

[0127] FIG. 13 shows a photo of the junction zone between MCF-7 cells treated with the compound at 100 nM and irradiated at 540 nm (B) or not (A). The living cells are visible owing to the purple marking following the MTT treatment.

[0128] FIG. 14 shows a graph illustrating the percentage of MCF-7 cells after treatment for 72 hours with a compound according to the invention at the doses indicated. Data are means of three experiments±standard deviation.

[0129] FIG. 15 is a bar graph showing the effectiveness of single-photon photodynamic therapy at 540 nm. Keratosis cells were incubated with the compound of formula

##STR00020##

[0130] for 20 min at doses of 0 nM (A), 10 nM (B), 25 nM (C) or 50 nM (D) and irradiated (black columns) or not (grey columns) at 530 nm. Two days later, the cells were subjected to a colorimetric cell viability assay (MTT). The experiment was carried out 3 times. The y-axis represents the percentage of living keratosis cells.

[0131] FIG. 16 shows the photos of the area not treated with the laser (1.) or treated with the laser (2.) at doses of 0 nM (A), 10 nM (B), 25 nM (C) or 50 nM (D) of compound of formula

##STR00021##

[0132] FIG. 17 is a bar graph showing the effectiveness of single-photon photodynamic therapy at 540 nm. Keratosis cells were incubated with the compound of formula:

##STR00022##

[0133] for 20 min h at doses of 0 nM (A), 10 nM (B), 25 nM (C) or 50 nM (D) and irradiated (black columns) or not (grey columns) at 530 nm. Two days later, the cells were subjected to a colorimetric cell viability assay (MTT). The experiment was carried out 3 times. The y-axis represents the percentage of living keratosis cells.

[0134] FIG. 18 shows the photos of the area not treated with the laser (1.) or treated with the laser (2.) at doses of 0 nM (A), 10 nM (B), 25 nM (C) or 50 nM (D) of compound of formula:

##STR00023##

[0135] FIG. 19 shows the absorption spectra of the phenazinium compounds according to the invention (A and B) and of the phenazine compounds (C and D).

[0136] FIG. 20 shows a graph illustrating the internalization of the compound of formula

##STR00024##

[0137] by live MCF-7 cancer cells. Cells were treated with 0.5 nM compound for 1, 3, 6 or 24 h and red fluorescence was measured by flow cytometry. The results show the mean of two experiments+/−the standard deviation. The x-axis represents the incubation time in hours and the y-axis the percentage of red fluorescent cells.

[0138] FIG. 21 shows a graph illustrating the internalization of the compound of formula

##STR00025##

[0139] by healthy living cells of the fibroblast type. Cells were treated with 0.5 nM compound for 1, 3, 6 or 24 h and red fluorescence was measured by flow cytometry. The results show the mean of two experiments+/−the standard deviation. The x-axis represents the incubation time in hours and the y-axis the percentage of red fluorescent cells.

EXAMPLES

[0140] Commercial analytical-grade reagents were obtained from suppliers and used directly without further purification. The .sup.1H and .sup.13C NMR spectra are recorded in CDCl.sub.3, CD.sub.2Cl.sub.2, CD.sub.3CN, acetone-d.sub.6 and DMSO-d.sub.6, determined with a Brucker AC250 spectrometer operating at 250 MHz or with a Jeol ECS400 spectrometer operating at 400 MHz. Chemical shifts are expressed in ppm and coupling constants (J) are in hertz. Separation patterns are designed in the form of s, singlet; br s, broad singlet; d, doublet; dd, doublet of doublets; t, triplet; td, triplet of doublets; qt, quintet.

[0141] The elemental and MS (mass spectrometry) analyses were carried out by the Spectropole de Marseille. ESI mass spectral analyses are recorded with a mass spectrometer 3200 QIRAP (Applied Biosystems SCIEX). High-resolution mass spectral analyses are recorded with a SYNAPT G2 HDMS mass spectrometer (Waters)

[0142] The preparative flash column chromatographies were carried out using silica gel G60 230-240 mesh (Merck).

Example 1—Syntheses of Compounds According to the Invention

[0143] 1—Synthesis Method No. 1—Compound of Formula III (1a)

##STR00026##

[0144] Compound A: 1-Octylamine (v=830 μL, 2.05 equiv.) and N,N-diisopropylethylamine (DIPEA) (v=874 μL, 2.05 equiv.) was added to a solution of 1,5-difluoro-2,4-dinitrobenzene (DFNB) (m=500 mg, 1.00 equiv.) in ethanol (v=50 mL). The solution was heated to reflux for 1.5 h. After cooling to room temperature, the resulting solid in suspension was isolated by filtration, rinsed with EtOH and dried under vacuum to obtain compound A (m=1.04 g, quantitative yield) in an orange crystalline form.

[0145] .sup.1H NMR (250 MHz, CDCl.sub.3): δ 9.24 (s, 1H), 8.32 (br s, 2H), 5.65 (s, 1H), 3.27 (td, J.sub.t=7.0 Hz, J.sub.d=5.3 Hz, 4H), 1.82-1.71 (m, 4H), 1.54-1.29 (m, 20H), 0.89 (t, J=6.8 Hz, 6H). .sup.13C NMR (63 MHz, CDCl.sub.3): δ 148.5, 129.5, 124.0, 90.0, 43.3, 31.7, 29.2, 29.1, 28.4, 27.1, 22.6, 14.0. ESI-MS: m/z [M+H].sup.+ 423.3 (100%), [M+Na].sup.+ 445.3 (6%), [M+K].sup.+ 461.3 (4%); [MH].sup.− 421.3 (100%). Elemental analysis for C.sub.22H.sub.38N.sub.4O.sub.4: calculated. C, 62.53, H, 9.06, N, 13.26, O, 15.15. found C, 62.58, H, 9.19, N, 13.21.

[0146] Compound B: Boc.sub.2O (m=1.94 g, 8.89 mmol, 3.7 equiv.) and 4-dimethylaminopyridine (DMAP) (m=50 mg, 0.41 mmol, 17 mol %) were added to a solution of compound A (m=1.03 g, 2.43 mmol, 1.0 equiv.) in THF (10 mL). The solution was refluxed for 4 h. The solvent was removed under vacuum. The crude product was purified by flash chromatography (silica F60, DCM 100) to obtain compound B (m=1.52 g, 2.44 mmol, quantitative yield) in the form of a yellow solid.

[0147] .sup.1H NMR (250 MHz, CDCl.sub.3): δ 8.54 (s, 1H), 7.24 (s, 1H), 3.69 (m, 4H), 1.68-1.25 (m, 42H), 0.87 (t, J=6.5 Hz, 6H). .sup.13C NMR (63 MHz, CDCl.sub.3): 152.1, 142.5, 141.0, 127.8, 122.8, 82.9, 50.9, 31.7, 29.2, 29.2, 28.7, 27.9, 26.9, 22.6, 14.0. MS: ESI-MS: m/z [M+NH.sub.4].sup.+ 640.4 (100%), [M+Na].sup.+ 645.4 (12%), [M+K].sup.+ 661.3 (5%). Elemental analysis for C.sub.32H.sub.54N.sub.4O.sub.8: calculated. C, 61.71, H, 8.74, N, 9.00, O, 20.55. found C, 61.68, H, 8.96, N, 8.82.

[0148] Compound 8b: Compound B (m=5.15 g, 8.28 mmol, 1.0 equiv.) and hydrazine monohydrate (2.3 mL, 47.1 mmol, 5.7 equiv.) were added to a suspension of Pd on carbon carbon 5% (m=180 mg, 0.085 mmol, 1% mol) in EtOH (80 mL). The mixture was heated to reflux for 1.5 h. The Pd/C was removed by filtration through Celite 545, and the solid phase was rinsed with dichloromethane (3×100 mL). The combined organic phase was washed with water (3×150 mL) and brine (100 mL), dried with anhydrous MgSO.sub.4, filtered, concentrated and dried under vacuum to obtain compound 8b (m=4.43 g, 7.87 mmol, 96% yield) as a yellow solid.

[0149] .sup.1H NMR (250 MHz, CDCl.sub.3): δ 6.55 (br s, 1H), 6.09 (s, 1H), 3.58 (br s, 6H), 3.27 (m, 2H), 1.51-1.24 (m, 42H), 0.86 (t, J=6.5 Hz, 6H). .sup.13C NMR (63 MHz, CDCl.sub.3): 155.2, 142.7, 142.5, 129.5, 129.1, 118.9, 102.0, 79.2, 57.6, 48.4, 31.5, 29.1, 29.0, 28.0, 26.6, 22.3, 18.1, 13.8. HRMS (ESI-TOF): m/z [M+H].sup.+ for C.sub.32H.sub.59N.sub.4O.sub.4.sup.+ calculated. 563.4531. found 563.4532. err.<1 ppm; m/z [M+NH.sub.4].sup.+ for C.sub.32H.sub.62N.sub.5O.sub.4.sup.+ calculated. 580.4796. found 580.4803. err.<2 ppm.

[0150] Compound 12b: TFA at 0° C. was added to a solution of compound 8b (m=303 mg, 0.538 mmol) in dichloromethane (v=5 mL), HCl (12N, v=2 mL). This mixture was stirred under argon overnight. The resulting solid in suspension was collected by filtration, rinsed with CH.sub.2Cl.sub.2 (v=20 mL), and dried under vacuum to obtain compound 12b (m=196 mg, 0.449 mmol, 84% yield) in the form of a light pink solid. This raw product was used directly without further purification.

[0151] .sup.1H NMR (250 MHz, DMSO-d.sub.6): δ 6.85 (br s, 1H), 6.44 (br s, 1H), 3.06 (t, .sup.3J.sub.HH=7.2 Hz, 4H), 1.62 (m, 4H), 1.35-1.26 (m, 20H), 0.86 (t, .sup.3J.sub.HH=6.8 Hz, 6H). No .sup.13C NMR spectrum could be recorded owing to the poor stability in solution. MALDI-TOF MS: m/z M.sup.+. for C.sub.22H.sub.42N.sub.4..sup.+ calculated. 362.3. found 362.3 (100%).

##STR00027##

[0152] Compound 13b: Compound 12b (m=306 mg, 0.703 mmol, 1.0 equiv.) was added to a solution of DFDNB (m=258 mg, 1.27 mmol, 1.8 equiv.) in MeCN (v=25 mL). The flask was closed with a septum and the solution was cooled in an ice water bath and degassed. N(iPr).sub.2Et (v=735 μL, 4.22 mmol, 6.0 equiv.) was then added dropwise using a syringe under argon. The solution was stirred at 0° C. for 2 hours, then at room temperature for an additional two hours. The solution was concentrated in vacuo, and the residue taken up with EtOH (v=30 mL) and MeCN (v=10 mL). The solid obtained in suspension was recovered by filtration, rinsed with EtOH (v=100 mL) and Et.sub.2O (v=20 mL), and dried under vacuum to obtain compound 13b in the form of an orange powder (m=365 mg, 0.499 mmol, 79% yield).

[0153] .sup.1H NMR (250 MHz, CDCl.sub.3): δ 9.29 (br s, 2H), 9.15 (d, .sup.4J.sub.HF=7.8 Hz, 2H), 6.84 (s, 1H), 6.52 (d, .sup.3J.sub.HF=13 Hz, 2H), 6.07 (s, 1H), 3.97 (br s, 2H), 3.19 (t, .sup.3J.sub.HH=7.0 Hz, 4H), 1.66-1.26 (m, 24H), 0.88 (t, .sup.3J.sub.HH=7.0 Hz, 6H). .sup.1H NMR (250 MHz, Acetone-d.sub.6): δ 9.60 (br s, 2H), 9.01 (d, .sup.4J.sub.HF=8.0 Hz, 2H), 7.08 (s, 1H), 6.70 (d, .sup.3J.sub.HH=14.3 Hz, 2H), 6.20 (s, 1H), 5.13 (br s, 2H), 3.24 (t, .sup.3J.sub.HH=7.0 Hz, 4H), 1.66-1.55 (m, 4H), 1.29-1.26 (m, 20H), 0.87 (t, .sup.3J.sub.HH=7.0 Hz, 6H). .sup.13C NMR (63 MHz, CDCl.sub.3): δ 159.9 (d, .sup.1J.sub.CF=267 Hz), 150.0, 149.8, 146.1, 127.9, 127.6, 127.3 (d, .sup.2J.sub.CF=10.1 Hz), 109.8, 103.7 (d, .sup.2J.sub.CF=27.7 Hz), 93.4, 43.5, 31.7, 29.28, 29.23, 29.18, 27.1, 22.6, 14.0. HRMS (ESI-TOF): m/z [M+H].sup.+ for C.sub.34H.sub.45N.sub.8O.sub.8F.sub.2.sup.+ calculated. 731.3323. found 731.3323. err.<1 ppm.

[0154] Compound 3: Compound 12b (m=115 mg, 0.263 mmol, 1.2 equiv.) was added to a solution of compound 13b (m=160 mg, 0.219 mmol, 1.0 equiv.) in anhydrous MeCN (v=30 mL). The flask was closed, degassed and N(iPr).sub.2Et was added dropwise (m=370 μL, 2.12 mmol, 9.6 equiv.) using a syringe under argon. The mixture was stirred at room temperature for 2 h with stirring, then refluxed overnight. After concentration of the solvent under vacuum, the residue was taken up with a mixture of acetone (v=5 mL) and ethanol (v=5 mL). The resulting solid product was isolated by filtration, washed with EtOH and dried under vacuum to obtain Compound 3 (m=155.5 mg, 0.148 mmol, 68% yield) as an orange powder.

[0155] .sup.1H NMR (250 MHz, CDCl.sub.3): δ 9.26 (s, 2H), 8.85 (br s, 4H), 6.58 (s, 2H), 5.82 (s, 2H), 5.49 (s, 2H), 3.91 (br t, .sup.3J.sub.HH=5.4 Hz, 4H), 3.09-2.99 (m, 8H), 1.53-1.28 (m, 48H), 0.89 (t, .sup.3J.sub.HH=6.8 Hz, 12H). .sup.13C NMR (63 MHz, CDCl.sub.3): δ 149.9, 146.4, 129.2, 129.0, 125.4, 110.6, 95.7, 92.6, 43.8, 31.8, 29.49, 29.46, 29.33, 27.3, 22.7, 14.1. HRMS (ESI-TOF): m/z [M+H].sup.+ for C.sub.56H.sub.85N.sub.12O.sub.8.sup.+ calculated. 1053.6608. found 1053.6608. err.<1 ppm.

##STR00028##

[0156] Compound 1a: SnCl.sub.2.2H.sub.2O (343 mg, 1.52 mmol, 32 equiv.) and HCl (12M, 0.13 mL) were added to a solution of macrocycle 3 (50 mg, 0.05 mmol, 1 equiv.) in absolute ethanol (50 mL). The mixture was stirred at reflux overnight and neutralized with NaHCO.sub.3 before adding ethanol (30 mL) and water (20 mL). After evaporation of the solvent under reduced pressure, the residue was extracted with a dichloromethane/ethanol mixture (3/1, v/v). The red organic layer was washed with an aqueous solution of HPF.sub.6 (1 wt. % in water, 4×150 mL) and brine (100 mL), dried with MgSO.sub.4 and concentrated in vacuo to obtain compound 1a [PF.sub.6] as a dark red solid (35 mg, 62% yield).

[0157] .sup.1H NMR (400 MHz, CD.sub.3CN): δ 7.82 (d, J=9.3 Hz, 1H), 7.23 (dd, J=9.3 Hz, J=2.2 Hz, 1H), 7.18 (s, 1H), 6.99 (s, 1H), 6.55 (d, J=2.2 Hz, 1H), 6.33 (br t, J=5.2 Hz, 1H), 6.12 (br s, 2H), 4.84 (br s, 2H), 4.57 (t, J=8.2 Hz, 2H), 3.37 (td, J=6.9 Hz, J=6.0 Hz, 2H), 1.72 (quint, J=7.3 Hz, 2H), 1.61 (quint, J=7.8 Hz, 2H), 1.47-1.27 (m, 20H), 0.91-0.87 (m, 6H). HRMS (ESI-TOF): m/z [M+NH.sub.4].sup.+ for C.sub.28H.sub.44N.sub.5.sup.+ calculated. 450.3591. found 450.3592. err.<1 ppm.

[0158] 2—Synthesis Method No. 2—Compound of Formula IV (1c)

##STR00029##

[0159] Compound 4: 2,4-difluoronitrobenzene (6.5 mL, 0.059 mol, 1 equiv.), 1-octylamine (40 mL, 0.243 mol, 4.1 equiv.) and DIPEA (18 mL, 0.101 mol, 1.7 equiv.) were introduced into a pressure canister, which was closed with a Teflon seal cap. The mixture was heated to 145° C. for 3 h. After cooling to room temperature, 15 mL of ethanol was added. This suspension was triturated by ultrasound. The resulting solid product in suspension was isolated by filtration, rinsed with hot water, and dried under vacuum to obtain compound 4 in the form of a yellow powder (21.6 mg, 96% yield).

[0160] .sup.1H NMR (250 MHz, CDCl.sub.3): δ 8.52 (br s, 1H), 7.99 (d, J=9.3 Hz, 1H), 5.89 (dd, J=9.3 Hz, J=2.3 Hz, 1H), 5.62 (d, J=2.3 Hz, 1H), 4.52 (br s, 1H), 3.23-3.14 (m, 4H), 1.75-1.62 (m, 4H), 1.42-1.28 (m, 20H), 0.91-0.85 (m, 6H). .sup.13C NMR (63 MHz, CDCl.sub.3): 154.4, 148.6, 129.2, 123.6, 104.7, 89.8, 43.3, 42.9, 31.8, 31.7, 29.3, 29.2, 29.1, 28.8, 27.1, 27.0, 22.6, 14.0. MS: ESI-MS: m/z [M+H].sup.+ 378.3 (100%); [MH].sup.− 376.3 (100%), [M+CH.sub.3COO].sup.− 436.3 (48%). Elemental analysis for C.sub.22H.sub.39N.sub.3O.sub.2.⅕C.sub.2H.sub.5OH: calculated. C, 69.56, H, 10.48, N, 10.86, O, 9.10. found C, 69.41, H, 10.39, N, 10.92.

[0161] Compound 12h: a solution of compound 4 (628 mg, 1.66 mmol, 1 equiv.) in THF (25 mL) was hydrogenated (40 bars) overnight in the presence of Pd/C (5 wt. %, 36 mg, 0.02 mmol, 1 mol %). After reducing the pressure, the solution was degassed under sonication for 5 min. 1,5-difluoro-2,4-dinitrobenzene (320 mg, 1.58 mmol, 0.95 equiv.) was added to the solution with stirring at 0° C. The solution was left at this temperature for a further 10 min and the completion of the reaction was monitored by TLC. Then DIPEA (301 μL, 1.66 mmol, 1 equiv.) was added to neutralize the solution. The Pd/C was removed by filtration through celite. The crude product was purified by flash chromatography on silica gel using a dichloromethane/cyclohexane mixture (1/1) as eluent to obtain compound 12h in the form of a red solid (620 mg, 75% yield).

[0162] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 9.31 (s, 1H), 9.15 (d, J=7.7 Hz, 1H), 6.83 (d, J=8.4 Hz, 1H), 6.53 (d, J=13.4 Hz, 1H), 6.00 (dd, J=8.4, 2.3 Hz, 1H), 5.95 (d, J=2.1 Hz, 1H), 3.77 (s, 1H), 3.67 (s, 1H), 3.11 (td, J=14.1, 8.4 Hz, 4H), 1.65 (quintet, J=7.2 Hz, 2H), 1.56 (quintet, J=7.1 Hz, 2H), 1.36 (m, 20H), 0.89 (t, J=6.7 Hz, 3H), 0.87 (t, J=6.7 Hz, 3H). .sup.13C NMR (100 MHz, CDCl.sub.3): δ 159.9 (d, J.sub.CF=270.7 Hz), 150.5, 150.3, 150.2, 145.1, 128.5, 127.7, 127.7, 127.6, 126.9 (d, J.sub.CF=10.2 Hz), 110.7, 103.9 (d, J.sub.CF=27.4 Hz), 101.6, 95.1, 43.9, 43.4, 31.8, 31.8, 31.8, 29.5, 29.4, 29.3, 29.3, 29.3, 29.3, 29.2, 29.2, 27.2, 27.1, 22.7, 22.6, 14.1, 14.1. HRMS (ESI-TOF): m/z [M+H].sup.+ for C.sub.28H.sub.43FN.sub.5O.sub.4.sup.+ calculated. 532.3294. found 532.3281. err.<2 ppm.

[0163] Compound 13d: a solution of compound 4 (628 mg, 1.66 mmol, 1 equiv.) in THF (25 mL) was hydrogenated (40 bars) overnight in the presence of Pd/C (5 wt. %, 36 mg, 1 mol %). After decreasing the pressure, the solution was degassed by sonication for 5 min, then cooled to 0° C. in an ice tray, and 1,5-difluoro-2,4-dinitrobenzene (320 mg, 1.58 mmol, 0.95 equiv.) was added to the solution with stirring. The reaction was maintained at 0° C. for 10 min. Then 1-octylamine (286 μL, 1.93 mmol, 1.1 equiv.) and DIPEA (289 μL, 1.66 mmol, 1 equiv.) were added. The mixture was stirred at room temperature for 3 days. After filtration through celite and concentration, the raw product was purified by flash chromatography on silica gel using a dichloromethane/cyclohexane mixture (50/50 to 55/45) as eluent to obtain compound 13d in the form of a red solid (498 mg, 49% yield).

[0164] TLC: R.sub.f=0.27 (SiO.sub.2 F60, dichloromethane/cyclohexane, 7/3). .sup.1H NMR (400 MHz, CDCl.sub.3): δ 9.27 (s, 1H), 9.12 (br s, 1H), 8.21 (br t, J=4.8 Hz, 1H), 6.87 (d, J=8.3 Hz, 1H), 6.00 (dd, J=8.3, 2.2 Hz, 1H), 5.96 (d, J=2.2 Hz, 1H), 5.69 (s, 1H), 3.77 (br s, 1H), 3.68 (br s, 1H), 3.13 (t, J=7.2 Hz, 2H), 3.13-3.05 (m, 2H), 3.00 (q, J=6.1 Hz, 2H), 1.68-1.52 (m, 6H), 1.45-1.23 (m, 30H), 0.90-0.84 (m, 9H). .sup.13C NMR (100 MHz, CDCl.sub.3): δ 149.5, 149.0, 148.4, 145.2, 129.4, 128.6, 124.8, 124.3, 112.0, 101.5, 95.2, 93.0, 44.1, 43.5, 43.1, 31.8, 31.8, 31.7, 29.6, 29.4, 29.4, 29.3, 29.2, 29.2, 29.2, 29.1, 28.2, 27.2, 27.1, 26.9, 22.6, 22.6, 22.6, 14.1, 14.1, 14.0. HRMS (ESI-TOF): m/z [M+I].sup.− for C.sub.36H.sub.60N.sub.6O.sub.4].sup.− calculated. 767.3726. found 767.3725. err.<2 ppm.

[0165] Compound 1c: A solution of compound 13d (200 mg, 0.31 mmol, 1 equiv.) in methanol (40 mL) was hydrogenated (40 bars) overnight in the presence of Pd/C (5 wt. %) and HCl (12M, 0.1 mL). Then the mixture was stirred in air for 24 hours. The Pd/C was removed by filtration through celite. After removal of the solvent under reduced pressure, the resulting solid was taken up with dichloromethane (80 mL), washed with a solution of aqueous HPF.sub.6 (1 wt. % in water, 2×50 mL) then distilled water (50 mL) and in fine concentrate. The residue was purified by flash chromatography on standard alumina 90 using a dichloromethane/cyclohexane mixture (100/0 to 99/1) as eluent to obtain compound 1c [PF.sub.6.sup.−] as a red solid (192 mg, 87% yield).

[0166] .sup.1H NMR (400 MHz, CD.sub.3CN): δ 7.78 (d, J=9.2 Hz, 1H), 7.19 (dd, J=9.2 Hz, 1.7 Hz, 1H), 6.96 (s, 1H), 6.89 (s, 1H), 6.52 (d, J=1.7 Hz, 1H), 6.24 (br t, J=5.2 Hz, 1H), 6.18 (br s, 2H), 4.78 (br s, 1H), 4.54 (t, J=8.1 Hz, 2H), 3.34 (td, J=6.7 Hz, 6.0 Hz, 2H), 3.28 (td, J=6.7 Hz, 5.2 Hz, 2H), 1.79-1.67 (m, 4H), 1.64-1.56 (m, 2H), 1.51-1.30 (m, 30H), 0.91-0.88 (m, 9H). .sup.13C NMR (100 MHz, DMSO-d.sub.6): δ=152.6, 150.3, 138.7, 138.3, 134.9, 132.2, 131.3, 130.2, 102.5, 92.3, 47.1, 43.1, 42.5, 31.2, 31.2, 28.8, 28.8, 28.7, 28.6, 28.0, 27.6, 26.7, 26.6, 26.2, 26.0, 22.0, 13.9, 13.9. HRMS (ESI-TOF): m/z [M+H].sup.+ for C.sub.36H.sub.60N.sub.5.sup.+ calculated. 562.4843. found 562.4855. err.<3 ppm.

[0167] Synthesis of Compounds where R2 is Hydrogen

[0168] PR4 Compound

Compound 12: 5-fluoro-2-nitroaniline

[0169] ##STR00030##

[0170] DFDNB (1.0 mL, 9.11 mmol, 1.0 eq), NH.sub.4Cl (975 mg, 18.2 mmol, 2.0 eq) and triethylamine (9.0 mL) were introduced in a pressure bomb. The bomb was closed with a Teflon cap. The mixture was allowed to heat up to 110° C. for 40 hrs. After cooling to room temperature, the raw product was placed in a mixture of DCM (150 mL) and water (150 mL). While stirring, 12N HCl was added dropwise until the pH of the aqueous phase reached approximately 1. The organic phase was separated and washed with water (150 mL), dried with MgSO.sub.4. Additional heptane (40 mL) was added to this solution. The solvent was removed by lowering the pressure. The residues were dried under vacuum to obtain compound 12 (1.25 g, 8.01 mmol, 88% yield) as a yellow powder. .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.184 (dd, J=9.6 Hz, J=5.6 Hz, 1H), 6.449-6.41 (m, 2H), 6.200 (br s, 2H).sup.13C NMR (100 MHz, CDCl.sub.3): δ 168.2 (d, J=260 Hz), 146.7, 146.6, 129.4 (d, J=12 Hz), 105.9 (d, J=25 Hz), 103.8 (d, J=26 Hz). MS: ESI-MS: m/z [M+Li].sup.+ 157.1 (26%), [M+Li].sup.+ 163.1 (100%); [MH].sup.− 154.9 (100%). Elemental analysis for C.sub.6H.sub.5FN.sub.2O.sub.2: calculated. C, 46.16, H, 3.23, F, 12.17, N, 17.94, W, 20.50. found C, 46.63, H, 3.15, N, 17.90.

Compound 13: 4-Nitro-N.SUP.1.-octylbenzene-1,3-diamine

[0171] ##STR00031##

[0172] Compound 12 (808 mg, 5.18 mmol, 1.0 eq) and 1-octylamine (3.0 mL, 18.2 mmol, 3.5 eq) were introduced into a pressure bomb. The bomb was closed with a Teflon cap. The mixture was stirred at 140° C. for 1 hour. After cooling to room temperature, heptane (10 mL) was added. The resulting solid in suspension was isolated by filtration, purified by chromatography (60F silica, DCM, 100) to obtain compound 13 (1.23 g, 4.64 mmol, 90% yield) as a yellow solid.

[0173] .sup.1H NMR (250 MHz, CDCl.sub.3): δ 7.96 (d, J=9.3 Hz, 1H), 5.97 (dd, J=9.3 Hz, J=2.5 Hz, 1H), 5.71 (d, J=2.5 Hz, 1H), 3.15 (t, J=7.1 Hz, 2H), 1.67 (quint, J=7.1 Hz, 2H), 1.40-1.28 (m, 10H), 0.90 (t, J=7.1 Hz, 3H). .sup.13C NMR (63 MHz, CDCl.sub.3): δ 153.9, 147.9, 128.4, 124.2, 106.2, 94.8, 43.4, 31.7, 29.2, 29.1, 29.0, 27.0, 22.6, 14.0 MS: ESI-MS: m/z [M+H].sup.+ 266.3 (100%), [M+Li].sup.+272.3 (19%), [M+Na].sup.+ 288.3 (4%); [MH].sup.− 264.1 (100%). Elemental analysis for C.sub.14H.sub.23N.sub.3O.sub.2: calculated. C, 63.37, H, 8.74, N, 15.84, O, 12.06. found C, 63.65, H, 8.83, N, 15.66.

Compound 14: N.SUP.4.-octylbenzene-1,2,4-triamine

[0174] ##STR00032##

[0175] A solution of compound 13 (975 mg, 3.67 mmol) in MeOH (75 mL) was hydrogenated (40 bars) in the presence of Pd/C (5%) overnight. The Pd/C was then removed by filtration through Celite. After concentration under reduced pressure and drying under vacuum, compound 14 was obtained in the form of a deep green solid (870 mg, 3.69 mmol, quantitative yield). .sup.1H NMR (400 MHz, CDCl.sub.3): δ 6.60 (d, J=8 Hz, 1H), 6.07 (d, J=2.4 Hz, 1H), 6.03 (dd, J=8 Hz, J=2.4 Hz, 1H), 3.11 (br s, 5H), 3.03 (t, J=7.2 Hz, 2H), 1.61 (quint, J=7.2 Hz, 2H), 1.38-1.28 (m, 10H), 0.90 (t, J=6.8 Hz, 3H)

[0176] .sup.13C NMR (100 MHz, CDCl.sub.3): 143.5, 137.4, 124.8, 119.4, 104.7, 102.1, 45.1, 31.8, 29.6, 29.4, 29.3, 27.2, 22.6, 14.1. Elemental analysis for C.sub.14H.sub.25N.sub.3: calculated. C, 71.44, H, 10.71, N, 17.85. found C, 71.23, H, 10.82, N, 17.88.

Compound 20: N.SUP.1.-(2,4-dinitro-5-(octylamino)phenyl)-M-octylbenzene-1,2,4-triamine

[0177] ##STR00033##

[0178] DFDNB (671.5 mg, 3.29 mmol, 0.90 eq) was added to a solution of compound 14 (860 mg, 3.65 mmol, 1.00 eq) in THF (40 mL). The flask was sealed and degassed by 3 pump-argon cycles. Degassed DIPEA (640 μL, 3.67 mmol, 1.00 eq) was added dropwise using a syringe under argon. The mixture was stirred at room temperature overnight. 1-Octylamine (604 μL, 3.65 mmol, 1.00 eq) and additional DIPEA (660 μL, 3.79 mmol, 1.04 eq) were added. The solution was heated to reflux for 3 h and cooled to room temperature. After concentration of the solvent in vacuo, the resulting solid product was isolated by filtration before the addition of EtOH (40 mL), rinsed with EtOH (6×10 mL) and dried in vacuo to obtain compound 20 (1.36 g, 2.57 mmol, 79% yield) as a yellow powder. .sup.1H NMR (400 MHz, CDCl.sub.3): δ 9.27 (s, 1H), 9.19 (br s, 1H), 8.23 (br t, J=4.8 Hz, 1H), 6.89 (d, J=8.4 Hz, 1H), 6.11 (dd, J=8.4 Hz, J=2 Hz, 1H), 6.06 (d, J=2 Hz, 1H), 5.71 (s, 1H), 3.69 (br s, 1H), 3.65 (br s, 2H), 3.11 (t, J=7.2 Hz, 2H), 3.06 (td, J=6.8 Hz, J=5.6 Hz, 2H), 1.67-1.57 (m, 4H), 1.42-1.27 (m, 20H), 0.90-0.87 (m, 6H). .sup.13C NMR (100 MHz, CDCl.sub.3): δ 149.3, 148.8, 148.5, 143.8, 129.5, 129.0, 124.9, 124.3, 112.3, 104.6, 99.0, 92.9, 44.0, 43.1, 31.8, 31.7, 29.5, 29.4, 29.3, 29.2, 29.1, 28.3, 27.2, 26.9, 22.6, 14.1. HRMS (ESI-TOF): m/z [M+H].sup.+ for C.sub.28H.sub.45N.sub.6O.sub.4.sup.+ calculated. 529.3497. found 529.3493. err.<1 ppm.

Compound PR4 According to the Invention: N.SUP.2.,N.SUP.7.-dioctylphenazine-2,3,7-triamine

[0179] ##STR00034##

[0180] A solution of compound 20 (302 mg, 0.571 mmol) in MeOH (30 mL) was hydrogenated (20 bars) in the presence of Pd/C (5%) and HCl (12M, 0.5 mL) overnight. After addition of MeOH (30 mL), the solution was stirred under air for 24 h. The Pd/C was removed by filtration under Celite, and the solid phase was rinsed with

[0181] MeOH (400 mL). The solution was neutralized with NaHCO.sub.3 to pH 9 before adding water (100 mL). The solvent was concentrated to about 100 mL under reduced pressure. After cooling at 5° C. overnight, the resulting suspended solid was isolated by filtration and washed with water (2×30 mL), dried under vacuum to obtain compound PR4 (238 mg, 0.529 mmol, 93% yield) as a red powder. .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 7.59 (d, J=9.2 Hz, 1H), 7.11 (dd, J=9.2 Hz, J=2.4 Hz, 1H), 6.79 (s, 1H), 6.59 (s, 1H), 6.58 (d, J=2.4 Hz, 1H), 6.22 (br t, J=4.8 Hz, 1H), 5.98 (br s, 2H), 5.65 (br t, J=4.4 Hz, 2H), 3.22 (td, J=6.8 Hz, J=5.2 Hz, 2H), 3.13 (td, J=6.8 Hz, J=5.6 Hz, 2H), 1.73-1.60 (m, 4H), 1.43-1.27 (m, 20H), 0.57-0.56 (m, 6H). .sup.13C NMR (100 MHz, DMSO-d.sub.6): 147.6, 143.0, 141.2, 140.2, 138.5, 135.5, 128.2, 121.1, 102.5, 99.9, 99.4, 43.2, 42.8, 31.2, 28.9, 28.2, 27.9, 26.8, 26.8, 22.1, 13.9. HRMS (ESI-TOF): m/z [M+H].sup.+ for C.sub.28H.sub.44N.sub.5.sup.+ calculated. 450.3591. found 450.3590. err.<1 ppm.

[0182] PR5 Compound

Compound 21: N.SUP.1.-(2,4-dinitro-5-(3,4,5-trimethoxyphenylamino)phenyl)-N.SUP.4.-octylbenzene-1,2,4-triamine

[0183] ##STR00035##

[0184] A solution of compound 13 (1.50 mg, 5.83 mmol, 1.0 eq) in THF (50 mL) was hydrogenated (20 bars) in the presence of 5% Pd/C (124 mg, 0.058 mmol, 1 mol %) overnight. Then the mixture was degassed by sonication for 5 min. After addition of DFDNB (1.10 g, 5.39 mmol, 0.92 eq), the reaction was stirred at room temperature under argon for 2 days. Then 3,4,5-trimethoxylaniline (2.20 g, 12 mmol, 2.23 eq) and DIPEA (600 μL, 3.44 mmol, 0.64 eq) were added to the reaction. The mixture was kept two more days at room temperature. The Pd/C was removed by filtration through Celite. After concentration in vacuo, the raw product was taken up in EtOH. The filtrate was concentrated and purified by column chromatography (silica 60F, AE/CH, 40/60) to obtain compound 21 (1.34 g, 2.30 mmol, 43% yield) in the form of a red solid. .sup.1H NMR (400 MHz, CDCl.sub.3): δ 9.73 (br s, 1H), 9.31 (s, 1H), 9.15 (br s, 1H), 6.75 (d, J=8.4 Hz, 1H), 6.37 (s, 2H), 6.28 (s, 1H), 5.95 (dd, J=8.4, 2.5 Hz, 1H), 5.90 (d, J=2.5 Hz, 1H), 3.79 (s, 3H), 3.76 (s, 6H), 3.61 (dps, 2H), 3.58 (dps, 1H), 3.00 (t, J=7.2 Hz, 2H), 1.58 (quintet, J=7.2 Hz, 2H), 1.40-1.29 (m, 10H), 0.90 (t, J=6.9 Hz, 3H). .sup.13C NMR (100 MHz, CDCL.sub.3): δ153.7, 149.4, 149.0, 146.7, 143.7, 136.3, 133.0, 129.3, 128.7, 125.23, 125.16, 111.8, 104.3, 101.7, 98.6, 95.4, 60.9, 56.1, 43.9, 31.8, 29.49, 29.39, 29.25, 27.2, 22.6, 14.1. HRMS (ESI-TOF): m/z [M+H].sup.+ for C.sub.29H.sub.39N.sub.6O.sub.7.sup.+ calculated. 583.2875. found 583.2878. err.<1 ppm.

PR5 Compound: N.SUP.7.-octyl-N.SUP.2.-(3,4,5-trimethoxyphenyl)phenazine-2,3,7-triamine

[0185] ##STR00036##

[0186] A solution of compound 21 (150 mg, 0.257 mmol) in MeOH (75 mL) was hydrogenated (20 bars) in the presence of Pd/C (5%) and HCl (12M, 0.5 mL) throughout the night. After addition of MeOH (30 mL), the solution was stirred under air for 24 h. The Pd/C was removed by filtration under Celite, and the solid phase was rinsed with MeOH (400 mL). The solution was neutralized with NaHCO.sub.3 to pH 9 before adding water (100 mL). The solvent was concentrated to about 100 mL under reduced pressure. After cooling at 5° C. overnight, the resulting solid in suspension was isolated by filtration and washed with water (2×30 mL), dried under vacuum to obtain compound PR5 (110 mg, 0.218 mmol, 85% yield) as a red powder. .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 7.79 (d, J=9.2 Hz, 1H), 7.60 (br s, 1H), 7.23 (s, 1H), 7.05 (dd, J=9.2, 2.5 Hz, 1H), 6.89 (d, J=2.5 Hz, 1H), 6.38 (s, 2H), 5.56 (s, 1H), 4.28 (br s, 2H), 4.19 (br t, J=5.3 Hz, 1H), 3.85 (s, 3H), 3.82 (s, 6H), 3.28 (td, J=6.9, 5.5 Hz, 2H), 1.72 (quintet, 7.2 Hz, 2H), 1.48-1.25 (m, 10H), 0.89 (t, J=6.8 Hz, 3H). .sup.13C NMR (100 MHz, DMSO-d.sub.6): δ 153.3, 148.4, 144.30, 144.17, 142.1, 138.4, 137.0, 136.26, 136.06, 132.5, 128.7, 122.0, 108.2, 103.5, 98.7, 97.8, 60.1, 55.76, 55.59, 42.7, 31.2, 28.82, 28.68, 28.2, 26.7, 22.0, 13.9. HRMS (ESI-TOF): m/z [M+H].sup.+ for C.sub.29H.sub.38N.sub.5O.sub.3.sup.+ calculated. 504.2969. found 504.2972. err.<1 ppm.

3—Synthesis Method No. 3—Compound of Formula II (1b)

[0187] ##STR00037##

[0188] Compound 13c: A solution of compound 4 (625 mg, 1.66 mmol, 1 equiv.) in THF (35 mL) was hydrogenated (40 bars) overnight in the presence of Pd/C (5 wt. %, 36 mg, 1 mol %). After decreasing the pressure, the solution was degassed by sonication for 5 min, then cooled to 0° C. in an ice tray, and 1,5-difluoro-2,4-dinitrobenzene (320 mg, 1.57 mmol, 0.95 equiv.) was added to the solution with stirring. Then tert-butylamine (736 μL, 6.98 mmol, 4.2 equiv.) and DIPEA (602 μL, 3.46 mmol, 2.1 equiv.) were added. The mixture was stirred at room temperature for 4 days. After filtration through celite via dichloromethane and evaporation of the solution, the raw product was purified by flash chromatography on silica gel using dichloromethane/cyclohexane (1/1 to 6/4) as eluent to obtain the compound 13c as a red solid (575 mg, 63% yield).

[0189] TLC: R.sub.f=0.25 (SiO.sub.2 F60, dichloromethane/cyclohexane, 7/3). .sup.1H NMR (400 MHz, CDCl.sub.3): δ 9.26 (s, 1H), 9.00 (br s, 1H), 8.40 (br s, 1H), 6.86 (d, J=8.3 Hz, 1H), 5.99 (dd, J=8.3, 2.4 Hz, 1H), 5.95 (d, J=2.4 Hz, 1H), 5.90 (s, 1H), 3.81 (br s, 1H), 3.66 (br s, 1H), 3.12 (t, J=7.1 Hz, 2H), 3.07-3.03 (m, 2H), 1.66-1.59 (m, 2H), 1.56-1.50 (m, 2H), 1.44-1.22 (m, 29H), 0.89 (t, 6.9 Hz, 3H), 0.86 (t, 6.9 Hz, 3H). .sup.13C NMR (100 MHz, CDCl.sub.3): δ 149.7, 148.4, 147.4, 145.4, 129.5, 129.0, 125.5, 124.0, 112.1, 101.5, 95.6, 95.1, 52.0, 44.1, 43.5, 31.8, 31.8, 29.5, 29.5, 29.4, 29.3, 29.3, 29.2, 29.0, 27.2, 27.1, 22.7, 22.6, 14.1, 14.1. HRMS (ESI-TOF): m/z [M+H].sup.+ for C.sub.32H.sub.53N.sub.6O.sub.4.sup.+ calculated. 585.4123. found 585.4123, err.<1 ppm.

[0190] Compound 1b: A solution of compound 13c (1 g, 1.71 mmol, 1 equiv.) in methanol (60 mL) was hydrogenated (20 bars) in the presence of Pd/C (5% by mass) and HCl (12M, 0.5 mL) for 6 hours. Then the mixture was stirred under air for 16 h. The Pd/C was removed by filtration through celite (AW) that had been rinsed several times with methanol and dichloromethane. After removal of the solvent under reduced pressure, the resulting solid was taken up in dichloromethane and washed with an aqueous solution of HPF.sub.6 (5% by mass in water, 2×60 mL) and distilled water (60 mL). The organic layer was dried with Na.sub.2SO.sub.4, filtered and evaporated under reduced pressure. The residue finally obtained was precipitated in pentane and filtered to obtain product 1 b [PF.sub.6.sup.−] in the form of a red solid (1.05 g, 95% yield).

[0191] .sup.1H NMR (250 MHz, CD.sub.3CN, diluted): δ 7.85 (d, J=9.3 Hz, 1H), 7.24-7.20 (m, 2H), 6.99 (s, 1H), 6.57 (d, J=1.8 Hz, 1H), 6.28-6.20 (br m, 3H), 4.58 (d, J=8 Hz, 2H), 4.37 (br s, 1H), 3.37 (td, J=7.0 Hz, J=6.2 Hz, 2H), 1.75-1.55 (m, 4H), 1.52 (s, 9H), 1.46-1.31 (m, 20H), 0.92-0.87 (m, 6H). .sup.1H NMR (250 MHz, CD.sub.3CN, concentrated): δ 7.76 (d, J=9.3 Hz, 1H), 7.20-7.15 (m, 2H), 6.96 (s, 1H), 6.49 (d, J=1.8 Hz, 1H), 6.34-6.29 (br m, 3H), 4.50 (d, J=8 Hz, 2H), 3.33 (m, 2H), 1.76-1.30 (m, 33H), 0.91-0.86 (m, 6H). .sup.13C NMR (63 MHz, CD.sub.3CN, concentrated): 154.3, 152.4, 139.3, 137.8, 137.6, 134.1, 133.2, 131.3, 121.7, 109.6, 94.7, 90.3, 53.1, 48.8, 44.2, 32.5, 32.5, 30.0, 30.0, 29.9, 29.2, 29.1, 27.8, 27.5, 27.2, 23.3, 23.3, 14.3, 14.3. HRMS (ESI-TOF): m/z [M+H].sup.+ for C.sub.32H.sub.52N.sub.5.sup.+ calculated. 506.4217. found 506.4220. err.<1 ppm.

Example 2—Physicochemical Properties of the Compounds According to the Invention

[0192] The inventors have identified the following different properties of the compounds according to the invention:

[0193] a. Solubility

[0194] The versatility of the synthesis method allows the introduction of various substituents ad infinitum to modulate the solubility properties. Depending on the hydrophilic/hydrophobic nature of R1, R2, R3 and R4, it is thus possible to solubilize type II compounds in polar, apolar, protic or aprotic solvents.

[0195] It is noted that although the compounds are more soluble in organic solvents, these exhibit the property of solubility in water, a property of major interest for use in vitro and in vivo on biological and cellular samples.

[0196] a. Characterization of Protonated Forms

[0197] The inventors have characterized the different protonated forms of compounds of formula I where R.sub.1 and R.sub.2 are C.sub.8H.sub.17, R.sub.3 is tert-butyl and R.sub.4 is H, of the following formula:

##STR00038##

[0198] as well as the mono, di and tri protonated forms of the following respective formulas

##STR00039##

The results obtained are shown in FIG. 1.

[0199] The .sup.1H NMR spectra of the compound

##STR00040##

[0200] confirmed this hypothesis in particular with the presence of three protons, Ha, Hb and Hc, in the aromatic region with coupling constants traditionally encountered within a 1,2,4-trisubstituted benzene. .sup.1H NMR data also showed the presence of two magnetically non-equivalent octyl chains. All of these data suggest the formation of a phenazinium derivative

[0201] In addition, the proton signals H.sub.a and H.sub.e—respectively at 5=7.17 and 7.00 ppm—experience a very strong shielding effect (δ.sub.Hd=6.25 ppm and (δ.sub.He=5.91 ppm) after addition of NaOD (40% w/w in D.sub.2O), an effect that is unusually observed within an aromatic system but already seen within certain triaminophenazines (δ=6.5-6.1 ppm and 5.9-5.4 ppm) described by Roy. This observation can be explained by a deprotonation reaction inducing a break in aromaticity in favor of a quinoidal-type structure

[0202] a. Optical Properties

[0203] In addition to characterizing the protonated forms, the inventors also tested the absorption and emission properties of the various compounds according to the invention.

[0204] i) Absorption Properties

[0205] The inventors tested the absorption of the four more or less protonated compounds mentioned above and evaluated ε (M.sup.−1cm.sup.−1) as a function of the wavelength λ (in nm).

[0206] FIG. 2 shows the absorption spectrum of the type I molecule. The absorption spectrum reflects the presence of an uncharged type I species for which the degree of delocalization/conjugation is lower (hypsochrome effect) than for the type II cationic species.

[0207] FIG. 3 shows the absorption spectrum of the type II molecule. The absorption spectrum reflects the presence of a mono-charged type II species for which the degree of delocalization/conjugation is greater (batochromic effect) than for the neutral type I species.

[0208] FIG. 4 shows the absorption spectrum of the C-type molecule. A “super” acid, triflic acid (HOTf) (pKa.sub.(MeCN)=0.70), was used to protonate II in the UV cell (C≈1.44×10.sup.−5 M, in MeCN). When acid is added (from 0.1 to 16 equiv.), the intensity of the initial bands located at 267, 308, 465 and 552 nm decreases in favor of the appearance of new bands at 274, 322, 507 and 686 nm. This spectral evolution reflects the disappearance of the starting compound II in favor of the formation of a single type C species whose absorption bands are of higher energy. The first protonation step is complete after adding 16 equiv. of HOTf

[0209] FIG. 5 shows the absorption spectrum of the D-type molecule. Beyond the addition of 16 equiv. of triflic acid, a new species appears (new bands at 268, 287, 370, 475 and 506 nm) at the same time as the C cation disappears. Double protonation of II is complete after adding plus 2600 equiv. of HOTf. The C band at 686 nm has completely disappeared in favor of the spectrum of a D trication having a narrow band at 506 nm and equipped with a shoulder characteristic of a cyanine-type structure.

[0210] ii) Emission Properties

[0211] The inventors tested the fluorescence emission as a function of the wavelength λ (in nm) for the following compound taken up in acetonitrile

##STR00041##

The results obtained are shown in FIG. 6.

[0212] The UV-Vis absorption spectrum of II showed the presence of two main bands located at λ.sub.max=265 nm and 549 nm and with respective shoulders located at λ=295 nm and 578 nm. Molecule II is also fluorescent and emits at λ.sub.em=642 nm (excitation at λ=550 nm). This emission is comparable to that produced by the “neutral red” cationic analogue compound reported in the literature (λ.sub.abs=534 nm and λ.sub.em=616 nm)

[0213] iii) Fluorescence Yield and Emission

[0214] The inventors then set out to measure the value ϕf of the quantum yield of fluorescence of the following compound taken up in acetonitrile

##STR00042##

[0215] To do this, the inventors measured the absorbance (relative intensity) as a function of the wavelength (in nm) in the presence of increasing doses of 1,8-DiazaBicyclo[4.3.0]Undec-7-ene (DBU—0 to 2 equivalents).

The results are shown in FIG. 7.

[0216] Phenazinium II and its II-H conjugate base fluoresce in MeCN at neutral or basic pH. Conversely, no luminescence property was observed in an acid medium. FIG. 7 shows the spectral evolution of phenazinium II during the addition of DBU (excitation at 483 nm). This evolution clearly reflects the disappearance of the starting compound (decrease in the band at 637 nm) in favor of the formation of a single II-H species possessing an emission at higher energy much lower than that of II (increase in the band at 550 nm). Calculations of quantum yields indeed show that the deprotonated form [II-H] produces less fluorescence (ϕ.sub.f=0.08 at 550 nm) than the starting form II (ϕ.sub.f=0.76 at 637 nm). The reference used for the calculation of the fluorescence quantum yield is tetraphenylporphyrin in acetonitrile (ϕ.sub.f=0.15).

[0217] The emission maximum is at a localized wavelength in the far red at 645 nm.

[0218] The coefficient ϕf found is 0.76, which attests to a very high fluorescence yield and a high brightness of about 50000.

[0219] The brightness (B) is proportional to the amount of light emitted by fluorescence at a given excitation light according to the relationship B=ε×ϕ (with ε=molar extinction coefficient and ϕ the emission quantum yield).

[0220] The calculation of ε is carried out using a spectrophotometer measuring the absorbance A (quantity without unit) of a dilute solution of known concentration C in a tank of thickness I.

[0221] The fundamental relation used in spectrophotometry is presented in the form: A=ε.Math.I.Math.c (A being the absorbance or optical density)

[0222] Calculation of ϕ: It is determined by measuring the emission intensity of a solution of known concentration; the reference used to calculate the fluorescence quantum yield here is tetraphenylporphyrin in acetonitrile (ϕ.sub.f=0.15).

[0223] The quantum yield is defined by:

[0224] ϕ=number of photons emitted/number of photons absorbed

[0225] iv) Fluorescence In Vivo

[0226] Prior to fluorescent labeling tests on cell lines, the inventors tested the toxicity of the compounds according to the invention.

[0227] MCF-7 cancer lines were seeded in 96-well plates at a concentration of approximately 5000 cells/well in 200 μl of culture medium and left in culture for 24 hours. Then, the cells were incubated for 72 h, with or without the compound to be tested (from 1 nM to 1 μM). After incubation with the compounds, an MTT test was carried out in order to test the cytotoxicity of the compounds. The cells were briefly incubated in the presence of 0.5 mg mL.sup.−1 of MTT for 4 h in order to measure mitochondrial activity. Then, the MTT precipitates were dissolved in 150 μL of an ethanol/DMSO (1:1) mixture solution and the absorbance was read at 540 nm.

[0228] The inventors came to the conclusion that the 100 nM dose did not significantly affect cell survival, as shown in FIG. 14. It is observed very clearly that the cytotoxicity in the dark is low at a concentration of 100 nM (FIG. 14)

[0229] The inventors tested the fluorescence of the compound of the following formula

##STR00043##

[0230] on MCF-7 breast cancer cells by single-photon (excitation at 514 nm) or two-photon (excitation at 790 or 810 nm) microscopy.

[0231] One- and two-photon imaging

[0232] The MCF-7 human breast cancer cells were seeded in petri dishes (World Precision Instrument, Stevenage, UK) having a glass plate at the bottom, in 2 mL of culture medium. The cells were then incubated for 16 h with the compound according to the invention at a concentration of 0.1 μM or 0.5 μM. 15 minutes before the end of the incubation, the cells were incubated with Hoechst 33342 (Invitrogen, Cergy Pontoise, France) at a final concentration of 5 μg.Math.mL.sup.−1 in order to label the cell nuclei. Then the cells were washed twice with culture medium.

[0233] One-photon fluorescence imaging was performed on live cells at a wavelength of 514 nm using a Carl Zeiss Confocal Microscope (LSM780). Two-photon fluorescence imaging was performed at wavelengths of 790 nm or 810 using the Chameleon laser available on the same microscope. All images were taken with the same objective, at the same magnification (63×/1.4 OIL DIC Plan-Apo).

[0234] The results obtained in single-photon microscopy are presented in FIG. 8, and in two-photon microscopy in FIG. 9.

[0235] These compounds showed remarkable one- and two-photon imaging properties (FIGS. 3 and 4). It clearly appears that the compounds are easily identifiable and that their localization is only cytoplasmic (the co-localizations are perfectly conclusive). It is interesting to note that the clearest and most intense markings are obtained with the lowest concentrations. Indeed, the markings are clearly finer and reveal intense cytoplasmic granules, which tends to show that a more precise identification of cytoplasmic targets is possible under these conditions. In particular, intense fluorescence foci obtained in perinuclear regions may correspond to the endoplasmic reticulum.

[0236] The inventors also tested the internalization of the compounds according to the invention over time.

[0237] The internalization kinetics were carried out with a CLARIOstar reader in order to quantify the internalization of the compound in the MCF-7 tumor cells. The values, corresponding to the ratio of the residual fluorescence/the total fluorescence, are presented in the form of means of three experiments, ±the standard deviation.

[0238] It is clearly seen (FIG. 10) that 10% of the fluorescent compound (II) penetrates the cell in less than 24 hours, allowing observation of very high-quality images.

[0239] The kinetics of incorporation by MCF-7 cells is shown in FIG. 10.

[0240] a. .sup.1O.sub.2 Output

[0241] The absorption spectra were measured with a Perkin-Elmer double beam UV-visible spectrophotometer (Lambda EZ 210). The fluorescence spectrum was measured with a Fluorolog FL3-222 spectrofluorimeter (Horiba Jobin Yvon, Longjumeau, France) equipped with a 450 W xenon arc lamp, a thermostated compartment (25° C.), a photomultiplier UV-visible R928 (HAMAMATSU Japan) and an InGaAs infrared detector cooled with liquid nitrogen (DSS-16A020L Electro-Optical System Inc, Phoenixville, Pa., USA). The excitation beam is separated by an SPEX dual grating monochromator (1200 lines/mm blazed at 330 nm). The fluorescence was measured by the UV-Visible detector via the SPEX double grating emission monochromator (1200 lines/mm blazed at 500 nm). The production of singlet oxygen was measured by the infrared detector via the SPEX double grating emission monochromator (600 lines/mm blazed at 1 μm). All spectra were measured using 4-sided quartz cuvettes. The absorbance values at the excitation wavelength of the references and samples have been adjusted to approximately 0.2.

[0242] By this method, the inventors were able to measure the quantum yield ϕ.sub.Δ, which is 0.11 for the compound

##STR00044##

[0243] This low efficiency is expected, since the vast majority of absorbed photons are converted into light, about 76% of photons.

Example 3—Use of the Compounds According to the Invention in Photodynamic Therapy In Vitro

[0244] The inventors tested the effect of the compounds according to the invention in photodynamic therapy. MCF7 cells were incubated with the compound of formula

##STR00045##

[0245] for 5 h and irradiated (or not) at 530 nm for 20 minutes. Two days later, the cells were subjected to a colorimetric cell viability assay (MTT).

[0246] The results are presented in FIGS. 12 and 13.

[0247] The results obtained under one-photon irradiation (λ.sub.irrad.=514 nm) are exceptional, since 98% of tumor cells are killed at very low concentrations (C=100 nM).

[0248] As can be seen in FIG. 13, at a concentration of 100 nM, two zones are easily distinguished owing to the violet crystals of MTT, which only stain living cells (not irradiated).

[0249] Two-photon photodynamic therapy (λ.sub.irrad.=810 nm) has shown very encouraging results, since nearly 50% of tumor cells are killed without optimization (irradiation for only 5 seconds at a very low concentration of 100 nM of compound according to the invention).

[0250] The inventors also tested the effect of the compounds according to the invention in photodynamic therapy on other models. Keratosis treatment tests were carried out at different concentrations of a photosensitizing compound (PS): either that used on the MCF-7 cells, or the compound of formula

##STR00046##

[0251] on cultured keratinocytes. PS was added to the cells for 20 minutes at doses of 0 nM (A), 10 nM (B), 25 nM (C) or 50 nM (D) and the latter were irradiated (black columns) or not (gray columns) at 530 nm (FIGS. 15 and 17). Two days later, the cells are subjected to a colorimetric cell viability assay (MTT) (FIGS. 16 and 18).

Example 4—Internalization of the Compounds According to the Invention

[0252] The inventors evaluated the capacity of the cells to internalize the compounds according to the invention.

[0253] To do this, MCF-7 cells or healthy donor fibroblasts have been treated or not with 0.5 nM of compound of formula

##STR00047##

[0254] for 1, 3, 6 or 24 h, and cell fluorescence was assessed by flow cytometry by detecting the number of cells with red fluorescence.

[0255] The results can be seen in FIGS. 20 and 21.

[0256] At an equal concentration (0.5 nM) after 6 h of incubation with the compound according to the invention, 46% of the cancerous cells (MCF-7) have internalized the compound, but only 18% of the healthy cells (fibroblasts) have. Similarly, 90% of cancer cells have internalized the compound according to the invention after 24 h compared with only 24% for healthy cells.

[0257] These results show that the compound according to the invention enters cancerous cells more rapidly than it enters healthy cells.

Example 5—Comparison Absorption of Phenazinium According to the Invention and Phenazine Described in the Prior Art

[0258] ##STR00048##

[0259] The cationic phenaziniums ([12].sup.+; [23].sup.+) are much more soluble than the neutral phenazines (24 and 25). The latter are indeed insoluble in alcohols and poorly soluble (C<10.sup.−4 M) in MeCN, or acetone, whereas cationic phenaziniums ([12].sup.+; [23].sup.+) are soluble in all common solvents (i.e. toluene, Et.sub.2O, CH.sub.2CL.sub.2, CHCl.sub.3, acetone, MeCN, MeOH, EtOH DMF, DMSO) due to their amphiphilic character (the charged part being hydrophilic and the alkylated part being hydrophobic).

[0260] The absorption spectra of the compounds ([12].sup.+; [23].sup.+) are almost identical and show a band located in the region visible at λ.sub.max=553 nm (ε.sup.543=43400 M.sup.−1 cm.sup.−1 and ε.sup.543=41200 M.sup.−1cm.sup.−1) with a shoulder around 465 nm. Two absorption bands located in the ultraviolet at 265 nm and 320 nm complete these absorption spectra.

[0261] The absorption characteristics of the phenazine compounds 24 and 25 are similar to those of the cationic phenazinium compounds with, however, a 100 nm blue shift, their absorption appearing respectively at λ.sub.max=472 nm (ε.sup.472=16100 M.sup.−1cm.sup.−1) and λ.sub.max=472 nm (ε.sup.472=14800 M.sup.−1cm.sup.−1). The corresponding intensities are conversely much weaker (35 to 45% of the intensity of the main peak of the cationic phenazinium compounds). Furthermore, compounds 24 and 25 show several additional absorption bands located between 220 and 300 nm.

The data is shown in FIG. 19.

[0262] This demonstrates that phenazinium compounds are more suitable for photon therapy than neutral phenazines because they are more soluble and can be irradiated with a lower-energy laser (longer excitation wavelengths).