Multi Metal Ion Recognizing Unsymmetrical tetra Dentate Schiff bases associated with Antifungal Activity

Introduction

Researchers are interested to develop selective chemosensors to identify the trace amount of transition metal ions involved in many biological processes. Not only have that, the influence of metal ions having redox behavior in the environment also captivated the attraction of scientist  to synthesize sensor compounds capable of identifying different metal ions¹.   Qualities like  non-destructive nature, easiness in synthesis, selectiveness towards target entities, quick retention time and capability to diagnose biological samples² project chemosensors to unique place  rather than the  other instrumental method of analysis³ 

Preparation of sensors proficient in identifying cations in solution and converting the action of recognition in to documentable signal are of flourishing field with enormous amount of published works⁴. Interlocking ability of the synthesized compounds with hazardous heavy metal cations and anions hinge on the π electrons cloud available on C=N group which in turn is influenced by heterocyclic  aromatic part with nitrogen and inductive effect of substituent groups⁵. Metal-to- Ligand charge-transfer and intramolecular charge transfer may emerge for the union of sensor and targeted ions⁶.

Copper ions control the biological activity of cytochrome c oxidase, superoxide dismutase and tyrosinase enzymes.  Despite, higher insertion modify the functions of enzymes and lead to antagonistic  actions like, lethargy, hike in blood pressure, nausea and Alzheimer’s  diseases⁷. Solubility of Hg²⁺ in water makes it to penetrate  through  membrane of the cell leading to  malfunction  of brain, letdown  of kidney  functions,  infirmity of  neuro system and  Minamata disease⁸. Consumption of chocolate, milk and cookies made of milk, canned food and hydrogenated oils induces Ni²⁺ ion in biological systems. Development of cancer in respiratory organs, pneumonia, asthma and malfunctioning of nervous system has been reported⁹for the excess intake of  Ni²⁺ ions.

Consumption of Cd²⁺ions toxicity prompts productivity, hepatic and cardiovascular dysfunctions¹⁰.  Harmfulness of Pb²⁺ ion exposure include procreative malady in human, neurological dysfunctions and loss of strength at bone joints¹¹. Health hazards persuaded by manganese toxicity include damage to immune system, central nervous system, kidney function and exocrine & endocrine function of pancreas¹².

Here in we report the synthesis of new unsymmetrical Schiff bases N‘-((E)-2-nitrobenzylidene)-2-((E)-2-(ferrocenylidene)hydrazine-1-carbothiohydrazide and N‘-((E)-2-hydroxy-5-nitrobenzyliden-2-((E)-2-(ferrocenylidene) hydrazine-1-carbothiohydrazide containing aromatic part at one end and ferrocene compound at the other end of the main frame structure. Spectroscopic and  redox studies exposes that synthesized  receptor  possess sensing  aptitude  towards metal ions like, Hg²⁺, Mn²⁺ ,Pb²⁺, Cd²⁺,Ni²⁺ and Cu²⁺. 

Reported literature¹³ discloses that the biological and chemical activities of Schiff bases depend upon the sp² hybridizednitrogen donor   atom of the azomethine group.  The proteins present in microorganisms find suitability to form hydrogen bond with the active site of azomethine group containing high electro negative nitrogen atom, which in turn is responsible for the anticancer, antibacterial and antifungal activities¹⁴.  Prokaryotic nature of bacteria (unicellular organism without nucleus, cell wall &organellesand survives on the host entities) helps to develop enormous amount of antibacterial commixture whereas eukaryotic nature of fungi (multicellular organism with nucleus, cell wall &organelles and endures independently) prevent the   formulation of antifungal agents¹⁵. The newly synthesized sensor by us exhibit better antifungal activity rather than antibacterial activities.

Experimental

Materials

Chemicals [AR grade] such as Carbon disulfide, 2-hydroxy-5-nitrobenzaldehyde, ferrocenecarboxaldehyde, hydrazinehydrate, 2-nitrobenzaldehyde and silica gel were purchased from E.Merck industry. They were used without further purification. Analytical gradeNiCl₂, MnCl₂, CuCl₂,HgCl₂, Pb(OAc)₂ and Cd(OAc)₂ used in the electronic spectral   and CV studies were procured  from Sigma–Aldrich. Acetonitrile [HPLC grade] obtained from E-Merck and absolute ethanol [spectral grade] acquired from Commercial Alcohols, Canada was used for spectral studies. [99+%] secured from Chemical Center, Mumbai was used as such without purification.

Instruments

Bruker Daltonics esquire 3000 spectrometer was used to record mass spectra.  BRUKER AVANCE spectrometer [500 MHz] engaging C₂D₅OD solvent was adopted to document proton NMR spectra. Perkin-Elmer 337 spectrometer was engaged to register FTIR spectra in the range of 400-4000 cm^-1 using KBr pellets. SHIMADZU MODEL UV-1800 240V spectrophotometer was affianced to observe UV–visible spectral studies between 200 and 800 nm.  CHI electrochemical analyzer 1200B model was employed to draw cyclic voltammograms using platinum as counter electrode, Ag/AgCl as reference electrode and glassy carbon as working electrode. The C,H and N contents were analyzed with Herarus C-H-N rapid analyzer.

Synthesis of N‘-((E)-2-Nitrobenzylidene)-2-((E)-2-(ferrocenylidene) hydrazine-1- carbothiohydrazide  [R1]

Hydrazinehydrate and carbon disulphide in 3:1 molar ratio was refluxed for ten hours at 80ºC along with 0.15 mole of 2- chloroethanol as catalyst to prepare   the precursor compound thiocarbohydrazide¹⁶. To a clear solution [0.01 mole/25 ml] of purified thiocarbohydrazide in ethanol, a mixture of 2-nitrobenzaldehyde (0.01mol) and ferrocenecarboxaldehyde (0.01 mol) in 180 mL ethanol was added. After half an hourstirring, the reaction mixture was refluxed for 6-7 hours. Thin layer chromatographic technique was used to check the progress of the reaction at various time intervals. Filtration was carried out after cooling and the filtrate was concentrated to get reddish yellow coloredN‘-((E)-2-Nitrobenzylidene)-2-((E)-2-(ferrocenylidene) hydrazine-1-carbothiohydrazide. Column having silica gel as stationary phase was used for the purification of crude sample. Ethanol was used as eluent. Color: Dark reddish orange. Yield: 0.538 g (91%), m.p. 69^oC.

Synthesis of N‘-((E)-2-hydroxy-5-nitrobenzylidene-2-((E)-2-(ferrocenylidene)hydrazine-1-carbothiohydrazide[R2]

Solution containing 0.01 mole of thiocarbohydrazide in 25 ml of ethanol was added with stirring to another solution having 0.01 mole of 2-hydroxy-5- nitrobenzaldehyde and 0.01 mole of ferrocenecarboxaldehyde in 180 ml ethanol. The reaction mixture was stirred for another half an hour and then refluxed for 6-7 hours. The progress of the reaction at various time intervals was checked using thin layer chromatographic technique. The reaction mixture was filtered after cooling. Greenish yellow color solid was obtained after concentrating the filtrate. The product was further purified by column chromatography using silica gel as stationary phase and ethanol as eluent. Yield: 0.5442 g, (91%), Color: reddish yellow, m.p. 70℃.

Scheme of Synthesis

Scheme 1: Scheme of Synthesis Click here to view scheme

In-vitro activities for microorganisms

Antimicrobial studies were carried out in triplicate (in-vitro) for the synthesized ligands by standard method¹⁷ against four bacteria at 37 °C and two fungi at room temperature.

Molecular docking studies

The molecular docking study was carried out using Auto dock version 4.2.6¹⁸ to investigate the binding mode of the synthesized compounds R1 and R2 with the target protein. The 3D crystal structure with Protein Data Bank PDB code 1PTF (Streptococcus faecalis), 6KVQ (Staphylococcus aureus), 7BU2 (Escherichia coli), 4YXB (Salmonella typhimurium) 3K4Q (Aspergillusniger),6TZ6 Candida albicans) was used as target protein and it was extracted from Research Collaboratory for Structural Bioinformatics (www.RCSB.org). Molecular Graphics Laboratory (MGL) tools of Auto dock were employed to get the docking score. Engaging 3D optimization tool, the structures of the compounds R1 and R2 were drawn using ChemSketch and converted to 3D structure. Geometrical optimization of ligands using ligand module was implemented using Molecular Mechanics Force Field 94(MMFF94) as implemented in the software. The analysis was done by docking the prepared ligand with the selected protein for its affinity towards the particular amino acid residue present and calculated the H-bond interaction and binding energy (K Cal/mole).

Elemental and Mass Spectral analysis

The data obtained on elemental analysis of the synthesized compounds matches very well with the theoretical values.  R1 (Found: C, 50.61; H, 3.73; N, 15.51; Fe, 12.18; Calc. for C₁₉H₁₇N₅O₂SFe: C, 50. 66; H, 3.77; N, 15.55; Fe, 12.20 %). R2 (Found: C, 50.59; H, 3.70; N, 15.47; Fe, 12.16; Calc. for C₁₉H₁₇N₅O₃SFe: C, 50.66; H, 3.77; N, 15.55; Fe, 12.20 %).

On mass spectral analysis, theadvent of molecular peak (ESI) m/z at 434 and 450 respectively for the compounds N‘-((E)-2-Nitrobenzylidene)-2-((E)-2-(ferrocenylidene) hydrazine-1-carbothiohydrazide &N‘-((E)-2-hydroxy-5-nitrobenzylidene-2-((E)-2-(ferrocenylidenehydrazine-1-carbothiohydrazide confirm the formation of expected receptors.

FTIR Spectral analysis

In the  FTIR spectrum of compound R1(Fig.1), the peak observed   around 500 cm^-1 and 830 cm^-1  are  assigned   to ferrocenecyclopentadienyl ring tilt stretching vibration and   C-H out of plane bend vibrations respectively¹⁹. The peak positioned between 900 cm^-1 to 1080 cm^-1 are allocated to the δ-C-C-H bending vibration in the penta cyclic ring.  The peak at 1104 cm^-1 is allotted for breathing ring deformation²⁰ vibration.  The  peaks appeared at 1340 cm^-1, 1519cm^-1 and  1567cm^-1 are assigned for  C=S  group stretching vibration, C-C stretching vibration of pentacyclic  ring and   NO₂ group vibration respectively.  The appearance of  –C=N stretching vibration  peak at 1650 cm^-1 confirms the formation of Schiff base and is lower than the vibration frequency  of –C=O group of the –CHO group present in ferrocene (1678 cm^-1)¹⁹. The absorption peak emerged at 2059 cm^-1 has been earmarked for aromatic stretching vibration. The peaks observed between 3200 -3400 cm^-1 is attributed to stretching vibration of secondary amine and water of hydration. Compound R2   also give the above mentioned peaks and the  stretching vibrational modes of  phenolic-OH appears along with secondary amine and water of hydration peaks in 3200 – 3400 cm^-1region itself ²¹.

Figure 1: FTIR spectrum of R1. Click here to view figure

NMR Spectral analysis

The proton  NMR spectrum  of  R1 in C₂D₅OD solvent ( Fig.2) contains relevant peaks and are assigned  accordingly δ,(ppm) 8.4(s, 2H, NCH),7.8(m, 4H aromatic), 4.8(m, 2H, (cp(subt.)), 4.4(m, 2H, (cp(subst.)), 4.2(s, 5H,( cp(unsubst.)), 1.19(s, 2H, 2NH). For compound R2 alike spectral peaks 8.4(m, 2H, NCH), 8.3 (s, 1H, aromatic), 8.1(s, 1H, aromatic), 7.0 (s, 1H,Ar),  4.4(m, 4H, (cp( subst.)),  4.2 (m, 2H, (cp(unsubst.)), 3.9(s, 5H,cpunsubst), 1.14(s,2H, NH),along with a prominent singlet at δ5.0(s, phenolic-OH) appear in the spectrum.

Figure 2: Proton NMR spectrum of R1. Click here to view figure

Results and Discussion  

Investigation of sensing nature of receptors 

Exploration of the ability of receptors to imprisonment with the various metal ions was carried out by titration method while recording the UV-Visible spectra. Twenty μL aliquots of metal solutions (10^-2M) were added to 2.5 ml of receptor solution (10^-5 M) taken in the quartz cell.  Since, chloride salts of copper, mercury and nickel are soluble in acetonitrile, solution of the receptor in acetonitrile was used for the above three metal salt solutions. Alcoholic solution of receptors was used for the chloride salts of manganese and acetate salts of lead & cadmium as these salts are soluble in ethanol.

Inacetonitrile R1 shows two shoulders around 259 nm and 313 nm (Fig.3a). Alcoholic solution of R1 displays three peaks near 205 nm, 243 nm and 313 nm (Fig.3b).  Aromatic ring π-π* transitions are assigned for above observation²².

Figure 3: Electronic spectra of R1 in a) acetonitrile b) ethanol. Click here to view figure

Effective coordination of Cu²⁺ ions with receptor is exposed by the development of   new prominent peaks around 305 nm, 350 nm and 460 nm (Fig. 4a).  The 460 nm peak (Fig.4b) has been assigned²³for MLCT band which has developed after the coordination of Cu²⁺ ions with receptor.  Development of additional peaks near 305 nm and 350 nm (Fig.4c) at the expenses of the shoulder peaks of receptor also ascertain the sensing capacity of R1.

Figure  4: Spectral changes noticed for the addition of Cu2+ ion to R1 a) Overall changes b) Formation of MLCT band c) Development additional peaks near 305 nm and 350 nm Click here to view figure

Successive addition of Hg²⁺  ions generate new peak around 237 nm (Fig. 5a) and  Pb²⁺ ions cause  overall blue shift for all the base peaks (Fig. 5b) of  the receptor R1.  Similarly consecutive addition of   Ni²⁺ ions gives   a shoulder around 269 nm (Fig. 5c).  Increase in absorbance value is noticed in the overall wavelength region for cumulative addition of Mn²⁺ and for Cd²⁺ ions along with the disappearance of shoulder at 243 nm ²².

Figure  5: Spectral changes noticed for R1 with the addition of a) Hg2+ ions b) Pb2+ ions c) Ni2+ions Click here to view figure

Aromatic ring π-π* transition of   R2 appear as a shoulder around 298 nm in acetonitrile and as a prominent peak at 313 nm in ethanol (Fig. 6).

Figure 6: UV-Visible spectrum of R2 in a) acetonitrile b) ethanol. Click here to view figure

Spectral changes observed for the addition of Cu²⁺ ions to R2 also generate  peaks near 303 nm,  354 nm and 460 nm (Fig.7a,b,c), which  confirms that  R2  is efficiently  sensing the Cu²⁺ ions.

Figure 7: Change in the absorbance spectrum of R2 with Cu2+ ions a) overall changes b) formation of MLCT band c) generation of new peaks  at 303 nm and 354 nm. Click here to view figure

Discerning ability of R2 towards Hg²⁺ , Ni²⁺ and Pb²⁺ ions is exposed by the formation of new peak at 230 nm  for Hg²⁺(Fig.8a),  blue shift of 298 nm shoulder  to 278 nm for Ni²⁺ (Fig.8b)  and conversion   of 313 nm peak to a  broad shoulder at the same wavelength (Fig.8c). 

Figure 8: Discerning ability of R2  towards a) Hg2+ions, b) Ni2+ ions, c) Pb2+ ions. Click here to view figure

Interaction studies with cyclic voltammetry

Responses to the applied potential were documented in cyclic voltammetry to establish the sensing priority order. Increasing   ΔE_P, I_pa and I_pc values  (Table 1) noticed  in the  voltammograms recorded  with   different scan  rate (20, 50 & 100 mV/ sec)  for  metal  free R1 (Fig.9) and over blowed ΔE_p values  (99-140 mV other than the expected 59 mV) emphasized the  quasi-reversible one-electron redox process²⁴.

Figure 9: Cyclic voltammograms of R1 (1X10-3 M)  with different scan rate in a) acetonitrile b) ethanol. Click here to view figure

Table 1: Electrochemical parameters for R1

Scan Rate- mV/ sec	Epa                (V)	Epc           (V)	ΔEp         (V)	E1/2         (V)	Ipa x10-5                (μA)	Ipcx10-5                    (μA)
Solvent -Acetonitrile
20	0.749	0.634	0.115	0.692	-0.928	0.224
50	0.760	0.632	0.128	0.696	-1.346	0.495
100	0.764	0.623	0.140	0.694	-1.818	0.774
Solvent – Ethanol
20	0.743	0.636	0.107	0.689	-0.371	0.090
50	0.750	0.651	0.099	0.700	-0.627	0.259
100	0.768	0.638	0.130	0.703	-1.059	0.487

 

The detected positive potential shift for oxidation peak and negative potential  shift for reduction peak^25-26in the voltammograms logged in the CV titration (to 10 ml of 10^-3 molar R1 solution 20 μL of 10^-3 molar metal solution were added up to 7eq) under equimolar (10^-3 R1/10^-3M²⁺) and multimolar (10^-3R1/10^-1M²⁺) concentration reveals that the synthesized receptors is capable of sensing deferent metal ions. Figure 10 chronicled for the addition of Cd²⁺ ions is presented here as a reference.

Figure 10: CV titration study of R1 and Cd2+ ions [50 mV/s] under a) equimolarb) multimolar conditions Click here to view  figure

The changes   noticed in the I_pa values (Table 2) for the addition of different metal ions with 10^-3M concentration (Fig.11) and experiential magnified ΔE_pamount(111- 138 mV) discloses  the different binding ability   of metal cation and also the effect of  electrostatic repulsion operated  between the  oxidized  ferrocene moiety  and  bonded metal cations²⁷.Accessing  the differences (ΔI_pa%)  between  the I_pa values noticed for the Fe^II/Fe^III oxidation wave of  receptor solution and different metal ions added  receptor solutions, uncover the  coordination order of R1 as Hg²⁺ (81) >Pb²⁺ (17) > Ni²⁺ (15.7) >  Mn²⁺(12.8) > Cd²⁺(4.2)>  Cu²⁺(3.7).

Figure 11: Cyclic voltammograms recorded for various metal ions with R1 [50 mV/s]  in a) acetonitrile b) ethanol. Click here to view figure

Table 2: Electrochemical data for equimolar titration (R1,10^-3 M / M²⁺, 10^-3 M) (Scan Rate 50 mV/ sec)

Addition	Epa (V)	Epc (V)	ΔEp (V)	E1/2 (V)	Ipa x10-5   (μA)	Ipcx10-6   (μA)
Solvent – Acetonitrile
Receptor	0.766	0.630	0.136	0.698	-1.352	4.959
Hg2+	0.754	0.622	0.132	0.688	-7.458	1.752
Cu2+	0.766	0.628	0.138	0.697	-1.386	4.112
Ni2+	0.762	0.619	0.142	0.691	-1.139	3.217
Solvent  –  Ethanol
Receptor	0.756	0.644	0.111	0.700	-7.303	2.627
Cd2+	0.754	0.640	0.113	0.697	-7.624	3.591
Mn2+	0.745	0.624	0.121	0.685	-6.361	2.985
Pb2+	0.748	0.624	0.124	0.686	-8.869	4.353

 

For multimolar  concentration, witnessed (Table 3)  binding  power  based on  declining in ΔI_pa (%) of  oxidation tendency  of metal ion  coupled with  R1 is Cu²⁺(86.07)> Hg²⁺(85.6) > Ni²⁺(85.47) > Cd²⁺(15.4) > Mn²⁺(81.96) > Pb²⁺(83.15). Comparison of  sensing priority  of R1 towards  several   metal ions  at  homo and hetero molecular concentrations  divulge R1 is effective  towards Cu, Hg, and Ni ions at  higher concentration  of metal salts   and  at lower concentration adept lead for  Hg, Pb and Ni ions (Fig. 12).

Figure 12: Comparison of sensing ability of R1 and metal ion concentration. Click here to view figure

Like R1, R2 also display same trend (Table 4) in the values of ΔE_P, I_pa and I_pc upon scanning with different scan rate

Table 4: Electrochemical parameters for R2.

Scan Rate- mV/ sec	Epa (V)	Epc (V)	ΔEp (V)	E1/2 (V)	Ipa x10-5          (μA)	Ipcx10-6       (μA)
Solvent -Acetonitrile
20	0.787	0.683	0.103	0.735	-0.746	2.822
50	0.795	0.685	0.109	0.740	-1.137	4.846
100	0.797	0.683	0.113	0.740	-1.628	7.525
Solvent – Ethanol
20	0.741	0.648	0.092	0.695	-0.397	0.681
50	0.750	0.651	0.099	0.700	-0.638	2.531
100	0.766	0.642	0.124	0.704	-1.037	5.328

 

Homo molar (10^-3, R2/10^-3, M²⁺) and hetero molar (10^-3, R2 /10^-1, M²⁺) titration studies (Fig.13) exemplify similar sensing behavior to metal ions. Calculated   ΔI_pa(%) values depict,   fastening trend for R2 under same molar condition  as  Pb²⁺ (81.3) > Cu²⁺ (76.8)> Mn²⁺ (73.35)> Ni²⁺(23.6) > Cd²⁺(22.12) > Hg²⁺(8.4) (Table 5) and for different molar it is  Cd²⁺ (82.1) > Mn²⁺(80.4) >Pb²⁺(79) > Cu²⁺(26.1) > Ni²⁺ (22.3) > Hg²⁺(18.1)  (Table 6). Above observation relate that R2 shows better recognition to Pb, Cu and Mn ions at lower concentration.  Higher quantity of Cd²⁺ is requisite for finding (Fig.14).

Figure 13: Changes in CV of R2 upon addition of different metal ion [50 mV/s] a) acetonitrile b) ethanol. Click here to view figure

Table 5: CV data for homo molar titration (R2,10^-3 M / M²⁺, 10^-3 M) (Scan Rate-50 mV/ sec).

Addition	Epa (V)	Epc (V)	ΔEp (V)	E1/2 (V)	Ipa x10-5            (μA)	Ipcx10-6             (μA)
Acetonitrile- solvent
Receptor	0.799	0.685	0.113	0.742	-1.143	4.888
Hg2+	0.785	0.675	0.109	0.730	-1.248	5.426
Cu2+	0.799	0.685	0.113	0.742	-1.152	3.961
Ni2+	0.803	0.681	0.121	0.742	-1.498	4.849
Ethanol – solvent
Receptor	0.750	0.651	0.099	0.700	-6.277	2.593
Cd2+	0.752	0.642	0.109	0.697	-8.057	4.050
Mn2+	0.781	0.613	0.167	0.697	-1.672	8.890
Pb2+	0.774	0.634	0.140	0.704	-1.172	4.565

 

Figure 14: Comparison of binding ability of R2 and metal ion concentration. Click here to view figure

Table 6: CV data for hetero molar titration (R2,10^-3 M / M²⁺, 10^-1 M) (Scan Rate-50 mV/ sec).

Addition	Epa (V)	Epc (V)	ΔEp (V)	E1/2 (V)	Ipa x10-5             (μA)	Ipcx10-6            (μA)
Solvent -Acetonitrile
Receptor	0.799	0.685	0.113	0.742	-1.143	4.888
Hg2+	0.795	0.659	0.136	0.727	-1.397	5.451
Cu2+	0.797	0.661	0.136	0.729	-1.548	5.785
Ni2+	0.777	0.659	0.117	0.718	-1.473	5.653
Solvent -Ethanol
Receptor	0.750	0.651	0.099	0.700	-6.277	2.593
Cd2+	0.777	0.6221	0.154	0.699	-1.117	5.803
Mn2+	0.756	0.624	0.132	0.690	-1.226	5.649
Pb2+	0.768	0.619	0.148	0.694	-1.306	7.112

 

Antimicrobial Studies

Disc diffusion method (Mueller Hinton Agar base) was adopted to discover antibacterial activity of R1 & R2 against Streptococcus faecalise, Staphylococcuse aureuse,  Salmonella typhimurium and Escherichia coli(Fig.15). Likewise, anti-fungal studies for fungi Candida albicans and Aspergillusniger was done using Sabouraud’s Dextrose agar as base(Fig.16). Table-7 highlights the zone of inhibition inmmperceived for the synthesized compounds R1 and R2 in antimicrobial analysis.

Figure 15: Zone of inhibition for a) Streptococcus faecalise, b) Staphylococcuse aureuse, c) Salmonella typhimurium and d) Escherichia coli. Click here to view figure

Figure 16: Zone of inhibition for a) Candida albicans.  b) Aspergillusniger. Click here to view figure

Defense  mechanism rendered by R1 and R2  to prevent the growth of   fungus  aspergillusniger is nearly 150 to 160 %  higher than that of the value  witnessed for  the  standard  Ketoconazole, which is  unusual. Fungus Candida albicans progress is also prevented up to 50% of the standard value. Above result discloses that the compound R1 can be examined for antifungal agents formulation as there are only minimum numbers of antifungal agents available in the market¹⁵.  Retardant nature of R1 & R2 displayed for two gram positive and two gram negative bacteria are on par with standard Ciprofloxacin.

Table 7: In-vitro antimicrobial studies data.

S.No	Microorganisms	Control	R1	R2	Ciprofloxacin/ Ketoconazole
zone of  inhibition in mm for bacteria
1	Staphylococcus aureus	–	10	08	25
2	Streptococcus faecalis	–	06	–	24
3	Escherichia coli	–	08	07	12
4	salmonella typhimurium	–	08	06	27
zone of  inhibition in mm for fungi
1	Candida albicans	–	10	10	25
2	Aspergillusniger	–	12	13	08

 

Molecular docking studies

The purpose of molecular docking is to determine the mode of interaction of the complex protein -ligand. Docking results arrived are presented in the table -8. For selected fungi and bacteria,   ligand R1 binding 3D and 2D views (Fig. 17) and ligand R2 binding 3D and 2D views (Fig. 18) are presented.  The binding scores for both the compounds fall between -3.61 to – 7.45 Kcal mol^-1. Compound R1 exhibited better binding affinity with the protein 6KVQ (-6.52 Kcal mol^-1) and R2 showed higher binding affinity with proteins 3K4Q and 7BU2 with -7.32 and – 7.45 Kcal.mol^-1 respectively. Proteins 3K4Q & 6TZ6 are present in fungi, whereas protein 7BU2 is present in bacteria.  Both the compounds R1 and R2 tested were involved in H-bond against active site residue 47 ILE, 50 PHE, 59 VAL, 60 ILE and 155 THR.  Above results confirm that such type of ligand would represent a promising class for further development of a new class of antimicrobial agents which deserves further exploration.

Table 8: Results obtained from molecular docking studies.

PDB	Free binding energy, kcal mol-1		R1		R2
1PTF	R1	R2	Hydrogen bonds with receptor amino acids	Distance(Å)	Hydrogen bonds with receptor amino acids	Distance(Å)
	-3.61	-4.69	30-TYR 50-PHE 59-VAL 60-ILE 114-PHE	3.93 3.69 3.88 3.98 3.23	23-VAL 24-GLN 28-LYS 47-ILE	3.44 3.95 3.80 3.66
3K4Q	-4.18	-7.32	45-LYS 47-ILE	3.04 2.15	27-GLN 277-LYS 278-LYS	3.91 3.55 3.37
4YXB	-5.21	-6.54	22-VAL 155-THR 157-GLU 190-LEU 225-LEU	3.90 3.19 3.56 3.70 3.30	26-ALA 28-ILE 29-PRO 46-ILE 51-ARG	3.67 3.83 3.78 3.27 3.23
6KVQ	-6.52	-5.26	153-LYS 155-THR 192-ASN 198-GLY	2.27 3.05 3.27 3.76	188-ASN 189-VAL	3.32 3.50
6TZ6	-5.23	-6.30	103-ASP 142-ARG 142-ARG	2.95 2.73 2.79	50-PHE 59-VAL 60-ILE 97-TYR	3.84 3.74 3.65 3.72
7BU2	-5.48	-7.45	50-PHE 59-VAL 60-ILE	3.84 3.74 3.65	42-HIS 180-LEU 240-ASN 332-ARG	3.64 3.85 3.31 3.75

 

Figure 17: Docked pose view of R1 with a) 3K4Q b) 6TZ6 c) 7BU2 Click here to view figure

Figure 18: Docked pose view of R2 with a) 3K4Q  b) 6TZ6 c) 7BU2 Click here to view figure

Conclusion

Synthesis of unsymmetrical Schiff base compounds having ferrocencarboxaldehyde azomethine at one end and imine with aromatic aldehyde at the other end has not been reported so far. Our team  have  overawed  the hurdles  faced  by the scientist by synthesizing  Schiff  bases N‘-((E)-2-nitrobenzylidene)-2-((E)-2-(ferrocenylidene)hydrazine-1- carbothiohydrazide and N‘-((E)-2-hydroxy -5-nitrobenzylidene – 2-((E)-2-(ferrocenylidene)hydrazine-1-carbothiohydrazide. Spectral analysis by FTIR, ¹HNMR and Mass spectrum authenticate   the formation of desired sensors.  Multi-metal ions sensing competency of newly prepared materials have been uncovered in UV-Visible spectral studies. Results elucidated from electrochemical studies are    harmonized   with the data of electronic spectral studies.  Assessment of difference in anodic current perceived (ΔI_pa) throwback the relation between the concentration of metal ions and receptors for appropriatebinding. Exaggerated antifungal activities identified in in-vitro studies and high free binding energy values observed in molecular docking studies for fungus   Aspergillusniger,    provoke   the rhythm of pharmaceutical research to be under taken.

Acknowledgement

The authors acknowledge the support from Dr. K. Pandian, Professor of Inorganic Chemistry & Controller of Examinations, University of Madras for the UV-Visible spectral studies free of cost. The research scholar D.Saranya wishes to record her thanks to the State Government of Tamil Nadu, India for the annual research assistant grant.

Conflicts of interest

There are no conflicts to declare.

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