Homo- and heteroleptic Zinc(II) carboxylates: Synthesis, structural characterization, and assessment of their biological significance in in vitro models

Article abstract of DOI:10.1016/j.ica.2020.119849

A series of five new zinc(II) complexes of 2-phenylbutyric acid comprising of one homoleptic (1) and four heteroleptic (2–5) complexes were synthesized under open beaker conditions. The behavior of the complexes in the solid and solution state was explored through FT-IR and NMR (¹H and ¹³C) spectroscopy, respectively. Crystallographic data have revealed that complexes 2 and 3 crystallized in monoclinic and triclinic crystal system, respectively, with a distorted octahedral geometry, whereas complex 4 crystallized in monoclinic crystal system with a pentagonal geometry around the zinc(II) atom. All the complexes have been screened for enzyme (alkaline phosphatase) inhibition, in vitro hemolysis and antileishmanial activity, DNA and cetyltrimethylammonium bromide (CTAB) interaction. The increase in the concentration of complexes 1, 2, 4 and 5 remarkably decreases the activity of enzyme alkaline phosphatase, whereas complex 3 was found inactive. The behavior of the complexes as a potent drug is clear from the results of hemolysis and antileishmanial activity against promastigote. The UV–Vis spectroscopic data of complexes–DNA interactions suggested a surface binding mode whereas the interaction of CTAB with complexes, studied through conductometry, confirmed strong interacting ability of complexes with CTAB.

Full text of DOI:10.1016/j.ica.2020.119849

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Homo- and Heteroleptic Zinc(II) Carboxylates: Synthesis, Structural Charac‐
terization, and assessment of their biological significance in in vitro models
Saba Naz, Noor Uddin, Kaleem Ullah, Ali Haider, Amna Gul, Sulaiman
Faisal, Akhtar Nadhman, Memoona Bibi, Sammer Yousuf, Saqib Ali
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Reference:
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https://doi.org/10.1016/j.ica.2020.119849
ICA 119849
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Please cite this article as: S. Naz, N. Uddin, K. Ullah, A. Haider, A. Gul, S. Faisal, A. Nadhman, M. Bibi, S.
Yousuf, S. Ali, Homo- and Heteroleptic Zinc(II) Carboxylates: Synthesis, Structural Characterization, and
assessment of their biological significance in in vitro models, Inorganica Chimica Acta (2020), doi: https://
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Homo- and Heteroleptic Zinc(II) Carboxylates: Synthesis, Structural Characterization,
and assessment of their biological significance in in vitro models
Saba Naza, Noor Uddina, Kaleem Ullaha, Ali Haidera*, Amna Gulb, Sulaiman Faisalb, Akhtar
Nadhmanb, Memoona Bibic, Sammer Yousufc, Saqib Alia*
a Department of Chemistry, Quaid-i-Azam University, Islamabad, 45320, Pakistan
bDepartment of Biotechnology, Institute of Integrative Biosciences, CECOS University,
Hayatabad, Peshawar, Pakistan
cH.E.J. Research Institute of Chemistry, International Centre for Chemical and Biological
Sciences, University of Karachi, Karachi 75270, Pakistan
*For correspondence: ahaider@qau.edu.pk (A. Haider); saqibali@qau.edu.pk (S. Ali)
ABSTRACT
A series of five new zinc(II) complexes of 2-phenylbutyric acid comprising of one homoleptic
(1) and four heteroleptic (2–5) complexes was synthesized under open beaker conditions. The
behavior of the complexes in the solid and solution state was explored through FT-IR and NMR
13
(1H and C) spectroscopy, respectively. Crystallographic data have revealed that complexes 2
and 3 crystallized in monoclinic and triclinic crystal system respectively with a distorted
octahedral geometry, whereas complex 4 crystallized in monoclinic crystal system with a
pentagonal geometry around the zinc(II) atom. All the complexes have been screened for enzyme
(alkaline phosphatase) inhibition, in vitro hemolysis and antileishmanial activity, DNA and cetyl
trimethyl ammonium bromide (CTAB) interaction. The increase in the concentration of
complexes 1, 2, 4 and 5 remarkably decreases the activity of enzyme alkaline phosphatase,
whereas complex 3 was found inactive. The behavior of the complexes as a potent drug is clear
from the results of hemolysis and antileishmanial activity against promastigote. The UV–Vis
spectroscopic data of complexes–DNA interactions suggested a surface binding mode whereas
the interaction of CTAB with complexes, studied through conductometry, confirmed strong
interacting ability of complexes with CTAB.
Keywords;
1

Zinc(II) carboxylates, single-crystal X-ray diffraction, alkaline phosphatase, hemolysis,
antileishmanial activity, DNA interaction, CTAB interaction
1. Introduction
There is an ongoing interest in the synthesis and development of transition metal complexes due
to their structural diversity that are linked directly to the potential applications in diverse fields
like catalysis, bio-medicine, photoluminescence etc. Therefore, the selection of ligand and
central metal ion describes the topology and applied features of transition metal complexes [1].
The carboxylic acids/derivatives with substituted aromatic rings as ligands are of special interest
because they adopt flexible coordination behavior like terminal bidentate, terminal monodentate,
and bridging etc. around the metal center. The complexes formed by these ligands proved
attractive topological features, with interesting bio-medical applications [2].
Short chain fatty acids such as phenylbutyric acids are of great significance in biology. They
have the potential to suppress the growth of tumor cells, acting as a potent compound for the
nuclear receptors protein (PPARs) thus, delaying the paralysis of the hind limbs in the ts1-
infected mice [3]. It is helpful in fighting against diabetes [4] and acts as a growth inhibitor or
activator depending on their concentration [5]. 2-phenylbutyric acid was also proved to be the
best for hypocholesterolemic and choleretic among a number of phenyl substituted aliphatic acid.
They are first synthetic pyrethroids which although lack cyclopropane carboxylate group but
resemble them sterically [6]. Complexation of such kinds of drugs can enhance their activities
for example tin(IV) based organometallic compounds of 2-phenylbutyric were tested against
different plant pathogens and showed significant results. They also showed excellent binding
interaction with DNA which is suggestive of their significance as a drug for diseases which are
threatening human [7].
Zinc(II) with its strong binding ability and Lewis acidity (small size and positive charge) is the
suitable choice as a metal part in coordination complexes and its chemistry can be easily
followed via spectroscopic techniques [8, 9]. Zinc plays important structural, functional and
catalytic roles in biology [10]. It is an integral part of enzymes such as alkaline phosphatase,
carbonic anhydrase, carboxypeptidase etc which are responsible for the proper functioning of the
body and also helps in the regulation of the cell process [11]. Zinc is helpful in the structural
2

stabilization of the insulin as well as in DNA rewinding [12, 13]. The Zn(II) complexes have
been used as antibacterial and antidiabetic agents [14]. The important feature of Zn(II) ion is its
ability to form diverse coordinative complexes i.e. heteroleptic complexes with more than one
type of ligands. Here different ligands can adopt the same or different binding modes around
Zn(II) center, introducing fascinating topological features into the complex [15]. Heterocycle N-
donor ligands like 2,2ʹ-bipyridyl and 1,10-phenanthroline are capable of coordinating in a
fascinating way [16]. On the basis of their proficient physical as well as chemical properties they
showed high stability and reactivity and are therefore employed extensively for various
applications. Literature review revealed that complexation of aromatic compounds with zinc(II)
and the presence of N-donor heterocycle ligands results in an increased antioxidant ability of
such compounds against free radicals [17].
Leishmaniasis is among the six main diseases which have been considered as infectious by
WHO. The bite of sandflies of genus Leishmania and fly of genus Phlebotomus is responsible for
the spread of Leishmaniasis in the new and old world respectively [18]. Statistical data showed
that there are about 1.5 million new cases of leishmaniasis in 98 countries and cause 20,000–
40,000 deaths annually. The symptoms of the disease vary depending on its four different forms
which include visceral, cutaneous, mucocutaneous and anthroponotic cutaneous leishmaniasis.
Although a number of antileishmanial drugs are in use however their toxicity, adverse effects
and resistance demands for the development of new semi synthetic and synthetic drugs [19]. In
the present study zinc(II) complexes of 2-phenylbutyric acid have been evaluated against
Leishmania tropica promastigotes which is the cause of CL.
Alkaline phosphatase is a class of metallo-enzyme which is involved in a number of important
physiological functions. Although the exact structural chemistry is not fully explored yet, it is
generally accepted that it consists of two zinc ions, one magnesium and a phosphorylatable
serine [20]. ALP is involved in important cellular functions like rearrangement of the cell during
the growth of an embryo, programmed cell death and the phosphorylation of the protein.
An increased level of ALP is consistent with a number of diseases like puberty disorders, paget
disease, liver cell damage, viral infection etc. There is an ongoing interest in the development of
small drug like molecules to understand its role in health and disease [21, 22]. A number of
compounds such as levamisole, polyoxomolybdates and oxovanadate etc have been developed
3

for the inhibition study. Levamisole has physiological role other than ALP inhibition so it is
uncertain that physiological effect brought out by levamisole is the result of ALP inhibition or
other characteristic. Similarly oxovanadate compounds has associated toxicity whereas
polyoxomolybdates are effected by the water solubility which however has been resolved by the
use of polymer bounded polyoxomolybdates. [21, 23-25].
DNA is one of the important bio macro-molecules which is actively involved in the genetic
processing. Small drug like molecules can interact non-covalently with DNA through
intercalation, groove and electrostatic binding causing an alteration in its primary structure. DNA
is therefore the target of cancerous drugs and chemotherapeutic agent. Among a number of
compounds studied so far zinc(II) possess special place in DNA interaction study due to its
involvement in the hydrolytic cleavage of DNA [26, 27].
Herein, synthesis of zin(II) based homo- and heteroleptic complexes of 2-phenylbutyric acid,
their physiochemical investigation, and potential application are discussed. The results indicates
that synthesized complexes were not only active for leishmaniasis and ALP inhibition but also
showed efficient binding ability with DNA and CTAB.
2. Experimental
2.1. Chemicals and Methodology
2-phenylbutyric acid and other precursors like zinc nitrate hexahydrate, sodium bicarbonate,
Nitrogen-sulfur donor ligands like 2,2ʹ-bypiridine, 1,10-phenanthroline, neocuproine (2,9-
dimethyl-1,10-phenanthroline), thiourea and cetyl trimethylammonium bromide were acquired
from Sigma–Aldrich (USA). All the solvents were purchased from Merck (Germany) and by
following the reported method they were dried before use [28]. Gallenkamp (UK) was used to
record the melting point of the complexes. FT-IR spectrophotometer of Bruker TENSOR II
(Germany) was used to obtain the FT-IR spectra in the range of 4000–400 cm-1. Whereas a 300
1
13
MHz Bruker Advanced Digital instrument was used to record the H and C spectra of ligand
and complexes at room temperature. The values of chemical shifts and coupling constants are
1
given in ppm and Hz, respectively. In H NMR data, the chemical shift values are explained
along with the multiplicities of the signals as s = singlet, d = doublet, t = triplet and m =
4

multiplet. UV–Visible spectrophotometer (Shimadzu 1700) was used to record the absorption
spectra of the complexes.
2.2. Synthetic procedure for Zn (II) carboxylates
2.2.1. Complex 1 [Zn (L)2]
Under constant stirring at room temperature, a 10 mL solution of NaHCO3 (0.l mol, 0.084g) in
water was added dropwise to the 10 mL methanolic solution of 2-phenylbutyric acid (0.1 mol,
0.164g). After 30 min of stirring, 5 mL aqueous solution of zinc nitrate hexahydrate (0.05M,
0.074g) was added dropwise. The temperature of the system was raised to 50 °C and stirred for
5–6 h (Scheme 1). The solvent was evaporated through rotary evaporation and the final product
was purified by washing several times with water and then air-dried.
Yield = 68%; m.p = 50–56oC; Mol. Wt. = 391.78 g/mol; FT-IR bands (4000–400 cm–1): 1548
(COO- asymm.), 1416 (COO- symm.), 132 (Δν), 436 (Zn–O); 1H NMR (in DMSO): 3.28 (t,2H,
H2, 3J(1H–1H) = 7.8 Hz), 7.14–7.32 (m, 10H, H4–8), 1.51–1.65 (m, 2H, H9), 1.85–1.99 (m, 2H,
3
H9ʹ), 0.81 (t, 6H, H10, J(1H−1H) = 7.2 Hz ); 13C NMR (in DMSO): 179.1 (C1), 54.9 (C2),
142.4, 128.3, 128.4, 126.5 (C3–8), 28.0 (C9), 12.9 (C10).
2.2.2. Complex 2 [Zn (L)2(bipy)]
In order to synthesize complex 2, same procedure was adopted as for complex 1 with the
addition of 5 mL methanolic solution of 2,2-bipyridine (0.05 mol, 0.039 g) after the addition of
zinc nitrate hexahydrate solution (Scheme 1). The reaction mixture was stirred for 5–6 h at 50 °C
and the solvent was then removed through rotary evaporation. Resulting light pink product was
washed with water and dried in air. Shiny pinkish crystals suitable for XRD were obtained by
recrystallization in the mixture of methanol and n-hexane (1:1).
Yield = 86%; m.p = 116–117°C; Mol. Wt. = 547.93 g/mol; FT-IR bands (4000–400cm-1): 1556
1
(COO- asymm.), 1410 (COO- sym), 146 (Δν), 510 (Zn–N), 416 (Zn–O); H NMR (in DMSO):
3
3.23 (t,2H, H2, J(1H−1H) = 7.2 Hz ), 7.21–7.38 (m 10H, H4–8), 1.48–1.56 (m, 2H, H9), 1.81–
3
1.87 (m, 2H, H9ʹ), 0.73(t, 6H, H10, J(1H−1H) = 6.3 Hz ), 8.65 (br, 2H, Hα,αʹ), 7.60 (br, 2H,
13
Hβ,βʹ), 8.14 (br, 2H, Hγ,γʹ), 8.54 (br, 2H, Hδ,δʹ); C NMR (in DMSO): 177.7 (C1), 55.9 (C2),
5

142.5, 128.2, 128.3, 126.4 (C3–8), 27.9 (C9), 12.3 (C10), 149.3 (2C, Cα,αʹ), 122.1 (2C, Cβ,βʹ),
136.8 (2C, Cγ,γʹ), 125.6 (2C, C δ, δʹ), 158.2 (2C, Cε,εʹ).
2.2.3. Complex 3 [Zn(L)2(phen)(H2O)]
The synthetic procedure for 3 was same as for complex 2 except the addition of methanolic
solution of 1,10-phenanthroline monohydrate (0.05 mol, 0.0495 g/ 5) instead of 2,2-bipyridine.
White shiny crystals suitable for XRD were obtained by recrystallization of product in the
mixture of methanol and n-hexane (1:1).
Yield = 71%; m.p = 191–193°C; Mol. Wt. = 589.96 g/mol; FT-IR bands (4000–400cm-1): 1515
1
(COO-asymm.), 1392 (COO-sym.), 123 (Δν), 508 (Zn–N), 422 (Zn–O); H NMR (in CDCl3):
3
3.49 (t,2H, H2, J(1H−1H) = 7.5 Hz ), 7.11–7.31 (m 10H, H4–8), 1.66–1.81 (m, 2H, H9); 2.02–
3
2.16 (m, 2H, H9ʹ), 0.86 (t, 6H, H10, J(1H–1H) = 7.3 Hz ), 8.91 (br, 2H,Hα,αʹ), 7.62 (br, 2H,
13
Hβ,βʹ), 8.29 (br, 2H, Hγ,γʹ), 7.72 (br, 2H, Hε,εʹ), 3.28 (s, 1H, Hwater); C NMR (in CDCl3):
183.0 (C1), 55.0 (C2), 140.5, 128.1, 128.4, 126.1 (C3–8), 27.4 (C9), 12.5 (C10), 149.7 (2C,
Cα,αʹ), 125.0 (2C, Cβ,βʹ), 138.4 (2C, Cγ,γʹ), 128.0 (2C, Cδ,δʹ), 126.5 (2C, Cε,εʹ), 141.7 (2C, Cζ,
ζʹ).
2.2.4. Complex 4 [Zn(L)2(2,9-Me2phen)]
Complex 4 was synthesized under same reaction conditions as that for complex 2 except 2,9-
dimethyl-1,10-phenanthroline (0.05 mol, 0.052 g/mol) was added instead of 2,2ʹ-bipyridine.
Brown colored shiny crystals for XRD were obtained on recrystallization in DMSO.
Yield = 87%; m.p = 146–148°C; Mol. Wt. = 600.03 g/mol; FT-IR bands (4000–400cm-1): 1504
1
(COO-asymm.); 1363 (COO-symm.), 141 (Δν), 504 (Zn–N); 435 (Zn–O); H NMR (in CDCl3):
3
3.49 (t,1H, H2, J(1H−1H) = 7.5 Hz ), 7.10–7.33 (m 5H, H4–8), 1.66–1.77 (m, 1H, H9), 2.04–
3
3
2.13 (m, 1H, H9ʹ), 0.88 (t, 3H, H10, J(1H−1H) = 7.2 Hz), 7.51(d, 2H, Hβ,βʹ, J(1H−1H) =
3
13
8.4Hz), 8.19(d, 2H, Hγ, γʹ, J(1H−1H) = 8.4 Hz), 7.66 (s, 2H, Hε, εʹ), 2.85 (s, 1H, HMethyl); C
NMR (in CDCl3): 181.0 (C1), 55.3 (C2), 140.2, 127.9, 128.1, 126.1 (C3–8), 27.7 (C9), 12.6
(C10), 160.5 (2C, Cα,αʹ), 125.5 (2C, Cβ,βʹ), 142.2 (2C, Cγ, γʹ), 126.8 (2C, Cδ,δʹ), 126.0 (2C,
Cε,εʹ), 138.9 (2C, Cζ,ζʹ), 24.5 (2C, CMethyl).
2.2.5. Complex 5 [Zn(L)2(THU)]
6

Complex 5 was synthesized under same reaction conditions as that for complex 2 except
methanolic solution of thiourea (0.05 mol, 0.0190 g) was added instead of 2,2ʹ-bipyridine. The
light yellow product was washed several times with water, air dried and recrystallized from
chloroform/n-hexane mixture.
Yield = 62%; m.p = 123–125°C; Mol. Wt. = 497.9 g/mol; FT-IR bands (4000–400 cm-1): 1574
1
(COO- asymm.), 1382 (COO-sym.), 192 (Δν), 461 (Zn–O), 566 (Zn–S); H NMR (in DMSO):
3
3.27 (t,2H, H2, J(1H−1H) = 7.8 Hz ), 7.14–7.31 (m 10H, H4–8), 1.53–1.62 (m, 2H, H9), 1.87–
3
1.96 (m, 2H, H9ʹ), 0.81 (t, 6H, H10, J(1H−1H) = 7.2 Hz), 7.66 (s, 2H, Hα); 13C NMR (in
DMSO): 178.2 (C1), 55.2 (C2), 142.5, 128.3, 128.4, 126.4 (3–8), 28.0 (C9), 12.9 (C10), 182.2
(2C, Cα).
7

Synthesis of Homo-heteroleptic Zinc(II) complexes (1-5)
[Zn(L)2]
(1)
[Zn(L)2(bpy)]
(2)
50 °C
bpy (soln)
50 °C
CH3OH/H2O
phen·H2O (soln)
NaL
HL NaHCO3
ZnNO3·6H2O (soln)
[Zn(L)2(phen)(H2O)]
room temperature
50 °C
(3)
Me2phen 50 °C
(soln)
thiourea (soln)
50 °C
Zn(L)2(Me2phen)]
(4)
[Zn(L)2(thiourea)]
(5)
Numbering of the ligand HLand nitrogen donor ligands attached to Zinc for NMR
7
7
1
COOH
8
2



N
N
S
3



4
5
4
5
C


H2N
NH2



6
Thiourea
2,2-bipyridine
HL

H3C
CH3






N
N
N
N






·H2O











1,10-phenonthroline monohydrate
2,9-dimethyl-1,10-phenonthroline
Scheme1: Synthesis of zinc(II) carboxylates together with numbering pattern.
2.3. Crystal structure analysis
Complexes 2, 3 and 4, were characterized and structurally elucidated by selecting the suitable
crystal of sizes 0.230 0.080  0.060 mm3, 0.150  0.120  0.030 mm3, and 0.080  0.090 
0.120mm3, respectively, via compound microscope. To collect the data sets Bruker Smart Apex
8

II and D8 Venture diffractometer equipped with Molybdenum (λ= 0.71073Å) and copper Kα
radiation (λ= 1.5406Å) respectively, and a CCD detector through φ–ω scan at 293 K. Moreover,
SAINT program was used for the reduction of each data set [29]. The structures of complexes 2,
3 and 4 were solved through direct method of SHELXL–97 whereas full-matrix least-square
calculations on F2 were done to accomplished refinement [30]. During structural solution, the
riding model with bond distance approximation (C–H = 0.93 A°) was used to monitor hydrogen
atom bonded to a carbon atom and all were found to be at their calculated position, while all
other atoms were refined with anisotropic temperature parameters. Difference Fourier maps were
used to locate the position of hydrogen on hetero atom whereas thermal parameter Uiso (H) = 1.2
Ueq (N), Uiso (H) = 1.5 Ueq (O), were used to refine them. The figures of complexes i.e. Figure
1 Figure 3 and Figure 5 were plotted using ORTEP [31]. Table 1 presents the essential
experimental details for all crystals.
Table 1: Crystallographic data for complexes 2, 3 and 4.
Complex ID
Empirical formula
Formula weight
Temperature
2
3
4
C30H30N2O4Zn
547.93
C32H32N2O5Zn
589.96
C34H34N2O4Zn
600.00
293(2) K
100(2) K
Monoclinic
C2/c
100(2) K
Triclinic
Crystal system
Space group
Monoclinic
P-1
P 21/n
a = 22.1723(8) Å α= 90°.
b = 8.3066(3) Å β= 111.87°
c = 15.0836(6) Å  = 90°
a = 11.1515(3) Å
α=71.690(10)° a = = 10.493(2) Å
α= 90°
Unit cell dimensions
b = 11.9391(3) Å β=87.2530(10)° b = 10.877(2) Å
c = 12.0735(3) Å  = 67.4310(10)° c = 12.0735(3) Å
β=90.81(3)°
 = 90°
Volume
3
3
3
2578.35(17) Å
1405.06(6) Å
2
3087.8(11) Å
4
Z
4
Density
3
3
3
1.412 Mg/m
1.392 Mg/m
1.291 Mg/m
Absorption coefficient
-1
-1
-1
1.640 mm
1.573 mm
0.834 mm
F(000)
1144
616
1256
Crystal size
3
3
3
0.230x 0.080 x 0.060 mm
0.15 x 0.12 x 0.03 mm
0.08 x 0.09 x 0.12 mm
Theta range for data 5.744 to 68.374°.
collection
4.23 to 68.40°.
2.018 to 25.698°.
Index ranges
-26<=h<=26, -10<=k<=10,
-18<=l<=18
-13<=h<=13, -14<=k<=14,
-14<=l<=14
-12<=h<=12, -13<=k<=13,
-32<=l<=32
9

Independent reflections 2355 [R(int) = 0.0395]
5146 [R(int) = 0.0302]
99.7 %
5658 [R(int) = 0.0804]
99.5 %
Completeness to theta 99.7 %
= 25.242°
Refinement method
Full-matrix least-squares on
2
2
Full-matrix least-squares on F
Full-matrix least-squares on F
2
F
Data
/
restraints
/
2355 / 0 / 168
5146 / 0 / 394
5658 / 0 / 389
parameters
2
1.001
1.042
1.001
Goodness-of-fit on F
Final
[I>2sigma(I)]
R
indices R1 = 0.0659, wR2 = 0.1779
R1 = 0.0374,
wR2 = 0.0923
R1 = 0.0642, wR2 = 0.1671
R indices (all data)
R1 = 0.0673, wR2 = 0.1793
R1 = 0.0395,
wR2 = 0.0938
R1 = 0.0909, wR2 = 0.1857
Largest diff. peak and
hole
-3
-3
-3
2.693 and -0.686e.Å
0.826 and -0.421 e.Å
0.271 and -0.471 e.Å
CCDC#
1974951
1974952
1974953
2.4. Interaction with CTAB
Interaction study of the complexes with CTAB (as the model cell membrane) was performed
through conductometry and their CMC (critical micelles concentration) was calculated. For this
purpose LR 01/T cell coupled to a Microprocessor Precision Conductivity Meter LF3000
(WTW) was used. The instrument was calibrated with the aqueous solution of KCl before
measurements. The temperature of the cell was kept constant around ± 0.01 K by using a
circulating thermostated liquid. During study we kept the concentration of the complex solutions
constant whereas that of the CTAB was changed gradually. In the results, accuracy and
uncertainty was expected to be within ± 0.5 and ± 0.0005 respectively for composition and ± 0.1
K for temperature.
2.5. Complex–DNA Interaction study
To perform the interaction study of the DNA with ligand HL and complexes 1–5, solution of
salmon sperm DNA, i.e., SS–DNA was prepared in distilled water by stirring overnight. The
solution showed absorption at 259 nm and the ratio of absorption at 260 nm/280 nm (i.e.,
nucleotide to protein ratio) was found to be about 1.7 indicating its purity. Using the value of
molar absorption coefficient of SS–DNA equal to 6600 M-1 cm-1, Beer lambert’s law was
10

employed to calculate the concentration of DNA and was found to be 1.210-4 M [32, 33]. The
solutions of ligand and complexes were prepared in ethanol, and during the experiment,
concentration of complexes was kept constant whereas that of DNA was varied. The DNA
solution was added both in the sample as well as in the reference cell during the experiment just
to cancel out the effect of DNA concentration. Before taking the measurements, the incubation
period of ten minutes was provided at room temperature. The absorption of this complex–DNA
solution was then noted in between 210 nm to 800 nm.
2.6. Enzyme phosphatase inhibition study
Enzyme phosphatase is involved in catalysis of the hydrolysis process of phosphate monoester
and produced alcohol as well as an inorganic phosphate (Scheme 2). The non specific nature of
the enzyme, as well as non-biological nature of the substrate (p-nitrophenyl phosphate) are two
factors responsible for the significance of assay [34]. Working substrate was made by mixing
four parts of reagent A [Diethanolamine (pH 9.8), 2 mol/dm3 and magnesium chloride 0.5
mmol/dm3] and one part of reagent B [p-nitrophenyl phosphate (50 mmol/dm3)], 2.5 mL of
reagent solution was taken in a cuvette, to this 40 μL of human serum containing ALP enzyme
was added and then incubated for 1 minute. The product p–nitrophenolate showed absorption at
405 nm which was noted by using Shimadzu 1700 UV–Visible Spectrophotometer at 25 °C [35].
This data was used to calculate the activity of the enzyme which was found to be 264 IU/L. To
this, different concentrations (20, 40, 60, 80 and 100 μL) of each complex were added
periodically from their respective 25 mM stock solutions and incubated again for 3–4 minutes.
Then, the absorption was noted after every one minute; after 8–10 readings, their average was
used to calculate the percentage inhibition [36]. All ALP studies were carried out in triplicate.
11

H
O
O-
-O
O-
O
O-
N+
N+
N+
O
P
-O
O-
ALP
+
Hydrolysis
O-
O
O-
O
O
P
O-
-O
Inorganic Phosphate
p-nitrophenolate
p-NPP
Scheme 2: Hydrolysis of p-PNPP by alkaline phosphatase
2.7. In vitro cytotoxicity
For this study, fresh blood of human was taken and washed with saline buffer i.e., sterile
Dulbecco's PBS. The washing was done several times and after each washing, the red blood
corpuscles in the pellet were collected at the acceleration rate of 1000 G for 5 minutes whereas
the supernatant was discarded. Final red blood corpuscles were diluted 30 times (v/v) using a
sterile saline buffer. After that in the solutions of complexes with different concentrations, a
fixed amount of the suspended RBC (200 μL) was mixed. Buffered saline (PBS) was used as a
negative control whereas DMSO and 0.1 percent Triton- 100 (as triplets) as a positive control.
The solutions were incubated for one hour at 37 °C and then centrifuged for 5 minutes under the
acceleration speed of 1000 G. Supernatant contains hemoglobin which was seeded in 96 well
plate (200 μL/per well) and by using Thermo Scientific (USA) spectrophotometer absorption was
checked at 576 nm. The induced % hemolysis was calculated using the following equation:
2.8. Antileishmanial Assay
For the current study, 10% fetal bovine serum, (FBS), Pen-Strep, 30 mM HEPES buffer along
with M199 media was used to culture Leishmania tropica promastigotes. Each well of the 96-
well plate was seeded with Log. phase promastigotes (2×106/mL). Afterward, 20 μL of each
12

complex in different concentrations, i.e., 500, 250, 100, 50, 10, 1, and 0.1 μg/mL (see Tale S4 for
molar concentration) was added to the wells of 96-well plate for the entire assay. Glucantime and
DMSO were used as positive and negative control respectively; then the system was incubated
for 24 h at 24 °C. For assessment, about 13 μL of each dilution was pipetted on a Neubauer
chamber and was counted under a microscope. The IC50 was calculated by Graphpad Prism 6.
3. Results and discussion
3.1. FT-IR Spectroscopic study
To confirm the complex formation and mode of coordination FT-IR spectra of the ligand HL and
synthesized complexes were recorded and compared in the range of 4000–400 cm-1. All vibration
frequencies and ∆ν calculations that reflect the complex formation and geometry around Zn(II)
atom are listed in Table S1. The formation of Complexes (1–5) is supported by the
disappearance of broad OH band in between 3300–2500 cm-1 [10] and the appearance of new
bands in the region around 410–440 cm-1 for Zn–O bond as well as by a shift in the COO
stretching frequencies [37]. In the spectra of all complexes, the signals for COOasym appeared
around 1738–1575 cm-1 and for COOsym appeared around and 1360–1434 cm-1. The difference
in symmetric and asymmetric absorption frequencies of the carboxylate group gives an
indication of its mode of coordination with metal. A rule has been established that if the
difference ∆v [ѵ(COO)asym-ѵ(COO)sym] in complexes is lower than 250 cm-1, the complex
will adopt a bidentate bridging or chelating mode of coordination but if it is higher, the ligand
will behave as monodentate [7]. The ∆ν value for complexes 1, 2 and 5 lower than 250 cm-1, i.e.,
132, 146 and 192 cm-1 respectively is indicative that the mode of coordination of carboxylate
group is bidentate with Zn+2 [38]. On the other hand, the appearance of pair of symmetric and
asymmetric vibration of COO- for complexes 3 and 4 with respective ∆ν values of 371, 181 cm-1
and 345, 136 cm-1 is an indication that monodentate and bidentate mode of coordination co-
exists. This kind of behavior is also supported by the literature where di and tetranuclear
complexes of zinc(II) with carboxylates and phenolate adopt monodentate as well as bidentate
mode of coordination [39]. The vibrational bands in the region of 500–515 cm-1 attributed to Zn–
N bond as well as C–H (out of plane) vibrational bands for the heterocycle aromatic rings at 774
cm-1 (bipy in 2), 726 cm-1 (phen in 3) and 734 cm-1 (Me2phen in 4) confirmed the attachment of
2,2ʹ-bpy, 1,10-phen, 2,9-Me2phen in complexes 2–4 [40, 41]. Moreover, the coordination of
13

thiourea to the Zn(II) center in complex 5 is evidenced from the presence of metal sulphur bond
at 566 cm-1 as well as by the presence of pair of bands due to νas(NH2) and νsy(NH2) in the range
of 3330–3196 cm-1 [42, 43].
3.2. NMR (1H and 13C) study
3.2.1. 1H NMR spectroscopic study
1
Further confirmation of the complexes is evident from the H NMR spectra data given in Table
S2. The absence of resonance signal at 12.3 ppm originating from the –OH group, as well as an
expected shift in the resonance of ligand’s proton, confirmed the synthesis of complexes 1–5
[44]. The attachment of bipyridine and phenanthroline in the complexes 2 and 3 was confirmed
by the presence of four signals in the region of 7.60–8.91 ppm which were attributed to the
equivalent protons of the heterocyclic moiety. Moreover, all were broadened and shifted
compared to respective free ligands due to probable intra or intermolecular interaction and
chemical exchange process, therefore, their accurate interpretation is difficult [45, 46]. The
attachment of water is evident from the singlet appearing at 3.28 ppm for the respective proton in
the complex 3 which may be contributed from the hydrated precursor i.e., monohydrated
penanthroline or solvent employed during the course of reaction i.e., methanol. This observation
is further supported by singal crystal x-ray analysis. The attachment of 2,9-dimethyl-1,10-
phenanthroline was apparent by the appearance of three signals attributed to two β, βʹ proton at
3
7.51 ppm as doublet with J[1H─1H] = 8.4, two γ, γʹ proton as doublet at 8.19 ppm with
3J[1H─1H] = 8.4 and two ε, εʹ proton as singlet at 7.66 ppm. The proton of methyl group at 2 and
9 position of 1,10-phenanthroline appeared as singlet at 2.85 ppm [47]. The equivalent proton of
thiourea resonate around 7.66 ppm and appeared as a broad signal in the spectrum of complex 5.
Srinivasan and co-workers during their study on thiourea zinc sulphate also reported the
appearance of broad signal for equivalent proton of thiourea [48].
3.2.2. 13C NMR spectroscopic study
A 13C NMR study of complexes reveals that carboxyl carbon (C1) show a downfield shift of 2–8
ppm as compared to that of HL (see Table S3). This is in accordance with the previousy reported
zinc(II) complexes of 4-(2-methoxy-5-nitrophenylamino)-4-oxobutanoic acid and 4-(2-nitro-4-
methoxyphenylamino)-4-oxobutanoic acid. The shift is a result of decrease in electron density
14

when ligand gets coordinated to the electropositive zinc(II) center [49]. Furthermore, the
presence of additional five signals (121.2 ppm–158.2 ppm) in the spectrum of complex 2 and six
signals (125.0 ppm–160.5 ppm) in the spectra of complex 3 and 4 are attributed to the equivalent
carbon of N-donor heterocycle rings to zinc(II) center [36]. The carbon atom of the substituted
methyl group of the 2,9-dimethyl-1,10-phenanthroline resonate at about 24.5 ppm in the
spectrum of complex 4. In complex 5, the signal appearing at 182.0 ppm attributed to C=S is an
indication of thiourea coordination with zinc(II) [50].
3.3. Crystal structure description
3.3.1. Crystallographic analysis of complex (2)
Single crystal x-ray diffraction analysis of complex 2 revealed that it has a plane of symmetry
incorporated along Zn1–N1–C15 and Zn1–O2–C8–O1 bonds crystallizing in monoclinic system
with space group C2/c. The unit cell of crystal lattice contained half occupancy of zinc(II) center
as an asymmetric unit and a hexa-coordinate environment. As shown in Figure 1 (ORTEP view),
the bidentate ligands i.e. 2-phenylbutanoate and 2,2'-bipyridine coordinated to Zn(II) in a way to
adopt distorted octahedral geometry with bond angles (Table 5) of 78.52 (17)º (N1–Zn1–N1A),
105.74(12)º (N1–Zn1–O2), 103.00(11)º (N1A–Zn1–O2), 142.60(18)º (O2–Zn1–O2A),
158.30(12)º (N1–Zn1–O1) and 95.3(13)º (O2A–Zn1–O1). The distortion of the octahedral
geometry is clear from the substended angels around zinc(II) center (in the range of 60.45(13) to
105.76 (12)). Such distortion in geometry was also observed in the reported crystal of (2,2′-
Bi-pyridyl-κ2N,N′)-bis-(salicyl-ato-κ2O,O′)-zinc(II). The deviancy from perfect octahedral
geometry is the result of steric strains imposed by the bulky nature of attached phenyl and
bipyridine ligands [51]. In addition, the atoms O1, O1A, N1, N1A are bonded at the basal sites
with bond length ranging from 2.090(3)–2.224(3) Å while atoms O2 and O2A at apical sites.
These bond lengths for Zn–O and Zn–N are in accordance to that of reported zinc(II)
carboxylates with bipyridine as hetero-ligand [52]. Various supramolecular interactions that led
the self assembling of complexes in three dimension pattern (Figure 2) includes C12–H12⋯O2
and C14–H14⋯O1 interactions, with donor–acceptor bond distance 3.464(6) Å, and 3.099(5) Å,
respectively (Table 2).
15

Figure 1. ORTEP diagram of complex 2 with 40% thermal ellipsoid probability.
Figure 2. Packing diagram of complex 2 with C14–H14…O3 and C12–H12…O2 inter–
molecular interactions.
Table 2: Data representing the hydrogen bond for complex 2 [Zn (L)2(bipy)] (Å and °).
d(D...A)
<(DHA)
D–H...A
d(D–H)
d(H…A)
C14–H14…O1
0.95
2.40
3.099(5)
130.3
16

C12–H12…O2
0.95
2.56
3.464(6)
159.0
3.3.2. Crystallographic analysis of complex 3
The structural parameters of complex 3 examined by x-ray crystallography revealed that
complex 3 is grown in the triclinic system with space group P-1. In the asymmetric unit, central
Zn(II) atom coordinated with mono-dentate (2-phenylbutanoate, water), and bi-dentate (2-
phenylbutanoate, 1,10-phenanthroline) ligands, in a way to adopt distorted octahedral geometry
as depicted in ORTEP Figure 3. The chelation pattern form four and five-membered chelates
around central Zn metal with bond angles of 58.13 (7)º (O3–Zn1–O4) and 77.10(7)º (N3–Zn1–
N1). The water molecule occupies the sixth coordination site of complex 3, placed at the distance
of 2.047 Å (Zn1–O5). Notably, a comprehensive structural study revealed the positional disorder
in C17, C18 of bidentate 2-phenylbutanoate ligand with bond distances of 1.32(7) Å (C17–C18)
and 1.156(7) Å (C17'–C18'). Furthermore, the peripheral coordination site around the central
zinc(II) atom was occupied by ligands at a coordinated distance of 2.082(16) Å – 2.388(2) Å.
These findings are in accordance with the previousy reported zinc(II) complexes of 5-hydroxy-
isophthalic acid and 1,10-phenanthroline [53]. The inter-molecular hydrogen bandings engaged
the crystal lattice in unit cell packing, exhibiting the 3D network included O5–H5…O4, C1–
H1…O2, C12–H12…O3, and C28–H28…O5 with donor-acceptor distances 2.671(3)Å,
3.417(4)Å, 3.363(4)Å, and 3.201(3)Å, respectively (Figure 4, Table 3).
17

Figure 3. ORTEP diagram showing molecular structure of complex 3 with 40% thermal
ellipsoid probability.
Figure 4. Packing diagram of complex 3 with O5–H5…O4, C1–H1…O2, C12–H12…O3, C28–
H28…O5 and inter-molecular interactions.
Table 3: Data representing hydrogen bond for complex 3 [Zn (L)2(phen)(H2O)] (Å and °).
d(D...A)
<(DHA)
D–H...A
d(D–H)
0.79
d(H…A)
1.89
O5–H5…O4
C1–H1…O2
2.671(3)
3.417(3)
3.363(3)
3.202(3)
175
150
148
144
0.95
2.56
C12–H12…O3 0.95
C28–H28…O5 0.95
2.52
2.38
3.3.2. Crystallographic description of complex 4
Single crystal x-ray diffraction analysis of complex 4 revealed that it crystallizes in a monoclinic
crystal system with P21/n space group. The data show that heteroleptic ligand, i.e,. 2,9-dimethyl-
1,10-phenanthroline, one of the two 2-phenylbutanoate ligands behave as bidentate whereas
others as monodentate to the zinc(II) center. ORTEP view (Figure 5) suggests a distorted square
planer pyramidal geometry. This low coordination number as compared to complex 3 is the
result of steric demand of bulky ligand [54]. The chelators 2-phenylbutanoate, 2,9-dimethyl-
18

1,10-penanthroline at basal sites around zinc(II) center adopted pentagonal geometry with
coordination bond distances of N1–Zn1 [2.060(4) Å], Zn1–N2 [2.103(3) Å], O1–Zn1 [2.201(5)
Å], Zn1–O2 [2.161(4) Å] and bond angles of 81.58 (14)º(N1–Zn1–N2), and 58.25(17)º (O1–
Zn1–O2). The mean value of Zn–N bond length i.e. 2.081 Å is comparable to reported
complexes like Zn(II) indole-2-carboxylate with 2,9-dimethyl-1,10-phenanthroline but are
shorter than the complex 3 [55]. The shorter Zn–N bond length and a large N–Zn–N bond angle
as compared to complex 3 (data given in Table 6 ad 7) are the result of steric strains imposed by
the substituted methyl group of 2,9-dimethyl-1,10-penanthroline ligand. These findings are in
accordance with the reported 2,9-dimethyl-1,10-penanthroline complexes with zinc as well as
other metals like copper and manganese [56-58] The O4 atom of monodentate 2-phenylbutanoate
ligand (O3/O4/C21–C28/C33/C34) lies at the distance of 2.803 Å, i.e. not equal to the sum of
van der Waals radii of involved atom. Thus, Zn1–O4 bond has not observed in this case. The
Zn–O bond distance for the attached carboxylate group through monodentate and bidentate
coordination mode is in agreement with that of reported (Acetato-O,O')(aeetato-O)(2,9-dimethyl-
1,10-phenanthroline-N,N')zinc(II) compound [59]. A positional disorder in C14, C31 atoms of
bidentate ligand was observed with the bond distances of 1.229(15) Å (C14A–C31A) and
1.238(16) Å (C14B–C31B). Each lattice point in unit cell packing connected through the C3–
H3…O1, C8–H8…O4, C28–H28…O4 and C30–H30…O4 inter- and intra-molecular hydrogen
bonds with donor-acceptor distances of 3.375(7)Å, 3.176(7)Å, 3.439(6)Å and 3.152(7)Å
respectively, led to the formation of the 3D pattern in crystal lattice (Table 4, Figure 6).
19

Figure 5. ORTEP diagram showing molecular structure of complex 4 with 30% thermal
ellipsoid probability.
Figure 6. Packing diagram of complex 4 with C3–H3…O1, C8–H8…O4, C28–H28…O4 and
C30–H30…O2 inter-molecular interactions.
Table 4: Data representing the hydrogen bond in complex 4 [Zn (L)2(2,9-Me2phen)] (Å and °).
D–H...A
d(D–H)
d(H…A)
d(D...A)
<(DHA)
20

C3–H3…O1
C8–H8…O4
C28–H28…O4
C30–H30…O2
0.93
0.93
0.93
0.96
2.58
2.32
2.52
2.40
3.375(7)
3.176(7)
3.439(6)
3.152(7)
143.1
152.6
170.9
135.3
Table 5: Selected bond length/Å and bond angles/º for complex 2
Bond length/Å
Zn(1)–N(1)
Zn(1)–O(2)
Zn(1)–O(1)
Zn(1)–C(8)
O(1)–C(8)
2.090(3)
2.091(3)
2.224(3)
2.488(4)
1.245(6)
Zn(1)–N(1)#1
Zn(1)–O(2)#1
Zn(1)–O(1)#1
Zn(1)–C(8)#1
O(2)–C(8)
2.090(3)
2.091(3)
2.224(3)
2.488(4)
1.263(6)
Bond angles/º
N(1)–Zn(1)–N(1)#1
N(1)–Zn(1)–O(2)
78.52(17)
105.76(12)
103.00(11)
103.00(11)
O(2)–Zn(1)–O(1)#1
O(2)#1–Zn(1)–O(1)#1
N(1)#1–Zn(1)–O(1)
N(1)–Zn(1)–O(1)
95.35(13)
60.45(13)
92.10(12)
158.31(12)
60.45(13)
95.35(13)
102.83(17)
N(1)#1–Zn(1)–O(2)
N(1)–Zn(1)–O(2)#1
N(1)#1–Zn(1)–O(2)#1 105.76(12)
O(2)#1–Zn(1)–O(1)#1
O(1)#1–Zn(1)–O(2)
O(2)#1–Zn(1)–O(2)
O(2)–Zn(1)–O(2)#1
N(1)–Zn(1)–O(1)#1
142.60(18)
158.30(12)
Table 6: Selected bond length/Å and bond angles/º for complex 3.
Bond length/Å
21

Zn(1)–O(2)
Zn(1)–O(5)
Zn(1)–O(3)
Zn(1)–O(4)
O(3)–C(20)
2.0468(14)
2.067(17)
2.0818(16)
2.3882(18)
1.264(3)
O(4)–C(20)
O(1)–C(10)
O(2)–C(10)
Zn(1)–N(3)
Zn(1)–N(1)
1.248(3)
1.240(3)
1.256(3)
2.1224(17)
2.1926(17)
Bond angles/º
O(2)–Zn(1)–O(5)
O(2)–Zn(1)–O(3)
O(5)–Zn(1)–O(3)
O(2)–Zn(1)–N(3)
O(5)–Zn(1)–N(3)
O(3)–Zn(1)–N(3)
O(2)–Zn(1)–N(1)
O(5)–Zn(1)–N(1)
90.20(6)
94.30(6)
147.10(6)
90.32(6)
106.54(6)
106.00(6)
167.02(6)
90.35 (7)
O(3)–Zn(1)–N(1)
N(3)–Zn(1)–N(1)
O(2)–Zn(1)–O(4)
O(5)–Zn(1)–O(4)
O(3)–Zn(1)–O(4)
N(3)–Zn(1)–O(4)
N(1)–Zn(1)–O(4)
92.27(6)
77.10(6)
102.71(6)
89.08(7)
58.13(7)
159.73(7)
90.2(6)
Table 7: Selected bond length/Å and bond angles/º for complex 4.
Bond length/Å
Zn(1)–O(3)
Zn(1)–N(2)
Zn(1)–O(2)
O(2)–C(13)
1.925(3)
2.103(3)
2.161(4)
1.222(7)
Zn(1)–N(1)
Zn(1)–O(1)
O(1)–C(13)
O(4)–C(21)
2.060(3)
2.202(5)
1.222(7)
1.196(5)
22

O(3)–C(21)
N(1)–C(11)
1.246(5)
1.375(5)
N(1)–C(10)
N(2)–C(12)
1.345(5)
1.335(5)
Bond angles/º
O(3)–Zn(1)–N(1)
O(3)–Zn(1)–N(2)
N(1)–Zn(1)–N(2)
O(3)–Zn(1)–O(2)
N(1)–Zn(1)–O(2)
N(2)–Zn(1)–O(2)
O(3)–Zn(1)–O(1)
119.66(15)
120.85(13)
81.59(14)
99.24(17)
93.33(16)
136.54(16)
108.34(16)
N(1)–Zn(1)–O(1)
N(2)–Zn(1)–O(1)
O(2)–Zn(1)–O(1)
O(3)–Zn(1)–C(13)
N(1)–Zn(1)–C(13)
N(2)–Zn(1)–C(13)
O(2)–Zn(1)–C(13)
127.79(15)
91.27(15)
58.25(17)
105.21(15)
113.03(17)
115.70(17)
29.13(18)
3.4. Complex–DNA interaction study through absorption spectroscopy
The interaction ability of HL and the complexes 1–5 with DNA was studied using UV-Visible
spectroscopy. The representative absorption spectra of complexes 2 and 3 with and without
addition of DNA are presented in Figures 7 and 8 respectively. In the absence of DNA,
absorption values between 230 nm to 320 nm were attributed to the intra-ligand π–π* transitions.
[60-62]. The spectra recorded after the successive addition of DNA generally reveals the mode
of interaction of compounds with DNA [58, 59]. As reported in our previous paper, ligand (HL)
shows a red shift coupled with hypchromic effect indicating an inercalative mode of interaction
[7]. Herein, complexes 2 and 3 shows a decrease in absorption intensity (hypochromic effect)
coupled with 2–4 nm decrease in wavelength (blue shift), supporting that the binding of
complexes with DNA occurs through surface along major or minor groove by the involvement of
hydrogen bonding as well as van der Waals forces [26, 63]. Such spectral features and mode of
binding are also supported by the work of Zhang and co-workers [64]. The presence of isosbetic
point in the spectrum of complex 3 is indicative of absence of any foreign specie and that an
equilibrium is established between free and DNA binded complex. The binding affinity of the
complexes was calculated quantitavely applying Benesi-Hildebrand equation:
23

A°
 G

G

1

+
x

-
G
K[DNA]

–
G
G
A A°
G
In above equation, Ao is the absorption of free complexes, A is complex–DNA absorbance, εG is
the absorption coefficient for free complexes and εH–G are the absorption coefficients of DNA
bonded complexes. From the plot of A°/A – A° vs. 1/ [DNA] the ratio of intercept-to-slope was
used to calculate binding constant K (M-1) [65]. The values of K for ligand and complexes are
given in Table 8. Importantly, the lower values of binding constant than that of classical metallo-
intercallator also support that the interaction occur through surface binding. However, these
values are higher than the previously reported zinc(II) complexes of ciprofloxacin and 2-(4-2-
(hydroxyimino)acetyl)phenoxy) acetic [66, 67]. The spontaneity of interaction was decided by
the Gibbs’s free energy which was calculated through the following equation:
ΔG = -RT lnK
In the above equation R represents general gas constant (8.314 J M−1 K−1), T represents
temperature (298 K). The spontaneity of the process is obvious from large negative values of
ΔG.
Table 8: Binding constant (K), Gibb’s free energy (ΔG) and λ max for ligand HL complexes 1–5.
Compound
HL
ΔG(kj/mol)
K (M-1)
λmax (nm)
1.09×104
2.26 × 104
4.07×104
4.24×104
3.54×104
3.28×104
-23.04
259
1
2
-24.83
-26.29
257
285
3
4
5
-26.40
-25.95
-25.76
266
261
240
24

Figure 7. Effect of DNA addition (15–225 μM) on the absorption spectra of complex 2. Whereas
K and G were calculated from graph between A0/A –A0 vs 1/ [DNA] (μM)−1.
Figure 8. Effect of DNA addition (15–195 μM) on the UV–Vis absorption spectra of complex 3,
where K and G were calculated from graph between A0/A –A0 vs 1/ [DNA] (μM)−1.
3.5. Interaction study with surfactant (CTAB)
25

To reach the site of action, a drug has to pass through cell membrane. Nature of the cell
membrane and the micelles formed by CTAB are similar, as both have a polar outer surface and
an inner hydrophobic core. This results in a net charge on the interacting molecules to create a
solubilization environment. Therefore, by studying compound behavior in aqueous-miceller
system, we can picture the compound’s movement through cell membrane. Moreover, micelle-
compound interaction not only reduces the side effect of a compound but also enhance the
activity by increasing its bioavailability [68]. In order to study the bioavailability of HL and
synthesized complexes, their interaction behavior with CTAB was determined through
conductometry. The experiments were performed at a fixed concentration of complexes (1 mM
in methanol) with varying concentrations of CTAB solution, i.e., 0–7 mM in water. The CTAB+
and Br─ ions generated in solution move easily at their low concentration (small size and less
interaction) causing a decrease in the surface tension and increase in the conductance. At the
inflection point or critical micelle concentration (CMC), which is different for each compound,
the monomers of the CTAB assemble and hence conductance decreases. Now, these are just the
Br─ ions moving from one micelle’s surface to others and causes conductance. Critical micelle
concentration of the free surfactant solution is lower as compared to that of the complex–CTAB
solution (see Table 9) because of interaction with compounds under study. This CTAB–Complex
interaction reduces the charge density and self-building of CTAB+ monomer causing an increase
in the resulting entropy of mixing ΔGm. This high value of CMC is an indicative of high
bioavailability of the synthesized complexes [69, 70]. Change in Gibb’s free energy (Gm)
involved for micelle formation is determined through equation given below:
Δ Gm = 2.303(1 + β) RT logXcmc
In the equation, Xcmc represents the CMC value in term of mole fraction. For aqueous solution it
is calculated as follows:
Xcmc = CMC/55.55
β represents the degree of counter ion binding and is calculated as,
β = 1– α
Where α is the extent of the detachment of ions and is calculated as,
26

α = S2/S1
S1 and S2 are slopes of the graph plotted between concentration vs conductivity before and after
CMC [70]. To calculate CMC values, the graphs were ploted between varying concentration of
CTAB vs specific conductance and such graphs for representative complexes 1 and 3 are
presented in Figure 9. The higher CMC values for all complexes compared to that of HL and
CTAB alone, is a clear indication of their higer bioavailability [7]. The Table 9 represents values
for CMC, InXcmc and ΔGm for HL and complexes 1–5. Importantly, the negative values of ΔGm
is an evidence of spontaneous CTAB–compound interaction process.
Figure 9. Graphs between CTAB–complex vs specific conductance presenting the inflection
point (CMC values) for represented complexes 1 and 3.
Table 9: Data about the different parameters of CTAB interaction study.
Complex
ΔG(KJ/mol)
-33.96
CMC [M]
0.0018
0.0050
0.0031
0.0035
0.0025
0.0037
-InXcmc
10.31
9.31
HL
1
-37.09
2
-36.52
9.90
3
-36.41
9.72
4
-37.20
9.82
5
-38.60
9.62
3.6. Enzyme inhibition activities
27

ALP Inhibition potential of the synthesized complexes was investigated and results depicted a
concentration dependent inhibitory effect. Alkaline phosphatase helps in the hydrolysis of a
number of groups like oxyphosphate monoester, O– and S–phoslhorothioates, phosphoramidates,
thiophosphate, and phosphate. The general kinetic mechanism suggests that during hydrolysis,
phosphoseryl intermediate is formed by the coordination of serine 102 with the zinc(II). Where
second zinc activates the nucleophilic attack of phosphate acceptor (water or alcohol) at a pH
around 8 [71]. This intermediate adopts a five coordinated geometry which is either square
pyramidal or trigonal bipyrimad . It was thought that the compounds like vanadium which
mimics this transition geometry can inhibit the function of enzyme [72]. Later on investigation
suggests that six and even seven coordinated complexes are also potent inhibitors, so the
conclusion was made that the inhibition is the result of combined structural and electronic factors
[73]. In the present study, an indirect relationship between the structure of the complex and the
inhibition is observed (Figure 10), i.e., as the structure becomes complicated activity decreases.
The order being 1 > 5 > 2 > 4 > 3. The complex 1 was found to be an effective inhibitor due to a
simple, less sterically hindered structure and binds easily with the enzyme’s active site. There is
a little decrease in activity from complex 1 to 5, 2 and 4 due to the presence of additional ligands
attached to the zinc center which cause hindrance in an effective binding of the complexes with
the enzyme. The complex 3 is found to be inactive due to the presence of two additional groups,
i.e., 1,10–phenonthroline and –OH, occupying all coordination sites, thus, making it difficult to
interact with the enzyme. Structure activity relationship has been observed for the
peroxidovandioum complexes, where pV(phen) shows less inhibition as compared to pV(bipy)
on the basis of spacial hindrance [74]. Similarly, another finding suggests that pV(bipy) shows
low inhibition activity as compared to the vanadate on the basis of spacial hindrance [75]. It is
clear from the Figure 10 that inhibition increases with an increase in concentration of complexes
1-5 from 195.3 μL to 946.96 μL. It may result from the displacement of the ion from the active
site of enzyme by the inhibitors at high concentration. The similar relationship between
inhibition and concentration of compounds has also been observed for the zinc(II) complexes of
coumarins-3-carboxylates [45] and o-methyl carbonodithioat [73]. S.Hussain and others also
observed same behavour for the heterobimetallic complexes of Sn(IV) and Pd(II) [76].
28

100
80
60
40
20
0
Complex 1
Complex 2
Complex 3
Complex 4
Complex 5
0
200
400
600
800
1000
Concentration of Inhibitor (μM)
Figure 10. Graph representing the inhibition of ALP’s under the influence of varying
concentrations of complexes.
3.7. Antileishmanial activity
Antileishmanial in vitro efficacy of the ligand and zinc (II) complexes at various concentrations
was examined using Leishmania tropica promastigotes. Promastigotes viability was checked in
both treated and controlled samples after 24 h of incubation. Interestingly, a significant decrease
in viability has been assessed in treated samples (Figure 11) as compared to controlled ones. The
IC50 values were 1.98±0.015, 0.75±0.021, 1.63±0.022, 1.12±0.03, 0.41±0.01 and 2.82±0.016
μg/mL, for 1, 2, 3, 4, 5 and HL respectively (see Table S5 fo values in micromole concetrations).
The results were promising and comparative to the standard drug Glucantime (5.4 μg/mL)
indicating that our synthesized complexes show marked activity against leishmaniasis.
29