Diapositiva 1

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.
Tris(imidazolyl)borates
Tetrakis(imidazolyl)borates
Bis(imidazolyl)borates
Poly(triazolyl)borates
Poly(benzotriazolyl)borates
Bis(3-R-2-thioxo-imidazolyl)borates
4
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
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
  coldmaterial (the natural isotopic mixture of
  185Re and 187Re) can be advantageously
  manipulated instead of the radioactive nuclide
  99gTc (t1/2 2.12 105 y, Eß 292 keV).
• Rhenium has two ß- emitters isotopes 186Re (ß-max
  1.07 MeV t1/2 90 h) and 188Re (ß-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
The metal - fragment strategy
M
Stable building -block
8
Re(V) complexes
Metal fragment
9
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
11
Marina Porchia,Grazia Papini, Carlo Santini,
Giancarlo Gioia Lobbia, Maura Pellei, Francesco
Tisato, Giuliano Bandoli, Alessandro Dolmella
Inorganica Chimica Acta 359 (2006) 25012508.
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) 26612666.
16
(No Transcript)
17
MIXTURE OF UNCHARACTERIZABLE PRODUCTS
NBu4ReNCl4
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
Ru(p-cymene)Cl22
21
Copper and Ruthenium derivatives
Ru(p-cymene)Cl22
Ru(p-cymene)Cl22
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

isotope half-life imaging (emission, energy, abundance) therapy (emission, energy, range in tissue) application
Cu-60 20 min PET (b, 873 keV, 93) Radiolabelling small molecules
Cu-61 3.3 h PET (b, 527 keV, 62) Radiolabelling small molecules
Cu-62 9.7 min PET (b, 1315 keV, 98) Radiolabelling small molecules
Cu-64 12.7 h PET (b, 278 keV, 19) Radiolabelling small molecules, peptides and antibodies
Cu-66 5.4 min (b-, 190 keV 0.95 mm) Radiolabelling small molecules for therapy
Cu-67 62.0 h SPECT (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
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
CuP4 tipe species
Cu(CH3CN)4PF6 4 thp ? Cu(thp)4PF6
Cu(CH3CN)4PF6 2 bhpe ?
Cu(bhpe)2PF6
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
Compound IC50 (µM) S.D. IC50 (µM) S.D. IC50 (µM) S.D. IC50 (µM) S.D. IC50 (µM) S.D. IC50 (µM) S.D. IC50 (µM) S.D. IC50 (µM) S.D. IC50 (µM) S.D.
Compound HL60 A549 MCF-7 Daudi HepG2 A375 CaCo2 HCT-15 HeLa
Cu(thp)4PF6 0.600.02 9.112.71 11.080.52 6.940.18 1.260.10 4.582.41 1.080.12 2.000.03 8.211.50
Cu(bhpe)2PF6 47.402.92 57.602.19 49.712.03 65.51.22 78.231.11 68.211.23 52.500.81 57.361.31 62.411.33
thp 68.632.44 72.912.44 64.234.29 gt100 98.713.63 88.703.88 gt100 gt100 gt100
bhpe 83.723.23 gt100 gt100 gt100 71.711.64 73.211.22 84.112.22 91.714.01 gt100
KPF6 gt100 gt100 gt100 gt100 gt100 gt100 gt100 gt100 gt100
Cisplatin 15.911.51 29.211.92 19.041.51 23.972.51 21.501.41 20.331.33 35.421.40 25.341.31 10.501.51
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)4PF6 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-resistan
ce 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.
Compound 2008 IC50 µM C13 IC50 µM R.F.
Cu(thp)4PF6 1.480.21 2.881.07 1.9
Cisplatin 12.691.72 89.184.50 7.02
Human cervix squamous carcinoma cells
Compound A431 IC50 µM A431/Pt IC50 µM R.F.
Cu(thp)4PF6 14.371.41 13.260.80 0.92
Cisplatin 22.061.62 57.761.81 2.61
Human colon adenocarcinoma cells
Compound LoVo IC50 µM LoVo-MDR IC50 µM R.F.
Cu(thp)4PF6 1.540.03 2.90.1 1.88
Doxorubicin 1.462.30 44.890.90 30.74
28
Comparison of IC50 values detected by MTT, NR and
TB test after incubation of 2008 cells with
Cu(thp)4PF6 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
----------2008 untreated cells ----------2008
cells treated with IC50 of Cu(thp)4PF6
24h 24h 24h 48h 48h 48h
Ctr Complex 3 p-Value Ctr Complex 3 p-Value
Apoptosis 4.240.71 2.430.66 lt0.001 1.210.73 15.390.96 lt0.001
G1 70.921.82 60.91.35 lt0.001 65.571.21 41.031.39 lt0.001
G2/M 20.291.11 33.51.28 lt0.001 32.621.46 37.031.12 lt0.001
Percentage of cells in different cell cycle
phases as function of time exposure of
Cu(thp)4PF6, 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)4PF6
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 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
1. n-BuLi 2. RX
36
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. 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
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
Perspectives
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 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.

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)