Synthesis, structural, spectral and biological evaluation of metals endowed 1,2,4-Triazole

Article abstract of DOI:10.4314/bcse.v34i2.11

Biologically active triazole Schiff base ligand 2,4-dichloro-6-[(1H-1,2,4-triazol-3-ylimino)methyl]phenol (A) has been synthesized by the condensation reaction of an equimolar amount of 1H-1,2,4-triazole-3-amine and 3,5-dichlorosalicylaldehyde and then it coordinated with salts of metals [VO(IV), Fe(II), Co(II), Ni(II), Cu(II) and Zn(II)] in 2:1 molar ratio to derive a series of transition metal chelates (1)-(6). All the compounds were characterized by various physical, spectral, analytical techniques and elemental analysis. Spectral characterization and magnetic moment data of complexes revealed square pyramidal geometry for vanadium complex and octahedral for remaining (2)-(6) complexes. Quantum chemical calculation has been carried out to explore optimized geometry and electronic structure of the ligand. Density functional theory (DFT) with B3LYP/6-311+g(d,p) method was performed to determine vibrational bands, frontier molecular orbitals (FMOs) and natural bond analysis (NBO) of the ligand. NBO analysis showed that the ligand bears higher molecular stability because of hyper conjugate interactions. Computational study results revealed that there was a close interaction of theoretical and experimental spectroscopic data. Global reactivity descriptors calculated by the energies of FMOs, indicated ligand to be bioactive. The synthesized compounds were studied for antibacterial, antifungal, antioxidant and antiglycation activity and the results revealed that ligand has remarkable activity which considerably increased upon chelation.

Full text of DOI:10.4314/bcse.v34i2.11

Bull. Chem. Soc. Ethiop. 2020, 34(2), 335-351.
 2020 Chemical Society of Ethiopia and The Authors
DOI: https://dx.doi.org/10.4314/bcse.v34i2.11
ISSN 1011-3924
Printed in Ethiopia
SYNTHESIS, STRUCTURAL, SPECTRAL AND BIOLOGICAL EVALUATION OF
METALS ENDOWED 1,2,4-TRIAZOLE
Sajjad Hussain Sumrra1*, Muhammad Anees1, Awais Asif2, Muhammad Nadeem Zafar1, Khalid
Mahmood3, Muhammad Faizan Nazar1, Muhammad Khalid4, Muhammad Arif Nadeem5 and
Muhammad Usman Khan6
1Department of Chemistry, University of Gujrat, Gujrat-50700, Pakistan
2Department of Biochemistry, Nawaz Sharif Medical College, University of Gujrat,
Gujrat-50700, Pakistan
3Institute of Chemical Sciences, Bahauddin Zakariya University, Multan-60800, Pakistan
4Department of Chemistry, Khwaja Fareed University of Engineering and Information
Technology, Rahim Yar Khan-64200, Pakistan
5Department of Chemistry, Quaid-i-Azam University, Islamabad-45320, Pakistan
6Department of Applied Chemistry, Government College University, Faisalabad-38000,
Pakistan
(Received November 29, 2019; Revised July 28, 2020; Accepted July 29, 2020)
ABSTRACT. Biologically active triazole Schiff base ligand 2,4-dichloro-6-[(1H-1,2,4-triazol-3-
ylimino)methyl]phenol (A) has been synthesized by the condensation reaction of an equimolar amount of 1H-
1,2,4-triazole-3-amine and 3,5-dichlorosalicylaldehyde and then it coordinated with salts of metals [VO(IV),
Fe(II), Co(II), Ni(II), Cu(II) and Zn(II)] in 2:1 molar ratio to derive a series of transition metal chelates (1)-(6). All
the compounds were characterized by various physical, spectral, analytical techniques and elemental analysis.
Spectral characterization and magnetic moment data of complexes revealed square pyramidal geometry for
vanadium complex and octahedral for remaining (2)-(6) complexes. Quantum chemical calculation has been
carried out to explore optimized geometry and electronic structure of the ligand. Density functional theory (DFT)
with B3LYP/6-311+g(d,p) method was performed to determine vibrational bands, frontier molecular orbitals
(FMOs) and natural bond analysis (NBO) of the ligand. NBO analysis showed that the ligand bears higher
molecular stability because of hyper conjugate interactions. Computational study results revealed that there was a
close interaction of theoretical and experimental spectroscopic data. Global reactivity descriptors calculated by the
energies of FMOs, indicated ligand to be bioactive. The synthesized compounds were studied for antibacterial,
antifungal, antioxidant and antiglycation activity and the results revealed that ligand has remarkable activity which
considerably increased upon chelation.
KEY WORDS: Triazole ligand, Transition metal chelates, Antibacterial, Antifungal, Antioxidant,
Computational study
INTRODUCTION
For the last two decades, Schiff bases were in continuous surfacing due to their ease in
preparation and variety of chemical reactions [1]. Schiff bases form an important class of
extensively used organic compounds and have various applications in different fields including
biological, analytical and inorganic chemistry [2]. Literature revealed that the reactivity of
Schiff bases enhanced significantly in the presence of OH group, primarily when the OH group
is attached at ortho position [3]. Schiff bases exhibit various biological activities and these
activities were increased by coordination with metal ions [4]. Various transition metals
coordinated with bi, tri and tetradentate Schiff base ligands having sulphur, nitrogen and oxygen
donor atoms which have played significant role in biological systems [5]. In contrast to 4d and
5d transition metal complexes, 3d metal complexes have revealed valuable imperative properties
as they can easily enter to the microbial cell membrane and have low toxicity [6].
__________
*Corresponding author. E-mail: sajjadchemist@uog.edu.pk
This work is licensed under the Creative Commons Attribution 4.0 International License

336
Sajjad Hussain Sumrra et al.
Microbial infection is the common growing health problem in contemporary medicinal field
and the use of antibiotic drugs is inevitable. Regardless of the availability of several antibiotic
and chemotherapeutic drugs, the emergence of various old and new antibiotic resistant bacterial
species created a substantial need for the innovative classes of antibacterial agents [7]. Design
and development of new metal-based drugs is an effective approach to fight against the current
threat of anticancer, antimicrobial and antioxidant resistance [8]. Therefore, compounds which
show activity against resistant strains, with enhanced safety and pharmacokinetic profiles are the
main goals for the current and future researchers in this area [9].
Triazole is a heterocyclic ring that is considered as pharmacologically imperative scaffold
and certainly used in many drugs associated with various biological activities [10]. Triazole
based transition metal complexes have shown substantial pharmaceutical and biological
activities such as antibacterial [11], antifungal [12], anticancer [13], herbicidal [14], cytotoxic
[15], antidiabetic [16], DNA binding and cleavage [17], anti-inflammatory and have abilities to
reduce the radical scavenging as well as oxidative properties [18]. Motivated by the above
mentioned facts and in the continuation of our research work [19] for biologically potent
nitrogen and oxygen endowed transition metal complexes; in the present paper we wish to report
the facile synthesis of another novel nitrogen based triazole Schiff base, 2,4-dichloro-6-[(1H-
1,2,4-triazol-3-ylimino)methyl]phenol by the condensation reaction of 1H-1,2,4-triazol-3-amine
and 3,5-dichlorosalicylaldehyde and its transition metal complexes [VO(IV), Fe(II), Co(II),
Ni(II), Cu(II) and Zn(II)] (Scheme 1). In addition to the synthesis of these compounds,
elucidation of their chemical structures was done by different spectroscopic techniques and DFT
based calculations. Furthermore, the investigated compounds were screened for antibacterial,
antiglycation, antifungal and antioxidant activities.
EXPERIMENTAL
Analytical grade chemicals purchased from authentic sources were used in this research; Sigma
Aldrich and Merck. Distilled ethanol and methanol were used in synthetic reaction of ligand and
its transition metal complexes, respectively. Stuart instrument was used for the determination of
melting point of synthesized ligand and its metal complexes. A high tech Shimadzu UV-4000
Spectrophotometer was used for UV-Vis analysis. Infrared spectroscopic technique was used for
the purpose of recording the wavelength of solid sample at 600-4000 cm-1 with the help of
1
Nicolet FT-IR Impact 400D infra-red spectrometer. H and 13CNMR of synthesized ligand was
performed by using Bruker Advance 300 MHz (Bruker Biospin GMBH Rheinstetten,
Germany). For the mass spectrometric analysis, ligand was dissolved in MeOH and injected
directly through direct insertion pump into the LTQ XL Linear Ion Trap Mass Spectrometer
(Thermo Scientific, USA) equipped with an Electro Spray Ionization (ESI) probe. Ligand was
analyzed in both positive as well as negative ion modes with a 10 µL min-l flow rate. Capillary
temperature and sheath gas flow (N2) were set at 350 °C and 30 arbitrary units, respectively.
Then, data was attained at both negative and positive total ion full scan mode with a range of
mass scan of m/z 50–700 using Xcalibur software. The full scan ESI-mass spectrum was used to
characterize the ligand, which gave specific m/z peaks by producing [M+Na]+, [M-H]- and
[M+H]+ ions. Moreover, the ESI-MS/MS fragmentation pattern of recognized peaks was used to
produce the daughter ions to confirm the molecular identity of ligand. All the antibacterial and
antifungal activities were performed at Department of Biochemistry, Nawaz Sharif Medical
College, University of Gujrat, Gujrat, Pakistan and antioxidant activity was carried out at
Department of Chemistry, University of Gujrat, Gujrat, Pakistan.
Synthesis of ligand
To synthesize ligand (A), magnetically refluxed 10 mL ethanolic solution of 3,5-
dichlorosalicylaldehyde (0.191 g, 1 mmol) was added in double neck flask with continuous
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Synthesis, structural, spectral and biological evaluation of metals endowed 1,2,4-triazole 337
stirring. After half an hour of refluxing, 1H-1,2,4-triazol-3-amine (0.084 g, 1 mmol) was added.
The reaction was continuously monitored by performing thin layer chromatography (TLC). The
pale-yellow precipitates of the product were obtained after refluxing of 6 hours (Scheme 1). The
precipitated product was cooled at room temperature overnight followed by filtration, washing
and drying. The obtained product was recrystallized in a mixture of ethanol and dioxane at room
temperature to get pure compound.
2,4-Dichloro-6-[(1H-1,2,4-triazol-3-ylimino)methyl]phenol (A)
Yield (94 %); melting point 231 °C; color pale yellow; IR (KBr, cm-1): 3180 (OH), 3070 (NH),
1
1640 (HC=N, azomethine), 1585 (C=N, triazole), 1380 (C-O), 1020 (N-N); H NMR (DMSO-
d6): δ 7.76 (d, C9-H), 7.92 (d, C12-H), 8.64 (s, C1-H), 9.41 (s, C13-H),13.85 (s, C8-OH), 14.29 (s,
C1-NH); 13C NMR (DMSO-d6, δ, ppm): 121.10 (C7), 122.05 (C10), 123.29 (C11), 131.56 (C9),
133.30 (C8), 135.50 (C12), 145.18 (C1), 155.66 (C5), 164.73 (C13); EIMS m/z (%): 257.08 [M+],
256.83 (19), 238.67 (75), 208.75 (6), 194.83 (78), 180.17 (100), 166.25 (46), 157.25 (12),
122.33 (9); anal. calcd. forC9H6Cl2N4O(257.08) %: C, 42.05; H, 2.35; N, 21.79; found: C, 41.80;
H, 2.26; N, 21.60.
Synthesis of transition metal complexes
All the transition metal chelates were synthesized by using standard procedure with molar ratio
of ligand and metal salts 2:1. In this method, (2.57 g, 10 mmol) of ligand was mixed with
methanol in double neck flask and refluxed for half an hour. After that, respective metallic salt
(5 mmol) was added and reaction was monitored by using TLC. Different metallic salts were
used at different time for complex formation. After 5-7 hours of refluxing, precipitates of metal
complexes were formed; precipitates were separated by filtration and were cooled at room
temperature over night. The produced metal complexes were recrystallized in a mixture of
methanol and dioxane at room temperature to get pure compound.
Scheme 1. Synthetic scheme of triazole ligand (A) and its metal complexes (1)-(6).
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Sajjad Hussain Sumrra et al.
Biological activities
All the newly synthesized metal based triazole complexes were evaluated for antibacterial,
antifungal, antioxidant and antiglycation activities.
In-vitro antibacterial activity
The novel synthesized ligand (A) and its transition metal complexes were tested against four
bacterial species (Staphylococcus aureus, Subtilis bacillus, Escherichia coli and Pseudomonas
syringae) through disc diffusion method [20]. The materials used in antibacterial activity
including nutrient broth, Petri-dishes and filter paper discs were autoclaved at 121 °C for 30
min. After heating the semi-liquid media of nutrient broth, it was poured into Petri plates and
kept for solidification in fume hood, then the prepared inoculum was dispersed on solidified
media by using glass loop spreader. Filter paper discs were kept on solidified bacterial inoculum
after dipping from standard and samples. All the solutions were prepared in dimethyl sulfoxide
(DMSO). Concentration of samples and standard solution was kept 5 mg/mL. Augmentin syrup
was used as standard drug because of its broad spectrum activity. After that, the Petri dishes
were kept in incubator at 37 °C and then the inhibition area was determined after 48 hours.
In-vitro antifungal activity
The antifungal activity of newly synthesized ligand (A) and its transition metal chelates was
performed by using disc diffusion method [20] against two fungal strains (Alterneria alternate
and Drechslera australensis). Terbicil capsule was used as standard drug. All the samples and
standard solutions were prepared in DMSO by using micropipette. After keeping the Petri dishes
in incubator at 37 °C for 72 hours, the inhibition zones were noted.
Antioxidant activity
By using DPPH method, reducing power method and concentration of total phenolic content,
anti-oxidant activity of synthesized compounds was determined.
DPPH method
Antioxidant activity of newly synthesized compounds was determined by adopting
recommended procedure by using DPPH (1,1-diphenyl-2-picryl hydrazyl) [21]. The procedure
was carried out by using different concentrations (2 mg/mL and 1 mg/mL) of sample solutions
and 20 mg/100 mL solution of DPPH radical. The reaction mixture was prepared by mixing 1
mL of DPPH solution in 4 mL of methanol and 100 µL of sample solution in test tubes. The
tubes were wrapped with aluminum foil and kept in incubator (37 °C) for half an hour.
Butylatedhydroxytoluene (BHT) was used as the standard. The absorption of synthesized
compounds was measured at 517 nm and the percentage inhibition was calculated by using
following formula:
(
)
ꢀꢁꢂꢃꢄꢅꢆꢂꢇꢈꢁꢉ
Percentage (%) inhibition =
× 100
(1)
ꢀꢁꢂꢃꢄ
Reducing power method
Total antioxidant capability of triazole ligand and its metal complexes was determined by using
standard procedure [22]. In this method, 1 mL sample solutions having concentration of 1
mg/mL were tested. The sample solutions were prepared in pure DMSO and mixed with 2.5 mL
of 2 M sodium phosphate buffer having pH 6.6 and 5 mL of 1% potassium ferricyanide. Then
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Synthesis, structural, spectral and biological evaluation of metals endowed 1,2,4-triazole 339
the reaction mixture was incubated at 50 °C for 30 min. After that, 5 mL of 10% trichloroacetic
acid was added and centrifuged at 1000 RPM for 10 min. After centrifugation, two separate
layers were formed. From upper layer, 5 mL sample solution was taken and mixed with 5 mL of
distilled water and 1 mL of 1% ferric chloride. At the end, absorbance was measured at 700 nm
with the help of spectrophotometer.
Concentration of total phenolic content
The absorbance of total phenolic content of ligand and its transition complexes was checked by
using standard procedure [23]. In this method, 0.1 mL of sample solutions having concentration
2 mg/mL was mixed with 2 mL of sodium carbonate (7.5%), 1 mL of 10% Folin reagent and 4
mL of methanol in seven different test tubes (one test tube for ligand and six for complexes).
Test tubes were vortexed for 30 s and kept the whole reaction mixture at 37 °C for half an hour.
Finally, after incubation, the absorbance was measured at 760 nm by UV-Vis
spectrophotometer. Gallic acid was used as a standard in this procedure. Quantifications of the
sample triplicate were made by using the standard calibration curve of the gallic acid.
Measurement was carried out for triplicate samples for each complex.
Antiglycation activity of ligand and its metal complexes
In this method glucose working solution having 1 M concentration was prepared in distilled
water. A buffer was formed by using 3.56 g of di-sodium-hydrogen phosphate and 2.76 g of
sodium-di-hydrogen phosphate in 100 mL of deionized water. In order to maintain the pH of
buffer solution at 7.4; mixed the 38.5 mL of di-sodium-hydrogen phosphate with 11.5 mL of
sodium-di-hydrogen phosphate. Similarly, 2 mg/mL solutions of BSA (bovine serum albumin),
TCA (trichloro acetic acid) and rutin were prepared accordingly in distilled water. Rutin was
used as standard. Three different solutions were prepared, i.e. positive (BSA + Buffer +
Glucose), negative (BSA + Buffer) and standard (BSA + Rutin + Glucose). All the sample
solutions were prepared in methanol with concentration of 2 mg/mL. The reaction was carried
out in small plastic tubes having 1 mL volume by using micropipette [24]. All the tubes were
kept at 37 °C in an incubator for seven days. After seven days reaction was stopped by adding
the 200 μL solution of TCA having concentration of 2 mg/mL in each tube. Then centrifuge all
the tubes at 8000 rpm for 5 min in order to form the pellets. At the end, added 100 μL buffers in
each tube and the fluorescence results were checked by using nano-flourometer instrument.
Excitation and emission wavelengths of nano-fluorometer were 330 and 440 nm, respectively.
Computational procedure
Density functional theory (DFT) study of synthesized triazole compounds was performed using
Gaussian program. The input files were recorded to use Gauss View [25]. Ultraviolet-visible
analysis was performed in combination of B3LYP/6-311+g (d,p) in gas phase. Avogadro,
Chemcraft and Gauss View softwares were used to check the output files [26].
RESULTS AND DISCUSSION
The condensation of 1H-1,2,4-triazole-3-amine with 3,5-dichlorosalicylaldehyde in an
equimolar ratio resulted to form a new triazole Schiff base which was an air and moisture stable
yellow colored solid compound. The synthesized ligand was soluble in methanol, ethanol,
dioxane, DMSO and DMF at room temperature. The ligand readily reacted with salts of
transition metals [VO(IV), Fe(II), Co(II), Ni(II), Cu(II) and Zn(II)] in methanol to form their
metal complexes by using ligand and metallic salt in 2:1 molar ratio, respectively. All the
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Sajjad Hussain Sumrra et al.
synthesized metal complexes were intensely colored except Zn(II) complex (6) that was
colorless. The metal complexes decomposed without melting. The spectral data showed that the
ligand coordinated with the metal ions in bidentate and tridentate manner via NO and NNO
donor sites. The spectral data of the prepared ligand and its metal complexes shown in Table 1,
was in good agreement with their structures indicating the high purity of all the compounds.
Table 1. Physical, analytical and elemental data of ligand (A) and metal complexes (1)-(6).
No Compounds
Yield (%)
color
M.W (g/mol)
chemical formula
[257.08]
C9H6Cl2N4O
[579.07]
C18H10Cl4N8O3V
[567.98]
C18H10Cl4FeN8O2
[571.06]
M.P
(oC)
231
Calculated % (found)
C
42.05
H
N
M
-
(A)
(1)
(2)
(3)
(4)
(5)
(6)
Ligand
VO(A)2
Fe(A)2
Co(A)2
Ni(A)2
Cu(A)2
Zn(A)2
94
2.35
21.79
Pale yellow
76
Green
89
Dark brown
91
Gray
86
Brown
92
(41.80) (2.26) (21.60)
37.33 1.74 19.35
(37.25) (1.68) (19.24)
38.06 1.77 19.73
(38.01) (1.71) (19.64)
37.86 1.77 19.62
(37.79) (1.72) (19.54) (10.26)
37.87 1.77 19.63 10.28
(37.67) (1.73) (19.56) (10.23)
236
246
252
290
240
256
8.80
(8.74)
9.83
(9.77)
10.32
C18H10Cl4CoN8O2
[570.82]
C18H10Cl4NiN8O2
[575.68]
37.55
1.75
(37.37) (1.71)
19.46
(19.4)
19.40
11.04
(10.97)
11.33
Dirty green
95
C18H10Cl4CuN8O2
[577.54]
37.43
1.75
Off white
C18H10Cl4N8O2Zn
(37.24)
(1.71) (19.35) (11.26)
1H-NMR spectrum
1H-NMR spectrum of triazole Schiff base ligand (A) was recorded in DMSO solvent to
determine the structural properties. The inspection of spectrum presented the representative
peaks of N-H, (HC=N) and C-H protons of synthesized triazole compound at 14.29, 9.41 and
8.64 ppm, respectively as a singlet. Furthermore, two signals; doublets at 7.76 and 7.92 ppm for
aromatic protons in benzene ring were observed. Higher chemical shift values of these two
protons were due to the attachment of more electronegative chloro group with neighbouring
carbon atoms. The proton signal at 7.92 ppm was due to C-H groups which are in between the
two chloro attached carbon atoms. The value of proton of OH group in ligand moved downfield
and was observed at 13.85 ppm due to the possibility of hydrogen bonding of oxygen atom and
proton of azomethine carbon [27]. The examination of all observed signals confirmed the
formation of triazole Schiff base ligand (A) and its proposed structure.
13C-NMR spectrum
13C-NMR spectrum of ligand was recorded in DMSO solvent. In 13C-NMR spectral data, all the
numbers of carbon atoms were found in their expected regions. The 13C-NMR data showed the
azomethine carbon (C13) peak at 164.73 ppm which confirmed the condensation reaction. Due to
the possibility of hydrogen bonding between oxygen of OH group and proton of azomethine
carbon, aromatic ring carbon (C8) displayed peak at 133.30 ppm. Carbon atom (C1) which is
present between the two nitrogen atoms in triazole ring, exhibited peak at 145.18 ppm, from
which confirmed the formation of ligand (A). Furthermore (C9), (C10), (C11), (C12) and (C13)
exhibited their respective peaks at 131.56, 122.05, 123.29, 135.50 and 164.73 ppm, individually
which are in close agreement to their experimental values and compromised well with the
number of carbon atoms present in the proposed structure of ligand.
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Synthesis, structural, spectral and biological evaluation of metals endowed 1,2,4-triazole 341
Mass spectrum
The mass spectral data and fragmentation pattern of the triazole Schiff base ligand (A) intensely
confirmed the formation of the ligand, its proposed structure along with its bonding pattern. It
was concluded from the mass spectrum of the ligand that different peaks of various fragments
from parent ligand molecule at different intensities gave the idea of molecular ion peak and base
peaks of synthesized compound. The molecular ion peak was noted at mass to charge ratio (m/z)
of 257.07 which indicated the formation of ligand. The base peak of most stable fragment of
parent ligand molecule was observed at m/z = 180.17. Fragmentation pattern of ligand resulted
from the breakdown of C-N, C=N, C-O, O-H, C-Cl and C-C.
IR spectra
The main IR vibrational bands of the triazole Schiff base and its metal complexes were found in
their expected regions. IR spectrum of triazole ligand (A) confirmed the formation of new
functional group, azomethine (HC=N) at 1640 cm-1 that strongly indicated the condensation
reaction. Ligand also showed band at 3180 cm-1 due to the presence of -OH group [28]. The
appearance of peaks at 1585, 1380 and 1020 cm-1 indicated the presence of C=N of triazole, C-O
and N-N groups, respectively [29]. There was a close agreement between both values of
experimental results taken from IR spectrum and theoretically computed data from IR spectrum
of ligand. After complex formation, band of azomethine vibration at 1640 cm-1 shifted to 1620-
1625 cm-1 thus indicating the coordination of the azomethine-N to the transition metals (V, Fe,
Co, Ni, Cu, Zn) [30]. Similarly, band of C-O at 1380 cm-1 was shifted to1355-1360 cm-1 and
disappearance of OH band at 3180 cm-1 hence confirming the deprotonation and coordination of
salicylidene–OH [31]. On the other hands, there was no shifting of N-N and N-H bands showing
their non-involvement in the coordination [32] and confirmed the proposed structures of the
metal complexes.
Molar conductance and magnetic moment measurements
The concentration of 0.01 M solution of DMF was used to determine the molar conductance of
synthesized metal complexes by using Inolab Cond 720 Conducto-meter at room temperature.
The conductance values were observed in the range of 12-20 -1cm2mol-1, hence confirming the
non-electrolytic nature of the metal complexes [33]. The magnetic moments of newly
synthesized compounds were determined by using Gouy balance. Some of compounds showed
high magnetic moments while some showed low magnetic moments depending upon the
magnetic power of unpaired and paired electrons. VO(IV) complexes exhibited magnetic
moment value of 4.42 BM that was indicative of one unpaired electron, and supporting its
square-pyramidal geometry. Magnetic moment values of Cu(II) and Fe(II) complexes were
found to be 1.75 and 5.59 BM [19], which indicated the presence of one and four unpaired
electrons of copper and iron, respectively. Both complexes showed octahedral geometry which
is most stable. Co(II) complex exhibited the magnetic moment value of 4.39 BM that clued the
presence of three unpaired electrons and octahedral geometry of the complex. The measured
magnetic moment value of Ni(II) complex was found to be 3.60 BM which confirmed the
presence of two unpaired electrons and octahedral geometry of the complex. Zn(II) did not show
magnetic moment value indicating the diamagnetic nature of the zinc complex [34].
Natural bond orbital (NBO) analysis
The NBO analysis of ligand (A) was done by using B3LYP/6-311+g(d,p) method to determine
the perturbation theory. The data obtained from NBO analysis was helpful to find the
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Sajjad Hussain Sumrra et al.
conjugation as well as the i
n
n
teraction of charge transfer in the molecule. The inter
a
a
ctions
gth of
h was
sis [35]. A lot of information was obtained from calculatioons of
sis, here just promising transitions are explained which ccontain
between contributors and ac
electron donors and electron acceptors was easily affected by stabilization energy, whi
achieved by using NBO anal
ligand by using NBO analy
cceptors gave massive amount of stabilization energy. Stren
cc
y
s
higher value of stability. The conjugation of the entire framework can also be explained on the
basis of second-order stabiliization energy. The stabilization energy E(2) is determi
equation;
nn
ed by
ꢓ
ꢒ
ꢊ(ꢋ) = ꢌꢍ ꢎꢏ
(2)
ꢐ.ꢑ
ꢔ ꢅꢔ
ꢑ
ꢐ
where qi, is the contributor orbital occupancy, F(i.,j) is the off diagonal NBO Fock
element whereas, εj and εi are he diagonal elements, respectively. NBO study exhibited b
er (ICT) interactions. The charge transformations take place
rominent atoms in which charge interactions take place are π (C5
mmatrix
tt
uunch of
intra-molecular charge trans
ff
between different atoms. The
pp
;;
– C6) → π* (C1 –C2), π (C5 – C6) → π* (C3 – C4) and π (C1 – C2) → π* (C5 – C6) have higher
stabilization energies 52.57, 4.68 and 5.729 kcal/mol, respectively as compared to other atoms
present in the molecule. The assignment of charge between the lone pairs are; O12 → σ (C3–
H4), Cl15 → σ* (C1– C2), N17 π* (N19– C20), leads higher stabilization energies 4.073, 2.909,
1.240 kcal/mol, respectively a compared to that of other atoms which contain lone pairs. All the
44
**
→
s
s
shown conjugated interactions of different atoms show charge transformations among each
other. The calculated NBO analysis exhibited high interactions of atoms with each other,
explaining the stability of co
Mulliken atomic charges (MA
Mulliken atomic charge analysis is a significant portion in quantum chemistry to determ
m
m
pound and gave an authentic idea about the stabilization en
eergy.
CC) analysis
i
i
ne the
chemical action in molecular structure; as the atomic charges are highly affected by ele
configuration, their polarity a d characteristics of molecules [36]. The Mulliken atomic c
of ligand (A) were determine by using B3LYP/6-311+g(d,p). The measured values of
charges exhibited the presenc
N = -0.155209), (18 N = -0.1
charge is distributed on the ccarbon atoms while all types of hydrogen atoms possess p
cctronic
n
h
arges
d
d
aatomic
e
7
of highly electronegative atoms such as (12 O = -0.2473377), (17
5012) and (19 N = -0.088723) from which it is clear that p sitive
sitive
oo
oo
charge. Histogram of Mullike
n
atomic charges is given in Figure 1.
Figure 1. Histogram for Mulli
k
k
en atomic charge distribution of the ligand (A).
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Synthesis, structural, spectral and biological evaluation of metals endowed 1,2,4-triazole 343
Experimental FT-IR results of ligand
The resulted experimental and computational data of ligand (A) including its intensity and
vibrations is given in Table 2. Theoretical study was carried out by B3LYP/6-311+g(d,p) basis
set and results were in close agreement with experimental band of IR.
Table 2. Experimental and theoretical frequencies of FT-IR for ligand (A).
Frequency scaled
726.5050
Experimental
730
IIR
Vibrational assignments
(s)C-Claliphatic
(s)C-Claliphatic+ C-Caromatic
(s)C-Naromatic
ꢕ(s)C-Haromatic
ꢕ(s)C-Haromatic + ꢕ(b)C-Caromatic
(s)C-Oaliphatic
(s)C-Oaliphatic + O-Haliphatic
(v)C-OHaliphatic
(v)C-OHaliphatic
(s)C-Naromatic
(as)C-Naromatic
(s)C-Caromatic
(s)C-Caromatic + (as)C-Caromatic
(v)C=Naromatic
(as)C=Naromatic
(s)N-Haromatic
(as)N-Haromatic
19.9516
07.0719
12.6818
17.0374
13.4856
115.5129
23.1238
12.9349
38.5510
23.7137
17.4504
138.5775
14.2577
26.5723
35.6012
07.3995
00.9429
759.6710
820.6385
838.6090
860.4536
830
850
1115.4025
1177.4153
1269.6629
1292.7173
1330.5198
1392.4886
1428.5505
1482.7146
1588.7302
1603.4793
3325.0352
3338.6076
1100
1195
1330
1450
1600
3400

= stretching, ꢕ = rocking, b = bending, s = symmetric, as = asymmetric. Intensity (I) in kJ/mol and frequency
(ꢖ) in cm-1.
UV-Visible analysis
UV-Visible spectral study for ligand (A) was performed by using time dependent density
fractional theory (TD-DFT) in the gas phase using B3LYP/6-311+g(d,p) to explain the
characteristics of vertical electronic transitions [19]. Two transitions of electrons were obtained
at 328.5 and 273 nm having oscillator strength of 0.1186 and 0.0006 respectively that are very
close to experimental variation values 328 and 291 nm, respectively.
Frontier molecular orbital (FMO) analysis
The stability, electrical properties and reactivity of any compound can be determined by energy
difference of frontier molecular orbitals [37]. The highest occupied molecular orbitals (HOMO)
and lowest unoccupied molecular orbitals (LUMO) behave as electron donors and the electron
acceptors, respectively. Furthermore, electronic excitations that take place from HOMO to
LUMO of ligand (A) areas illustrated in Figure 2. The data obtained from DFT calculations
showed that the ligand (A) exhibited 403 orbitals, from which the numbers of HOMO and
LUMO orbitals were 65 and 66, respectively. The appearance of golden color indicates the
positive and blue color indicates the negative part of orbitals. The energy values of HOMO and
LUMO orbitals obtained from DFT study were -6.966 and -2.621 eV, respectively. Furthermore,
energy values of HOMO-1 and LUMO+1 were -7.248 and -1.225 eV, respectively. In the
similar way -7.805 eV energy was found for HOMO-2 and -1.054 eV was found as the energy
of LUMO+2. While the energies of HOMO-3 and LUMO+3 were obtained as -8.050 eV and -
0.732 eV, respectively. The energy differences were 4.345, 6.023, 6.751 and 7.318 eV for
HOMO→LUMO; HOMO-1→LUMO+1; HOMO-2→LUMO+2 and HOMO-3→LUMO+3,
respectively.
Bull. Chem. Soc. Ethiop. 2020, 34(2)

344
Sajjad Hussain Sumrra et al.
Figure 2. Frontier molecular orbitals of the ligand (A).
Global reactivity descriptors
The energies of HOMO and LUMO are used to gain insight into the promising global reactivity
descriptors and summarized in Table 3. Koopmans’s theorem has been used to determine the
electronegativity (χ), chemical hardness (ƞ) and chemical potential (μ) [38], which are defined
from the following equations as;
IP = -EHOMO
EA = -ELUMO
(3)
(4)
[
]
ꢘꢙꢚꢛꢜ
ꢋ
ꢚ ꢝ
ꢋ
ꢞꢟꢠꢡ
ꢞꢟꢠꢡ
ꢢꢡꢠꢡ
ꢗ =
ꢣ =
ꢤ =
= − ꢝ
(5)
(6)
(7)
[
]
ꢘꢙꢅꢛꢜ
ꢋ
ꢅ ꢝ
ꢋ
= − ꢝ
ꢢꢡꢠꢡ
ꢝ
ꢚ ꢝ
ꢋ
ꢞꢟꢠꢡ
ꢢꢡꢠꢡ
where IP = ionization potential (a.u), EA = electron affinity (au).
Global softness (σ) is defined by following relationship;
ꢥ = ꢦ ꢣ
(8)
(9)
ꢋ
And electrophilicity index (ω) can be defined by below equation;
ꢓ
ꢨ
ꢧ =
ꢋꢩ
This parameter is considered as a measure of the electrophilic strength of a compound.
Electronegativity (χ) has been defined as negative of the chemical potential. Usually, the
chemical behavior of any species can be evaluated with the help of global softness, global
hardness, electronegativity and chemical potential parameters. Molecules containing small
energy gap are recognized as soft compounds and the molecules which contain large energy
gap, are recognized as hard compounds with less chemical reactivity and greater kinetic
Bull. Chem. Soc. Ethiop. 2020, 34(2)

Synthesis, structural, spectral and biological evaluation of metals endowed 1,2,4-triazole 345
stability. In the current study, the larger magnitude of ionization potential is observed, which
suggests high reactivity of the title molecule (A). The value of softness is higher as compared to
that of hardness, suggesting its high reactivity and less stability (Table 3). However, the
molecule has the negative value of chemical potential. On the basis of the obtained information,
it can be concluded that the molecule might be highly reactive and reasonably stable.
Table 3. Ionization potential (I), electron affinity (A), electronegativity (χ), chemical hardness (ƞ), chemical
potential (µ), global softness (ϭ) and electrophilicity index (ω).
Combinations
A
B
C
D
Ionization potential (I)
Electron affinity (A)
Electronegativity (χ)
Chemical hardness (ƞ)
Chemical potential (µ)
Global softness (ϭ)
6.967
2.622
4.794
2.217
-4.794
0.225
5.180
7.249
1.225
4.230
3.012
-4.230
0.166
2.970
7.805
1.055
4.430
3.375
-4.430
0.148
2.907
8.050
0.732
4.394
3.659
-4.391
0.136
2.634
Electrophilicity index (ω)
A = HOMO→LUMO, B = HOMO-1→LUMO+1, C = HOMO-2→LUMO+2, D = HOMO-3→LUMO+3; units in
eV.
Molecular electrostatic potential (MEP) analysis
Molecular electrostatic potential (MEP) chart symbolizes the three-dimensional distribution of
charge magnitude of a molecule. MEP is used to determine the interaction of charge transfer in
single atom with each other. It is recognized as a helpful factor, which illustrates the shape, size,
negative, positive zone without charge of an inspected system, which is generally assessed with
standard shading. In terms of charge distribution, the order of code colors is: red > orange >
yellow > green > blue. For that reason, the red region can be the favored destination for
electrophilic attack whereas the blue region can be the favored destination for nucleophilic
attack. The experimental data and computational theories can be used to determine the MEP,
because it is known as one of the significant physical properties [32]. In Figure 3, all the
hydrogen atoms of ligand (A) having blue color, suggest the comparative nonattendance of the
electrons around hydrogen atoms. On the other hand, the more electronegative atoms like
oxygen and nitrogen having red color show comparatively more magnitude of electron density,
subsequently, it can be proved as a favored site for encounter of electrophilic attack.
Figure 3. The molecular electrostatic potential for ligand.
Bull. Chem. Soc. Ethiop. 2020, 34(2)

346
Sajjad Hussain Sumrra et al.
In-vitro antibacterial activity
Antibacterial activity of triazole based ligand and its transition metal complexes was checked
against four types of bacterial strains, two Gram-positive (Subtilis bacillus and Staphylococcus
aureus) and two Gram-negative (Pseudomonas syringae and Escherichia coli). The inhibition
zones (millimeter) of ligand and its metal complexes were checked by comparing with the
inhibition zone of amoxicillin which was used as standard drug. The results of all compounds
and standard drug are shown in Table 4 and Figure 4. Amoxicillin exhibited greater inhibition as
compared to other newly synthesized compounds. It was also concluded from results that ligand
(A) showed less activity as compared to standard drug (amoxicillin) but comparably exhibited
higher inhibition than metal complexes. Among metal chelates, zinc complex showed highest
inhibition against Gram negative bacteria (Pseudomonas syringae and Escherichia coli), copper
complex exhibited highest activity against Bacillus subtilis. Furthermore, complexes of copper
and cobalt showed highest inhibition against Staphylococcus aureus as compared to other metal
chelates [39].
Table 4. Antibacterial and antifungal activities of ligand and its metal complexes.
Compound
No.
Bacterial strains
Fungal strains
Subtilis Pseudomonas
Escherichia
Staphylococcus
Alterneria
Drechslera
bacillus
15
08
05
07
05
08
07
syringae
13.5
09
07
08
05
07
10
-
coli
08
05
00
08
04
06
13
-
aureus
07
08
06
08
05
08
08
alternate
australensis
(A)
15
19
05
07
13
10
17
-
10
09
14
08
07
07
05
-
VO(A)2
Fe(A)2
Co(A)2
Ni(A)2
Cu(A)2
Zn(A)2
DMSO
SD
-
-
25
22
21
21
23
22
Figure 4. Antibacterial and antifungal activities of Schiff base ligand and metal complexes.
In-vitro antifungal activity
The antifungal activity of newly synthesized ligand (A) and its transition metal chelates was
performed by using disc diffusion method against two fungal strains (Alterneria alternate,
Drechslera australensis). The inhibition zones (mm) of ligand and its metal complexes were
Bull. Chem. Soc. Ethiop. 2020, 34(2)

Synthesis, structural, specctral and biological evaluation of metals endowed 1,2,4-triazolle 347
checked by comparing with teerbicil, which was used as standard drug. The results of inh
zones (mm) of all the synthesized compounds and standard drug are presented in Table 4 and
Figure 4. Overall triazole liga d displayed significant results against both fungal strains. AAmong
the metal complexes, vanadium and zinc complexes possessed highest inhibition gainst
Alterneria alternate. Howeve , all the complexes were active against both fungal strains.
iibition
n
aa
rr
In-vitro antioxidant activity
The synthesized compounds sshowed antioxidant activity which was checked by using DDPPH
radical scavenging, reducing ppower method and concentration of total phenolic content.
DPPH Method
The diphenylpicrylhydrazide ((DPPH) is free radical and have ability to check out the poteenncy of
radical scavenging of triazole ligand and it transition metal chelates. Two sample concentrrations
(1 mg/mL and 2 mg/mL) were used to check the antioxidant activity of triazole ligand and its
metal chelates. Butylatedhyd
r
oxytoluene (BHT) was used as standard. Results showeed that
among 1 mg/mL sample con
c
centrations, nickel complex exhibited [40] highest (52.62%%) and
iron complex showed lowest (8.04%) activity. While among 2 mg/mL sample concentr
zinc complex showed highest (18.85%) and copper complex exhibited lowest (10.32%) acctivity
as depicted in Table 5 and Fig re 5.
aations,
u
Table 5. Antioxidant and antiglycationactivity results of ligand (A) and metal complexes.
Comp-
ound
Antioxidant Activity
Reducing power
Absorbance
Antiglycation
activity
% activity
DPPH method
% inhibition % inhibition
Total phenolic contents
Absorb-
ance (nm)
Concentrations
(2 mg/mL)
(1 mg/
m
m
L)
(nm)
0.25
1.68
0.37
0.79
0.25
0.33
0.18
-
(ppm)
468
304
267
247
203
693
269
-
A
17.53
16.38
17.32
19.18
15.71
10.32
18.85
64.38
20.
40.
08.
16.
52.
46.
44.
55.
2
7
2
4
5
2
5
3
6
0.56
0.39
0.36
0.34
0.29
0.79
0.35
-
84.98
83.54
82.62
83.85
69.79
75.04
63.68
88.19
VO(A)2
Fe(A)2
Co(A)2
Ni(A)2
Cu(A)2
Zn(A)2
Standard
99
00
99
66
99
88
55
Figure 5. Antioxidant and antigglycation activities of ligand and its metal complexes.
Bull. Chem. Soc. Ethiop. 2020, 34(2)

348
Sajjad Hussain Sumrra et al.
Reducing power method
Total antioxidant capability of triazole Schiff base and its metal complexes was determined. The
results revealed that all the transition metal complexes exhibited high absorbance than that of
their parent compound except zinc complex, which displayed least (0.18 nm) absorbance and
vanadyl complex experienced highest absorbance of 1.68 nm (Table 5).
Concentration of total phenolic content
Concentrations of total phenolic contents of the ligand and its transition metal complexes were
studied by UV-Vis spectrophotometer. Results were measured in absorbance and
concentrations. The triazole ligand has more promising results as compared to metal complexes
except copper complex which has shown better results out of the metal complexes (Table 5).
Antiglycation activity of ligand and its metal complexes
Antiglycation activity of synthesized ligand and its metal complexes was determined by using
standard reported method. The triazole ligand and its complexes demonstrated promising results
as shown in (Figure 5 and Tables 5). The triazole ligand displayed highest activity of 84.98%
and cobalt complex possessed maximum activity 83.85% while zinc complex showed lowest
activity of 63.68%.
CONCLUSION
In this study, we have reported the synthesis of a newly synthesized triazole ligand, 2,4-
dichloro-6-[(1H-1,2,4-triazol-3-ylimino)methyl]phenol (A) along with its VO(IV), Fe(II),
Co(II), Ni(II), Cu(II) and Zn(II) complexes. Synthesized compounds were characterized by
physical, spectroscopic and elemental analysis. The results of DFT study of ligand displayed
very close relationship between experimental values obtained from IR, UV and computed data
obtained theoretically. All the compounds were assessed for their antimicrobial, antioxidant and
antiglycation properties. Results of these activities showed that complexes have higher
biological activities than that of parent ligand molecule. Finally, these promising results are
helpful in different biological applications in the future endeavors.
ACKNOWLEDGEMENT
The authors are thankful to the Higher Education Commission (HEC) of Pakistan for providing
financial support through the NRPU Project # 7800.
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