Design and Synthesis of a Bombesin Peptide-Conjugated Tripodal Phosphino Dithioether Ligand Topology for the Stabilization of the fac-[M(CO)3]+ Core (M = 99 mTc or Re)

A new tumor-seeking tridentate topology consisting of a phosphino dithioether ((HOCH2)2PCH2CH2S(CH2)nCH2SR; PS2) ligand framework for the production of kinetically inert and in vivo stable facial [99mTc(CO)3(PS2)]+ or [Re(CO)3(PS2)]+ is described. The X-ray crystal structure of fac-Re(CO)3(PS2)PF6 is reported. The bioconjugation strategies for incorporating bombesin (BBN) peptides on to the PS2 tripodal framework and, thereby, de novo designing of GRP receptor-seeking Tc(PS2–BBN)(CO)3 are developed. INTRODUCTION Advances in metal-based molecular imaging and therapy agents will hinge upon discoveries in ligand architecture and coordination chemistry of specific transitionmetals.1 For example, phosphine-based ligating frameworks have been shown to impart in vivo stability to radioactive metals through chelate interactions, and such agents have been utilized for biomedical applications.2 It is very well established that the steric and electronic properties of phosphines exert a major influence on bonding interactions with transition metals of biological interest, thus, creating a compelling scenario for the de novo design of new functionalized phosphines.3 Specifically, water-soluble phosphines functionalized with peptides or other biomolecules serve as biological markers for the in vivo targeting of receptors overexpressed in tumors for diagnostic/therapeutic use.3 Surprisingly, despite the importance of peptide–phosphine conjugates in biomedical applications, little effort has been directed toward developing the synthetic strategies for the immobilization of biomolecules on water-soluble phosphine ligands. Technetium-99m occupies a special position in inorganic radiopharmaceuticals1,4,5 because of the following reasons: (i) optimal nuclear properties (t1/2 = 6.02 h, E(γ) =140 keV) for molecular imaging; (ii) easy availability through the transportable 99Mo/99mTc generator system; and (iii) conversion to clinically useful, ligand exchangeable inorganic complexes for biomolecule conjugation. More importantly, 99mTc has a versatile inorganic chemistry, spanning different oxidation states, to produce a variety of ligand exchangeable coordination complexes.1 Among several precursor complexes of technetium, the kinetically inert [99mTc(CO)3(H2O)3]+ complex has attracted much attention in recent years due to its commercial availability as the Isolink kit and ease of generation under clinical settings.5 In fact, three water molecules in the complex can be replaced by a chelating ligand conjugated to a biomolecule such as a tridentate ligand or a “2+1” mixed ligand system, to yield a stable complex for targeted in vivo imaging applications. Alberto and co-workers have examined a library of N and S donor ligands for stabilizing the fac-[M- (CO)3]+ moiety.6 Their investigations showed that by judicious selection of the ligand system, effective labeling of biomolecules could be achieved.7 Likewise, Smith and co-workers have shown that the combination of bidentate N-donor ligands with a water-soluble monophosphine was effective in stabilizing the tricarbonyl core.8 On the inorganic chemistry front, novel tridentate N-donor ligand systems were developed by Marzilli and coworkers for the effective stabilization of the M(CO)3 moiety. They have shown that organic soluble metal precursors can serve as synthons for effective radiolabeling.9 In addition, electron-rich bidentate O-donor ligands (β-diketones acetylacetone and curcumin) along with monodentate nitrogen ligands have been explored to generate stable fac-[M(CO)3(OO)(N)]+ complexes. 10 However, precise knowledge of ligand design is not completely understood—more specifically, which combinations of hard bases vs soft donors would lead to enhanced in vivo stability of the [99mTc(CO)3]+ moiety in order to engineer the design and development of organ-specific (or tumor-specific) radiopharmaceuticals. In this respect, as part of our ongoing drug discovery program,11 we have investigated pharmacophore motifs of a library of ligands encompassing hard nitrogen donors, phosphine π acids, and sulfur-containing soft donors. We, herein, report the development of ligand frameworks spanning the tripodal coordination sites of [99mTc(CO)3(H2O)3]+. The results reported herein encompass our successful chemistry for the synthesis of the phosphino dithioether ((HOCH2)2PCH2CH2S(CH2)nCH2-SCH3; PS2 ) tripodal ligand backbone, its conjugation with bombesin peptide, and the “proof of principle” that such a π-acid phosphine (P) and soft σ-donor thioether (S) topology will produce in vivo stable imaging agents. As a first step toward establishing the utility of the PS2 ligand framework for in vivo biomedical applications, we focused our efforts on the synthesis of bifunctional ligand systems, bioconjugation with receptor-avid bombesin peptide, complexation with 99mTc and Re carbonyl complexes, and in vivo stability studies in mice. Bombesin (BBN), a 14-amino-acid peptide, was first isolated from the skin of the frog amphibian Bombina.12 BBN functions as a potent autocrine or paracrine growth factor for cells.13 In the past decade, a plethora of information has been generated on BBN/receptor expression and physiological processes.14 BBN shows high affinity for the gastrin-releasing peptide (GRP) receptor subtype BB2.14 GRP receptors are overexpressed in many cancers, including prostate, breast, and small cell lung cancer.15 Analogues of bombesin with modified structures have exhibited a similar or even higher affinity for these receptors. Synthetic peptides can be readily generated through automated solid phase techniques. For our studies, we have synthesized and utilized the seven-amino-acid truncated bombesin analogue (BBN) as a vehicle to target GRP receptors. The results reported in this paper include (i) synthesis and characterization of PS2 ligand systems, (ii) synthesis and X-ray crystallographic investigation of the [Re(CO)3(PS2)]+ complex, (iii) synthesis of the bifunctional PS2 ligand system and bioconjugation with the truncated bombesin peptide, (iv) synthesis and in vivo stability analysis of [99mTc(CO)3(PS2)]+ in mice, and (v) synthesis and in vivo stability analysis of [99mTc(CO)3(PS2–BBN)]+ in mice.