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Table 1. Analytical Data for Homobivalent Derivatives 3 and 4Compound Formula Calculated [ion] Found t R (purity)3 C 119 H 170 N 36 O 24 1244.6 [M+2H] 2+ 1244.6 15 min (98%)4 C 122 H 170 N 36 O 27 Re 920.6 [M+3H] 3+ 920.6 15.5 min (98%)The binding affinity of 3 (IC 50 = 0.33 ± 0.27 nM) to MC1R is very high and of the sameorder as NAPamide (IC 50 = 0.78 ± 0.03 nM) and the monovalent ligand pz-NAPamide(IC 50 = 0.66 ± 0.13 nM) prepared previously [3]. The metallation of 3 led to a complex 4with slightly lower affinity (IC 50 = 1.43 ± 1.54 nM).The radioactive complex 4a was prepared in high yield and radiochemical purity(98%) by reacting 3 with [ 99m Tc(CO) 3 (H 2 O) 3 ] + (Scheme 1). The highly hydrophiliccomplex 4a (log P o/w = - 1.82 ± 0.02) was characterized/identified comparing its RP-HPLCchromatogram with the one obtained for 4, synthesized as surrogate (Figure 1).ESI-MS[M+5H] 5+552.8[M+4H] 4+690.74a4[M+3H] 3+920.6Fig. 1. HPLC chromatograms of 4a (γ - detection)/ 4 (UV-vis detection) and ESI-MSspectrum of 4.In vitro studies have shown that 4a is stable in fresh human serum (6h, 37°C).Internalization studies in B16F1 murine melanoma cells revealed that over 45% of thecell-associated activity was internalized after 4 h, at 37°C.In conclusion, we have synthesized and characterized the novel homobivalentfac-[M(CO) 3 ] + complexes 4/4a (M = Re/ 99m Tc) containing the NAPamide. Preliminarystudies have shown that the radioactive complex is enzymatically stable, displayed highaffinity to MC1R and high cellular uptake. The biological evaluation of 4a in tumourbearingmice is currently underway.AcknowledgmentsM. Morais thanks FCT for a PhD grant (SFRH/BD/48066/2008). Covidean is acknowledged for theIsoLink ® Kits. Dr. J. Marçalo is acknowledged for performing the ESI-MS analyses. The QITMSinstrument was acquired with the support of the Programa Nacional de Reequipamento Científico(Contract REDE/1503/REM/2005 - ITN).References1. Miao, Y., Quinn, T. Crit. Rev. Oncol/Hematol. 67, 213-228 (2008).2. Raposinho, P.D., Correia, J.D.G., Cristina, M.C., Santos, I. Biopolymers: Peptide Science (2010).3. Raposinho, P.D., Correia, J.D.G., Alves, S., Botelho, M.F., Santos, A.C., Santos, I. Nucl. Med. Biol.35, 91-99 (2008).4. Vagner, J., Handl, H.L., Gillies, R., Hruby, V.J. Bioorg. Med. Chem. Lett. 14, 211-215 (2004).531