Exploiting the potential of aryl acetamide derived Zn(II) complexes in medicinal chemistry: Synthesis, structural analysis, assessment of biological profile and molecular docking studies

Article abstract of DOI:10.1039/c5nj03531g

In the medical arena, advancements in the rational design of metal-based therapeutic agents showcase increasingly significant research efforts towards the development of new compounds with fewer toxic side effects. In this context, our present manuscript explicitly encapsulates the design and synthesis of Zn(ii) complexes derived from different aryl acetamides, as potential frontline enzyme inhibitors as well as antileishmanial and anticancer agents. The structures of the synthesized metal complexes were established on the basis of spectro-analytical data and, in the case of 4c, by single crystal X-ray diffraction analysis. The X-ray structure of the Zn(ii) complex, dichlorido-bis[N-(4-methoxyphenyl)acetamide-O]-zinc(ii), 4c, showed that the zinc atom and the chloride ligands lie on a mirror plane, with the acetamide ligands in general positions. The coordination geometry of the zinc atom was tetrahedral, with the N-(4-methoxyphenyl)acetamide ligands bound to zinc via the acetamide oxygen atoms. The designed coordination complexes were analysed for their enzyme inhibition potential, and anticancer and antileishmanial efficacy. Detailed kinetic studies for complex 4b, the most active carbonic anhydrase and alkaline phosphatase inhibitor, indicated competitive and uncompetitive modes of inhibition against carbonic anhydrase and tissue non-specific alkaline phosphatase, respectively. The bioactivity results and molecular docking analysis revealed that the synthesized coordination complexes (4a-c) have great potential as enzyme inhibitors, in addition to being anticancer and anti-parasitic drug candidates.

Full text of DOI:10.1039/c5nj03531g

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Exploiting the potential of aryl acetamide derived Zn(II) complexes in medicinal
chemistry: Synthesis, structural analysis, assessment of biological profile and molecular
docking studies
Kishwar Sultanaa†, Sumera Zaibb†, Najm ul Hassan Khanc, Imtiaz Khand, Khadija Shahide, Jim
Simpsonf and Jamshed Iqbalb*
aFaculty of Pharmacy, The University of Lahore, Lahore, Pakistan
bCentre for Advanced Drug Research, COMSATS Institute of Information Technology,
Abbottabadꢀ22060, Pakistan
cIslam College of Pharmacy, Pasrur Road, Sialkot, Pakistan
dDepartment of Chemistry, QuaidꢀiꢀAzam University, Islamabadꢀ45320, Pakistan
eRiphah Institute of Pharmaceutical Sciences, Riphah International University Islamabadꢀ44000
Pakistan
fDepartment of Chemistry, University of Otago, PO Box 56, Dunedin 9054, New Zealand
†These authors contributed equally to this work.
To whom Correspondence should address:
Prof. Dr. Jamshed Iqbal
Centre for Advanced Drug Research, COMSATS Institute of Information Technology,
Abbottabadꢀ22060, Pakistan
Tel.: +92ꢀ992ꢀ383591/96; Fax: +92ꢀ992ꢀ383441. Eꢀmail: drjamshed@ciit.net.pk

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Abstract
In the medical arena, advancements in the rational design of metalꢀbased therapeutic agents
showcase increasingly significant research attempts towards the development of new compounds
with fewer toxic side effects. In this context, our present manuscript explicitly encapsulates the
design and synthesis of Zn(II) complexes derived from different aryl acetamides, as potential
frontline enzyme inhibitors as well as antileishmanial and anticancer agents. The structures of
synthesized metal complexes were established on the basis of spectroꢀanalytical data and, in case
of 4c, by single crystal Xꢀray diffraction analysis. The Xꢀray structure of Zn(II) complex,
dichloridoꢀbis[Nꢀ(4ꢀmethoxyphenyl)acetamideꢀO]ꢀzinc(II), 4c, showed the zinc atom and the
chlorido ligands to lie on a mirror plane with the acetamide ligands in general positions. The
coordination geometry of the zinc atom was tetrahedral with the Nꢀ(4ꢀmethoxyphenyl)acetamide
ligands bound to zinc via the acetamide oxygens. The designed coordination complexes were
analysed for their enzyme inhibition potential, anticancer and antileishmanial efficacy. Detailed
kinetics studies for complex 4b, the most active carbonic anhydrase and alkaline phosphatase
inhibitor, indicated a competitive and uncompetitive mode of inhibition against carbonic
anhydrase and tissue nonꢀspecific alkaline phosphatase, respectively. The bioactivity results and
molecular docking analysis revealed that the synthesized coordination complexes (4aꢀc) hold
great potential to be developed as promising enzyme inhibitors in addition to the anticancer and
antiꢀparasitic drug candidates.
Introduction
Since the discovery of cisplatin and its derivatives as potential anticancer agents, coordination
complexes gained much attention of the medicinal scientific community and set a platform for
the development of metalꢀbased compounds/drugs for the treatment of a diverse range of

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disorders.1 In this quest, several coordination compounds have been synthesized involving
different metal center like copper, zinc, cobalt, nickel, gold and antimony.2,3 In addition, a variety
of mononuclear metal complexes with different ligands have been shown to be effective for
cancer therapy both in vitro and in vivo.4‒8 Transition metal complexes with platinum, gold,
palladium and ruthenium metal centers are under various stages of clinical trials.9,10 Interest in
zinc complexes stems from their wide and increasing utility in medicinal chemistry. In addition,
zinc is considered as the most abundant trace intracellular element with its substantial occurrence
in the nucleus.11 Furthermore, Zn(II) ions are present in zinc finger proteins that recognize and
bind to DNA.12,13 Several Zn(II) and other metal complexes with a variety of ligands have been
reported in the literature with different coordination modes displaying a unique spectrum of
biological functions including antitumor, antioxidant, xanthine oxidase, anticonvulsant,
antimicrobial and antileishmanicidal activities.14 Also, various metal complexes with different
coordination ligands have emerged as inhibitors of carbonic anhydrases,15 cholinesterase,16 and
alkaline phosphatases.17 However, there is still an unmet need for the development of
coordination complexes with unique and multifaceted biological profile.
Hence, a combination of all components discussed above appeared to be a very promising aim
with respect to the development of alternative treatments and ailments for different biological
disorders. Based on these literature findings, and our interest in the development of coordination
complexes as potential biological drug candidates,18 we aimed to explore a library of
unprecedented zinc complexes based on the aryl acetamide ligands which were screened for the
inhibition of carbonic anhydrase and alkaline phosphatase enzymes. The designed compounds
were also found as promising anticancer and antileishmanial agents.
Results and discussion

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Synthesis of ligands and their Zn(II) complexes
The synthetic pathway leading to the preparation of Zn(II) complexes is depicted in Scheme 1.
The key ligands (aryl acetamides, 3aꢀc) were synthesized using the literature reported procedure
by the condensation reaction of various anilines (1aꢀc) and acetic anhydride (2) under neat
reaction conditions without using any solvent or catalyst.19 All the synthesized aryl acetamides
were obtained in good yields. The preparation of Zn(II) complexes was achieved by the reaction
of corresponding aryl acetamides with zinc(II) chloride.20 The newly synthesized complexes
were afforded in good yields.
Spectroscopic characterization
The synthesized ligands (3aꢀc) and their corresponding Zn(II) complexes (4aꢀc) were
1
13
characterized by readily available spectroscopic techniques including FTIR, H and C NMR
spectroscopy and mass spectrometry and, in case of 4c, by single crystal Xꢀray diffraction
analysis. The purity of the compounds was ascertained by elemental analysis.
In case of aryl acetamides, the key functional groups including the carbonyl and amine were
appeared in the range of 1661‒1645 cm‒1 and 3291‒3240 cm‒1, respectively. The other related
stretching frequencies were also observed in the appropriate region of infraꢀred spectra. 1H NMR
spectroscopic investigations also revealed the formation of ligands (3aꢀc) where the dominant
signals for N‒H functional group appeared in the range of 7.96‒7.39 ppm in addition to the
acetyl group observed in the range of 2.23‒2.16 ppm. The 13C NMR data has also confirmed the
formation of ligands 3aꢀc. All the characterization data were consistent with those observed
previously in the literature.21
The synthesized Zn(II) complexes were also characterized by spectroscopic data. The type of
binding of the ligands to the metal ion was elucidated by comparing the IR spectra of the

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complexes with the spectra of free ligands. In this case, the bands appeared due to carbonyl
vibrations were observed in the range of 1649‒1636 cm‒1. The shifting of the acetyl carbonyl to
lower stretching frequencies indicated the involvement of these carbonyl groups in the
coordination of the metal ions. New far–IR absorption bands in the range of 429–417 cm‒1 in
Zn(II) complexes indicated the complexation of the metal to ligands through acetyl carbonyl
moiety.18b,22
1H and 13C NMR spectroscopy also aided in the characterization of the newly synthesized Zn(II)
1
complexes. In H NMR spectra, the most significant functional groups including N‒H moiety
(9.67‒7.98 ppm), acetyl protons (2.16‒1.97 ppm), and the methoxy substituents (3.87‒3.66 ppm)
present on the aryl ring appeared in their respective regions with slight change in chemical shift
values. In case of free ligands, these functional groups were observed at 7.76‒7.04, 2.20‒2.16,
13
and 3.88‒3.79 ppm, respectively.21 In C NMR spectra, the carbonyl group resonated in the
range of 167.3‒164.8 ppm, whereas their corresponding ligands appeared at 168.5‒168.3 ppm.21
The elemental composition of the ligand and Zn(II) complexes were unambiguously determined
and reported which suggested the proposed composition for all compounds. The representative
Zn(II) complex (4c) has been subjected for its mass spectral studies. The electronꢀionization
mass spectrum of Zn(II) complex is depicted in Figure S1, shows a molecular ion peak at m/z
466 (28.2%) which corresponds to its molecular weight. The fragment ion observed at m/z 429
(50%) was due to the expulsion of chlorine ligand from the molecular ion which further
fragmented to ion observed at m/z 264 (68.3%) by the loss of an acetamide ligand. The fragment
at m/z 166 (100%) was due to the protonated acetamide ligand which was also the base peak.
This fragmentation pattern is consistent with its structure (Figure S2).

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R
H
N
NH2
Me
R
O
H
N
Me
1a c
ZnCl2
heat
R
Cl
Zn
Cl
+
O
EtOH, reflux, 2 h
O
O
3a c
O
Me
O
Me
2
Me
HN
R
4a c
R = H, 2-OMe, 4-OMe
Scheme 1. Synthesis of Zn(II) complexes.
Xꢀray crystallography
Structural description
In parallel, for a full structural revelation, the Xꢀray diffraction measurements were carried out
for the synthesized complex 4c. The crystal and instrumental parameters used in the unit cell
determination, the data collection, and structure refinement parameters are presented in the Table
1 with selected bond lengths and angles and hydrogenꢀbond geometry shown in Table 2 and 3. A
thermal ellipsoid plot at the 50% probability level for dichloridoꢀbis[Nꢀ(4ꢀ
methoxyphenyl)acetamideꢀO]ꢀzinc(II) (4c) is presented in Fig. 1.

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Fig. 1. The structure of 4c showing the atom numbering with ellipsoids drawn at the 50%
probability level. Numbered atoms are related to unꢀnumbered atoms by the symmetry operation.
‒x+1,y,z.

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Table 1. Crystal data and structure refinement for 4c
Empirical formula
Formula weight
Temperature
C18H22Cl2N2O4Zn
466.64
150(2) K
Wavelength
1.54178 Å
Orthorhombic
C m c 21
Crystal system
Space group
Unit cell dimensions
a = 16.8955(6) Å
b = 10.3515(4) Å
c = 11.5475(5) Å
2019.59(14) Å3
4
α = 90°
β = 90°
γ = 90°
Volume
Z
Density (calculated)
Absorption coefficient
F(000)
1.535 mg/m3
4.350 mm‒1
960
Crystal size
0.25 × 0.12 × 0.05 mm3
Theta range for data collection
Index ranges
5.010 to 69.611°
‒20<=h<=20, ‒12<=k<=12, ‒14<=l<=11
18993
Reflections collected
Independent reflections
Completeness to theta = 67.679°
Absorption correction
Max. and min. transmission
Refinement method
Data / restraints / parameters
Goodnessꢀofꢀfit on F2
Final R indices [I>2σ(I)]
R indices (all data)
Absolute structure parameter
Largest diff. peak and hole
1754 [R(int) = 0.0486]
100 %
Multiꢀscan
0.8045 and 0.4720
Fullꢀmatrix leastꢀsquares on F2
1754 / 2 / 133
1.057
R1 = 0.0256, wR2 = 0.0664
R1 = 0.0257, wR2 = 0.0665
0.08(2)
0.440 and ꢀ0.275 e.Å‒3

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Table 2. Selected bond lengths [Å] and angles [°] for 4c
Bond lengths [Å]
Zn1ꢀO1
Zn1ꢀO1i
Zn1ꢀCl1
Zn1ꢀCl2
O1ꢀC8
1.9834(18)
1.9835(18)
2.2212(10)
2.2263(9)
1.248(3)
O2ꢀC4
O2ꢀC9
1.373(3)
1.436(3)
1.332(3)
1.432(3)
0.791(13)
N1ꢀC8
N1ꢀC1
N1ꢀH1
Bond angles [°]
O1iꢀZn1ꢀO1
O1iꢀZn1ꢀCl1
O1ꢀZn1ꢀCl1
O1iꢀZn1ꢀCl2
O1ꢀZn1ꢀCl2
94.12(11)
107.23(6)
107.23(6)
116.52(5)
116.52(5)
Cl1ꢀZn1ꢀCl2
113.27(4)
136.68(16)
117.8(2)
C8ꢀO1ꢀZn1
C4ꢀO2ꢀC9
C8ꢀN1ꢀC1
123.9(2)
Symmetry transformation used to generate equivalent atoms: i = ‒x+1,y,z
Table 3. Hydrogenꢀbond geometry for 4c (Å, °)
D—H…A
D—H
H…A
D…A
D—H…A
C7ꢀH7C…Cl2
0.98
2.95
3.714(3)
136
N1ꢀH1…O2ii
0.791(13)
0.98
2.124(14)
2.62
2.912(3)
3.458(3)
3.883(3)
3.451(4)
173(4)
144
C7ꢀH7B…O2ii
C9ꢀH9A…Cl2iii
C7ꢀH7B…Cgiv
0.98
2.96
158
0.98
2.90
117
Symmetry codes: (ii) 1/2‒x, 3/2‒y, 1/2+z; (iii) 1‒x, 2‒y, ‒1/2+z; (iv) –x, 2‒y, 1/2+z; Cg1 is the centroid of the
C1…C6 benzene ring.

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The structure reveals a fourꢀcoordinate Zn(II) complex in which the Zn1 atom and two chlorido
ligands lie on a mirror plane with two Nꢀ(4ꢀmethoxyphenyl)acetamide ligand in general
positions. These bind to the Zn(II) atom through the acetamide O1 atoms to give an
approximately tetrahedral coordination geometry about the Zn1 cation. A more precise
description of the geometry can be obtained by calculating the τ4 index.23
360-(α+β)
τ4 = _______________
141
α and β are the two largest L—M—L angles for the 4ꢀcoordinate complex. In this instance τ4 =
0.92, very close to the value 1.0 required for a perfect tetrahedron. Both of the Nꢀ(4ꢀ
methoxyphenyl)acetamide ligands point in the same direction away from the Zn1—Cl2 vector
resulting in an overall Vꢀshape for the complex molecule. The methoxy substituent lies close to
the benzene ring plane, maximum deviation 0.141(6) Å. The ring plane subtends an angle of
52.11(13)° to the N1,C8,C7,O1 acetamide plane. The phenyl ring planes of the two ligands are
inclined at 23.15(13)° to one another. Weak, intramolecular C7—H7C…Cl2 hydrogen bonds
with a C7…Cl2 separation of 3.713(3) Å affect the overall conformation of the molecule.
The novelty of the complex reported here is revealed by a search of the Cambridge database
(Version 5.36, November 2014 plus 2 updates).24 This shows that while the structure of the
ligand has been reported previously,25,26 no structures of transition metal coordination complexes
of this ligand are reported. Nonetheless four coordinate Zn(II) complexes with ZnO2Cl2
coordination spheres abound, with 147 examples in the database. Also, the Zn—O and Zn—Cl
distances found here, Table 2, are closely similar to the mean of the Zn—Cl, 2.23(3) Å, and
Zn—O 1.99(4) Å distances over this extensive series of compounds. The bond lengths and

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angles in the coordinated ligand are also closely similar to those in the free Nꢀ(4ꢀ
methoxyphenyl)acetamideꢀO with the exception of a slight extension of the O1—C8 bond on
coordination to 1.248(3) Å compared to 1.227(4) in the free ligand.
Only one structure of a closely related acetamide complex, dichloridoꢀbis(N,Nꢀ
dimethylacetamideꢀO)ꢀzinc, is found.27‒29 Here again, the metrical data for these determinations
are comparable to those found for 4c.
The crystal packing for 4c is dominated by N1—H1…O2 hydrogen bonds augmented by C7—
H7B…O2 contacts that form zigꢀzag chains of molecules along a (Fig. 2). Chains also form
along c as a result of weaker C7—H7B…Cg1 contacts (Cg1 is the centroid of each C1…C6
benzene ring) strengthened by C2—H2…Cl1 and C9—H9A…Cl2 hydrogen bonds (Fig. 3), and
these contacts combine to generate an extensive three dimensional network of molecules stacked
along the b axis direction (Fig. 4).
Fig. 2. Chains of molecules of 4c formed along a.

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Fig. 3. Chains of molecules of 4c formed along c. C—H…π contacts are shown as dotted green
lines. Cg1 is the centroid of each C1…C6 benzene ring.
Fig. 4 Overall packing for 4c viewed along the b axis direction.

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Molar conductivity
All the complexes are nonꢀelectrolytes30 as indicated by their low molar conductance values
(7.35–12.54 ꢁ‒1 cm2 mol‒1) of 10‒3 M solutions in DMSO at room temperature.
Pharmacological applications
Metalꢀbased compounds exhibit remarkable physicoꢀchemical properties, which are rather
unique and of high interest in the medicinal chemistry research for the synthesis of lead
compounds and drug candidates, and spectacular advances have been recently made in this field
of research. A few of the metal complexes are under clinical trials which possibly possess the
potential in various areas of medicines to act as anticancer and antileishmanial agents in addition
to some other targets. In this contribution, we have developed some Zn(II) complexes based on
aryl acetamide moiety as possible inhibitors of carbonic anhydrase and alkaline phosphatase
enzymes. We have also exemplified the antileishmanial and antiproliferation potential of these
coordination compounds.
Carbonic anhydrase inhibition
The synthesized ligands (3aꢀc) and their Zn(II) complexes (4aꢀc) were screened for their ability
to act as carbonic anhydrase (CA) inhibitors against bovine CA II (bCAꢀII) isozyme.31,32
Acetazolamide was used as a standard inhibitor with an IC50 value of 0.96 ± 0.18 µM. IC50
values were determined for all the compounds and the inhibition data is reported in Table 4.
According to the inꢀvitro studies, the IC50 values of the carbonic anhydrase activity of the ligands
and their corresponding Zn(II) complexes were found in the range of 7.57‒0.81 µM. A careful
insight into these results revealed that Zn(II) complexes are much better inhibitors of carbonic
anhydrase as compared to their corresponding ligands which showed less inhibition efficacy,

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whereas, in certain cases, the synthesized complexes demonstrated higher inhibition as compared
to the standard inhibitor, acetazolamide.
Table 4. Carbonic anhydrase inhibition of compounds 3aꢀc and 4aꢀc
Carbonic anhydrase inhibition
Entry
Substituent (R)
IC50 ± SEM (µM)a
H
7.57 ± 0.16
4.21 ± 0.30
5.48 ± 0.51
0.91 ± 0.52
0.81 ± 0.18
4.72 ± 0.25
0.96 ± 0.18
3a
2ꢀOMe
4ꢀOMe
H
3b
3c
4a
2ꢀOMe
4ꢀOMe
—
4b
4c
Acetazolamide
aSEM = Standard error mean (experiments run in triplicate)
The IC50 values obtained from the CA inhibitory activity of Zn(II) complexes showed promising
results. The most active compound was 4b which demonstrated an IC50 value of 0.81 ± 0.18 µM,
a considerably higher inhibition as compared to the standard inhibitor (IC50 = 0.96 ± 0.18 µM).
This coordination complex possesses an electronꢀrich methoxy substituent attached to the orthoꢀ
position of the aromatic ring. The same effect has also been observed for the complex 4a
(unsubstituted phenyl ring) which also showed better inhibition as compared to the standard
drug. Whereas, moving the methoxy group from orthoꢀ to paraꢀposition (complex 4c) resulted in
markedly reduced inhibitory potential (IC50 = 4.72 ± 0.25 µM).
Alkaline phosphatase inhibition

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Alkaline phosphatase inhibitory potential of the synthesized aryl acetamide ligands (3aꢀc) and
their corresponding Zn(II) complexes (4aꢀc) was determined using potassium dihydrogen
phosphate as a standard inhibitor (IC50 = 2.41 ± 0.03 µM).33,34 The inhibitory activity results are
summarized in Table 5 in the form of IC50 values. The careful observation of the bioactivity
results revealed that the Zn(II) complexes displayed good alkaline phosphatase inhibitory
activity, and in case of 4b, comparable to the standard inhibitor. Moreover, the parent ligands
were observed to possess less inhibitory effect. This many fold increase in bioactivity could
possibly be attributed to the metal interaction to the amino acid residues in proteins. Among the
tested derivatives, complex 4a was also highlighted as a good inhibitor of alkaline phosphatase
with an IC50 value of 2.91 ± 0.37 µM which is slightly higher than the positive control.
Moreover, the least active complex was 4c, where an IC50 value of 3.18 ± 0.76 µM was
observed. This compound incorporates a paraꢀmethoxy group on the aryl ring.
Table 5. Alkaline phosphatase inhibition of compounds 3aꢀc and 4aꢀc
Alkaline phosphatase inhibition
Entry
Substituent (R)
IC50 ± SEM (µM)a
H
11.23 ± 0.43
7.65 ± 0.62
5.59 ± 0.52
2.91 ± 0.37
2.54 ± 0.58
3.18 ± 0.76
2.41 ± 0.03
3a
3b
2ꢀOMe
4ꢀOMe
H
3c
4a
2ꢀOMe
4ꢀOMe
—
4b
4c
KH2PO4
aSEM = Standard error mean (experiments run in triplicate)

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Antileishmanial activity
The antileishmanial activity of synthetic derivatives (3aꢀc, and 4aꢀc) was measured by MTT
assay,35,36 and the activity results in terms of their IC50 values are presented in Table 6.
Amphotericin B was used as a standard drug for comparison purpose which demonstrated an
IC50 value of 0.29 ± 0.05 µM. Examination of the antileishmanial activity results unfolded that
some of the synthesized coordination complexes possess good inhibition as compared to their
respective ligands. The most active analogue was 4b, Zn(II) complex, with an IC50 value of 0.78
± 0.31 µM. This derivative incorporates an orthoꢀmethoxy substituent on the aryl ring. The
activity strength was reduced when the substituent at the aryl ring is removed (compound 4a;
IC50 = 1.42 ± 0.45 µM). In general, the obtained results indicated that this type of metal
complexes may need further attention to be developed as therapeutic agents to treat
leishmaniasis.
Table 6. Antileishmanial activity of compounds 3aꢀc and 4aꢀc
Antileishmanial activity
Entry
Substituent (R)
IC50 ± SEM (µM)a
H
5.12 ± 0.38
3.05 ± 0.19
4.03 ± 0.56
1.42 ± 0.45
0.78 ± 0.31
1.85 ± 0.09
0.29 ± 0.05
3a
2ꢀOMe
4ꢀOMe
H
3b
3c
4a
2ꢀOMe
4ꢀOMe
—
4b
4c
Amphotericin B
aSEM = Standard error mean (experiments run in triplicate)

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Anticancer activity
Antiproliferative activity of the synthesized ligands (3aꢀc) and their Zn(II) complexes (4aꢀc) was
measured inꢀvitro using Skehan et al. protocol37 by the cell growth inhibition against lung
carcinoma (Hꢀ157) and kidney fibroblast (BHKꢀ21) cell lines. Vincristine was used as a standard
drug with an IC50 value of 1.08 ± 0.09 µM for both cell lines. Table 7 summarizes the anticancer
activity results where it appears that metal complexes exhibited low cytotoxicity at Vero cells
and significant activity against Hꢀ157 and BHKꢀ21 cancer cells as compared to their parent aryl
acetamide ligands. Vero cells are normal epithelial kidney cells extracted from African green
monkey and used here as control to determine the safety. The analysis of the tabulated results
suggested that the presence/absence of “R” group influence the anticancer activity. From the
tested series, complex 4b with an electronꢀrich methoxy group at an orthoꢀposition of the aryl
ring showed highest cytotoxicity with an IC50 value of 1.52 ± 0.21 µM against Hꢀ157 cell lines.
This anticancer potential is comparable to the standard drug (vincristine). When the position of
“R” group is changed or it is removed completely, a decrease in activity has been observed. The
activity results of Zn(II) complexes against BHKꢀ21 cell lines were also promising where
complex 4c with a paraꢀmethoxy substituent displayed activity results with an IC50 value of 1.24
± 0.98 µM. The complex 4a was relatively less active.
As the investigated complexes are unprecented, the activity results were compared with Zn(II)
complexes (based on different ligands) and some other coordination compounds.18b‒d,38 On the
basis of the biological potential of the coordination compounds displayed herein, we strongly
believe that these examples will contribute enormously towards the identification of a new area
in the design of metalꢀbased therapeutic agents.

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Table 7. Anticancer potential of compounds 3aꢀc and 4aꢀc
Hꢀ157 cell lines BHKꢀ21 cell lines
Vero cell lines
% inhibition
Entry
Substituent (R)
IC50 ± SEM (µM)a
H
3.38 ± 0.31
2.35 ± 0.17
6.41 ± 0.34
4.32 ± 0.29
4.17 ± 0.38
2.01 ± 0.57
1.24 ± 0.98
1.08 ± 0.09
21.9 ± 2.2
11.1 ± 1.7
19.5 ± 2.5
16.2 ± 2.7
18.8 ± 2.4
14.1 ± 1.9
11.7 ± 1.09
3a
2ꢀOMe
4ꢀOMe
H
8.02 ± 0.23
6.11 ± 0.56
2.19 ± 0.18
1.52 ± 0.21
1.73 ± 0.49
1.08 ± 0.09
3b
3c
4a
4b
2ꢀOMe
4ꢀOMe
—
4c
Vincristine
aSEM = Standard error mean (experiments run in triplicate)
Kinetic studies
In order to determine the type of inhibition, detailed kinetics studies were carried out for
compound 4b, the most active carbonic anhydrase and alkaline phosphatase inhibitor. The results
indicated the competitive inhibition of bovine erythrocyte carbonic anhydrase II (bCAꢀII) (Fig.
5), and uncompetitive inhibition of tissue nonspecific alkaline phosphatase by compound 4b
(Fig. 6).

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Fig. 5. Doubleꢀreciprocal plots of the inhibition kinetics of bovine erythrocyte carbonic
anhydrase II (CAꢀII) indicating competitive inhibition by compound 4b. Changes in the initial
velocities of the reaction were measured at different concentrations of the inhibitor 4b using
substrate 4ꢀnitrophenyl acetate.

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Fig. 6. Doubleꢀreciprocal plots of the inhibition kinetics of tissue nonspecific alkaline
phosphatase indicating uncompetitive inhibition by compound 4b. Changes in the initial
velocities of the reaction were measured at different concentrations of the inhibitor 4b using
substrate 4ꢀnitrophenyl phosphate.
Molecular docking studies
Molecular docking studies of Nꢀ(aryl)acetamide derivatives were performed using LeadIT to
investigate the binding modes of inhibitors as well as interactions in the active site. Validation of
docking methodology was done by reꢀdocking the ligand of human carbonic anhydrase (3K34)
in the active site of bovine carbonic anhydrase (1V9E) and it reproduced successfully the ligand
with an RMSD of < 1.5 Å.
Molecular docking studies revealed that the phenyl ring having methoxy substituent are involved
in aromatic and hydrophobic interactions within the active site residues like Thr198, His118,
Val141, Val120, Gln91, Asn61 and Asn66. Additionally, the hydrogen bonding interaction was
observed between the oxygen and hydrogen of the inhibitors with residues His63, His93, His95
and Thr197. N–Phenyl moiety incorporating methoxy substituent at 2ꢀ and 4ꢀ position are found
to inhibit the enzyme by fitting in the active pocket towards the hydrophilic portion. Whereas,
acetamide part was flanking towards the hydrophobic pocket. After Hyde assessment of 10
lowest energy docked conformations, the pose of compounds 3b and 3c with the most favorable
binding mode was selected and are shown in Fig. 7 and 8, respectively.

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Fig. 7. Putative binding mode of 3b bound to the active site of bovine carbonic anhydrase II.
Carbon atoms of 3b are colored magenta and that of protein are blue.

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Fig. 8. Putative binding mode of 3c bound to the active site of bovine carbonic anhydrase II.
Carbon atoms of 3c are colored green and that of protein are blue.
In general, both the compounds presented similar interactions with the active site residues but
more specifically, methoxy side chain of 3b was more towards the residues His118, Val120,
Val141 and Gln91, whereas for 3c, it is inclined towards residues Phe65, Asn66 and Asn61. The
phenyl ring for both the compounds was found adjacent to residues His63, His95 and His 93.
Conclusions
Metalꢀbased therapeutics is an emerging class of drugs in medicinal research with unique and
beneficial properties. In the present study, we aimed to demonstrate clearly that coordination
complexes possess outstanding biological potential to be used in medicinal chemistry
development. In this regard, the synthesized Zn(II) complexes based on aryl acetamide moiety
were tested for their carbonic anhydrase and alkaline phosphatase inhibition. The activity results
were remarkably good and several coordination complexes displayed excellent inhibition profile.
The designed compounds were also evaluated for their anticancer and antiꢀparasitic properties.
The analysis of the activity results also suggested that the biological profile of the synthetic
complexes is much more improved as compared to their corresponding aryl acetamide ligands.
Molecular docking studies of the ligands were also carried out to give insights into the binding
site interactions of the enzyme.
Experimental section
Materials
Unless otherwise noted, all the chemicals and reagents were of analytical grade and were
purchased from sigma Aldrich and Merck chemical companies. All solvents were purified by

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distillation, dried using the appropriate drying agents, saturated with nitrogen, stored over 4 Å
molecular sieves, and degassed before use.
Instrumentation and methods
Thin layer chromatography (TLC) was performed on Merck DFꢀAlufoilien 60F254 0.2 mm
precoated plates. Product spots were visualized under UV light at 254 nm. Melting points were
recorded on a digital Gallenkamp (SANYO) melting point apparatus using open capillary tubes
and are uncorrected. Infraꢀred (FTIR) spectra were recorded on a Bruker AlphaꢀP FTIR
1
spectrometer (4000‒400 cm‒1). H NMR spectra were recorded on a Bruker AV400 (400 MHz)
spectrometer. Chemical shifts (δ) are quoted in parts per million (ppm) downfield of
tetramethylsilane, using residual deuterated solvent as internal standard (DMSOꢀd6 at 2.50 ppm).
Abbreviations used in the description of resonances are: s (singlet), d (doublet), t (triplet), m
13
(multiplet). Coupling constants (J) are quoted to the nearest 0.1 Hz. Protonꢀdecoupled C NMR
spectra were recorded on a Bruker AV400 (100.6 MHz) spectrometer. Chemical shifts (δ) are
quoted in parts per million (ppm) downfield of tetramethylsilane, using deuterated solvent as
internal standard (DMSOꢀd6 at 39.52 ppm). Mass spectral data were recorded using the
electrospray ionization (ESI) technique. Molar conductance of the synthesized complexes was
determined in DMSO (10‒3 M) at room temperature.
Synthesis of ligands and complexes
General procedure for the preparation of aryl acetamides (3aꢀc)
The aryl acetamide ligands (3aꢀc) were synthesized by the reaction of corresponding aniline (1aꢀ
c) (1.0 mmol) with acetic anhydride (2) (1.0 mmol). Initially, the low melting solid was heated to
its melting point followed by the addition of other reactant with continuous stirring. The reaction
mixture was heated until a homogenous phase of both reactants is formed. The mixture was

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cooled to room temperature and triturated with chloroform to afford the desired aryl acetamide
compounds (3aꢀc).19 The spectroꢀanalytical data obtained for compounds 3aꢀc was consistent
with the literature reports.21
General procedure for the preparation of Zn(II) complexes (4aꢀc)
The corresponding aryl acetamide (1.0 mmol) was dissolved in ethanol (10 mL). The zinc
chloride (0.6 mmol) was dissolved separately in ethanol (10 mL) and added to the aryl acetamide
solution slowly. After complete addition, the mixture was heated at reflux for 2 h. After cooling
to room temperature, the excess solvent was removed under reduced pressure. The crude mass
was triturated with DCM/hexane to precipitate the corresponding complexes. The precipitated
solid was recrystallized (50% hexane:chloroform) and left at room temperature for slow
evaporation of solvent which affords the crystals of corresponding Zn(II) complexes (4aꢀc).20
Zinc(II) complex of Nꢀphenylacetamide (4a)
Yield (73%). m.p. 60‒65 °C; IR (ATR, cm‒1) 3461 (N‒H), 1649 (C=O), 1586, 1493 (C=C), 717,
1
661 (monoꢀsubstitutedꢀAr), 429 (Zn‒O); H NMR (400 MHz, DMSOꢀd6) δ 8.93 (2H, s, NH),
7.55 (4H, d, J = 8.4 Hz, ArH), 7.34 (4H, d, J = 8.4 Hz, ArH), 7.13 (2H, t, J = 6.7 Hz, ArH), 2.16
(6H, s, CH3); 13C NMR (100.6 MHz, DMSOꢀd6) δ 167.3, 137.5, 127.7, 123.5, 122.2, 27.2; EIMS
(m/z) 406, 369, 234, 136; Analysis calcd. for C16H18Cl2N2O2Zn: Calculated (%); C, 47.26; H,
4.46; N, 6.89. Found (%); C, 46.91; H, 4.63; N, 6.69.
Zinc(II) complex of Nꢀ(2ꢀmethoxyphenyl)acetamide (4b)
Yield (73%). m.p. 118‒121 °C; IR (ATR, cm‒1) 3384 (N‒H), 1643 (C=O), 1590, 1484 (C=C),
1
747 (orthoꢀsubstitutedꢀAr), 421 (Zn‒O); H NMR (400 MHz, DMSOꢀd6) δ 7.98 (2H, s, NH),
7.06‒7.00 (8H, m, ArH), 3.87 (6H, s, OCH3), 2.07 (6H, s, CH3); 13C NMR (100.6 MHz, DMSOꢀ
d6) δ 164.8, 155.7, 127.6, 147.2, 123.7, 120.3, 119.6, 54.8, 27.2; EIMS (m/z) 466, 429, 264, 166;

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Analysis calcd. for C18H22Cl2N2O4Zn: Calculated (%); C, 46.32; H, 4.75; N, 6.00. Found (%); C,
46.09; H, 4.90; N, 5.79.
Zinc(II) complex of Nꢀ(4ꢀmethoxyphenyl)acetamide (4c)
Yield (89%). m.p. 123‒126 °C; IR (ATR, cm‒1) 3290 (N‒H), 1636 (C=O), 1606, 1509 (C=C),
826 (paraꢀsubstitutedꢀAr), 417 (Zn‒O); 1H NMR (400 MHz, DMSOꢀd6) δ 9.67 (2H, s, NH), 7.38
(4H, d, J = 8.4 Hz, ArH), 6.82 (4H, d, J = 8.0 Hz, ArH), 3.66 (6H, s, OCH3), 1.97 (6H, s, CH3);
13C NMR (100.6 MHz, DMSOꢀd6) δ 165.5, 156.2, 131.8, 121.4, 114.3, 55.6, 29.9; EIMS (m/z)
466, 429, 264, 166; Analysis calcd. for C18H22Cl2N2O4Zn: Calculated (%); C, 46.32; H, 4.75; N,
6.00. Found (%); C, 46.02; H, 4.56; N, 5.72.
Biological protocols
In-vitro carbonic anhydrase inhibition
A series of aryl acetamides and their Zn(II) complexes (3aꢀc and 4aꢀc) were screened for their
inhibitory activity against carbonic anhydrase (CA). The activity was measured by determining
the amount of pꢀnitrophenol formed during the hydrolysis of pꢀnitrophenyl acetate by using
standard protocol.31,32 The compounds were initially screened at 1 mM concentration.
Compounds which exhibited >50% inhibition were further diluted at 8ꢀ10 concentrations.
Results of the active compounds in the form of IC50 values are presented in the Table 4.
Alkaline phosphatase inhibition
The synthesized compounds (3aꢀc and 4aꢀc) were tested against calf intestinal alkaline
phosphatase (CIAP). The compounds were initially tested against these enzymes at 1 mM
concentration to further make a doseꢀresponse curve of potential inhibitors of synthesized
compounds by using standard protocol.33,34 The compounds which showed ≥50% inhibition were
selected for further determination of doseꢀresponse curves against all compounds. For this

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purpose 7ꢀ9 serial dilutions of each compound spanning three orders of magnitude were prepared
in enzyme assay buffer and their dose response curves were obtained by adopting the same
methods used for initial screening. All experiments were performed in triplicate. Results reported
are mean of three independent experiments (± SEM) and expressed as percent inhibitions
calculated by the formula:
Inhibition (%) = [100 ‒ (Abs of test compound/Abs of control) × 100]
IC50 values of potential inhibitors (≥50%) were obtained with the help of nonꢀlinear regression
analysis program of Graph Pad prism 5.0 Software Inc., San Diego, California, USA.
Antileishmanial activity
Parasite and culture
Leishmania major promastigotes were cultured at 25 ± 1 °C to logarithmic phase in DꢀMEM/Fꢀ
12 medium (Gibco BRL) without phenol red, supplemented by 10% heat inactivated fetal bovine
serum (FBS), 100 IU/mL penicillin and 100 ꢂg/mL streptomycin, then washed 3 times with
phosphateꢀbuffered saline (PBS). The cells were centrifugated at 1500 rpm, for 10 min at room
temperature and reꢀsuspended at a concentration of 2.5 × 106 parasites/mL in medium.
Antileishmanial activity assay (MTT assay)
Inꢀvitro antileishmanial activity of the compounds was evaluated against the promastigote forms
of Leishmania major using a MTT (3ꢀ(4,5ꢀdimethylthiazolꢀ2yl)ꢀ2,5ꢀdiphenyltetrazolium
bromide). The MTT assay used was originally described by Mosmann35 and later modified by
Niks and Otto.36 A stock solution of MTT (Sigma Chemical Co., St. Louis, Mo.) was prepared in
PBS at 5 mg/mL and stored in the dark at 4 °C. For the antileishmanial activity assay, 100
ꢂL/well of the culture which contained 2.5 × 106 cells/mL promastigotes was seeded in 96ꢀwell
flatꢀbottom plates. Then, 10 ꢂL/well from various concentrations of compounds were added to

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triplicate wells and plates were incubated for 72 h at 25 ± 1 °C. The well containing only 100 ꢂL
culture medium without any compound, drug or parasite, was taken as blank. Amphotericin B
was used as a standard drug. At the end of incubation, 10 ꢂL of MTT was added to each well and
plates were again incubated for 3 h at 25 ± 1 °C. Enzyme reaction was then stopped by the
addition of 100 ꢂL of 50% isopropanol and 10% sodium dodecyl sulfate (0.1 N HCl). The plates
were incubated for an additional 30 min under agitation at room temperature. Relative optical
density (OD) was then measured at a wavelength of 570 nm using a 96ꢀwell microplate reader
(BioꢀTek ELx 800TM, Instruments, Inc. Winooski, VT, USA). The background absorbance of
plates was measured at 690 nm and subtracted from 570 nm measurement. The absorbance of the
formazan produced by the action of mitochondrial dehydrogenases of metabolically active cells
was correlated with the number of viable cells.35,36 All experiments were performed in triplicate.
Results reported were the mean of three independent experiments (± SEM). IC50 values of
potential inhibitors (≥50%) were obtained with the help of nonꢀlinear regression analysis
program of Graph Pad prism 5.0 Software Inc., San Diego, California, USA.
Anticancer activity
Cell lines and cell cultures
Lung carcinoma (Hꢀ157), (ATCC CRLꢀ5802), kidney fibroblast (BHKꢀ21), (ATCC CCLꢀ10)
and African green monkey kidney normal cell line (Vero), (ATCC CCLꢀ81) were kept in RPMIꢀ
1640, having heatꢀinactivated fetal bovine serum (10%) glutamine (2 mM), Pyruvate (1 mM),
100 U/mL penicillin and 100 µg/mL streptomycin, in Tꢀ75 cm2 sterile tissue culture flasks in a
5% CO2 incubator at 37 °C.39 For experiment, 96ꢀwell plates were used for growing Hꢀ157,
BHKꢀ21 and Vero cells by inoculating 5 × 104 cells per 100 µL per well and plates were

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incubated at 37 °C for 24 h in a humidified atmosphere containing 5% CO2. Within 24 h, a
uniform monolayer was formed which was used for experiments.
Cytotoxicity analysis by sulforhodamine B (SRB) assay
To perform cytotoxicity assay, with Hꢀ157, BHKꢀ21 and Vero cells, a previously described
method by Skehan et al.,37 was adopted with minor modifications. Briefly, cells were cultured in
different 96 well plates for 24 h. The compounds in different concentrations were inoculated in
test wells. Furthermore, positive control vincristine was prepared in DMSO. The well containing
culture media with cells having no compound or drug was taken as blank. Vero cells were treated
at 100 µM test compounds to check the toxicity against normal cell lines. The plates were then
incubated for 48h. After that, cells were fixed with 50 µL of 50% ice cold trichloroacetic acid
solution (TCA) and plates were incubated at 4 °C for 1 h. Subsequently, plates were washed five
times with phosphateꢀbuffered saline (PBS) and air dried. Fixed cells were further treated with
0.4% w/v sulforhodamine B dye (prepared in 1% acetic acid solution) and left at room
temperature for 30 min. After that the plates were rinsed with 1% acetic acid solution and
allowed to dry. In order to solubilize the dye, the dried plates were treated with 10 mM Tris base
solution for 10 min at room temperature. The absorbance was measured at 490 nm subtracting
the background measurement at 630 nm.40 All experiments were performed in triplicate. Results
reported are the mean value of three independent experiments (± SEM). IC50 values of potential
inhibitors (≥50%) were obtained with the help of nonꢀlinear regression analysis program of
Graph Pad prism 5.0 Software Inc., San Diego, California, USA.
Docking protocols
Structure selection and preparation

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In order to perform docking studies, high resolution crystal structures of hCAꢀII (PDB ID: 3K34,
0.9 Å)41 and bCAꢀII (PDB ID: 1V9E, 1.95 Å)42 were obtained from the Protein Data Bank. Prior
to molecular modeling experiments, the structures of the enzyme and the compounds were
prepared as follows. The enzyme structures were protonated with the Protonate3D43 algorithm
implemented in the molecular modeling tool MOE.44 The structure was energy minimized using
Amber99 force field including all crystallographic solvent molecules. The backbone atoms were
restrained with a small force in order to avoid collapse of the binding pockets during energy
minimization calculations. After minimization, the water molecules were removed. As 1V9E did
not contain any bound ligand, the validity of docking methodology was assessed by reꢀdocking
the coꢀcrystalized ligand of hCAꢀII (3K34) in the active site of 1V9E. The docking method was
able to reproduce the experimentally bound conformation of ligand in the active site with an
RMSD of < 1.5 Å.
Compounds preparation
The 3D structural coordinates were generated for all the compounds using MOE followed by
assignment of protonation and ionization states in physiological pH range by using the “wash”
module. Afterwards, the compounds structures were energy minimized with the MMFF94x force
field for docking studies.44
Docking studies
Docking studies of the compounds (3aꢀc) were performed using LeadIT from BioSolveIT,
GmbH Germany.45 Receptor was loaded by Load or Prepare Receptor utility of the LeadIT
software, while selecting the metal ions as part of the receptor. The binding site for 1V9E was
defined in 8.0 Å spacing of the amino acid residues surrounding the Zn ions.