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School of Advanced Studies Doctorate course in Chemical Sciences Cycle XX Scientific-Sector CHIM/03 Dr.ssa Grazia Papini New metal complexes supported.

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Presentation on theme: "School of Advanced Studies Doctorate course in Chemical Sciences Cycle XX Scientific-Sector CHIM/03 Dr.ssa Grazia Papini New metal complexes supported."— Presentation transcript:

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

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

3 Nitrogen heterocycles other than pyrazole can be used, such as imidazole, triazole, benzotriazole, thioimidazole, ecc. Bis(imidazolyl)borates Tris(imidazolyl)borates Tetrakis(imidazolyl)borates Bis(3-R-2-thioxo-imidazolyl)borates Poly(triazolyl)boratesPoly(benzotriazolyl)borates μ 2 -N,N μ 4 -N 4 μ 3 -N,N,N 3 -S,S,B-H 2 -S,S

4 Scorpionate ligands with EWG substituents Scorpionate ligands with EWG substituents [H 2 B(pz COOEt,Me ) 2 ] - [H 2 B(tz NO2 ) 2 ] - [H 2 B(pz NO2 ) 2 ] - [H 2 B(pz CF3 ) 2 ] - [H 3 B(pz CF3 )] - H. V. R. Dias, S. Alidori, G. Gioia Lobbia, G. Papini, M. Pellei, C. Santini Inorg. Chem. (2007) M. Pellei, S. Alidori, G. Papini, G. Gioia Lobbia, J. D. Gorden, H. V. Rasika Dias, C. Santini, Dalton Trans. (2007) 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) M. Pellei, F. Benetollo, G.Gioia Lobbia, S. Alidori, and C. Santini, Inorg. Chem., (2005) Other modifications include changing the substituents on the heterocyclic ring.

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

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 coldmaterial (the natural isotopic mixture of 185 Re and 187 Re) can be advantageously manipulated instead of the radioactive nuclide 99g Tc (t 1/2 = 2.12 · 10 5 y, E β = 292 keV). Rhenium has two β - emitters isotopes 186 Re (β - max = 1.07 MeV; t 1/2 = 90 h) and 188 Re (β - max = 2.10 MeV; t 1/2 = 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 M The metal - fragment strategy Stable building -block Labile groups

8 Re(V) complexes Metal fragment

9 N N N N Re O OR Cl SO 2 O N N N N Re O OR Cl CO O ROH(Et 3 N) 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 Mixed coordination sphere complexes E= CO,SO 2 n= 2, 3 Structure of the complex [Re(O)(bdmpza)(OCH 2 CH 2 O)] Structure of the complex [Re(O)(bdmpza)(OCH 2 CH 2 CH 2 O)] M. Porchia, G. Papini, C. Santini, G. Gioia Lobbia, M. Pellei, F. Tisato, G. Bandoli, A. Dolmella, Inorg. Chem. 44 (2005) 4045

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.

16

17 [NBu 4 ][ReNCl 4 ] MIXTURE OF UNCHARACTERIZABLE PRODUCTS

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 Rhenium derivatives NBu 4 ReNCl 4 CH 2 Cl 2 NBu 4 ReNCl 4 CH 2 Cl 2 [Ru(p-cymene)Cl 2 ] 2

21 Copper and Ruthenium derivatives [Ru(p-cymene)Cl 2 ] 2 CH 2 Cl 2 [Ru(p-cymene)Cl 2 ] 2 CH 2 Cl 2 Cu(SMe 2 )Br CH 3 CN Cu(SMe 2 )Br CH 3 CN

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 isotopehalf-lifeimaging (emission, energy, abundance) therapy (emission, energy, range in tissue) application Cu-6020 minPET (b +, 873 keV, 93%) Radiolabelling small molecules Cu hPET (b +, 527 keV, 62%) Radiolabelling small molecules Cu minPET (b +, 1315 keV, 98%) Radiolabelling small molecules Cu hPET (b +, 278 keV, 19%) Radiolabelling small molecules, peptides and antibodies Cu min(b -, 190 keV; 0.95 mm)Radiolabelling small molecules for therapy Cu hSPECT (g, 185 keV, 48%) (b -, 190 keV; 0.95 mm)Radiolabelling small molecules, peptides and antibodies Fichna et al, Bioconjugate Chem., 14 (2003) 3-17

23 Copper(I) derivatives P(CH 2 OH) 3 Cells line of ovarian carcinoma (2008) and cis-platino resistent carcinoma cells (C13) 49 C. Marzano, M. Pellei, D. Colavito, S. Alidori, G. Gioia Lobbia, V. Gandin, F. Tisato, and C. Santini, J. Med. Chem., 49 (2006) 7317

24 CuP 4 tipe species [Cu(CH 3 CN) 4 ][PF 6 ] + 4 thp [Cu(thp) 4 ][PF 6 ] [Cu(CH 3 CN) 4 ][PF 6 ] + 2 bhpe [Cu(bhpe) 2 ][PF 6 ] [Cu(thp) 4 ][PF 6 ] [Cu(bhpe) 2 ][PF 6 ] 31 P-NMR = (q), (septet) [Cu(thp) 4 ] + m/z = 560 (6) [Cu(thp) 3 ] + m/z = 436 (65) [Cu(thp) 2 ] + m/z =312 (100) 31 P-NMR = (dbr), (septet) [Cu(bhpe) 2 ] + m/z = 492 (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)

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)

26 Citotoxic activities Compound IC 50 (µM) ± S.D. HL60A549MCF-7DaudiHepG2A375CaCo2HCT-15HeLa [Cu(thp) 4 ][PF 6 ] 0.60± ± ± ± ± ± ± ± ±1.50 [Cu(bhpe) 2 ][PF 6 ] 47.40± ± ± ± ± ± ± ± ±1.33 thp 68.63± ± ±4.29> ± ±3.88>100 bhpe 83.72±3.23> ± ± ± ±4.01>100 KPF 6 >100 Cisplatin 15.91± ± ± ± ± ± ± ± ±1.51 A549 = lung cancer CaCo2, HCT-15 = colon cancer Hela = cervix cancer MCF-7 = breast cancer HL60 = leukemia Daudi = lymphoma HepG2 = epatoma A375 = melanoma IC 50 values represent the drug concentrations that reduced the mean absorbance at 570 nm to 50% of those in the untreated control wells.

27 Compound 2008 IC 50 [µM] C13 IC 50 [µM] R.F. [Cu(thp) 4 ][PF 6 ]1.48± ± Cisplatin12.69± ± Compound A431 IC 50 [µM] A431/Pt IC 50 [µM] R.F. [Cu(thp) 4 ][PF 6 ]14.37± ± Cisplatin22.06± ± Compound LoVo IC 50 [µM] LoVo-MDR IC 50 [µM] R.F. [Cu(thp) 4 ][PF 6 ]1.54± ± Doxorubicin1.46± ± Human ovarian adenocarcinoma cells Human cervix squamous carcinoma cells Human colon adenocarcinoma cells Cytotoxic activity of [Cu(thp) 4 ][PF 6 ] 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 IC 50 values calculated for the resistant cells and those arising from the sensitive ones.

28 Comparison of IC 50 values detected by MTT, NR and TB test after incubation of 2008 cells with [Cu(thp) 4 ][PF 6 ] 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 Cell cycle phases G1 = GAP 1 S = Synthesis (DNA replication) G2 = GAP 2 M = mitosis (nuclear and cytoplasmic division) I = Interphase Percentage of cells in different cell cycle phases as function of time exposure of [Cu(thp) 4 ][PF 6 ], vs control untreated cells 3 h 12 h 24 h 48 h 3 h 12 h 24 h 48 h untreated cells cells treated with IC 50 of [Cu(thp) 4 ][PF 6 ] 24h48h CtrComplex 3p-ValueCtrComplex 3p-Value Apoptosis % 4.24± ±0.66< ± ±0.96<0.001 G1 % 70.92± ±1.35< ± ±1.39<0.001 G2/M% 20.29± ±1.28< ± ±1.12<0.001

30 untreated cells cells treated with IC 50 of [Cu(thp) 4 ][PF 6 ] Forward scattering (index of cell size) vs side scattering (index of cell granularity) as a function of time in 2008 cells Flow cytometric profiles of 2008 cells untreated (panel A) and treated with (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. Mitochondrial energization of treated tumor cells as the retention of a mitochondrial selective cationic fluorescent probe, tetramethyl rhodamine methyl ester (TMRM). 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 Sodium acetate buffer TPA 64 Cu(II)Cl 2 THP Sodium acetate buffer Sodium acetate buffer Ligand In vitro cell experiments Cell uptake behavior of complexes 1-4 into EMT-6 mammary carcinoma cells. Error bars not seen are within symbols. (2) (1) (3)(4)

32 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 64 Cu 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 64 Cu- ATSM and 64 Cu-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 [ 64 Cu((EtOCH 2 CH 2 ) 2 PCH 2 CH 2 P(CH 2 CH 2 EtO) 2 )] + Biodistribution was carried out on 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)

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 LiAlH 4 1. n-BuLi 2. RX

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

38 64 Cu complexes Biodistribution data The retention of activity in tissues is similar to that observed with 64 Cu-cyclam and 64 Cu-monooxo-tetrazamacrocyclic complexes, but, on comparison with 64 Cu-TETA and 64 Cu-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 64 Cu is sequestered by proteins, and remaining trapped in these tissues hindering clearance.

39 Perspectives M BFCA

40 Conclusions The monooxo Re(V) core is conveniently stabilized by tripodal scorpionate ligands comprising carboxylate or sulfonate tails, giving a series of intermediate Re(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 64 Cu(I) monophosphine complexes were evaluated as a basis for a new class of copper radiopharmaceuticals. [ 64 Cu(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 64 Cu labeled to this form (N2S2) macrocyclics. Although the in vivo biodistribution of complexes suggests dissociation of the 64 Cu from the chelates, these new ligands platform offers the potential as a basis for further development to improve the in vivo stability.

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


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