阅读说明：本技术 新型偶氮苯衍生物、其制备方法及其在与电离辐射联合的治疗性治疗中的用途 (Azobenzene derivatives, preparation method and use thereof in therapeutic treatment in combination with ionizing radiation ) 是由 纪尧姆·博尔特 帕特里克·库夫勒尔 西蒙娜·米拉 弗雷德里克·普祖莱 于 2020-02-07 设计创作，主要内容包括：本发明涉及通过电离辐射可活化的新型衍生物、其制备方法及治疗用途。(The present invention relates to novel derivatives activatable by ionising radiation, to a process for their preparation and to their therapeutic use.)

1. A compound of formula (I):

wherein:

m is 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 (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) and Bi (III);

n is 1,2, 3,4, 5, 6 or 7;

v and V', which may be the same or different, are hydrogen atoms or a linear or branched C1-C10 alkyl or alkoxy chain, or a C1-C10 alkyl chain joined together to form a ring, said C1-C10 alkyl chain containing one or more heteroatoms selected from N, O or S and optionally substituted with one or more substituents independently selected from halogen atoms and nitriles, nitro, thio, amino, amido, aryl, heteroaryl, hydroxyl, carboxylic acid or carboxyl groups;

r1, R1', R2, R2', which may be identical or different, are hydrogen atoms or a linear or branched C1-C10 alkyl or alkoxy chain optionally substituted by one or more substituents independently selected from halogen atoms and nitrile, nitro, thio, amino, amido, aryl, heteroaryl, hydroxyl, carboxylic acid or carboxyl groups;

r3 and R3', which may be the same or different, are hydrogen atoms, or a linear or branched C1-C10 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 carboxyl groups, or R3 and R3' are linked together to form a 5-to 14-membered heterocyclic or heteroaryl group;

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 the same or different, are independently selected from H; a halogen atom; alkoxy, alkyl or cycloalkyl optionally substituted by one or more heteroatoms or groups COOH, CONH₂、COSH、OH、NH₂SH interruption or substitution; a 5-to 12-membered aryl or heteroaryl group, optionally substituted with one or more COOH, CONH₂COSH group, COOH or NH₂Substituted by groups;

z represents optionally substituted by one or more heteroatoms or COOH, CONH₂、COSH、OH、NH₂Alkyl interrupted by or substituted by SH groups; optionally substituted by one or more COOH, CONH₂5-to 12-membered aryl or heteroaryl substituted with a COSH group; or COOH or NH₂A group;

w and W' may be the same or different and independently represent CH₂A group; or an aryl or cycloalkyl group; oxygen or nitrogen atoms (secondary or tertiary); an amide bond; an ester linkage; a thioether bond;

u and U ', which may be the same or different, represent a CH or NH group, with the understanding that the double bond U ═ U' is cis or trans;

t represents CH₂A group; -a C (═ O) NH group; alkoxy, alkyl or cycloalkyl, optionally substituted by one or more heteroatoms or COOH, CON₂、COSH、OH、NH₂SH group interruption or substitution; a 5-to 12-membered aryl or heteroaryl group containing one or more heteroatoms and/or optionally substituted with one or more groups selected from COO alkyl, CONH alkyl, COSH alkyl;

r represents H, or a linear or branched C1-C18 alkyl or alkoxy chain, said C1-C18 alkyl or alkoxy chain being optionally substituted by one or more substituents independently selected from halogen atoms and nitrile, nitro, thio, amino, amido, aryl, heteroaryl, hydroxy, carboxylic acid or carboxy groups;

p and q are integers from 0 to 6, especially from 1 to 6;

it will be appreciated that, in order to ensure neutrality of the molecule, the n cationic charges of M may be neutralized by from 0 to n carboxyl groups (-COOH) optionally substituted on R1, R1', R2, R2', R3, R3', V, V' and/or, if necessary, by from 0 to n counterions present in the solution;

the compounds of formula (I) are in the form of cis and/or trans isomers and mixtures thereof.

2. A compound of formula (I) according to claim 1, having formula (IA):

wherein:

m, n, R1, R2, R3, V, M, R1', R2', R3', V ', M ', p are as defined for formula (I), and

x is selected from hydrogen and halogen atoms;

r is a linear or branched C1-C12 alkyl group;

n ═ N double bonds are cis or trans;

it will be appreciated that, to ensure neutrality of the molecule, the n cationic charges of M may be neutralized by from 0 to n carboxyl groups (-COOH) substituted on R1, R1', R2, R2', R3, R3', V, V' and/or, if necessary, by from 0 to n counterions present in the solution.

3. The compound of claim 1 or 2, having the general formula (IB):

wherein:

m, n, p, X and R are as defined in claim 1 or 2.

4. The compound according to any one of the preceding claims, having the general formula (IC):

wherein:

m, n, X are as defined in the preceding claims.

5. A method of making a compound of any one of the preceding claims, comprising:

-a step of complexing a compound of formula (II) with a precursor of a metal M:

wherein:

r1, R2, R3, V, m, R1', R2', R3', V ', m ', Z, T, q, p, W ', X, X ', X ", X '", Y, Y ', Y ", Y '", R, U, U ' are as defined in claims 1 to 4,

the compounds of formula (II) are in the form of cis and/or trans isomers and mixtures thereof; and

possible UV irradiation of the product obtained, so as to obtain mainly the cis isomer.

6. The process according to claim 5, wherein the compound of formula (II) is obtained by coupling a compound of formula (III) with a compound of formula (IV):

wherein:

r1, R2, R3, V, m, R1', R2', R3', V ', m ', Z, T, q, p, W ', X, X ', X ", X '", Y, Y ', Y ", Y '", R, U, U ' are as defined in claims 1 to 4; and

e is a linear or branched C1-C10 alkyl or alkoxy chain, cycloalkyl or aryl optionally comprising one or more heteroatoms selected from N, O or S, and optionally interrupted or substituted by one or more substituents independently selected from halogen atoms and anhydrides, carboxyl, nitrile, nitro, thio, amino, amido, aryl, heteroaryl, hydroxyl, ester, carboxylic acid or carboxyl groups, with the proviso that E contains a nucleophilic substituent such as hydroxyl, thiol or primary or secondary amine functional group to effect coupling to molecule (III);

g is a linear or branched C1-C10 alkyl or alkoxy chain, cycloalkyl or aryl optionally containing one or more heteroatoms selected from N, O or S, and optionally substituted with one or more substituents independently selected from halogen atoms and anhydrides, carboxyl, nitrile, nitro, thio, amino (primary or secondary), amido, aryl, heteroaryl, hydroxyl, carboxylic acid or carboxyl groups, it being understood that G contains an electrophilic substituent, such as an activated carboxylic acid function or an anhydride function, to effect coupling to molecule (IV).

7. A pharmaceutical composition comprising a compound of formula (I) as defined in any one of claims 1 to 4, predominantly in the cis form, and at least one pharmaceutically acceptable excipient.

8. A compound of formula (I) as defined in any one of claims 1 to 4 for use in the treatment of cancer, such as lung cancer, pancreatic cancer, liver cancer, spleen cancer, small cell lung cancer, 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 cancer, bone cancer, myeloma, plasmacytoma, germ cell cancer, uterine cancer, leukemia, lymphoma, neuroblastoma, osteosarcoma, retinoblastoma, central nervous system cancer, wilms' tumor.

9. The compound of formula (I) for use according to claim 8, comprising administering compound (I) predominantly in the cis form and irradiating the tumor with ionizing radiation, such as in particular those selected from X-rays, gamma rays, electrons and intense beams, such as protons or carbon ions.

10. The compound of formula (I) for use according to any one of claims 8 to 9, further comprising monitoring the treatment by in vivo medical imaging such as MRI, PET, X-ray or SPECT.

11. The compound of formula (I) for use according to any one of claims 8 to 10, further comprising the administration of one or more anti-cancer agents, such as chemotherapeutic or immunotherapeutic agents, such as immune checkpoint inhibitors against CTLA4, PD-L1 and PD 1.

Description of the drawings:

[ FIG. 1] A]FIG. 1 shows absorption spectra of cis-GdAzo (a) and cis-GdFAzo (d) under gamma irradiation in PBS. PPS 1: comprising a light steady state of predominantly cis-isomer. b. e: histogram (b) showing the molecular activation of (n ═ 3) cis-GdAzo and cis-Azo (control molecules without Gd) or histogram (e) showing the molecular activation of cis-GdFAzo and cis-FAzo (control molecules without Gd) determined by HPLC under gamma irradiation in PBS and calculated by the difference in the trans isomer ratio in the medium. c: molecular activation of cis-GdAzo and cis-GdFAzo under X-ray, gamma and linear accelerator linear electron accelerators. f: histogram showing the molecular activation of (n ═ 3) cis-MAzo (M is the metal indicated on the X axis) determined by HPLC under gamma irradiation in PBS and calculated by the difference in the trans isomer ratio in the medium. Gy: grey (J.L)^-1). XR: and (4) irradiating X-rays. GR: gamma ray radiation. E: radiation through a linear accelerator (Linac) linear electron accelerator. NI: not radiated. The standard deviation is plotted on a chart. P<0.001 (two-way analysis of variance with post hoc tests of Bonferroni).

[ FIG. 2]]Figure 2 shows microscope images of cancer cells (Panc-1, human pancreatic cancer) in the presence of propidium iodide and the inactive compound cis-GdAzo (1,2) or active compound trans-GdAzo (3,4) at a concentration of 500 μ M, either before (1,3) or after (2,4)30 minutes of compound introduction. Superposition of white light and fluorescence images (propidium iodide in nuclei appears red and shows membrane permeabilization). b: in propidium iodide and concentrationMicroscopic images of cancer cells (Panc-1) 30 minutes before (1,3) or after (2,4) irradiation of the medium with gamma radiation (2Gy) in the presence of 250 μ M (1,2) or 500 μ M (3,4) of the inactive compound cis-GdAzo. c: histograms comparing the effect of gamma radiation (2Gy) on cell permeabilization (Panc-1) in the presence of propidium iodide and cis-GdAzo (n ═ 3) were obtained. d. e: histogram comparing the effect of gamma radiation (2Gy) on mortality of Gem resistant cancer cells (CCRF-CEM ARAC-8C, human acute lymphoblastic leukemia) in the presence of cis-GdAzo (d) or cis-GdAzo and Gem (0.1 μ M) for 1 hour, 4 days post-treatment. f: is compared withOrAnd a histogram of the effect of gamma radiation (2Gy) on mortality of Gem-resistant cancer cells (CCRF-CEM ARAC-8C) in the presence of Gem (0.1. mu.M). Gy: grey (J.L)^-1). GR: gamma ray radiation. NI: not radiated. The standard error of the mean is plotted in graph c and the standard deviation is plotted in graphs d-f. P<0.05，**P<0.01，***P<0.001 (two-way analysis of variance with post hoc tests of Bonferroni).

Detailed Description

Examples

I. Analytical chromatography

Method A

On a 1565 binary HPLC pump (Waters), PAD2998 reader (Waters) and an Agilent Eclipse XDB-C18 reverse phase column (length: 250mm, diameter: 4.6mm, stationary phase: 5 μm), a 0.05% water-TFA/0.05% acetonitrile-TFA gradient system was used; 0'(98/2), 5' (98/2), 25'(0/100), 27' (0/100), 29'(98/2), 35' (98/2), at a flow rate of 1mL.min^-1(30. mu.L injection) was analyzed by HPLC (high performance liquid chromatography) and the data was processed on Empower.

Method B

On an Alliance 2695 system (Waters) equipped with an Xbridge C18 reverse phase column (length: 150mm, diameter: 2.1mm, stationary phase: 3.5 μm)A 0.1% water-FA/acetonitrile gradient system was used; 0'(95/5), 20' (0/100) at a flow rate of 0.25mL.min^-1(injection 10. mu.L) for LCMS (liquid chromatography mass spectrometry) analysis. The mass analyzer is TOF LCT Premier (Waters). The capillary voltage was 2.8 kV. The cone voltage was 35V. The source temperature was 120 ℃ and the desolvation temperature was 280 ℃. Data processing was performed on MassLynx.

Method C

On a 2525 binary HPLC pump (Waters) coupled to a 515HPLC pump (Waters), a PAD 2996 reader (Waters) and an Xbridge C18 reverse phase column (length: 100mm, diameter: 3.0mm, stationary phase: 3.5 μm), a 0.05% water-TFA/acetonitrile isocratic system was used with a flow rate of 0.75mL.min^-1(10. mu.L injected) for HPLC analysis. The detection wavelength corresponds to the isoabsorption point of the compound studied in the mobile phase used. Data processing was performed on MassLynx.

II.Summary of the invention

Summary of the invention

All reagents were purchased at highest purity from Sigma-Aldrich or Alfa Aesar and used without further purification. DOTAGA anhydride was purchased from CheMatech and used without further purification. Silica gel for flash chromatography (Aldrich 717185Si 60,40-63 μm) was purchased from VWR. RP-18 reverse phase flash chromatography was carried out on a CombiFlash instrument (Biotage). Thin layer chromatography (detection by 254nm UV lamp or ninhydrin) was performed using aluminum foil coated with 60F254 silica gel.¹H、¹³C and¹⁹f NMR spectra were obtained on a Bruker 300MHz or 400MHz spectrometer at ambient temperature. Chemical shifts δ are in ppm using solvent as reference. The coupling constant J is measured in hertz. The coupling distribution is described by the abbreviations d (doublet), t (triplet) and m (multiplet). Unless otherwise stated, a time-of-flight mass analyzer (Waters) equipped with a TOF-LCT Premier mass spectrometer was used at 3/7H₂High Resolution Mass Spectrometry (HRMS) experiments were performed in positive mode by electrospray ionization in O/methanol mixtures. Analytical chromatographic methods (HPLC and LCMS) are described in the special section. Purity of the synthesis intermediate is determined by¹H NMR or reverse phase HPLC. The purity of the final product was determined by reverse phase HPLC and confirmed as>95％。

The following compounds were synthesized according to the following synthetic schemes:

[ chemical formula 17]

Wherein X is H or F,

wherein M is Cu, Ga, Y, In, Eu, Gd, Yb or Bi,

wherein n is 2 or 3,

[ chemical formula 18]

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

4-Butoxyaniline (2.00mL,12.0mmol) and sodium nitrite (0.854g,12.0mmol,1.0 equiv.) were dissolved in 1:1EtOH/H₂O mixture (24mL) and the medium is cooled to 0 ℃. Ice (12g) was added to the medium before careful addition of cc HCl (2.6 mL). A solution of a pre-prepared aqueous solution (6.3mL) of phenol (1.14g, 12.0mmol,1.0 equiv.) and NaOH (0.960g, 24.0mmol, 2.0 equiv.) cooled to 0 deg.C was carefully introduced into the 0 deg.C medium. The medium is stirred for 20 minutes AT 0 ℃ and then for 70 minutes AT Ambient Temperature (AT). After adjusting the pH to 1(cc HCl), the medium was allowed to stand at ambient temperature for 30 minutes before filtration. The precipitate was washed with water (4 x 50mL), dissolved in DCM and the organic phase was over MgSO₄Dried and then concentrated. 4-hydroxy-4' -butoxyazobenzene 1(2.76g,10.2mmol, 85%) was isolated as a black amorphous powder.

¹H NMR(400MHz；CDCl₃)δ(ppm):7.87(d；J＝8.9Hz；2H)；7.82(d；J＝8.8Hz；2H)；6.98(d；J＝8.9Hz；2H)；6.91(d；J＝8.8Hz；2H)；4.03(t；J＝6.5Hz；2H)；1.85-1.75(m；2H)；1.58-1.45(m；2H)；0.99(t；J＝7.4Hz；3H)。

¹³C NMR(75MHz；CDCl₃)δ(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。

HRMS(m/z):C₁₆H₁₉N₂O₂Calculated as 271.1447([ M + H)]⁺). Actually 271.1440.

R_f0.36 (silica; DCM; UV).

Synthesis of 4- (N- (tert-butoxycarbonyl) -ethoxyamine) -4' -butoxyazobenzene (2)

4-hydroxy-4' -butoxyazobenzene 1(2.00g,7.40mmol) and K₂CO₃(1.53g,11.1mmol,1.5 equiv.) was dissolved in acetone (25 mL). After stirring for 30min under AT and argon, 2- (Boc-amino) ethyl bromide (4.98g,22.2mmol,3.0 equiv.) was introduced into the medium. After stirring at reflux for 18 hours, the medium was filtered off while hot and the precipitate was washed with hot acetone (75 mL). The filtrate was allowed to stand for 30 minutes AT before filtration, and the resulting precipitate 1 was washed with cold acetone. The filtrate was allowed to stand at 0 ℃ for 3 hours before filtration. The resulting precipitate 2 was washed with cold acetone and precipitate 1 was added. The filtrate was concentrated and purified by flash chromatography on silica gel (DCM). 4- (N- (tert-Butoxycarbonyl) -ethoxyamine) -4' -butoxyazobenzene 2(2.27g, 5.49mmol, 74%) was isolated as a yellow amorphous powder (57% precipitate, 17% flash chromatography).

¹H NMR(400MHz；CDCl₃)δ(ppm):7.86(2d；J＝8.9Hz；4H)；6.99(2d；J＝8.9Hz；4H)；4.10(t；J＝5.0Hz；2H)；4.04(t；J＝6.5Hz；2H)；3.57(m；2H)；1.87-1.74(m；2H)；1.64-1.40(m；11H)；1.00(t；J＝7.4Hz；3H)。

¹³C NMR(100MHz；CDCl₃)δ(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。

HRMS(m/z):C₂₃H₃₂N₃O₄Calculated as 414.2393([ M + H)]⁺). Actually 414.2396.

R_f0.90 (silica; DCM/methanol 98: 2; UV).

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

4- (N- (tert-Butoxycarbonyl) -ethoxyamine) -4' -butoxyazobenzene 2(1.26g,3.04mmol) was dissolved in a 4:1DCM/TFA mixture (63mL) and the medium was stirred AT AT for 1.5 h. After concentration, diethyl ether (250mL) was added and the precipitate formed solidified for 1 h, then filtered and washed with diethyl ether (4 x 50 mL). 4-Aminoethoxy-4' -butoxyazobenzene 3(1.25g,2.93mmol, 96%, TFA salt) was isolated as a yellow amorphous powder.

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

¹³C NMR(75MHz；MeOD)δ(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。

HRMS(m/z):C₁₈H₂₄N₃O₂Calculated as 314.1869([ M + H)]⁺). Actually 314.1864.

HPLC (method a); t is t_R17.92 min (cis) -20.35 min (trans).

R_f0.70 (silica; DCM/methanol 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)

4-aminoethoxy-4' -butoxyazobenzene 3(259mg,0.606mmol, TFA salt) was dissolved in anhydrous DMF (4.3 mL). After introduction of triethylamine (253. mu.L, 1.81mmol,3.0 eq.) the medium is stirred for 5 minutes under AT argon. DOTAGA anhydride (278mg,0.606mmol,1.0 equiv.) was then introduced and the medium was stirred at 70 ℃ under argon for 21 hours. After concentration, diethyl ether (100mL) was added and the precipitate formed solidified for 30 minutes, then filtered and washed with diethyl ether (2 x 50 mL). By reverse phase flash chromatography (RP-18, Biotage, gradient H)₂O0.05％FA/CH₃CN 0.05% FA1:0-2:8) purification of the crude product. 4- (N- (1,4,7,10-tetraazacyclododecane-1, 4, 7-triacetic acid-10-glutaryl) -ethoxyamine) -4' -butoxyazobenzene Azo (294mg,0.381mmol, 63%) was isolated by freeze drying as a yellow electrostatic powder.

HRMS(m/z):C₃₇H₅₄N₇O₁₁Calculated as 772.3881([ M + H)]⁺). Actually 772.3883.

LCMS (method B); t is t_R12.357 min (cis) -14.437 min (trans).

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

2.6-difluoro-4-butoxyaniline (2.20g,10.9mmol) and sodium nitrite (0.755g,10.9mmol,1.0 equiv.) were dissolved in 1:1EtOH/H₂O mixture (22mL) and the medium is cooled to 0 ℃. Ice (11g) was added to the medium before careful addition of cc HCl (2.37 mL). An aqueous solution (5.7mL) of 3, 5-difluorophenol (1.42g, 10.9mmol,1.0 equiv.) and NaOH (0.876g, 21.9mmol, 2.0 equiv.) prepared in advance was cooled to 0 deg.C and carefully introduced into the medium at 0 deg.C. The medium is stirred AT 0 ℃ for 20 minutes and then AT for 70 minutes. After adjusting the pH to 1(cc HCl), the medium is left AT for 30 minutes before filtration. The precipitate was washed with water (4 x 50mL), dissolved in DCM and the organic phase was over MgSO₄Dried and then concentrated. The residue (black viscous liquid) was concentrated under vacuum manifold (3.15g) and used for the next step without further purification.

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

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

¹⁹F NMR (376 MHz; DMSO; decoupled) delta (ppm): 118.99; -119.27.

¹⁹F NMR(376MHz；DMSO)δ(ppm):-118.99(d；J_F-H＝12.1Hz)；-119.27(d；J_F-H＝12.0Hz)。

HRMS(m/z):C₁₆H₁₅F₄N₂O₂Calculated as 343.1070([ M + H)]⁺). Actually 343.1060.

R_f0.20 (silica; cyclohexane/ethyl acetate 9: 1; UV).

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

2, 6-difluoro-4-hydroxy-2 ',6' -difluoro-4 ' -butoxyazobenzene 4(2.18g crude, considered 6.37mmol) and K₂CO₃(1.32g, 9.57mmol, 1.5 equiv.) was dissolved in acetone (22 mL). After stirring for 30min under AT and argon, 2- (Boc-amino) ethyl bromide (4.29g, 19.1mmol, 3.0 equiv.) was introduced into the medium. After stirring at reflux for 18 hours, the medium is concentrated and purified by flash chromatography on silica gel (cyclohexane/ethyl acetate, gradient 1/0-7/3). 2, 6-difluoro-4- (N- (tert-butoxycarbonyl) -ethoxyamine) -2',6' -difluoro-4 ' -butoxyazobenzene 5(624mg,1.28mmol, 17% in 2 steps) was isolated as an amorphous yellow powder.

¹H NMR(400MHz；DMSO)δ(ppm):7.04(s；1H)；6.95(d；JH-F＝11.8Hz；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＝7.4Hz；3H)。

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

¹⁹F NMR (376 MHz; DMSO; decoupled) delta (ppm): 118.79; -118.85.

¹⁹F NMR(376MHz；DMSO)δ(ppm):-118.79(d；J_F-H＝12.0Hz)；-118.85(d；J_F-H＝11.9Hz)。

HRMS(m/z):C₂₃H₂₈F₄N₃O₄Calculated as 486.2016([ M + H)]⁺). Actually 486.2015.

R_f0.30 (silica; cyclohexane/ethyl acetate 8: 2; UV).

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

2, 6-difluoro-4- (N- (tert-butoxycarbonyl) -ethoxyamine) -2, 6-difluoro-4' -butoxyazobenzene 5(879mg,1.81mmol) was dissolved in a 4:1DCM/TFA mixture (37mL) and the medium was stirred AT for 1.5 h. After concentration, diethyl ether (200mL) was added and the precipitate 1 formed was coagulated for 1 hour, then filtered and washed with diethyl ether (4 x 100 mL). The filtrate was concentrated and a sticky precipitate 2 formed in n-hexane (200mL), filtered and washed with n-hexane (3 x 200 mL). Precipitates 1 and 2 were dried under vacuum manifold. 2, 6-difluoro-4-aminoethoxy-2 ',6' -difluoro-4 ' -butoxyazobenzene 6(858mg, 1.72mmol, 95%, TFA salt) was separated into an amorphous yellow powder (precipitate 1, 70%) and a yellow oil (precipitate 2, 25%).

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

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

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

¹⁹F NMR(376MHz；DMSO)δ(ppm):-73.46(s)；-118.60(d；J_F-H＝11.9Hz)；-118.73(d；J_F-H＝11.8Hz)。

HRMS(m/z):C₁₈H₂₀F₄N₃O₂Calculated as 386.1492([ M + H)]⁺). Actually 386.1491.

R_f0.33 (silica; DCM/methanol 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)

2, 6-difluoro-4-aminoethoxy-2 ',6' -difluoro-4 ' -butoxyazobenzene 6(267mg, 0.535mmol, TFA salt) was dissolved in anhydrous DMF (3.8 mL). After introduction of triethylamine (223 μ L,1.60mmol,3.0 equivalents), the medium is stirred under argon for 5 minutes AT. DOTAGA anhydride (245mg, 0.533mmol, 1.0 equiv.) was then introduced and the medium was stirred at 70 ℃ under argon for 21 hours. After concentration, diethyl ether (100mL) was added and the precipitate formed solidified for 30 minutes, then filtered and washed with diethyl ether (2 x 50 mL). By reverse phase flash chromatography (RP-18, Biotage, gradient H)₂O 0.05％FA/CH₃CN 0.05% FA1:0-2:8) purification of the crude product. 2.6-difluoro-4- (N- (1,4,7, 10-tetraazacyclododecane-1, 4, 7-triacetic acid-10-glutaryl) -ethoxyamine) -2, 6-difluoro-4' -butoxyazobenzene FAzo (92.1mg,0.109mmol, 20%) was isolated by freeze-drying in the form of a yellow electrostatic powder.

HRMS(m/z):C₃₇H₅₀F₄N₇O₁₁Calculated as 844.3504([ M + H)]⁺). Actually 844.3495.

LCMS (method B); t is t_R14.107 min (cis) -14.894 min (trans).

General scheme for Azo and FAzo complexation

The Azo or FAzo and the metal reagent being dissolved in H₂O (26 mM). After adjusting the pH to 5.5, the medium is stirred at 50 ℃ for 17 hours, ensuring that the pH is maintained in the range 5.5-6.0. Then purified by freeze-drying the concentrated medium and reverse phase flash chromatography (RP-18, Biotage, gradient H)₂O/CH₃CN1:0-0:1) was preceded by adjusting the pH to 6.5. The final product was isolated as an electrostatic powder by freeze-drying.

[ Table 1]

*: 250mM in 6N HCl.

TABLE 1 Experimental conditions for the complexation reaction.

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

HRMS(m/z):C₃₇H₅₁N₇NaO₁₁Cu was calculated as 855.2840([ M + Na ]]⁺). Actually 855.2854.

LCMS (method B); t is t_R13.550 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)

HRMS(m/z):C₃₇H₅₁N₇O₁₁Ga is calculated as 838.2902([ M + H)]⁺). Actually 838.2906.

LCMS (method B); t is t_R12.840 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)

HRMS(m/z):C₃₇H₄₉N₇Na₂O₁₁Y is calculated as 902.2344([ M-H +2 Na)]⁺). Actually 902.2349.

LCMS (method B); t is t_R15.957 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)

HRMS(m/z):C₃₇H₄₉N₇Na₂O₁₁In was calculated as 928.2324([ M-H +2 Na)]⁺). Actually 928.2319.

LCMS (method B); t is t_R15.451 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)

HRMS (m/z) (negative ion mode): C₃₇H₄₉N₇O₁₁Eu calculated as 920.2702([ M-H)]^-). Actually 920.2694.

LCMS (method B); t is t_R13.419 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)

HRMS(m/z):C₃₇H₄₉N₇Na₂O₁₁Gd was calculated to be 971.2527([ M-H +2 Na)]⁺). Actually 971.2529.

LCMS (method B); t is t_R15.956 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)

HRMS(m/z):C₃₇H₄₅F₄N₇Na₂O₁₁Gd was calculated to be 1043.2150([ M-H +2 Na)]⁺). Actually 1043.2158.

LCMS (method B); t is t_R18.900 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)

HRMS(m/z):C₃₇H₄₉N₇Na₂O₁₁Yb was calculated as 987.2674([ M-H +2 Na)]⁺). Is actually 987.2683。

LCMS (method B); t is t_R16.112 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)

HRMS(m/z):C₃₇H₄₉N₇Na₂O₁₁Bi is calculated as 1022.3089([ M-H +2 Na)]⁺). Actually 1022.3093.

LCMS (method B); t is t_R14.235 min (cis) -19.055 min (trans).

III.Ionizing radiation and cell experiments

Summary of the invention

Buffered media such as Phosphate Buffered Saline (PBS), Dulbecco's Modified Eagle medium-high glucose (DMEM), Rossville Park Memorial Institute (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 seimer feishel Scientific, france, and gemcitabine hydrochloride (Gem) was purchased from Sequoia Research Products (uk).

Cell lines

Human pancreatic cancer (PANC-1) and human acute lymphoblastic leukemia (CCRF-CEM) cells were purchased from ATCC (US) and maintained according to the supplier's recommendations. Gem resistant human acute lymphoblastic leukemia cells (CCRF-CEM ARAC 8C, hENT-1 receptor not expressed) were provided by Drddy Ullmann's University of Oregon Health Sciences (Oregon Health Sciences University) friend. 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 culture media were supplemented with penicillin (50 u.ml)^-1) And streptomycin (0.05 mg.mL)^-1). Cells were maintained at 37 ℃ and 5% CO₂In a humid atmosphere. Cells were used before reaching the eighteenth passage and harvested at 70-80% confluence.

Ionizing radiation source

And (4) UV irradiation. UV irradiation in a CN-15.LC Chamber (Vilber Lourmat) equipped with two 15W tubes (365nm) to induce isomerization of the trans isomer to the cis isomer, which provided 0.817mW.cm at the irradiation site^-2Radiation (determined by a Cole-Parmer VLX-3W microprocessor controlled radiometer calibrated to 365nm, Vilber Lourmat).

And (4) irradiating X-rays. X-ray irradiation was performed using an X-ray generator (Xrad 320 Dx) providing photons with a mean energy of 80keV (range 0-200keV) at a dose rate of about 1Gy/min (200kV, 20 mA). The radiation dose is shown in grey.

And (4) irradiating gamma rays. Gamma radiation was applied with a cesium 137 source (IBL637) to provide 662keV photons at a dose rate of about 1 Gy/min. The radiation dose is indicated in grey (1Gy ═ 1J/L).

And (4) irradiating an electron beam. Electron beam irradiation was carried out using a linear electron accelerator (Linac, Kinetron) to deliver electrons at 4.5MeV of energy at a dose rate of about 4 Gy/min. The radiation dose is shown in grey.

Quantification of ionizing radiation induced isomerization by absorption spectrophotometry and HPLC (FIG. 1)

Gadolinium-containing compounds (GdAzo or GdFAzo) and control compounds (Azo or FAzo) (50 μ M, 200 μ L, PBS) were introduced into two 96-well microplates (10 wells for each compound). Two microplates were first irradiated under uv light (365nm,0.817mw. cm)^-25 minutes). One microplate (plate 1) was then kept in the dark and used as an unirradiated control, while the second microplate (plate 2) was irradiated with increasing doses of ionizing radiation (2Gy, 3Gy, 5Gy and 10 Gy). After each irradiation, the non-irradiated (plate 1) and irradiated (plate 2) compounds were analyzed by absorbance spectrophotometry and HPLC injection (method C). A 26 minute time delay between UV irradiation of plate 1 and plate 2 was used in order to simultaneously analyze the unirradiated control compound (plate 1) and the ionising irradiated compound (plate 2) after UV irradiation. The complete experiment was performed within 4.6 hours. The absorption spectra are plotted by the average of three replicates. The relative amounts of each isomer were obtained by HPLC (detection at isosbestic wavelength under elution conditions), molecular activation(%) was determined from the difference in the proportion of trans isomers in the medium (3 independent experiments).

Confocal microscope without ionizing radiation (FIG. 2a)

24h before the start of the experiment, cells (PANC-1) were transferred to an imaging plate (ibiTreat 8-well microscope slides, purchased from Ibidi, Germany) at 37 ℃ and 5% CO₂Was maintained in medium (33000 cells, 200. mu.L/well). Immediately before the experiment, the cell culture medium was replaced with FBS-and phenol red-free medium (100 μ L), and then IP (5 μ L, final concentration of 1 μ M) was added to the medium. 15 minutes after the addition of IP, a first set of images was acquired. cis-GdAzoor, trans-GdAzo compounds (100. mu.L, final concentration 0. mu.M, 250. mu.M or 500. mu.M) were then introduced into the cell culture medium. The imaging plate was then kept in the dark and images were taken every 5 minutes for 1 hour. By UV irradiation (365nm,0.817 mW.cm)^-230min) Compound Trans GdAzo (105. mu.L, 500. mu.M or 1mM, cis GdAzo) in 96-well microplates>85%) to yield the compound cis-GdAzo. Cells were observed using a Leica TCS SP8 gated STED inverted microscope (Leica, germany) equipped with a Fluotar CS 210 x/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 560-650nm and transmission images were obtained using the same light and PMT-trans detector. The pinhole was set at 1.0Airy (diameter 73.19 μm). A 16-digit digital image was obtained using Leica SP8 LAS X software (version 2.0.1, Leica, germany).

Confocal microscope with ionizing radiation present (FIGS. 2b-c)

24 hours before the start of the experiment, cells (PANC-1) were transferred to an imaging plate (Lab-Tek chamber slide System, glass, 8-well, from Thermo Fisher Scientific, France) and incubated at 37 ℃ and 5% CO₂Was maintained in a medium (10,000 cells, 200. mu.L/well). Just prior to the experiment, the cell culture medium was replaced with PBS (100 μ L) and IP (5 μ L, final concentration 1 μ M) was added to the medium. 15 minutes after PI addition, cis GdAzo (100. mu.L, final concentration 0. mu.M, 250. mu.M, 500. mu.M or 850. mu.M) was introduced into the cell culture medium. At this stage a first set of images is acquired, then the imaging plate is illuminated (gamma ray)Line, 2 Gy). The imaging plate was then kept in the dark at 37 ℃ (plate warmer) and images were acquired every 5 minutes for 30 minutes and then every 10 minutes for 90 minutes. Similar procedure was used for the non-irradiated control experiment except that the imaging plate was not irradiated with gamma rays. The cis GdAzo compound was irradiated by UV (365nm,0.817 mW.cm)^-230min) Trans GdAzo Compound (105. mu.L, 500. mu.M, 1mM or 1.7mM, cis GdAzo) in 96-well plates>85%) obtained. Cells were observed using an X10 second objective, N.A.0.4, and Nikon inverted microscope (Nikon Instruments Inc., Tokyo, Japan) equipped with a Yokogawa CSU-X1 head. The instrument was equipped with a laser diode with an excitation wavelength of 561 nm. The red fluorescence emission was collected over a bandwidth of 598-672nm and transmission images were obtained with a white diode. The pinhole was set to 50 μm and the magnifying lens to 1.2. The images were recorded by an e-Volves-CMOS camera (Photometrics). Four images were recorded per well, two wells for each concentration of cis GdAzo. The 16 digital images were analyzed using ImageJ software (version 1.50i with a tunable watershed plug-in). The number of cells in the images obtained before and after irradiation was determined manually and the number of fluorescent cells in the images obtained before and after irradiation was calculated automatically using a script encoded on ImageJ. Cell permeability was determined by the difference in the proportion of fluorescent cells 30 minutes before and after gamma irradiation (3 independent experiments).

Therapeutic Effect under ionizing radiation (FIGS. 2d-f)

Just prior to the experiment, cells (CCRF-cemamac-8C) were dispersed in PBS and transferred to 48-well microplates (TPP cell culture microplates, purchased from Thermo Fisher Scientific, france) (40,000 cells, 80 μ L/well). Gem (20. mu.L, final concentration 0.1. mu.M) or PBS (20. mu.L) and cis GdAzo compound (100. mu.L, final concentration 0. mu.M, 250. mu.M, 500. mu.M or 850. mu.M) were added (2Gy) immediately prior to gamma irradiation of the medium. The medium was incubated at 37 ℃ and 5% CO in dark and humid atmosphere₂The reaction was maintained for 1 hour. Next, media (600 μ Ι _) was added to each well and the cells were washed by three centrifugation cycles (300G, 5 min, 800 μ Ι _, per wash). Finally dispersing the cells in the presence or absence of Gem(final concentration 0. mu.M or 0.1. mu.M) in medium (600. mu.L) at 37 ℃ and 5% CO₂For 4 days in a humid environment. The number of viable cells was determined by cell counting in the presence of 1:1(v/v) trypan blue (in triplicate). The experiment was independently repeated 3 times. Cell viability is expressed as the ratio of the number of viable cells after treatment to the number of viable cells without any treatment (no radiation, absence of Gem and cis GdAzo). A similar procedure was used for the non-irradiated control experiments, except that the 48-well plates were not irradiated with gamma rays. In addition to usingIn place of cis GdAzo, a similar procedure was usedControl experiment (fig. 2 f). By ultraviolet irradiation (365nm,0.817 mW.cm)^-230min) Trans GdAzo Compound (105. mu.L, 500. mu.M, 1mM or 1.7mM, cis GdAzo) in 96-well microplates>85%) to obtain a cis-GdAzo compound.

These results show that:

(i) a new concept for activating therapeutic molecules by using ionizing radiation (X-rays, gamma rays, electrons) (fig. 1 a-f). This new activation concept has strong clinical application potential due to the very high penetration capacity of these radiations in living tissue. All organic molecules described in the literature require the use of ultraviolet to near-infrared radiation, which does not penetrate deeply into biological tissues (which can penetrate several hundred microns under optimal conditions).

(ii) Depending on the metal used and the radiation source, MAzo compounds (M ═ Cu, Ga, Y, In, Eu, Gd, Yb, Bi) can be activated by ionizing radiation of different efficiencies (fig. 1a-c, f). The GdFAzo compounds may also be activated by ionizing radiation (FIGS. 1 c-e). This compound is characterized by a sufficiently slow thermal relaxation to allow for studies in vivo. Although the activation of compound GdFAzo is relatively low under the experimental conditions used in vitro (figure 1c compared to compound GdAzo), it is difficult to predict the molecular activation efficiency of this compound in vivo.

(iii) The type of metal 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 (the atomic number of yttrium) to allow consistent activation (greater than 30% at 5 Gy). In fact, 6 compounds containing metals with a size greater than or equal to yttrium showed such activation, while 2 compounds containing smaller metals (Cu, Z29 and Ga, Z31) showed low activation (less than 10% under 5 Gy) (fig. 1 f).

(iv) 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 1keV to 4.5MeV) (fig. 1c), and therefore, a group of radiotherapy devices currently in clinical use should be able to efficiently produce the described molecular activation.

(v) The compound GdAzo was able to induce permeability of cancer cell membranes under low doses of ionizing radiation (GdAzo at concentrations of 250 μ M, 500 μ M and 850 μ M, respectively, 6.9% (relative to 1.2% non-irradiated), 8.2% (relative to 2.8% non-irradiated) and 12.4% (relative to 7.5% non-irradiated) by gamma-ray of 2Gy) (fig. 2a, b, c).

(vi) Compound GdAzo was able to induce toxicity in gemcitabine resistant cancer cell lines (human acute lymphoblastic leukemia) in the absence or presence of gemcitabine (fig. 2d, e), and after 4 days of treatment cell viability was reduced to 23% (relative to 79% non-irradiated), 18% (relative to 42% non-irradiated) and 3.0% (relative to 27% non-irradiated) at GdAzo without gemcitabine, respectively, after treatment with 2Gy γ radiation. Cell viability after 4 days of treatment was reduced to 15% (relative to 49% non-irradiated), 9.1% (relative to 30% non-irradiated) and 1.2% (relative to 16% non-irradiated) at concentrations of 250 μ M, 500 μ M and 850 μ M, respectively, in the presence of gemcitabine. This study showed that the compound is active under ionizing radiation alone and that the presence of the surrounding active substances only slightly enhances the therapeutic effect of the compound (fig. 2d, e).

(vii) Due to commercial control compounds(gadolinium chelate alone) had no therapeutic effect under ionizing radiation, so the presence of azobenzene or stilbene (stillbene) motifs on the molecule was necessary to produce a therapeutic effect (fig. 2 f).