Synthesis, DFT, computational exploration of chemical reactivity, molecular docking studies of novel formazan metal complexes and their biological applications

Article abstract of DOI:10.1002/aoc.5444

The computational exploration of chemical reactivity and molecular docking of the synthesized formazan compounds (S1-S6) were studied. Further, their antimicrobial activity against bacterial strains (S. epidermidis, B. cereus, K. pneumoniae and P. aeruginosa) and against fungal strains (T. mentagrophytes, C. albicans, A. niger, S. cerevisiae and C. glabrata) using agar diffusion method and antioxidant activity following DPPH inhibition assays were evaluated. Anticancer activity was executed in vitro model of human breast carcinoma (MCF-7) cell line. The superior and enhanced antibacterial and antimycotic activities were exhibited by formazan compound (S4) by presenting maximum ZOIs and MICs values. While enhanced antioxidant in terms of percentage inhibition of DPPH and cytotoxic effect on human breast carcinoma-cells demonstrated by formazan compound (S1) which was further validated by the results of molecular docking studies of (S1) with the human estrogen receptor protein. In order to compute quantum chemical reactivity descriptors from conceptual density functional theory (CDFT) point of view of this system, including chemical potential (μ), chemical hardness (η), electrophilicity (ω), condensed Fukui function and dual descriptors are calculated at the same level of calculation. The most active sites of these molecules are determined and correlated with experimental data. The present investigation displays that formazans compounds could be potential drug candidate that constrains the growth of microbial strains, possess ability to cause cytotoxic effect on carcinoma cells and act as effective scavenger for free radical species.

Full text of DOI:10.1002/aoc.5444

Received: 6 September 2019
DOI: 10.1002/aoc.5444
Revised: 18 November 2019
Accepted: 26 November 2019
F U L L P A P E R
Synthesis, DFT, computational exploration of chemical
reactivity, molecular docking studies of novel formazan
metal complexes and their biological applications
Shakeel Ahmad Khan1,2
Mahmoud A. Noamaan4 | Tahir Rasheed5
| Komal Rizwan3 | Sammia Shahid1
|
| Hira Amjad1
1Department of Chemistry, School of
The computational exploration of chemical reactivity and molecular docking
of the synthesized formazan compounds (S1-S6) were studied. Further, their
antimicrobial activity against bacterial strains (S. epidermidis, B. cereus,
K. pneumoniae and P. aeruginosa) and against fungal strains
(T. mentagrophytes, C. albicans, A. niger, S. cerevisiae and C. glabrata) using
agar diffusion method and antioxidant activity following DPPH inhibition
assays were evaluated. Anticancer activity was executed in vitro model of
human breast carcinoma (MCF-7) cell line. The superior and enhanced
antibacterial and antimycotic activities were exhibited by formazan compound
(S4) by presenting maximum ZOIs and MICs values. While enhanced antioxi-
dant in terms of percentage inhibition of DPPH and cytotoxic effect on human
breast carcinoma-cells demonstrated by formazan compound (S1) which was
further validated by the results of molecular docking studies of (S1) with the
human estrogen receptor protein. In order to compute quantum chemical reac-
tivity descriptors from conceptual density functional theory (CDFT) point of
view of this system, including chemical potential (μ), chemical hardness (η),
electrophilicity (ω), condensed Fukui function and dual descriptors are calcu-
lated at the same level of calculation. The most active sites of these molecules
are determined and correlated with experimental data. The present investiga-
tion displays that formazans compounds could be potential drug candidate
that constrains the growth of microbial strains, possess ability to cause cyto-
toxic effect on carcinoma cells and act as effective scavenger for free radical
species.
Science, University of Management and
Technology, Lahore, 54770, Pakistan
2Center of Super Diamond and Advanced
Films (COSDAF), Department of
Chemistry, City University of Hong Kong,
83 Tat Chee Avenue, Kowloon, China
3Department of Chemistry, Government
College Women University, Faisalabad,
38000, Pakistan
4Mathematics Department, Faculty of
Science, Cairo University, Giza, 12613,
Egypt
5School of Chemistry and Chemical
Engineering, State Key Laboratory of
Metal Matrix Composites, Shanghai Jiao
Tong University, Shanghai, 200240, China
Correspondence
Shakeel Ahmad Khan, Department of
Chemistry, School of Science, University
of Management and Technology, Lahore-
54770, Pakistan.
Email: shakilahmadkhan56@gmail.com
Komal Rizwan, Department of Chemistry,
Government College Women University,
Faisalabad, 38000, Pakistan.
Email: komal.rizwan45@yahoo.com
K E Y W O R D S
anticancer, antimicrobial, DFT studies, formazan, molecular docking
1 | INTRODUCTION
emerging issue. Besides, some fungi for example Candida
species are also displaying resistance to various antifun-
Now days, development of multi-drug resistance in sev-
eral bacterial strains including salmonella, streptococci,
gonococci, enterococci, staphylococci etc. has become an
gal drugs.[1] Numerous antimicrobial drugs have been
developed however; because of the emergence of
multidrug-resistance in pathogens, clinical-efficiency of
Appl Organometal Chem. 2020;e5444.
wileyonlinelibrary.com/journal/aoc
© 2020 John Wiley & Sons, Ltd.
1 of 24
https://doi.org/10.1002/aoc.5444

2 of 24
KHAN ET AL.
the existing drugs is being vulnerable.[2] According to
World Health Organization (WHO), if we could not
develop efficient drugs to control or destroy microbes, the
death caused by microbial diseases may rise to millions
by 2020. Regarding this, it has become obligatory to find
out alternate routes to tackle these multi-drug resistant
microbes.[1] In addition to deaths due to microbial dis-
eases, cancer is also one of the prime factor for the deaths
in human beings. Literature survey reveals that interna-
tional cancer burden has become twice during the period
of 1975 to 2000, and will double again by 2020.[3] Even
though, significant advancements have been taken place
in cellular and molecular biology which have improved
the chemotherapeutic treatments of cancer but persistent
determination is still obligatory to discover new-fangled
anticancer drugs.[4–7]
medium for the reaction of formaldehyde and
β-ketosulfones.[32] Meglumine is a promising contender
as it have eco-friendly properties such as physiological
inertness and biodegradability. Moreover, meglumine is
amino sugar derivative of sorbitol, which could be
employed as an excipient in different pharmaceutical for-
mulations and with the combination of iodinated com-
pounds because of its low toxicity. Meglumine is
economical, non-toxic, stable to moisture and air, and
can be easily availed from commercial market.[33]
Recently, meglumine has widely explored as an efficient
catalyst for the synthesis of several organic compounds
such as pyrazoles, dihydro-pyridines, pyrazolo-pyrano-
pyrimidines, pyranopyrazole derivatives and 2-amino-
4H-pyrans.[34–38]
We aimed to synthesize metal complex formazans
by following simple, efficient and environment friendly
approach. For this, first we have synthesized the aro-
The azomethine and its derivatives have been consid-
ered as a protuberant research objects due to their signifi-
cant complexometric behavior and pharmacological
physiognomies.[8] Azomethine derivatives perform note-
worthy role by demonstrating numerous biological activi-
ties such as anticonvulsant, antiviral, antimicrobial,
antioxidant, antitubercular, anticancer, anti-HIV, anthel-
mintic and anti-inflammatory. Azomethines are to be
considered as an important intermediate for the develop-
ment of different kinds of biological active derivatives
viz. formazans, pyrazolines, thiazolidinones, azetidinones
and isoxazolines.[8–12] Formazans and their derivatized
molecular structures are highlighted a lot because of their
noteworthy pharmaceutical and therapeutic potentials as
an antifertility,[13] antioxidants,[14] antibacterial,[9,10]
matic
hydrazone
(coupling
agent)
employing
meglumine (biocompatible) as a catalyst, which further
reacted with aromatic diazonium salt to produce
formazans. Upon metallization reaction with different
metal salts, metal complex formazans were synthesized
which were then evaluated for their biomedical appli-
cations such as antibacterial, antifungal, and antican-
cer. The structural properties of formazans were also
studied through DFT calculations and molecular dock-
ing studies.[39]
2 | EXPERIMENTAL
2.1 | Chemicals
antifungal,[10]
antiviral,[15]
antiparkinsonian,[16]
antitubercular,[17] anticonvulsant,[18] anti HIV,[19]
anticancer,[20] antihyperglycemic,[21] antiproliferative,[22]
analgesic and anti-inflammatory agents.[23–27] Several
reports are available on formazans and their derivatives
for biological applications, however very few reports are
on formazan metal complexes.[28]
The chemicals and reagents exploited in this work have
been purchased from Sigma Aldrich and.
Fluka Chemische (Switzerland). They are all of ana-
lytical grade.
Formazans are typically synthesized in two steps;
diazotization of aromatic amines followed by coupling
with the hydrazones (coupler agent).[29,30] Hydrazones
are synthesized by the condensation reaction of hydra-
zines and aldehydes in the presence of catalysts
i.e. NaOH, Na2CO3, Betaine HCl, TsOH etc. under high
temperature conditions with prolonged reaction time
(1 hr to 3 hr approximately). The limitations of this
approach are high temperature, long reaction time, mod-
est yield, environmental incompatibility, and the usage of
toxic chemical entities. Now days, much attention has
been considered for exploiting biocompatible or bio-
nature materials as catalyst or solvent in organic reac-
tions.[31] In this regards, Gu et al. has introduced the
solution of meglumine and gluconic acid as promoting
2.2 | Generalized protocol for the
synthesis of hydrazone 4-[(2Z)-2-
benzylidenehydrazinyl] benzene sulfonic
acid (BASAH)
The hydrazone (BASAH) was prepared by the reduction
of diazonium salt (SAD) of 0.1 mole of 4-
aminobenzenesulphonic
acid
(SA)
into
4-hydrazinylbenzenesulfonic acid (SAH) which upon
condensation with benzaldehyde (BA) in the presence of
meglumine (0.15 mole), and C2H5OH/H2O (1:1, 400 ml)
at room temperature yielded resultant product (BASAH)
as presented in Scheme 1.

NOVEL FORMAZAN METAL COMPLEXES AND THEIR BIOLOGICAL APPLICATIONS
3 of 24
and Found: S = 17.01, N = 14.81, O = 25.41, H = 4.25,
C = 38.23%.
2.2.2 | Synthesis of hydrazone compound
(BASAH)
The hydrazone (BASAH) was synthesized by the con-
densation reaction of compound (SAH) and benzalde-
hyde (Scheme 1).[33] In typical procedure, 0.1 mole of
compound (SAH) (93% purity) dissolved in
C2H5OH/H2O (1:1, 400 ml). Subsequently, 0.15 mole of
meglumine was added in the reaction mixture. After-
wards, 0.1 mole of benzaldehyde was added dropwise in
the reaction mixture in 10 min. The reaction mixture was
stirred at room temperature for reaction completion. The
reaction was monitored continuously through thin layer
chromatography (TLC). The reaction was completed
within 30 min. After reaction completion, C2H5OH/H2O
(1:1, 50 ml) was added and the final product was
extracted using C2H5COOH (30 ml). The anhydrous
Na2SO4 was used for the amputation of organic phase
and solvent was removed under vacuum conditions. The
final product was purified by recrystallization process
described by.[29,30]
SCHEME 1 Synthesis of formazan and metallized formazan
complexes (S1-S6)
Pale yellow powder. Yield = 96%. FTIR (KBr,
Cm−1); 1405 (SO3H), 3451 (N-H), 1601 (aromatic
C=C), 1521 (H-C=N), 3020 (aromatic C-H). 1H-NMR
(D2O, 400 MHz) δ: 6.74 to 7.68 = Ar-H (m 9H),
11.31 = SO3H (s 1H), 4.70 = N-H (s 1H), 8.10 = H-
C=N (s 1H). 13C-NMR (D2O, 75 MHz) δ: 115.3, 118.1,
120.1, 128.9, 128.9, 129.2, 129.2, 130.9, 131.1, 133.8,
144.4, 150.6 (Aromatic carbons), 155.9 (C=N). Anal.
Calc. for C13H12N2O3S (Molecular weight = 276.31
gmol−1): S = 11.60, N = 10.14, O = 17.37, H = 4.38,
C = 56.51% and Found: S = 11.55, N = 10.09,
O = 17.32, H = 4.35, C = 56.49%.
2.2.1 | Synthesis of diazonium salt (SAD)
and 4-hydrazinylbenzenesulfonic acid
(SAH)
The
diazonium
salt
(SAD)
and
4-hydrazinylbenzenesulfonic acid (SAH) was synthesized
following the procedure as described by[9,10] with slight
modifications. For the synthesis of diazonium salt
(SAD), the aqueous solution of 0.1 mole of (SA) was
diazotized in the presence of NaNO2 (7.3 g) and H2SO4
(20 ml) at 0–5 ꢀC. Further, the hydrazine compound
(SAH) was obtained by the reduction of already synthe-
sized moist diazonium salt (SAD) in the presence of
Na2SO3 (56.5 g) and H2SO4 (33.2 ml) at 0–5 ꢀC
(Scheme 1). Purification of the hydrazine compound
(SAH) was accomplished following the protocol.[29,30]
Compound (SAH); Off white powder. Yield = 95%.
Melting point = 286 ꢀC. FTIR (KBr, Cm−1); 1385 (SO3H),
3486 (N-H), 1604 (aromatic C=C), 3012 (aromatic C-H).
1H-NMR (D2O, 400 MHz) δ: 6.96 to 7.68 = Ar-H (m 4H),
11.19 = SO3H (s 1H), 4.20 = N-H (s 1H), 1.40 = N-H
(s 2H). 13C-NMR (D2O, 75 MHz) δ: 114.5, 114.5, 128.6,
128.6, 140.2, 154.8 (Aromatic carbons). Anal. Calc. for
2.3 | Synthesis of formazan compound 4-
(2-(((2-hydroxy-5-nitrophenyl) diazenyl)
(phenyl) methylene) hydrazinyl) benzene
sulfonic acid (S1)
The formazan compound (S1) was synthesized by the
azo coupling reaction of diazonium salt (NAPD) of
2-amino-4-nitrophenol
(NAP)
and
synthesized
hydrazone (BASAH) (Scheme 1). The diazonium salt
(NAPD) was prepared by diazotizing of aqueous solu-
tion of 0.1 mole of 2-amino-4-nitrophenol (NAP) in
the presence of NaNO2 (7.4 g) and H2SO4 (20 ml) at
10–15 ꢀC. Further, the diazonium salt (NAPD) was
coupled to coupler agent hydrazone (BASAH) in
C6H8N2O3S (Molecular weight
=
188.2 gmol−1):
S = 17.04, N = 14.88, O = 25.50, H = 4.28, C = 38.29%

4 of 24
KHAN ET AL.
alkaline media (pH ꢁ 8–9) at 0–5 ꢀC. The coupling
reaction was monitored by TLC and the reaction was
completed within 1 hr. The isolation of the formazan
compound (S1) was achieved employing salting out
process as described by.[9,10] Further, formazan com-
pound (S1) was purified by recrystallization process
described by.[29,30]
2.4.1 | Formazan cu metal complex (S2)
Greyish-black powder. Yield
=
91%. Melting
point = 420 ꢀC. λmax in nm (log ε) in ethanol: λmax1 = 455
(0.45), λmax2 = 320 (0.89), λmax3 = 285 (0.25). FTIR (KBr,
Cm−1); 3390 (O-H), 640 (Cu-O), 1380 (Ar-NO2), 1285
(SO3H), 839 (CN=NC), 1589 (aromatic C=C), 1316
(N=N), 1501 (C=N), 3020 (aromatic C-H). 1H-NMR
(CDCl3, 400 MHz) δ: 6.79 to 8.60 = Ar-H (m 12H),
11.18 = SO3H (s 1H). 13C-NMR (CDCl3, 75 MHz) δ:
117.6, 118.8, 126.6, 126.8, 127.1, 128.5, 129.6, 132.1, 132.9,
135.5, 140.1, 141.7, 149.6, 156.6, 159.9 (Aromatic car-
bons), 155.4 (C=N). Anal. Calc. for C19H19CuN5O9S
(Molecular weight = 556.932 gmol−1): Cu = 11.41,
S = 5.76, N = 12.57, O = 25.85, H = 3.44, C = 40.97% and
Found: Cu = 11.35, S = 5.70, N = 12.51, O = 25.80,
H = 3.40, C = 40.91%.
Reddish-brown powder. Yield
= 90%. Melting
point = 390 ꢀC. λmax in nm (log ε) in ethanol:
λmax1 = 469 (0.51), λmax2 = 339 (0.71), λmax3 = 289
(0.31). FTIR (KBr, Cm−1); 1340 (Ar-NO2), 1415 (SO3H),
1129 (Ar-OH), 3421 (N-H), 761 (CN=NC), 1615 (aro-
matic C=C), 1358 (N=N), 1510 (C=N), 3015 (aromatic
C-H). 1H-NMR (CDCl3, 400 MHz) δ: 6.49 to
7.59 = Ar-H (m 12H), 11.30 = SO3H (s 1H), 4.33 = N-
H (s 1H), 5.40 = Aromatic-OH (s 1H). 13C-NMR
(CDCl3, 75 MHz) δ: 104, 106.5, 118.5, 126.2, 127.1,
128.2, 129.6, 132.3, 135.5, 140.1, 141.3, 147.6, 165.3
(Aromatic carbons), 155.5 (C=N). Anal. Calc. for
C19H15N5O6S (Molecular weight = 441.417 gmol−1):
2.4.2 | Formazan Fe metal complex (S3)
S
= 7.26, N = 15.87, O = 21.75, H = 3.43,
C = 51.70% and Found: S = 7.19, N = 15.81,
O = 21.69, H = 3.40, C = 51.63%.
Reddish-brown powder. Yield
=
92%. Melting
point = 450 ꢀC. λmax in nm (log ε) in ethanol: λmax1 = 449
(0.40), λmax2 = 330 (0.99), λmax3 = 291 (0.30). FTIR (KBr,
Cm−1); 3421 (O-H), 691 (Fe-O), 1358 (Ar-NO2), 1391
(SO3H), 828 (CN=NC), 1591 (aromatic C=C), 1435
(N=N), 1531 (C=N), 3030 (aromatic C-H). 1H-NMR
(CDCl3, 400 MHz) δ: 6.81 to 8.31 = Ar-H (m 12H),
11.22 = SO3H (s 1H). 13C-NMR (CDCl3, 75 MHz) δ:
112.3, 117.3, 118.4, 126.2, 128.1, 128.5, 129.6, 132.1, 135.5,
138.1, 140.1, 150.1, 151.3, 160.3, (Aromatic carbons),
155.7 (C=N). Anal. Calc. for C19H19FeN5O9S (Molecular
weight = 549.292 gmol−1): Fe = 10.17, S = 5.84,
N = 12.75, O = 26.21, H = 3.49, C = 41.54%. Found:
Fe = 10.11, S = 5.80, N = 12.71, O = 26.20, H = 3.41,
C = 41.50%.
2.4 | Synthesis of formazan metal
complexes (S2-S6)
The formazan compound (S1) is a tridentate ligand
and have ability to form metal complexes. Therefore,
we further employed formazan compound (S1) for the
synthesis of different metal complexes following the
procedure described by[9,10] with slight modifications.
In typical procedure, formazan compound (S1) was
prepared adopting the methodology described in
scheme 1. After, formazan metal complexes (1:1) of
copper (S2), iron (S3), cobalt (S5) and nickel (S6)
were prepared by the metallization reaction of 0.1
mole of tridentate ligand (formazan compound S1)
with different metal salts (0.1 mole) such as
2.4.3 | Formazan Cr metal complex (S4)
CuSO4.5H2O,
NiCl2.6H2O
FeSO4.5H2O,
respectively
CoSO4.7H2O,
slightly
and
acidic
Greyish-brown powder. Yield
=
93%. Melting
at
point = 460 ꢀC. λmax in nm (log ε) in ethanol:
λmax1 = 471 (0.46), λmax2 = 341 (1.01), λmax3 = 279
(0.29). FTIR (KBr, Cm−1); 635 (Cr-O), 1351 (Ar-NO2),
1399 (SO3H), 849 (CN=NC), 1599 (aromatic C=C),
1465 (N=N), 1516 (C=N), 3035 (aromatic C-H). 1H-
NMR (CDCl3, 400 MHz) δ: 6.70 to 8.01 = Ar-H
(m 24H), 11.29 = SO3H (s 2H). 13C-NMR (CDCl3,
75 MHz) δ: 117.6, 118.4, 128.1, 126.4, 127.1, 128.4,
132.6, 133.3, 134.6, 134.8, 136.4, 138.6, 140.0, 141.3,
149.3, 159.9 (Aromatic carbons), 150.3, 155.3 (C=N).
pH ꢁ 6.50–6.70 at 65–70 ꢀC. On the other hand,
formazan metal complexes (2:1) of chromium (S4) was
synthesized by treating 0.1 mole of formazan com-
pound (S1) with 0.05 mole of chromium salt
(Cr3(OH)2(OOCCH3)7 at slightly acidic pH ꢁ 6.70 at
elevated temperature 90–100 ꢀC. The metallization
reaction was completed within 2–3 hr for (1:1) metal
complexes while 3–4 hr for (2:1). The formazan metal
complexes (S2-S6) was isolated through acidification
and salting out methods.[9,10] Therein after, they were
purified by recrystallization process described by.[29,30]
Anal.
Calc.
for
C38H26CrN10O12S2
(Molecular
weight = 930.799 gmol−1): Cr = 5.59, S = 6.89,

NOVEL FORMAZAN METAL COMPLEXES AND THEIR BIOLOGICAL APPLICATIONS
5 of 24
N = 15.05, O = 20.63, H = 2.82, C = 49.03% and
Found: Cr = 5.55, S = 6.80, N = 14.99, O = 20.60,
H = 2.80, C = 49.01%.
Standard antioxidant “Vitamin C" was employed as a
positive control. The reaction mixture was incubated in
dark condition for 30 min at room temperature
[44,45]
ꢀ
(25–30 C),
and the changes in color was recorded
by measuring absorption maxima (λmax) at 517 nm
through an ELISA reader. The free radical-scavenging
activity was calculated as percentage inhibition of DPPH
employing following equation.
2.4.4 | Formazan co metal complex (S5)
Dark-brown powder. Yield = 90%. Melting point = 410 ꢀC.
λmax in nm (log ε) in ethanol: λmax1 = 480 (0.48),
λ
À
Á
max2 = 349 (1.20), λmax3 = 281 (0.33). FTIR (KBr, Cm−1);
Percentage inhibition of DPPH = ½ Acontrol
−
Asample
3345 (O-H), 641 (Co-O), 1315 (Ar-NO2), 1231 (SO3H),
799 (CN=NC), 1599 (aromatic C=C), 1324 (N=N), 1501
(C=N), 3010 (aromatic C-H). 1H-NMR (CDCl3, 400 MHz)
δ: 6.89 to 8.67 = Ar-H (m 12H), 11.21 = SO3H (s 1H).
13C-NMR (CDCl3, 75 MHz) δ: 112.3, 117.3, 118.4, 126.2,
128.1, 128.5, 129.6, 132.2, 135.5, 138.1, 140.4, 150.1, 151.3,
160.1 (Aromatic carbons), 155.9 (C=N). Anal. Calc. for
C19H19CoN5O9S (Molecular weight = 552.380 gmol−1):
Co = 10.67, S = 5.68, N = 12.68, O = 26.07, H = 3.47,
C = 41.31% and Found: Co = 10.60, S = 5.63, N = 12.59,
O = 26.01, H = 3.39, C = 41.30%.
=Acontrolꢂ × 100:
where Asample is the absorbance of the DPPH solution
with synthesized non-metal and complex formazans
(S1-S6) while Acontrol is the absorbance of the DPPH
solution without synthesized formazan compounds
(S1-S6). The experiments were performed in triplicates
with a mean standard error.
2.6 | Antibacterial activity
2.4.5 | Formazan Ni metal complex (S6)
The synthesized metal complex formazans (S1-S6)
were scrutinized for their antibacterial propensity
against different bacteriological strains such as Staphy-
lococcus epidermidis (ATCC 12228), Bacillus cereus
(ATCC 11778), Klebsiella pneumoniae (ATCC BAA-
1705™) Pseudomonas aeruginosa (ATCC 15442).[46–49]
These bacteriological strains were acquired from
Pakistan Chemical Scientific Industrial Research
(PCSIR) laboratories complex, Lahore, Pakistan. The
bactericidal propensity of synthesized metal complex
formazans (S1-S6) was deliberated on agar plates by
well-known agar well diffusion method as described
by.[50–52] For each type of microbial species, the strep-
tomycin and neomycin were employed as a standard
antibiotic drug (positive control).[53–55]
Greenish-brown powder. Yield
=
91%. Melting
point = 435 ꢀC. λmax in nm (log ε) in ethanol: λmax1 = 475
(0.41), λmax2 = 343 (1.30), λmax3 = 291 (0.27). FTIR (KBr,
Cm−1); 3446 (O-H), 641 (Ni-O), 1338 (Ar-NO2), 1315
(SO3H), 861 (CN=NC), 1609 (aromatic C=C), 1401
(N=N), 1499 (C=N), 3040 (aromatic C-H). 1H-NMR
(CDCl3, 400 MHz) δ: 6.88 to 8.71 = Ar-H (m 12H),
11.30 = SO3H (s 1H). 13C-NMR (CDCl3, 75 MHz) δ:
112.6, 117.9, 118.4, 126.2, 128.1, 128.5, 129.6, 132.3, 135.5,
138.4, 140.1, 150.9, 151.3, 160.1 (Aromatic carbons), 155.3
(C=N). Anal. Calc. for C19H19NiN5O9S (Molecular
weight = 552.140 gmol−1): Ni = 10.63, S = 5.81,
N = 12.68, O = 26.08, H = 3.47, C = 41.33% and Found:
Ni = 10.60, S = 5.80, N = 12.60, O = 26.01, H = 3.40,
C = 41.27%.
2.7 | Antimycotic activity
2.5 | Antioxidant activity
The antimycotic propensity of the synthesized metal
complex formazans (S1-S6) were evaluated against differ-
ent mycological strains such as Trichophyton men-
tagrophytes (ATCC 9533), Candida albicans (ATCC
10231), Aspergillus niger (ATCC 16404), Saccharomyces
cerevisiae (ATCC 9763) and Candida glabrata (ATCC
90030) adopting the standard protocol described by.[56,57]
The employed fungal microorganisms were acquired
from the microbiology laboratory of University of Veteri-
nary and Animal Science Lahore, Pakistan. Terbinafine
and Fluconazole were served as standard antimycotic
The synthesized formazan compounds (S1-S6) were
screened for their antioxidant propensity employing a
modified-protocol
based
on
Brand–William's
procedure.[39–43] In a typical procedure, the 20 μl of
methanolic suspensions of each synthesized formazan
compounds (S1-S6) were mingled with 180 μl of 1 mM
methanolic DPPH solution. The different concentrations
of each synthesized formazan compounds (S1-S6)
(75, 150, 250, 500 and 1000 μg/ml) were selected.

6 of 24
KHAN ET AL.
drugs (positive control) for the comparative study of anti-
mycotic propensity against different fungal strains.
(MCF-7) cell line, they were further screened for docking
studies to know on what level and how they can combine
with breast cancer causing protein (The human estrogen
receptor).
2.8 | MTT cell proliferation assay
By employing 3–(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-
2H tetrazolium bromide (MTT) (Life Technologies,
Eugene, OR) assay, the cell viability of human breast car-
cinoma (MCF-7) cell line treated by synthesized metal
complex formazans (S1-S6) were assessed. The human
2.9.1 | Recuperate of protein and ligand
formation
For docking studies of synthesized formazan compounds
(S1-S6) with the breast carcinoma cells, the human estro-
gen receptor protein was used as three dimensional struc-
ture and was taken from protein data bank with PDB ID:
2IOK and resolution of 2.4 Å.[58] The excluding entities
like water was removed in Accelrys discovery studio visu-
alizer.[59] On the basis of experiment and literature, syn-
thesized formazan compounds (S1-S6) were chosen and
made in chem draw for the docking process. The Doxoru-
bicin was used as standard ligand for further analysis due
to its reviews as better anticancer drug. It was taken from
PubChem with CID: 31703.[60] All the ligands were made
in.sdf file, which was later converted into.pdb file using
discovery studio visualizer.
ꢀ
breast carcinoma (MCF-7) cell line at 37 C in a moist-
ened thermosphere comprising of 95% air and 5% CO2
was cultured in Dulbecco's modified Eagles medium
(DMEM) (Gibco-BRL, Grand Island, NY) subsequently
they were seeded at a density of 1 × 104 per well in a
96-well plate (Corning Costar, Lowell, NY, USA). Used
Dulbecco's modified Eagles medium (DMEM) was sup-
plemented with 1% penicillin/streptomycin (P/S) and
10% foetal bovine serum (FBS). Afterwards, the wells
containing cultured human breast carcinoma (MCF-7)
cell line in a 96-well plate were subjected to treat with
different concentrations (1, 10, 20, 30, 40 and 50 μg/ml)
of standard (Doxorubicin), and synthesized formazans
(S1-S6) at 37 ꢀC for 48 hr at 90% confluency. Subse-
ꢀ
quently, the wells were further incubated at 37 C for
2.9.2 | Docking
4 hr after the addition of 10 μl of MTT (5 mg/ml) in PBS
solution. Moreover, to dissolve the unsolvable formazan
crystals into the colored solution, 100 μl of dimethyl
sulphoxide (DMSO) was added in each well. An enzyme-
linked immunesorbent assay (ELISA) reader (Bio-Tek
Instruments, Inc., Vinooski, VT, USA) was employed to
measure the absorption maxima of reaction mixture in
each well. Results were expressed as the percentage of
the control (without compound set at 100%). The percent-
age of cell survival was calculated as OD value of cells
treated with the test compound−OD value of culture
medium/(OD value of control cell−OD value of culture
medium) × 100%. The experiment was performed in trip-
licates with a mean standard error.
In order to find out the action of ligands (S1-S6) over the
protein, docking was executed and the results with differ-
ent ligands were compared. The process was executed
using autodock and vina autodock and later analyzed by
using discovery studio resulting 3d representation.
Autodock was also used for charge addition both in estro-
gen receptor protein and the ligands. The protein recep-
tor was added with kollman charge while the ligands had
gesteiger charges with the minimization of torsions.
Thus, the ligands and receptor were converted into.pdbqt
format after the above operations.[61,62]
2.9.3 | Physico-chemical properties
analysis
2.9 | Molecular docking studies
Drug designing is resulted by a number of experimental
and computational processes where physico-chemical
properties play an important role as they indicates the
drug capability to interact with the biological process
along with the environmental process and the physical
hazards. Here the physico-chemical properties include
molecular weight, total surface area, polar surface area,
druglikeness, clogP and clogS. The druglikeness follows
Lipinski rule of five for the drug analysis. All the proper-
ties were found out using Data warrior.[63]
Computational biology is the ultimate source of attrac-
tion in drug designing due to its low coast and quick
results leading a new pathway in drug discovery. For bet-
ter drug designing and prediction, molecular docking is a
great tool and success that not only mention the active
site for the reaction of drug but also its binding affinity
along with other biological effects. After the evolution of
synthesized formazan compounds (S1-S6) for their cyto-
toxic potential in vitro on human breast carcinoma

NOVEL FORMAZAN METAL COMPLEXES AND THEIR BIOLOGICAL APPLICATIONS
7 of 24
2.10 | Computational methods
3.2 | Antibacterial activity
All computations were carried out using Gaussian
09 W software package.[64] The molecular geometry for
the studied compounds were fully optimized using
Density functional theory B3LYP method,[65–67] by
implementing LANL2DZ basis set for Cu, Fe, Cr, Co
and Ni-atoms[68,69] and 6–31 + G(d,p)[70] basis set for
the rest of atoms. No symmetry constrains were
applied during the geometry optimization.[71,72] NBO
calculations have been performed at the same level of
calculation by using NBO 3.1 program as implemented
in the Gaussian 09 W software package. The optimized
The antibacterial propensity of the synthesized formazan
compounds (S1-S6) was executed against Staphylococcus
epidermidis, Bacillus cereus (Gram-positive bacteria),
Klebsiella pneumoniae and Pseudomonas aeruginosa
(Gram-negative bacteria) and their outcomes are shown
in Figure S2 in the form of their zone of inhibitions
values (ZOIs) while their results of minimum inhibitory
concentration values (MICs) in μg/mL are presented in
Table S1. ZOIs results were demonstrated that the syn-
thesized formazan compound (S1) was found to exhibit
least antibacterial propensity by presenting minimum
structure was visualized using Chemcraft version 1.6
package[73] and GaussView version 5.0.9.[74] Further-
more, the Multiwfn v3.7 software program was used[75]
in order to compute quantum chemical descriptors
from CDFT point of view.
ZOIs values (21
15 0.04 mm, 17
0.08 mm, 19
0.08 mm) against Staphylococcus
0.02 mm,
epidermidis, Bacillus cereus, Klebsiella pneumoniae and
Pseudomonas aeruginosa respectively compared to metal
complex formazan compounds (S2-S6) and employed
standard drugs. On the other hand, the synthesized
formazan compounds (S2, S3, S5 and S6) were demon-
strated the moderate to good antibacterial propensity
(Figure S2). Moreover, substantial and enhanced
antibacterial propensity was exhibited by the formazan
compound (S4) with the maximum ZOIs values
3 | RESULTS AND DISCUSSION
3.1 | Antioxidant activity
(33
27
0.04 mm, 32
0.05 mm) against of S. epidermidis, B. cereus,
0.09 mm, 26
0.01 mm,
The newly synthesized formazan compounds (S1-S6)
were scrutinized for the assessment of their antioxidant
potential by DPPH free radical scavenging assay and
their antioxidant results in terms of percentage inhibi-
tion of DPPH are presented in Figure S1. It has been
anticipated from the results that maximum and supe-
rior antioxidant propensity in terms of percentage inhi-
bition of DPPH free radical (73.5%, 80%, 83%, 86%,
90%) was unveiled by all the concentrations (75 μg/ml,
150 μg/ml, 250 μg/ml, 500 μg/ml, 1000 μg/ml) of
formazan compound (S1) respectively, which were
analogous to the employed standard antioxidant
“Ascorbic acid”. While the formazan compounds (S2,
S3, S5, S6) demonstrated the moderate to good antioxi-
dant propensity by scavenging the DPPH free radical
as presented in Figure S1. The formazan compound
(S4) exhibited least antioxidant propensity. Further,
results were also attributed concentration dependent
antioxidant activity as increasing concentration of the
formazan compounds (S1-S6) exhibited effective and
enhanced percentage inhibition of DPPH free radical.
Literature data demonstrates that antioxidants combine
with a stable free radical DPPH to transform it to
2,2-diphenyl-1- picryl hydrazine which results in the
decoloration of DPPH. Therefore, the scavenging apti-
tudes of the tested compounds were determined from
the degree of decoloration of DPPH (more degree of
coloration, more will be scavenging and vice versa).
K. pneumoniae and P. aeruginosa respectively in compari-
son to metal complex formazan compounds (S2, S3, S5
and S6) and standard drugs. It has been also anticipated
from the MICs results that formazan compound (S4)
were found to unveil the similar MICs results to that of
standard antibiotics drugs (streptomycin and neomycin)
while the other formazan compounds (S1, S2, S3, S5, S6)
were found to exhibit least MICs results (Table S1). The
fact due to which formazan compounds (S1-S6) pres-
ented moderate to noteworthy and substantial
antibacterial propensity was the structural moiety
i.e. substitution of electron-withdrawing group (-SO3H
and -NO2) on the para position of benzene ring
(Scheme 1), which enable them to unveil significant and
effective bactericidal potential. Moreover, literature sur-
vey also exposes that antibacterial propensity depends on
the position and existence of functional groups within
the molecular structure[9,10,48] and electron-withdrawing
groups (–NO2, –CN, -SO3H, –CHO, –C=O) are responsi-
ble to upsurge the antibacterial propensity of the mole-
cule compared to electron-donating groups (–O−, –NH2,
–NHR, –OR, –NHCOR). Furthermore, ortho and para
positions are more favorable to augment the bactericidal
potential than meta position as more stabilization of the
molecular structure can be achieved when electron-
withdrawing groups positioned on these two positions of
benzene ring.[9,10] Henceforth, it was ostensible and

8 of 24
KHAN ET AL.
established from the outcomes of MICs and ZOIs that the
nature of formazan compounds (S1-S6) is bacteriostatic
as well as bactericidal, which empower them to demon-
strate substantial antibacterial propensity against Gram-
positive and Gram- negative bacteria.
been anticipated from the cytotoxicity results that human
breast carcinoma (MCF-7) cell line treated with the
formazan compounds (S1-S6) was found to exhibit con-
centration dependent anticancer propensity. The maxi-
mum cytotoxic effect (49%, 55%, 62%, 74%, 79%, 84%) on
human breast carcinoma (MCF-7) cell line was
experimented with the different concentrations (50 μg/ml,
40 μg/ml, 30 μg/ml, 20 μg/ml, 10 μg/ml, 1 μg/ml) of
formazan compound (S1) respectively in contrast to
other formazan compounds (S2, S3, S4, S5, S6) but was
comparable to that of employed standard anticancer drug
“Doxorubicin”. Moreover, the other formazan com-
pounds (S2, S3, S5, S6) along with their different concen-
trations were demonstrated the moderate level cytotoxic
effect on human breast carcinoma (MCF-7) cell line how-
ever, among all, the least cytotoxicity on breast carci-
noma cells was perceived by the formazan compound
(S4) as presented in Figure S5. From these findings, we
assumed that more percentage cytotoxic effect results in
decreasing the cell viability and vice versa. According to
the results of MTT cell proliferation assay, formazan
compounds have possess the capability to impede the
breast cancer cells proliferation. Conclusively, it was evi-
dent and confirmed from the cytotoxicity results that
formazan compounds (S1-S6) have potential towards to
act as anticancer.
3.3 | Antimycotic activity
The antimycotic propensity of the synthesized formazan
compounds (S1-S6) was evaluated against Trichophyton
mentagrophytes, Candida albicans, Aspergillus niger, Sac-
charomyces cerevisiae, and Candida glabrata. The out-
comes of antimycotic propensity in the form of minimum
inhibitory concentration (MICs in μg/mL) and zone of
inhibitions values (ZOIs) are manifested in Figure S3 and
S4 respectively. ZOIs results were demonstrated that sig-
nificant and superior antimycotic propensity was
unveiled by the formazan compound (S4) with the maxi-
mum ZOIs values (33
0.04 mm, 29
0.09 mm,
39 0.01 mm, 36 0.05 mm, 30
0.05 mm) against
T. mentagrophytes, C. albicans, A. niger, S. cerevisiae and
C. glabrata respectively in contrast to formazan com-
pounds (S2, S3, S5 and S6) and standard drugs
(Terbinafine and Fluconazole). While the synthesized
formazan compound (S1) against each fungal strains
were demonstrated the least antimycotic propensity in
contrast to formazan compounds (S2-S6) and employed
standard drugs. On the other hand, the synthesized
formazan compounds (S2, S3, S5 and S6) were demon-
strated the moderate to good antimycotic propensity.
Moreover, It has been also anticipated from the MICs
results that formazan compound (S4) were found to
unveil the similar MICs results to that of standard antibi-
otics drugs while the other formazan compounds (S1, S2,
S3, S5, S6) were found to exhibit least MICs results
(Figure S3). Hence, it was apparent and proven from the
outcomes of MICs and ZOIs that formazan compounds
(S1-S6) are fungicidal in nature, which endow them to
demonstrate substantial antifungal propensity against
employed fungal strains.
3.5 | Docking studies of the synthesized
formazan compounds (S1-S6)
The docking studies indicates the binding of human
estrogen receptor with different ligands (S1-S6) along
with the doxorubicin, which is the standard drug for the
breast cancer to analyze the protein binding activity com-
putationally. The docking process provided the binding
affinity and inhibitory constant which indicated the
almost all the ligands had good values except ligand
(formazan compound S4) (Table S2). The Ki value of all
(S1, S2, S3, S5, S6) except the already mentioned (S4) are
more better than the standard doxorubicin indicating bet-
ter binding of ligands at active site of protein and thus
indicating better IC50 value that plays a better role mode
of action of drug in body. The less the Ki value, the better
IC50 value resulting in better drug activity.[58–61] As far as
bonding is concerned, the presence of benzene rings and
amine groups provided the pi bond in high frequency
while the hydrogen bonds are less in number though pre-
sent. The binding sites in ligands (S1-S6) revealed some
common residues to bind with the receptor protein which
3.4 | MTT cell proliferation assay
The human breast carcinoma (MCF-7) cell lines as in
vitro models for cancerous cells were employed to deter-
mine the cytotoxicity potential of the formazan com-
pounds (S1-S6) and their cytotoxicity results are
manifested in Figure S5. The cytotoxicity potential of the
formazan compounds (S1-S6) is profoundly influenced
by the substitution of electron-donating groups (-OH,
-NH2, OCH3, -CH3) in their molecular structures. It has
are Glu323, Pro324, Ile326, Met343, Leu346, Thr347, Leu349
Ala350, Asp351, His373, His377, Glu380, Trp383, Leu387,
Trp393, Arg394, Glu397, Phe404, Met421, Ile424, Phe445
,
,

NOVEL FORMAZAN METAL COMPLEXES AND THEIR BIOLOGICAL APPLICATIONS
9 of 24
Arg515, Ser516, Ser518, Asn519, Met522, Glu523, Leu525,
Tyr526, Pro535, Leu536 and Tyr537 (Figures S6-S11).
metal ions coordinated to the ligand via N and O atoms
forming the complexes. One ligand moiety used in the
coordination with Cu (II) (S2), Fe (II) (S3), Co (III) (S5)
and Ni (II) (S6) forming [ML(H2O)3] complexes and two
ligand moiety in case of Cr (III) (S4) forming [CrL2].
3.6 | Physico-chemical properties
analysis
The physico-chemical properties were analyzed revealing
molecular weight, total surface area, polar surface area,
druglikeness, clogP and clogS of different ligands
(Table 1). The most important Lipinski rule of five for
druglikeness indicates that the molecular weight for the
drug must be greater than 500 Da and calculated logP
must be greater than 5.[58–60] The given results indicated
that the most ligands were having more than 500 Da
molecular weight except ligand (S1) while the logP value
is not in range for all ligands even for the standard drug.
The ratio of total surface area and polar surface area rev-
ealed almost all the ligands (S1-S6) have mostly polar
surface area making them almost the polar ligand. As far
as calculated druglikeness is concerned the standard drug
has the positive value while all the other ligands (S1-S6)
were having negative values for druglikeness. The values
for logS is almost in between −6.23 to 0.907 while the
standard range of logS is between −2 to −5. Thus, only
ligand (formazan compound, S1) and doxorubicin are in
the standard range.[61]
3.7.2 | Geometry of the complexes
Tables 2 and Figure 1 present the optimized geometry,
numbering system, vector of the dipole moment, ener-
getic, bond lengths, bond angles and dihedral angles of
all metal complexes studied in this work. In Cu (II), Fe
(II), Co (III), Ni (II) and Cr (III) complexes, the metal ion
coordinates with O7 and N14 from the ligand to form a
five-member ring with the sequences O7,C6,C1,N14 there
are another chelation with N14 and N18 from a six-
member ring with the sequences N14,N15,C16,N17,N18
and three water molecule in all complexes except Cr (III)
which contained two ligand molecules to form two five-
and two six-membered rings with sequences O7,C6,C1,
N14, O52,C51,C46,N59, and N14,N15,C16,N17,N18,
N59,N60,C61,N62,N63 respectively. Therefore, form reg-
ular octahedral geometry is expected for all the studied
complexes except in case of Cu (II) distortion of octahe-
dral geometry. Most M-N and M-O bonds show elonga-
tion upon chelation. The length of the coordinate
covalent bonds between metal and ligand site, i.e. M-N
and M-O, are too long compared to the typical MX bond
lengths.[76] The too long M-O and M-N bonds in the stud-
ied complexes mean that the ionic character of these
bonds is small which is vary between 1.89 to 2.07 Å
(Table 2). The calculated values of bond angles between
metal ion and binding sites (Tables 2) C6O7M, O7MN14,
O7MO49(O52), O7MO49(N59) and N14MN18 vary
between 80 and 112 degree, which compare nicely with
the experimental data as obtained from X-ray analysis for
OH complexes,[72] which indicate a regular octahedral
geometry is expected for all the studied complexes except
in case of Cu (II) distortion of octahedral geometry. The
3.7 | Density functional theory (DFT)
studies
3.7.1 | Structural properties
The optimized geometrical parameters (bond lengths,
bond angles and dihedral angles), natural charges on
active centers, reactivity descriptors, molecular electro-
static potential maps and energetic of the ground state
for the studied complexes were computed and analyzed.
From the elemental analysis and spectroscopic data,
TABLE 1 The physico-chemical properties analysis of formazan compounds (S1-S6) towards human estrogen protein receptor for drug
designing
Formazan Compounds
Molecular weight
442.431
Total surface area
317.58
Polar surface area
176.92
Drug likeness
−13.682
clogP
clogS
−3.131
0.745
S1
S2
S3
S4
S5
S6
−1.1844
−1.3126
−1.3126
−2.2736
−1.3126
−1.3126
554.337
339.96
209.36
−6.3567
562.038
339.96
209.36
−4.5067
0.745
935.850
625.56
331.02
−25.31
−6.235
0.745
557.158
339.96
209.36
−3.8167
557.425
339.96
209.36
−3.9367
0.745
Values are mean SD triplicate assays.

10 of 24
KHAN ET AL.
TABLE 2 The selected bond length (Å), bond angles and dihedral angles, (degree) of H2L (S1), Cu (II) (S2), Fe (II) (S3), Cr (II) (S4), Co
(III) (S5) and Ni (II) (S6) complexes B3LYP/GENECP level of theory
H2L (S1)
1.42
Cu (II) (S2)
1.44
Fe (II) (S3)
1.43
Co (III) (S5)
1.44
Ni (II) (S6)
1.44
Cr (III) (S4)
1.44
R(C1-C6)
R(C1-N14)
1.41
1.40
1.42
1.39
1.41
1.40
R(C6-O7)
1.36
1.31
1.32
1.30
1.31
1.31
R(O7-M45)
R(−,H45)
1.277
0.97
1.03
1.99
1.96
1.93
2.03
1.20
R(N14-N15)
1.27
1.29
1.29
1.28
1.30
R(N14-M45)
R(−,H46)
1.32
1.98
1.87
1.89
2.01
2.02
R(C16-N17)
1.33
1.34
1.37
1.34
1.32
R(N17-N18)
1.32
1.30
1.32
1.29
1.31
1.31
R(N18-C19)
1.39
1.41
1.42
1.43
1.42
1.41
R(N18-M45)
R(−,H46)
1.03
1.97
1.89
1.94
2.04
2.07
R(M45-O46)
2.64
2.36
2.32
2.17
R(−,O52)
R(−,N59)
R(−,N63)
118.6
1.20
2.02
2.07
R(M45-O49)
4.67
4.62
2.37
2.22
R(M45-O52)
2.10
2.07
2.05
2.19
A(C1-C6-O7)
117.7
109.9
120.0
110.9
111.0
129.6
126.4
84.2
118.0
111.0
112.7
130.3
127.4
85.3
118.2
111.0
111.8
130.4
126.2
85.7
120.1
111.1
111.3
130.6
124.7
83.1
A(C6-O7-M45)
A(C1-N14-M45)
A(N15-N14-M45)
A(N17-N18-M45)
A(O7-M45-N14)
A(O7-M45-O46)
A(O7-M45-O49)
A(O7-M45-O52)
A(N14-M45-N18)
A(N18-M45-O46)
A(N18-M45-O49)
A(N18-M45-O52)
D(N-14-C1-C6-C5)
D(N14-C1-C6-O7)
D(C6-C1-N14-N15)
D(C6-O7-M45-N14)
D(C6-O7-M45-O46)
D(C6-O7-M45-O49)
D(C6-O7-M45-O52)
D(M45-N14-N15-C16)
D(C1-N14-M45-N18)
D(N15-N14-M45-N18)
D(N15-N14-M45-O46)
D(N15-N14-M45-O49)
D(N15-N14-M45-O52)
112.7
112.1
127.8
119.3
79.5
71.7
77.4
87.2
87.0
A(−,-,O52)
A(−,-,N59)
A(−,-,N63)
76.2
180.0
103.1
85.8
99.7
87.6
86.8
100.5
83.4
81.6
82.7
79.6
88.3
90.8
90.8
88.8
103.7
87.4
83.9
92.8
93.1
A(−,-,O52)
A(−,-,N59)
A(−,-,N63)
176.5
83.4
103.8
180.0
102.1
91.5
100.9
85.8
109.2
85.5
105.2
178.5
−2.2
−178.9
−0.6
−118.5
155.6
147.7
179.9
179.2
−1.2
−179.0
0.3
178.9
−0.4
−177.4
4.1
179.5
−0.3
−177.1
3.6
−0.02
−179.9
1.5
155.2
−20.8
−111.0
151.0
155.6
0.9
−92.6
99.2
−93.0
98.1
D(−,-,-,O52)
D(−,-,-,N59)
D(−,-,-,N63)
22.5
−22.2
159.3
84.7
−178.7
1.5
−178.9
2.0
178.6
−3.2
−107.9
75.7
175.5
−6.8
−99.9
86.1
177.0
−5.5
−99.9
87.5
121.1
−8.6
−112.9
71.4
−52.2
D(−,-,-,O52)
D(−,-,-,N59)
D(−,-,-,N63)
28.3
37.6
107.1
127.8
Values are mean SD triplicate assays.

NOVEL FORMAZAN METAL COMPLEXES AND THEIR BIOLOGICAL APPLICATIONS
11 of 24
FIGURE 1 The optimized
geometry, numbering system, vector of
dipole moment of H2L (S1), Cu (III)
(S2), Fe (II) (S3), Cr (III) (S4), Co (III)
(S5) and Ni (II) (S6) complexes using
B3LYP/GENECP level of calculation
computed values of dihedral angles around metal ion
represent in Table 2, i.e. N14C1C6C5, C6C1N14N15,
C6O7MN14, MN14N15N18 and C1N14MN18, are close
from 0 or 180o which indicate that the metal ion is in the
same molecular plane of the ligand except Cr-molecule
out of plane with perfect octahedral structure and other
coordination with water are close to 95o except in case of
Cu (II) complex there are large deviation from regular
octahedral structure.
describes the distribution of electrons in various sub-
shells of their atomic orbits. The natural charges on the
coordinating sites in the core, valence and Rydberg sub-
shells and natural electronic configuration of the metal
in the studied complexes are presented in Tables 3 and
S3. The most electronegative centers are accumulated on
O7, N14, N18, O46, O49 and O52. These electronegative
atoms have a tendency to donate electrons. Whereas, the
most electropositive atoms are Cu, Fe, Co, Ni and Cr
have a tendency to accept electrons and incase of Cr the
most electronegative centers are localized on O7, N14,
N18, O52, N59 and N63 because Cr coordinated with two
ligand molecules. The central metal ion in the Cu, Fe,
Co, Ni and Cr complexes received 1.11e, 1.23e, 2.09e,
1.31e and 2.6e from the donating active sites of the
3.7.3 | Natural charges and natural
population
The natural population analysis performed on the elec-
tronic structures of the studied complexes clearly

12 of 24
KHAN ET AL.
TABLE 3 Natural charge on coordinated atoms of H2L (S1), Cu (II) (S2), Fe (II) (S3), Cr (III) (S4), Co (III) (S5) and Ni (II) (S6)
complexes using B3LYP/GENECP level of theory
Atom
O7
H2L (S1)
−0.679
−0.215
−0.236
Cu (II) (S2)
−0.75
Fe (II) (S3)
−0.718
Co (III) (S5)
−0.656
Ni (II) (S6)
−0.771
−0.29
Cr (III) (S4)
−0.599
−0.173
−0.218
O52
N14
N17
O46
O49
O52
−0.257
−0.208
−0.992
−1.012
−1.006
−0.232
−0.154
−0.238
−0.208
−0.227
−0.952
−0.962
−0.967
−0.976
−0.97
−0.599
−0.174
−0.218
−1.038
−0.977
N59
−0.985
−0.928
N62
Values are mean SD triplicate assays.
ligands with d-orbit electronic configuration 3d9.44, 3d6.63
,
indicating its lowest stability and high reactivity and
other derivative gradient decreasing order as
S2 < S4 < S6 < S1. The two properties like I (potential
ionization) and A (electron affinity) are so important, the
determination of these two properties allow us to calcu-
late the global reactivity descriptors. These two parame-
ters are related to the one-electron orbital energies of the
HOMO and LUMO respectively. The more I is lower, the
molecule will be the better electron donor. The more A is
larger the molecule will be the better electron acceptor
From Table 4, S5 have the highest values of both I and A
3d8.36, 3d7.65, 3d4.88 from the active sites of the ligands
respectively.
3.7.4 | Frontier molecular orbitals
(FMOs) analysis
The values of the calculated quantum chemical parame-
ters such as the energy of the highest occupied molecular
orbital (EHOMO), energy of the lowest unoccupied molec-
ular orbital (ELUMO), energy gap (ΔEgap), ionization
energy (I), electron affinity (A)[77,78] are presented in
Table 4. The value of HOMO energy determines the abil-
ity of electron to be donated. When the value of EHOMO is
high, it indicates the ease of donating electron to the
unoccupied orbital of the receptor molecule. When the
value of ELUMO is small, this means that it has a small
resistance to accept electrons so it will be more able to
accept electron. The HOMO and LUMO energy values
are related to the ionization potential (I = − EHOMO) and
electron affinities (A = − ELUMO). The difference between
HOMO and LUMO energy values gives the HOMO-
LUMO energy gap. The compound S3 showed highest
energy gap (ΔEgap = 2.64) which indicates its higher sta-
bility and less reactivity. The molecule that have the low-
est energy gap is the molecule S5 (ΔEgap = 2.14 eV)
verses other derivatives with order of
I
as
S1 > S2 > S3 > S6 > S4 in case A, order is
S2 > S1 > S6 > S3 > S4. From Figure 2, it is cleared that
the dispersion of isodensities of HOMOs and LUMOs is
almost similar in all the compounds, which dispersed on
the phenyl rings of ligand and the coordination active
centers.
3.7.5 | Global reactivity descriptors
Chemical reactivity can be simply defined as the ten-
dency of a chemical matter to undergo chemical reaction
with another chemical matter. It is well-known that the
understanding of the nature of chemical interactions and
the prediction of chemical reactivity of atoms, ions or
TABLE 4 Molecular orbital energies and other properties of synthesized derivatives (S1-S6)
EHOMO
Compounds (eV)
ELUMO
(eV)
Energy gap ΔE
(eV)
Ionization Potential,
I (eV)
Electron Affinity,
A (eV)
Dipole moment
debye (D)
H2L (S1)
−6.13
−5.90
−5.77
−3.72
−9.62
−5.74
−3.62
−3.76
−3.13
−1.34
−7.78
−3.35
2.51
2.14
2.64
2.38
1.85
2.40
6.13
5.90
5.77
3.72
9.62
5.74
3.62
3.76
3.13
1.34
7.78
3.35
2.76
7.00
8.19
0.00
1.85
2.92
Cu (II) (S2)
Fe (II) (S3)
Cr (III) (S4)
Co (III) (S5)
Ni (II) (S6)
Values are mean SD triplicate assays.

NOVEL FORMAZAN METAL COMPLEXES AND THEIR BIOLOGICAL APPLICATIONS
13 of 24
FIGURE 2 Frontier molecular
orbitals of the newly synthesized
formazan compounds (S1-S6)
molecules are some of the challenging issues in chemis-
try, which are important to explain the reactivity of syn-
thesized formazan compounds (S1-S6). In the CDFT,
quantum chemical descriptors like chemical hardness
(η), electronic chemical potential (μ), and electronegativ-
ity (χ) are usually considered. μ and η are defined as the
first derivative of the electronic energy and chemical
potential with respect to the electron number (N) at con-
stant external potential, v(r), respectively.[79,80]
ꢀ
ꢁ
∂E
∂N
μ =
ð1Þ
vðrÞ

14 of 24
KHAN ET AL.
chemical system becomes saturated by adding electrons.
It plays an excellent role in describing the chemical reac-
tivity of a system. The results from Table 5 indicate that
S4 has the lowest electrophilicity index value (0.0 eV)
and is nucleophilic in nature, whereas S3 has the highest
value i.e., of 25.41 eV and is strongly electrophilic in
nature and other order is S2 > S6 > S1 > S5.
The electronegativity (X), describes the ability of a
molecule to attract electrons towards itself in a covalent
bond. Thus, compound S5 possesses higher electronega-
tivity value (8.7 eV) than all compounds so; it is the best
electron acceptor and other derivatives ordered
S1 > S2 > S6 > S3 > S4. Global softness of a molecule
represents reactivity of compounds. S5 complex showed
highest softness values (0.54 eV) and showed high reac-
tivity, While S3 showed the least value of softness
ꢀ
ꢁ
ꢀ
ꢁ
1
2
∂2E
∂N2
1
∂μ
η =
=
ð2Þ
2 ∂N
vðrÞ
vðrÞ
Within the framework of finite differences approxima-
tion, the following expressions based on HOMO and
LUMO energy values for complexes at B3LYP/GENECP
level of calculation as follows[77,78]
:
η = ðEHOMO −ELUMOÞ=2
μ = −ðEHOMO + ELUMOÞ=2
ω = μ2=2η
ð3Þ
ð4Þ
ð5Þ
ð6Þ
ð7Þ
(0.38
eV)
with
other
substituted
order
χ = ðEHOMO + ELUMOÞ=2
S = 1=2η
S2 > S4 = S6 > S1 > S3.
3.7.6 | Local reactivity descriptor
The values of all the important reactivity descriptors of
the compounds under study are given in the Table 5.
These descriptors are important to explain the reactivity
and stability of studied compounds.
The concepts of local and global reactivity descriptors
have been widely used to understand the chemical reac-
tivity and site selectivity[81,82] and to analyze local molec-
ular site selectivity of the molecules. Yang and Mortier
(1986) defined the Fukui function as the first derivative
of the electronic density ρ(r) of a system with respect to
the number of electrons (N) at a fixed external potential
ν(r)[83] as given these Equation:
From Table 5, it is clear that among all derivatives, S3
has highest value of η equal to 1.32 eV with the chemi-
cally hardest compound among all, whereas S5 has the
lowest value (0.92 eV) and are chemically soft and more
reactive. Other synthetized derivatives order of hardness
is S2 > S4 > S6 > S2. These findings are consistent with
the HOMO-LUMO band gaps of all synthesized deriva-
tives. The electronic chemical potential (μ) value gives an
idea about the charge transfer within any compound in
its ground state. From Table 5, it is clear that S4 has the
highest chemical potential value (−2.53 eV), while S5 has
the lowest chemical potential (−8.7 eV) and other with
order S3 > S6 > S2 > S1.
ꢀ
ꢁ
ꢀ
ꢁ
∂ρðrÞ
∂N
1
2
∂μ
f k =
=
ð8Þ
∂vðrÞ
vðrÞ
vðrÞ
Parr and Yang[84] define local descriptors such as electro-
philic and nucleophilic Fukui functions. Thus, calculat-
ing Fukui functions can enable us to determine the active
sites of a molecule, based on the electronic density
changes experienced by the molecule during a reaction.
The electrophilicity index (ω) is a thermodynamic
property that measures the changes in energy when a
TABLE 5 Reactivity indices of synthesized formazan compounds (S1-S6)
Chemical hardness, Chemical potential, Electrophilicity
Electronegativity
X, eV
Global softness
S, eV
Compounds η (eV)
μ (eV)
−4.87
−4.83
−4.45
−2.53
−8.70
−4.55
Index, ω (eV)
H2L (S1)
1.26
1.07
1.32
1.19
0.92
1.20
3.03
4.87
4.83
4.45
2.53
8.70
4.55
0.40
0.47
0.38
0.42
0.54
0.42
Cu (II) (S2)
Fe (II) (S3)
Cr (III) (S4)
Co (III) (S5)
Ni (II) (S6)
22.94
25.41
0.00
1.85
3.56
Values are mean SD triplicate assays.

NOVEL FORMAZAN METAL COMPLEXES AND THEIR BIOLOGICAL APPLICATIONS
15 of 24

16 of 24
KHAN ET AL.
Fukui functions f k− , f k+ , f ko and Δfk are calculated for three
highest ωαk corresponds to the most electrophilic site in a
molecule. In addition, Lee et al.[89] proposed different
local softness, which can be used to describe the reactiv-
ity of atoms in molecules, which can be defined as in
Equation (10).
chemical situations, using the following equations
as[85–87]
:
f k− = qkðNÞ−qkðN−1Þ≈ρHOMOðrÞ for electrophilic attack
f k+ = qkðN + 1Þ−qkðN Þ≈ρLUMOðrÞ for nucleophilic attack
f ok = 1 ½qkðN + 1Þ−qkðN−1Þꢂ≈ 1½ρHOMOðrÞ + ρLUMOðrÞꢂ for
2
2
sαk = sf αk
ð10Þ
Radical attack
Where qk(N) is the atomic population on the kth atom
for the neutral molecule, while qk(N+1) and qk(N − 1) are
the atomic population on the kth atom for its anionic and
cationic species, respectively. As it is known, the concept
of generalized philicity have been introduced by
Chattaraj et al.[88] they defined a local quantity called
philicity associated with a site k in a molecule with the
assistance of corresponding condensed-to-atom variants
of Fukui function, f αk as in Equation (9).
Where α = +, − and 0 represents local softness quan-
tities describing nucleophilic, electrophilic and radical
attacks respectively. In addition to the information con-
cerning electrophilic and nucleophilic capacity of a given
atomic site in the molecule, Morell and Labbe et al.[90]
proposed another Dual descriptor (Δfk) which is given
by:
Δf k = f k+ −f k−
ð11Þ
ωαk = ωf αk
ð9Þ
if Δfk > 0, then the site is favored for a nucleophilic
attack, whereas if Δfk < 0, then the site may be favored
for an electrophilic attack. The calculation of Fukui func-
tions indices, local philicity, local softness and Dual
Where α = +, − and 0 corresponds to local philic
quantities describing nucleophilic, electrophilic and radi-
cal attacks, respectively. In the light of Equation (9), the
TABLE 7 Fukui and Dual descriptors of synthesized formazan compound Cr (III) (S4) at B3lyp/GENECP level
−
+
−
+
Atoms
C1
fk
fk
Δfk
Atoms
C39
Cr45
C46
C47
C48
C49
C50
C51
O52
N59
N60
C61
N62
N63
C64
C65
C66
C67
C68
C79
C80
C84
fk
fk
Δfk
0.006
0.006
0.001
0.014
0.001
0.002
0.007
0.034
0.013
0.055
0.045
0.069
0.026
0.023
0.002
0.022
0.000
0.008
0.014
0.002
0.018
0.001
0.001
0.050
0.003
0.012
0.009
0.010
0.002
0.070
0.096
0.009
0.083
0.023
0.004
0.010
0.010
0.021
0.004
−0.013
0.005
0.005
0.001
0.005
−0.005
0.044
0.010
0.199
0.006
0.006
0.001
0.014
0.001
0.002
0.007
0.034
0.013
0.055
0.045
0.069
0.026
0.023
0.002
0.022
0.000
0.008
0.014
0.010
0.005
0.044
0.001
0.050
0.003
0.012
0.009
0.010
0.002
0.070
0.096
0.009
0.083
0.023
0.004
0.010
0.010
0.021
0.004
−0.013
0.005
0.005
−0.005
−0.155
−0.005
0.044
C2
C3
0.002
C4
−0.003
0.008
C5
0.002
C6
0.009
−0.003
0.008
O7
−0.005
0.037
N14
N15
C16
N17
N18
C19
C20
C21
C22
C23
C34
C35
C36
C37
C38
0.009
0.083
−0.005
0.037
−0.046
0.038
0.083
−0.046
−0.022
−0.013
0.007
−0.046
0.038
−0.046
−0.022
−0.013
0.007
−0.002
0.004
−0.021
−0.009
0.003
−0.002
0.004
−0.022
−0.009
−0.005
−0.017
0.004
Values are mean SD triplicate assays.

NOVEL FORMAZAN METAL COMPLEXES AND THEIR BIOLOGICAL APPLICATIONS
17 of 24

18 of 24
KHAN ET AL.

NOVEL FORMAZAN METAL COMPLEXES AND THEIR BIOLOGICAL APPLICATIONS
19 of 24

20 of 24
KHAN ET AL.

NOVEL FORMAZAN METAL COMPLEXES AND THEIR BIOLOGICAL APPLICATIONS
21 of 24
FIGURE 3 MEP surfaces of the newly
synthesized formazan compounds (S1-S6)
TABLE 11 The MEP analysis of synthesized formazan compounds (S1-S6)
Compounds
H2L (S1)
+ve potential
0.095
-ve potential
−0.095
Cu (II) (S2)
Fe (II) (S3)
Cr (III) (S4)
Co (III) (S5)
Ni (II) (S6)
0.084
−0.084
0.085
−0.085
0.111
−0.111
0.177
−0.177
0.088
−0.088
Values are mean SD triplicate assays
descriptor of the studied molecules S1-S6 at the level
B3LYP/GENECP is given in Table 6–10.
conclusion can be reached considering the Dual descrip-
tor Δfk regarding electrophilic and nucleophilic attack.
These results are completely changed when the chelation
formed with central metal ions. In case of synthesized
formazan compounds S2, S3 and S6, the most electro-
philic active sites are located on C1, C6, O7, C16, C34,
C35, C37 and M45. While nucleophilic attacks are on C2,
From the values of Fukui functions f k− and f k+ , it can
be stated that the most electrophilic active site in ligand
molecule is located on C16, N18, C34, C35 and C37. Like-
wise, the active sites susceptible for nucleophilic attacks
are C1, C2, C4, C6, O7, N14, N15 and N17. The same

22 of 24
KHAN ET AL.
C4, N14, N15 and N17, the reason of these characteristic
differences is the redistribution of electron density inside
molecules due to chelation form, high electronegativity
of N and O atoms in coordination centers also the effect
of -NO2, -SO3H substituted groups.
given in Table 11, which indicate the same results
from NBO population and local Fukui descriptors
analysis.
In case of S5 compounds, the Co45 and N18 pattern
changed from electrophilic to nucleophilic attack due to
back donation of Co atom to the center atoms. These
results mixed with the same last in S1, S3 and S6. In case
of S4, the most electrophilic active sites are located on
C1, O7, C16, N18, Cr45 and N63 while nucleophilic
attacks are N14, N15, N17, N59, N60 and N62, which is
almost results in all complexes except S5 chelation. These
results are in agreement with the last population analysis
part with computed HOMO and LUMO.
To complete the picture, the condensed local soft-
ness, local electrophilicity/nucleophilicity index, and
relative electrophilicity/nucleophilicity have been also
calculated for each atoms in the studied molecules
from CDFT point of view by using implemented code
in Multiwfn v3.7 software program.[91] A close inspec-
tion would reveal that all the molecules had the
donating and the back-donation process at their che-
lated center (C1, C6, O7, M, N14, N15, C16, N17 and
N18) in agreement with the frontier orbital results
obtained. According to these results, one can conclude
that proposed molecules will have many active centers
to interact with pocket protein surface, through donat-
ing electrons to the metal ions 3d orbitals and back
donation process. Finally, the above local descriptors
reveal that the theoretical variation of the efficiencies
of the investigative molecules agrees with the available
experimental data in the same work.
4 | CONCLUSION
The superior antibacterial and antimycotic activities
were exhibited by formazan compound (S4) by pre-
senting maximum ZOIs and MICs values. While sub-
stantial and enhanced antioxidant in terms of
percentage inhibition of DPPH and cytotoxic effect on
human breast carcinoma cells was demonstrated by
formazan compound (S1) which was further validated
by the results of molecular docking studies of (S1)
with the human estrogen receptor protein. To explore
the structural properties, Density functional theory
(DFT) investigations on all synthesized complexes
(S1-S6) were performed. Frontier molecular orbitals
(FMO) analysis reveals that the spread of isodensity
was mainly concentrated on the phenyl ring of ligand
and groups directly attached. The computational results
also show that C16, N18, C34, C35 and C37 are the
most preferred sites for electrophilic attack. Likewise,
the active sites susceptible for nucleophilic attacks are
C1, C2, C4, C6, O7, N14, N15 and N17. The
HOMO/LUMO band gap studies and MEP analysis
revealed the reactivity and the binding of complexes as
a drug to the protein actives sites. The formazans com-
pounds could be potential drug candidate that con-
strains the growth of microbial strains, possess ability
to cause cytotoxic effect on carcinoma cells and act as
effective scavenger for free radical species.
ACKNOWLEDGMENTS
3.7.7 | Molecular electrostatic potential
(MEP)
This work was supported by the Department of Chemis-
try, School of Science, University of Management and
Technology, Lahore, Pakistan and Shafi Reso Chemical
Lahore, Pakistan.
MEP analysis with the help of quantum mechanical
methods has been used extensively to explain the reac-
tive sites within compounds (S1-S6). MEP is found to
be a useful descriptor to predict reactive sites for elec-
trophilic and nucleophilic attack reactions as well as
hydrogen-bonding interactions.[92,93] With MEP analy-
sis, the reactive sites can be located by different color
codes, such as the red color in a MEP graphic indi-
cates an electron-rich site, blue color indicates an
electron-deficient site, while the green color is indica-
tive of a neutral region. The MEP analysis of com-
pounds viz. S1-S6 was carried out. Figure 3 displayed
the MEP surfaces of the newly synthesized derivatives
S1-S6, while the corresponding +ve and –ve potential
values from the MEP analysis of these compounds are
CONFLICT OF INTEREST
No conflict of interest associated with this work.
CONTRIBUTIONS OF AUTHORS
The authors declare that this work was done by the
authors named in this article. The authors Shakeel
Ahmad Khan has done the experimental work and
Sammia Shahid supervised the whole research work.
Mahmoud A. Noamaan, Komal Rizwan, Tahir Rasheed
and Hira Amjad assisted in the docking and DFT with
computational exploration of chemical reactivity stud-
ies of new synthesized formazn compounds
respectively.

NOVEL FORMAZAN METAL COMPLEXES AND THEIR BIOLOGICAL APPLICATIONS
23 of 24
[29] K. Hunger, Industrial Dyes: Chemistry, Properties, Applications,
Wiley-VCH, Germany 2003 1.
DECLARATIONS
[30] F. E. H. David, L. Blangey, Fundamental processes of dye chem-
istry, Interscience publisher's ltd., London 1949 1.
[31] Y. Gu, F. Jérôme, Chem. Soc. Rev. 2013, 42, 9550.
[32] J. Yang, H. Q. Li, M. H. Li, J. J. Peng, Y. L. Gu, Adv Synth
Catal. 2012, 354, 688.
ORCID
Shakeel Ahmad Khan https://orcid.org/0000-0002-
7594-1222
Tahir Rasheed https://orcid.org/0000-0001-9265-6303
[33] M. Zhang, Z. R. Shang, X. T. Li, J. N. Zhang, Y. Wang, K. Li,
Y. Y. Li, Z. H. Zhang, Synth. Commun. 2017, 47, 178.
[34] R. Y. Guo, Z. M. An, L. P. Mo, R. Z. Wang, H. X. Liu,
S. X. Wang, Z. H. Zhang, ACS Comb. Sci. 2013, 15, 557.
[35] R. Y. Guo, Z. M. An, L. P. Mo, S. T. Yang, H. X. Liu,
S. X. Wang, Z. H. Zhang, Tetrahedron 2013, 69, 9931.
[36] X. T. Li, A. D. Zhao, L. P. Mo, Z. H. Zhang, RSC Adv. 2014, 4,
51580.
[37] H. S. Chen, R. Y. Guo, Monatsh. Chem. 2015, 146, 1355.
[38] X. T. Li, Y. H. Liu, X. Liu, Z. H. Zhang, RSC Adv. 2015, 5,
25625.
[39] P. Geerlings, F. De Prof, W. Langenaeker, Chem. Rev. 2003,
103, 1793.
REFERENCES
[1] K. S. Sunil, C. Usha, Asian J. Pharm. Clin. Res. 2018, 11, 398.
[2] A. Subramanian, N. Kannan, P. Mani, Int. J. Curr. Pharm. Res.
2017, 9, 85.
[3] N. Mulcahy, Cancer to Become Leading Cause of Death
Worldwide by 2010. Medscape Medical News, 2008.
[4] L. Hong, G. P. Schroth, H. R. Matthews, P. Yau,
E. M. Bradbury, J. Biol. Chem. 1993, 268, 305. PMID: 8416938
[5] H. Patel, P. Sinha, Curr. Sci. 2001, 81, 641.
[6] R. Marti-Centelles, R. Cejudo-Marin, E. Falomir, J. Murga,
M. Carda, J. A. Marco, Bioorg. Med. Chem. 2013, 21, 3010.
[7] M. Mahdavi, K. Pedrood, M. Safavi, M. Saeedi, M. Pordeli,
S. K. Ardestani, S. Emami, M. Adib, A. Foroumadi, A. Shafiee,
Eur. J. Med. Chem. 2015, 95, 492.
[40] F. Ijaz, S. Shahid, S. A. Khan, W. Ahmad, S. Zaman, Trop J
Pharm Res. 2017, 16, 743.
[41] S. A. Khan, F. Noreen, S. Kanwal, A. Iqbal, G. Hussain, Mater.
Sci. Eng. C 2018, 82, 46.
[42] S. A. Khan, N. Rasool, M. Riaz, R. Nadeem, U. Rashid,
K. Rizwan, M. Zubair, I. H. Bukhari, T. Gulzar, Asian J. Chem.
2013, 25, 7457.
[8] A. S. Shawali, N. A. Samy, J Adv Res. 2015, 6, 241.
[9] S. A. Khan, S. Shahid, S. Kanwal, G. Hussain, Dyes Pigm. 2018,
148, 31.
[10] S. A. Khan, S. Shahid, S. Kanwal, K. Rizwan, T. Mahmood,
K. Ayub, J. Mol. Struct. 2018, 1175, 73.
[43] S. A. Khan, S. Shahid, Z. A. Khan, A. Iqbal, Int. J. Sci. And Res.
Pub. 2016, 6, 529.
[11] D. E. Berry, R. G. Hicks, J. E. Gilroy, J. Chem. Educ. 2009,
86, 76.
[44] S. A. Khan, F. Noreen, S. Kanwal, G. Hussain, Dig.
J. Nanomater. Biostruct. 2017, 12, 877.
[12] S. B. Chavan, S. B. Zangade, Y. Archana, Y. B. VIbhute,
J Chem Pharm Res. 2012, 3, 262.
[45] A. Khalid, S. Shahid, S. A. Khan, S. Kanwal, A. Yaqoob,
Z. G. Rasool, K. Rizwan, Trop J Pharm Res. 2018, 17, 1531.
[46] S. A. Khan, S. Shahid, W. Ahmad, S. Ullah, Int J Pharm Sci
and Res. 2017, 2, 22.
[47] S. A. Khan, S. Shahid, M. R. Sajid, F. Noreen, S. Kanwal, Int.
J. Adv. Res. 2017, 5, 925.
[48] S. A. Khan, S. Kanwal, A. Iqbal, W. Ahmad, Int. J. Adv. Res.
2017, 5, 1350.
[49] S. A. Khan, M. Jameel, S. Kanwal, S. Shahid, Int J Pharm Sci
and Res. 2017, 2, 29.
[50] S. A. Khan, S. Shahid, M. Jameel, A. Ahmad, Int J Pharma
Chem. 2016, 6, 107.
[51] S. A. Khan, W. Ahmad, K. S. Munawar, S. Kanwal, Indian
J. Pharm. Sci. 2018, 80, 480.
[52] A. Iqbal, Z. A. Khan, S. A. Shahzad, M. Usman, S. A. Khan,
A. H. Fauq, A. Bari, M. A. Sajid, Turk. J. Chem. 2018, 42, 1518.
[53] S. A. Khan, S. Shahid, S. Jabin, S. Zaman, M. Sarwar, Dig.
J. Nanomater. Biostruct. 2018, 13, 285.
[54] S. Shahid, S. A. Khan, W. Ahmad, U. Fatima, S. Knawal,
Indian J. Pharm. Sci. 2018, 80, 173.
[13] M. Kidwai, N. Negi, S. D. Gupta, Chem. Pharm. Bull. 1994, 42,
2363.
[14] J. D. Bhosale, A. R. Shirolkar, U. D. Pete, C. M. Zade,
D. P. Mahajan, C. D. Hadole, S. D. Pawar, U. D. Patil,
R. Dabur, R. S. Bendre, J Pharm Res. 2013, 7, 582.
[15] D. D. Mukerjee, S. K. Shukla, B. L. Chowdhary, Arch Pharm
(Weinheim). 1981, 314, 991.
[16] R. Khanna, G. Palit, V. K. Srivastava, K. Shanker, Indian
J. Chem. 1990, 29B, 556.
[17] J. P. Raval, P. Patel, S. Pradip, P. S. Patel, Int J Pharm Tech
Res. 2009, 1, 1548.
[18] A. Srivastava, A. V. Kumar, Indian J. Pharm. Sci. 2001,
65, 358.
[19] S. D. Bhardwaj, Asian J. Chem. 1998, 10, 39.
[20] S. D. Bhardwaj, V. S. Jolly, Asian J. Chem. 1997, 9, 48.
[21] H. A. Mahdi, K. S. Abbas, H. M. Dagher, Chem Mat Res. 2015,
7, 72.
[22] A. S. Yaglıoglu, H. Senoz, Hacettepe J Biol Chem. 2013, 41, 371.
[23] R. Kaisi, K. Pande, T. N. Bhalla, J. P. Barthwal, G. P. Gupta,
S. S. Parmar, J. Pharm. Sci. 1990, 79, 317.
[55] M. A. Qamar, S. Shahid, S. A. Khan, S. Zaman, M. Sarwar,
Dig. J. Nanomater. Biostruct. 2017, 12, 1127.
[56] S. A. Khan, S. Shahid, W. Bashir, S. Kanwal, A. Iqbal, Trop J
Pharm Res. 2017, 16, 2331.
[24] D. J. Knowles, Trends Microbiol. 1997, 5, 379.
[25] C. R. Harris, A. Thorarensen, Curr. Med. Chem. 2004, 11, 2213.
[26] I. Chopra, J. Hodgson, B. Metcalf, G. Poste, JAMA 1996,
275, 401.
[57] W. Ahmad, S. A. Khan, K. S. Munawar, A. Khalid, S. Kawanl,
Trop J Pharm Res. 2017, 16, 1137.
[27] N. Singh, S. K. Bhati, A. Kumar, Eu J Med Chem. 2008, 43,
2597.
[58] K. D. Dykstra, L. Guo, E. T. Birzin, W. Chan, Y. T. Yang,
E. C. Hayes, C. A. DaSilva, L. Y. Pai, R. T. Mosley, B. Kraker,
[28] P. Thangavelu, S. Chellappa, S. Thangavel, Int J Pharm Pharm
Sci. 2018, 10, 56.

24 of 24
KHAN ET AL.
P. M. Fitzgerald, F. DiNinno, S. P. Rohrer, J. M. Schaeffer, Bio-
[82] P. K. Chattaraj, D. R. Roy, Chem. Rev. 2007, 107, PR46.
org. Med. Chem. Lett. 2007, 17, 2322.
[59] Dassault Systèmes BIOVIA, Biovia Discovery Studio Visualizer,
v16.1.0, San Diego, Dassault Systèmes 2016.
[60] O. Tacar, P. Sriamornsak, C. R. Dass, J. Pharm. Pharmacol.
2013, 65, 157.
[61] G. M. Morris, R. Huey, W. Lindstrom, M. F. Sanner,
R. K. Belew, D. S. Goodsell, A. J. Olson, J. Comput. Chem.
2009, 30, 2785.
[83] W. Yang, W. J. Mortier, J. Am. Chem. Soc. 1986, 108, 5708.
[84] R. G. Parr, W. Yang, J. Am. Chem. Soc. 1984, 106, 4049.
[85] R. G. Parr, R. G. Pearson, J. Am. Chem. Soc. 1983, 105, 7512.
[86] P. K. Chattaraj, S. Giri, J. Phys. Chem. A 2007, 111, 11116.
[87] R. R. Contreras, P. Fuentealba, M. Galvan, P. Perez, Chem.
Phys. Lett. 1999, 304, 405.
[88] R.
Parthasarathi,
J.
Padmanabhan,
M.
Elango,
V. Subramanian, P. K. Chattaraj, Chem. Phys. Lett. 2004,
[62] O. Trott, A. J. Olson, J. Comput. Chem. 2010, 31, 455.
[63] T. Sander, J. Freyss, M. von Korff, C. Rufener, J. Chem. Inf.
Model. 2015, 55, 460.
394, 225.
[89] C. T. Lee, W. T. Yang, R. G. Parr, J. Mol. Struct.: THEOCHEM
1988, 40, 305.
[64] A. D. Becke, Phys. Rev. a 1988, 38, 3098.
[90] C. Morell, A. Grand, A. Toro-Labbe, J. Phys. Chem. A 2005,
[65] A. D. Becke, J. Chem. Phys. 1993, 98, 5648.
[66] B. G. Johnson, M. J. Frisch, Chem. Phys. Lett. 1993, 216, 133.
[67] C. Lee, W. Yang, R. G. Parr, Phys. Rev. B 1988, 37, 785.
[68] A. Schafer, C. Huber, R. Ahlrichs, J. Chem. Phys. 1994, 100,
5829.
109, 205.
[91] J. Padmanabhan, R. Parthasarathi, V. Subramanian,
P. K. Chattaraj, J. Phys. Chem. A 2006, 110, 2739.
[92] F. J. Luque, J. M. Lopez, M. Orozco, Theor. Chem. Acc. 2000,
103, 343.
[69] A. D. McLean, G. S. Chandler, J. Chem. Phys. 1980, 72, 5639.
[70] R. Ditchfield, W. J. Hehre, J. A. Pople, J. Chem. Phys. 1971,
54, 724.
[93] F. Weigend, R. Ahlrichs, Phys. Chem. Chem. Phys. 2005, 7,
3297.
[71] S. E. Ulic, C. O. D. Vedova, A. Hermann, H.-G. Mack,
H. Oberhammer, J. Phys. Chem. A 2008, 112, 6211.
[72] A. E. Reed, F. Weinhold, J. Chem. Phys. 1983, 78, 4066.
[73] http://www.chemcraftprog.com.
[74] R. Dennington, T. Keith, J. Millam, GaussView, Version 5,
Semichem Inc., Shawnee Mission, KS 2009.
SUPPORTING INFORMATION
Additional supporting information may be found online
in the Supporting Information section at the end of this
article.
[75] L. Tian, C. Feiwu, J. Comput. Chem. 2012, 33, 580.
[76] S. S. Zumdahl, Chemistry for Chemical and Biological Sciences,
University Science Books, Sausalito, CA 2000.
[77] R. G. Pearson, Proc. Natl. Acad. Sci. 1986, 83, 8440.
[78] A. K. Chandra, T. Uchimara, J. Phys. Chem. A 2001, 105, 3578.
[79] S. B. Liu, Acta Phys.-Chim. Sin. 2009, 25, 590.
[80] J. Frau, D. Glossman-Mitnik, Front. Chem. 2017, 5, 16.
[81] P. Geerlings, F. De Proft, W. Langenaeker, Chem. Rev. 2003,
103, 1793.
How to cite this article: Khan SA, Rizwan K,
Shahid S, Noamaan MA, Rasheed T, Amjad H.
Synthesis, DFT, computational exploration of
chemical reactivity, molecular docking studies of
novel formazan metal complexes and their
biological applications. Appl Organometal Chem.
2020;e5444. https://doi.org/10.1002/aoc.5444