Synthesis, spectroscopic characterization, DFT optimization and biological activities of Schiff bases and their metal (II) complexes

Article abstract of DOI:10.1016/j.molstruc.2017.05.098

A Novel Schiff base, 3-(((4-chlorophenyl)imino)methyl)benzene-1,2-diol (HL¹) was successfully synthesized along with a structurally similar Schiff base 3-(((4-bromophenyl)imino)methyl)benzene-1,2-diol (HL²). Both the Schiff bases were used to synthesize their zinc (II) and cobalt (II) complexes. These compounds were characterized by FTIR, ¹H NMR, ¹³C NMR and elemental analysis. Metal complexes were confirmed by TGA. Crystals of Schiff bases were also characterized by X-ray analysis and experimental parameters were found in line with the theoretical parameters. Quantum mechanical approach was also used to fine useful structural parameters and to ensure the geometry of metal complexes. The photometric behaviors of all the synthesized compounds were investigated in a wide pH range using BR buffers. The appearance of isosbestic points indicated the existence of Schiff bases in more than one isomeric form. Moreover, these compounds were screened for enzyme inhibition; antibacterial, cytotoxic and in vivo antidiabetic activities and compounds were found active against one or other activity. Results indicate that ZnL₂² is a good inhibitor of alkaline phosphatase enzyme and possess highest potential against diabetes, blood cholesterol level and cancer cells. This effort just provides preliminary data for some biological properties. Further investigations are required to precisely determine mechanistic pathways of their use towards drug development.

Full text of DOI:10.1016/j.molstruc.2017.05.098

Accepted Manuscript
Synthesis, spectroscopic characterization, DFT optimization and biological activities
of Schiff bases and their metal (II) complexes
Abdur Rauf, Afzal Shah, Khurram Shahzad Munawar, Abdul Aziz Khan, Rashda
Abbasi, Muhammad Arfat Yameen, Asad Muhammad Khan, Abdur Rahman Khan,
Irfan Zia Qureshi, Heinz-Bernhard Kraatz, Zia-ur-Rehman
PII:
S0022-2860(17)30709-3
10.1016/j.molstruc.2017.05.098
MOLSTR 23839
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 22 March 2017
Revised Date: 18 May 2017
Accepted Date: 21 May 2017
Please cite this article as: A. Rauf, A. Shah, K.S. Munawar, A.A. Khan, R. Abbasi, M.A. Yameen, A.M.
Khan, A.R. Khan, I.Z. Qureshi, H.-B. Kraatz, Zia-ur-Rehman, Synthesis, spectroscopic characterization,
DFT optimization and biological activities of Schiff bases and their metal (II) complexes, Journal of
Molecular Structure (2017), doi: 10.1016/j.molstruc.2017.05.098.
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ACCEPTED MANUSCRIPT
Synthesis, spectroscopic characterization, DFT optimization and biological
activities of Schiff bases and their metal (II) complexes
Abdur Raufa,*, Afzal Shahb,*, Khurram Shahzad Munawarc, Abdul Aziz Khand, Rashda
Abbasie, Muhammad Arfat Yameenf, Asad Muhammad Khana, Abdur Rahman Khana, Irfan
Zia Qureshid, Heinz-Bernhard Kraatzg, Zia-ur-Rehmanb
a. Department of Chemistry, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan
b. Department of Chemistry, Quaid-i-Azam University, 45320 Islamabad, Pakistan
c. Department of Chemistry, School of Science, University of Management and Technology, Lahore, Pakistan
d. Department of Animal Sciences, Quaid-i-Azam University, 45320 Islamabad, Pakistan
e. Institute of Biomedical and Genetic Engineering Islamabad, Pakistan
f. Department of Pharmacy, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan
g. Department of Physical and Environmental Sciences, University of Toronto, 1265 Military Trail, Toronto M1C
1A4, Canada
*To whom correspondence should be addressed
E-mails: raufrazi41@41hotmail.com (A. Rauf),
afzals_qau@yahoo.com (A. Shah)
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Graphical Abstract
Two Schiff bases and their Zinc (II) and cobalt (II) complexes were synthesized and
confirmed. Their DFT optimizations, pH dependent photometry and biological activities were
evaluated in detail. Some of the compounds being good antidiabetic, antibacterial and
cytotoxic agents were found astonishing candidate for drug development.
Abstract
A Novel Schiff base, 3-(((4-chlorophenyl)imino)methyl)benzene-1,2-diol (HL1) was
successfully synthesized along with a structurally similar Schiff base 3-(((4-
bromophenyl)imino)methyl)benzene-1,2-diol (HL2). Both the Schiff bases were used to
synthesize their zinc (II) and cobalt (II) complexes. These compounds were characterized by
1
FTIR, H NMR, 13C NMR and elemental analysis. Metal complexes were confirmed by
TGA. Crystals of Schiff bases were also characterized by X-ray analysis and experimental
parameters were found in line with the theoretical parameters. Quantum mechanical approach
was also used to fine useful structural parameters and to ensure the geometry of metal
complexes. The photometric behaviors of all the synthesized compounds were investigated in
a wide pH range using BR buffers. The appearance of isosbestic points indicated the
existence of Schiff bases in more than one isomeric form. Moreover, these compounds were
screened for enzyme inhibition; antibacterial, cytotoxic and in vivo antidiabetic activities and
compounds were found active against one or other activity. Results indicate that ꢀꢁꢂꢃꢃ is a
good inhibitor of alkaline phosphatase enzyme and possess highest potential against diabetes,
blood cholesterol level and cancer cells. This effort just provides preliminary data for some
2

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biological properties. Further investigations are required to precisely determine mechanistic
pathways of their use towards drug development.
Keywords: Schiff Base; Metal-Schiff base complexes; Spectroscopy; X-ray analysis;
antibacterial activity, antidiabetic activity, cytotoxicity
3

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1.
Introduction
Schiff bases are the organic compounds containing azomethine/ imine (–C=N–)
functional group. They were first reported by Hugo Schiff and can be synthesized by the
condensation reaction of amines with carbonyl compounds [1, 2]. Plethora of Schiff base
derivatives is also present in natural and natural-derived compounds like ancistrocladidine
and chitosan derived Schiff bases [3]. Numerous Schiff bases have been reported to exhibit
remarkable biological activities containing antibacterial, antifungal, antimalarial, anti-
proliferative, anti-inflammatory, antiviral, and antipyretic properties [4] and the imine group
present in such compounds is thought to be responsible for such properties [5-7]. Cleiton et
al. have explored antibacterial activities of several compounds [3]. Pandeya et al. reported
strong antibacterial activities of 5-chloro-salicylaldehyde derived and Isatin-derived Schiff
bases [8]. N-(salicylidene)-2-hydroxyaniline has been reported to show inhibitory action
against Mycobacterium tuberculosis H37Rv [6]. Similarly, 2,4-dichloro-5-fluorophenyl
derivatives was reported to inhibit the growth of E. coli, S. aureus, Klebsiella pneumoniae,
Aspergillus fumigatus, Aspergillus flavus, Trichophyton mentagrophytes, Penicillium
marneffei and Pseudomonas aeruginosa [9]. Very small amount of N-(Salicylidene)-2-
hydroxyaniline was reported to significantly inhibit the growth of fungi [10]. Literature
survey reveals that chitosan derived Schiff bases were efficiently used as inhabitant against
the growth of Botrytis cinerea and Colletotrichum lagenarium [7]. 1-amino-3-
hydroxyguanidine tosylate-derived Schiff bases were indicated to be very effective against
mouse hepatitis virus (MHV), inhibiting its growth [11]. Vitamin B6, a Schiff base derivative
of pyridoxine is reported to abolish the enzyme activities of proteins [12]. Beside inhibitory
action, catalytic properties of Schiff bases have been reported like plant hormone and plant
growth regulatory activity towards cytokine and auxin [13-15]. Their photometric and
thermochemical properties make them useful in modern applications including solar filters,
photodetectors, photostabilizers, microelectronic devices and optical switches [16-20].
Due to the presence of lone pair on sp2 hybridized N atom, Schiff bases are able to
produce metal complexes, polycyclic quinoline derivatives and numerous heterocyclic
compounds [16, 21-25]. Their reactivity increases exponentially in the presence of –OH
groups especially when the –OH group is present at –ortho position [26-31]. Research in this
field has received utmost attention because induction of metals to Schiff base structures
robust their properties manifold and makes them more applicable. They are good catalysts
and catalyst producing agents because of their greater redox activities [32-34]. Due to their
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structural diversity they are used in stereo chemistry [35, 36] and medicinal applications [37].
They are used to produce dyes and pigments [3]. DNA interacting modes of these metal
complexes make them useful as new therapeutic agents [38]. Transition metal complexes of
bipyridine and phenanthroline are used in petroleum industry [39]. Hamada and Kawamoto
reported the use of metal complexes as an effective emitting layer and luminescent
compounds [40-42]. Schiff bases and their metal complexes have been well explored and
reviewed. Although a number of reviews are available to disclose the effect of metal on the
properties of Schiff bases. Silva et al. proposed that Schiff bases derived from
salicylaldehyde may provide a platform to produce useful compounds with desirable
biological and industrial properties [11, 43], therefore in this research work, salicylaldehyde
derivative is used to synthesize Schiff bases and metallic complex. An effort is made to
explore its physicochemical properties.
2.
Experimental
2.1
Materials and Reagents
2,3-Dihydroxybenzaldehyde, 4-chloroaniline and 4-bromoaniline were received from Sigma
Aldrich, USA. Zinc acetate and cobalt acetate were obtained from Fluka, Switzerland. The
analytical grade solvents like, chloroform, n-hexane, ethanol and dimethyl sulfoxide were
obtained from Merck, Germany. All BRB solutions were prepared according to reported
protocol [44, 45] using analytical-grade reagents and doubly distilled water with
(conductivity ≤0.1 µScm−1). All experiments were carried out at room temperature (25±1 ºC).
2.2
Instrumentation and measurement
Thermo Nicolet 6700 FTIR spectrophotometer was used to obtain IR spectra in the range of
1
4000–100 cm−1. H and 13C NMR were recorded on Bruker AC Spectrometers with
tetramethylsilane (TMS) as internal reference. Absorption spectra were recorded on
Shimadzu 1601 spectrophotometer and the pH measurements were carried out with a Crison
micro pH 2001 pH-meter with an Ingold combined glass electrode. GAUSSIAN 09 software
was used to find geometry of Schiff bases and their metal complexes using 6-31++G** and
6-31G++ basis sets, respectively. Summary of the different calculated parameters is given in
Table 6.
2.3
Synthesis of Schiff base ligands
3-(((4-chlorophenyl)imino)methyl)benzene-1,2-diol (HL1)
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Schiff bases were prepared by mixing equimolar amounts of 2,3-dihydroxybenzaldehyde and
respective 4-haloaniline in freshly dried ethanol. After refluxing for 2 hours, red crystalline
solid product was appeared which was cooled, filtered and further processed for
crystallization in a mixture of chloroform and n-hexane (3:1).
o
C13H10ClNO2: Mol. Wt. 247.68 gmol-1, Yield 87 %, M.P. 127.5 C; FT-IR (KBr, υ/cm-1):
3445 (OH phenolic), 3032 (CH Aromatic), 1617 (HC=N Schiff base), 1463 (C=C Aromatic),
1
Anal. Calc.: C, 62.73; H, 4.06; N, 5.63. Found: C, 61.52; H, 3.91; N, 5.49 %. H NMR
(DMSO-d6, ppm, 400 MHz) δ: 8.58 (s, 1H, CH=N), 12.52 (s, 1H, OH), 9.32 (s, 1H, OH),
13
6.84 (t, 1H), 7.05 (dd, 1H), 6.95 (dd, 1H), 7.52 (d, 2H), 7.02 (d, 2H), C NMR (DMSO-d6,
ppm, 101 MHz) δ: 163.42 (CH=N), 149.5 (C-OH), 149.1 (C-OH), 145.3 (C-N), 119.6-132.8
(Aromatic Carbons).
3-((4-bromophenyl)imino)methyl)benzene-1,2-diol (HL2)
o
C13H10BrNO2: Mol. Wt. 292.13 gmol-1, Yield 83 %, M.P. 150.2 C; FT-IR (KBr, υ/cm-1):
1620 (HC=N Schiff base), 3448 (OH phenolic), 3080 (CH Aromatic), 1462 (C=C Aromatic),
1
Anal. Calc.: C, 53.40; H, 3.46; N, 4.79. Found: C, 53.28; H, 3.30; N, 4.65 %. H NMR
(DMSO-d6, ppm, 400 MHz) δ: 8.56 (s, 1H, CH=N), 11.08 (s, 1H, OH), 9.87 (s, 1H, OH),
13
6.83 (t, 1H), 7.07 (dd, 1H), 6.92 (dd, 1H), 7.75 (d, 2H), 7.15 (d, 2H), C NMR (DMSO-d6,
ppm, 101 MHz) δ: 162.52 (CH=N), 149.4 (C-OH), 149.0 (C-OH), 150.3 (C-N), 119.6-132.9
(Aromatic Carbons).
2.4
Synthesis of transition metal (II) complexes
All the metal complexes were prepared by the following method. Corresponding metal (II)
acetate salt was added to hot ethanol solution of Schiff base ligand in molar ratio 2:1
respectively. In few minutes, the colored complex was precipitated out in each case. The
complexes were filtered and washed with ethanol.
bis(2-(((4-chlorophenyl)imino)methyl)-6-hydroxyphenoxy)zinc (ꢀꢁꢂꢄꢃ)
o
C26H18Cl2N2O4Zn: Mol. Wt.: 558.72 gmol-1 Yield: 74 %, M.P. 278.3 C; FT-IR (KBr,
υ /cm-1): 1574 (HC=N Schiff base), 3060 (CH Aromatic), 1443 (C=C Aromatic), 450 (Zn-O),
428 (Zn-N); Anal. Calc.: C, 56.12; H, 3.27; N, 5.04. Found: C, 55.97; H, 3.01; N, 4.88 %. 1H
NMR (DMSO-d6, ppm, 400 MHz) δ: 8.43 (s, 1H, CH=N), 9.82 (s, 1H, OH), 6.82 (t, 1H),
7.06 (dd, 1H), 6.91 (dd, 1H), 7.51 (d, 2H), 7.78 (d, 2H), 13C NMR (DMSO-d6, ppm, 101
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MHz) δ: 158.7 (CH=N), 145.4 (C-O), 147.24 (C-OH), 151.7 (C-N), 119.6-132.8 (Aromatic
Carbons).
bis(2-(((4-bromophenyl)imino)methyl)-6-hydroxyphenoxy)zinc (ꢀꢁꢂꢃꢃ)
o
C26H18 Br2N2O4Zn: Mol. Wt.: 647.62 gmol-1 Yield: 78 %, M.P. 243.6 C; FT-IR (KBr,
υ /cm-1): 1576 (HC=N Schiff base), 3018 (CH Aromatic), 1446 (C=C Aromatic), 443 (Zn-O),
418 (Zn-N). Anal. Calc.: C, 48.18; H, 2.81; N, 4.32. Found: C, 48.02; H, 2.63; N, 4.09 %. 1H
NMR (DMSO-d6, ppm, 400 MHz) δ: 8.39 (s, 1H, CH=N), 9.78 (s, 1H, OH), 6.81 (t, 1H),
7.20 (dd, 1H), 6.86 (dd, 1H), 7.20 (d, 2H), 7.75 (d, 2H), 13C NMR (DMSO-d6, ppm, 101
MHz) δ: 158.5 (CH=N), 145.5 (C-O), 147.38 (C-OH), 152.8 (C-N), 119.6-132.9 (Aromatic
Carbons).
bis(2-(((4-chlorophenyl)imino)methyl)-6-hydroxyphenoxy)cobalt (ꢅꢆꢂꢄꢃ)
o
C26H18 Cl2N2O4Co: Mol. Wt.: 552.27 gmol-1 Yield: 79 %, M.P. > 300 C; FT-IR (KBr,
υ /cm-1): 1578 (HC=N Schiff base), 3019 (CH Aromatic), 1441 (C=C Aromatic), 541 (Co-O),
466 (Co-N); Anal. Calc.: C, 56.49; H, 3.29; N, 5.07. Found: C, 56.25; H, 3.06; N, 4.89 %.
bis(2-(((4-bromophenyl)imino)methyl)-6-hydroxyphenoxy)cobalt (ꢅꢆꢂꢃꢃ)
o
C26H18 Br2N2O4Co: Mol. Wt.: 641.17 gmol-1 Yield: 82 %, M.P. 291.5 C; FT-IR (KBr,
υ /cm-1): 1572 (HC=N Schiff base), 3022 (CH Aromatic), 1452 (C=C Aromatic), 538 (Co-O),
458 (Co-N). Anal. Calc.: C, 48.66; H, 2.84; N, 4.37. Found: C, 48.55; H, 2.60; N, 4.15 %.
TGA data of metal complexes is given in Table 1.
2.5
X-Ray Structure Determination
Single crystals of HL1 and HL2 were used to investigate structural arrangements using
Microstar diffractometer at 100 K. Charge Flipping program was used to solve the structure
with olex2 [46] and refined with XL [47] refinement package using least squares
minimization. Data and details of crystal structure are given in Tables 2-4.
2.6
Assay of alkaline phosphatase activity
Synthesized Schiff bases and their metal complexes were investigated for inhibition of
alkaline phosphatase (ALP) enzyme as the reported procedure [47]. p-Nitrophenyl phosphate
was hydrolyzed in the presence of ALP, Mg2+ ions and phosphate buffer of pH 9.8 to produce
yellow colored p-nitrophenol (Scheme 1) and the progress was monitored by measuring its
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absorbance values at 405 nm. Finally different amounts of the synthesized compounds were
added periodically from stock DMSO solution and absorbance was measured again after
incubation. Each experiment was repeated thrice and average values were used to calculate
percentage inhibition.
ALP
+2
p-Nitrophenyl phosphate + Mg + H2O
p-Nitrophenol + Pi
Scheme 1. Hydrolysis of alkaline phosphatase
where Pi is inorganic phosphate.
2.7
Quantitative antibacterial activity assay by minimum inhibitory concentration
In vitro Serial dilution microplate method was used for determination of MIC on
Staphylococcus aureus and Escherichia coli bacteria according to previously described
methods with some modifications [48, 49]. The test samples were dissolved in
DMSO/Mueller Hinton Broth (MHB). The final concentration of DMSO was kept lower than
2.5% so it may not affect the bacterial growth. The prepared test samples (100µl) were added
to the first well of a 96-well microtiter plate and serially diluted two fold. Overnight grown
bacterial cultures were adjusted to 0.5 McFarland standards and 100µl were added to each
well. The plates were incubated at 37°C for 18 hrs. The assay was performed in triplicate.
Wells containing MHB with 100µl of inoculum and DMSO to a final concentration of 2.5%
served as negative control. The MIC of samples was measured by the addition of 40 µl of
1 mg/5 mL of p-iodonitrotetrazolium violet (INT) and incubated at 37°C for 30 min. The
lowest sample concentration that prevent the color change of the medium by the addition of
INT, demonstrated the complete inhibition of microbial growth [48]. Microplates with
experimental results can be seen in Fig. S1-S4 and MIC values are given in Table 7.
2.8
In vitro anti-cancer activity
2.8.1 Cell culture
Human hepatocellular carcinoma HepG2 (ATCC HB-8065™) cell line was cultured in
DMEM supplemented with FBS (10%), Na-pyruvate (1 mM), L-glutamine (2 mM), penicillin
(100 U/ml) and streptomycin (100 µg/ml). The cultures were maintained at 37°C and 5% CO2
under humidified conditions.
2.8.2 Cytotoxicity analysis
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In vitro cytotoxicity analysis of the compounds was done by sulforhodamine B (SRB) assay
as described earlier [50]. In short, actively growing HepG2 cells (10000 cells/well) were
exposed to the compounds at 200 µM concentration for 24 hrs under standard culture
conditions. Untreated and DMSO (solvent) treated cultures were included as experimental
control. Cultures were fixed using pre-chilled trichloroacetic acid (50%) for 1 h at 4°C,
washed thoroghly with deionized water and air dried. The cells were then stained with SRB
solution (0.05%) for 30 minutes, washed with acetic acid (1%), dried overnight and
photographed with a Olympus IMT-2 inverted microscope equipped with digital camera. The
incorporated dye was solubilized in 10 mM Tris and absorbance (OD) was measured on
microplate reader (AMP PLATOS R-496) at the wavelength of 565 nm. Percent (%) viability
was calculated relative to the untreated sample using the following formula
Percent (%) viability = [Abs(565)test sample-Abs(565)compound
Abs(565)blank] × 100
/ Abs(565)untreated
-
sample
control
Where, Abs(565)test sample and Abs(565)untreated sample is OD for cultures treated and untreated
samples, respectively. Abs (565)compound control and Abs (565)blank is the OD measured in
compounds only and media only samples.
2.8.3 Statistical analysis
The results presented as mean ± SD were analyses by ANOVA and two tailed t-test.
Differences between untreated and treated samples were investigated. Further, metal
complexes were compared with their respective Schiff bases. Results were considered
significant when p<0.05.
2.9
In vivo Antidiabetic Activity
2.9.1 Animals Maintenance and Treatment
A total of fifty healthy adult male Sprague–Dawley rats (average body wt = 255 ± 19g)
obtained from the National Institute of Health Islamabad and were maintained at the Animal
House Facility of the Animal Sciences Department, Quaid-i-Azam University Islamabad.
Five rats were housed per single cage and had free access to standard rodent diet and drinking
water maintained, while photoperiod conditions were 12hL:12hD. A total of eight groups (n
= 5) were made diabetic through a single dose (150 mg/kg b.w) of alloxan monohydrate
(Sigma Aldrich, USA) administered intraperitonaly to overnight fasted rats. Six groups were
treated with six different compounds; ꢇꢂꢄ, ꢇꢂꢃ, ꢀꢁꢂꢃꢄ, ꢀꢁꢂꢃꢃ, ꢅꢆꢂꢃꢄ and ꢅꢆꢂꢃꢃ with acute
dose of 35.0 mg/kg b.w while the control groups, positive and negative were treated with
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antidaibetic compound glibenclamide (10mg/kg b.w) dissolved in distilled H2O, (Euglucon,
Roche Pharma) and normal saline respectively [51].
2.9.2 Glucose, Triglycerides and Cholesterol Determination
Glucose level was determined from blood samples collected from the caudal vein at different
times. Two samples were drawn before treatments with compounds (dissolved in DMSO) (-1
pre alloxan sample and 0 post alloxan sample) and seven samples were drawn at one hour
interval (1, 2, 3, 4, 5, 6 and 7hr) post treatment with compounds. The plasma level of glucose
was determined with a glucometer (Accu-Check Active,Roche) and dextrostix strips.
Triglycerides and cholesterol level were determined through commercially available kits
obtained from the Globe Diagnostic (Italy), following the standard procedure given in along
with each kit.
2.9.3 Statistical Analysis
Data were analyzed through one-way analysis of variance (ANOVA) to compare the means
between groups, followed by Post hoc Bonferroni tests for multiple comparisons among
different groups. Data are presented by line or bar graphs as mean ± S.D, while P < 0.05 was
considered statistically significant difference.
3.
Results and Discussion
3.1
Structural Characterization
FTIR spectra of HL1 and HL2 showed a strong absorption band in the range 1617-1620 cm-1
due to azomethine group indicating the formation of Schiff bases. Moreover, appearance of
1
resonance signals related to azomethine proton and carbon in H and 13C NMR spectra
respectively confirmed the formation of Schiff bases. Absence of primary amine peak
revealed their purity. Zn metal complexes were confirmed by the shifting of azomethine
signal to longer wavelength (1574-1576 cm-1) in FTIR spectra and by downfield shifting of
azomethine resonance signals in the NMR spectra. Disappearance of proton resonance signal
related to ortho-hydroxyl group also ensured the coordination of ligand molecules with zinc
ions. Thermogravimetric analysis of metal complexes was carried out from ambient
temperature to 1000 °C in nitrogen atmosphere. The summary of decomposed fragments
against various temperature ranges is summarized in the Table 1. These observations are in
line with elemental analyses. No weight loss around 100oC ensured absence of water
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molecules in coordinated structures. In all cases fragmentation took place in one step with the
loss of ligands and metal oxides left in the form of residue.
3.2
Crystal structures description
Crystal structure of HL1 is depicted in Fig. 1 along with crystal packing and numbering
scheme. Summary of structural refinements and crystal data can be seen in Table 2. Selected
bond distances and bond angles for HL1 are presented in Table 3 and compared with DFT
calculated parameters. Critical review of the data reveals that the novel Schiff base HL1
belongs to triclinic crystal system and have non-planar geometry as reported for HL2 [52].
Smaller N-C7 bond length as compared to N-C8 ensures double bond character in the N-C7
bond and existence of molecule in enolic form as supported by computational results.
Distorted hexagonal rings can be justified by the presence of attached groups. There is a
possibility of intermolecular as well as intramolecular hydrogen bonding as shown in Table 4
and Fig. 1. Experimental parameters were found in good agreement with the calculated
values. In case of HL2, results were found similar as reported by Shuja et al. [52].
3.3
Electronic absorption spectroscopy
Fig. 2 depicts absorption spectra of 13.8 µM solutions of ligands and their metal complexes
in 9:1 mixture of ethanol and DMSO. HL1 exhibits an intense band at 222 nm and a less
intense doublet with maxima at 250 and 266 nm. The intense band is thought to be due to π–
π* transitions of aromatic rings while less intense doublet can be assigned to π–π* transitions
of –C=N group and –C=O group which arises due to tautomerism [47]. In case of HL2 these
bands shifted to 225, 284 and 318 nm, respectively. This red shift can be attributed to the
increase in electron density due to less electronegative nature of –Br group hence decreasing
the required energies for transitions [47]. Further observation of Fig. 2 reveals that all the
metal complexes have different photometric behavior as compared to ligands due to change
in their orbital energies and band gap values shown by the computational results (Table 6).
Moreover, appearance of new peaks in the range of 215-235 nm and 370-500 nm offers the
strong evidence of complexation. Shifting of signals related to azomethine and carbonyl
groups towards larger wavelength suggests a significant charge transfer from metal orbitals to
ligand orbitals (LMCT) [51]. Appearance of (less intense) new peaks after 450 nm can be
assigned to d-d transitions which were used to find the geometry of the complexes by
calculating their extinction coefficients. 554 and 811 m2 mol-1 ɛ values suggest ꢀꢁꢂꢃꢃ and
ꢅꢆꢂꢃꢃ to have tetrahedral geometries [53, 54]. Geometries of ꢀꢁꢂꢃꢄ and ꢅꢆꢂꢄꢃ can’t be
predicted due to absence of charge transfer band.
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The absorption spectra of 20 µM HL1 and HL2 in different pH media are shown in Fig.3.
Observation of the spectra reveals that pH of the medium strongly affects the photometric
behavior of both the ligands. Increase in the alkalinity of the medium results in bathochromic
shift. This shift can be explained by the removal of proton from –OH group leaving negative
charge on oxygen atom which makes the transition more feasible [55]. Appearance of
isosbestic points in the range of 253-351 nm indicates the existence of equilibrium between
different mesomeric forms of the molecules. In case of metal complexes no significant
change was observed with increase in pH probably due to M-O bonds. λmax, ɛ and pKa values
for ligands and their complexes are summarized in Table 5.
3.4
Computational Studies
Structures of Schiff bases and their metal complexes were optimized using 6-31++G** and
6-31G++ basis sets, respectively due to complexity of metal complexes. Results reveal that
both the ligands possess non planar structures as reported for such molecules [47]. Bond
lengths obtained from DFT studies were compared with experimental results obtained from
X-ray analysis and found in good agreement (Table 3). Metal complexes were found to
possess tetrahedral geometries as shown in Fig. 4. The most stable confirmation was also
used to calculate different useful parameters like total energy, dipole moment, EHOMO, ELUMO
,
band gap energy (∆E = EHOMO - ELUMO), ionization energy, electron affinity,
Electronegativity (Table 6). These calculations ensure about the geometries of metal
complexes and offer better explanation to the experimental findings.
3.5
Inhibition of ALP
Alkaline phosphatases (ALPs) are dimeric metallic enzymes having zinc metal ions in each
active site [47] . In bacteria they are present in periplasmic space while in humans they are
present in all body tissues but mainly in kidney, placenta and intestinal mucosa [56]. They are
one of the clinical important enzymes as they act as catalysts in dephosphorylation and their
abnormal amount causes several diseases including anemia, hypophosphatasia,
hypothyroidism, osteoporosis, leukemia, lymphoma, breast cancer and sarcoidosis [57, 58].
Metal complexes of Schiff bases are found to be best inhibitors of phosphatase enzymes [59]
as they can block their active cites, therefore synthesized compounds were screened for
inhibitory action. All the compounds were found to reduce the activity of enzyme in
concentration dependent manner (Fig. 5); however presence of metal ions enhanced this
12

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inhibitory action as expected. ꢀꢁꢂꢃꢃ has shown best results with 80% inhibition at 500 µM
concentration.
3.6
Antibacterial activity
Compounds ꢇꢂꢄ, ꢀꢁꢂꢄꢃ and ꢇꢂꢃ have shown moderate antibacterial activity (<100µg/ml)
against E. coli while ꢇꢂꢄ and ꢅꢆꢂꢃꢄ have moderate antibacterial activity against S. aureus.
Rest of all the compounds have low antibacterial activity (>100µg/ml) against both the
bacteria [60]. Microplates with experimental results can be seen in Fig. S1-S4 and MIC
values are summarized in Table 7.
3.7
In vitro Cytotoxicity analysis
The Schiff bases and their metal complexes were investigated for cytotoxic potential against
HepG2 cell line (Fig. 6 & 7). Cultures were exposed to the compounds (200 µg/ml) for 24
hours, and then analyzed by SRB assay. Data showed that both ꢀꢁꢂꢃꢃ (relative viability =
26.67 ± 3.97) and ꢅꢆꢂꢃꢃ (relative viability = 32.53 ± 2.54) were biologically active against
HepG2 cells, whereas ꢇꢂꢃ exhibited slight toxicity against the cultures. On the other hand
ꢇꢂꢄ (relative viability = 36.78 ± 2.85) was more toxic when compared to ꢀꢁꢂꢃꢄ and ꢅꢆꢂꢄꢃ.
Furthermore, ꢀꢁꢂꢃꢃ and ꢅꢆꢂꢃꢃ also induced morphological changes in the cells causing
cellular clumping and blebbing thus leading to apoptosis [61, 62].
3.8
In vivo Antidiabetic Activity
Compound ꢇꢂꢄ showed high level of diabetic activity. The rats treated with compound ꢇꢂꢄ
had higher blood glucose level as compared to positive control group (P ˂ 0.001), while no
significant difference was observed in comparison with compounds ꢀꢁꢂꢄꢃ, ꢅꢆꢂꢃꢄ and the
negative control group. Compound ꢀꢁꢂꢃꢄ and ꢅꢆꢂꢄꢃ aslo drastically increased glucose level
as compared to positive control group (P ˂ 0.001), (Fig. 8A).
In contrast compound ꢀꢁꢂꢃꢃ had better antidiabetic potential than ꢇꢂꢃand ꢅꢆꢂꢃꢃ compounds.
The blood glucose level of the rats treated with compound ꢀꢁꢂꢃꢃ was lower as compared to
ꢇꢂꢃ treated group, ꢅꢆꢂꢃꢃ treated group and negative control group (P ˂ 0.001 in all cases).
The antidiabetic potential of ꢀꢁꢂꢃꢃ was almost similar to the reference antidiabetic compound
(Fig. 8B).
A high level of similarity was found in the level of diabetic potential between ꢇꢂꢄ and ꢀꢁꢂꢃꢄ
compounds and between ꢅꢆꢂꢄꢃ and ꢅꢆꢂꢃꢃ compounds. The blood glucose level of the treated
13

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rats with these four compounds was significantly higher than the positive control group (P ˂
0.001), while comparable with the negative control group (P ˃ 0.5; Fig. 8 A&B).
Compound ꢇꢂꢄ treatment significantly increased the serum triglycerides concentration
compared to HLꢈ treatment (P ˂ 0.01) and both negative and positive control groups (P ˂
0.001) but the concentration was found decreased as compared to ꢅꢆꢂꢄꢃ treated animals (P ˂
0.001). Likewise, compound ꢇꢂꢃ led to significant increase concentration of triglycerides as
compared to ꢀꢁꢂꢃꢃ, ꢅꢆꢂꢃꢃ (P ˂ 0.001), and positive control group (P ˂ 0.01). In contrast the
concentration of serum triglycerides was drastically decreased with ꢀꢁꢂꢃꢃ treatment in
comparison with ꢀꢁꢂꢄꢃ treated animals and negative control group (P ˂ 0.001), while
treatment with compound ꢅꢆꢂꢃꢃ caused a decrease in serum triglycerides concentration in
comparison with ꢅꢆꢂꢄꢃ, negative (P ˂ 0.001) and positive control groups (P ˂ 0.5), (Fig. 8C).
A highly significant decrease was observed in the cholesterol level of the animals treated with
compounds ꢇꢂꢄ (P ˂ 0.01), ꢀꢁꢂꢄꢃ (P ˂ 0.05), ꢅꢆꢂꢃꢄ (P ˂ 0.05), ꢇꢂꢃ (P ˂ 0.001), and ꢅꢆꢂꢃꢃ
(P ˂ 0.01) in comparison with the negative control group animals. The concentration of
cholesterol did not decrease with ꢀꢁꢂꢃꢃ treatment in comparison with negative control group
rats (P ˃ 0.5), however highly significant increase as compared to ꢇꢂꢃ (P ˂ 0.01) and ꢅꢆꢂꢃꢃ
(P ˂ 0.5) compounds (Fig. 8D).
4
Conclusion
Two Schiff bases and their four metal complexes were synthesized and characterized by
various techniques. FTIR, NMR, elemental analysis and TGA ensured the formation of
compounds. XRD results indicated the existence of H-bonding in Schiff base molecules and
DFT results also supported these findings. Computational results were also used to explain
ensure the geometry of metal complexes and finding useful energy parameters. Photometric
signature of Schiff bases was found to be strongly pH dependent. Furthermore, metal
complexes were found good inhibitors of alkaline phosphatase enzyme than Schiff bases.
Results of antibacterial, anticancer and antidiabetic activities reveal that some of these
compounds have good potential to be used in drug development. ꢀꢁꢂꢃꢃ was found to be most
active against cancer cell as well as diabetes. HL2 have shown markable decrease in blood
cholesterol level. Further efforts should be made to precisely explore the possible mechanistic
pathways of their activity in vivo as well as in vitro.
Acknowledgements
14

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The authors gratefully acknowledge the financial support of the Higher Education
Commission of Pakistan through project number 21-1209, CIIT Abbottabad and Quaid-i-
Azam University Islamabad.
Appendix A. Supplementary material
CCDC reference number 210655. See http://www.rsc.org/suppdata/dt/b3/b305443h/ for
crystallographic data in CIF or other electronic format.
15

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Table 1. Thermogravimetric Analysis data of metal (II) complexes
% Weight loss
Observed Calculated Observed Calculated
% Residue
Evolved
components
Temp.
Complexes
range oC
ZnO2
ꢀꢁꢂꢄꢃ
ꢀꢁꢂꢃꢃ
ꢅꢆꢂꢄꢃ
ꢅꢆꢂꢃꢃ
320-358 C26H18Cl2N2O2 82.78
375-420 C26H18Br2N2O2 84.20
540-580 C26H18 Cl2N2O3 86.92
380-431 C26H18 Cl2N2O3 88.92
88.57
84.96
86.43
88.31
17.22
ZnO2
16.80
CoO
13.08
CoO
17.43
15.03
13.56
11.68
11.08

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Table 2. Crystal data and structure refinement for HL1
HL1
Identification code
Empirical formula
Formula weight
Temperature/K
Crystal system
Space group
a/Å
C13H10NO2Cl
247.67
100
triclinic
P-1
6.0176(6)
8.7051(8)
11.6778(11)
85.304(4)
80.991(4)
70.664(4)
569.81(10)
2
b/Å
c/Å
α/°
β/°
γ/°
Volume/Å3
Z
ρcalcmg/mm3
m/mm-1
1.444
2.877
F(000)
Crystal size/mm3
256.0
0.25 × 0.2 × 0.04
2Θ range for data collection 7.668 to 140.152°
Index ranges
-7 ≤ h ≤ 7, -10 ≤ k ≤ 10, -14 ≤ l ≤ 14
Reflections collected
Independent reflections
Data/restraints/parameters
Goodness-of-fit on F2
19056
2134[R(int) = 0.0471]
2134/0/163
1.058
Final R indexes [I>=2σ (I)] R1 = 0.0367, wR2 = 0.1008
Final R indexes [all data] R1 = 0.0370, wR2 = 0.1012
Largest diff. peak/hole / e Å-3 0.24/-0.31

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Table 3 Selected experimental and calculated geometric parameters for HL1
HL1 (exp) HL1 (calc) ∆
Bond Distance (A˚)
Cl1
O1
O2
N1
N1
C1
C11
C2
C3
C7
C8
C7
1.743(15) 1.757
1.359(17) 1.351
1.369(18) 1.361
1.287(19) 1.289
1.416(19) 1.407
0.014
-0.008
-0.008
0.002
-0.009
0.001
1.448(2)
1.449
Bond Angles (˚)
C7
N1
C1
C1
C2
C3
C7
C8
C8
C11
C8
C7
C2
C1
C4
C1
C13
N1
Cl1
119.69(12) 121.25
120.54(13) 120.56
119.05(14) 119.02
121.87(13) 123.39
118.74(13) 120.32
121.58(13) 122.26
119.46(14) 118.84
118.49(13) 118.04
119.10(12) 119.52
1.56
0.02
-0.03
1.52
1.58
0.68
-0.62
-0.45
0.42
C2
C6
O1
O2
N1
C9
C13
C10
Table 4. Hydrogen-bonding geometry (A, °) for HL1
D H A d(D-H)/Å
d(H-A)/Å
d(D-A)/Å
D-H-A/°
O1 H1 N1 0.92(2)
O2 H2 O1 0.80(3)
1.73(2)
2.08(3)
2.5761(16)
2.7789(15)
152(2)
146(2)

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Table 5. Selected photometric parameters of ligands and metal complexes
(M-1cm-1) × 10-4
Compounds
λmax (nm)
pKa
ɛ
ꢇꢂꢄ
3.42
2.03
5.61
4.32
5.58
6.07
222
226
219
232
241
245
7.0
ꢇꢂꢃ
6.0
ꢀꢁꢂꢄꢃ
ꢀꢁꢂꢃꢃ
ꢅꢆꢂꢄꢃ
ꢅꢆꢂꢃꢃ
-
-
-
-
Table 6. Selected computational parameters of ligands and metal complexes calculated by
RB3LYP set using DFT method in GAUSSIAN 09 software
ꢇꢂꢄ
ꢇꢂꢃ
ꢀꢁꢂꢄꢃ
ꢀꢁꢂꢃꢃ
ꢅꢆꢂꢄꢃ
ꢅꢆꢂꢃꢃ
Charge
0
0
0
0
0
0
Spin
Singlet
4.400
Singlet
4.815
Singlet
6.140
Singlet Doublet Doublet
6.543 6.233 6.660
Dipole Moment (D)
Total Energy (kev)
EHOMO (ev)
ELUMO (ev)
∆E gap (ev)
-31.581 -88.829 -111.319 -225.815 -100.579 -215.075
-0.207
-0.089
0.118
-0.205
-0.086
0.119
-0.217
-0.088
0.129
-0.214
-0.085
0.129
-0.216
-0.094
0.122
-0.212
-0.086
0.126
Ionization Energy (kJmol-1)
Electron Affinity (kJmol-1)
Electronegativity (kJmol-1)
Point Group
19.972
8.587
-0.148
C1
19.779
8.298
-0.146
C1
20.937
8.491
-0.152
C1
20.648
8.201
-0.150
C1
20.841
9.070
-0.155
C1
20.455
8.298
-0.149
C1

ACCEPTED MANUSCRIPT
Table 7. MIC of Schiff bases and metal complexes against E. coli and S. aureus bacteria
Compounds
E. coli (µg/ml)
S. aureus (µg/ml)
ꢇꢂꢄ
16
64
128
64
>128
>128
32
ꢀꢁꢂꢄꢃ
ꢅꢆꢂꢄꢃ
ꢇꢂꢃ
>128
128
64
>128
>128
0.25
ꢀꢁꢂꢃꢃ
ꢅꢆꢂꢃꢃ
Standard Control (Ceftriaxone) 0.5
DMSO concentration was less than 2.5%. The activity of compounds is significant when MIC<10 mg/mL, moderate
when 10<MIC<100 mg/mL and low when MIC>100 mg/mL [60].

ACCEPTED MANUSCRIPT
Fig. 1. Crystal packing of HL1 along with numbering scheme and unit cell dimensions
1.00
1
HL
0.60
2
HL
0.45
0.75
0.30
0.15
0.50
0.00
210
280
350
420
490
560
ZnL12
ZnL22
CoL12
CoL22
0.25
0.00
210
280
350
420
490
560
Wavelength (nm)
Fig. 2. UV-Visible spectra of 13.8 µM HL1
,
HL2, ꢀꢁꢂꢃꢄ, ꢅꢆꢂꢄꢃ, ꢀꢁꢂꢃꢃ and ꢅꢆꢂꢃꢃ

ACCEPTED MANUSCRIPT
pH-3.0
pH-4.0
pH-5.0
pH-6.0
pH-7.4
pH-9.0
pH-3.0
(A)
(B)
pH-5.0
pH-6.0
pH-7.0
pH-9.0
pH-10.0
pH-11.0
0.8
0.6
0.4
0.2
0.0
0.8
0.6
0.4
0.2
0.0
Isosbestic Points
Isosbestic Points
280
350
420
490
560
280
350
420
490
560
Wavelength (nm)
Wavelength (nm)
Fig. 3. UV-Vis spectra of 20 µM HL1 (A) and HL2 (B) in different pH media
35
30
25
A
20
15
10
1.0
1.5
2.0
2.5
3.0
Fig. 4. Geometries of metal complexes (A) ꢀꢁꢂꢄꢃ (B) ꢅꢆꢂꢄꢃ (C) ꢀꢁꢂꢃꢃ (D) ꢅꢆꢂꢃꢃ optimized using
DFT calculation

ACCEPTED MANUSCRIPT
ꢇꢂꢄ
80
60
40
20
0
ꢀꢁꢂꢄꢃ
ꢅꢆꢂꢄꢃ
ꢇꢂꢃ
ꢀꢁꢂꢃꢃ
ꢅꢆꢂꢃꢃ
0
100
200
300
400
500
Concentration (µM)
Fig. 5. Inhibition profile of alkaline phosphatase at different concentrations of ligands and their
complexes
Fig. 6. Morphological effects of the synthesized Schiff bases and their metal complexes (ꢇꢂꢄ,
ꢇꢂꢃ, ꢀꢁꢂꢄꢃ, ꢀꢁꢂꢃꢃ, ꢅꢆꢂꢃꢄ and ꢅꢆꢂꢃꢃ) on HepG2 cells. Unexposed, DMSO (negative control) and
Doxorubicin (positive control) were included as experimental controls.