Synthesis, pH dependent photometric and electrochemical investigation, redox mechanism and biological applications of novel Schiff base and its metallic derivatives

Article abstract of DOI:10.1016/j.saa.2017.01.018

A novel Schiff base, 1-((2, 4-dimethylphenylimino)methyl)naphthalen-2-ol abbreviated as (HL) and its four metallic complexes were synthesized and confirmed by ¹H and ¹³C NMR, FTIR, TGA and UV–Visible spectroscopy. Schiff base was also characterized by X-ray analysis. The photometric and electrochemical responses of all the synthesized compounds were investigated in a wide pH range. Structures of the compounds were optimized computationally for the evaluation of different physico-chemical parameters. On the basis of electrochemical results the redox mechanistic pathways of the compounds were proposed. The cytotoxicity analysis on Hela cells revealed that HL and its complexes inhibit cell growth as revealed from their IC50 values (HL):106.7?μM, (L₂VO): 40.66?μM, (L₂Sn): 5.92?μM, (L₂Zn): 42.82 and (L₂Co): 107.68?μM. The compounds were tested for anti-diabetic, triglyceride, cholesterol, anti-microbial, anti-fungal and enzyme inhibition activities. The results revealed that HL and its complexes are promising new therapeutic options as these compounds exhibit strong activity against cancer cells, diabetics, fungal and microbial inhibition.

Full text of DOI:10.1016/j.saa.2017.01.018

Accepted Manuscript
Synthesis, pH dependent photometric and electrochemical
investigation, redox mechanism and biological applications of
novel Schiff base and its metallic derivatives
Abdul Rauf, Afzal Shah, Abdul Aziz Khan, Aamir Hassan Shah,
Rashda Abbasi, Irfan Zia Qureshi, Saqib Ali
PII:
DOI:
Reference:
S1386-1425(17)30019-7
doi: 10.1016/j.saa.2017.01.018
SAA 14871
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
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3 October 2016
7 January 2017
7 January 2017
Please cite this article as: Abdul Rauf, Afzal Shah, Abdul Aziz Khan, Aamir Hassan
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and electrochemical investigation, redox mechanism and biological applications of novel
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ACCEPTED MANUSCRIPT
Synthesis, pH dependent photometric and electrochemical
investigation, redox mechanism and biological applications of novel
Schiff base and its metallic derivatives
Abdul Rauf a, Afzal Shah a*, Abdul Aziz Khan a, Aamir Hassan Shaha,b*, Rashda Abbasi c,
Irfan Zia Qureshid and Saqib Alia
a Department of Chemistry Quaid-i-Azam University, 45320, Islamabad, Pakistan
bUniversity of Chinese Academy of Sciences, Beijing 100049, China.
cInstitute of Biomedical and Genetic Engineering Islamabad, Pakistan
dDepartment of Animal Sciences, Quaid-i-Azam University, 45320 Islamabad, Pakistan
*To whom correspondence should be addressed
Tel: +92-5190642110
Fax: +92-5190642241
E-mails: afzals_qau@yahoo.com (Dr. Afzal Shah) aamir@nanoctr.cn (Dr. Aamir Hassan Shah)
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Abstract
A novel Schiff base, 1-((2, 4-dimethylphenylimino)methyl)naphthalen-2-ol abbreviated as (HL)
1
13
and its four metallic complexes were synthesized and confirmed by H and C NMR, FTIR,
TGA and UV–Visible spectroscopy. Schiff base was also characterized by X-ray analysis. The
photometric and electrochemical responses of all the synthesized compounds were investigated
in a wide pH range. Structures of the compounds were optimized computationally for the
evaluation of different physico-chemical parameters. On the basis of electrochemical results the
redox mechanistic pathways of the compounds were proposed. The cytotoxicity analysis on Hela
cells revealed that HL and its complexes inhibit cell growth as revealed from their IC50 values
(HL):106.7 µM, (L2VO): 40.66 µM, (L2Sn): 5.92 µM, (L2Zn): 42.82 and (L2Co): 107.68 µM.
The compounds were tested for anti-diabetic, triglyceride, cholesterol, anti-microbial, anti-fungal
and enzyme inhibition activities. The results revealed that HL and its complexes are promising
new therapeutic options as these compounds exhibit strong activity against cancer cells,
diabetics, fungal and microbial inhibition.
Keywords: Schiff base; Metal-Schiff base complexes; Redox mechanism; Charge transfer;
Biological activities
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1.
Introduction
Schiff bases are nitrogenous class of organic compounds, also known as imines or azomethines.
Their antibacterial, anticancer, antitumor, anti-inflammatory, anti-tuberculosis, insecticidal,
antimicrobial and anticonvulsant activities are extensively reported [1-3] as they find
applications against a number of organisms including plasmopora viticola, candida albicans,
bacillus polymxa, mycobacteria, trychophyton gypseum, erysiphe graminis and escherichia coli
staphylococcus aureus. Aromatic Schiff bases have a wide variety of applications in medicinal
and inorganic chemistry [4]. They are used for enhancing the sensitivity and selectivity of
electrochemical and optical sensors due to their redox and optical properties [5-7]. Their metal
complexes serve as models for anticancer and herbicidal applications [8, 9]. O-phenylenediamine
based Schiff bases are bestowed with clinical properties. Schiff base obtained from p-toluidene
and furylglyoxal has been reported for its excellent inhibitory action against bacillus subtilis,
escherichia coli, staphylococcus aureus and proteus vulgaris [10]. Schiff bases of 4-
dimethylamine benzaldehyde are potent antibacterial agents. Isatin Schiff bases possess antiviral,
antiprotozoal, anti-HIV, anticonvulsant and anthelmintic activities. Some Schiff bases are also
used as antibodies and anti-inflammatory agents [11-14]. Based on the above mentioned broad
range applications, pharmacologists and biochemists are paying the utmost attention to the
synthesis of new medicinally important Schiff bases [15, 16].
Schiff bases can act as good chelating agents, [17, 18] especially when –OH or –SH
functional groups are present in their structures. A plethora of biological, industrial, analytical
and environmental applications of metal complexes of Schiff base ligands have been reported
[16], so, synthesis of new candidates of this class and detailed investigations of their optical and
redox activities related to their biological role seems conceivable. With this aim we synthesized a
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novel Schiff base, 1-((2, 4-dimethylphenylimino)methyl)naphthalen-2-ol and its complexes with
VO, Sn, Zn and Co. In electrochemical sensors, Schiff bases are used as components of the
recognition layer for the selective and sensitive detection of metal ions [19, 20]. So, we
investigated the redox behavior of our synthesized Schiff base and its metal complexes for
getting insights about their medicinal, biological and metal ions sensing role. Computational
studies were also performed and interestingly the theoretical calculations were found to support
the experimental outcomes.
2.
Experimental
2.1. Chemicals
Acetic acid, ethylenediaminetetraacetic acid disodium salt dihydrate (Na2 EDTA), 2-hydroxy-1-
naphthaldehyde, 2,4-dimethylaniline, dibutyltindichloride, L-glutamine, penicillin-G, pyruvic
acid, sodium chloride, sodium dodecyl sulfate (SDS), streptomycin sulfate, sulforhodamine B
(SRB), trichloroacetic Acid (TCA), trypsin/EDTA (5%), vanadyl(V) isopropoxide, zinc acetate,
cobalt acetate and magnesium chloride were received from Aldrich, USA. The solvents like
toluene, chloroform, n-hexane, ethanol and dimethyl sulfoxide were obtained from Merck,
Germany and dried before use. Diethanolamine and p-nitrophenyl phosphate hexahydrate were
used as received from Sigma Aldrich. Human serum was used as a source of alkaline
phosphatase.
2.2. Synthesis
2.2.1 1-((2, 4-dimethylphenylimino)methyl)naphthalen-2-ol (HL)
Stoichiometric amounts of 2,4-dimethylbenzenamine and 2-hydroxy-1-naphthaldehyde (5 mmol
of each) were added to freshly dried ethanol. The mixture was refluxed for 2 hrs. The obtained
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yellow crystalline solid was cooled, filtered and crystallized in a mixture of chloroform and pet
ether (3:1) (Scheme 1). The formation of HL was ensured from the following data.
Yield: 85 %, M.P.: 155.5 oC; Mol. Wt.: 275.33, IR υ/cm-1: 3446 (OH), 1615 (C=N), 1299 (C-
1
O). H NMR (CDCl3, 300 MHz) δ: 15.09 (s, 1H, -OH), 9.27 (s, 1H, H9, HC=N), 7.24 (d, 1H,
H3, J = 1.2 Hz) 7.09-7.12 (m, 3H, H5, H6, H12), 2.35 (s, 3H, H7), 2.44 (s, 3H, H8), 8.07 (d, 1H,
H13 J = 8.8 Hz), 7.67 (d, 1H, H15, J = 8.8 Hz), 7.30 (d, 1H, H16, J = 8.0 Hz), 7.50 (d, 1H, H17,
13
J = 8.0 Hz), 7.69 (d, 1H, H18, J = 8.0 Hz); C NMR (75 MHz) δ: 140.6 (1C, C1), 130.7 (1C,
C2), 133.3 (1C, C3), 136.8 (1C, C4), 127.8 (1C, C5), 118.6 (1C, C6), 18.1 (1C, C7), 21.0 (1C,
C8), 171.7 (1C, C9), 108.6 (1C, C10), 153.1 (1C, C11), 116.9 (1C, C12), 131.8 (1C, C13), 129.3
(1C, C14), 128.0 (1C, C15), 122.7 (1C, C16), 127.0 (1C, C17), 123.4 (1C, C18), 136.4 (1C,
C19).
By using HL as a ligand, its four metal complexes were also synthesized according to Scheme 2.
2.2.2 Oxovanadium(IV) complex of 1-((2,4-dimethylphenylimino) methyl)naphthalen-2-ol
(L2VO)
Oxovanadium complex (L2VO) was synthesized by treating the vanadyl(V) isopropoxide with
HL in 1:2 using acetonitrile as a solvent at room temperature as shown in Scheme 2. The green
colored product (L2VO) was precipitated out immediately, filtered, washed with acetonitrile and
then air dried.
o
Complex (L2VO) : Yield: 76 %, M.P.:> 300 C; Mol. Wt.: 615.61, IR υ/cm-1: 1599 (C=N),
1301 (C-O), 978 (V=O), 572 (V-O). μeff (BM) 2.04. Anal. Calc. for [C38H32N2O3V] (%); C,
74.14; H, 5.24; N, 4.55; O, 7.80; Found: C, 74.55; H, 5.20; N, 4.52; O, 8.01
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2.2.3 Dibutyltin complex of 1-((2,4-dimethylphenylimino)methyl) naphthalen-2-ol (L2Sn)
Dibutyltin complex (L2Sn) was synthesized by refluxing the dibutyltindichloride with HL in 1:2
ratio using toluene as a solvent at elevated temperature for four hours. The yellow solution was
concentrated by the process of evaporation under reduced pressure to get the precipitates of
(L2Sn).

CH2-CH2-CH2-CH3



Sn
O
Complex (L2Sn) : Yield: 70 %, M.P.: 249.3oC; Mol. Wt.: 781.61 , IR υ/cm-1: 1590 (C=N),
1
1303 (C-O), 540 (Sn-C), 551 (Sn-O), 452 (Sn-N). H NMR (CDCl3, 300 MHz) δ: 9.28 (s, 1H,
H9, HC=N), 7.28 (s, 1H, H3), 7.11-7.15 (m, 3H, H5, H6, H12), 2.39 (s, 3H, H7), 2.49 (s, 3H,
H8), 8.10 (d, 1H, H13, J = 8.4 Hz), 7.72 (d, 1H, H15, J = 7.8 Hz), 7.35 (t, 1H, H16, J = 8.1 Hz),
7.54 (t, 1H, H17, J = 8.1 Hz), 7.82(d, 1H, H18, J = 8.4 Hz); 1.40-1.48 (m, 2H, αH) 1.78-1.85 (m,
13
4H, βH, H), 0.97 (t, 3H, H, J = 7.2 Hz). C NMR (75 MHz) δ: 140.2 (1C, C1), 130.7 (1C,
C2), 133.4 (1C, C3), 137.2 (1C, C4), 127.8 (1C, C5), 118.6 (1C, C6), 18.1 (3C, C7), 21.0 (3C,
C8), 172.6 (1C, C9), 117.0 (1C, C10), 154.0 (1C, C11), 118.6 (1C, C12), 131.9 (1C, C13), 129.4
(1C, C14), 128.2 (1C, C15), 123.1 (1C, C16), 127.0 (1C, C17), 123.5 (1C, C18), 136.6 (1C,
1
C19), 26.8 (2C, αC), 26.9 (2C, βC), 26.3 (2C, C), 13.5 (3C, C), J = 423.75/405.0 Hz
(
119/117Sn, 13C).
2.2.4 Zinc complex of 1-((2,4-dimethylphenylimino)methyl) naphthalen-2-ol (L2Zn)
Zinc complex (L2Zn) was synthesized by adding zinc acetate in HL solution in 1:2 using ethanol
as a solvent at room temperature. The yellow colored product (L2Zn) was precipitated out in 5-10
minutes, filtered, washed with ethanol and then air dried.
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Complex (L2Zn) : Yield: 78 %, M.P.:173 oC; Mol. Wt.:614.06 , IR υ/cm-1: 1591 (C=N), 1310
(C-O), 474 (Zn-O), 465 (Zn-N). 1H NMR (CDCl3, 300 MHz) δ: 9.30 (s, 1H, H9, HC=N), 7.30
(s, 1H, H3), 7.13-7.16 (m, 3H, H5, H6, H12), 2.35 (s, 3H, H7), 2.44 (s, 3H, H8), 8.09 (d, 1H,
H13, J = 8.4 Hz), 7.70 (d, 1H, H15, J = 7.8 Hz), 7.33 (t, 1H, H16, J = 8.1 Hz), 7.56 (t, 1H, H17,
13
J = 8.1 Hz), 7.80(d, 1H, H18, J = 8.4 Hz). C NMR (75 MHz) δ: 140.1 (1C, C1), 130.6 (1C,
C2), 133.3 (1C, C3), 136.9 (1C, C4), 127.8 (1C, C5), 118.6 (1C, C6), 18.1 (3C, C7), 21.0 (3C,
C8), 171.8 (1C, C9), 108.5 (1C, C10), 155.1 (1C, C11), 117.0 (1C, C12), 129.5 (1C, C13), 129.3
(1C, C14), 128.1 (1C, C15), 122.8 (1C, C16), 127.0 (1C, C17), 123.4 (1C, C18), 136.4 (1C,
C19).
2.2.5 Cobalt complex of 1-((2,4-dimethylphenylimino)methyl) naphthalen-2-ol (L2Co)
Cobalt complex (L2Co) was synthesized by treating cobalt acetate with HL in 1:2 ratio using
ethanol as a solvent at room temperature according to Scheme 2. The reddish brown colored
product (L2Co) was precipitated out in few minutes, filtered, washed with ethanol and then air
dried. The formation of oxovanadium and cobalt complex was ensured from the following
thermo gravimetric analysis (TGA) data shown in Table 1.
Complex (L2Co) : Yield: 80 %, M.P.:183 oC; Mol. Wt.: 607.61 , IR υ/cm-1: 1600 (C=N), 1305
(C-O), 474 (Co-O), 507 (Co-N). μ eff (BM) 1.81. Anal. Calc. for [C38H32N2O2Co] (%); C, 75.12;
H, 5.31; N, 4.61; O, 5.27; Found: C, 75.35; H, 5.22; N, 4.75; O, 5.43.
(TGA data is given in Table 1)
2.3. X-ray structure determination and UV-Vis spectroscopic measurements
Suitable single crystal HL was mounted on a Microstar diffractometer. Data were collected by
keeping the temperature constant at 100 K. The structural calculations were solved with the
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olex2 using Charge Flipping program and refined with XL [21] refinement packages using the
least squares minimization. Crystal data and details of the parameters are summarized in Tables
S1-S3. Shimadzu 1601 spectrophotometer was used for UV-visible spectroscopic studies. Crison
micro pH 2001 pH-meter was used for pH measurements. Before each experiment fresh solution
was prepared from stock solution using 50% ethanol and 50% BR buffers.
2.4. Anti-diabetic Activity
Fifty BALB/c healthy adult male mice with an average body weight of 35.0±5.0 g were procured
from the National Institute of Health Islamabad. To reduce stress and avoid overcrowding, five
animals were housed per cage. All animals had free access to standard diet of rodents and
drinking water. Standard temperature of 28 °C and 12hL: 12hD photoperiod was maintained
throughout. Animals were subjected to diabetic disease through alloxan monohydrate (Sigma
Aldrich, USA) using a single intraperitoneal injection (i.p.) at the dose of 150 mg/kg b.w.
Diabetic level was achieved at glucose concentration > 200 mg/dl. A total of seven experimental
groups were constituted and each containing five animals. All groups were made diabetes’s
above. Five groups were treated with five different compounds; HL, 1, 2, 3, and 4 at the dose of
35.0 mg/kg b.w. The remaining two groups; positive control group and negative control group
were treated with glibenclamide (10mg/kg b.w in distilled H2O), (Euglucon, Roche Pharma) and
normal saline respectively. Blood was extracted through caudal vein at nine different times i.e. -1
(prealloxan), 0 (post alloxan) and 1-7 hrs after treatment with doses. Plasma glucose level was
measured with dextrostix strips using glucometer (Accu-Check Active,Roche). Serum
triglycerides and cholesterol were measured through commercially kits which were obtained
from Globe Diagnostic (Italy), using the standard procedure of colorimetry. One-way analysis of
variance (ANOVA) was used for data analysis using Sigma Plot (Version 12.0), Post hoc Tukey-
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kramer test, and where test for normality failed ANOVA on ranks test was applied. Statistically
significant difference was considered where P<0.05. Data are presented as line or bar graphs.
2.5.
Cytotoxicity
2.5.1. In-Vitro studies: Cell line and cell culture
Human cervix adenocarcinoma (HeLa, ATCC CCl-2) cell line was cultured in Dulbecco's
Modified Eagle Medium (DMEM) supplemented with GPPS (2 mM L-glutamine, 1 mM Na-
pyruvate, 100 U/ml penicillin and 100 µg/ml streptomycin) and 10 % fetal calf serum under a
humidified atmosphere at 5% CO2 and 37 °C. Cells were harvested by trypsinization (0.5 mM
trypsin/EDTA) for 60 sec at 25°C.
2.5.2. Cytotoxicity analysis on HeLa
Cytotoxicity caused by the compounds was tested on HeLa cells by the SRB assay as described
earlier [22]. Briefly, pre-seeded cultures (>90% viability; 1.5×105 cells/ml) were exposed to the
different compound concentrations (150, 100, 50, 10 and 5 µM) for 24 h. Untreated cultures,
solvent (5% DMSO in ethanol) treated and non-cellular background comprising of ‘media-only’
and ‘compounds-only’ samples were also included in each of the experiment. Cultures were
fixed by adding gently, 50% pre-chilled TCA and incubation at 4 °C for 1 h followed by washing
with deionized water and air-drying. Plates were stained with 0.4% SRB solution, rinsed with
1% acetic acid and then air-dried. Samples were photographed with Olympus IMT-2 inverted
microscope (magnification 60X) equipped with digital camera. The incorporated dye was
dissolved in 10 mM Tris (pH 8.0) and absorbance was measured on AMP PLATOS R-496
microplate reader at 565 nm wavelength.
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Percent viability of the treated and untreated samples was calculated relative to the untreated
control sample using the following formula
Percent viability = [Abs (565)Test samples - Abs (565)Compound only / Abs (565)Untreated control - Abs
(565)Media only] × 100
Where Abs (565)Test samples and Abs (565)Untreated control represent the OD at 565 nm for the treated
samples and untreated control samples, respectively. Abs (565)Compound only and Abs (565)Media only
represent background OD and was measured in non-cellular background samples.
2.6.
Assay of alkaline phosphatase activity
HL and its four complexes were applied to investigate their inhibitory action against the enzyme
Alkaline Phosphatase by using the reported procedure [21]. Reagent A (Diethanolamine pH 9.82
along with 0.5mmol/dm3 magnesium chloride) and reagent B (50 mmol/dm3 p-nitrophenyl
phosphate) were mixed with 4:1 to make substrate. After incubation of 300 sec at 25 °C, 40 µL
of human serum was added in 2 mL of the substrate. Absorbance was recorded after incubation
of 60 sec to check the activity of enzyme. ALP produced yellow colored product (p-nitrophenol)
by hydrolyzing the p-NPP (Scheme 3). Various amounts of HL (20 µL, 40 µL, 60 µL, 80 µL)
were added periodically from the stock solution, incubated for 180 sec and absorbance was
recorded after 1 to 5 minutes. Percentage inhibition was calculated from the average of these
measurements. The same procedure was repeated for each metal complex.
2.7. Antimicrobial activities
The fungicidal and bactericidal activities of HL and its metallic complexes were carried out by
disc diffusion method [23]. The nutrient agar and potato dextrose agar were used as growth
culture media for antibacterial and antifungal tests, respectively. The biologically active samples
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formed clear zones of inhibition around the discs. The zones were measured in millimeters using
a zone reader.
2.9. Electrochemical studies
Electrochemical investigations were carried using Autolab running with GPES 4.9 software,
Eco-Chemie, the Netherlands. Glassy carbon, Ag/AgCl (3 M KCl) and platinum wire were used
as working, reference and counter electrodes. The working electrode was centrally placed to
keep a minimum distance between the counter and reference electrodes for minimizing the IR
drop effect. The surface of working electrode was polished using diamond spray (particle size 1
m) followed by thorough rinsing with doubly distillated water before every experimental assay
[24]. All electrochemical experiments were carried out under nitrogen saturated solutions.
3.
Results and Discussion
3.1. Spectroscopic characterization of the synthesized compounds
In the FTIR spectrum the strong absorption band at 1615 cm-1 due to azomethine group shows
the formation of Schiff base (HL). Moreover, the appearance of resonance signals at 9.27 ppm
1
13
and 172.1 ppm in H and C NMR spectra confirms the formation of HL. Absence of any
resonance peak related to primary amine suggests the purity of the synthesized product. In case
of metal complexes, the absorption bands corresponding to azomethine group in the FTIR
spectra appearing at lower wavelengths in the range of 1591-1600 cm-1 suggests the coordination
of azomethine nitrogen with metal atoms. Data obtained from elemental analysis and thermo
gravimetric analysis of L2VO and L2Co were found in good agreement and indicate the
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formation of these complexes. For tin and zinc complexes, downfield shifting of azomethine
group (HC=N) resonance signals was observed and disappearance of proton resonance signal of
hydroxyl group confirmed the bonding of ligand anion with the metallic cationic parts.
Moreover, Sn-C coupling constant for dibutyltin complex was calculated from the satellite peaks
that appeared at 23.9 and 29.6 ppm in 13C NMR spectrum.
The molecular structure of HL along with crystallographic numbering scheme is shown in Fig.
S1. Summary of crystal data and details of structural refinements can be seen in Table S1.
Selected bond distances and bond angles of HL are listed in Table 2. It is evident from the data
that Schiff base has monoclinic structure. Double bond between C and N can be ensured from
the shorter N-C11 bond length as compared to N-C12 bond length. Position of H1 represents that
HL exists in zwitterionic form as well as in enolic form (Fig. S1). Intramolecular hydrogen
bonding is present between H atom of hydroxyl group and N atom in enolic as well zwitterionic
forms (Table 3). Tortional angles reveal that the molecular geometry is non-planar. The aromatic
rings are not in the form of regular hexagons due the attached moieties.
UV-visible spectra of HL, L2VO, L2Sn, L2Zn and L2Co in a 9:1 mixture of ethanol and
DMSO are displayed in Fig. 1. HL shows four bands at 318, 350, 442 and 461 nm. The first band
around 318 nm can be related to π-π* transition of the aromatic chromophore containing
azomethine group. While the second less intense band at 350 nm may be attributed to n- π*
transition of the non-bonding electrons present on the nitrogen of –C=N group [25]. The signals
at 442 and 461 nm are possible to emerge as a result of π-π* and n- π* transitions of the aromatic
chromophore containing -C=O group. This attribution is in line with the crystal studies which
offer evidence about the presence of keto group in the structure of HL. Further observation of
Fig. 1 reveals that the orbital energies of HL change upon complexation with metals. The
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appearance of a band in the wavelength range of 260-270 nm in the spectra of complexes and its
absence in the spectrum of HL offers a convincing proof for the formation of metal-HL
complexes. This band may emerge due to possible charge transfer (CT) from a metal centered
orbital to a ligand centered orbital (MLCT) or vice versa (LMCT) [25-27]. The decrease in the
intensity of signals corresponding to azomethine nitrogen and carbonyl oxygen suggests the
charge transfer bands of L2VO, L2Zn and L2Co to be LMCT in nature. While the increase in
absorbance of these chromophores in the UV-Vis spectrum of L2Sn points to the MLCT
characteristic of the charge transfer band corresponding to this complex. Such a behavior also
highlights the importance of back bonding/donation [27]. The molar extinction coefficient (ε)
values of L2VO, L2Zn, L2Sn and L2Co evaluated from their CT bands are 4.15×104, 3.90×103
3.50×103, 3.35×103 m2 mole-1 respectively. These values suggest that among the studied
complexes, VO and Co are the strongest and weakest acceptors of electrons from HL. Further
observation of the spectra shown in Fig. 1 reveals the appearance of broad bands around 500 nm.
These less intense bands compared to CT signals are attributable to d-d transition [28]. The
extinction coefficients evaluated from signals corresponding to d-d transition can help in
predicting the geometry of complexes. The expected ε values of octahedral complexes remain
less than 10 m2 mol-1, for square planar complexes their range is around 10-500, while for
tetrahedral complexes, molar extinction coefficient exceeds 500 m2 mol-1 [29]. Based on these
criteria, we speculate our synthesized complexes L2VO and L2Co (ε values 250 and
450 m2 mol-1) to have square planar geometry. However, the geometry of L2Zn and L2Sn cannot
be predicted as no d-d band appears in the spectra of these complexes.
Fig. S2-A represents the absorption spectra of HL in different pH media. HL has shown an
intense doublet band at 321 nm. Increase in pH of the medium results in hypochromic effect and
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a new band appears at 414-461 nm which further shifts to 396 nm at pH > 9. The appearance of
this new band can be related to the deprotonation of HL. An isosbestic point at 371 nm also
appears in the spectra which suggest the transformation of HL into different isomers. Tin
complex shows similar photometric response with two isosbestic points at 298 and 374 nm, thus,
offering clues about the existence of its different mesomeric forms (Fig. S2-B). No significant
change was observed in the photometric studies of complexes (L2VO), (L2Zn) and (L2Co) in
different pH media.
3.2. Electrochemical studies
Cyclic voltammetric behavior of I mM solution of HL and its complexes was examined at a scan
rate of 100 mV s-1 in media different of pH using GCE as working electrode. In solution of pH
7.0, HL registered two oxidation signals at +0.76 V and +1.01 V in the forward scan with no
peak in the reverse scan, thus, ensuring irreversibility of both the oxidation processes. The
oxidation signals were found independent of each other as shown in Fig. S3. While scanning the
potential from zero to negative going direction, one reduction peak of HL at -0.94 V was
observed in solution conditions of pH 7.
In order to investigate the influence of medium on the redox behavior of HL, cyclic
voltammograms were recorded in different pH media. It was found that redox nature of HL is
strongly pH dependent as shown in Fig. 2. In acidic solution (pH-4.0) there was no change in
oxidation peaks while the reduction peak of HL splitted into three well defined peaks. In basic
medium (pH-10.0), the two oxidation peaks overlapped and converted into a single broader peak
at +0.83 V. While no significant shift in reduction peak was observed with change in pH.
However in acidic medium a new reduction peak at -1.13 V came to sight. Fig. S4 depicts the
cyclic voltammograms of HL and its complexes in a buffered medium of pH 7.0. In case of all
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metal complexes, the two oxidation peaks overlap and result into one broader peak at +0.76 V,
+0.79 V, +0.74 V and +0.79 V for L2VO, L2Sn, L2Zn and L2Co respectively. In the negative
potential window instead of one sharp cathodic signal, two overlapping peaks appeared for
L2VO, L2Sn and L2Co. Compound L2Zn registered a sharp reduction peak at -0.44 V and the
reduced product showed irreversible oxidation at -0.24 V (Fig. S5). The shifting of peaks with
the change in pH of solutions offers a clear evidence of the pH dependent nature of all these
redox processes (Fig. 2 & S5).
The effect of scan rate on the cyclic voltammetric response was monitored for all the synthesized
compounds in pH-7.0. By using Randles-Sevcik equation, diffusion coefficients were calculated
from the slopes of the plots between Ip and υ1/2. The straight lines of the plots suggested the
diffusion controlled nature of the redox processes [30-33]. CVs of different concentrations of
analytes were obtained and used for the calculation of electron transfer rate constant (ks) using
the following equation [34].
Ip= nFACks
Where n represents number of electrons, calculated from DPV studies and A the surface area of
working electrode. ks was calculated from the slope of Ip versus concentration plot (Fig. S6). The
values of diffusion coefficients and ks for HL, L2VO, L2Sn, L2Zn and L2Co are given in Table 4.
To ensure the reversible/irreversible nature of the redox processes of HL and its
complexes, square wave voltammetric response was observed. The same direction of forward
and backward current components of all the redox processes (Fig. S7) offered clues about their
irreversible nature. These results are consistent with the cyclic voltammetric findings. Due to its
sensitive nature, SWV was used for the estimation of the limit of detection (LOD) and the limit
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of quantification (LOQ) by observing the effect of concentration on peak current values of the
analytes (Fig. 3). The LOD and LOQ values are given in Table 5.
As the redox processes were found pH dependent, so their differential pulse voltammetric
response was investigated in a wide pH range of 2-12. The no. of electrons was calculated from
half peak width values for irreversible processes using the following equation [35].
3.52 푅푇
푊
1/2
=
훼푛퐹
Fig. 4 shows the DPVs for oxidation of HL and its cobalt complex. In strong acidic media, two
well defined pH dependent oxidation peaks are observed. Intensity of 1st oxidation peak started
decreasing with the increase in pH and finally it disappeared pH higher than 4.0, while the 2nd
oxidation peak intensified and became broader with increasing pH. In all other metal complexes,
the same two oxidation signals were observed but peak 3a was not present in all cases.
Vanadium complex of HL showed a new pH dependent oxidation signal at low potential value of
+0.26 V in strongly acidic solutions. Shifting of peak 2a with pH is significant up to pH-5.0,
after that it shows no involvement of proton during electron transfer; hence it was used to
calculate the pKa values of the compounds [36]. Shifting of peak potential towards more
negative potential explains the facile oxidation of the analytes with increase in pH. A new pH
independent and broad oxidation peak in the range of pH 4.0-9.0 ensured another one electron
oxidation step of HL without involvement of proton. Number of protons involved in each
process was calculated using the following equation [35].
푑퐸푝
0.059
⁄
⁄
=
훼푛
푑푝퐻
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Slopes of Ep vs pH plots were used to calculate the number of protons involved in the oxidation
processes of HL and its complexes (Fig. 5).
Fig. S8 and S9 show DP voltammograms of reduction processes of HL and its complexes. In the
negative potential window, HL registered two pH dependent signals in the acidic pH range while
no signal in basic media. Shifting of peak potential to more negative values can be attributed to
the difficulty of reduction process due to de-protonation of the electroactive moiety of HL.
Complex (L2VO) registered two reduction peaks both of which are independent of pH. Peak 1c
remained the same in the whole pH range while intensity of 2nd peak decreased with increase in
pH and disappeared in the basic region. Tin complex (L2Sn) also exhibited two step reductions in
which first process was just an electron transfer while in the second process proton transfer
involved along with the electron transfer. Cobalt complex (L2Co) registered only one pH
independent peak in acidic media and no peak in basic pH range. Zinc complex (L2Zn) exhibited
a reduction process at a very low potential value of 0.0 V in strongly acidic solution of pH-2.0
which showed significant shifting by increasing pH of the medium. The reduced product of zinc
complex was found to oxidize in an irreversible manner as its oxidation signal also appears in the
reverse scan (Fig. S10). These findings are in agreement with the cyclic voltammetric response.
Another reduction signal appeared at -0.67 V in acidic range which diminished with increase in
pH. Number of electrons and protons involved in all these reduction processes were also
calculated and applied for suggesting the mechanistic pathways of these processes (Fig. S11 &
S12).
3.3. Biological Activities
3.3.1. Antidiabetic Activity
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Mice treated with ligand HL showed significant decrease in blood glucose level as compared to
the negative control group but showed increase as compared to positive control group (p < 0.001;
q = 8.329 and p < 0.001; q = 7.722; Fig. 6A). Serum triglycerides concentration decreased
significantly as compared to negative and positive control groups (p < 0.001; q = 8.717; and p <
0.001; q = 15.415; Fig. 7A), with no significant effects on serum cholesterol concentration (Fig.
7B). Animals treated with oxovanadium complex (L2VO) significantly increased the blood
glucose concentration as compared to the positive control group (p < 0.001; q = 12.907; Fig. 6B),
a decrease in serum triglycerides concentration as compared to both positive and negative control
groups (p < 0.001; q = 13.336 and p < 0.001; q= 6.638; Fig. 7A), while significant decrease
occurred in serum cholesterol concentration as compared to both positive and negative control
groups (p < 0.05; q = 5.172 and p < 0.05; q =4.233; Fig. 7B). L2Sn treated group demonstrated a
decrease in blood glucose concentration as compared to the negative control group (p < 0.001; q
= 18.037; Fig. 6C), while a significant decrease occurred in serum triglycerides concentration
relative to positive and negative control groups (p < 0.001; q= 14.455 and p < 0.001; q = 7.758;
Fig. 7A). Serum cholesterol concentration decreased as compared to the positive control group (p
< 0.05; q = 4.430; Fig. 7B). L2Zn treated group showed a significant decrease in blood glucose
level as compared to the negative control groups (p < 0.001; q = 16.161; Fig. 6D), a significant
decrease in serum triglycerides concentration relative to positive and negative control groups (p
< 0.001; q= 13.416 and p < 0.05; q = 6.718), while a decrease was noticeable in serum
cholesterol concentration as compared to the positive group (p < 0.05; q = 4.408). L2Co treated
group showed significant decrease in blood glucose level as compared to negative control (p <
0.001; q = 19.361; Fig. 6E), while no effect was evident on serum triglycerides concentration and
cholesterol concentrations (Figs. 7A & 7B).
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3.3.2. In Vitro studies: Cytotoxicity analysis on HeLa
The synthesized Schiff base and its complexes were tested for their cytotoxic and anticancer
activity against HeLa cell line at different concentrations (150, 100, 50, 10, and 5 µM) using
SRB assay. SRB stained samples were visualized with Olympus IMT-2 inverted microscope
(magnification 60X) and photographed (Fig 8 A-Q). The viability curves (Fig. 9) were
generated. The IC50 values of the complexes were calculated and listed in Table 6. HL (Fig 8 C,
D, E) was found toxic to the cells in comparison to the untreated culture. A significant decrease
in relative viability (p ˂ 0.05) was observed with an IC50 value of 106.7 µM. L2VO showed a
strong influence on the cellular morphology and cells became more fiber like (Fig. 8 F-H). A
strong decrease in relative viability (p ˂ 0.001) of the treated cultures was observed and this
complex showed two times higher toxicity to the cells compared to HL, with an IC50 value of
40.66 µM. Cells were found very sensitive to L2Sn as only a few live cells existed at 100 and 50
µM (Fig 8J, K). The complex induced a highly significant decrease in relative viability (p ˂
0.001) of the cultures. The IC50 value of L2Sn was 5.92 µM, thus cells showed 18 times more
sensitivity to this complex in comparison to HL. L2Zn showed a strong effect similar to L2VO on
the relative viability (p ˂ 0.001) of the cultures with an IC50 value of 42.82 (p ˂0.001; Fig 8 L,
M, N). When compared to HL, L2Co was found less toxic to the cultures with an IC50 value of
107.68 (p ˃ 0.05; Fig 8 O, P, Q).
3.3.3. Inhibition of ALP
Literature survey reveals that Schiff bases show ALP inhibition activity and their complexes
further enhance this activity. They can block the active sights of enzyme. Alkaline phosphatase
uses a wide variety of phosphomonoesters to catalyze the transfer of phosphate groups to water
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[21]. Experimental observations show that inhibition activity of HL is quite insignificant while
the presence of metals enhances the deactivation of enzyme. The minimum activity of dibutyltin
complex may be due to long alkyl chains having non-polar nature while Zn complex has shown
excellent inhibition of nearly 90%. Inhibition profile is presented in Fig. 10.
3.3.4. Antimicrobial activities
Inhibitory action of HL and its metallic complexes was investigated against various strains of
bacteria (Bacillus subtilis, escherichia coli, pasturella multocida and staphlocuccus aureus) and
fungi (Ganoderma lucidum aspergillus niger, alternaria alternate and penicillium notatum) by
disc diffusion method [37]. Streptomycin and fluconazole were selected as positive controls for
antibacterial and antifungal screening tests, respectively. A recommended concentration of a test
sample/reference drug (1mg/1mL of solvent) was introduced into the discs. The biologically
active samples inhibited the bacterial/fungal growth around discs and the zones of inhibition
were measured. The data have been summarized in Tables 7 & 8. The free ligand (HL) was
almost inactive against the tested organisms. The coordination of ligand with metals (V, Sn, Zn,
Co) induced antimicrobial activities in the complexes. Activity of the synthesized complexes was
found in good agreement with their structures. The results demonstrate that each of the
coordinated metals (V, Sn, Zn, Co) plays an important role in biological actions of these
complexes [38]. The zinc complex (L2Zn) was found the most potent inhibitor of bacteria/fungi
while the vanadyl product (L2VO) displayed the least activity among all the metal coordinated
products. The coordination of ligand with the dibutyltin(IV) moiety appreciably enhanced the
antibacterial/antifungal activity of L2Sn. The inhibitory action of organotin(IV) compounds can
be related to their interaction ability with DNA and proteins. After zinc, the highest antimicrobial
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potential was observed for ligand-Co complex. The metal complexes were found more potent
inhibitors of fungal culture as compared to bacterial growth [39].
3.4. Computational study
The DFT optimized structures of the HL and its complexes were obtained using 6-311++G** and
6-311G++ basis sets, respectively. Numbering scheme of HL used for computational calculations
is given in Fig. S13. Mullikan charges, dipole moments, optimization energies, spin multiplicity
and point groups were calculated from the optimized structures (see Table S2). Comparison of
bond lengths obtained from computational studies with that of X-ray analysis showed a good
agreement with each other (see Table 2). NMR spectra of HL was calculated theoretically using
6-311++G** basis set and compared with the experimental spectra (see Table S3). The
computational findings reveal that the zwitter ionic form of HL is more stable than its enolic
form. This structure is also consistent with its x-ray studies. Charge distribution values indicate
that O atom of hydroxyl group and N atom of azomethine group are more negative in charge thus
suggesting their preferred coordination with metal ions.
3.5
Redox mechanism of HL and complexes
The number of electrons and protons involved in redox processes were calculated from half peak
width and slope values of Ep versus pH plots. The HL exhibited three oxidation peaks in positive
potential window. Peak 1a was found to involve loss of one electron and one proton. On the
basis of voltammetric results it is suggested that one electron and one proton are lost from the
hydroxyl group at ortho position which may then convert to a free radical and possibly gets
stabilized by dimerization or by adsorption at the electrode surface. It may either form a five
membered ring with N atom or get converted to keto form with free radical resonating in the
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whole benzene ring. It can also get adsorb on the GCE surface or dimerize as shown in Scheme
4. Similarly, peak 2a involves the loss of two electrons and two protons. This peak is assigned to
azomethine group of HL present in keto form as shown in Scheme 5. On the basis of DPV
results, the formation of 1-((2,4-dimethyl phenylimino)methylene)naphthalen-2(1H)-one is
proposed.
Reduction of HL exhibited two separate peaks. W1/2 value of peak 1c suggested loss of three
protons along with four electrons per two molecules. This peak can be related to the conversion
of azomethine group into -C-N bond (Scheme 6). Reduction peak 2c involves the reduction of -
C=O moiety (keto form) by the gain of one electron and one proton. Oxidation of oxovanadium
complex occurs in three different steps. DPV results show that peak 1a involves loss of two
electrons along with two protons. Peak 2a remains independent in the whole studied pH range
showing no involvement of proton while loss of three protons and four electrons per two
molecules for peak 3a was calculated from DPV results. Organotin complex of HL was found to
undergo oxidation in two steps in a similar manner. Electrochemical investigations demonstrated
that pH independent peak 1a involves the loss of one electron while peak 2a involves loss of one
proton along with one electron. Cobalt and zinc complexes were also found to oxidize in a
similar manner but in case of zinc complex pH independent peak 2a appeared only in the basic
pH regime. In case of HL, the two peaks are assigned to the oxidizable moieties –OH and –C=N
present in the molecule.
4.
Conclusion
A Schiff base HL and its four metallic complexes were synthesized and characterized by several
techniques. FTIR, single crystal XRD, computational studies and spectroscopic results confirmed
the existence of HL in zwitter ionic form along with usual enolic form. Photometric and redox
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behavior of HL and complexes were found to be strongly pH dependent and irreversible. The
computational studies were used as helping tools in suggesting the redox mechanism of the
electrophores of the analytes. Schiff base and its complexes exhibit excellent biological activities and
toxicity to cancer cells. These studies provide preliminary screening data for diabetic, fungal, microbial
and cancer treatment. Further studies are required to precisely evaluate the underlying mechanism of their
activity in vitro as well as in vivo.
Acknowledgements
The authors gratefully acknowledge the financial support of Quaid-i-Azam University,
Islamabad and Higher Education Commission of Pakistan.
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