Targeting nitric oxide synthase with 99mTc/Re-tricarbonyl complexes containing pendant guanidino or isothiourea moieties

The visualization of inducible nitric oxide synthase (iNOS) in vivo with specific radioactive probes could provide a valuable insight into the diseases associated with upregulation of this enzyme. Aiming at that goal, we have synthesized a novel family of conjugates bearing a pyrazolyl-diamine chelating unit for stabilization of the fac-[M(CO)3]+ core (M = 99mTc, Re) and pendant guanidino (L1 = guanidine, L2 = N-hydroxyguanidine, L3 = N-methylguanidine, L4 = N-nitroguanidine) or S-methylisothiourea (L5) moieties for iNOS recognition. L1–L5 reacted with fac-[M(CO)3(H2O)]+, yielding complexes of the type fac-[M(CO)3(k3-L)]+ (M = Re/99mTc; 1/1a, L = L1; 2/2a, L = L2; 3/3a, L = L3; 4/4a, L = L4; 5/5a, L = L5), which were fully characterized by the usual analytical methods in chemistry and radiochemistry, including X-ray diffraction analysis in the case of 1. The rhenium complexes 1–5 were prepared as “cold” surrogates of the 99mTc(I) complexes. Enzymatic assays with murine purified iNOS demonstrated that L1, L2, 1 and 2 are poor NO-producing substrates. These assays have also shown that metallation of L4 and L5 (Ki > 1000 μM) gave complexes with increased inhibitory potency (4, Ki = 257 μM; 5, Ki = 183 μM). The organometallic rhenium complexes permeate through LPS-treated RAW 264.7 macrophage cell membranes, interacting specifically with the target enzyme, as confirmed by the partial suppression of NO biosynthesis (ca. 20% in the case of 4 and 5) in this cell model. The analog 99mTc(I)-complexes 1a–5a are stable in vitro, being also able to cross cell membranes, as demonstrated by internalization studies in the same cell model with compound 4a (4h, 37 °C; 33.8% internalization). Despite not being as effective as the α-amino-acid-containing metal-complexes previously described by our group, the results reported herein have shown that similar 99mTc(I)/Re(I) organometallic complexes with pendant amidinic moieties may hold potential for targeting iNOS expression in vivo.

Graphical abstract99mTc/Re-tricarbonyl complexes with pendant N-nitroguanidine (4/4a) or S-methylisothiourea (5/5a) moieties permeate through LPS-activated RAW 264.7 macrophage cell membranes, suppressing NO biosynthesis (4 and 5: ca. 20%). These complexes may hold potential for targeting inducible Nitric Oxide Synthase in vivo.Figure optionsDownload full-size imageDownload as PowerPoint slideResearch highlights► 4/4a and 5/5a permeate through LPS-activated RAW 264.7 macrophage cell membranes. ► 4 and 5 suppress NO biosynthesis (ca. 20%) in LPS-activated macrophages. ► The stable 99mTc(I)-complexes 4a and 5a may hold potential for targeting iNOS in vivo.