Synthesis, Characterization, and Stability of Iron (iii) Complex Ions Possessing Phenanthroline-based Ligands

It has previously been demonstrated that phenan-throline-based ligands used to make gold metallo-therapuetics have the ability to exhibit cytotoxicity when not coordinated to the metal center. In an effort to help assess the mechanism by which these ligands may cause tumor cell death, iron binding and removal experiments have been considered. The close linkage between cell proliferation and intracellular iron concentrations suggest that iron deprivation strategies may be a mechanism involved in inhibiting tumor cell growth. With the creation of iron (III) phen complexes , the iron binding abilities of three polypyridal ligands [1,10-phenanthroline (phen), 2,9-dimethyl-1, 10-phenanthroline (methyl phen), and 2,9-di-sec-butyl-1, 10-phenanthroline (sec-butyl phen)] can be tested via a competition reaction with a known iron chelator. Therefore, iron (III) complexes possessing all three ligands were synthesized. Initial mass spectrometric and infrared absorption data indicate that iron (III) tetrachloride complex ions with protonated phen ligands (R phenH +) were formed: [phenH][FeCl 4 ], [ methyl phenH][FeCl 4 ], [ sec-butyl phenH][FeCl 4 ]. UV-Vis spectroscopy was used to monitor the stability of the complex ions, and it was found that the sec-butyl phen-iron complex was more stable than the phen and methyl phen analogues. This was based on the observation that free ligand was observed immediately upon the addition of EDTA to the [phenH][FeCl 4 ] and [ methyl phenH] [FeCl 4 ] complex ions.


INTRODUCTION
Cancer is the 2nd leading cause of death in the United States.Though a wide variety of organic-based molecular compounds have been used to treat tumors, the metallotherapy cisplatin is one of the most successful anti-cancer drugs.Despite its success, cisplatin therapy has several drawbacks, including the development of resistance to the drug by tumors and the occurrence of severe side-effects during treatment [1].It is well known that gold (III) complexes are potential alternatives to the platinum (II) based drugs [2].Even though gold (III) complexes with polypyridyl based ligands [e.g., 1,10phenanthroline (phen), 2,2'-bipyri-dine (bipy), and 2,2'-6',2''-terpyridine (terpy)] have shown promise as anticancer agents, it has also been shown that the polypyridyl ligands can have antitumor activity on their own; therefore the antiproliferative properties of the ligands must be considered when evaluating the antitumor activity of the corresponding metal-based complexes [1,3,4].
We have previously reported the synthesis of a gold (III) complex bearing a 2,9-di-sec-butyl-1,10-phenanthroline ( sec-butyl phen)ligand {[( sec-butyl phen) AuCl 3 ]} [5].The anti-tumor activity of this gold complex compound is currently being studied, therefore it is desired to determine the potential activity of the sec-butyl phen ligand.It has been previously hypothesized that chelating ligands can impact tumor cell death via iron depletion [6].Additionally, Satterfield and Brodbelt reported that alkyl substituents on phenanthroline ligands have an impact on metal binding [7], and this data predicts that sec-butyl phen should exhibit stronger metal binding than unsubstituted phen.To test whether alkyl substituted phenanthroline based ligands have different iron binding abilities, we synthesized and characterized iron complex ions with a series of phenanthroline-based ligands [phen, sec-butyl phen, and 2,9-dimethyl-1,10-phenanthroline ( methyl phen)], and evaluated the relative stability of these three complex ions.

General Experimental Procedures
sec-butyl phen was synthesized according to a previously published protocol [8]; phen and methyl phen were purchased from Sigma-Aldrich and used without purification, as were all other reagents and solvents used in the course of characterizing the iron (III) complex ions.UV-Vis spectra were recorded on a Varian Cary 50 UV-Vis spectrophotometer, and IR spectra were obtained using a Perkin Elmer Spectrum One FT-IR Spectrophotometer fitted with a Universal ATR sampling accessory.Mass spectrometric analyses were performed using a Waters GCT (2008) high resolution mass spectrometer, using Electrospray Ionization (ESI) and Liquid Injection Field Desorption Ionization (LIFDI) techniques.

EDTA Competition Reaction
The EDTA binding reaction was modeled on a previously published protocol [10].Stock solutions were prepared in 5 mL of acetonitrile using 10.0 mg of the phenanthroline iron complexes {2.94 × 10 ].These stock solutions were then diluted in acetonitrile to create solutions with a final concentration of 3.43 × 10 −5 M. A 0.028 M solution of EDTA in distilled water was prepared for the binding experiment.The EDTA solution was then added to the iron (III) complex ions in a 20:1 EDTA:iron molar ratio, and the UV-Vis absorption spectra were collected every minute for thirty minutes.

Synthesis
The phen-iron complexes were synthesized by combining the phenanthroline ligand with FeCl 3 in a 1:1 molar ratio, in the presence of 10 M aqueous hydrochloric acid and water (Scheme 1).The resulting precipitates were then filtered, dried under vacuum, and recrystallized from ethanol to obtain the final complex ions.The products obtained were the complex ions comprised of the protonated phen ligand cation ( R phenH + ) and the FeCl 4 − anion.It is noted that previous work describing a similar synthetic protocol reported the formation of an iron (III) phen complex where the ligand was directly coordinated to the iron (III) metal center.However, the yield of this compound was extremely low, and was likely a minor side product in the reaction [9].Khavasi and coworkers also report that the complex ion possessing [phenH] + and [FeCl 4 ] − was isolated after the reaction between phen and FeCl 3 in hot aqueous solution [11].Other studies do demonstrate that complexes possessing direct coord-

Infrared Spectroscopy
IR results for compounds 1-3 show a single resonance at 3350 cm −1 , 3375 cm −1 , and 3200 cm −1 , respectively, indicating the presence of a protonated secondary amine and thereby providing additional evidence that the complex ions proposed in Scheme 1 were obtained.The presence of the R phen ligands is confirmed by the C−H aromatic resonances at 3051 cm −1 (1), 3051 cm −1 (2), and 3068 cm −1 (3), as well as C-N bands at 1345 cm −1 (1), 1345 cm −1 (2), and 1342 cm −1 (3).Though the previous reports of the [phenH][FeCl 4 ] complex ion do not provide IR data for the N-H stretch of the protonated ligand, previous reports with similar [ R phenH][AuCl 4 ] complex ions also indicate that the N-H stretch for the protonated R phen ligands is observed in the IR spectrum (at approximately 3180 cm −1 ) [5].

Mass Spectrometry
In addition to the elemental analysis and IR data, mass spectrometry indicates that [ R phenH][FeCl 4 ] complex ions were isolated.This is evidenced by the fact that electrospray ionization (ESI) only produced the individual [ R phenH] + ion (observed in positive ion mode), or the [FeCl 4 ] − ion (observed in negative ion mode; see Supplemental Data Figure 1 for representative data).In order to determine if the complex ions could be ionized without breaking the ion pair, Liquid Introduction Field Desorption Ionization (LIFDI) was subsequently used.LIFDI is known for being a soft ionization technique [14], thus the complex ions were expected to remain associated during the ionization process.Upon ionization via LIFDI positively charged molecular ions composed of [( R phen)FeCl 2 ] + fragments, formed upon the loss of two chloride ligands, were indeed detected (see Figure 1 and Supplemental Data Figures 2 and 3).It is also noted that compounds 2 and 3 exhibited fragment peaks in the LIFDI mode corresponding to the [ R phenH] + protonated ligands, further corroborating the final structural assignments for this class of compounds (compound 1 had a [(phen)FeCl 2 ] + fragment with m/z = 306; compound 2 had a [( methyl phen)FeCl 2 ] + fragment with m/z = 334 and a [ methyl phenH] + fragment with m/z = 209; compound 3 had a [( sec-butyl phen)FeCl 2 ] + fragment with m/z = 418 and a [ sec-butyl phenH] + fragment with m/z = 293).

Ultraviolet Visible (UV-Vis) Spectroscopy
Compounds 1-3 were characterized by UV-Vis spec-  troscopy.Although direct coordination of the ligand with the iron metal center was not achieved using the synthetic protocol described herein, the complex ions have absorption spectra that confirm the presence of both the protonated R phen ligand and [FeCl 4 ] − anion.In fact, in the absence of the elemental analysis, IR, and mass spectrometry data, the absorption profiles for the [ R phenH] [FeCl 4 ] complex ions could be misinterpreted as evidence for direct metal binding of the phen ligand; the π  π* charge transfer of the ligand red shifts from 260 -270 nm for the free ligand (265 nm for phen; 270 nm for methyl phen; 270 nm for sec-butyl phen) to 270 -290 nm in the complex ions (275 nm for [phenH][FeCl 4 ]; 285 nm for [ methyl phenH][FeCl 4 ]; 285 nm for [ sec-butyl phenH] [FeCl 4 ]), and two absorption bands assigned as a ligand-to-metal charge transfer are observed at approximately 315 -320 nm and 360 -370 nm (see Figure 2).The red shift in the π  π* charge transfer hasbeen previously reported as a result of the phen ligand coordinated to the metal iron, as have the presence of similar ligand-to-metal charge transfer bands in phenanthroline iron (III) chloride com-plexes [11].The similarity between UV-Vis absorption spectra for complex ions possessing protonated phen ligands and coordination complexes possessing coordinated phen ligands has been noted before, specifically for [ R phenH][AuCl 4 ] complex ions and [( R phen)AuCl 3 ] coordination complexes [5].

EDTA Stability Studies
Even though the R phen ligands did not directly coordinate to the iron (III) metal centers, competition binding experiments were carried out to determine if the substitution on the phen ligand might impact the stability of the [FeCl 4 ]+ cation in solution.In order to test the stability of the complex ions, compounds 1-3 were treated with an excess of EDTA and the UV-Vis spectra monitored for a 30-minute period.The un-substituted [phenH][FeCl 4 ] complex ion (compound 1) and methyl substituted [ methyl phenH][FeCl 4 ] complex ion (compound 2) showed an immediate dissociation of the free ligand upon addition of EDTA, evidenced by the blue shift of the π  π* charge transfer band, and the complexation of the iron (III) metal center, evidenced by the elimination of the ligand-to-metal charge transfer bands at 315 -320 nm and 360 -370 nm (see Figures 3(a) and (b)).The decomposition of compounds 1 and 2 in the presence of EDTA was essentially instantaneous, as these spectral changes were observed within the first minute upon adding the EDTA solution.
Conversely, the sec-butyl substituted [ sec-butyl phenH] [FeCl 4 ] complex ion (compound 3) displayed behavior that was significantly different from the other two compounds in the series.Although the π  π* charge transfer band immediately shifts back to the absorbance maximum observed in the free ligand, there is a gradual decrease in the ligand-to-metal charge transfer bands at approximately 320 nm and 370 nm (see Figure 3(c)).These results suggest that the sec-butyl phen ligand stabilizes the [ R phenH][FeCl 4 ] complex ion.This could potentially be attributed to the fact that sec-butyl phen is a stronger base than phen and methyl phen, thereby inhibiting the deprotonation of the ligand by EDTA.However, given the fact that the ligand-to-metal charge transfer bands are eliminated more slowly over time compared to the [phenH] [FeCl 4 ] and [ methyl phenH][FeCl 4 ] complex ions, it appears that the sec-butyl phen ligand may provide some protection to iron (III) metal center.Satterfield and Brodbelt have previously quantified the ability of substituted phen ligands to bind metal cations, and their studies indicate that the larger sec-butyl substituent on the 2,9-positions of the phen ligand enhance metal binding [7].Thus, the fact the sec-butyl phen ligand provides some protection of the iron (III) metal center is not completely unexpected.The LIFDI mass spectrometry data seem to provide fur-ther evidence of an interaction between the R phen ligands and iron (III) metal center in solution, given the fact that the molecular ions for compounds 1-3 are comprised of a [( R phen)FeCl 2 ]+ cation.

SUMMARY
It has been shown that reacting phen and 2,9-di-alkylsubstituted phen ligands with iron (III) chloride in the presence of aqueous acid results in the isolation of complex ions comprised of protonated R phen ligands and anions.Even though direct coordination of the R phen ligands was not observed, EDTA stability studies indicate that the [ sec-butyl phenH][FeCl 4 ] complex ion (compound 3) is more stable than the unsubstituted phen and methyl phen congeners, and LIFDI mass spectrometry analyses provide evidence of an interaction between the R phen ligands and the iron (III) metal center in solution.Future work will be focused on developing a synthetic protocol that results in the direct coordination between sec-butyl phen ligand and iron (III) metal center, and then comparing its binding strength to the analogous and methyl phen coordination complexes.Ultimately, this data will lend more insight about the potential role of sec-butyl phen as an iron chelator in the course of tumor cell antiproliferative studies.

Figure 1 .Figure 2 .
Figure 1.Representative LIFDI mass spectrometry data for the [ R phen H][FeCl 4 ] complex ions.The spectrum shown is for the [ sec-butyl phenH][FeCl 4 ] complex ion (compound 3).The molecular ion at m/z = 418 corresponds to the complex ion after the loss of two Cl − ligands; the fragment peak at m/z = 293 corresponds to the protonated sec-butyl phen ligand.