The mono(catecholamine) derivatives as iron chelators: synthesis, solution thermodynamic stability and antioxidant properties research

There is a growing interest in the development of new iron chelators as novel promising therapeutic strategies for neurodegenerative disorders. In this article, a series of mono(catecholamine) derivatives, 2,3-bis(hydroxy)-N-(hydroxyacyl)benzamide, containing a pendant hydroxy, have been synthesized and fully characterized by nuclear magnetic resonance, Fourier transform infrared spectroscopy and mass spectrum. The thermodynamic stability of the chelators with FeIII, MgII and ZnII ions was then investigated. The chelators enable formation of (3 : 1) FeIII complexes with high thermodynamic stability and exhibited improved selectivity to FeIII ion. Meanwhile, the results of 1,1-diphenyl-2-picryl-hydrazyl assays of mono(catecholamine) derivatives indicated that they all possess excellent antioxidant properties. These results support the hypothesis that the mono(catecholamine) derivatives be used as high-affinity chelator for iron overload situations without depleting essential metal ions, such as MgII and ZnII ions.


RP, 0000-0002-2517-0853
There is a growing interest in the development of new iron chelators as novel promising therapeutic strategies for neurodegenerative disorders. In this article, a series of mono(catecholamine) derivatives, 2,3-bis(hydroxy)-N-(hydroxyacyl)benzamide, containing a pendant hydroxy, have been synthesized and fully characterized by nuclear magnetic resonance, Fourier transform infrared spectroscopy and mass spectrum. The thermodynamic stability of the chelators with Fe III , Mg II and Zn II ions was then investigated. The chelators enable formation of (3 : 1) Fe III complexes with high thermodynamic stability and exhibited improved selectivity to Fe III ion. Meanwhile, the results of 1,1-diphenyl-2-picrylhydrazyl assays of mono(catecholamine) derivatives indicated that they all possess excellent antioxidant properties. These results support the hypothesis that the mono(catecholamine) derivatives be used as high-affinity chelator for iron overload situations without depleting essential metal ions, such as Mg II and Zn II ions.

Introduction
Iron is essential to the proper functioning of most organisms, but it is toxic when present in excess. In    The mono(catecholamine) derivatives were fully characterized by nuclear magnetic resonance (MNR), Fourier transform infrared spectroscopy (FTIR) and mass spectrum (MS). The FTIR spectra of the derivatives showed distinct absorption peaks at 1639-1647 cm −1 of benzamide carbonyl stretching vibration, 1583-1593 and 1540-1548 cm −1 of benzene ring skeleton vibration. The 1 H NMR signals of the catecholamine aromatic protons of the chelators in (CD 3 ) 2 CO were identified as doublets at 7.22-7.28 ppm (d, J = 7.8 Hz, 1H, Ar-H), doublet of doublets at 6.96 ppm (dd, J = 7.8, 1.2 Hz, 1H, Ar-H) and triplets at 6.69- 6.71 ppm (t, J = 7.8 Hz, 1H, Ar-H). The signals of methylene of benzyl at 5.15-5.16 ppm (s, 9H, O-CH 2 -Ar) and 5.09-5.10 ppm (s, 9H, O-CH 2 -Ar) disappeared. Meanwhile, the 13 C NMR signals of methylene of benzyl also disappeared in the mono(catecholamine) derivatives. Results indicated that the structure of mono(catecholamine) derivatives is as we expected.

Solution thermodynamics
In its neutral form, mono(catecholamine) derivatives (hereafter also designed as L 1−3 H 2 ) have two dissociable protons, corresponding to catecholamine moieties. Because catecholamine moieties require deprotonation for efficient metal chelation, their metal affinity is necessarily pH dependent. In the presence of dissolved metal ions (M a+ ) and protonated chelator (LH i , where L is a chelator with i removable protons), the pH-dependent metal-chelator complex with a general formula M m L l H h forms. The relative amount of each species in solution is determined by equation (2.1), whose rearrangement provides the standard formation constant notation of log β mlh (equation (2.2)). The log β mlh value describes a cumulative formation constant. For convenience, these are discussed as stepwise association constants, either for complex formation log K 1n0 (equation (2.3)) or ligand protonation log K 01 h (equation (2.4)). When addressing protonation constants, the stepwise formation constants are commonly reported as protonation constants (log K h H , h = 1, 2, 3, . . . ): The log K h H were determined from a combination of potentiometric and spectrophotometric titration.
The obtained data were analysed using HYPSPEC 2014 and HYPQUAD 2013 [27,28]. The determined log K h H of L 1−3 H 2 and different phenolate type chelators are listed in table 1. All the chelators contain two basic sites from the phenolate oxygen atoms of the catechol moieties. Therefore, L 1−3 H 2 were both treated as diprotic acids for data analysis. The potentiometric titration curves of L 1−3 H 2 are shown in figure 3, the a value is the mol ratio of added base per chelators. The deprotonation of phenolic hydroxy in an orthoposition relative to amide gives rise to the buffer region at low pH (pH < 9.0), and its inflection points at a = 1, which indicated the orthophenolic hydroxy complete dissociation. These potentiometric titrations data were used to obtain the log    titration data were required to accurately determine the higher log K 1 H values. The spectrophotometric titration spectrogram of L 1 H 2 was selected for illustration because it is similar to that of L 2−3 H 2 , as shown in figure 4. The spectrograms of L 2-3 H 2 are shown in the electronic supplementary material, figures S1-S2. The initial absorbance peaks at 310 nm shifted to 330 nm with the increase of pH (5.3-9.0), which was attributed to the deprotonation of the phenolic hydroxy in an orthoposition relative to amide. Subsequently, the peaks width gradually broadened, and the intensity was gradually decreasing with the increase of pH (9.0-12.5), which was attributed to the further deprotonation. comparable with the corresponding values for N-methyl-2,3-dihydroxybenzamide (MDHB) [29], N,Ndimethyl-2,3-dihydroxybenzamide (DMB) [5] and catechol [30] ( The spectrophotometric titration spectrogram of the Fe III -L 1 H 2 complex was selected for illustration because it is similar to that of L 2−3 H 2 , as shown in figure 6. The spectrograms of L 2-3 H 2 are shown in the electronic supplementary material, figures S5-S6. In the titration spectrogram, intense ligand-to-metal charge transfer (LMCT) bands of the Fe III complexes allow the proton-dependent equilibria to be monitored spectrophotometrically. All of the chelator L 1−3 H 2 possess at least three spectrophotometrically distinguishable species that can be assigned by comparison to the well-defined Fe III -catechol system [34][35][36]. For the sake of clarity, we will use the FeL n notation to ignore the charge state of the complexes, which varies across our series of chelator. In all cases, a light green solution was observed under acidic conditions, corresponding to FeL forms. Upon addition of alkali, a blue solution was observed, which is indicative of FeL 2 predominates. The solution converts, under more alkaline conditions, to red that is assigned as FeL 3 . So, we proposed the complexation processes are that complete deprotonation bidentate gradually replaced solvent molecules with the increased pH value (figure 7). The specific species and the LMCT band wavelengths with their corresponding extinction coefficients for each FeL n spectral are compiled in table 2.
The affinities of chelator L 1−3 H 2 with metal ions were determined by spectrophotometric titration data, which were analysed using the program HYPSPEC 2014 [27]. The species distribution diagrams of metal-complexes were obtained using the simulation program HYSS [37]. The species distribution diagrams of Fe III complexes are shown in figure 8 [39]. d DTPA in [40].
that the pFe III value of L 1 H 2 is higher than that of L 2−3 H 2 indicated a shorter pendant hydroxide group with lesser steric hindrance, which favours higher Fe III affinity.
Metal affinity studies have focused on the Fe III ion; however, the presence of Mg II and Zn II ions in biological systems leads us to evaluate the affinity of the chelators with Mg II and Zn II ions. The Mg II and Zn II ions affinities of L 1−3 H 2 were determined through spectrophotometric titrations under the same conditions above. The spectrophotometric titration spectrograms of Mg II -L 1−3 H 2 and Zn II -L 1−3 H 2 complexes are shown in the electronic supplementary material, figures S9-S14. The log β mlh , pMg II and pZn II values of L 1−3 H 2 and related compounds are listed in table  The pMg II and pZn II values of L 1−3 H 2 were significantly lower than those of the efficient chelator diethylenetriaminepentaacetic acid (DTPA) [40]. The low pMg II and pZn II values were similar to those of catechol chelators [8,10,[40][41][42][43][44], indicating the formation of chelators L 1−3 H 2 with weak Mg II and Zn II affinity, as predicted.

Antioxidant activity
1,1-Diphenyl-2-picryl-hydrazyl (DPPH) is a stable free radical [45], which is widely used to monitor the free radical scavenging ability of various antioxidants [46][47][48]. The assays were carried out in methanol, and the results were expressed as EC 50 , which represented the antioxidant concentration required to decrease the initial DPPH concentration by 50%. Low EC 50 values indicate a highly radical scavenging capacity. This parameter is widely used to measure antioxidant capacity but does not consider the reaction time. The time needed to reach the steady state to the concentration corresponding at EC 50 (T EC50 ) was calculated, and antiradical efficiency (AE) was introduced as a parameter to characterize antioxidant compounds [46]. AE was determined by the following equation: 50 . (2.5) The EC 50 value of antioxidant L 1−3 H 2 and their Fe III complexes were obtained from the curves of the percentage of remaining DPPH at the steady state against the concentration n, where n is the molar ratio of antioxidant to DPPH (mol AH/mol DPPH). The percentage of remaining DPPH was determined from the kinetic curves of different concentrations n, figure 9 and electronic supplementary material, figures S15-S19. The EC 50 and AE values of L 1−3 H 2 , complexes Fe III -L 1−3 H 2 , (BHA) butylated hydroxyanisole, catechol and ascorbic acid [46,49] are listed in table 3 ( a Antioxidant. b BHA and ascorbic acid in Ref. [46]. c Catechol in Ref. [49]).
The structures of phenolic derivatives have great influence on antioxidant activity [50,51]. L 1−3 H 2 , BHA and catechol possess similar bisphenolic structures, which lead them with approximate EC 50 values.
For the results of L 1−3 H 2 and Fe III -L 1−3 H 2 , the increase in AE values can be explained by mechanism of the DPPH's reaction with phenols (ArOH). The antioxidant capacity of antioxidant L 1−3 H 2 was influenced by amide group and coordination reaction. DPPH reacted with ArOH through an electrontransfer process from ArOH or its phenoxide anion (ArO − ) to DPPH (electron transfer mechanism) [52]. The electron-transfer process from ArO − to DPPH was fast. The presence of amide group in L 1−3 H 2 increased the quantity of ArO − and the observed values of the reaction rate, which conferred high AE values. The AE values of the complexes Fe III -L 1−3 H 2 reduce generally, the Fe III coordination reaction produced acids and ArO-M complex. The acids and coordination reaction all reduced the quantity of ArO − and antioxidant capacity. The results were as expected.

Conclusion
In this article, a series of mono(catecholamine) derivatives, 2,3-bis(hydroxy)-N-(hydroxyacyl)benzamide, containing a pendant hydroxy, were synthesized and fully characterized. The results of thermodynamic stability with a set of metal ions showed that the chelators possess with high thermodynamic stability and exhibited improved selectivity to the Fe III ion. Meanwhile, the results of DPPH assays of mono(catecholamine) derivatives indicated they all possess excellent antioxidant properties. These results also indicated that mono(catecholamine) derivatives have a potential application prospect as chelator for the iron overload situations without depletion of essential metal ions such as Mg II and Zn II ions. 4. Experimental section 4. 1

. General
The organic reagents used were pure commercial products from Aladdin. The solvents were purchased from Chengdu Kelong Chemical Reagents Co. The 300-400 mesh silica gels were purchased from Qingdao Hailang Chemical Reagents Co. 1 H NMR and 13 C NMR spectra were recorded on a Bruker Avance III 600 MHz, CDCl 3 and CD 3 OD were used as the solvents, tetramathylsilane as the internal standard. The FTIR spectra were obtained from a Nicolet 380 FTIR spectrophotometer (Thermo Fisher Nicolet, USA) with a resolution of 4 cm −1 from 400 cm −1 to 4000 cm −1 . The ultraviolet (UV)-vis spectrophotometer (Thermo Scientific Evolution 201, USA) used had a double-beam light source from 190to 1100 nm. Mass spectral analysis was conducted using a Varian 1200 LC/MS. Synthesis of 2,3-bis(benzyloxy)-N-(hydroxyethyl)benzamide (4a). A solution of 2,3-bis(benzyloxy)benzoic acid 2 (1.67 g, 5.0 mmol), HOBt (0.12 g, 0.9 mmol) and DCC (1.24 g, 6.0 mmol) in CH 2 Cl 2 (50 ml) was stirred for 30 min at room temperature. Ethanolamine (0.34 g, 5.5 mmol) was added dropwise over 3 min and the mixture stirred for 10 h. The solution was filtered to remove the dicyclohexyl urea. The filtrate was concentrated in vacuo and the residue purified by flash column chromatography (volume ratio of acetone/hexane 2 : 3) to give the product 4a as clear oil (80%). R f = 0. 4