Synthesis of polydentate, multi metal ion sensing, unsymmetrical Schiff bases with complimented antifungal activity

Polydentate, unsymmetrical, and multi metal ion sensing Schiff bases comprised of ferrocenecarboxaldehyde attached azomethine group at one side and aromatic aldehyde linked imine on the other side have been synthesized. Cumulative addition of different metal salts solution to receptors solution, changes the electronic spectra contrarily and for the addition of Cu2+ ions, generation of MLCT charge transfer band responsible for the coordination of metal ion with a receptor is observed. Electrochemical data (ΔEp) arrived from the cyclic voltammograms suggest a quasi-reversible process. The modest concentration of metal ions required for effective sensing by the sensory material is calculated from the Ipa values observed for metal ion added and metal free sensor solutions. Inhibition zones noticed in in vitro analysis for two fungi, two gram positive and two gram negative bacterial stains interpret that the new compounds possess high antifungal activity. Binding energy calculation using molecular docking software also ascertains the antifungal bustle.

2-nitrobenzaldehyde, 2-hydroxy-5-nitrobenzaldehyde, dimethylmalonate, hydrazinehydrate, and silica gel were procured from E. Merck industry. HPLC grade acetonitrile acquired from E-Merck and spectral grade ethanol got from Commercial Alcohols, Canada was employed in sensing studies. Supporting electrolyte Tetrabutylammoniumperchlorate (99+% purity) received from Chemical Center, Mumbai was utilized (under care) as such in CV studies.

Instruments
Perkin-Elmer 337 spectrometer was used to record FTIR spectrum with KBr pellets in the range of 400-4000 cm -1 . Bruker Daltonics esquire 3000 spectrometer was engaged to observe the mass spectra. BRUKER AVANCE spectrometer (500 MHz) was employed to detect the 1 H NMR spectra using C 2 D 5 OD as solvent. Bruker Avance 400 MHz NMR spectrometer was utilized to record the 13 C NMR spectrum in DMSO solvent. SHIMADZU MODEL UV-1800 240V spectrophotometer was utilized to perceive the UV-visible spectra between 200 and 800 nm. CHI electrochemical analyzer 1200B model having Ag/AgCl as reference electrode, glassy carbon as working electrode and platinum as the counter electrode was exploited to identify the cyclic voltammograms. CV studies were carried out in nitrogen atmosphere using 0.1 M tetrabutylammonium perchlorate as a supporting electrolyte. Perking -Elmer 2400 series CHSN/O analyzer was hired to estimate the C, H, and N contents.

In vitro antimicrobial activity
Standard procedure reported in the literature [19] for in vitro antimicrobial studies was exploited to expose the antifungal activity against fungi and antibacterial activity against two gram positive & two gram negative bacteria. An average value obtained from three different antimicrobial processes was considered for analysis.

Molecular docking studies
To investigate the binding mode of R1 and R2 with the target proteins, Autodock 4.2.6 version [20] running on Windows 7 was used. Protein extracted from Research Collaboratory for Structural Bioinformatics (www.RCSB.org) data Bank (PDB) provided the enzymes used for docking studies. MGL tools (Molecular Graphics Laboratory) of Autodock were used to get the docking score. The structures of compounds R1 and R2 were drawn using ChemSketch and converted to 3D structure with the help of 3D optimization tool. Geometrical optimization of the ligands structures was carried out by ligand module of Molecular Mechanics Force Field 94 (MMFF94). Thereafter, the docking position of prepared ligands with preferred proteins of the enzymes was computed by engaging Autodock tools and best docked pose was analyzed.  Figure S1 and S2) on mass spectral analysis also ascertain the formation of expected receptors.

FTIR spectral analysis
Ferrocene cyclopentadienyl ring tilt stretching vibration and C-H out of plane bend vibration appear at 472 cm -1 and 822 cm -1 respectively in the FTIR spectrum of R1 ( Figure 1). The -C-C-H bending vibration in the cyclopentadienyl ring emerges between 939 cm -1 to 1227 cm -1 [21]. Stretching of -NO 2 , -CH=N (imine) and amide -C=O arises at 1344 cm -1 , 1515 cm -1 , and 1668 cm -1 respectively [22,23]. Peaks responsible for water of hydration and stretching vibration of secondary amine ascends near 3225 cm -1 and 3090 cm -1 . FTIR spectrum of R2 ( Figure S3) also contains all the above mentioned peaks and the phenolic-OH group stretching vibration appears along with the stretching vibrations of secondary amine and water of hydration peaks appeared around 3090 cm -1 to 3225 cm -1 region itself [24].

Exploration of the captivity of metal ions
The UV-visible spectrum of R1 in acetonitrile contains two peaks near 249 nm and 283 nm ( Figure 3a) and in ethanol in addition to the two peaks seemed around 246 nm & 286 nm, a shoulder emerges close to 353 nm. (Figure 3b). Aromatic  ring π-π* transition has been assigned [25] for the peaks and n-π* charge transfer transition has been allocated [26] for the shoulder that appeared in the near UV region.
The difference in solubility of metal salts splits the sensing studies into two parts. Alcoholic solutions for MnCl 2 , Pb(OAc) 2 & , Cd(OAc) 2 and acetonitrile solution for CuCl 2 , HgCl 2 & NiCl 2 were used. To conduct the titration studies in UV-visible spectrophotometer, 20 μL aliquot of 1.25 × 10 -3 M metal salt solutions were added to 2.5 mL of 1 × 10 -5 M receptor solution taken in the cuvette. Spectral changes noted for the addition of Cu 2+ ions are given in Figure 4a. The development of a new peak around 457 nm (Figure 4b) is earmarked for the formation of a metal-to-ligand charge transfer band responsible for the combination of metal ions with receptor [25].
The disappearance of π-π* transition peaks with simultaneous formation of the shoulder around 348 nm also exposes the binding aptitude of R1 towards Cu 2+ ions.
Generation of the new peak near 237 nm for the addition of Hg 2+ ions (Figure 5a), the conversation of π-π* transition peaks into shoulders at the same wavelength upon increasing the concentration of Ni 2+ ions ( Figure 5b) and blue shift of ligand peaks (246 nm to 238 nm and 286 nm to 283 nm) for the hike of Pb 2+ ion ( Figure 5c) concentration expose the binding aptitude of R1. Cumulative addition of Cd 2+ and Mn 2+ ions increases absorbance value at the λ max of sensor peaks for the first two additions and thereafter a decrease in absorbance was noticed.
The electronic spectrum of R2 in acetonitrile (solvent) shows a peak at 272 nm and two shoulders around 300 nm & 363 nm (Figure 6a). In solvent ethanol two prominent peaks near 238 nm & 356 nm and a broad peak closer to 298 nm have been observed ( Figure 6b). Observations made in the lower wavelength region have been assigned for π-π* transition and higher wavelength area have been allotted for n-π* transition [27].   Coordination of R2 with Cu 2+ ions is exposed by the generation of MLCT [25] absorption peak (459 nm) along with the development of new peak near 357 nm and a shoulder around 248 nm (Figure 7a). Incarceration of Hg 2+ and Pb 2+ ions by the receptor is revealed upon the formation of new peak at 237 nm ( Figure 7b) and gradual disappearance of 298 nm broad peak (Figure 7c) respectively. Imprisonment of Ni 2+ is exposed by the transformation of π-π* transition peaks into shoulder at the same wavelength for the addition of Ni 2+ ions. Captivity of Cd 2+ and Mn 2+ is realized by the increase in absorbance value noticed in all wavelength regions for the successive addition of Cd 2+ and Mn 2+ ions.

Sensing analysis with electrochemical redox studies
Sensing priority analysis is investigated against applied potential by comparing the electrochemical data obtained for metal free and metal added receptor solutions. Data extracted from the voltammograms (Figure 8a & 8b) recorded for R1 with different scan rate is associated with increased ΔE P , I pa & I pc values ( Table 1). Noticed enhanced ΔE p values (78-126 mV instead of 59 mV) emphasize the quasi-reversible one-electron redox behavior of ferrocene moiety [28].
Voltammograms registered for the titration carried out in the three electrode cell compartment by keeping 10 mL of 1 × 10 -  The difference in coordination ability of the various metal ions with the newly synthesized sensor compound is reflected in the observed varied amount of I pa and ΔE p values ( Table 2) incurred from the electrochemical data. Further, the difference in the ΔI pa ( %) calculated from the I pa values noticed for the oxidation wave of receptor solution and different metal ion added receptor solution unearth the binding ability sequence of R1 as Cu-84.4 > Hg-30.3 > Cd-22.8 > Pb-22.8 > Mn-7.1 > Ni-2.83. It is proposed that the first factor for the difference in the obtained I pa and ΔE p values might be the variation in repulsive force operating between the oxidized ferrocene unit and sensed metal cations. The second factor might be the affinity acting between the sensor and metal ions [31].
In the CV titration with 1 × 10 -3 M receptor solution and 1 × 10 -1 M metal salts solution, the assessed percentage decrease in ΔI pa , which in turn estimated from anodic current (I pa ) values noticed (Table 3) (Figure 11) .
The I pa , I pc, and ΔE P values noted for R2 (Table 4) with various scan rate (Figure 12a & 12b) follow the similar trend observed for R1 and reflect the quasi-reversible reduction process.

Antimicrobial studies
The ability of the newly synthesized compounds to prevent the growth of bacteria was investigated against Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, and Streptococcus faecalise in Mueller Hinton agar base by disc diffusion method ( Figure 14). Likewise, the capability of R1 & R2 to restrict the progress of fungi was carried out in the base    Sabouraud's Dextrose agar for Candida albicans and Aspergillus niger ( Figure 15). Average values of growth inhibition distances obtained from three different experiments are given in Table 7. Compounds R1 and R2 avert the growth of fungus Aspergillus niger strongly to a level of 100% and 87.5% respectively like that of the standard material ketoconazole. Progression of Candida albicans is also prohibited up to 35%. Observed antibacterial activities are on par with ciprofloxacin (standard material).The above results demand more focused research by pharmacists to develop new formulations for fungi using the new compounds R1 and R2.

Molecular docking
The way of interaction of the complex protein and ligand is investigated using molecular docking methods. Results obtained in the above analysis are given in Table 8. The 3D and 2D view binding of R1 & R2 are denoted in Figure 16