School of Advanced StudiesDoctorate course in Chemical Sciences

Cycle XXScientific-Sector CHIM/03

Dr.ssa Grazia Papini

New metal complexes supported by scorpionate and macrocyclic ligands:

chemistry and biological studies

TutorProf. Giancarlo Gioia Lobbia

Well-known Scorpionate Ligands

Bis(pyrazolyl)borates Tris(pyrazolyl)borates Tetrakis(pyrazolyl)borates

“Nitrogen heterocycles other than pyrazole can be used, such as imidazole, triazole, benzotriazole, thioimidazole, ecc.”

Bis(imidazolyl)borates

Tris(imidazolyl)borates

Tetrakis(imidazolyl)borates

NN N

B

NNN

H

H NN N

NN N

B

NNN

H

N

N NB

N

NN

H

H NNN

N

N NB

N

NN

H

Bis(3-R-2-thioxo-imidazolyl)borates

Poly(triazolyl)borates Poly(benzotriazolyl)borates

N

N

N

N

B

H

H

R5

R4 R4

R5

μ2-N,N’

N

N

N

N

BR5

R4 R4

R5

N

NR4

R5

N

N

R5

R4

μ4-N4

N

N

N

N

B

H

R5

R4 R4

R5

N

NR4

R5

μ3-N,N’,N”

NN N NB

H

H

S S

R R

3-S,S’,B-H 2-S,S’

Scorpionate ligands Scorpionate ligands with EWG with EWG

substituentssubstituents

[H2B(pzCOOEt,Me)2]-

N

NB

N

N

OO

OO

H H

[H2B(tzNO2)2]-

NN

NNN

N

BHH

O2N NO2K+

[H2B(pzNO2)2]-

NN

NN

BHH

O2N NO2K+

[H2B(pzCF3)2]-

NN

NN

BHH

F3C CF3K+

[H3B(pzCF3)]-

B

H

H

H

NN

CF3

Na

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.”

“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, ”

Si

NN

NN

N N

Me

R R'

R

R'

R'

RP

N

N

NN

O

RR

NN

R

R' R'R'C

N

N

NN

R

R3R3

NN

R3

R5 R5R5

R4 R4

RC(pzx)3 RSi(pzx)3(pzx)3PO

NN

NN

CH

CO

O

bdmpza

NN

NN

CH

CS

S

bdmpzta bdmpzs

NN

NN

C HSO

OLiO

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

Bioactive moleculeLinkerM

The “metal - fragment” strategy

Stable building -block

Labile groups

Re(V) complexes

NN

NN

CH

CO

OLi

NN

NN

C HSO

OLiO

N N

NN

CO2Na

Cl

ReCl Cl

Cl

O-

Metal fragment

X Y

N

ReN Cl

Cl

O

O

biomolecule

N

ReN X

Y

O

O

S

NHNH

O

OH

O

NN

NN

Re O

Cl

Cl

MeOO

MeO [ReOCl4][NBu4]

CH3CN (Et3N)

N

N

N

N

Re

OClCl

COO

MeOH (Et3N)

[ReOCl4][NBu4]

NN

NN

ReO

ORCl

SO2ON

N

N

N

Re

OORCl

COO

N N

NN

O

OH

NN

NN

CH

CO

OLi

NN

NN

C HSO

OLiO

[ReOCl4][NBu4]

ROH(Et3N)

[ReOCl4][NBu4]

ROH (Et3N)

Metal Fragments

M. Porchia, G. Papini, C. Santini, G. Gioia Lobbia, M. Pellei, F. Tisato, G. Bandoli, A. Dolmella, Inorg. Chem. 44 (2005) 4045

Mixed coordination sphere complexes

CH2Cl2 (Et3N)

[ReOCl4][NBu4]

NN

NNE

LiO

HO(CH2)nOH

E= CO,SO2

n= 2, 3

N

N

N

N

Re

O OO

EO

(CH2)n

N

N

N

N

Re

OOMeCl

EO

N

N

N

N

Re

OClCl

EO

N

N

N

N

Re

OOEtCl

EO

Et3N

OH(CH2)nOH

Structure 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

N

N

N

N

Re

OOMeCl

COO

N

N

N

N

Re

OClCl

COO

N

N

N

N

Re

OOEtCl

COO

Et3N

HOOC(CH2)COOH

N

N

N

N

Re

OOO

COO

O O

N N

N NCO2LiCH2Cl2 (Et3N)

[ReOCl4][NBu4]

HOOC(CH2)COOH

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.

Potential Nitridorhenium complexes

N

N

N

N

N

N

B

H

RR

R

N

N

N

N

NN

N

NN

B

H

RR

R

:

:

:

N

N

N

NB

H H

R R

N

N

N

N

NN

: :

BH H

R R

ClRe

Cl Cl

Cl

N

Nitridorhenium precursors

Re H2O2

H2ORe2O7 ReCl3PPh3

PPh3, [PhNHNH2]HClHCl PPh3

EtOH

Re H2O2

H2ORe2O7 [NBu4][ReO4] HCl g

NaN3

[NBu4]OH

Cl

ReCl Cl

Cl

N

Cl

RePh3P Cl

PPh3

N

Pre-carbene ligands

2 BzBr

X

N

N

N

XN

B

H H

Bz Bz

Br-

KBH4 +

XN

NH

2135°C X

N

N

N

XN

B

H H

K+

KBH4 +

XN

NH

3170°C X

N

N

N

XN

N

XN

B

H

K+X

N

N

N

XN

N

XN

B

BzBz

Bz

H 2

2Br-3 BzBr

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

R2

OO

R1

+ H

O

H

+ R3 NH2

NH4Ac

N

NR1

R2

R3

MeOH/H2O

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

H

OO

H

+ H

O

H 2) Mes-NH2, NH4AcN

N

Mes

1) CH3COOH, 75°C

H

OO

H

+ tBu-NH2

2) HCOH, NH3AcN

N

t-Bu

1) MeOH/H2O, 70°C

N

N

24h

N

N

N

N

N

N

B

H

Mes

Mes Mes

+ (CH3)2S.HBBr2

CH2Cl22Br -

N

N24h

N

N

N

N

N

N

B

H

t-Bu

t-Bu t-Bu

+ (CH3)2S.HBBr2

CH2Cl22Br -

X

N

N

N

XN

B

H H

Bz Bz

Li +

THF, n-BuLi

- 78°C

X

N

N

N

XN

B

H H

Bz Bz

Br-

N

N

N

N

N

N

B

RR

R

:

:

:

H

Li +THF, n-BuLi

- 78°CN

N

N

N

N

N

B

RR

R

H 2

2Br-

[NBu4][ReNCl4] MIXTURE OF UNCHARACTERIZABLE

PRODUCTS

R = Bz, t-Bu, Mes

Silver(I) carbene complexes

CH2Cl2, 24h

N

N

N

N

N

N

B

H

R

R R

Ag2O

NN

NN

B

H

R R

NN

NN

B

H

RR

AgAg

NN

R

NN

R

Ag Br

R = Bz, t-Bu, Mes

2Br-

N

N

N

N

NN

BH H

Br

Bz Bz

Ag2O

CH2Cl2

N

N

N

N

NN

BH

H

Bz

Bz

N

N

N

N

NN

BH

H

Bz

Bz

Ag

Ag

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

Carbene transfer reactions

CH2Cl2, 2h

Au(SMe2)Cl

NN

NN

B

H

R R

NN

NN

B

H

RR

AgAg

NN

R

NN

R

Ag Br

R = Bz, t-Bu, Mes

NN

NN

B

H

R R

NN

NN

B

H

RR

AuAu

NN

R

NN

R

Au Br

N

N

N

N

NN

BH

H

Bz

Bz

N

N

N

N

NN

BH

H

Bz

Bz

Ag

Ag

CH2Cl2, 2h

Au(SMe2)Cl

N

N

N

N

NN

BH

H

Bz

Bz

N

N

N

N

NN

BH

H

Bz

Bz

Au

Au

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

Rhenium derivatives

NBu4ReNCl4

N N

N

NN

N

B

H

H

R

RNN

N

N N

N

B

H

H

R

R

Re

N

N N

N

NN

N

B

H

H

R

R

Re

NCl

ClN

N

N

N

NN

BH

H

Bz

Bz

N

N

N

N

NN

BH

H

Bz

Bz

Ag

AgCH2Cl2

B

N

N

N

NN

H

N

R

R

R

Re

Cl

N

NBu4ReNCl4N

NN

N

B

H

R R

NN

NN

B

H

RR

AgAg

NN

R

NN

R

Ag Br CH2Cl2

Copper and Ruthenium derivatives

NN

NN

B

H

R R

NN

NN

B

H

RR

AgAg

NN

R

NN

R

Ag Br

N

N

N

N

NN

BH

H

Bz

Bz

N

N

N

N

NN

BH

H

Bz

Bz

Ag

Ag

N N

N

NN

N

B

H

H

Bz

Bz

Ru

Cl [Ru(p-cymene)Cl2 ]2CH2Cl2

N

N

N

N

BH

Bz

Bz

N

N

Bz

RuCl

[Ru(p-cymene)Cl 2] 2

CH 2Cl 2

N

N

N

N

NN

BH

H

Bz

Bz

N

N

N

N

NN

BH

H

Bz

Bz

Cu

Cu

Cu(SMe 2)Br

CH 3CN

NN

NN

B

H

R R

NN

NN

B

H

RR

CuCu

NN

R

NN

R

Cu Br

Cu(SMe2 )BrCH

3CN

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 moleculesfor 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

Copper(I) derivatives

CuP P

HO OHHO

HO OH

OH

NCCH3CH3CN

PF6

HO P

OH

OH

P(CH2OH)3

Cells line of ovarian carcinoma (2008) and cis-platino resistent carcinoma cells (C13)

NN N

N

Cu

P PHO

HO

HO

OHOH

OH

C=OO

O2N NO2

H

NNN N

NN

Cu

P PHO

HO

HO

OHOH

OH

C=OO

O2N NO2

H

NNN N

NN

Cu

P PHO

HO

HO

OHOH

OH

C=OO

H

NN N

N

Cu

P PHO

HO

HO

OHOH

OH

C=OO

H

NN N

N

Cu

P PHO

HO

HO

OHOH

OH

C=SS

H

C. Marzano, M. Pellei, D. Colavito, S. Alidori, G. Gioia Lobbia, V. Gandin, F. Tisato, and C. Santini, J. Med. Chem., 4949 (2006) 7317

“CuP4” tipe species

[Cu(CH3CN)4][PF6] + 4 thp [Cu(thp)4][PF6]

[Cu(CH3CN)4][PF6] + 2 bhpe [Cu(bhpe)2][PF6]

P POH

OH

HO

HO

P POH

OH

HO

HOCu

HO OH

OHHO

[Cu(thp)4][PF6]

P POH

OH

HO

HO

P POH

OH

HO

HOCu

[Cu(bhpe)2][PF6]

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)

31P-NMR = + 9.67 (dbr), - 144.05 (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) 798-808.

bhpe

PO

O O

Br

Br+

P

P

OO O

OOO

+ P

Br

OO O

P OO O

+

H2P PH2 PPHO

OH

HO

OHHO

OH

P PHO

HO

OH

OH

180°C

LiAlH4

HCOH, HCl

EtOH

NaHCO3

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.

Citotoxic activities

Compound

IC50 (µM) ± S.D.

HL60 A549 MCF-7 Daudi HepG2 A375 CaCo2 HCT-15 HeLa

[Cu(thp)4][PF6] 0.60±0.02 9.11±2.71 11.08±0.52 6.94±0.18 1.26±0.10 4.58±2.41 1.08±0.12 2.00±0.03 8.21±1.50

[Cu(bhpe)2][PF6] 47.40±2.92 57.60±2.19 49.71±2.03 65.5±1.22 78.23±1.11 68.21±1.23 52.50±0.81 57.36±1.31 62.41±1.33

thp 68.63±2.44 72.91±2.44 64.23±4.29 >100 98.71±3.63 88.70±3.88 >100 >100 >100

bhpe 83.72±3.23 >100 >100 >100 71.71±1.64 73.21±1.22 84.11±2.22 91.71±4.01 >100

KPF6 >100 >100 >100 >100 >100 >100 >100 >100 >100

Cisplatin 15.91±1.51 29.21±1.92 19.04±1.51 23.97±2.51 21.50±1.41 20.33±1.33 35.42±1.40 25.34±1.31 10.50±1.51

A549 = lung cancer CaCo2, HCT-15 = colon cancer Hela = cervix cancerMCF-7 = breast cancerHL60 = leukemiaDaudi = lymphomaHepG2 = epatomaA375 = melanoma

IC50 values represent the drug concentrations that reduced the mean absorbance at 570 nm to 50% of those in the untreated control wells.

Compound2008

IC50 [µM]C13

IC50 [µM] R.F.

[Cu(thp)4][PF6] 1.48±0.21 2.88±1.07 1.9

Cisplatin 12.69±1.72 89.18±4.50 7.02

CompoundA431

IC50 [µM]A431/Pt

IC50 [µM] R.F.

[Cu(thp)4][PF6] 14.37±1.41 13.26±0.80 0.92

Cisplatin 22.06±1.62 57.76±1.81 2.61

CompoundLoVo

IC50 [µM]LoVo-MDRIC50 [µM] R.F.

[Cu(thp)4][PF6] 1.54±0.03 2.9±0.1 1.88

Doxorubicin 1.46±2.30 44.89±0.90 30.74

Human ovarian adenocarcinoma cells

Human cervix squamous carcinoma cells

Human colon 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.

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.

Cell cycle phases

G1 = GAP 1S = Synthesis (DNA replication)G2 = GAP 2M = mitosis (nuclear and cytoplasmic division)I = Interphase

Percentage of cells in different cell cycle phases as function of time exposure of

[Cu(thp)4][PF6], vs control untreated cells

3 h

12 h

24 h

48 h

3 h

12 h

24 h

48 h

----------2008 untreated cells----------2008 cells treated with IC50 of [Cu(thp)4][PF6] 24h 48h

Ctr Complex 3 p-Value Ctr Complex 3 p-Value

Apoptosis % 4.24±0.71 2.43±0.66 <0.001 1.21±0.73 15.39±0.96 <0.001

G1 % 70.92±1.82 60.9±1.35 <0.001 65.57±1.21 41.03±1.39 <0.001

G2/M% 20.29±1.11 33.5±1.28 <0.001 32.62±1.46 37.03±1.12 <0.001

----------2008 untreated cells----------2008 cells treated with IC50 of [Cu(thp)4][PF6]

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 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.

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.

Potential Cu(I) radiopharmaceuticals

0.0

2.0

4.0

6.0

8.0

10.0

0 20 40 60 80 100 120

% C

ell A

ssoc

iate

d R

adio

activ

ity

Time (min)

13

4

2

Sodium acetatebuffer

TPA64Cu(II)Cl2

64Cu

P

HO

HOOH

P

OH

OHHO

P PHO

OH OH HOHOOH

NNP

NNN

PN

NN

PN NN

P

N

64Cu

NN

NN

64Cu

P

CO2

HO

HOOH

P

OH

OHHO

NN

NNN

N

64Cu

P

CO2

HO

HOOH

P

OH

OHHO

THPSodium acetatebuffer

Sodium acetatebuffer

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)

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 themonocationic 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)]+

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

blood lung liver(all) spleen kidney muscle heart brain bone tumor

%ID

/g

Organ

1 hour

4 hour

24 hour

64Cu

P

HO

HOOH

P

OH

OHHO

P PHO

OH OH HOHOOH

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.

axial

coronal

1 h 2 h 24 h

ScaleOff-ScaleHigh

Low

ScaleOff-ScaleHigh

Low

0

1

2

3

4

5

0 5 10 15 20 25

SU

V

Time (h)

TumorMuscleLiverKidneyHeart

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

New N-, P- donor ligands

N N

P

P

P

P

O

O

O

O

O

O

O

O

O

O

O

O

NH

NH

P P

O OO

O

O

O

NN

N N

P

OO

O

CH3

CH3 P

O O

O

CH3

CH3

P

O

O

O

CH3

CH3P

O

O

OCH3

CH3

NN

P

OO

O

CH3

CH3 P

O O

O

CH3

CH3

N N

P

O

O

O

CH3

CH3P

O

O

OCH3

CH3

H2NNH2

P

OO

O

P

OO

O Br

PO

O

O

PO

O

O Br

Cyclam

Cyclen

NN

P

OO

O

CH3

CH3 P

O O

O

CH3

CH3

N N

P

O

O

O

CH3

CH3P

O

O

OCH3

CH3

NN

P

P

N N

P

P

OHOH

OH

OH

OH

OH

OH

OH

1. HCHO, HCl, EtOH

2. NaHCO3

NN

PH2

PH2

N N

PH2

PH2

LiAlH4

1. n-BuLi

2. RX

N

NN

N

P

PRP

P

R

R

R

R

RR

R

New macrociclic ligands

N H 2HO O C

SH

H CH O

H 2O

HNH O O C

S

1) Na/NH3

2) NH4Cl, pH = 2

NHHOOC

SH

HN COOH

HS

N H 2HO O C

SH

H CH O

H 2O

HNH O O C

S

1) Na/NH3

2) NH4Cl, pH = 2

NHHOOC

SH

HN COOH

HS

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

NH

HOOC

SH

HN

COOH

HS

1) NaHCO3

N+HOOC

S

N+COOH

S

+2) HCl, pH = 2

(CH2)3BrBr

13-membered NEC-SP

N+HOOC

S

N+COOH

S

12-membered NEC-SE

NH

HOOC

SH

HN

COOH

HS

1) NaHCO3+

2) HCl, pH = 2(CH2)2

BrBr

NH

HOOC

SH

HN

COOH

HS

1) MeOH/H2O/NaOH+

2) HCl, pH = 2(CH2)4

BrBr

14-membered NEC-SB

N+HOOC

S

N+COOH

S

H

H

H

H

H

HH

H

H

H

H

H

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

Cu(CH3COO)2

H2O

Cu(CH3COO)2

H2O

Cu(CH3COO)2

H2O

N+HOOC

S

N+COOH

S

13-membered NEC-SP

N+HOOC

S

N+COOH

S

12-membered NEC-SE

14-membered NEC-SB

H

H

H

H

H

HH

H

Cu(NEC-SP)

Cu(NEC-SE)

Cu(NEC-SB)

S S

NHNH

CuOO O O

S S

NHNH

CuOO O O

S S

NHNH

CuOO O O

N+HOOC

S

N+COOH

S

H

H

H

H

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.

64Cu(II)Cl2

Sodium acetate buffer

Macrociclic ligands

N+HOOC

S

N+COOH

S

H

HH

H

64Cu-Complexes

S S

NHNH

64CuOO O O

0

2

4

6

8

10

12

14

16

18

7 8 9 Cu-64-Cyclam Cu-64-TETA Cu-64-DOTA Cu-CB-TE2A

%ID

/org

an

Complex

Blood

Liver

Kidney

Perspectives

1) NaOH, pH = 5.5

2) sulfo-NHS, EDC

NHHOOC

S

HN COOH

S

NH

S

HN

S

O

ON

O

O

SO3-

O

ON

O

O

SO3-

1) pH = 7.5

2) BM-NH2 . HCl

NH

S

HN

S

OO

OH

HO

H

HO

H

OHNHH

H

HOO H

OH

H

OH

H

HOHN H

H

OH

CuAcH2O

N

S

N

S

OO

OH

HO

H

HO

H

OHNHH

H

HOO H

OH

H

OH

H

HOHN H

H

OH

Cu

M

BFCA

NHHOOC

SH

HN COOH

HS

NHHOOC

S

HN COOH

S

X

XCl Cl

NHHOOC

S

HN COOH

S

NHROOC

S

HN COOR

S

ROH, HCl

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.

Partners and AcknowledgementsPartners and Acknowledgements

Prof. Giuliano BandoliProf. Alessandro Dolmella

Dr.ssa Cristina MarzanoDip. di Scienze Farmaceutiche

Università di Padova

Dr. Franco BenetolloICIS-CNR, Padova

Prof. Rasika Dias Department of Chemistry and BiochemistryThe University of Texas at Arlington (USA)

Dr. Francesco Tisato Dr.ssa Marina PorchiaDr. Fiorenzo Refosco, Dr.ssa Cristina BolzatiICIS-CNR, Padova

Prof. Giancarlo Gioia LobbiaProf. Carlo Santini

Dr.ssa Maura PelleiDr. Simone Alidori

Prof. Jason S. LewisCarolyn J. Anderson