Presentation on theme: "New metal complexes supported by scorpionate and macrocyclic ligands: chemistry and biological studies Dr.ssa Grazia Papini School of Advanced Studies."— Presentation transcript:
1New metal complexes supported by scorpionate and macrocyclic ligands: chemistry and biological studiesDr.ssa Grazia PapiniSchool of Advanced StudiesDoctorate course in Chemical SciencesCycle XXScientific-Sector CHIM/03TutorProf. Giancarlo Gioia Lobbia1
3“Nitrogen heterocycles other than pyrazole can be used, such as imidazole, triazole, benzotriazole, thioimidazole, ecc.”μ3-N,N’,N”μ2-N,N’μ4-N4Tris(imidazolyl)boratesBis(imidazolyl)boratesTetrakis(imidazolyl)borates3-S,S’,B-H2-S,S’Poly(triazolyl)boratesPoly(benzotriazolyl)boratesBis(3-R-2-thioxo-imidazolyl)borates3
4Scorpionate ligands with EWG substituents “Other modifications include changing the substituents on the heterocyclic ring.”[H2B(pzCOOEt,Me)2]-[H3B(pzCF3)]-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[H2B(tzNO2)2]-[H2B(pzNO2)2]-[H2B(pzCF3)2]-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“In addition, tripodal ligands can have central atoms other than boron, such as carbon, phosphorus, or silicon….”RC(pzx)3RSi(pzx)3(pzx)3PO“…..and bearing a coordinating moiety (R') such as acetate, dithioacetate, sulfonate, ethoxide, ”bdmpzabdmpztabdmpzs5
6Rhenium complexesVersatile chemistry: several oxidation states accessible (from -I to VII); different coordination numbers (from 4 to 8); various donor set availableThe 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
7The “metal - fragment” strategy BioactivemoleculeLinkerLabile groupsMStable building -block
9Metal FragmentsROH(Et3N)ROH (Et3N)NReOClNReOClS2M. Porchia, G. Papini, C. Santini, G. Gioia Lobbia, M. Pellei, F. Tisato, G. Bandoli, A. Dolmella, Inorg. Chem. 44 (2005) 4045
10Mixed coordination sphere complexes E= CO,SO2n= 2, 3Structure of the complex[Re(O)(bdmpza)(OCH2CH2O)]Structure of the complex [Re(O)(bdmpza)(OCH2CH2CH2O)]M. Porchia, G. Papini, C. Santini, G. Gioia Lobbia, M. Pellei, F. Tisato, G. Bandoli, A. Dolmella, Inorg. Chem. 44 (2005) 4045
11Structure 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.
21[Ru(p-cymene)Cl2]2 [Ru(p-cymene)Cl2]2 Copper and Ruthenium derivatives Cu(SMe2)BrCH3CN[Ru(p-cymene)Cl2]2CH2Cl2[Ru(p-cymene)Cl2]2CH2Cl2Cu(SMe2)BrCH3CN21[Ru(p-cymene)Cl2]2[Ru(p-cymene)Cl2]2
22Fichna et al, Bioconjugate Chem., 14 (2003) 3-17 Copper derivativesIt is an essential trace metal for living organismsCopper complexes’ activity is extremely wideCopper has a well-documented coordination chemistrySeveral radioactive copper isotopes are available nowadays for biomedical purposes both for radioimaging and targeted radiotherapyisotopehalf-lifeimaging(emission, energy, abundance)therapy(emission, energy, range in tissue)applicationCu-6020 minPET(b+, 873 keV, 93%)Radiolabelling small moleculesCu-613.3 h(b+, 527 keV, 62%)Cu-629.7 min(b+, 1315 keV, 98%)Cu-6412.7 h(b+, 278 keV, 19%)Radiolabelling small molecules, peptides and antibodiesCu-665.4 min(b-, 190 keV; 0.95 mm)for therapyCu-6762.0 hSPECT(g, 185 keV, 48%)Fichna et al, Bioconjugate Chem., 14 (2003) 3-1722
23Copper(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) 7317P(CH2OH)3Cells line of ovarian carcinoma (2008) and cis-platino resistent carcinoma cells (C13)23
2431P-NMR = + 9.67 (dbr), - 144.05 (septet) “CuP4” tipe species[Cu(CH3CN)4][PF6] + 4 thp [Cu(thp)4][PF6][Cu(CH3CN)4][PF6] + 2 bhpe [Cu(bhpe)2][PF6][Cu(thp)4][PF6][Cu(bhpe)2][PF6]31P-NMR = (dbr), (septet)[Cu(bhpe)2]+ m/z = 492 (100)31P-NMR = (q), (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)24
25bhpeC. Marzano, V. Gandin, M. Pellei, D. Colavito, G. Papini, G. Gioia Lobbia, M. Porchia, F. Tisato and C. Santini,J. Med. Chem. 1 (2008)25
26Citotoxic activitiesCompoundIC50 (µM) ± S.D.HL60A549MCF-7DaudiHepG2A375CaCo2HCT-15HeLa[Cu(thp)4][PF6]0.60±0.029.11±2.7111.08±0.526.94±0.181.26±0.104.58±2.411.08±0.122.00±0.038.21±1.50[Cu(bhpe)2][PF6]47.40±2.9257.60±2.1949.71±2.0365.5±1.2278.23±1.1168.21±1.2352.50±0.8157.36±1.3162.41±1.33thp68.63±2.4472.91±2.4464.23±4.29>10098.71±3.6388.70±3.88bhpe83.72±3.2371.71±1.6473.21±1.2284.11±2.2291.71±4.01KPF6Cisplatin15.91±1.5129.21±1.9219.04±1.5123.97±2.5121.50±1.4120.33±1.3335.42±1.4025.34±1.3110.50±1.51A549 = lung cancerCaCo2, HCT-15 = colon cancerHela = cervix cancerMCF-7 = breast cancerHL60 = leukemiaDaudi = lymphomaHepG2 = epatomaA375 = melanomaIC50 values represent the drug concentrations that reduced the mean absorbance at 570 nm to 50% of those in the untreated control wells.26
27Human cervix squamous carcinoma cells Human colon adenocarcinoma cells Human ovarian adenocarcinoma cellsCytotoxic 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.Compound2008IC50 [µM]C13R.F.[Cu(thp)4][PF6]1.48±0.212.88±1.071.9Cisplatin12.69±1.7289.18±4.507.02Human cervix squamous carcinoma cellsCompoundA431IC50 [µM]A431/PtR.F.[Cu(thp)4][PF6]14.37±1.4113.26±0.800.92Cisplatin22.06±1.6257.76±1.812.61Human colon adenocarcinoma cellsCompoundLoVoIC50 [µM]LoVo-MDRR.F.[Cu(thp)4][PF6]1.54±0.032.9±0.11.88Doxorubicin1.46±2.3044.89±0.9030.74
28Comparison of IC50 values detected by MTT, NR and TB test after incubation of 2008 cells with [Cu(thp)4][PF6] for different exposure timesTB test reveals damage to cell membraneMTT test mainly reflects damage to mitochondriaThe NR assay indicates damage to lysosomes and Golgi apparatusLysosomes/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.
293 h12 h24 h48 hCell cycle phasesG1 = GAP 1S = Synthesis (DNA replication)G2 = GAP 2M = mitosis (nuclear and cytoplasmic division)I = Interphaseuntreated cellscells treated with IC50 of [Cu(thp)4][PF6]24h48hCtrComplex 3p-ValueApoptosis %4.24±0.712.43±0.66<0.0011.21±0.7315.39±0.96G1 %70.92±1.8260.9±1.3565.57±1.2141.03±1.39G2/M%20.29±1.1133.5±1.2832.62±1.4637.03±1.12Percentage of cells in different cell cycle phases as function of time exposure of [Cu(thp)4][PF6], vs control untreated cells
30Forward scattering (index of cell size) vs side scattering (index of cell granularity) as a function of time in 2008 cellsMitochondrial 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 (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.untreated cellscells 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.
31Potential Cu(I) radiopharmaceuticals TPA64Cu(II)Cl2Sodium acetatebufferSodium acetatebufferTHP(2)In vitro cell experiments(1)Sodium acetatebufferLigandCell uptake behavior of complexes 1-4 into EMT-6 mammary carcinoma cells. Error bars not seen are within symbols.(3)(4)31
32Biodistribution Studies 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. SantiniJournal of Biological Inorganic Chemistry, 13 (2008)The uptake and retention of activity was high in many non-target tissues lung and liverPoor 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 themonocationic nature of the complexTumor 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 1Tumor uptake of complex 1 is significantly higher than that for [64Cu((EtOCH2CH2)2PCH2CH2P(CH2CH2EtO)2)]+32
33Small 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 pointsStandard 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
36New macrociclic ligands 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) submittedP. Blondeau, C. Berse, D. Gravel, Can. J. Chem. 45 (1967) 49.36
37Copper(II) complexesG. 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) submitted37
3864Cu 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 with64Cu-TETA and 64Cu-DOTA, the uptakeand 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.38
40ConclusionsThe 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’ 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 development to improve the in vivo stability.
41Prof. Alessandro Dolmella Dr.ssa Cristina Marzano Partners and AcknowledgementsProf. Giancarlo Gioia LobbiaProf. Carlo SantiniDr.ssa Maura PelleiDr. Simone AlidoriProf. Jason S. LewisCarolyn J. AndersonDr. Franco BenetolloICIS-CNR, PadovaProf. Giuliano BandoliProf. Alessandro DolmellaDr.ssa Cristina MarzanoDip. di Scienze FarmaceuticheUniversità di PadovaDr. Francesco TisatoDr.ssa Marina PorchiaDr. Fiorenzo Refosco,Dr.ssa Cristina BolzatiICIS-CNR, PadovaProf. Rasika DiasDepartment of Chemistry and BiochemistryThe University of Texas at Arlington (USA)41