Azobenzene derivatives, process for their preparation and their use for therapeutic treatment associated with ionizing radiations

Abstract

The present invention relates to new ionizing radiation-activatable derivatives, their preparation process and their therapeutic uses.

Claims

1. A compound having the formula (IA): ##STR00019## in which: M represents a metal atom selected from Ce(III), Pr(III), Nd(III), Sm(III), Eu(III), Gd(III), Tb(III), Dy(III), Ho(III), Er(III), Tm(III), Yb(III), Mg(II), Ca(II), Mn(II), Fe(II), Fe(III), Cu(II), Zn(II), Ga(III), Y(III), Zr(III), Tc(IV), Tc(VI), Tc(VII), Ru(II), Ru(III), Ru(IV), Pd(II), Ag(I), In(III), Hf(IV), Re(VI), W(II), W(III), W(IV), W(V), W(VI), Os(III), Os(IV), Ir(III), Ir(IV), Pt(II), Au(I), Au(III), Tl(III), Zr(IV), Nb(III), Bi(III); n is 1, 2, 3, 4, 5, 6, or 7; V and V, which may be identical or different, are hydrogen atoms or linear or branched C1-C10 alkyl or alkoxy chains, or C1-C10 alkyl chains linked together and comprising one or more heteroatoms selected from N, O, or S, optionally substituted with one or more substituents independently selected from halogen atoms, and nitrile, nitro, thio, amino, amido, aryl, heteroaryl, hydroxyl, carboxylic acid or carboxylate groups, to form a ring; R1, R1, R2, R2, which may be identical or different, are hydrogen atoms, or linear or branched C1-C10 alkyl or alkoxy chains optionally substituted with one or more substituents independently selected from halogen atoms, and nitrile, nitro, thio, amino, amido, aryl, heteroaryl, hydroxyl, carboxylic acid or carboxylate groups; R3 and R3, which may be identical or different, are hydrogen atoms or linear or branched C1-C10 alkyl or alkoxy chains optionally substituted with one or more substituents independently selected from halogen atoms, and nitrile, nitro, thio, amino, amido, aryl, heteroaryl, hydroxyl, carboxylic acid or carboxylate groups, or R3 and R3 are linked together to form a 5- to 14-membered heterocycle or heteroaryl; m and m, which may be identical or different, are equal to 1 or 2; p is an integer between 0 and 6; X is selected from hydrogen and halogen atoms; R is a linear or branched C1-C12 alkyl group; the NN double bond is in cis or trans form; wherein the n cationic charges of M are optionally neutralized by 0 to n carboxylate groups (COOH) substituted on R1, R1, R2, R2, R3, R3, V, V, and/or by 0 to n of the counterions present in solution, and cis and/or trans isomers and mixtures thereof.

2. A compound having the general formula (IB): ##STR00020## in which M represents a metal atom selected from Ce(III), Pr(III), Nd(III), Sm(III), Eu(III), Gd(III), Tb(III), Dy(III), Ho(III), Er(III), Tm(III), Yb(III), Mg(II), Ca(II), Mn(II), Fe(II), Fe(III), Cu(II), Zn(II), Ga(III), Y(III), Zr(III), Tc(IV), Tc(VI), Tc(VII), Ru(II), Ru(III), Ru(IV), Pd(II), Ag(I), In(III), Hf(IV), Re(VI), W(II), W(III), W(IV), W(V), W(VI), Os(III), Os(IV), Ir(III), Ir(IV), Pt(II), Au(I), Au(III), Tl(III), Zr(IV), Nb(III), Bi(II); n is 1, 2, 3, 4, 5, 6, or 7; p is an integer between 0 and 6; X is selected from H; halogen atoms; alkoxy, alkyl or cycloalkyl groups optionally interrupted or substituted with one or more heteroatom(s) or group(s) COOH, CONH.sub.2, COSH, OH, NH.sub.2, SH; 5- to 12-membered aryl or heteroaryl groups optionally substituted with one or more COOH, CONH.sub.2, COSH groups; COOH or NH.sub.2 groups; and R represents H, or linear or branched C1-C18 alkyl or alkoxy chain, optionally substituted with one or more substituents independently selected from halogen atoms, and nitrile, nitro, thio, amino, amido, aryl, heteroaryl, hydroxyl, carboxylic acid or carboxylate groups.

3. A compound having the general formula (IC): ##STR00021## in which M represents a metal atom selected from Ce(III), Pr(III), Nd(III), Sm(III), Eu(III), Gd(III), Tb(III), Dy(III), Ho(III), Er(III), Tm(III), Yb(III), Mg(II), Ca(II), Mn(II), Fe(II), Fe(III), Cu(II), Zn(II), Ga(III), Y(III), Zr(III), Tc(IV), Tc(VI), Tc(VII), Ru(II), Ru(III), Ru(IV), Pd(II), Ag(I), In(III), Hf(IV), Re(VI), W(II), W(III), W(IV), W(V), W(VI), Os(III), Os(IV), Ir(III), Ir(IV), Pt(II), Au(I), Au(III), Tl(III), Zr(IV), Nb(III), Bi(III); n is 1, 2, 3, 4, 5, 6, or 7; X may be selected from H; halogen atoms; alkoxy, alkyl or cycloalkyl groups optionally interrupted or substituted with one or more heteroatom(s) or group(s) COOH, CONH.sub.2, COSH, OH, NH.sub.2, SH; 5- to 12-membered aryl or heteroaryl groups optionally substituted with one or more COOH, CONH.sub.2, COSH groups; COOH or NH.sub.2 groups; and cis and/or trans isomers and mixtures thereof.

4. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of formula (I): ##STR00022## in which: M represents a metal atom selected from Ce(III), Pr(III), Nd(III), Sm(III), Eu(III), Gd(III), Tb(III), Dy(III), Ho(III), Er(III), Tm(III), Yb(III), MO(II, Ca(II), Mn(II), Fe(II), Fe(III), Cu(II), Zn(II), Ga(III), Y(III), Zr(III), Tc(IV), Tc(VI), Tc(VII), Ru(II), Ru(III), Ru(IV), Pd(II), Ag(I), In(III), Hf(IV), Re(VI), W(II), W(III), W(IV), W(V), W(VI), Os(III), Os(IV), Ir(III), Ir(IV), Pt(II), Au(I), Au(III), Tl(III), Zr(IV), Nb(III), Bi(III); n is 1,2,3,4,5,6, or 7; V and V, which may be identical or different, are hydrogen atoms or linear or branched C1-C10 alkyl or alkoxy chains, or C1-C10 alkyl chains linked together and comprising one or more heteroatoms selected from N, O, or S, optionally substituted with one or more substituents independently selected from halogen atoms, and nitrile, nitro, thio, amino, amido, aryl, heteroaryl, hydroxyl, carboxylic acid or carboxylate groups, to form a ring; R1, R1, R2, R2, which may be identical or different, are hydrogen atoms, or linear or branched C1-C10 alkyl or alkoxy chains optionally substituted with one or more substituents independently selected from halogen atoms, and nitrile, nitro, thio, amino, amido, aryl, heteroaryl, hydroxyl, carboxylic acid or carboxylate groups; R3 and R3, which may be identical or different, are hydrogen atoms or linear or branched C1-C10 alkyl or alkoxy chains optionally substituted with one or more substituents independently selected from halogen atoms, and nitrile, nitro, thio, amino, amido, aryl, heteroaryl, hydroxyl, carboxylic acid or carboxylate groups, or R3 and R3 are linked together to form a 5- to 14-membered heterocycle or heteroaryl; m and m, which may be identical or different, are equal to 1 or 2; X, X, X, X, Y, Y, Y, Y, which may be identical or different, are independently selected from H; halogen atoms; alkoxy, alkyl or cycloalkyl groups optionally interrupted or substituted with one or more heteroatom(s) or group(s) COOH, CONH.sub.2, COSH, OH, NH.sub.2, SH: 5- to 12-membered aryl or heteroaryl groups optionally substituted with one or more COOH, CONH.sub.2, COSH groups; COOH or NH.sub.2 groups; Z represents an alkyl group optionally interrupted or substituted with one or more heteroatom(s) or a COOH, CONH.sub.2, COSH, OH, NH.sub.2, SH group; a 5- to 12-membered aryl or heteroaryl group optionally substituted with one or more COOH, CONH.sub.2, COSH groups; or a COOH or NH.sub.2 group; W and W, which may be the same or different, independently represent a CH.sub.2 group; or an aryl or cycloalkyl group; an oxygen or nitrogen atom (secondary or ternary); an amide linkage; an ester linkage; a thioether linkage; U and U, which may be the same or different, represent the CH or NH group, it being understood that the double bond UU is in cis or trans form; T represents a CH.sub.2 group; a C(O)NH group; an alkoxy, alkyl or cycloalkyl group optionally interrupted or substituted with one or more heteroatom(s) or a COOH, CONH.sub.2, COSH, OH, NH.sub.2, SH group; a 5- to 12-membered aryl or heteroaryl group containing one or more heteroatom(s) and/or optionally substituted with one or more groups chosen from COOalkyl, CONHalkyl, COSHalkyl; R represents H, or linear or branched C1-C18 alkyl or alkoxy chain, optionally substituted with one or more substituents independently selected from halogen atoms, and nitrile, nitro, thio, amino, amido, aryl, heteroaryl, hydroxyl, carboxylic acid or carboxylate groups; p and q are integers between 0 and 6; wherein the n cationic charges of M are optionally neutralized by 0 to n carboxylate groups (COOH) optionally substituted on R1, R1, R2, R2, R3, R3, V, V, and/or by 0 to n of the counterions present in solution; and cis and/or trans isomers and mixtures thereof, wherein the cancer is selected from the group consisting of lung cancer, pancreatic cancer, liver cancer, spleen cancer, small cell lung carcinoma, prostate cancer, rhabdomyosarcoma, stomach cancer, gastrointestinal cancer, colorectal cancer, kidney cancer, breast cancer, ovarian cancer, testicular cancer, thyroid cancer, head and neck cancer, skin cancer, soft tissue sarcoma, bladder carcinoma, bone cancers, myeloma, plasmacytoma, germ cell cancer, uterine cancer, leukemia, lymphoma, neuroblastoma, osteosarcoma, retinoblastoma, central nervous system cancers, and Wilms' tumors.

5. The method according to claim 4, wherein the compound (I) is predominantly in cis form, and further comprising a step of irradiating a tumor caused by the cancer with ionizing radiation.

6. The method of claim 4, further comprising monitoring said treatment by in vivo medical imaging.

7. The method of claim 4, further comprising administering one or more chemotherapy and/or immunotherapy anti-cancer agents to the subject.

8. The method of claim 5, wherein the ionizing radiation is X-rays, gamma rays, electron beams or hadron beams.

9. The method of claim 8, wherein the hadron beams comprise protons and/or carbon ions.

10. The method of claim 6, wherein the in vivo medical imaging is MRI, PET, X-ray or SPECT.

11. The method of claim 7, wherein the one or more chemotherapy and/or immunotherapy anti-cancer agents include at least one immune checkpoint inhibitor.

12. The method of claim 11, wherein the at least one immune checkpoint inhibitor is anti-CTLA4, anti-PD-L1 or anti-PD1.

Description

FIGURES

(1) FIG. 1 shows the absorbance spectra of cis-GdAzo (a) and cis-GdFAzo (d) under gamma irradiation in PBS. PPS1: Photostationary state containing the predominantly cis isomer. b, e: Histograms representing the molecular activation of cis-GdAzo and cis-Azo (control molecule without Gd) (b) or of cis-GdFAzo and cis-FAzo control molecule without Gd) (e) under gamma irradiation in PBS determined by HPLC and calculated by the difference in the proportion of trans-isomer in the medium (n=3). c: Molecular activation of cis-GdAzo and cis-GdFAzo under X-ray, gamma and Linac linear electron accelerator. f: Histogram representing the molecular activation of cis-MAzo (M being the metal indicated on the X-axis) under gamma irradiation in PBS determined by HPLC and calculated by the difference in the proportion of trans isomer in the medium (n=3). Gy: Gray (J.L.sup.1). XR: X-ray irradiation. GR: Gamma ray irradiation. E: Irradiation by Linac linear electron accelerator. NI: Not irradiated. Standard deviations are plotted on the graphs. *** P<0.001 (Two-way Anova with Bonferroni post-test).

(2) FIG. 2 shows a: Microscopy images of cancer cells (Panc-1, human pancreatic cancer) in the presence of propidium iodide and the inactive compound cis-GdAzo (1, 2) or the active compound trans-GdAzo (3, 4) at a concentration of 500 PM before (1, 3) or 30 min after (2, 4) the introduction of the compounds. Superposition of white light and fluorescence images (propidium iodide in the nuclei appears in red and shows membrane permeabilization). b: Microscopy images of cancer cells (Panc-1) in the presence of propidium iodide and the inactive compound cis-GdAzo at a concentration of 250 M (1, 2) or 500 M (3, 4), before (1, 3) or 30 min after (2, 4) irradiation of the medium under gamma radiation (2 Gy). c: Histogram comparing the impact of gamma irradiation (2 Gy) on cell permeabilization (Panc-1) in the presence of propidium iodide and cis-GdAzo (n=3). d, e: Histograms comparing the impact of gamma irradiation (2 Gy) on the mortality of Gem-resistant cancer cells (CCRF-CEM ARAC-8C, human acute lymphoblastic leukemia) 4 days after treatment in the presence of cis-GdAzo (d) or in the presence of cis-GdAzo and Gem (0.1 M) (e) for a duration of 1 h. f: Histogram comparing the impact of gamma irradiation (2 Gy) on the mortality of Gem-resistant cancer cells (CCRF-CEM ARAC-8C) in the presence of Dotarem or Dotarem and Gem (0.1 M). Gy: Gray (J.L.sup.1). GR: Gamma ray irradiation. NI: Not irradiated. The standard errors of the mean are plotted in graph c and the standard deviations are plotted in graphs d-f. * P<0.05, ** P<0.01, *** P<0.001 (Two-way Anova with Bonferroni post-test).

EXAMPLES

1. Analytical Chromatography Methods

(3) Method A

(4) HPLC (high-performance liquid chromatography) analyses were performed on a 1565 binary HPLC pump (Waters), PAD 2998 reader (Waters), with an Agilent Eclipse XDB-C18 reversed-phase column (length: 250 mm, diameter: 4.6 mm, stationary phase: 5 m) using a 0.05% water-TFA/0.05% acetonitrile-TFA gradient system; 0 (98/2), 5 (98/2), 25 (0/100), 27 (0/100), 29 (98/2), 35 (98/2) at a flow rate of 1 mL.Math.min.sup.1 (30 L injected) The data was processed on Empower.

(5) Method B

(6) LCMS (liquid chromatography mass spectrometry) analyses were performed on an Alliance 2695 system (Waters) with an XBridge C18 reversed-phase column (length: 150 mm, diameter: 2.1 mm, stationary phase: 3.5 m) using a 0.1% water-FA/acetonitrile gradient system; 0 (95/5), 20 (0/100) at a flow rate of 0.25 mL.Math.min.sup.1 (10 L injected). The mass analyser was a TOF LCT Premier (Waters). The capillary voltage was 2.8 kV. The cone voltage was 35 V. The source temperature was 120 C. and the desolvation temperature was 280 C. The data was processed on MassLynx.

(7) Method C

(8) HPLC analyses were performed on a 2525 binary HPLC pump (Waters) coupled to a 515 HPLC pump (Waters), PAD 2996 reader (Waters), with an XBridge C18 reversed-phase column (length: 100 mm, diameter: 3.0 mm, stationary phase: 3.5 m) using a 0.05% water-TFA/acetonitrile isocratic system at a flow rate of 0.75 mL.Math.min.sup.1 (10 L injected). The detection wavelength corresponded to the isobestic point of the compound studied in the mobile phase used. The data was processed on MassLynx.

II. Summaries

(9) General

(10) All reagents were purchased from Sigma-Aldrich or Alfa Aesar with the highest purity available and were used without further purification. The DOTAGA anhydride was purchased from CheMatech and was used without further purification. The silica gel (Aldrich 717185 Si 60, 40-63 m) used for the flash chromatography was purchased from VWR. RP-18 reversed-phase flash chromatography was performed on a CombiFlash instrument (Biotage). Thin-layer chromatography was carried out using aluminium foil coated with 60F254 silica gel (detection by UV lamp at 254 nm or by ninhydrin). The .sup.1H, .sup.13C et .sup.19F NMR spectra were acquired on Bruker 300 MHz or 400 MHz spectrometers at ambient temperature. Chemical shifts are given in ppm using the solvent as a reference. The coupling constants J are measured in Hz. The coupling profiles are described by the abbreviations d (doublet), t (triplet) and m (mutiplet). High resolution mass spectrometry (HRMS) experiments were performed in a 3/7 H.sub.2O/methanol mixture by electrospray ionization in positive mode, unless otherwise indicated, using a time-of-flight mass analyser with a TOF-LCT Premier mass spectrometer (Waters). The analytical chromatography methods (HPLC and LCMS) are described in a dedicated paragraph. The purity of the synthetic intermediates was determined by .sup.1H NMR or reversed-phase HPLC. The purity of the final products was determined by reversed-phase HPLC and was confirmed to be >95%.

(11) The following compounds were synthesized:

(12) ##STR00016## Where X=H or F, Where M=Cu, Ga, Y, In, Eu, Gd, Yb or Bi, Where n=2 or 3 according to the following synthesis scheme:

(13) ##STR00017## ##STR00018##

Synthesis of 4-hydroxy-4-butoxyazobenzene (1)

(14) 4-Butoxyaniline (2.00 mL, 12.0 mmol) and sodium nitrite (0.854 g, 12.0 mmol, 1.0 equiv) were dissolved in a 1:1 EtOH/H.sub.2O mixture (24 mL) and the medium was cooled to 0 C. Ice (12 g) was introduced into the medium before the careful addition of cc HCl (2.6 mL). A previously prepared aqueous solution (6.3 mL) of phenol (1.14 g, 12.0 mmol, 1.0 equiv) and NaOH (0.960 g, 24.0 mmol, 2.0 equiv) cooled to 0 C. was carefully introduced into the medium at 0 C. The medium was stirred for 20 min at 0 C. and then for 70 min at ambient temperature (AT). After adjusting the pH to 1 (cc HCl), the medium was left to stand for 30 min at AT before filtration. The precipitate was washed with water (450 mL), solubilized in DCM and the organic phase was dried over MgSO.sub.4 before being concentrated. 4-hydroxy-4-butoxyazobenzene 1 (2.76 g, 10.2 mmol, 85%) was isolated as a black amorphous powder.

(15) .sup.1H NMR (400 MHz; CDCl.sub.3) (ppm): 7.87 (d; J=8.9 Hz; 2H); 7.82 (d; J=8.8 Hz; 2H); 6.98 (d; J=8.9 Hz; 2H); 6.91 (d; J=8.8 Hz; 2H); 4.03 (t; J=6.5 Hz; 2H); 1.85-1.75 (m; 2H); 1.58-1.45 (m; 2H); 0.99 (t; J=7.4 Hz; 3H)

(16) .sup.13C NMR (75 MHz; CDCl.sub.3) (ppm): 161.51; 158.38; 146.89; 146.63; 124.81; 124.56; 116.07; 114.92; 68.22; 31.35; 19.33; 13.96.

(17) HRMS (m/z): calculated for C.sub.16H.sub.19N.sub.2O.sub.2; 271.1447 ([M+H].sup.+). Found 271.1440.

(18) R.sub.f=0.36 (SiO.sub.2; DCM; UV).

Synthesis of 4-(N-(tert-butyloxycarbonyl)-ethoxyamine)-4-butoxyazobenzene (2)

(19) 4-Hydroxy-4-butoxyazobenzene 1 (2.00 g, 7.40 mmol) and K.sub.2CO.sub.3 (1.53 g, 11.1 mmol, 1.5 equiv) were dissolved in acetone (25 mL). After 30 min stirring at AT under argon, 2-(Boc-amino)ethyl bromide (4.98 g, 22.2 mmol, 3.0 equiv) was introduced into the medium. After stirring at reflux for 18 h, the medium was filtered off while hot and the precipitate was washed with hot acetone (75 mL). The filtrate was left to stand at AT for 30 min before filtration and the resulting precipitate 1 was washed with cold acetone. The filtrate was left to stand at 0 C. for 3 h before filtration. The resulting precipitate 2 was washed with cold acetone and added to precipitate 1. The filtrate was concentrated and purified by flash chromatography on silica (DCM). 4-(N-(tert-butyloxycarbonyl)-ethoxyamine)-4-butoxyazobenzene 2 (2.27 g, 5.49 mmol, 74%) was isolated as a yellow amorphous powder (57% by precipitation, 17% by flash chromatography).

(20) .sup.1H NMR (400 MHz; CDCl.sub.3) (ppm): 7.86 (2d; J=8.9 Hz; 4H); 6.99 (2d; J=8.9 Hz; 4H); 4.10 (t; J=5.0 Hz; 2H); 4.04 (t; J=6.5 Hz; 2H); 3.57 (m; 2H); 1.87-1.74 (m; 2H); 1.64-1.40 (m; 11H); 1.00 (t; J=7.4 Hz; 3H).

(21) .sup.13C NMR (100 MHz; CDCl.sub.3) (ppm): 161.46; 160.62; 156.03; 147.51; 147.07; 124.52; 124.48; 114.84; 114.82; 79.79; 68.18; 67.64; 40.27; 31.42; 28.55; 19.38; 13.98.

(22) HRMS (m/z): calculated for C.sub.23H.sub.32N.sub.3O.sub.4; 414.2393 ([M+H].sup.+). Found 414.2396.

(23) R.sub.f=0.90 (SiO.sub.2; DCM/MeOH 98:2; UV).

Synthesis of 4-aminoethoxy-4-butoxyazobenzene (3)

(24) 4-(N-(tert-butyloxycarbonyl)-ethoxyamine)-4-butoxyazobenzene 2 (1.26 g, 3.04 mmol) was dissolved in a 4:1 DCM/TFA mixture (63 mL) and the medium was stirred for 1.5 h at AT. After concentration, diethyl ether (250 mL) was introduced and the precipitate formed was concretized for 1 h before filtration and washing with diethyl ether (450 mL). 4-aminoethoxy-4-butoxyazobenzene 3 (1.25 g, 2.93 mmol, 96%, TFA salt) was isolated as a yellow amorphous powder.

(25) .sup.1H NMR (400 MHz; MeOD) (ppm): 7.88 (d; J=8.9 Hz; 2H); 7.85 (d; J=8.9 Hz; 2H); 7.15 (d; J=8.9 Hz; 2H); 7.04 (d; J=8.9 Hz; 2H); 4.32 (t; J=4.4 Hz; 2H); 4.06 (t; J=6.4 Hz; 2H); 3.41 (t; J=4.4 Hz; 2H); 1.86-1.73 (m; 2H); 1.60-1.46 (m; 2H); 1.00 (t; J=7.4 Hz; 3H).

(26) .sup.13C NMR (75 MHz; MeOD) (ppm): 163.03; 161.34; 148.97; 148.10; 125.42; 125.30; 115.99; 115.80; 69.12; 65.59; 40.24; 32.43; 20.28; 14.16.

(27) HRMS (m/z): calculated for C.sub.18H.sub.24N.sub.3O.sub.2; 314.1869 ([M+H].sup.+). Found 314.1864.

(28) HPLC (method A); t.sub.R 17.92 min(cis)-20.35 min(trans).

(29) R.sub.f=0.70 (SiO.sub.2; DCM/MeOH 95:5; UV or ninhydrin).

Synthesis of 4-(N-(1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid-10-glutaryl)-ethoxyamine)-4-butoxyazobenzene (Azo)

(30) 4-aminoethoxy-4-butoxyazobenzene 3 (259 mg, 0.606 mmol, TFA salt) was dissolved in anhydrous DMF (4.3 mL). After introduction of triethylamine (253 L, 1.81 mmol, 3.0 equiv), the medium was stirred for 5 min at AT under argon. The DOTAGA anhydride (278 mg, 0.606 mmol, 1.0 equiv) was then introduced and the medium was stirred for 21 h at 70 C. under argon. After concentration, diethyl ether (100 mL) was introduced and the precipitate formed was concretized for 30 min before filtration and washing with diethyl ether (250 mL). The crude was purified by reverse phase flash chromatography (RP-18, Biotage, gradient H.sub.2O 0.05% FA/CH.sub.3CN 0.05% FA 1:0 to 2:8). 4-(N-(1,4,7,10-tetraazacyclododecane-1,4,7-tri-acetic acid-10-glutaryl)-ethoxyamine)-4-butoxyazobenzene Azo (294 mg, 0.381 mmol, 63%) was isolated by freeze-drying in the form of a yellow electrostatic powder.

(31) HRMS (m/z): calculated for C.sub.37H.sub.54N.sub.7O.sub.11; 772.3881 ([M+H].sup.+). Found 772.3883.

(32) LCMS (method B); t.sub.R 12.357 min(cis)-14.437 min(trans).

Synthesis of 2,6-difluoro-4-hydroxy-2,6-difluoro-4-butoxyazobenzene (4)

(33) 2.6-difluoro-4-butoxyaniline (2.20 g, 10.9 mmol) and sodium nitrite (0.755 g, 10.9 mmol, 1.0 equiv) were dissolved in a 1:1 EtOH/H.sub.2O mixture (22 mL) and the medium was cooled to 0 C. Ice (11 g) was introduced into the medium before the careful addition of cc HCl (2.37 mL). A previously prepared aqueous solution (5.7 mL) of 3,5-difluorophenol (1.42 g, 10.9 mmol, 1.0 equiv) and NaOH (0.876 g, 21.9 mmol, 2.0 equiv) cooled to 0 C. was carefully introduced into the medium at 0 C. The medium was stirred for 20 min at 0 C. and then for 70 min at AT. After adjusting the pH to 1 (cc HCl), the medium was left to stand for 30 min at AT before filtration. The precipitate was washed with water (450 mL), solubilized in DCM and the organic phase was dried over MgSO.sub.4 before being concentrated. The residue (black viscous liquid) was concentrated under a vacuum manifold (3.15 g) and was used without further purification in the next step.

(34) .sup.1H NMR (400 MHz; DMSO) (ppm): 6.93 (d; JH-F=11.4 Hz; 2H); 6.64 (d; JH-F=11.4 Hz; 2H); 4.09 (t; J=6.5 Hz; 2H); 1.77-1.66 (m; 2H); 1.49-1.38 (m; 2H); 0.94 (t; J=7.4 Hz; 3H)

(35) .sup.13C NMR (101 MHz; DMSO) (ppm): 161.40 (t; J.sub.C-F=14.8 Hz); 161.11 (t; J.sub.C-F=14.8 Hz); 157.69 (dd; J.sub.C-F=39.1; 7.7 Hz); 155.13 (dd; J.sub.C-F=38.2; 7.8 Hz); 125.00 (t; J.sub.C-F=10.2 Hz) 124.05 (t; J.sub.C-F=10.3 Hz); 100.26 (dd; J.sub.C-F=22.6; 2.0 Hz); 99.68 (dd; J.sub.C-F=24.2; 2.1 Hz); 68.74 (s); 30.31 (s); 18.55 (s); 13.58 (s).

(36) .sup.19F NMR (376 MHz; DMSO; decoupled) (ppm): 118.99; 119.27.

(37) .sup.19F NMR (376 MHz; DMSO) (ppm): 118.99 (d; J.sub.F-H=12.1 Hz); 119.27 (d; J.sub.F-H=12.0 Hz).

(38) HRMS (m/z): calculated for C.sub.16H.sub.15F.sub.4N.sub.2O.sub.2; 343.1070 ([M+H].sup.+). Found 343.1060.

(39) R.sub.f=0.20 (SiO.sub.2; cyclohexane/ethyl acetate 9:1; UV).

Synthesis of 2,6-difluoro-4-(N-(tert-butyloxycarbonyl)-ethoxyamine)-2,6-difluoro-4-butoxyazobenzene (5)

(40) 2,6-difluoro-4-hydroxy-2,6-difluoro-4-butoxyazobenzene 4 (2.18 g crude, 6.37 mmol considered) and K.sub.2CO.sub.3 (1.32 g, 9.57 mmol, 1.5 equiv) were dissolved in acetone (22 mL). After 30 min stirring at AT under argon, 2-(Boc-amino)ethyl bromide (4.29 g, 19.1 mmol, 3.0 equiv) was introduced into the medium. After stirring at reflux for 18 h, the medium was concentrated and purified by flash chromatography on silica (cyclohexane/ethyl acetate, gradient 1/0 to 7/3). 2,6-difluoro-4-(N-(tert-butyloxycarbonyl)-ethoxyamine)-2,6-difluoro-4-butoxyazobenzene 5 (624 mg, 1.28 mmol, 17% on 2 steps) was isolated as an amorphous yellow powder.

(41) .sup.1H NMR (400 MHz; DMSO) (ppm): 7.04 (s; 1H); 6.95 (d; JH-F=11.8 Hz; 4H); 4.10 (m; 4H); 3.34-3.26 (m; 2H); 1.75-1.67 (m; 2H); 1.49-1.39 (m; 2H); 1.38 (s; 9H); 0.94 (t; J=7.4 Hz; 3H).

(42) .sup.13C NMR (101 MHz; DMSO) (ppm): 161.53 (t; J.sub.C-F=14.3 Hz); 161.16 (t; J.sub.C-F=14.2 Hz); 157.59 (t; J.sub.C-F=7.9 Hz); 155.64 (s); 155.03 (t; J.sub.C-F=7.7 Hz); 125.09 (t; J.sub.C-F=8.3 Hz); 124.90 (t; J.sub.C-F=8.3 Hz); 99.90 (dd; J.sub.C-F=11.3; 1.7 Hz); 99.67 (dd; J.sub.C-F=11.3; 1.7 Hz); 77.86 (s); 68.80 (s); 67.87 (s); 38.71 (s); 30.29 (s); 28.17 (s); 18.53 (s); 13.57 (s)

(43) .sup.19F NMR (376 MHz; DMSO; decoupled) (ppm): 118.79; 118.85.

(44) .sup.19F NMR (376 MHz; DMSO) (ppm): 118.79 (d; J.sub.F-H=12.0 Hz); 118.85 (d; J.sub.F-H=11.9 Hz).

(45) HRMS (m/z): calculated for C.sub.23H.sub.28F.sub.4N.sub.3O.sub.4; 486.2016 ([M+H].sup.+). Found 486.2015.

(46) R.sub.f=0.30 (SiO.sub.2; cyclohexane/ethyl acetate 8:2; UV).

Synthesis of 2,6-difluoro-4-aminoethoxy-2,6-difluoro-4-butoxyazobenzene (6)

(47) 2,6-difluoro-4-(N-(tert-butyloxycarbonyl)-ethoxyamine)-2,6-difluoro-4-butoxyazobenzene 5 (879 mg, 1.81 mmol) was dissolved in a 4:1 DCM/TFA mixture (37 mL) and the medium was stirred for 1.5 h at AT. After concentration, diethyl ether (200 mL) was introduced and the precipitate 1 formed was concretized for 1 h before filtration and washing with diethyl ether (4100 mL). The filtrate was concentrated and a viscous precipitate 2 was formed in n-hexane (200 mL), filtered and washed with n-hexane (3200 mL). Precipitates 1 and 2 were dried under a vacuum manifold. 2,6-difluoro-4-aminoethoxy-2,6-difluoro-4-butoxyazobenzene 6 (858 mg, 1.72 mmol, 95%, TFA salt) was isolated as an amorphous yellow powder (precipitate 1, 70%) and a yellow oil (precipitate 2, 25%).

(48) .sup.1H NMR (400 MHz; DMSO) (ppm): 7.98 (s; 3H); 7.00 (d; J.sub.H-F=11.3 Hz; 2H); 6.96 (d; J.sub.H-F=11.8 Hz; 2H); 4.30 (t; J=5.0 Hz; 2H); 4.11 (t; J=6.5 Hz; 2H); 3.26 (t; J=5.0 Hz; 2H); 1.80-1.63 (m; 2H); 1.53-1.33 (m; 2H); 0.94 (t; J=7.4 Hz; 3H).

(49) .sup.13C NMR (101 MHz; DMSO) (ppm): 161.76 (t; J.sub.C-F=14.4 Hz); 160.27 (t; J.sub.C-F=14.0 Hz); 157.57 (dd; J.sub.C-F=25.8; 7.6 Hz); 155.00 (dd; J.sub.C-F=25.9; 7.4 Hz); 125.57 (s); 124.86 (s); 100.07 (dd; J.sub.C-F=24.7; 2.7 Hz); 99.80 (dd; J.sub.C-F=24.7; 2.5 Hz); 68.85 (s); 65.82 (s); 38.06 (s); 30.29 (s); 18.54 (s); 13.57 (s)

(50) .sup.19F NMR (376 MHz; DMSO; decoupled) (ppm): 73.46; 118.60; 118.74.

(51) .sup.19F NMR (376 MHz; DMSO) (ppm): 73.46 (s); 118.60 (d; J.sub.F-H=11.9 Hz); 118.73 (d; J.sub.F-H=11.8 Hz).

(52) HRMS (m/z): calculated for C.sub.18H.sub.20F.sub.4N.sub.3O.sub.2; 386.1492 ([M+H].sup.+). Found 386.1491.

(53) R.sub.t=0.33 (SiO.sub.2; DCM/MeOH 95:5; UV or ninhydrin).

Synthesis of 2,6-difluoro-4-(N-(1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid-10-glutaryl)-ethoxyamine)-2,6-difluoro-4-butoxyazobenzene (FAzo)

(54) 2,6-difluoro-4-aminoethoxy-2,6-difluoro-4-butoxyazobenzene 6 (267 mg, 0.535 mmol, TFA salt) was dissolved in anhydrous DMF (3.8 mL). After introduction of triethylamine (223 L, 1.60 mmol, 3.0 equiv), the medium was stirred for 5 min at AT under argon. The DOTAGA anhydride (245 mg, 0.533 mmol, 1.0 equiv) was then introduced and the medium was stirred for 21 h at 70 C. under argon. After concentration, diethyl ether (100 mL) was introduced and the precipitate formed was concretized for 30 min before filtration and washing with diethyl ether (250 mL). The crude was purified by reverse phase flash chromatography (RP-18, Biotage, gradient H.sub.2O 0.05% FA/CH.sub.3CN 0.05% FA 1:0 to 2:8). 2.6-difluoro-4-(N-(1,4,7,10-tetraazacyclododecane-1,4,7-tri-acetic acid-10-glutaryl)-ethoxyamine)-2,6-difluoro-4-butoxyazobenzene FAzo (92.1 mg, 0.109 mmol, 20%) was isolated by freeze-drying in the form of a yellow electrostatic powder.

(55) HRMS (m/z): calculated for C.sub.37H.sub.50F.sub.4N.sub.7O.sub.11; 844.3504 ([M+H].sup.+). Found 844.3495.

(56) LCMS (method B); t.sub.R 14.107 min(cis)-14.894 min(trans).

(57) General Protocol for the Complexing of Azo and FAzo

(58) Azo or FAzo and the metal reagent were dissolved in H2O (26 mM). After adjusting the pH to 5.5, the medium was stirred for 17 h at 50 C., ensuring that the pH remained in the range 5.5-6.0. The pH was then adjusted to 6.5 before concentration of the medium by freeze-drying and purification by reversed-phase flash chromatography (RP-18, Biotage, gradient H2O/CH3CN 1:0 to 0:1). The final product was isolated by freeze-drying as an electrostatic powder.

(59) TABLE-US-00001 TABLE 1 Experimental conditions for complexation reactions. Reaction Amount scale (mol of Color Starting starting Type reagent Yield of final Metal material material of reagent (equiv) (%) product Cu Azo 104 Cu(OAc).sub.2 1.05 60 green Ga Azo 78.0 GaNO.sub.3 2.05 82 yellow Y Azo 90.0 YCl.sub.36H.sub.2O 1.05 31 yellow In Azo 80.0 InCl.sub.34H.sub.2O 1.05 27 yellow Eu Azo 130 EuCl.sub.36H.sub.2O 1.05 39 yellow Gd Azo 259 GdCl.sub.36H.sub.2O 1.05 88 yellow Gd FAzo 142 GdCl.sub.36H.sub.2O 1.05 97 yellow Yb Azo 110 YbCl.sub.36H.sub.2O 1.05 21 yellow Bi Azo 104 BiCl.sub.3* 1.05 73 yellow *250 mM in 6N HCl.

Copper 4-(N-(1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid-10-glutaryl)-ethoxyamine)-4-butoxyazobenzene (CuAzo)

(60) HRMS (m/z): calculated for C.sub.37H.sub.51N.sub.7NaO.sub.11Cu; 855.2840 ([M+Na].sup.+). Found 855.2854.

(61) LCMS (method B); t.sub.R 13.550 min(cis)-15.934 min(trans).

Gallium 4-(N-(1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid-10-glutaryl)-ethoxyamine)-4-butoxyazobenzene (GaAzo)

(62) HRMS (m/z): calculated for C.sub.37H.sub.51N.sub.7O.sub.11Ga; 838.2902 ([M+H].sup.+). Found 838.2906.

(63) LCMS (method B); t.sub.R 12.840 min(cis)-15.072 min(trans).

Yttrium 4-(N-(1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid-10-glutaryl)-ethoxyamine)-4-butoxyazobenzene (YAzo)

(64) HRMS (m/z): calculated for C.sub.37H.sub.49N.sub.7Na.sub.2O.sub.11Y; 902.2344 ([MH+2Na].sup.+). Found 902.2349.

(65) LCMS (method B); t.sub.R 15.957 min (cis)-19.660 min (trans).

Indium 4-(N-(1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid-10-glutaryl)-ethoxyamine)-4-butoxyazobenzene (InAzo)

(66) HRMS (m/z): calculated for C.sub.37H.sub.49N.sub.7Na.sub.2O.sub.11In; 928.2324 ([MH+2Na].sup.+). Found 928.2319.

(67) LCMS (method B); t.sub.R 15.451 min(cis)-18.899 min(trans).

Europium 4-(N-(1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid-10-glutaryl)-ethoxyamine)-4-butoxyazobenzene (EuAzo)

(68) HRMS (m/z) (negative mode): calculated for C.sub.37H.sub.49N.sub.7O.sub.11Eu; 920.2702 ([MH].sup.). Found 920.2694.

(69) LCMS (method B); t.sub.R 13.419 min(cis)-16.718 min(trans).

Gadolinium 4-(N-(1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid-10-glutaryl)-ethoxyamine)-4-butoxyazobenzene (GdAzo)

(70) HRMS (m/z): calculated for C.sub.37H.sub.49N.sub.7Na.sub.2O.sub.11Gd; 971.2527 ([MH+2Na].sup.+). Found 971.2529.

(71) LCMS (method B); t.sub.R 15.956 min(cis)-19.633 min(trans).

Gadolinium 2,6-difluoro-4-(N-(1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid-10-glutaryl)-ethoxyamine)-2,6-difluoro-4-butoxyazobenzene (GdFAzo)

(72) HRMS (m/z): calculated for C.sub.37H.sub.45F.sub.4N.sub.7Na.sub.2O.sub.11Gd; 1043.2150 ([MH+2Na].sup.+). Found 1043.2158.

(73) LCMS (method B); t.sub.R 18.900 min(cis)-20.524 min(trans).

Ytterbium 4-(N-(1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid-10-glutaryl)-ethoxyamine)-4-butoxyazobenzene (YbAzo)

(74) HRMS (m/z): calculated for C.sub.37H.sub.49N.sub.7Na.sub.2O.sub.11Yb; 987.2674 ([MH+2Na].sup.+). Found 987.2683.

(75) LCMS (method B); t.sub.R 16.112 min(cis)-19.968 min(trans).

Bismuth 4-(N-(1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid-10-glutaryl)-ethoxyamine)-4-butoxyazobenzene (BiAzo)

(76) HRMS (m/z): calculated for C.sub.37H.sub.49N.sub.7Na.sub.2O.sub.11Bi; 1022.3089 ([MH+2Na].sup.+). Found 1022.3093.

(77) LCMS (method B); t.sub.R 14.235 min(cis)-19.055 min(trans).

III. Ionizing Radiation and Cell Experiments

(78) General

(79) Buffer media such as phosphate buffered saline (PBS), Dulbecco's Modified Eagle Medium-high glucose (DMEM), Roswell Park Memorial Institute medium (RPMI), penicillin, streptomycin and trypan blue solution (0.4%) were purchased from Sigma Aldrich (France). Fetal bovine serum (FBS) was purchased from Gibco (France), propidium iodide (PI) was purchased from Thermo Fisher Scientific (France) and gemcitabine hydrochloride (Gem) was purchased from Sequoia Research Products (UK).

(80) Cell Lines

(81) Human pancreatic cancer (PANC-1) and human acute lymphoblastic leukemia (CCRF-CEM) cells were purchased from ATCC (US) and maintained as recommended by the supplier. Gem-resistant human acute lymphoblastic leukemia cells (CCRF-CEM ARAC 8C, hENT-1 receptor not expressed) were kindly provided by Dr. Buddy Ullmann (Oregon Health Sciences University). Briefly, PANC-1 cells were maintained in DMEM buffer supplemented with 10% (v/v) heat-inactivated FBS. CCRF-CEM and CCRF-CEM ARAC-8C cells were maintained in RPMI buffer supplemented with 10% (v/v) heat-inactivated FBS. All cell media were supplemented with penicillin (50 U.Math.mL.sup.1) and streptomycin (0.05 mg.Math.mL.sup.1). The cells were maintained in a humid atmosphere at 37 C. with 5% CO2. The cells were used before reaching the eighteenth passage and were harvested at 70-80% confluence.

(82) Ionizing Radiation Sources

(83) UV irradiation. UV irradiation to induce isomerization of the trans isomer to the cis isomer was carried out in a CN-15.Math.LCchamber (Vilber Lourmat) equipped with two 15W tubes (365 nm) which provided an irradiance of 0.817 mW.Math.cm.sup.2 at the irradiation position (determined by a Cole-Parmer VLX-3W microprocessor-controlled radiometer calibrated for 365 nm, Vilber Lourmat).

(84) X-ray irradiation. X-ray irradiation was performed with an X-ray generator (Xrad 320 Dx) providing photons at an average energy of 80 keV (range 0-200 keV) at a dose rate of approximately 1 Gy/min (operation at 200 kV, 20 mA). Radiation doses are expressed in Gray.

(85) Gamma ray irradiation. Gamma irradiation was performed with a Cesium-137 source (IBL 637) providing 662 keV photons at a dose rate of approximately 1 Gy/min. Radiation doses are expressed in Gray (1 Gy=1 J/L).

(86) Electron beam irradiation. Electron beam irradiation was performed with a linear electron accelerator (Linac, Kinetron) delivering electrons at an energy of 4.5 MeV at a dose rate of approximately 4 Gy/min. Radiation doses are expressed in Gray.

(87) Quantification of Ionizing-Radiation-Induced Isomerization by Absorbance Spectrophotometry and HPLC (FIG. 1)

(88) Gd-containing compounds (GdAzo or GdFAzo) and control compounds (Azo or FAzo) (50 M, 200 L, PBS) were introduced into two 96-well microplates (10 wells for each compound). Both microplates were first irradiated under UV light (365 nm, 0.817 mW.Math.cm.sup.2, 5 min). One microplate (plate 1) was then kept in the dark and used as a non-irradiated control while the second (plate 2) was irradiated with an increment of ionizing radiation doses (2 Gy, 3 Gy, 5 Gy and 10 Gy). After each irradiation, absorbance spectrophotometric and HPLC injection (method C) analyses were performed on the non-irradiated (plate 1) and irradiated (plate 2) compounds. A 26 min time lag between UV irradiation of plates 1 and 2 was used to allow analysis of the unirradiated control compounds (plate 1) at the same time as the ionizing irradiated compounds (plate 2) after UV irradiation. The complete experiment was performed in 4.6 h. The absorbance spectra were plotted by the mean of triplicates. The relative amount of each isomer was obtained by HPLC (detection at the isosbestic point wavelength under elution conditions) and the molecular activation (%) was determined by the difference in the proportion of trans isomer in the medium (3 independent experiments).

(89) Confocal Microscopy without Ionizing Radiation (FIG. 2a)

(90) The cells (PANC-1) were transferred to an imaging plate (ibiTreat 8-well micro-slide purchased from Ibidi, Germany) 24 h before the start of the experiment, and were maintained in culture medium (33000 cells in 200 L/well) under humid atmosphere at 37 C. with 5% CO2. Immediately prior to the experiment, the cell medium was replaced with culture medium without FBS and phenol red (100 L) and then IP (5 L, final concentration 1 M) was added to the medium. 15 min after the addition of the IP, a first series of images was acquired. Then the cis-GdAzoor trans-GdAzocompound (100 L, final concentration 0 M, 250 M or 500 M) was introduced into the cell medium. The imaging plate was then kept in the dark and images were acquired every 5 min for 1 h. The compound cis-GdAzo was obtained by UV irradiation (365 nm, 0.817 mW.Math.cm.sup.2, 30 min) of the compound trans-GdAzo (105 L, 500 M or 1 mM, cis-GdAzo>85%) in a 96-well microplate. The cells were observed with a Leica TCS SP8 gated-STED inverted microscope (Leica, Germany) using a Fluotar CS2 10/0.30 sec HC PL objective. The instrument was equipped with a white light laser (excitation wavelength 538 nm). The red fluorescence emission was collected over a bandwidth of 560-650 nm and transmission images were obtained with the same light line and a PMT-trans detector. The pinhole was set at 1.0 Airy (73.19 m diameter). The 16-bit digital images were obtained with the Leica SP8 LAS X software (version 2.0.1, Leica, Germany).

(91) Confocal Microscopy in the Presence of Ionizing Radiation (FIGS. 2b-c)

(92) Cells (PANC-1) were transferred to an imaging plate (Lab-Tek chamber slide system, glass, 8-well purchased from Thermo Fisher Scientific, France) 24 h before the start of the experiment, and were maintained in culture medium (10,000 cells in 200 L/well) in a humid atmosphere at 37 C. with 5% CO2. Just before the experiment, the cell medium was replaced with PBS (100 L) and IP (5 L, final concentration 1 M) was added to the medium. 15 min after the addition of PI, cis-GdAzo (100 L, final concentration 0 M, 250 M, 500 M or 850 M) was introduced into the cell medium. A first series of images was acquired at this stage, and then the imaging plate was irradiated (gamma rays, 2 Gy). The imaging plate was then kept in the dark at 37 C. (plate warmer) and images were acquired every 5 min for 30 min and then every 10 min for 90 min. A similar procedure was used for the control experiment without irradiation except that the imaging plate was not irradiated with gamma rays. The cis-GdAzo compound was obtained by UV irradiation (365 nm, 0.817 mW.Math.cm.sup.2, 30 min) of the trans-GdAzo compound (105 L, 500 M, 1 mM or 1.7 mM, cis-GdAzo>85%) in a 96-well microplate. Cells were observed with a Nikon inverted microscope (Nikon Instruments Inc., Tokyo, Japan) using a 10 sec objective, N.A. 0.4 and equipped with a Yokogawa CSU-X1 head. The instrument was equipped with a laser diode at 561 nm as excitation wavelength. The red fluorescence emission was collected over a bandwidth of 598-672 nm and the transmission images were obtained with a white diode. The pinhole was set at 50 m and the magnification lens at 1.2. The images were recorded by an e-Volve s-CMOS camera (Photometrics). Four images were recorded per well and two wells were used for each concentration of cis-GdAzo. Analysis of the 16-bit digital images was performed using ImageJ software (version 1.50i with the Adjustable Watershed plugin). The number of cells in the images acquired before irradiation was determined manually and the number of fluorescent cells in the images acquired before and after irradiation was calculated automatically using a script coded on ImageJ. Cell permeabilization was determined by the difference in the proportion of fluorescent cells before and 30 min after gamma irradiation (3 independent experiments).

(93) Therapeutic Effect Under Ionizing Radiation (FIGS. 2d-f)

(94) Just before the experiment, the cells (CCRF-CEM ARAC-8C) were dispersed in PBS and transferred to a 48-well microplate (TPP cell culture microplates purchased from Thermo Fisher Scientific, France) (40,000 cells in 80 L/well). Gem (20 L, final concentration 0.1 M) or PBS (20 L) and cis-GdAzo compound (100 L, final concentration 0 M, 250 M, 500 M or 850 M) were added just prior to gamma irradiation of the medium (2 Gy). The medium was maintained in the dark and in a humid atmosphere at 37 C. with 5% CO2 for 1 h. Next, culture medium (600 L) was added to each well and the cells were washed by three centrifugation cycles (300 G, 5 min, 800 L of culture medium used for each wash). The cells were finally dispersed in culture medium (600 L) containing or not containing Gem (final concentration 0 M or 0.1 M) and were maintained in a humid atmosphere at 37 C. with 5% CO2 for 4 days. The number of live cells was determined by cell count in the presence of 1:1 (v/v) trypan blue (triplicate). The experiment was repeated three times independently. Cell viability was expressed as the ratio of the number of living cells after treatment to the number of living cells without any treatment (without irradiation, in the absence of Gem and cis-GdAzo). A similar procedure was used for the control experiment without irradiation except that the 48-well microplate was not irradiated with gamma rays. A similar procedure was used for the control experiment with Dotarem (FIG. 2f) except that Dotarem was used instead of cis-GdAzo. The cis-GdAzo compound was obtained by UV irradiation (365 nm, 0.817 mW.Math.cm2, 30 min) of the trans-GdAzo compound (105 L, 500 M, 1 mM or 1.7 mM, cis-GdAzo>85%) in a 96-well microplate.

(95) These results show: (i) a new concept of activation of therapeutic molecules by the use of ionizing radiation (X-rays, gamma rays, electrons) (FIG. 1a-f). As these radiations have a very high penetration power in living tissues, this new activation concept has a strong potential for clinical use. All the organic molecules described in the literature today require the use of UV to near-IR radiation, which does not penetrate biological tissues in depth (it penetrates to a few hundred micrometres in the best of conditions). (ii) that it is possible to activate MAzo compounds (M=Cu, Ga, Y, In, Eu, Gd, Yb, Bi) by ionizing radiation with different efficiencies depending on the metal and irradiation source used (FIGS. 1a-c,f). The GdFAzo compound can also be activated by ionizing radiation (FIGS. 1c-e). This compound is characterized by a slow enough thermal relaxation to consider its study in vivo. Although the activation of the compound GdFAzo is relatively low under the experimental conditions used in vitro (compared to the compound GdAzo, FIG. 1c), it is difficult to predict the molecular activation efficiency that the compound would have in vivo. (iii) the type of metal to be used to enable activation of the organic unit. It has been shown that the atomic number Z of the metal must be greater than 39 (that of Yttrium) to allow for consistent activation (>30% under 5 Gy). Indeed, 6 compounds containing a metal of size greater than or equal to that of Yttrium showed such activation while 2 compounds containing smaller metals (Cu, Z=29, and Ga, Z=31) showed low activation (<10% under 5 Gy) (FIG. 1f). (iv) that molecular activation can be performed under various types of ionizing radiation (X-rays, gamma rays, electrons), with different particles (photons and electrons) and different energies (from 1 keV to 4.5 MeV) (FIG. 1c), and thus that the set of radiotherapy devices currently in clinical use should be effective at generating the described molecular activation. (v) that the compound GdAzo is able to induce the permeabilization of cancer cell membranes under ionizing radiation at low doses (6.9% (vs. 1.2% without irradiation), 8.2% (vs. 2.8% without irradiation) and 12.4% (vs. 7.5% without irradiation) at GdAzo concentrations of 250 M, 500 M and 850 M respectively by gamma rays at 2 Gy) (FIG. 2a,b,c). (vi) that the compound GdAzo is able to induce toxicity in a gemcitabine-resistant cancer cell line (human acute lymphoblastic leukemia), in the absence or presence of gemcitabine (FIG. 2d,e), after treatment with gamma radiation at 2 Gy In the absence of gemcitabine, cell viability 4 days after treatment was reduced to 23% (vs. 79% without irradiation), 18% (vs. 42% without irradiation) and 3.0% (vs. 27% without irradiation) at GdAzo concentrations of 250 M, 500 M and 850 M respectively. In the presence of gemcitabine, cell viability 4 days after treatment was reduced to 15% (vs. 49% without irradiation), 9.1% (vs. 30% without irradiation) and 1.2% (vs. 16% without irradiation) at GdAzo concentrations of 250 M, 500 M and 850 M respectively. This study shows that the compound is active alone under ionizing radiation and that the presence of a surrounding active substance only slightly enhances the therapeutic effect of the compound (FIG. 2d,e). (vii) that the presence of the azobenzene or stillbene motif on the molecule is necessary to generate the therapeutic effect since the commercial control compound Dotarem (gadolinium chelate alone) has no therapeutic effect under ionizing radiation (FIG. 2f).