Rhenium(I) Tricarbonyl Complexes of 1,10-Phenanthroline Derivatives with Unexpectedly High Cytotoxicity

Eight rhenium(I) tricarbonyl aqua complexes with the general formula fac-[Re(CO)3(N,N′-bid)(H2O)][NO3] (1–8), where N,N′-bid is (2,6-dimethoxypyridyl)imidazo[4,5-f]1,10-phenanthroline (L1), (indole)imidazo[4,5-f]1,10-phenanthroline (L2), (5-methoxyindole)-imidazo[4,5-f]1,10-phenanthroline (L3), (biphenyl)imidazo[4,5-f]1,10-phenanthroline (L4), (fluorene)imidazo[4,5-f]1,10-phenanthroline (L5), (benzo[b]thiophene)imidazo[4,5-f]1,10-phenanthroline (L6), (5-bromothiazole)imidazo[4,5-f]1,10-phenanthroline (L7), and (4,5-dimethylthiophene)imidazo[4,5-f]1,10-phenanthroline (L8), were synthesized and characterized using 1H and 13C{1H} NMR, FT-IR, UV/Vis absorption spectroscopy, and ESI-mass spectrometry, and their purity was confirmed by elemental analysis. The stability of the complexes in aqueous buffer solution (pH 7.4) was confirmed by UV/Vis spectroscopy. The cytotoxicity of the complexes (1–8) was then evaluated on prostate cancer cells (PC3), showing a low nanomolar to low micromolar in vitro cytotoxicity. Worthy of note, three of the Re(I) tricarbonyl complexes showed very low (IC50 = 30–50 nM) cytotoxic activity against PC3 cells and up to 26-fold selectivity over normal human retinal pigment epithelial-1 (RPE-1) cells. The cytotoxicity of both complexes 3 and 6 was lowered under hypoxic conditions in PC3 cells. However, the compounds were still 10 times more active than cisplatin in these conditions. Additional biological experiments were then performed on the most selective complexes (complexes 3 and 6). Cell fractioning experiments followed by ICP-MS studies revealed that 3 and 6 accumulate mostly in the mitochondria and nucleus, respectively. Despite the respective mitochondrial and nuclear localization of 3 and 6, 3 did not trigger the apoptosis pathways for cell killing, whereas 6 can trigger apoptosis but not as a major pathway. Complex 3 induced a paraptosis pathway for cell killing while 6 did not induce any of our other tested pathways, namely, necrosis, paraptosis, and autophagy. Both complexes 3 and 6 were found to be involved in mitochondrial dysfunction and downregulated the ATP production of PC3 cells. To the best of our knowledge, this report presents some of the most cytotoxic Re(I) carbonyl complexes with exceptionally low nanomolar cytotoxic activity toward prostate cancer cells, demonstrating further the future viability of utilizing rhenium in the fight against cancer.


S4
S5 Figure S2. a) Hydrogen bonding and weak interactions (indicated in pink dashed lines) and b) π-interactions observed in the structure of 3a.
S6 Figure S3. Illustration of the one dimensional infinite chain along the c-axis observed in the structure of 3a. Table S2. Summary of the hydrogen bonding interactions, weak interactions, and πinteractions observed in the structure of 3a.
Symmetry codes and transformations used to generate equivalent atoms: 1 2-x,1-y,1-z, 2 1-x,1-y,1-z. The intermolecular interactions in the crystal structure of 3a were quantified using Hirshfeld surface analysis and the extensive π-interactions observed in this structure is confirmed and well-illustrated in the shape index (a) and curvedness plot (b) in Figure S4. The blue and red triangles in Figure S4a are characteristic of π-π-interactions and represent the convex regions (due to ring carbon atoms for the molecule inside the surface) and concave regions (due to carbon atoms of the π-stacked molecule above it) respectively. The π-π-interactions is also confirmed in the curvedness surface as the large green area in Figure S4b which is indicative of a 'relatively flat' region. Figure S4. Hirshfeld surface of 3a mapped with a) shape index and b) curvedness, illustrating the extensive π-π-interactions observed in the structure of 3a.

S8
The one dimensional infinite chain formed by N3-H3A O5 and N5-H5A Br1 is illustrated ⋯ ⋯ in Figure S5. In the shape index plot ( Figure S5a), the red concave region around the acceptor atom O5 and the blue convex region around the donor at N5 is illustrated; this is confirmed in the d norm plot ( Figure S5b) with the red electronegative region around the acceptor O5 and the blue electropositive region around the donor atom N5.

Stability study
The stability study of the complexes (1 -8) was performed in DMSO, phosphate buffer pH 7.4, both in the presence and absence of NaCl ( Figure S7 -S10), and in the presence of FBS ( Figure   S11) by UV/Vis measurements for 6 hours.
In the DMSO solution, all complexes showed stability during measurements, except complexes 6 and 8. For complexes 6 and 8, multiple isosbestic points were observed, suggesting DMSO coordination with the Re centre by replacing the labile water molecule.
In the buffer solution, we observed a decrease in absorbance of the complex with time without any isosbestic point or new peak. This was due to the low solubility of the complexes causing precipitation. Any degradation of solvated complexes was not observed in buffer solution at pH 7.4, both in the presence or absence of chloride. The absence of a new isosbestic point of chloride ion suggests a stable Re-OH 2 bond.
In the presence of 10% FBS, a significant increase in the solubility of the complexes was observed. Surprisingly, no new isosbestic point was observed for any cases, suggesting no covalent interaction of the complex with FBS. Despite the increase in solubility, a little amount of precipitation was observed for complexes 1, 2, 3 and 6. A very little amount of precipitation was observed in our stability kinetics study, but in the cell culture medium, we did not find any visible precipitation at the highest concentration up to 24 hours.
The ESI-MS spectra of 6 and 8 are presented in Figure S12, while the 1 H NMR spectra of 6 and 8, illustrating the stability of the complexes in DMSO, are presented in Figure S13 and S14. S12 Figure S7. The stability of complexes 1 and 2 in DMSO, 10 mM Phosphate buffer pH 7.4 and 10 mM Phosphate buffer pH 7.4 in the presence of 4 mM saline, analysed using UV/Vis absorption spectra. S13 Figure S8. The stability of complexes 3 and 4 in DMSO, 10 mM Phosphate buffer pH 7.4 and 10 mM Phosphate buffer pH 7.4 in the presence of 4 mM saline, analysed using UV/Vis absorption spectra. S14 Figure S9. The stability of complexes 5 and 6 in DMSO, 10 mM Phosphate buffer pH 7.4 and 10 mM Phosphate buffer pH 7.4 in the presence of 4 mM saline, analysed using UV/Vis absorption spectra. S15 Figure S10. The stability of complexes 7 and 8 in DMSO, 10 mM Phosphate buffer pH 7.4 and 10 mM Phosphate buffer pH 7.4 in the presence of 4 mM saline, analysed using UV/Vis absorption spectra. S16 Figure S11. The stability of complexes 1 -8 in the presence of FBS, analysed using UV/Vis absorption spectra. S17 Figure S12. The ESI-MS spectra of complexes 6 and 8 in methanol after 24h incubation in DMSO. S18 Figure S13. 1

The stability of fac-[Re(CO) 3 (L3)(H 2 O)] + (3) and fac-[Re(CO) 3 (L6)(H 2 O)] + (6) in DMF:H 2 O
(7:3, v/v %) at pH 5.5 was monitored for 24 hours ( Figure S23 and S24). The UV/Vis spectra of 3 and 6 at different pH values (~pH 5.8 to ~pH 9.8) are given in Figures S25 and S26 as illustration of the change in absorbance with an increase in pH.  hours. Figure S17. UV/Vis spectra of 3 at selected pH values illustrating the change in absorbance with an increase in pH. Figure S18. UV/Vis spectra of 6 at selected pH values illustrating the change in absorbance with an increase in pH.

DNA binding study
The binding of 6 with guanosine was monitored by 1 H NMR. The different spectra of these experiments are presented in Figure S27 - Figure S30. In Figure S31, the stability of 6 in DMF is confirmed by mass spectrometry and the results of the mass spectrometry of the binding study of 6 with guanosine are provided. Figure S32 represents the binding study of 6 with ctDNA by displacing intercalated ethidium bromide (EB) to evaluate the ability of intercalation. Figure S19. The binding study of complex 6 with Guanosine was measured by 1 H NMR (Aromatic region).  Figure S20. The binding study of complex 6 with Guanosine was measured by 1 HNMR. Figure S21. 1 H NMR spectrum of guanosine and complex 6 binding study. The study was performed in a 1:2 molar ratio of the complex: guanosine in DMF-d7 at 37°C.

BSA binding
The Stern-Volmer and Scatchard plots for the BSA fluorescence quenching of 1 -8 are presented in Figure S33 - Figure S36 below. Figure S25. The Stern-Volmer and Scatchard plots for the BSA fluorescence quenching upon the addition of complexes 1 and 2. Figure S26. The Stern-Volmer and Scatchard plots for the BSA fluorescence quenching upon the addition of complexes 3 and 4. Figure S27. The Stern-Volmer and Scatchard plots for the BSA fluorescence quenching upon the addition of complexes 5 and 6. Figure S28. The Stern-Volmer and Scatchard plots for the BSA fluorescence quenching upon the addition of complexes 7 and 8.

S31
The percentage cell viability vs. log of concentration (nM) graphs for 3, 6, and cisplatin against human prostate adenocarcinoma (PC-3) cells are presented in Figures S37, S39, and S40. The plots of tirapazamine against PC-3 cells in normoxic and hypoxic conditions are given in Figure   S38 and the representative plots of cisplatin against PC-3 cells are provided in Figure S41.

Mitochondrial respiration test
The formation of vacuoles inside the PC3 cells that are treated with 3 indicates paraptosis and are illustrated in Figure S42. A representative image of DAPI stained cells are provided in Figure S43, normalized oxygen consumption rates and different respiration parameters of PC3 cell treated with 3 and 6 are provided in Figure S44, and a representation of the DCF fluorescence intensity is given in Figure S45. S35 Figure S35. One representative image of DAPI stained cells, which were imaged (20X) and counted in Gen 5 software programme using Cytation 5, imagining facility, BioTek. The number of cells was used to normalise the obtained data as per experimental requirements.