Pyrazolyl Derivatives as Bifunctional Chelators for Labeling Tumor-Seeking Peptides with the fac-[M(CO)3]+ Moiety (M = 99mTc, Re):  Synthesis, Characterization, and Biological Behavior

Radiolabeling of biologically active molecules with the [99mTc(CO)3]+ unit has been of primary interest in recent years. With this in mind, we herein report symmetric ( L 1 ) and asymmetric ( L 2 − L 5 ) pyrazolyl-containing chelators that have been evaluated in radiochemical reactions with the synthon [99mTc(H2O)3(CO)3]+ (1a). These reactions yielded the radioactive building blocks [99mTc(CO)3(k3-L)]+ (L = L 1 − L 5 , 2a−6a), which were identified by RP-HPLC. The corresponding Re surrogates (2 − 6) allowed for macroscopic identification of the radiochemical conjugates. Complexes 2a−6a, with log P o/w values ranging from −2.35 to 0.87, were obtained in yields of ≥90% using ligand concentrations in the 10-5-10-4 M range. Challenge studies with cysteine and histidine revealed high stability for all of these radioactive complexes, and biodistribution studies in mice indicated a fast rate of blood clearance and high rate of total radioactivity excretion, occurring primarily through the renal-urinary pathway. Based on the framework of the asymmetric chelators, the novel bifunctional ligands 3,5-Me2-pz(CH2)2N((CH2)3COOH)(CH2)2NH2 ( L 6 ) and pz(CH2)2N((CH2)3COOH)(CH2)2NH2 ( L 7 ) have been synthesized and their coordination chemistry toward (NEt4)2[ReBr3(CO)3] (1) has been explored. The resulting complexes, fac-[Re(CO)3(k3-L)]Br ( L 6 (7), L 7 (8)), contain tridentate ancillary ligands that are coordinated to the metal center through the pyrazolyl and amine nitrogen atoms, as observed for the other related building blocks. L 6 and L 7 were coupled to a glycylglycine ethyl ester dipeptide, and the resulting functionalized ligands were used to prepare the model complexes fac-[Re(CO)3(κ3-3,5-Me2-pz(CH2)2N(glygly)(CH2)2NH2)]+ (9/9a) and fac-[Re(CO)3(κ3-pz(CH2)2N(CH2)3(glygly)(CH2)2NH2)]+ (10/10a) (M = Re, 99mTc). These small conjugates have been fully characterized and are reported herein. On the basis of the in vitro/in vivo behavior of the model complexes (2a−6a, 9a, 10a), we chose to evaluate the in vitro/in vivo biological behavior of a new tumor-seeking Bombesin pyrazolyl conjugate, [( L 6 )-G-G-G-Q-W-A-V-G-H-L-M-NH2], that has been labeled with the [99mTc(CO)3]+ metal fragment. Stability, in vitro cell binding assays, and pharmacokinetics studies in normal mice are reported herein.