Cyclic Analogs of Desferrioxamine E Siderophore for 68Ga Nuclear Imaging: Coordination Chemistry and Biological Activity in Staphylococcus aureus

As multidrug-resistant bacteria are an emerging problem and threat to humanity, novel strategies for treatment and diagnostics are actively sought. We aim to utilize siderophores, iron-specific strong chelating agents produced by microbes, as gallium ion carriers for diagnosis, applying that Fe(III) can be successfully replaced by Ga(III) without losing biological properties of the investigated complex, which allows molecular imaging by positron emission tomography (PET). Here, we report synthesis, full solution chemistry, thermodynamic characterization, and the preliminary biological evaluation of biomimetic derivatives (FOX) of desferrioxamine E (FOXE) siderophore, radiolabeled with 68Ga for possible applications in PET imaging of S. aureus. From a series of six biomimetic analogs, which differ from FOXE with cycle length and position of hydroxamic and amide groups, the highest Fe(III) and Ga(III) stability was determined for the most FOXE alike compounds–FOX 2-4 and FOX 2-5; we have also established the stability constant of the Ga-FOXE complex. For this purpose, spectroscopic and potentiometric titrations, together with the Fe(III)–Ga(III) competition method, were used. [68Ga]Ga-FOXE derivatives uptake and microbial growth promotion studies conducted on S. aureus were efficient for compounds with a larger cavity, i.e., FOX 2-5, 2-6, and 3-5. Even though showing low uptake values, Fe-FOX 2-4 seems to be also a good Fe-source to support the growth of S. aureus. Overall, proposed derivatives may hold potential as inert and stable carrier agents for radioactive Ga(III) ions for diagnostic medical applications or interesting starting compounds for further modifications.


Synthetic procedures and characterization data for the prepared compounds
The general route for the synthesis of intermediates 1 and 2 is depicted in Scheme S1. BocHN  Scheme S1. The general route for the synthesis of 1 and 2.

General procedure for the synthesis of precursor for compounds 1 and 2
To a solution of tert-butyl benzyloxycarbamate (1.0 equiv.) in DMF (5 ml / 1 mmol), 50% NaH (1.1 equiv.) was added portionwise, and the reaction was stirred at RT for 30 min. After 30 min, appropriate bromoester (1.1 equiv.) was added. Reaction was heated at 70°C and its progress was monitored by LC-MS. Then poured into mixture of ice and water and the product was extracted three times by hexane. The hexane extracts were washed with brine, dried over MgSO 4 and concentrated in vacuo to give the respective product as yellowish oil with a yield of 80-90%, depending on bromoester used.
Next, the oil was diluted with small amount of dioxane and 3M HCl/dioxane (10 ml / 1 g of compound) was added. Reaction progress was monitored by LC-MS. Then the reaction mixture was concentrated under reduced pressure and evaporated thrice from hexane to give the amine in a quantitative yield.
To pivaloylic anhydride of Boc-β-alanine or Boc-γ-aminobutyric acid (1.0 equiv.) in THF (tetrahydrofuran) (5 ml / 1 mmol), the solution of amine obtained above (1.0 equiv.) in THF was added along with NMM (N-methylmorpholine, 3.0 eq). Reaction was stirred at RT and its progress was monitored by LC-MS. Then the reaction mixture was diluted with EtOAc, washed twice with H 2 O, twice with 2M HCl, twice with 5% NaHCO 3 , brine and dried over MgSO 4 .
The residue was purified using column flash chromatography (silica gel; EtOAc/hexanes) to give the pure precursor for 1 and 2 as an oil.

Results
Precursor with x = 2 and y = 2, methyl ester: Yield: 47%; LC-MS t R = 4.5 min, ESI-MS (m/z): To a solution of precursor for 1 and 2 (1.0 equiv.) in MeOH (5 ml / 1 mmol), 1M NaOH (2.0 equiv.) was added and the reaction was stirred at RT. Reaction progress was monitored by LC-MS. Then the reaction mixture was diluted with H 2 O, MeOH was evaporated and the residues was acidified with 2M HCl to pH~1-2 and extracted twice with EtOAc. The organic layers were combined, washed with brine, dried over MgSO 4 and concentrated in vacuo to give the respective product. The precursor for 1 and 2 was diluted with small amount of dioxane and 3M HCl/dioxane (10 ml / 1 g of compound) was added. Reaction progress was monitored by LC-MS. Then the reaction mixture was concentrated under reduced pressure and evaporated thrice from hexane to give the respective product. To a solution of the above compound (1.0 equiv.) in MeOH (5 ml / 1 mmol), 1M NaOH (2.0 equiv.) was added and the reaction was stirred at RT. Reaction progress was monitored by LC-MS. Then the reaction mixture was diluted with H 2 O, MeOH was evaporated and the residues was acidified with 2M HCl to pH~1-2 and extracted twice with EtOAc. The organic layers were combined, washed with brine, dried over MgSO 4 and concentrated in vacuo to give the respective acid.

Results
The above acid was diluted with small amount of dioxane and 3M HCl/dioxane (10 ml / 1 g of compound) was added. Reaction progress was monitored by LC-MS. Then the reaction mixture was concentrated under reduced pressure and evaporated thrice from hexane to give the respective product.
Results:  All peak assignments were based on the comparison between the calculated and experimental isotope patterns.

ESI-MS measurements
The ESI-MS spectra of the reaction mixture of Fe(ClO 4 ) 3 /ligand and Ga(ClO 4 ) 3 /ligand with a 1:1 molar ratio (Fig. 2) are characterized by the presence of a few major peaks successfully attributed to the mononuclear species (Table S2, Table S3). Experiments were performed in methanol-water system (50:50 v/v) for better solvent evaporation.

Ligand's protonation constants
The acid-base properties of investigated ligands were studied by potentiometric technique. The optimized logβ values defined by equations (S1) and (S2) were calculated using SUPERQUAD 1 or Hyperquad 2006 2 programs (Table S4). For the sake of simplicity charges are omitted in all chemical equilibria given here.
(S1) be ascribed unambiguously to either of the groups. Thus, to thoroughly determine the protonation processes of studied compounds, the pH-dependent NMR titrations would be needed. However, such a detailed analysis is not necessary for the determination of stability of iron complexes and therefore was not performed.

Complex formation equilibria with Fe(III)
During spectrophotometric titrations of Fe(III)-FOX systems, the color of the solutions changed from dark red in acidic pH to light yellow above pH 7. Spectral changes of LMCT bands (Fig. S3) (Fig. S3 g). Stability constants gathered in Table S5 as logβ[FeH m L n ], defined by equations (S3) and (S4), and spectroscopic characteristics of calculated spectra given in Table S6 were determined by refinement of the spectroscopic and potentiometric data.
The uncertainties in the logK values correspond to the added standard deviations in the cumulative constants.
Corresponding electronic spectra are presented in Fig. S3, while speciation plots are presented in Figure S7.  Values presented in Table S5 were calculated from data collected during measurements implementing two separate methods. As we can see above, this data correlates very well and further confirms our calculations.

The spectrophotometric competition experiments with EDTA
The spectrophotometric competition experiments with EDTA were performed at fixed pH 7.0, where fully formed complexes of Fe(III)-FOXE analogs were formed, characterized by strong bands at 430-440 nm. Those LMCT bands were gradually silenced after addition of fixed amount of EDTA ligand, from 0-10 molar equivalent for FOX 2-5 and 0-1.2 molar equivalent for rest of the ligands (Fig. S4). The competition data were refined to obtain the overall Fe(III)-FOX binding constant logβ FeL (Table S7) for analogs using a model involving two ligands and one metal. The protonation constants and Fe(III) formation constants for EDTA 4 and the protonation constants of the analogs (The acid-base properties of investigated ligands were studied by potentiometric technique. The optimized logβ values defined by equations (S1) and (S2) were calculated using SUPERQUAD 1 or Hyperquad 2006 2 programs (Table S4). For the sake of simplicity charges are omitted in all chemical equilibria given here.     For each complex the stability constant for Fe(II)-analog was calculated using eq. S10 (S10)