School of Advanced Studies Doctorate course in Chemical Sciences Cycle XX Scientific-Sector CHIM/03

1. New metal complexes supported by scorpionate and macrocyclic ligands: chemistry and biological studies Dr.ssa Grazia Papini School of Advanced Studies Doctorate course in Chemical Sciences Cycle XX Scientific-Sector CHIM/03 Tutor Prof. Giancarlo Gioia Lobbia

2. Well-known Scorpionate Ligands Tris(pyrazolyl)borates Tetrakis(pyrazolyl)borates Bis(pyrazolyl)borates

3. “Nitrogen heterocycles other than pyrazole can be used, such as imidazole, triazole, benzotriazole, thioimidazole, ecc.” μ3-N,N’,N” Tris(imidazolyl)borates μ2-N,N’ μ4-N4 Tetrakis(imidazolyl)borates Bis(imidazolyl)borates Poly(triazolyl)borates Poly(benzotriazolyl)borates 3-S,S’,B-H 2-S,S’ Bis(3-R-2-thioxo-imidazolyl)borates

4. [H2B(pzCOOEt,Me)2]- [H3B(pzCF3)]- [H2B(tzNO2)2]- [H2B(pzNO2)2]- [H2B(pzCF3)2]- “Other modifications include changing the substituents on the heterocyclic ring.” S. Alidori, M. Pellei, C. Pettinari, C. Santini, B. W. Skelton, A. H. White, Inorg. Chem. Commun., (2004). G. Bandoli, A. Dolmella, G. Gioia Lobbia, G. Papini, M. Pellei, C. Santini Inorg. Chim. Acta (2006) H. V. R. Dias, S. Alidori, G. Gioia Lobbia, G. Papini, M. Pellei, C. Santini Inorg. Chem. (2007) Scorpionate ligands with EWG substituents M. Pellei, F. Benetollo, G.Gioia Lobbia, S. Alidori, and C. Santini, Inorg. Chem., (2005) M. Pellei, S. Alidori, G. Papini, G. Gioia Lobbia, J. D. Gorden, H. V. Rasika Dias, C. Santini, Dalton Trans. (2007)

5. bdmpzs bdmpza bdmpzta “In addition, tripodal ligands can have central atoms other than boron, such as carbon, phosphorus, or silicon….” RC(pzx)3 (pzx)3PO RSi(pzx)3 “…..and bearing a coordinating moiety (R') such as acetate, dithioacetate, sulfonate, ethoxide, ”

6. Rhenium complexes • Versatile chemistry: several oxidation states accessible (from -I to VII); different coordination numbers (from 4 to 8); various donor set available • The similarity between technetium and rhenium chemistry, determined a widespread use of the latter as a technetium surrogate to perform macroscopic chemistry of potential radiopharmaceuticals. In this way, a ‘‘cold’’material (the natural isotopic mixture of 185Re and 187Re) can be advantageously manipulated instead of the radioactive nuclide 99gTc (t1/2 = 2.12 · 105 y, Eβ = 292 keV). • Rhenium has two β- emitters isotopes 186Re (β-max = 1.07 MeV; t1/2 = 90 h) and 188Re (β-max = 2.10 MeV; t1/2 = 17 h) which are of great interest to nuclear medicine due to their physical and nuclear properties finalized to a potential application in the radiopharmaceutical

7. Bioactive molecule Linker Labile groups The “metal - fragment” strategy M Stable building -block

8. Re(V) complexes Metal fragment

9. N N S O 2 O N N R e C l O R O ROH (Et3N) ROH(Et3N) N N C O O N N R e C l O R O Metal Fragments M. Porchia, G. Papini, C. Santini, G. Gioia Lobbia, M. Pellei, F. Tisato, G. Bandoli, A. Dolmella, Inorg. Chem. 44 (2005) 4045

10. E= CO,SO2 n= 2,3 M.Porchia, G. Papini, C. Santini, G. Gioia Lobbia, M. Pellei, F. Tisato, G. Bandoli, A. Dolmella, Inorg. Chem. 44 (2005) 4045 Structure of the complex [Re(O)(bdmpza)(OCH2CH2CH2O)] Structure of the complex [Re(O)(bdmpza)(OCH2CH2O)] Mixed coordination sphere complexes

11. Structure of the complex [Re(O)(bdmpza)(mal)] Marina Porchia,Grazia Papini, Carlo Santini, Giancarlo Gioia Lobbia, Maura Pellei, Francesco Tisato, Giuliano Bandoli, Alessandro Dolmella Inorganica Chimica Acta 359 (2006) 2501–2508.

12. Potential Nitridorhenium complexes

13. Nitridorhenium precursors

14. Pre-carbene ligands G. Papini, C. Santini, G. Gioia Lobbia, M. Pellei, G. Bandoli, A. Dolmella J. Organomet. Chem.(2008) submitted

15. Liu J., Chen J., Zhao J., Zhao Y., Li L., Zhang H., Synthesis 17 (2003) 2661–2666.

17. MIXTURE OF UNCHARACTERIZABLE PRODUCTS [NBu4][ReNCl4]

18. Silver(I) carbene complexes G. Papini, C. Santini, G. Gioia Lobbia, M. Pellei, G. Bandoli, A. Dolmella J. Organomet. Chem.(2008) submitted

19. Carbene transfer reactions G. Papini, C. Santini, G. Gioia Lobbia, M. Pellei, G. Bandoli, A. Dolmella J. Organomet. Chem.(2008) submitted

20. NBu4ReNCl4 CH2Cl2 NBu4ReNCl4 CH2Cl2 Rhenium derivatives [Ru(p-cymene)Cl2]2

21. Cu(SMe2)Br [Ru(p-cymene)Cl2]2 CH3CN CH2Cl2 [Ru(p-cymene)Cl2]2 Cu(SMe2)Br CH2Cl2 CH3CN Copper and Ruthenium derivatives [Ru(p-cymene)Cl2]2 [Ru(p-cymene)Cl2]2

22. Copper derivatives It is an essential trace metal for living organisms Copper complexes’ activity is extremely wide Copper has a well-documented coordination chemistry Several radioactive copper isotopes are available nowadays for biomedical purposes both for radioimaging and targeted radiotherapy Fichna et al, Bioconjugate Chem., 14 (2003) 3-17

23. P(CH2OH)3 Copper(I) derivatives C. Marzano, M. Pellei, D. Colavito, S. Alidori, G. Gioia Lobbia, V. Gandin, F. Tisato, and C. Santini, J. Med. Chem., 49 (2006) 7317 Cells line of ovarian carcinoma (2008) and cis-platino resistent carcinoma cells (C13)

24. [Cu(bhpe)2][PF6] [Cu(thp)4][PF6] “CuP4” tipe species [Cu(CH3CN)4][PF6] + 4 thp [Cu(thp)4][PF6] [Cu(CH3CN)4][PF6] + 2 bhpe [Cu(bhpe)2][PF6] 31P-NMR = + 9.67 (dbr), - 144.05 (septet) [Cu(bhpe)2]+ m/z= 492 (100) 31P-NMR = - 5.35 (q), -145.14 (septet) [Cu(thp)4]+ m/z = 560 (6) [Cu(thp)3]+ m/z = 436 (65) [Cu(thp)2]+m/z =312 (100) C. Marzano, V. Gandin, M. Pellei, D. Colavito, G. Papini, G. Gioia Lobbia, M. Porchia, F. Tisato and C. Santini, J. Med. Chem. 51 (2008) 798-808.

25. bhpe C. Marzano, V. Gandin, M. Pellei, D. Colavito, G. Papini, G. Gioia Lobbia, M. Porchia, F. Tisato and C. Santini, J. Med. Chem. 1 (2008) 798-808.

26. Citotoxic activities A549 = lung cancer CaCo2, HCT-15 = colon cancer Hela = cervix cancer MCF-7 = breast cancer HL60 = leukemia Daudi = lymphoma HepG2 = epatoma A375 = melanoma IC50 values represent the drug concentrations that reduced the mean absorbance at 570 nm to 50% of those in the untreated control wells.

27. Human ovarian adenocarcinoma cells Cytotoxic activity of [Cu(thp)4][PF6] onto three additional cell line pairs, two of which (2008/C13* ovarian cancer cells and A431/A431-Pt cervix carcinoma cells) selected for their resistance to cisplatin and one (LoVo/LoVoMDR) for its resistance to doxorubicin. Cross-resistance profiles were evaluated by means of the resistance factor (RF), which is defined as the ratio between IC50 values calculated for the resistant cells and those arising from the sensitive ones. Human cervix squamous carcinoma cells Human colon adenocarcinoma cells

28. Comparison of IC50 values detected by MTT, NR and TB test after incubation of 2008 cells with [Cu(thp)4][PF6]for different exposure times TB test reveals damage to cell membrane MTT test mainly reflects damage to mitochondria The NR assay indicates damage to lysosomes and Golgi apparatus Lysosomes/Golgi apparatus are more sensitive to complex treatment. On the contrary, the scarce permeability to vital dye indicates that plasma membrane function is still maintained until the late phase of cell death. Lysosomal damage represents the early cellular event associated with copper(I) complex cytototoxicity.

29. 3 h 3 h 12 h 12 h 24 h 24 h 48 h 48 h Cell cyclephases G1 = GAP 1 S = Synthesis (DNA replication) G2 = GAP 2 M = mitosis (nuclear and cytoplasmic division) I = Interphase ----------2008 untreated cells ----------2008 cells treated with IC50 of [Cu(thp)4][PF6] Percentage of cells in different cell cycle phases as function of time exposure of [Cu(thp)4][PF6], vs control untreated cells

30. Forward scattering (index of cell size) vs side scattering (index of cell granularity)as a function of time in 2008 cells Mitochondrial energization of treated tumor cells as the retention of a mitochondrial selective cationic fluorescent probe, tetramethyl rhodamine methyl ester (TMRM). Flow cytometric profiles of 2008 cells untreated (panel A) and treated with 3.125 (panel B) or 6.25 (panel C) µM of copper(I) complex for 24 h and stained with TMRM (10 nM). Copper(I) complex induced a massive increase of the TMRM fluorescence reflecting a dramatic alteration of mitochondrial membrane potential that might be correlated with the induction of a G2/M phase cell cycle arrest. ----------2008 untreated cells ----------2008 cells treated with IC50 of [Cu(thp)4][PF6] The coordination of mono-phosphine ligands to copper(I) gives rise to a metallodrug able to inhibit the growth of tumor cells via cell G2/M cell cycle arrest and paraptosis accompanied with the loss of mitochondrial transmembrane potential.

31. Potential Cu(I) radiopharmaceuticals TPA 64Cu(II)Cl2 Sodium acetate buffer (2) Sodium acetate buffer THP In vitro cell experiments (1) Sodium acetate buffer Ligand Cell uptake behavior of complexes 1-4 into EMT-6 mammary carcinoma cells. Error bars not seen are within symbols. (3) (4)

32. Biodistribution was carried out on 16-18 g female BALB/c mice implanted with EMT-6 cells subcutaneously into the left flank. Tumors were allowed to grow for 14 days (approx 0.3 – 0.7 cm3), at which time the animals received 0.20 MBq (~5 μCi) of complex 1 in 100 μL of saline via lateral tail vein injection. Mice were examined at 3 time points (n = 4 per group at 1, 4 and 24 hours).  S. Alidori, G. Gioia Lobbia, G. Papini, M. Pellei, M. Porchia, F. Refosco, F. Tisato, J.S. Lewis, C. Santini Journal of Biological Inorganic Chemistry, 13 (2008) 307-315. Biodistribution Studies The uptake and retention of activity was high in many non-target tissues lung and liver Poor blood clearance suggestes breakdown of the complex and binding of 64Cu to serum proteins in vivo. The heart uptake was high at all time points and there was no clearance from the myocardium over 24 h post-injection potentially due to the monocationic nature of the complex Tumor uptake of complex 1 was highest at 1 h and decreased slowly over 24 h. In the same EMT-6 tumor model, uptake of 64Cu-ATSM and 64Cu-PTSM (both of which are clinically tested agents) into the tumor at 40 min post-injection showed lower uptake than that of 1 Tumor uptake of complex 1 is significantly higher than that for [64Cu((EtOCH2CH2)2PCH2CH2P(CH2CH2EtO)2)]+

33. Small animal PET Imaging Selected axial and coronal images obtained using co-registration techniques demonstrating the uptake of 1 at 1, 2 and 24 h post injection in a mouse with an EMT-6 tumor (arrow) implanted on the flank.The EMT-6 tumors can be easily visualized at all time points Standard uptake values (SUVs) of 1 in selected organs in EMT-6 tumor bearing mice over 24 h (n = 4). The uptake in the EMT-6 tumor at 1 h which remained static over 24 h

34. New N-, P- donor ligands

35. LiAlH4 1. n-BuLi 2. RX

36. P. Blondeau, C. Berse, D. Gravel, Can. J. Chem. 45 (1967) 49. New macrociclic ligands G. Papini, S. Alidori, J. S. Lewis, D. E. Reichert M. Pellei,,G. Gioia Lobbia, G. B. Biddlecombe,C. J. Anderson, C. SantiniJ. Med. Chem. (2008) submitted

37. Copper(II) complexes G. Papini, S. Alidori, J. S. Lewis, D. E. Reichert, M. Pellei, G. Gioia Lobbia, G. B. Biddlecombe, C. J. Anderson, C. SantiniJ. Med. Chem. (2008) submitted

38. 64Cu complexes Biodistribution data The retention of activity in tissues is similar to that observed with 64Cu-cyclam and 64Cu-monooxo-tetrazamacrocyclic complexes, but, on comparison with 64Cu-TETA and 64Cu-DOTA, the uptake and retention of and are orders-of-magnitude higher. The poor clearance suggests that the complexes are rapidly degraded in blood and tissues and the 64Cu is sequestered by proteins, and remaining trapped in these tissues hindering clearance.

39. M BFCA Perspectives

40. The monooxo Re(V) core is conveniently stabilized by tripodal scorpionate ligands comprising carboxylate or sulfonate tails, giving a series of intermediateRe(O)(NNO)Cl(X) (X = Cl, OR). To these entities various bidentate ligands (BID) can be attached to produce "3 + 2" mixed ligand compounds. • Hydrophilic ‘cold’ Cu(I)-complexes have shown significant antiproliferative activity in vitro on a series of tumor cell lines, also resistance to cisplatin, showing a different pathway of action from that of cisplatin. • Hydrophylic ‘hot’ 64Cu(I) monophosphine complexes were evaluated as a basis for a new class of copper radiopharmaceuticals. [64Cu(thp)4]+ = building-block for new radiopharmaceuticals, perhaps the first time such a method has been used in the production of Cu-radiopharmaceuticals. • Novel macrocyclic ligands, based on the L,L-ethylenedicysteine skeleton, have been prepared in view of the attractive opportunity to use them as bifunctional chelators for copper nuclides. This is the first report of 64Cu labeled to this form (N2S2) macrocyclics.Although the in vivo biodistribution of complexes suggests dissociation of the 64Cu from the chelates, these new ligands platform offers the potential as a basis for further developmentto improve the in vivo stability. Conclusions

41. Partners and Acknowledgements Prof. Giancarlo Gioia Lobbia Prof. Carlo Santini Dr.ssa Maura Pellei Dr. Simone Alidori Prof. Jason S. Lewis Carolyn J. Anderson Dr. Franco Benetollo ICIS-CNR, Padova Dr. Francesco Tisato Dr.ssa Marina Porchia Dr. Fiorenzo Refosco, Dr.ssa Cristina Bolzati ICIS-CNR, Padova Prof. Giuliano Bandoli Prof. Alessandro Dolmella Dr.ssa Cristina Marzano Dip. di Scienze Farmaceutiche Università di Padova Prof. Rasika Dias Department of Chemistry and Biochemistry The University of Texas at Arlington (USA)