Synthesis and SAR of novel ^99mTc/Re(CO)₃ labeled benzenesulfonamide (BzSA) conjugates targeting carbonic anhydrase IX (CA IX)

Objectives CA IX is a marker of tumor hypoxia and is constitutively expressed in clear cell renal carcinoma (RC). To develop radiopharmaceuticals for molecular imaging of hypoxic tumors and RC, a series of BzSA based CA-IX inhibitors containing a variety of novel tridentate chelates for complexing the M(CO)₃ core (M = Re or ^99mTc), were synthesized and evaluated in hypoxic CA IX expressing HeLa cells.

Methods CA IX inhibitors were synthesized starting with a BzSA moiety tethered through a linker to pyridylmethylamine, functionalized bis-imidazolylmethylamine or functionalized bis-triazolylmethylamine derived chelates and evaluated for binding to hypoxic HeLa cells.

Results BzSA Re(CO)₃ complexes derived from 4-(2-Y-ethyl)benzenesulfonamide were prepared where Y is bis-pyridylmethylamine (DPA), pyridinylmethylamine monoacetic acid (PAMA), bis(1H-imidazole)diacetic acid (CIM), bis(1H-imidazole)tetraacetic acid (TIM), bis(1H-imidazole)hexacetic acid (HIM), bis(triazolylmethylamine)diacetic acid (CTR) and bis(triazolylmethylamine)diaminobutane (ATR). Variable binding affinity to CA IX expressing HeLa cells was observed for the 4-(2-Y-ethyl)benzenesulfonamide Re(CO)₃ complexes (IC₅₀ = 3 - 280 nM). An effect of the linker length and composition of the linker between BzSA and the chelators was observed. It was especially favorable for the bulkier HIM complexes to be spaced away from the BzSA ring (IC₅₀ : 4-(2-HIM-ethyl)BzSA (116 nM) > 4-(2-HIM-butoxy)BzSA (35 nM) and 4-(2-HIM-hexyloxy)BzSA (33 nM)). Non-metalated free ligands displayed poor affinity for CA IX suggesting that the metal complex is required for high affinity binding. 4-(2-TIM-ethyl)BzSA was radiolabeled with ^99mTc in >85% RCY and >90% RCP.

Conclusions A series of BzSA CA IX inhibitors containing novel tridentate chelates with the M(CO)₃ core (M = Re or ^99mTc) were prepared and evaluated. Several potent analogs are currently being investigated preclinically for the potential use in the imaging of hypoxic solid tumors and RC in man