ARTEMISININ-DERIVATIVE N-HETEROCYCLIC CARBENE GOLD(I) HYBRID COMPLEXES

Abstract

The present invention relates to compounds of formula (I), which are artemisinin-derivative N-heterocyclic carbene gold (1) hybrid complexes, and to their therapeutic uses.

Claims

1. Compound chosen from compounds of formula (I) and their isomers: ##STR00014## wherein each R is independently a C1-C6 alkyl, quinoline, benzyl or mesityl, X.sup.− is an anion, and n is a integer which is equal to 3, 4 or 5.

2. Compound according to claim 1, wherein it is chosen from compounds of formula (I′): ##STR00015## wherein each R is independently a C1-C6 alkyl, quinoline, benzyl or mesityl, X.sup.− is an anion, and n is a integer which is equal to 3, 4 or 5.

3. Compound according to claim 1, wherein the C1-C6 alkyl is a linear hydrocarbon group comprising from 1 to 6 carbon atoms, or a branched hydrocarbon group comprising from 3 to 6 carbon atoms.

4. Compound according to claim 1, wherein the R radicals are identical or different, and are chosen from methyl, quinolone, benzyl and mesityl radicals.

5. Compound according to claim 1, wherein both R radicals are identical.

6. Compound according to claim 1, wherein X.sup.− is an anion chosen from halogens, nitrate and hexafluorophosphate.

7. Compound according to claim 1, wherein the compound is chosen from the following compounds: ##STR00016##

8. Composition comprising, in a pharmaceutically acceptable medium, at least one compound of formula (I) according to claim 1.

9. (canceled)

10. A method for treating and/or preventing cancer, and/or for increasing the sensitivity of a cancer to a chemotherapeutic agent, and/or for decreasing the resistance of a cancer with respect to a chemotherapeutic drug, comprising administering to a subject in need thereof with an effective amount of at least one compound of formula (I) wherein the cancer is a solid or non solid.

11. Compound of formula (IV): ##STR00017## wherein: each R is independently a C1-C6 alkyl, quinoline, benzyl or mesityl, X.sup.− is an anion, and n is a integer which is equal to 3, 4 or 5.

12. A method for treating cancer, and/or for preventing cancer metastasis, and/or for preventing cancer recurrence, and/or for decreasing resistance to an additional therapy, in a subject, said method comprising administering to a subject in need thereof a combination product comprising: a) a compound according to claim 1, and b) at least one additional therapy, wherein the compound and the at least one additional therapy are administered simultaneously, separately or sequentially.

13. The method according to claim 12, wherein said at least one additional therapy b) is immunotherapy, chemotherapy and/or radiotherapy.

14. The method according to claim 12, wherein the subject is a human suffering from a cancer and resistant to chemotherapy.

15. A method for preventing and/or treating an inflammatory disease, comprising administering to a subject in need thereof an effective amount of at least one compound according to claim 1.

16. The compound according to claim 3, wherein the C1-C6 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl or n-hexyl.

17. The compound according to claim 5, wherein both R radicals are methyl.

18. The compound according to claim 6, wherein X.sup.− is chloride (Cl.sup.−) or nitrate (NO.sub.3.sup.−).

19. The method of claim 10, wherein the solid or non solid cancer is a colon cancer, a colorectal cancer, a melanoma, a bone cancer, a breast cancer, a thyroid cancer, a prostate cancer, an ovarian cancer, a lung cancer, a pancreatic cancer, a glioma, a cervical cancer, an endometrial cancer, a head and neck cancer, a liver cancer, a bladder cancer, a renal cancer, a skin cancer, a stomach cancer, a testis cancer, an urothelial cancer, an adrenocortical carcinoma, leukemia or lymphoma.

Description

[0111] The figures used in the present application are the following and serve as illustrative purposes only:

[0112] FIG. 1. Induction of ROS by dihydroartemisinin (DHA) and gold complexes (auranofin and complex 2a) on PC-3, A549, MCF-7 and HepG2 cells after different times of treatment. *p<0.05, **p<0.005, ***p<0.001, compared with ROS generation at 0 h.

[0113] FIG. 2. Impact of N-Acetyl-L-cysteine (NAC) and reduced glutathione (GSH) on the cytotoxicity of complex 2a. HepG2 cells were treated with complex 2a (1 mM) for 24 h in the absence or presence of different concentrations of NAC and GSH. Cell viability was measured by MTT assay. Data are presented as means±SEM of three independent experiments. *p<0.05, **p<0.005, ***p<0.001, compared with the cell viability of complex 2a alone.

[0114] FIG. 3. IC.sub.50 value of complex 2a towards isolated mammalian TrxR.

[0115] FIG. 4. NRF2 transcriptional activity.

[0116] The ARE Reporter Hep G2 cell line containing a firefly luciferase gene under the control of ARE stably integrated into Hep G2 cells was used to quantify NRF2 transcriptional activity after 16 hours of treatment with the indicated doses of the different complexes. The results are shown as fold induction of ARE luciferase reporter expression. Dashed line indicates a fold induction of 1 (values >1 mean activation and values <1 mean inhibition).

[0117] The cell line was validated for the response to the stimulation of tert-butylhydroquinone (tBHQ) according to the manufacturer's instructions (A).

[0118] Dose responses of ARE Reporter Hep G2 cells are shown to Auranofin (B), DHA (C), the compound 3 (D) and the compound 2a (E) where the “log(inhibitor) vs. Response” is represented in continuous light grey line (E).

[0119] FIG. 5. NF-kB transcriptional activity.

[0120] The NF-kB Reporter (Luc)—A549 Stable Cell Line was used to quantify the inhibitory effects of the indicated doses of the molecules of the invention on transcriptional activity of NF-kB activated by 1 ng/ml TNFα (7 hours of treatment).

[0121] Luminescence was read using a luminometer and readings were normalized to wells that only contain media to obtain the Relative Luminescence Units (RLUs).

[0122] Error bar=standard deviation (SD).

[0123] Dose responses of NF-kB Reporter—A549 cells activated by TNFα to Auranofin (A), DHA (B), the compound 3 (C) and the compound 2a (D) where the “log(inhibitor) vs. Response” is represented in continuous light grey lines (A to 0).

EXAMPLE

[0124] 1. Materials

[0125] All complexation reactions were performed under an inert atmosphere of dry nitrogen by using standard vacuum line and Schlenk tube techniques. Reactions involving silver compounds were performed with the exclusion of light. CH.sub.3CN was dried over CaH.sub.2 and subsequently distilled. 10β-(20-Bromopropoxy)dihydroartemisinin (DHA-C3) was synthetized according a modified literature procedure..sup.[1] All other reagents were used as received from commercial suppliers.

[0126] Human prostate cancer PC-3 and lung carcinoma A549 cell lines were obtained from DSMZ (Braunschweig, Germany). Human bladder cancer T24, human osteosarcoma U-2 OS, human breast cancer MCF-7, human hepatocarcinoma HepG2 cells, human normal epithelial prostate RPWE-1, human chronic myeloid leukemia LAMA, mouse osteoblasts MC3T3 and murine fibroblasts NIH3T3 were from ATCC-LGC Standards (Molsheim, France). All the cell culture medium, fetal bovine serum (FBS) and phosphate-buffered saline (PBS) were purchased from Thermo Fisher Scientific. N-Acetyl-L-cysteine (NAC), reduced Glutathione (GSH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich.

[0127] 2. Instrumentation

[0128] .sup.1H (300 or 400 MHz) and .sup.13C NMR spectra (75 or 101 MHz) and 2D experiments were recorded at 298 K on Bruker AV300, Bruker AV400 or Bruker Avance 500 spectrometers in CDCl.sub.3 as solvent. All chemical shifts for .sup.1H and .sup.13C are relative to TMS using .sup.1H (residual) or .sup.13C chemical shifts of the solvent as a secondary standard. All the .sup.1H and .sup.13C signals were assigned based on chemical shifts, spin-spin coupling constants, splitting patterns and signal intensities, and by using .sup.1H-.sup.1H COSY45, .sup.1H-.sup.13C HMBC and .sup.1H-.sup.13C HSQC/HMQC, experiments for complexes 2a-2c. Gradient-enhanced .sup.1H COSY45 was realised included 2 scans per increment. .sup.1H-.sup.13C correlation spectra using a gradient-enhanced HSQC/HMQC sequence (delay was optimised for .sup.1J.sub.CH of 145 Hz) was obtained with 2 scans per increment. Gradient-enhanced HMBC experiment was performed allowing 62.5 ms for long-range coupling evolution (8 scans were accumulated). Typically, 1024 t2 data points were collected for 256 t1 increments. High Resolution Mass Spectrometry (HRMS) analysis were performed with a Xevo G2 QTOF Waters spectrometer using electrospray ionization (ESI) by the “Service de Spectrométrie de Masse de Chimie UPS-CNRS (Toulouse)”. Elemental analyses were carried out by the “Service de Microanalyse du Laboratoire de Chimie de Coordination (Toulouse)”. The absorbance for MTT assay was measured using a Promega E7031 microplate reader.

[0129] 3. Synthesis of Proligands 1a-c and Complexes 2a-c

[0130] The general scheme for preparing the complexes 2a-c is the following:

##STR00010##

[0131] Complexes 2a-c are compounds of formula (I) according to the invention.

[0132] In order to fuse DHA and NHCs precursors the inventors used aliphatic linkers of different lengths C3 to C5. The synthesis (Scheme 1) starts with the formation of an ether, by reacting commercially DHA with a bromoalcohol in the presence of boron trifluoride etherate catalyst according to the procedure described by Haynes for the C3-derivative, leading to the single β-isomer DHA-C3 to DHA-C5 (see reference 1). The next step was the reaction between the bromoalkyl DHA derivatives and methyl imidazole in order to obtain the corresponding carbene precursors 1a to 1c with yields ranging from 39 to 92%. The formation of the target gold complexes has been achieved by two approaches. For the C3 derivative, the convenient transmetalation route involving the mild base Ag.sub.2O, followed by an ion exchange with AgNO.sub.3 and subsequent addition of Au(SMe.sub.2)Cl has been used. For the C4 and C5 derivatives, the direct metalation involving K.sub.2CO.sub.3 and Au(SMe.sub.2)Cl has been applied. The gold(I) complexes 2a-c were isolated after purification by chromatography as white solids with yields of 31 to 84%. All compounds were characterized by .sup.1H and .sup.13C NMR spectroscopy, high-resolution mass spectrometry and elemental analysis.

[0133] 3.1. Synthesis of Proligands 1a-c

[0134] The following picture describes the numbering of H (.sup.1H NMR) and C (.sup.13C NMR). These notations are used in the following experimental section.

##STR00011##

[0135] 10β-(20-Bromopropoxy)dihydroartemisinin (DHA-C3).sup.[1]

[0136] Under a nitrogen atmosphere, dihydroartemisinin (DHA) (2 g, 7.0 mmol) was dissolved in 200 mL Et.sub.2O. 3-Bromopropan-1-ol (0.76 mL, 8.4 mmol, 1.2 eq.) and BF.sub.3.Et.sub.2O (6 drops) were added and the reaction mixture was stirred for 4 h at room temperature. Then the solution was treated with a saturated solution of NaHCO.sub.3 and the product was extracted with Et.sub.2O (3×20 mL). The combined organic phases were dried over Na.sub.2CO.sub.3, filtered and the solvent was evaporated to dryness. The crude product was purified by column chromatography on silica using hexane-ethyl acetate as eluent (100/0 to 100/20) to give a white solid (1.277 g, 45% yield). .sup.1H NMR (400 MHz, CDCl.sub.3): δ=5.44 (s, 1H, H12), 4.82 (d, J=3.4 Hz, 1H, H10), 4.04-3.97 (m, 1H, H18), 3.54-3.47 (m, 3H, H18, H20), 2.70-2.60 (m, 1H, H9), 2.43-2.33 (m, 1H, H4), 2.15-2.06 (m, 2H, H19), 2.03-2.01 (m, 1H, H4), 1.94-1.85 (m, 1H, H5), 1.80-1.72 (m, 2H, H8), 1.68-1.62 (m, 1H, H7), 1.54-1.50 (m, 1H, H8a), 1.49-1.47 (m, 1H, H5), 1.46 (s, 3H, H14), 1.37-1.30 (m, 1H, H6), 1.29-1.23 (m, 1H, H5a), 0.97 (d, J=6.3 Hz, 3H, H15), 0.94-0.89 (m, 1H, H7), 0.92 (d, J=7.4 Hz, 3H, H16). .sup.13C NMR (75 MHz, CDCl.sub.3): δ=104.05 (1C, C3), 102.07 (1C, C10), 87.89 (1C, C12), 80.99 (1C, C12a), 65.66 (1C, C18), 52.56 (1C, C5a), 44.36 (1C, C8a), 37.42 (1C, C6), 36.40 (1C, C4), 34.62 (1C, C7), 32.52 (1C, C19), 30.86 (1C, C9), 30.57 (1C, C20), 26.16 (1C, C14), 24.65-24.49 (2C, C5, C8), 20.35 (1C, C15), 12.96 (1C, C16).

10β-(21-Bromobutoxy)dihydroartemisinin (DHA-C4)

[0137] Under a nitrogen atmosphere, dihydroartemisinin (DHA, 500 mg, 1.76 mmol) was dissolved in 200 mL Et.sub.2O. 4-Bromobutan-1-ol (398 mg, 2.6 mmol, 1.48 eq.) and BF.sub.3.Et.sub.2O (6 drops) were added and the reaction mixture was stirred for 4 h at room temperature. Then the solution was treated with a saturated solution of NaHCO.sub.3 and the product was extracted with Et.sub.2O (3×20 mL). The combined organic phases were dried over Na.sub.2CO.sub.3, filtered and the solvent was evaporated to dryness. The crude product was purified by column chromatography on silica using hexane-ethyl acetate as eluent (100/0 to 100/20) to give a white solid (220.3 mg, 29% yield). Anal. Calcd. for C.sub.19H.sub.31BrO.sub.5: C, 54.42; H, 7.45. Found: C, 54.36; H, 7.38. .sup.1H NMR (400 MHz, CDCl.sub.3): δ=5.40 (s, 1H, H12), 4.80 (d, J=3.2 Hz, 1H, H10), 3.9-3.88 (m, 1H, H18), 3.50-3.36 (m, 3H, H18, H21), 2.71-2.59 (m, 1H, H9), 2.45-2.32 (m, 1H, H4), 2.08-2.03 (m, 1H, H4), 1.99-1.86 (m, 3H, H19, H5), 1.83-1.74 (m, 4H, H8, H20), 1.70-1.63 (m, 1H, H7), 1.56-1.53 (m, 1H, H8a), 1.51-1.49 (m, 1H, H5), 1.46 (s, 3H, H14), 1.38-1.32 (m, 1H, H6), 1.30-1.26 (m, 1H, H5a), 0.98 (d, J=6.3 Hz, 3H, H15), 0.97-0.95 (m, 1H, H7), 0.92 (d, J=7.4 Hz, 3H, H16). .sup.13C NMR (101 MHz, CDCl.sub.3): δ=104.10 (1C, C3), 102.02 (1C, C10), 87.92 (1C, C12), 81.10 (1C, C12a), 67.44 (1C, C18), 52.58 (1C, C5a), 44.42 (1C, C8a), 37.49 (1C, C6), 36.44 (1C, C4), 34.64 (1C, C7), 33.63 (1C, C21), 30.90 (1C, C9), 29.83 (1C, C19), 28.34 (1C, C20), 26.22 (1C, C14), 24.69-24.51 (2C, C5, C8), 20.37 (1C, C15), 13.04 (1C, C16). HRMS (ES.sup.+): calcd. for C.sub.19H.sub.31BrNaO.sub.5 m/z=441.1253; found 441.1251.

10β-(22-Bromopentoxy)dihydroartemisinin (DHA-C5)

[0138] Under a nitrogen atmosphere, dihydroartemisinin (DHA, 1 g, 3.5 mmol) was dissolved in 200 mL Et.sub.2O. 5-Bromopentan-1-ol (601 mg, 3.6 mmol, 1.03 eq.) and BF.sub.3.Et.sub.2O (6 drops) were added and the reaction mixture was stirred for 4 h at room temperature. Then the solution was treated with a saturated solution of NaHCO.sub.3 and the product was extracted with Et.sub.2O (3×20 mL). The combined organic phases were dried over Na.sub.2CO.sub.3, filtered and the solvent was evaporated to dryness. The crude product was purified by column chromatography on silica using hexane-ethyl acetate as eluent (100/0 to 100/20) to give a white solid (314.4 mg, 21% yield). Anal. Calcd. for C.sub.20H.sub.33BrO.sub.5: C, 55.43; H, 7.68. Found: C, 55.49; H, 7.82. .sup.1H NMR (400 MHz, CDCl.sub.3): δ=5.41 (s, 1H, H12), 4.79 (d, J=3.2 Hz, 1H, H10), 3.87 (dt, J=9.8, 6.2 Hz, 1H, H18), 3.45-3.37 (m, 3H, H18, H22), 2.66-2.61 (m, 1H, H9), 2.39 (ddd, J=14.5, 13.4, 3.9 Hz, 1H, H4), 2.08-2.02 (m, 1H, H4), 1.94-1.87 (3H, H5, H21), 1.82-1.77 (m, 1H, H7), 1.67-1.48 (m, 8H, H5, H8, H8a, H19, H20), 1.50 (s, 3H, H14), 1.40-1.32 (m, 1H, H6), 1.29-1.22 (m, 1H, H5a), 0.97 (d, J=6.3 Hz, 3H, H15), 0.98-0.91 (m, 1H, H7), 0.92 (d, J=7.4 Hz, 3H, H16). .sup.13C NMR (101 MHz, CDCl.sub.3): δ=104.05 (1C, C3), 101.99 (1C, C10), 87.92 (1C, C12), 81.12 (1C, C12a), 68.01 (1C, C18), 52.58 (1C, C5a), 44.45 (1C, C8a), 37.46 (1C, C6), 36.44 (1C, C4), 34.66 (1C, C7), 33.81 (1C, C22), 32.41 (1C, C21), 30.92 (1C, C9), 28.82 (1C, C19), 26.21 (1C, C14), 24.96 (1C, C20), 24.68-24.48 (2C, C5, C8), 20.36 (1C, C15), 13.04 (1C, C16). HRMS (ES.sup.+): calcd. for C.sub.20H.sub.33BrNaO.sub.5 m/z=455.1409; found 455.1409.

##STR00012##

3′-methyl-1′-[10β-(20-propoxy)dihydroartemisin]1H-imidazol-3-ium bromide (1a)

[0139] To a stirred solution of DHA-C3 (304 mg, 0.75 mmol) in CH.sub.3ON (6 mL) under reflux, 1-methylimidazole (60 μL, 0.75 mmol) was added and the reaction mixture was stirred 3 days under reflux. The solvent was evaporated under reduced pressure and the viscous residue was purified by chromatography on silica with CH.sub.2Cl.sub.2-MeOH as eluent (1/0.1 to 1/0.25) to afford a white solid (238 mg, 65% yield). Anal. Calcd. for C.sub.22H.sub.35BrN.sub.2O.sub.5: C, 54.21; H, 7.24; N, 5.75. Found C, 54.12; H, 7.26; N, 5.68. .sup.1H NMR (400 MHz, CDCl.sub.3): δ=10.44 (s, 1H, H2′), 7.48 (s, 1H, H4′), 7.38 (s, 1H, H5′), 5.38 (s, 1H, H12), 4.79 (d, J=3.6 Hz, 1H, H10), 4.44 (t, J=7.2 Hz, 2H, H20), 4.14 (s, 3H, H6′), 3.92-3.86 (m, 1H, H18), 3.53-3.47 (m, 1H, H18), 2.68-2.61 (m, 1H, H9), 2.37 (ddd, J=14.6, 13.4, 4.0 Hz, 1H, H4), 2.29-2.22 (m, 2H, H19), 2.04 (ddd, J=14.6, 4.9, 2.9 Hz, 1H, H4), 1.93-1.86 (m, 1H, H5), 1.81-175 (m, 1H, H7), 1.72-1.61 (m, 2H, H8), 1.51-1.44 (m, 2H, H5, H8a), 1.42 (s, 3H, H14), 1.38-1.31 (m, 1H, H6), 1.29-1.23 (m, 1H, H5a), 0.97 (d, J=6.3 Hz, 3H, H15), 0.95-0.89 (m, 1H, H7), 0.92 (d, J=7.4 Hz, 3H, H16). .sup.13C NMR (101 MHz, CDCl.sub.3): δ=137.90 (1C, C2′), 123.37 (1C, C4′), 122.15 (1C, C5′), 104.23 (1C, C3), 102.20 (1C, C10), 87.96 (1C, C12), 80.92 (1C, C12a), 64.60 (1C, C18), 52.42 (1C, C5a), 47.48 (1C, C20), 44.16 (1C, C8a), 37.45 (1C, C6′), 36.90 (1C, C6), 36.33 (1C, C4), 34.45 (1C, C7), 30.77 (1C, C9), 30.60 (1C, C19), 26.10 (1C, C14), 24.63-24.57 (2C, C5, C8), 20.29 (1C, C15), 13.13 (1C, C16). HRMS (ES.sup.+): calcd. for C.sub.22H.sub.35N.sub.2O.sub.5 m/z=407.2545; found 407.2546.

3′-methyl-1′-[10β-(21-butoxy)dihydroartemisin]1H-imidazol-3-ium bromide (1 b)

[0140] To a stirred solution of DHA-C4 (75 mg, 0.18 mmol) in CH.sub.3CN (3 mL) under reflux, 1-methylimidazole (57 μL, 0.72 mmol, 4 eq.) was added and the reaction mixture was stirred 5 days under reflux. The solvent was evaporated under reduced pressure and the viscous residue was purified by chromatography on silica with CH.sub.2Cl.sub.2-MeOH as eluent (1/0.1 to 1/0.25) to afford a white solid (83 mg, 92% yield). Anal. Calcd. for C.sub.23H.sub.37BrN.sub.2O.sub.5: C, 55.09; H, 7.44; N, 5.59. Found C, 55.12; H, 7.56; N, 5.58. .sup.1H NMR (400 MHz, CDCl.sub.3): δ=10.38 (s, 1H, H2′), 7.55 (t, 1H, J=1.8 Hz, H4′), 7.42 (t, 1H, J=1.8 Hz, H5′), 5.34 (s, 1H, H12), 4.75 (d, J=3.4 Hz, 1H, H10), 4.37 (t, J=7.4 Hz, 2H, H21), 4.10 (s, 3H, H6′), 3.83 (dt, 1H, J=9.9, 6.0 Hz, H18), 3.40 (dt, 1H, J=9.9, 6.4 Hz, H18), 2.62-2.52 (m, 1H, H9), 2.37-2.29 (m, 1H, H4), 2.04-1.83 (m, 3H, H4, H20), 1.90-1.83 (m, 1H, H5), 1.73-1.59 (m, 5H, H7, H8, H19), 1.51-1.41 (m, 2H, H8a, H5), 1.39 (s, 3H, H14), 1.35-1.27 (m, 1H, H6), 1.25-1.17 (m, 1H, H5a), 0.94 (d, J=6.3 Hz, 3H, H15), 0.91-0.83 (m, 1H, H7), 0.87 (d, J=7.3 Hz, 3H, H16). .sup.13C NMR (101 MHz, CDCl.sub.3): δ=137.95 (1C, C2′), 123.41 (1C, C4′), 121.59 (1C, C5′), 104.13 (1C, C3), 102.12 (1C, C10), 87.91 (1C, C12), 81.03 (1C, C12a), 67.43 (1C, C18), 52.49 (1C, C5a), 49.88 (1C, C21), 44.29 (1C, C8a), 37.43 (1C, C6′), 36.82 (1C, C6), 36.38 (1C, C4), 34.51 (1C, C7), 30.84 (1C, C9), 27.28-26.44 (2C, C19, C20), 26.17 (1C, C14), 24.65-24.52 (2C, C5, C8), 20.32 (1C, C15), 13.09 (1C, C16). HRMS (ES.sup.+): calcd. for C.sub.23H.sub.37N.sub.2O.sub.5 m/z=421.2702; found 421.2704.

3′-methyl-1′-[10β-(22-pentoxy)dihydroartemisin]1H-imidazol-3-ium bromide (1c)

[0141] To a stirred solution of DHA-C5 (189 mg, 0.45 mmol) in CH.sub.3CN (6 mL) under reflux, 1-methylimidazole (143 μL, 1.80 mmol, 4 eq.) was added and the reaction mixture was stirred 3 days under reflux. The solvent was evaporated under reduced pressure and the viscous residue was purified by chromatography on silica with CH.sub.2Cl.sub.2-MeOH as eluent (1/0.1 to 1/0.25) to afford a white solid (91 mg, 39% yield). Anal. Calcd. for C.sub.24H.sub.39BrN.sub.2O.sub.5: C, 55.92; H, 7.63; N, 5.43. Found C, 55.86; H, 7.56; N, 5.38. .sup.1H NMR (400 MHz, CDCl.sub.3): δ=10.40 (s, 1H, H2′), 7.54 (t, 1H, J=1.8 Hz, H4′), 7.43 (t, 1H, J=1.8 Hz, H5′), 5.34 (s, 1H, H12), 4.73 (d, J=3.6 Hz, 1H, H10), 4.33 (t, J=7.5 Hz, 2H, H22), 4.12 (s, 3H, H6′), 3.79 (dt, 1H, J=9.8, 6.3 Hz, H18), 3.45 (dt, 1H, J=9.8, 6.5 Hz, H18), 2.62-2.54 (m, 1H, H9), 2.38-2.30 (m, 1H, H4), 2.04-1.92 (m, 3H, H4, H21), 1.89-1.83 (m, 1H, H5), 1.74-1.58 (m, 5H, H7, H8, H19), 1.53-1.37 (m, 4H, H5, H8a, H20), 1.40 (s, 3H, H14), 1.35-1.29 (m, 1H, H6), 1.27-1.18 (m, 1H, H5a), 0.94 (d, J=6.3 Hz, 3H, H15), 0.90-0.83 (m, 1H, H7), 0.86 (d, J=7.3 Hz, 3H, H16). .sup.13C NMR (101 MHz, CDCl.sub.3): δ=137.60 (1C, C2′), 123.51 (1C, C4′), 121.89 (1C, C5′), 104.07 (1C, C3), 101.99 (1C, C10), 87.88 (1C, C12), 81.09 (1C, C12a), 67.86 (1C, C18), 52.52 (1C, C5a), 50.00 (1C, C22), 44.37 (1C, C8a), 37.44 (1C, C6′), 36.77 (1C, C6), 36.40 (1C, C4), 34.56 (1C, C7), 30.88 (1C, C9), 30.06-29.01 (2C, C19, C21), 26.19 (1C, C14), 24.66-24.48 (2C, C5, C8), 23.00 (1C, C20), 20.35 (1C, C15), 13.05 (1C, C16). HRMS (ES.sup.+): calcd. for C.sub.24H.sub.39N.sub.2O.sub.5 m/z=435.2859; found 435.2861.

[0142] 3.2. Synthesis of Complexes 2a-c

[0143] Complex 2a

[0144] In a Schlenk tube, 1a (102 mg, 0.21 mmol) and Ag.sub.2O (25 mg, 0.11 mmol) were dissolved in CH.sub.3CN (3 mL) under a nitrogen atmosphere and protection of the light and stirred overnight at room temperature. A solution of AgNO.sub.3 (19 mg, 0.11 mmol) in 2 mL of CH.sub.3CN was added and the mixture was stirred for 2 h. Finally, Au(SMe.sub.2)Cl (32 mg, 0.11 mmol) was added and after stirring for 1 h at room temperature, the solution was filtered through a pad of celite and the solvent removed under reduced pressure to afford a white solid (95 mg, 84% yield). Crystals suitable for X-ray diffraction analysis were obtained by slow diffusion of Et.sub.2O in a CH3CN solution of this complex. Anal. Calcd. For C.sub.44H.sub.68AuN.sub.5O.sub.13: C, 49.30; H, 6.39; N, 6.53. Found C, 49.32; H, 6.45; N, 5.49. .sup.1H NMR (400 MHz, CDCl.sub.3): 57.26 (d, J=1.8 Hz, 1H, H4′), 7.16 (d, J=1.9 Hz, 1H, H5′), 5.37 (s, 1H, H12), 4.79 (d, J=3.5 Hz, 1H, H10), 4.28 (t, J=7.0 Hz, 2H, H20), 3.95 (s, 3H, H6′), 3.91-3.89 (m, 1H, H18), 3.46-3.43 (m, 1H, H18), 2.64-2.62 (m, 1H, H9), 2.37-2.34 (m, 1H, H4), 2.19-2.16 (m, 2H, H19), 2.04-2.02 (m, 1H, H4), 1.89-1.87 (m, 1H, H5), 1.73-1.71 (m, 2H, H8), 1.61-1.59 (m, 1H, H7), 1.48-1.46 (m, 1H, H8a), 1.46-1.43 (m, 1H, H5), 1.42 (s, 3H, H14), 1.32-1.30 (m, 1H, H6), 1.25-1.20 (m, 1H, H5a), 0.96 (d, J=6.2 Hz, 3H, H15), 0.95-0.92 (m, 1H, H7), 0.91 (d, J=7.4 Hz, 3H, H16). .sup.13C NMR (101 MHz, CDCl.sub.3): δ 183.71 (1C, C2′), 123.26 (1C, C4′), 121.84 (1C, C5′), 104.19 (1C, C3), 101.98 (1C, C10), 87.93 (1C, C12), 80.89 (1C, C12a), 64.81 (1C, C18), 52.46 (1C, C5a), 48.50 (1C, C20), 44.22 (1C, C8a), 38.21 (1C, C6′), 37.52 (1C, C6), 36.35 (1C, C4), 34.50 (1C, C7), 31.70 (1C, C19), 30.75 (1C, C9), 26.11 (1C, C14), 24.66-24.54 (2C, C5, C8), 20.31 (1C, C15), 13.10 (1C, C16) HRMS (ES.sup.+): calcd. for C.sub.44H.sub.68AuN.sub.4O.sub.10 m/z 1009.4601; found 1009.4591.

[0145] Complex 2b

[0146] Under a nitrogen atmosphere, potassium carbonate (27.7 mg, 0.20 mmol) was added to a mixture of 1 b (80 mg, 0.16 mmol) and Au(SMe.sub.2)Cl (26.5 mg, 0.09 mmol) in dry CH3CN (5 mL). The mixture was then heated to 60° C. and stirred for 2 h. After cooling to room temperature, the solution was filtered through a pad of celite and the solvent removed under reduced pressure. The complex was purified by preparative chromatography on silica plate with CH.sub.2Cl.sub.2-MeOH as eluent (100/8) to afford a white solid (68.7 mg, 80% yield). Anal. Calcd. For C.sub.46H.sub.72AuClN.sub.4O.sub.10. C, 51.47; H, 6.76; N, 5.22. Found C, 51.39; H, 6.68; N, 5.18. .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.37 (d, J=1.9 Hz, 1H, H4′), 7.22 (d, J=1.9 Hz, 1H, H5′), 5.33 (s, 1H, H12), 4.74 (d, J=3.2 Hz, 1H, H10), 4.25 (t, J=7.0 Hz, 2H, H21), 3.97 (s, 3H, H6′), 3.86-3.80 (m, 1H, H18), 3.42-3.37 (m, 1H, H18), 2.61-2.57 (m, 1H, H9), 2.38-2.30 (m, 1H, H4), 2.04-1.84 (m, 4H, H4, H5, H20), 1.76-1.58 (m, 5H, H7, H8, H19), 1.46-1.37 (m, 2H, H5, H8a), 1.40 (s, 3H, H14), 1.30-1.19 (m, 2H, H5a, H6), 0.94 (d, J=5.9 Hz, 3H, H15), 0.95-0.84 (m, 1H, H7), 0.86 (d, J=7.3 Hz, 3H, H16). .sup.13C NMR (101 MHz, CDCl.sub.3): δ 183.57 (1C, C2′), 123.45 (1C, C4′), 121.53 (1C, C5′), 104.10 (1C, C3), 101.99 (1C, C10), 87.87 (1C, C12), 80.97 (1C, C12a), 67.50 (1C, C18), 52.40 (1C, C5a), 51.04 (1C, C21), 44.27 (1C, C8a), 38.42 (1C, C6′), 37.49 (1C, C6), 36.35 (1C, C4), 34.53 (1C, C7), 30.79 (1C, C9), 28.34 (1C, C20), 26.73, (1C, C19), 26.16 (1C, C14), 24.65-24.48 (2C, C5, C8), 20.35 (1C, C15), 13.08 (1C, C16) HRMS (ES.sup.+): calcd. for C.sub.46H.sub.72AuN.sub.4O.sub.10 m/z 1037.4914; found 1037.4929.

[0147] Complex 2c

[0148] Under a nitrogen atmosphere, potassium carbonate (38.7 mg, 0.28 mmol) was added to a mixture of 1c (118.5 mg, 0.23 mmol) and Au(SMe.sub.2)Cl (35.3 mg, 0.12 mmol) in dry CH.sub.3CN (5 mL). The mixture was then heated to 60° C. and stirred for 2 h. After cooling to room temperature, the solution was filtered through a pad of celite and the solvent removed under reduced pressure. The complex was purified by preparative chromatography on silica plate with CH.sub.2Cl.sub.2-MeOH as eluent (100/8) to afford a white solid (39.3 mg, 31% yield). Anal. Calcd. For C.sub.48H.sub.76AuClN.sub.4O.sub.10. C, 52.34; H, 6.95; N, 5.09. Found C, 52.39; H, 6.98; N, 5.08. .sup.1H NMR (300 MHz, CDCl.sub.3): δ 7.32 (d, J=1.9 Hz, 1H, H4′), 7.22 (d, J=1.9 Hz, 1H, H5′), 5.36 (s, 1H, H12), 4.75 (d, J=3.6 Hz, 1H, H10), 4.25 (t, J=7.0 Hz, 2H, H22), 3.99 (s, 3H, H6′), 3.88-3.78 (m, 1H, H18), 3.41-3.33 (m, 1H, H18), 2.63-2.58 (m, 1H, H9), 2.43-2.32 (m, 1H, H4), 2.08-1.97 (m, 4H, H4, H5, H21), 1.78-1.59 (m, 5H, H7, H8, H19), 1.53-1.38 (m, 4H, H5, H8a, H20), 1.43 (s, 3H, H14), 1.30-1.24 (m, 2H, H5a, H6), 0.97 (d, J=6.1 Hz, 3H, H15), 0.97-0.84 (m, 1H, H7), 0.87 (d, J=7.4 Hz, 3H, H16). .sup.13C NMR (101 MHz, CDCl.sub.3): 183.59 (1C, C2′), 123.28 (1C, C4′), 121.65 (1C, C5′), 104.08 (1C, C3), 101.91 (1C, C10), 87.86 (1C, C12), 81.01 (1C, C12a), 67.85 (1C, C18), 52.49 (1C, C5a), 51.22 (1C, C22), 44.32 (1C, C8a), 38.38 (1C, C6′), 37.48 (1C, C6), 36.37 (1C, C4), 34.58 (1C, C7), 31.22 (1C, C21), 30.82 (1C, C9), 29.13 (1C, C19), 26.18 (1C, C14), 24.66-24.46 (2C, C5, C8), 23.23 (1C, C20), 20.37 (1C, C15), 13.02 (1C, C16) HRMS (ES.sup.+): calcd. for C.sub.48H.sub.76AuN.sub.4O.sub.10 m/z 1065.5221; found 1065.5226.

[0149] .sup.13C NMR spectroscopy unequivocally evidences the formation of the cationic gold(I) complexes with resonance of the carbenic carbons located at 183.6-183.7 ppm. HRMS spectra of 2a-c exhibit the classical peak for the cationic fragment [M-X.sup.−].sup.+ and elemental analysis correspond to the general [AuL.sub.2][X] formula.

[0150] 4. Crystallographic Data for 2a

[0151] Single crystals suitable for X-ray structure analysis have been obtained by gas phase diffusion from diethyl ether to a saturated solution of 2a in acetonitrile. In the solid state the gold(I) shows the typical linear coordination stabilized by two NHC ligands. The NHC planes are crossed around the C—Au—C axis with torsion angles from 116° to 138°. It is remarkable that the bulky DHA-derivative groups are on the same side of the central bisNHC gold motif.

[0152] This is due to an aurophilic interaction leading to a dimeric form of the complex with Au—Au distance of 345.0 pm.

[0153] All data were collected at low temperature using oil-coated shock-cooled crystals on a Bruker-AXS APEX II diffractometer with MoKa radiation (λ=0.71073 Å). The structure was solved by direct methods.sup.[2] and all non hydrogen atoms were refined anisotropically using the least-squares method on F.sup.2..sup.[3] The absolute structure parameters have been refined using the Flack-method..sup.[4]

[0154] Complex 2a: C.sub.44H.sub.68AuN.sub.5O.sub.13.12, Mr=1074.0, crystal size=0.40×0.30×0.05 mm.sup.3, orthorhombic, space group I222, a=10.644(2) Å, b=19.073(3) Å, c=48.320(8) Å, V=9809(3) Å.sup.3, Z=8, 59886 reflections collected, 6963 unique reflections (R.sub.int=0.1151), R1=0.0568, wR2=0.1300 [I>2σ(I)], R1=0.1309, wR2=0.1727 (all data), absolute structure factor x=−0.021(8), residual electron density=3.308 e Å.sup.−3.

[0155] 5. Cell Culture and Cell Viability Assay (MTT Assay)

[0156] PC-3 human prostate cancer cells, and LAMA chronic myeloid leukemia were cultured in RPMI 1640 containing 10% fetal bovine serum and 1% antibiotics (100 U/mL penicillin and 100 μg/mL streotimycin) at 37° C. in 5% CO.sub.2 humidified incubators. HL60 chronic myeloid leukemia were cultured in RPMI 1640 containing 15% fetal bovine serum and 1% antibiotics (100 U/mL penicillin and 100 μg/mL streotimycin) at 37° C. in 5% CO.sub.2 humidified incubators. HepG-2 human liver cancer cells were cultured in EMEM containing 10% of FBS, 1% of nonessential amino acids, 1 mM of Na-pyruvate, 1% of PenStrep antibiotics and 600 μg/mL of Geneticin. A549 human lung carcinoma cells, T24 human bladder carcinoma cells, MCF-7 human breast adenocarcinoma cells, U-2OS human osteosarcoma and NIH3T3 murine fibroblast cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% antibiotics at 37° C. in 5% CO.sub.2 humidified incubators. MC3T3 mouse osteoblast cells were cultured in MEM medium containing 10% fetal bovine serum and 1% antibiotics at 37° C. in 5% CO.sub.2 humidified incubators. RWPE-1 human prostate normal cells were cultured in K-SFM medium containing 0.05 mg/ml bovine pituitary extract (BPE) and 5 ng/ml epidermal growth factor (EGF) at 37° C. in 5% CO.sub.2 humidified incubators. The MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was used to determine cell death as originally described by Mosmaneland modified by Cuvillier et al..sup.[6] Briefly, cells were seeded 5,000 to 10,000 cells/well in 24-well plates depending on the cell type and allowed to attach overnight. All of the complexes were dissolved in DMSO. The concentration of the complexes was calculated according to the elemental composition of the complexes determined by the elemental analyses. Media in the presence of the tested complexes were added and serially diluted to various concentrations (from 5 μM to 0.01 μM). Next to these concentrations, for DHA 50, 20 and 10 μM and for the cationic bisNHC gold(I) complex 3 0.001 μM have been used. The maximum concentration of DMSO in media did not exceed 0.5% (v/v). After 72 h of treatment, cells were incubated at 37° C. and 5% CO.sub.2 with 25 μL MTT solution (5 mg/mL; Sigma-Aldrich) in 24-well plates for approximately 3 to 4 h. After solubilization with 500 μL of lysis buffer (DMSO), formazan was quantified by spectrophotometry with a microplate reader at 570 nm absorbance. The GI.sub.50 values corresponding to the concentration that caused 50% inhibition of cell proliferation were calculated from dose-response curves obtained by nonlinear regression analysis (Prism 8, Graphpad Software). All the results were calculated from data obtained in three independent experiments.

[0157] Precursors 1a-c and complexes 2a-c were evaluated for their in vitro cytotoxic abilities against PC-3 prostate cancer cell line and three non-cancer cell lines (fibroblast NIH3T3, osteoblast MC3T3 and epithelial prostate RPWE-1) (Table 1 below). Interestingly, the imidazolium salts 1a-c showed no cytotoxic effects (GI.sub.50>20 μM), while complexes 2a-c exhibited strong antiproliferative activities with GI.sub.50 values between 20 nM and 70 nM. The selectivity indexes (SI=G150 (non-cancer cell line)/G150 (cancer cell line)) of 2a-c gave nearly the same values concerning NIH3T3 cells ranging from 15.5 to 16.7, while RPWE-1 cells gave a more differentiated result with the highest SI=6.9 for 2a. As reference drugs, Auranofin, an anti-arthritis drug currently in clinical phase I and II trials as anticancer drug, and DHA were tested. Moreover a published cationic bisNHC gold(I) complex 3, containing a methyl and a quinoline substituents.sup.[7], and a mixture of 3 and DHA (1:2) were investigated in order to evaluate the potential synergetic effect of the hybrid complexes. The structure of the cationic bisNHC gold(I) complex 3 is as follows:

##STR00013##

[0158] Outstandingly, complexes 2a-c displayed 16 to 55-fold and 22 to 78-fold higher potency than Auranofin and DHA on PC-3 cells, respectively. Moreover, they are 6.2 to 16.7 more selective towards cancer cells than NIH3T3 compared to the two drug references. Remarkably, complex 2a shows an SI PC-3/RPWE-1 value close to that of DHA but 69 and 35 times higher than that obtained for both gold references, Auranofin and complex 3, respectively. Complex 3 showed 10 to 35-fold lower activity than 2a-c and the mixture of 3 and DHA has an efficiency between DHA and 3 with a low selectivity. Overall, these results highlight that linking a derivative of DHA on the NHC scaffold of a bisNHC gold(I) unit led to a synergy, expressed by a high cytotoxicity combined with a high selectivity.

[0159] Due to its better selectivity, the inventors chose complex 2a for further biological investigations. 2a has been tested on a panel of seven other representative human cancer cell lines, namely A549 (lung), MCF-7 (breast), T24 (bladder), U-2 OS (bone), LAMA (leukemia), HL60 (acute myeloid leukemia) and Hep-G2 (liver) (see Table 2). As in the case of PC-3 cells the G150 values for six cancer cell lines were in the lower nM range spanning from 22 to 175 nM. Complex 2a remains extensively more effective than the control molecules used in this study on all the tested cancer cell lines, even on hepatocellular carcinoma HepG-2, which are always more difficult to treat (the commercial drug Sorafenib currently used to treat hepatocarcinoma displays an IC.sub.50 of 6.4 μM on the HepG-2 cell line). Differential effects of arsenic trioxide on chemosensitization in human hepatic tumor and stellate cell lines (F. Rangwala, K. P. Williams, G. R. Smith, Z. Thomas, J. L. Allensworth, H. K. Lyerly, A. M. Diehl, M. A. Morse, G. R. Devi, BMC Cancer 2012, 12, 402), validating the concept of gold(I)-artemisinin like hybrid complexes.

TABLE-US-00001 TABLE 1 Cytotoxicity and selectivity of 1a-c and 2a-c on PC-3, NIH3T3 and RWPE-1 cell lines (GI.sub.50 [μM], 72 h, MTT assay)..sup.[a] Compound PC-3 NIH3T3/SI.sup.[b] MC3T3/SI.sup.[b] RWPE-1/SI.sup.[b] 1a >20 >20/—  >20/— 1b >20 >20/—  >20/— 1c >20 >20/—  3.74/—  2a 0.070 1.13/16.1  1.62/23.1 0.48/6.9 2b 0.042 0.70/16.7 0.11/2.6 2c 0.020 0.31/15.5 0.098/4.9  Auranofin 1.09 1.10/1.0  1.39/1.2 0.084/0.1  DHA 1.56 3.86/2.5  3.55/2.3 9.68/6.2 3 0.70 7.98/11.4  17.9/25.5 0.112/0.2  3/DHA (1:2) 1.09 2.85/2.6  1.76/1.6 0.22/0.2 .sup.[a]The GI.sub.50 values represent the concentration of compound causing 50% inhibition of cell growth. Mean of three independent experiments. .sup.[b]Selectivity index.

TABLE-US-00002 TABLE 2 Cytotoxicity of 2a on A549, MCF-7, T24, U2OS, LAMA, HL60 and HepG-2 lines (GI.sub.50 [μM], 72 h, MTT assay)..sup.[a] Compound A549 MCF-7 T24 U2OS HepG-2 LAMA HL60 2a 0.11 0.09 0.17 0.12 2.43 0.08 0.022 Auranofin 4.41 1.39 1.10 0.47 3.62 0.81 0.95 DHA 11.0 9.67 4.99 4.10 12.0 5.6 25 3 1.16 0.38 0.19 2.51 5.23 0.66 0.50 3/DHA 1.99 0.61 0.32 1.25 4.71 0.80 0.47 (1:2) .sup.[a]The GI.sub.50 values represent the concentration of compound causing 50% inhibition of cell growth. Mean of three independent experiments.

[0160] Table 3 below also presents the IC.sub.50 observed for each cell line, for the different tested molecules, which are: the compound (I) of the invention (compound 2a), auranofin, DHA, NHC-gold(I) complex 3 alone (without any artemisinin or DHA) and a mixture of NHC-gold(I) complex 3 alone and DHA alone, in a molar ratio of 1:2.

TABLE-US-00003 TABLE 3 IC.sub.50 of 2a, auranofin, DHA, NHC-gold(I) complex alone 3 and a mixture of 3 alone and DHA alone, in a molar ratio of 1:2 on cancerous cells A549, MCF-7, T24, U-2 OS, PC-3, LAMA and HL60, and on MC3T3, NIH3T3 and RWPE-1 non-cancerous cell lines. 3/DHA 2a DHA 3 (ratio 1/2) Auranofin Non tumoral cell models MC3-T3 (osteoblasts) 1.62 3.55 17.9 1.76 1.39 NIH3T3 (fibroblasts) 1.13 3.86 7.98 2.85 1.10 RWPE1 (prostatic epithelial cells) 0.48 9.68 0.112 0.218 0.084 MEAN 1.08 5.70 8.66 1.61 0.86 μM Tumor cell models PC-3 (prostate) 0.070 1.56 0.,695 1.09 1.09 A549 (lung) 0.115 11.04 1.16 1.99 4.41 U2OS (bone tumor) 0.122 4.10 2.51 1.25 0.474 T24 (bladder) 0.175 4.99 0.191 0.319 1.10 MCF-7 (breast) 0.089 9.67 0.380 0.610 1.39 LAMA (chronic myeloid leukemia) 0.079 5.60 0.662 0.800 0.809 HL60 (acute myeloid leukemia) 0.022 3.25 0.500 0.471 0.951 MEAN of 7 tumor cell models 0.096 5.74 0.87 0.93 1.46 μM Comparison of the specificity for tumoral cells versus non tumoral cells which come from a same tissue (e.g. prostate and bone)* Prostate: RWPE1 (normal 6.9 6.2 0.2 0.2 0.1 immortalized)/PC-3 (tumoral) Bone: MC3-T3 (normal 13.3 0.9 7.1 1.4 2.9 immortalized)/U2OS (tumoral) *shows the very high specificity of the Compound 2a

[0161] 6. Measurement of Intracellular Reactive Oxygen Species (ROS) and Determination of ROS Generation

[0162] The cellular ROS generation was shown by the increase of fluorescence intensity of DCF according to a previously reported protocol..sup.[8] Briefly, PC-3, A549, MCF-7 and HepG2 cells were seeded at a density of 50,000 cells/well in 24-well plates for 24 h. Cells were washed with PBS buffer and stained with DCFH-DA (final concentration 20 μM) for 45 min. Then cells were washed with PBS buffer and the culture medium without phenol red containing gold complexes were added to the cells. The fluorescence intensity of DCF (excitation/emission, 485/535 nm) was measured by fluorescence microplate reader at different time points.

[0163] For N-acetyl-cysteine (NAC) and reduced glutathione (GSH) treatment, the cells were pretreated with different concentrations of NAC and GSH (2, 5 and 10 mM) for 1 h, then gold complex 2a was added for incubation for 72 h. After that, cells were further incubated at 37° C. and 5% CO.sub.2 with 25 μL MTT solution (5 mg/mL; Sigma-Aldrich) in 24-well plates for approximately 3 h. The cytotoxicity was determined as described above.

[0164] The results are in FIGS. 1 and 2.

[0165] 7. Inhibition of Mammalian TrxR

[0166] To determine the inhibition of mammalian TrxR, an established microplate reader-based assay was performed with minor modifications..sup.[9] For this purpose, commercially available rat liver TrxR (from Sigma-Aldrich) was used and diluted with distilled water to achieve 3.5 U/mL. Complex 2a was freshly dissolved as stock solution in sterile DMSO. 25 μL Aliquot of the enzyme and either 25 μL potassium phosphate buffer (pH 7.0) containing complex 2a in graded concentrations or 25 μL buffer without the complex but DMSO (positive control) were added. The resulting solution (final DMSO concentration of 0.5% v/v) was incubated at 37° C. for 75 min with moderate shaking in a 96-well plate. To each well, 225 μL of the reaction mixture (1.0 mL reaction mixture consists of 500 μL 100 mM potassium phosphate buffer pH 7.0, 80 μL 100 mM EDTA solution pH 7.5, 20 μL 0.2% BSA, 100 μL of a 20 mM NADPH and 300 μL distilled water) was added and the reaction was initiated immediately by adding 25 μL of 20 mM DTNB solution. After thorough mixing, the formation of TNB was monitored by a microplate reader at 405 nm at 1 min intervals for 10 measurements. The increase of TNB concentration over time followed a linear tendency (r.sup.2≥0.99), and the enzymatic activities were calculated as the slopes (increase in absorbance per second). Non-interference with the assay components was confirmed by a negative control experiment using an enzyme-free test compound. 1050 values were calculated as the concentration of the compound decreasing the enzymatic activity of the untreated control by 50%. The results are in FIG. 3.

[0167] 8. NRF2 Transcriptional Activity

[0168] ARE-reporter-HepG-2 cells were harvested from culture in Growth Medium 1K and they were seeded at a concentration of 40,000 cells/well into white clear-bottom 96-well microplate in 45 μL of Growth Medium 1K without Geneticin. Cells were allowed to attach overnight and then treated with different concentrations of complex 2a (from 0.01 μM to 20 μM), auranofin (from 0.01 μM to 20 μM) or DHA (from 0.01 μM to 3, 5, 10, 20 and 50 μM), and complex (from 0.01 μM to 20 μM). After incubation at 37° C. and 5% CO.sub.2 for 16 hours, ONE-Step luciferase assay reagent (100 μL) was added and rock at room temperature for over 15 minutes. Then the luminescence of each well was determined by CLARIOstar microplate reader to quantify induction of ARE. Three independent experiments were performed as biological triplicates. The results are in FIG. 4.

[0169] 9. NF-kB Transcriptional Activity

[0170] The results are in FIG. 5.

[0171] 10. References [0172] [1] R. K. Haynes, H.-W. Chan, M.-K. Cheung, W.-L. Lam, M.-K. Soo, H.-W. Tsang, A. Voerste, I. D Williams, Eur. J. Org. Chem., 2002, 1, 113-132. [0173] [2] G. M. Sheldrick, Acta Crystallogr., 1990, A46, 467-473. [0174] [3] G. M. Sheldrick, Acta Crystallogr., 2008, A64, 112-122. [0175] [4] (a) H. D. Flack, Acta Crystallogr. 1983, A39, 876-881; (b) S. Parsons, H. D. Flack, T. Wagner, Acta Cryst. 2013, 869, 249-259. [0176] [5] T. Mosmann, J. Immunol. Methods, 1983, 65(1-2), 55-63. [0177] [6] O. Cuvillier, V. E. Nava, S. K. Murthy, L. C. Edsall, T. Levade, S. Milstien, S. Spiegel, Cell Death Differ., 2001, 2, 162-171. [0178] [7] C Hemmert, A. Fabië, A. Fabre, F. Benoit-Vical, H. Gornitzka, Eur. J. Med. Chem., 2013, 60, 64-75. [0179] [8] Z. Zhao, P. Gao, Y. You, T. Chen, Chem. Eur. J., 2018, 24, 3289-3298. [0180] [9] R. Rubbiani, I. Kitanovic, H. Alborzinia, S. Can, A. Kitanovic, L. A. Onambele, M. Stefanopoulou, Y. Geldmacher, W. S. Sheldrick, G. Wolber, A. Prokop, S. Wölfl, I. Ott, J. Med. Chem., 2010, 53, 8608-8618.