Fluorescent imaging probes of nitrogen-containing bone active drugs: Design, synthesis and applications.

摘要

Bisphosphonates (BPs) are therapeutic agents for treatment of bone disorders such as osteoporosis and Paget's disease. Several nitrogen-containing bisphosphonates (N-BPs) and phosphonocarboxylate (PC) analogues also have potential as anti-cancer agents. However, details of the skeletal distribution, cellular uptake and mechanisms of these drugs remain to be elucidated, stimulating the creation of imaging probes which mimic some or all of their pharmacological properties.;A fluorescent imaging "toolkit" with more than 20 probes derived from all three heterocyclic N-BP drugs (risedronate, zoledronate and minodronate) and related analogues have been successfully synthesized. A linking strategy with two routes was applied under exceedingly mild conditions and introduced a terminal amino group in drug-linker intermediates capable to conjugate with the commercially available activated ester of fluorescent dyes. All the fluorescent probes were prepared in good yield (50--77%) and high purity (> 95%), and are fully characterized by HPLC, UV-VIS and fluorescence emission spectroscopy, 1H and 31P NMR and high-resolution MS.;The "toolkit" contains a series of fluorescent probes ranging from visible to near infrared optical window and the probes generally retain substantial affinity for bone mineral reflecting the varying affinities of their parent drug components, depending on the structure of the conjugating fluorescent dye. In addition, we have obtained evidence that certain probes (e.g. the FAM- and ROX- conjugates) have anti-prenylation and anti-resorptive effects in vitro and in vivo, demonstrating that the probes could retain biological activities of the parent BP drugs. Due to their diverse spectroscopic and pharmacological properties, the fluorescent imaging probe "toolkit" has been successfully utilized in various biological researches including osteoclast imaging, drug distribution at bone skeleton and cellular level, mechanism studies of osteonecrosis of the jaw (ONJ), cancer imaging, et al., and several papers have been published based on the obtained results, demonstrating the versatility of the imaging probe "toolkit".;The first example of a bifunctional amino/azido-containing N-heterocyclic bisphosphonate (amino-azido-para-dRIS, compound 7 in Chapter 5) has been synthesized; and it has been successfully applied for the preparation of fluorescent probes via Cu(I) catalyzed alkyne-azide coupling (CuAAC) click reaction. The synthetic strategy developed based on para-dRIS, should be adaptable to other N-heterocyclic deoxy-bisphosphonates and related analogues, such as dRIS, dRISPC, et al. In addition, two functionalities, the azido and amino groups, were introduced sequentially to make dual-conjugation of the molecule possible, providing the opportunity for novel cleavable imaging agents and drug delivery system design.