Introduction
Iron chelators such as deferoxamine, deferiprone and deferasirox (Exjade® or ICL670) have been used in iron overload therapy.[1] In this work, we report on ESP atomic charge derivation, force field parameter development and Force Field Topology DataBase (FFTopDB) building for deferasirox- and calix[n]arenes-deferasirox-based Fe(III) chelators (structures 4-7, Figure 1a and structures 1-2, Figure 1b). These empirical force field data are used with the Cornell et al. Amber force field (latest version: Amber99SB)[2] to study the structure and dynamics of the reported Fe(III) chelators by molecular dynamics (MD) simulations in condensed phase. Complexes constituted of Fe(III) covalently bound (structures 5-6, Figure 1a) and ionically bound (structures 4-7, Figure 1a) to the six coordination sites of two deferasirox derivatives were both considered in MD simulations.

Computational details
The geometries of the different building blocks constituting the deferasirox- and calix[4]arenes-deferasirox-based Fe(III) chelators were optimized using the B3LYP/6-31G* theory level with the Gaussian 2003 (version E.01) program.[3] For each building block the lowest minimum found after a conformational search was selected except for 2-methoxyethanol, where two conformations were choosen. A minimum was considered only if no canonical intra-molecular hydrogen bond [donor (D)-acceptor (A) distance lower than 3.20 Å and the D-H...A angle between 120-180°] was observed in a structure. Molecular electrostatic potential (MEP) computation involved the Connolly surface algorithm, a radius of 1.8 Å for iron and the B3LYP/6-31G* theory level implemented in the Gaussian 2009 (version A.02) program. As defined by Steinhauser et al. the high spin state (S = 5/2) of Fe(III) was considered in DFT calculations.[4] For each building block two molecular orientations based on the rigid-body reorientation algorithm implemented in the R.E.D. program were involved in MEP computation ensuring the reproducibility of the charge values.[5] The molecular fragments required for MD simulations were constructed by setting specific intra- and inter-molecular charge constraints between fully characterized connecting groups during the charge fitting step (see Figure 1a). An additional intra-molecular charge constraint of +3.0 e was used to define iron ionically bound. ESP charge fitting was carried out using a standalone version of the RESP program and following a single stage fitting procedure.[6] A Relative Root Mean Square (RRMS) value of 0.0226 between the MEP calculated by quantum chemistry and that generated using the derived charge values was obtained for the charge fitting step. A highly similar RRMS value was also obtained in the absence of intra- and inter-molecular charge constraints. The relative small RRMS values as well as the small difference of RRMS between the charge fitting steps carried out with and without intra- and inter-molecular charge constraints demonstrate the accuracy of the fitting step performed in this work and the weak effect of the constraints used.

Charge derivation involving multiple orientations, multiple conformations and multiple molecules and FFTopDB building for deferasirox- and calix[4]arenes-deferasirox-based Fe(III) systems have been automatically carried out using the R.E.D. IV program (version January 2011) available in R.E.D. Server "Development". a) Description of the different building blocks involved in ESP charge derivation and FFTopDB building; dashed and gray boxes: intra-molecular charge constraints within each selected building block; plain and gray boxes: inter-molecular charge constraints defined between pairs of building blocks, b) building of the calix[4]arene-deferasirox Fe(III) chelators obtained from total synthesis using the FFTopDB, c) gif animated image: representative three-dimensional structure of the calix[4]arene-deferasirox Fe(III) chelator, experimentally witnessed, obtained from MD simulations (hydrogen atoms are omitted for clarity).

FFTopDB validation
The new force field libraries have been validated based on 50 nsec MD simulations using the SAJFAL structure taken from the Cambridge Structural Database (CSD).[4] RMSD mean values for heavy atoms and for the six connecting heteroatoms and Fe(III) of 0.840 ± 0.172 and 0.100 ± 0.018 [Fe(III) covalently bound complex] and of 0.862 ± 0.215 and 0.169 ± 0.016 Å [Fe(III) ionically bound complex] were calculated, respectively. Mean values for the gyration radius (Rg) of 5.204 ± 0.038 and 5.300 ± 0.046 Å were obtained for the covalently and ionically bound complexes, respectively. These Rg values calculated from MD simulations agree well with the experimental one measured on the SAJFAL structure (5.421 Å).

Calix[4]arene-deferasirox Fe(III) chelator structures
Three-dimensional structures of the calix[4]arene-deferasirox Fe(III) chelators were obtained from 50 nsec MD simulations. The structure of 1 (Figure 1b) was confirmed by geometry optimization using the Gaussian 2009 program and the B3LYP/6-31G* theory level.

The ESP charge values reported in this project can be compared to the corresponding RESP charges available in the F-89 R.E.DD.B. project.

[1] Liu and Hider Coord. Chem. Rev. 2002, 232, 151–171.
[2] Cornell et al. J. Am. Chem. Soc. 1995, 117, 5179–5197; Hornak et al. Proteins: Struct., Funct., Bioinf. 2006, 65, 712–725.
[3] IUPAC names of the building blocks considered in this work: 2-ethyl-6-methylphenol, 2-methoxyethanol, N-(2-methoxyethyl)acetamide, 4-[3,5-bis(2-hydroxyphenyl)-1,2,4-triazole-1-yl]benzamide and 3,5-bis(2-hydroxyphenyl)-1-phenyl-1,2,4-triazole complexed with Fe(III).
[4] Steinhauser et al. Eur. J. Inorg. Chem. 2004, 21, 4177–4192.
[5] Information about the atoms involved in the rigid-body reorientation algorithm can be obtained from the PDB files available in this project.
[6] Bayly et al. J. Phys. Chem. 1993, 97, 10269–10280, and here.