2.5. Synthesis and Characterization of BP Conjugated Antibiotics (BCAs)

The chemical structures of sitafloxacin, tedizolid, and the various BP and HBP conjugates used in this study are described in Figure 3 (Bisphosphonate-carbamate-sitafloxacin: BCS; α-hydroxybisphosphonate-carbamate-sitafloxacin: HBCS; Bisphosphonate-carbamate-tedizolid: BCT; α-hydroxybisphosphonate-carbamate-tedizolid: HBCT; Bisphosphonate-ester-tedizolid (BET)). The BPs used for conjugation are: (2-(4-hydroxyphenyl)ethane-1,1-diyl)bis(phosphonic acid) (HPBP); (2-(4-aminophenyl)ethane-1,1-diyl)bis(phosphonic acid) (APBP); 4-(2,2-diphosphonoethyl)benzoic acid (CPBP); and the HBPs used for conjugations are: (1-hydroxy-2-(4-hydroxyphenyl)ethane-1,1-diyl)bis(phosphonic acid) (HPHBP); (2-(4-aminophenyl)-1-hydroxyethane-1,1-diyl)bis(phosphonic acid) (APHBP). All of these compounds were synthesized similarly according to procedures as originally reported in Sedghizadeh et al. [28], and Sun et al. [25]. The compounds were characterized by NMR, mass spectrometry, combustion elemental analysis and HPLC, demonstrating >95% purity.

Molecular structure and empirical formula of antibiotics and conjugates: (1) HBCS, (2) BCS, (3) Sitafloxacin hydrate, (4) HPHBP, (5) PHBP, (6) HBCT, (7) BCT, (8) BET, (9) Tedizolid, (10) APHBP, (11) APBP, (12) CPBP were synthesized according to procedures reported by Sun et al. and Sedghizadeh et al. [28,31]. All compounds were characterized by NMR, mass spec, elemental analyses and HPLC demonstrating high purity. On cleavage of the carbamate linkage of (1), (2), (6), (7), the released bisphosphonates (4), (5), (10), (11) and CO₂ are pharmacologically inactive components. Since cleavage of the linker and release of antibiotic occurs mainly at sites of higher bone resorption caused by osteoclastic or bacterial osteolysis, extremely high local concentrations of antibiotic are generated beneath or in the proximity of infections on bone surfaces. Conjugate (8) utilizes an ester linkage which appears to cleave more readily than carbamate comparators in vitro and will be compared for efficacy in vivo.