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 [Tc-99m(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 [Tc-99m(H2O)(3)(CO)(3)](+) (1a). These reactions yielded the radioactive building blocks [Tc-99m(CO)(3)(k(3)-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-Me-2-pz(CH2)(2)N((CH2)(3)COOH)(CH2)(2)NH2 (L-6) and pz(CH2)(2)N((CH2)(3)COOH)(CH2)(2)NH2 (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)(k(3)-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)(kappa(3)-3,5-Me-2-pz(CH2)(2)N(glygly)(CH2)(2)NH2)](+) (9/9a) and fac-[Re(CO)(3)(kappa(3)-pz(CH2)(2)N(CH2)(3)(glygly)(CH2)(2)NH2)](+) (10/10a) (M = Re, Tc-99m). 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.