Synthesis and Antimicrobial Assessment of Fe 3 + Inclusion Complex of p-tert-Butylcalix [ 4 ] arene Diamide Derivative

Present study deals with the synthesis of the p-tert-butylcalix[4]arene diamide derivative as ligand (L) and its Fe complex, followed by its characterization using TLC and FT-IR, while UV-Vis and Job’s plot study were performed for complex formation. Antimicrobial activity of the derivative (L) and its metal complex was carried out by the disc diffusion method against bacteria (Escherichia coli and Staphylococcus albus) and fungi (R. stolonifer). Different concentrations of the derivative (L) (6, 3, 1.5, 0.75, and 0.37 μg/mL) and its Fe complex were prepared, and Mueller–Hinton agar was used as the medium for the growth of microorganisms. Six successive dilutions of the derivative (L) and Fe complex were used against microorganisms. Two successive dilutions (6 and 3 μg/mL) of the derivative (L) showed antibacterial action against both Gram-positive and Gramnegative bacteria. In addition, three successive dilutions (6, 3, and 1.5 μg/mL) of the derivative (L) showed antifungal activity. However, all of six dilutions of the Fe complex showed antimicrobial activity. Derivative (L) showed 3 and 1.5 μg/mL minimum inhibitory concentrations (MIC) against bacteria and fungi, respectively. On the contrary, its Fe complex showed 0.37 μg/mL value of MIC against bacteria and fungi. Hence, Fe complex of the derivative (L) was found to be a more effective antimicrobial agent against selected bacteria and fungi than the diamide derivative (L).


Introduction
During the past thirty years, calixarenes have been successfully merged into different fields because of their remarkable architectural distinctiveness.Sufficient literature has been published by researchers in which various applications of calixarenes have been explored like in catalysis, sensor technology, HPLC as the stationary phase, membrane technology, enzyme mimetic, chiral separation, nonlinear optics, and ion selective study and also as antimicrobial agents [1][2][3][4][5][6].
On the basis of their low mammalian toxicity, calixarenes were reported to have widespread applications in the field of pharmaceutical and biological sciences [7][8][9].A group of researchers first time published research on antibacterial action of calixarenes.In their research, calixarenebased compounds were used against tuberculosis and mycobacteriosis models [10].Another group of researchers showed the antibacterial activity of vancomycin-modified calix [4]arenes.ey explored their synthesized compounds for the antimicrobial activity against bacteria, i.e., Bacillus cereus, Staphylococcus epidermidis, and Staphylococcus aureus [11].In 2006, researchers designed novel antifungal agents hybrids of calix [4]arene and amphotericin B [12].
ey observed that the antifungal action of both compounds was either superior or equivalent to amphotericin B. e calixarene-based derivatives also proved to be excellent antifungal agents against selected fungi [13].Recently, in 2012, the bioactivity assessment of water-soluble calix [4] arene derivatives was performed [6].e compound showed good antimicrobial activity against selected microorganisms.Recently, some calix [4]arene-based diamide compounds as well as the Cu 2+ complex were explored for antimicrobial activity [14].
e current study addresses the synthesis and characterization of the diamide derivative of p-tert-butylcalix [4] arene (L) and its Fe 3+ complex and the assessment of relative antimicrobial studies of ligand and its complex.

Materials and Methods
Gallenkamp (UK) model was used to check the melting points on a M.F.B. 595.01 M, using conserved capillary tubes.FT-IR spectrophotometer ( ermo Nicollet AVATAR 5700) was also used (in the range 4000 to 400 cm −1 ) by applying the KBr pellet method.CHNS instrument model Flash EA 1112 elemental analyzer (20090.Rodano, Milan, Italy) was used for elemental analysis.PerkinElmer (Shelton, CT06484, USA) Lambda-35 double-beam spectrophotometer was used for UV-Vis analysis, and standard 1.00 cm quartz cells were used.
Analytical grade chemicals were used without further purification.
ey were obtained from different sources: Merck (Darmstadt, Germany), Alfa Aesar (Germany), and Sigma (St. Louis, MO, USA).Plates (obtained from Merck), precoated with silica gel, were used for thin layer chromatography analysis.Glass apparatus was carefully cleaned and washed with 5 M nitric acid and subsequently with deionized water prior to use.Millipore Milli-Q Plus water purification system (ELGA Model CLASSIC UVF, UK) was used to obtain deionized water for the preparation of aqueous solutions.For the antimicrobial activity, culture medium was obtained from Oxoid Ltd, Basingstoke, Hampshire, England (Batch no.CM0337).

Experimental Scheme.
e sequential scheme for the present study is given in Figure 1.

Synthesis.
Compounds 1 and 2 were synthesized by the reported methods [15,16].Derivative (L) and its metal complex were synthesized according to the procedure depicted below.
Synthesis of the derivative (L) by the modified method was carried out by adding 1 mmol of compound (2) in 15 mL of chloroform and stirred for 5-10 minutes at room temperature.50 mL solution of ethanolamine and methanol (1 : 3 v : v) was added dropwise to a flask containing compound (2), and the reaction was kept on stirring at room temperature.After 48 hours, white gummy material appeared, and the solvent was removed via the rotary evaporator.
e remaining residue was dissolved in methanol, and distilled water was added into it; after vigorous shaking, white precipitates appeared which were filtered and dried.Recrystallization in mixture of solvents (CH 2 Cl 2 and CH3OH) gave white crystals, and the yield was 69.89% [17].Derivative (L) was characterized with FT-IR and elemental.

UV-Spectroscopic Measurements.
Complexation study was carried out by using appropriate concentrations of the derivative (L) and metal cations.Stock solution of the derivative (L) (2.6 × 10 −3 M) was prepared in 10 mL ethanol, which was further diluted to 100 mL (2.58 × 10 −5 M) in ethanol. is method was used for spectroscopic analysis of the derivative (L).Using absorption cell of 1 cm, 10 equivalents of the essential metals were measured.2 mL of the derivative (L) (2.6 × 10 −5 M) and 2 mL of metal salts (2.6 × 10 −4 M) were mixed together in 5 mL glass test tubes separately.Spectral response of the derivative (L) before and after the mixing of metals was recorded.

Determination of Complex Stoichiometry.
Stoichiometric ratio of the derivative (L) and metal (Fe 3+ ) was determined using Job's plot method.For this method, equimolar solutions (2.6 × 10 −5 M) of both host and guest were added to each other in 1 : 9 to 9 : 1 varying ratios [18,19].3+ Complex.Iron complex was prepared according to Job's plot.e saturated solution of ligand was separately prepared followed by addition of stoichiometric amount of metal ions into the ligand solution.Subsequently, the solution was stirred at normal temperature for 24 hours.Finally, the solvent was evaporated, and the bright crystals obtained were dried in a vacuum oven.Complex was characterized with FT-IR and elemental analysis.

Microbial Study.
e modified disc diffusion method of Kirby-Bauer [20] was used to check the in vitro antimicrobial activity of the derivative (L) and its Fe 3+ complex.With the help of American Type Culture Collection (ATCC), antimicrobial activity was studied against different microorganisms.e growth of microorganism species including bacteria and a fungus was carried out using the Mueller-Hinton agar (MHA) medium.Under the instructions of manufacturer, the culture medium was prepared and used.
To check the antimicrobial activity of the derivative (L) and its Fe 3+ complex, five successive dilutions 6, 3, 1.5, 0.75, and 0.37 (μg/mL) were prepared in absolute ethanol.Discs used were of Whatman No. 3 (Filter paper discs) with a diameter size of 6 mm.ey were soaked in each prepared dilutions for a short period of time, and then under sterile circumstances, discs were dried (to get rid of residual solvent) at room temperature.After that, the discs were placed on microbial agar plates (made in streaking fashion) and incubated at 37 °C for 24 hours (for bacteria) and 48 hours (for fungi).At the end of incubation period, the antimicrobial activity of compounds against each microbial species was recorded by measuring the zone of the inhibition diameter in millimeter (mm), and MIC levels were calculated.e tests were carried out in triplicate as suggested [21].

UV-Vis Study. UV-Vis spectroscopic technique was
used to explore the complexation behavior of the derivative (L) against various essential metal ions, i.e., K + , Na + , Mg 2+ , Fe 3+ , Co 2+ , Zn 2+ , and Ni 2+ .10 eq. of various metal ions was used against the derivative (L) (2.6 × 10 −3 M) in absolute ethanol to check the complexation ability.Figure 2 shows the UV-Vis spectra of the derivative (L) after the addition of metal ions.
e result showed the prominent variations in peaks during complexation.e obtained spectra showed that the derivative (L) possesses good attraction towards the essential metal ions (showing the di erence in region 220-230 nm), but it showed noticeable interaction to Fe 3+ .e electronic spectra of the derivative (L), Fe 3+ , and Fe 3+ complex were recorded in ethanol, as given in Figure 3, and values of wavelength maximum are given in Table 1.e reported complex is novel having brownish color.Actually, the transition metals having incomplete d orbital absorb radiation in the UV-Vis region and show electronic transitions between di erent energy levels, whose separation is largely controlled by ligand species.e spectrum observed in Figure 3 for the derivative (L) indicates that 215 and 280 nm peaks are produced by intraligand transitions.In case of the Fe 3+ complex, red shifts were observed.In the complex, peaks observed at 250 and 300 nm are assigned to characteristic metal d-d transitions.It indicates that metal and ligand molecules are well incorporated to give observed spectral changes.Likewise, when metal and ligand are coordinated, ligand causes splitting within d orbitals of metal shifting it to longer wavelength.Spectrum of ligand has shown a bathochromic shift after coordination.
Iron gives octahedral geometry here because it produces six coordination environments.Since Fe 3+ gives the d 5   system, whereas Fe 2+ gives d 6 , the high charge density and small size of Fe 3+ make it a hard base, where O and N donor ligands appear to be small sized, and relatively nonpolarisable ligands are hard bases; hence, this type of hardhard type acid-base combination favors signi cantly stable Fe 3+ derivative (L) complex, whereas Fe 2+ is classi ed as borderline acid which will probably give less stable complex.
e complex has faint brownish color mostly due to the d-d transition that gives weak colors since it is a Laporte forbidden transition.However, transitions after complex formation may involve electronic rearrangements of valence electrons.Moreover, complex color is mostly dependent on metal, oxidation state of metal, and number of d electrons in metal, as Fe 2+ gives green color, while Fe 3+ produces brown color.Contrary to this, charge transfer is less probable here because internally it involves the oxidation-reduction process.LMCT and MLCT cause the redox changes and may give very strong/sharp peaks.Such change is not observed here, but instead weak peaks are observed here evidencing the peaks to be produced from d-d transitions.However, it is believed that variation in absorption peaks is associated with stable chelate complex formation in case of iron, which further supported the complexation.Furthermore, the study was carried out on the Fe 3+ complex.

Concentration Study.
In order to evaluate the complexation e ciency of the derivative (L), the concentration of Fe 3+ was steady increased from 1.0 to 10 equivalents.e results shown in Figure 4 indicate that adsorption intensity of the derivative (L) was increased linearly with the increasing dose of Fe 3+ ions.ese results suggest that derivative (L) possesses appreciable binding e ciency with Fe 3+ .3+ Complex.Technique of constant variation was performed for Job's plot using absorbance versus mole fraction (X) to know the stoichiometric ratio between the derivative (L) and Fe 3+ metal ion.In case of Job's plot study for the Fe 3+ complex, it is revealed that derivative (L) shows good complexation at 0.5 mole fraction (Figure 5), which re ects that derivative (L)and Fe 3+ possesses good relation in 1 : 1. erefore, the reaction was set between derivative (L) and Fe 3+ in absolute ethanol using indicated appropriate ratios for complex synthesis.3+ Complex.FT-IR study helps to con rm the complex formation by showing disappearance and appearance or shifting of some peaks present in the derivative (L) spectrum after complexation.Figure 6 shows the comparable spectra of the derivative (L) and its Fe 3+ complex in which reasonable changes are observed.A band 1666.49cm −1 for C O is shifted to 1659.76 cm −1 in the Fe 3+ complex spectrum.Also a band 1549.27cm −1 for NH bending is shifted to 1541.39 cm −1 after complexation.Two peaks at 1110.4 cm −1 and 1059.34 cm −1 for C-O stretch of hydroxyl of the ethanolamine group are shifted to 1104.2 cm −1 and 1051.6 cm −1 after complexation.e OH stretch peak at 3382.99 cm −1 is also shifted to 3378.9 cm −1 on complexation with Fe 3+ .ese remarkable changes suggest that Fe 3+ coordinates with nitrogen, oxygen of the carbonyl group, and oxygen of the hydroxyl group.In addition, these changes indicate the complex formation.

Antimicrobial Study.
Derivative (L) and its Fe 3+ complex are good antimicrobial agents, as shown in Tables 2 and 3. Table 1: λ max of various transitions in the derivative (L) and iron complex.

Compounds
Transitions (λ in nm) Derivative (L) 215 280 L-Fe 3+   Journal of Chemistry ey showed antimicrobial activity against selected microorganisms (S. albus, E. coli, and R. stolonifer).However, it is important to note that the Fe 3+ complex showed higher antimicrobial activity as compared to the derivative (L).Increase in the activity of complex can be explained on the basis of Tweedy's chelating theory [22] and overtone concept [23,24].A preliminary evaluation of the antimicrobial activity of the derivative (L) and Fe 3+ complex against S. albus and E. coli was performed and gauged according to the following four stage criteria: (+++) High antimicrobial activity; zone of inhibition is 12-17 mm (++) Relatively high antimicrobial activity; zone of inhibition is 8-11 mm (+) Weak antimicrobial activity; zone of inhibition is 1-7 mm (−) No antimicrobial activity 3.6.Antibacterial Activity.From Figures 7 and 8, it is clear that the Fe 3+ complex showed higher antimicrobial activity (+++) at 6 μg/mL against S. albus, and for E. coli, it showed higher antimicrobial activity (+++) at 6, and 1.5 μg/mL, while it showed relatively high antimicrobial activity (++) at 3 μg/mL against S. albus and at 0.75 μg/mL against E. coli.It showed weak antimicrobial activity (+) below 3 μg/mL against S. albus and at 0.37 μg/mL against E. coli.However, derivative (L) showed relatively high antimicrobial activity (++) at 6 μg/mL and weak antimicrobial activity (+) at 3 μg/mL against E. coli.It showed weak antimicrobial activity (+) at 6 and 3 μg/mL against S. albus.No antimicrobial study (−) was observed below 3 μg/mL for both bacterial strains.

Minimum Inhibitory Concentration (MIC).
e minimum concentration at which the growth of microorganisms (bacteria and fungi) is inhibited is usually known as in vitro minimum inhibitory concentration (MIC) [25].Di erent concentration level solutions (6, 3, 1.5, 0.75, and 0.37 μg mL −1 ) of the derivative (L) and its metal complex were prepared against bacteria and fungi (Tables 2 and 3).Figures 10 and 11 show that derivative (L) had the same MIC values (3 μg/mL) for both selected bacterial strains, which means it is less e ective against bacteria because at lower concentrations, its e ect was not observed.However, the Fe 3+ complex showed high activity against E. coli and S. albus with an MIC value of 0.37 μg/mL for both bacteria, because it showed antimicrobial action even at lower concentrations.
In case of antifungal action, Figure 12 shows that derivative (L) had an MIC value of 1.5 μg/mL, which means it is more e ective against R. stolonifer as compared to selected bacteria for which it showed an MIC value of 3 μg/mL.However, the Fe 3+ complex was equally e ective against the fungi and two bacterial species tested with an MIC value of 0.37 μg/mL.

Conclusions
e derivative (L) was synthesized and explored for the complexation study with essential metals.It was revealed that derivative (L) showed selective behavior toward the Fe 3+ ion.Job's plot study evaluated that derivative (L) and Fe 3+    6 Journal of Chemistry metal ion form good complex in 1 : 1 ratio.Finally, derivative (L) and its Fe 3+ complex were bioassayed for antimicrobial activity.On the basis of MIC values, it is concluded that the Fe 3+ complex of the derivative (L) is more e ective against selected bacteria and fungi, as compared to the derivative (L) because the Fe 3+ complex showed higher antimicrobial sensitivity towards selected bacteria and fungi by showing an MIC value of 0.37 μg/mL.us, the Fe 3+ complex of the derivative (L) may prove to be a good candidate for further studies as a pharmaceutical drug.

Figure 8 :
Figure 8: Antimicrobial activity of the derivative (L) and its Fe 3+ complex against E. coli (G −ve).

Table 2 :
Zones of inhibition for antimicrobial activities of the tested derivative (L).

Table 3 :
Zones of inhibition for antimicrobial activities of the tested Fe 3+ complex of the derivative (L).