Synthesis, characterization, biological screening and molecular docking studies of 2-aminonicotinaldehyde (ANA) and its metal complexes

Article abstract of DOI:10.1007/s11164-017-3089-y

The biological and optical importance of the o-aminoaldehyde family of ligands inspired us to evaluate the coordination properties and biological activities of 2-aminonicotinaldehyde (ANA). Here, we report the synthesis, characterization, biological screening and molecular docking studies of ANA and its metal complexes of Ni(II), Pd(II), Co(II) and Cu(II) using various analytical and spectroscopic techniques. The single crystal X-ray diffraction studies of ANA explain the solid-state assembly and an interesting supramolecular herring bone stacking pattern by classical N–HO/N intra/inter molecular and non-classical C–HO/N intermolecular H-bonding. ANA and its metal complexes were screened for in vitro anticancer, antimicrobial and anti-oxidant activities. Anticancer activity was tested against HeLa, MCF-7 and HEK293 human cancer cell lines. The [Ni(ANA)₂Cl₂] complex showed good activity against HeLa and MCF-7, the [Pd(ANA)₂Cl₂] and [Cu(ANA)₂Cl₂] complexes against HeLa, and the [Co(ANA)₂Cl₂] complex against MCF-7. In antimicrobial screening, the [Co(ANA)₂Cl₂] and [Cu(ANA)₂Cl₂] complexes were proved to be potent antibacterial and antifungal agents. The anti-oxidant activity of these complexes was investigated through DPPH radical assay, and it was found that all the complexes have good radical scavenging capability. Molecular docking studies were also carried for all the metal complexes against EGFR as a target protein by using Autodock, and the results strongly correlated with the anticancer activity.

Full text of DOI:10.1007/s11164-017-3089-y

Res Chem Intermed
DOI 10.1007/s11164-017-3089-y
Synthesis, characterization, biological screening
and molecular docking studies of
2-aminonicotinaldehyde (ANA) and its metal complexes
Ramaiah Konakanchi1 Ramachary Mallela2
•
•
Ramu Guda3 Laxma Reddy Kotha1
•
Received: 25 April 2017 / Accepted: 21 July 2017
Ó Springer Science+Business Media B.V. 2017
Abstract The biological and optical importance of the o-aminoaldehyde family of
ligands inspired us to evaluate the coordination properties and biological activities
of 2-aminonicotinaldehyde (ANA). Here, we report the synthesis, characterization,
biological screening and molecular docking studies of ANA and its metal com-
plexes of Ni(II), Pd(II), Co(II) and Cu(II) using various analytical and spectroscopic
techniques. The single crystal X-ray diffraction studies of ANA explain the solid-
state assembly and an interesting supramolecular herring bone stacking pattern by
classical N–HÁÁÁO/N intra/inter molecular and non-classical C–HÁÁÁO/N inter-
molecular H-bonding. ANA and its metal complexes were screened for in vitro
anticancer, antimicrobial and anti-oxidant activities. Anticancer activity was tested
against HeLa, MCF-7 and HEK293 human cancer cell lines. The [Ni(ANA)2Cl2]
complex showed good activity against HeLa and MCF-7, the [Pd(ANA)2Cl2] and
[Cu(ANA)2Cl2] complexes against HeLa, and the [Co(ANA)2Cl2] complex against
MCF-7. In antimicrobial screening, the [Co(ANA)2Cl2] and [Cu(ANA)2Cl2] com-
plexes were proved to be potent antibacterial and antifungal agents. The anti-oxi-
dant activity of these complexes was investigated through DPPH radical assay, and
it was found that all the complexes have good radical scavenging capability.
Molecular docking studies were also carried for all the metal complexes against
EGFR as a target protein by using Autodock, and the results strongly correlated with
the anticancer activity.
&
Laxma Reddy Kotha
laxmareddychem12@gmail.com
1
2
3
Department of Chemistry, National Institute of Technology, Warangal 506004, India
Department of Chemistry, Lal Bahadur College, Warangal 506007, India
Department of Chemistry, Kakatiya University, Warangal 506009, India
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R. Konakanchi et al.
Keywords Metal complexes Á 3D architecture Á Antimicrobial activity Á Anticancer
activity Á Anti-oxidant activity Á Docking studies
Introduction
In recent years, modern coordination chemistry has expanded its horizons to
emerging areas of metal organic frameworks [1–4], crystal engineering [5],
nonlinear optical materials [6–10] and other areas of interdisciplinary research.
Several classes of ligand architectures have been developed over the years to suit
appropriate applications. One such class of ligands with scarce literature citations
are the o-aminoaldehydes. Historically, o-aminoaldehydes have often been
employed as important building blocks for several pharmaceutically relevant
compounds like naphthyridine derivatives (for osteoporosis) [11], plant-antitumor
agents (camptothecin analogues) [12], antihistamine and pain-relieving agents
(mGlu5 receptor antagonist) [13] (Fig. 1). The reaction of pyridine in o-aminoben-
zaldehyde (2-aminonicotinaldehyde, ANA) with cyclic aliphatic diketones (1,3-
diketocyclohexane) yields fused polypyridyl compounds which have been exten-
sively used to prepare polymetallic complexes having interesting magnetic
properties [14] (Schemes 1). The metal binding affinities of o-aminoaldehydes
have not been extensively studied. Eichhorn and co-workers have reported the
Cu(II) complexes of o-aminobenzaldehyde. Preliminary spectral characterization
revealed a di-copper complex [15]. The coordination chemistry of ANA was quite
different from that of the parent ligand. Theoretically, there are three plausible metal
binding sites in ANA, pyridine N being the probable electron donor site, others
include the amine N and the aldehyde O as is the case with o-aminobenzaldehyde.
Usually, ancillary ligands have been employed along with ANA to satisfy the
coordination number of the metal. In all cases, pyridine N was found to be binding
to the metal center.
A detailed literature search on the coordination chemistry of ANA (without
ancillary ligands) did not produce any results. The only comparable complexes
reported were the Zn(II) complexes of 2-aminopyridine and 3-pyridinealdehyde
[16, 17]. These complexes were reported to show excellent non-linear optical
properties (Fig. 2). The structure of ANA is shown in Fig. 3. The option of having
more than one potential metal binding site makes the investigation of the
coordination chemistry of ANA very challenging. Equipped with the knowledge on
Fig. 1 Application of o-aminoaldehlydes as synthetic building blocks for pharmaceutical applications
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Synthesis, characterization, biological screening and…
Scheme 1 Application of o-aminoaldehydes for the synthesis of fused polypyridyl ligands
Fig. 2 M = Zn(II) complexes of 2-aminopyridine and 3-pyridinaldehyde
Fig. 3 Structure of 2-aminonicotinaldehyde (ANA)
the biological and optical importance of amino and aldehyde of pyridine analogues,
we set out to explore the coordination properties of ANA with metals like Ni(II),
Pd(II), Co(II) and Cu(II). Different analytical tools, UV–Vis, FTIR, docking studies
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R. Konakanchi et al.
and cyclic voltamograms, were employed to understand the metal binding affinity of
ANA in the metal complexes.
Experimental
General information
All reactions were performed using dry solvents and standard Schlenk techniques.
The ligand ANA was prepared as reported previously [18]. Nicotinamide and
ammonium sulfamate were purchased from Loba Chemie, and nickel(II) chloride
hexahydrate, palladium(II) chloride, copper(II) chloride, cobalt(II) chloride hex-
ahydrate and CDCl3 (99.9% D, containing 0.03% TMS) were purchased from
Aldrich (USA). Iron(II) triflate diacetonitrile was prepared according to known
procedures [19]. The drying agents employed at various stages of purification
procedures, i.e., anhydrous sodium sulfate, calcium hydride and sodium hydroxide
and hydrochloric acid of analytical reagent grade were obtained from Ranbaxy
(India). The nitrogen gas utilized in this study was obtained from Jainex Gases
(India). It was further purified by using a P2O5/KOH tower, and molecular sieves,
methanol and acetonitrile were of spectroscopic grade from Merck (India). They
were further purified by distilling over calcium hydride [20, 21].
Materials, methods and equipment
All the compounds synthesized during this study were characterized by recording
their melting point, nuclear magnetic resonance (1H and 13C), infrared and
electronic absorption spectra and by electrochemical (cyclic-voltammetry) methods
and docking studies. Melting points were measured by using Bucchi–Electrothermal
melting point apparatus and elemental analysis by using a Vario MICRO CHNS/
15082029 analyzer. The 1H NMR and 13C NMR spectra were recorded on a Bruker
Ascend-400 MHz spectrometer using DMSO-d6 as the solvent at ambient temper-
atures. Tetramethylsilane (TMS) was the internal standard employed while
recording these spectra. IR spectra were recorded on a Perkin-Elmer 100S
spectrophotometer by dispersing the solid samples with KBr pellets. The electronic
spectra of the ligands and their metal complexes were recorded on a Hewlett-
Packard 8452 diode-array spectrometer equipped with HP-chemstation software.
Cyclic-and differential-pulse voltammetric experiments (CH3CN, 0.1 M TBAP)
were performed on a CH Instruments model CHI 620A electrochemical analyzer
(working and auxiliary electrodes: Pt; reference electrode: Ag/AgCl). A Fc?/Fc
(Fc = ferrocene) couple was used to calibrate the redox potential values.
Crystal structure determination
A single crystal of the ligand ANA suitable for X-ray diffraction analysis was
obtained by slow evaporation from ethanol solution. The diffraction data were
collected on a Bruker SMART APEX-II CCD diffractometer, equipped with a fine
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Synthesis, characterization, biological screening and…
˚
focus 1.75-Kw sealed tube Mo-ka radiation (k = 0.71073 A) at 296 K, with
increasing omega width of 0.3° per frame at a scan speed of 5 s/frame. Data
integration and reduction were carried out with the help of the SAINT program [22].
Multiscan empirical absorption corrections were applied to the data using the
program SADABS [23]. Structures were solved by direct methods and refined with
full-matrix least squares on F2 using the SHELXL–97 program [24]. All non-
hydrogen atoms were refined anisotropically. The hydrogen atoms were located
from the Fourier maps and refined. Structural illustrations have been drawn with
Cameron and ORTEP software.
Synthesis
Synthesis of 2-aminonicotinaldehyde
The ligand ANA was prepared as per the procedure reported in the literature [18]
and, on recrystallization from ethanol,yielded yellow crystals suitable for X-ray
diffraction. Yield 40%. Anal. Calcd. For C6H6N2O (%): C, 58.95; H, 4.91; N, 22.93.
Found: C, 58.80; H, 4.80; N, 22.87. M.P 99 °C; IR (KBr, cm-1): 3396 [(N–H)], 1667
[t(c=0)], 1575 [t(c=N) (Py)]. 1HNMR (DMSO, ppm, 400 MHz): d 9.84 (s, 1H), 8.24 (d,
J = 8.0 Hz, 1H), 8.00 (d, J = 8.0 Hz, 1H), 7.55 (s, 2H, –NH2), 6.72–6.75 (m, 1H).
13CNMR (DMSO, ppm, 100 MHz): d 194.12, 158.70, 155.36, 145.02, 113.44,
112.69.
Nickel(II) complex of 2-aminonicotinaldehyde
This complex was prepared by direct reaction between the freshly purified ligand
(0.244 g, 2 mmol) and nickel(II) chloride hexahydrate NiCl2Á6H2O (0.2376 g,
1 mmol). It was dissolved in 50 mL of anhydrous methanol and refluxed for 3–4 h.
The color of the reaction mixture changed from pale yellow to reddish brown. At the
end of the reaction, a reddish brown solid was obtained which was recrystallized
from methanol to obtain 75% yield. Anal. Calcd. for [Ni(C6H6N2O)2Cl2] (%): C,
38.52; H, 3.21; N, 14.98. Found: C, 38.50; H, 3.20; N, 14.96. M.P 278–280 °C; IR
(KBr, cm-1): 3390 [t(N–H)], 1658 [t(C=O)], 1542 [t(C=N) (Py)].
Palladium(II) complex of 2-aminonicotinaldehyde
This complex was prepared by direct reaction between the freshly purified ligand
(0.244 g, 2 mmol) and palladium(II) chloride (0.1773 g, 1 mmol) which were
dissolved in 50 mL of anhydrous methanol and refluxed for 2–3 h. The color of the
reaction mixture changed from pink to yellow. Yield of the complex [Pd(C6H6
N2O)2Cl2] 85%. Anal. Calcd. for [Pd(C6H6N2O)2)Cl2] (%): C, 34.15; H, 2.84; N,
13.28. Found: C, 34.12; H, 2.80; N, 13.20. M.P 328–330 °C. IR (KBr, cm-1): 3389
[t(N–H)], 1653 [t(C=O)], 1550 [t(C=N) (Py)]. 1HNMR (DMSO, ppm, 400 MHz): d 9.85
(s, 2H, –CHO), 8.25 (d, J = 8.0, 2H), 8.01 (d, J = 8.0, 2H), 7.55 (s, 4H, –NH2),
6.73–6.76 (m, 2H). 13CNMR (DMSO, ppm, 100 MHz): d 194.12, 158.70, 155.38,
145.04, 113.47, 112.68.
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R. Konakanchi et al.
Cobalt(II) complex of 2-aminonicotinaldehyde
This complex was prepared by direct reaction between the freshly purified ligand
(0.488 g, 4 mmol) and Cobalt(II) chloride hexahydrate, CoCl2Á6H2O (0.474 g,
2 mmol) which were dissolved in 50 mL of anhydrous methanol and refluxed for
3–4 h. The color of the reaction mixture changed from pale yellow to reddish
brown. At the end of the reaction, a brown solid was formed which was
recrystallized from methanol, to obtain 75% yield. Anal. Calcd. For [Co(C6H6N2
O)2Cl2] (%): C, 38.49; H, 3.20; N, 14.97; found: C, 38.43; H, 3.17; N, 14.92. M.P
182–185 °C. IR (KBr, cm-1): 3385 [t(N–H)], 1654 [t(C=O)], 1512 [t(C=N) (Py)].
Copper(II) complex of 2-aminonicotinaldehyde
This complex was prepared by direct reaction between the freshly purified ligand
(0.488 g, 4 mmol) and copper(II) chloride, CuCl2 (0.2689 g, 2 mmol) which were
dissolved in 50 mL of anhydrous methanol and refluxed for 2–3 h. The color of the
reaction mixture changed from pale yellow to brown. At the end of the reaction, a
brownish black solid was obtained, which was recrystallized from methanol, yield
70%. Anal. Calcd. for [Cu(C6H6N2O)2Cl2] (%): C, 38.02; H, 3.16; N, 14.78; found:
C, 37.99; H, 3.12; N, 14.75. M.P 218–220 °C. IR (KBr, cm-1): 3391 [t(N–H)], 1660
[t(C=O)], 1542 [t(C=N) (Py)].
Bio-assay investigations
In vitro antibacterial and antifungal activity
In vitro antibacterial and antifungal activities of the ligand and their metal
complexes were tested by using the disc diffusion method. In vitro antibacterial
activities have been evaluated against 2 Gram-positive bacteria (Bacillus subtilis,
Staphylococcus aureus), 2 Gram-negative bacteria (Klebsiella pneumoniae and
Escherichia coli) and two fungi strains (Asperegillus niger, Pencillium notatum).
Using Whatmann No. 1 paper, 6-mm-diameter sterile antibiotic discs were placed
over nutrient agar medium. By using a micropipette, 100 lg/mL of the concentrated
compounds were transferred to each disc (compounds were dissolved initially in
DMSO). Subsequently, the bacteria and fungi were incubated overnight at 37 and
25 °C, respectively. The zone of inhibition was determined in mm and distinguished
with standard antibiotics. DMSO was used as a negative control while streptomycin
30 lg/disc (standard antibiotic) and standard antifungal drug Ketoconazole (10 lg/
disc) were used as positive controls. All the tests were carried out in triplicate, and
the average zones of inhibition were recorded. Minimum inhibitory concentration
(MIC) values for the tested compounds as well as standards were measured in lg/
mL.
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Synthesis, characterization, biological screening and…
In vitro DPPH free radical scavenging activity assay
The free radical scavenging activity of the synthesized ligand and its metal complexes
were determined by using DPPH (1,1-diphenyl-2-picryl hydrazyl) according to the
literature study [25], DPPH is a well-known radical and a scavenger for other radicals.
Therefore, the DPPH radical was reduced in the presence of an anti-oxidant. In brief,
0.2 mM solution of DPPH in 100 mL methanol was prepared. Then, 3 mL of
methanolic DPPH solution was added to 1 mL of ligand and its metal complexes in
DMSO at different concentrations (3, 10, 30, and 100 lM). The mixture was shaken
vigorously and kept in the dark at room temperature. After 30 min, the absorbance of
the test compounds was measured at 517 nm using a UV–Vis spectrophotometer;
methanol was used as blank. Ascorbic acid was used as standard and DPPH solution
was used as control without the test compounds. The percentage of scavenging
activity of DPPH free radicals was measured using the following formula.
A0 À A1
Scavenging activity ð%Þ ¼
 100:
A0
Here A0, is the absorbance of DPPH in the absence of an anti-oxidant and A1; is the
absorbance of DPPH in the presence of an oxidant. In addition, IC50 values were
calculated for all compounds.
Anticancer activity
In vitro anticancer activity of the synthesized ligand and its metal complexes was
evaluated against three human cancer cell lines, i.e., HeLa (human cervical carcinoma
cell line), MCF-7 (human breast carcinoma cell line) and HEK293T (human embryonic
kidney cell line) by using MTT colorimetric assay as per the ATCC protocol [26, 27].
Cis-platinwasusedasthestandarddrug. Theadherentcellsweretrypsinizedasstatedby
the protocol and, after centrifugation, they were resuspended in newly prepared
medium. By careful pipetting, the cell suspension was mixed to obtain a homogeneous
single cell suspension. Different concentrations of drug solutions were prepared in
media with the final concentration of the DMSO at less than 1%. In each well of a
96-well plate, 100 lL of the cell suspension was transferred into 100 lL of 1% drug
solution. This plate was incubated at 37 °C for 72 h in a CO2 incubator. After 72 h of
incubation, 20 lL of MTT was added to each well. The plate was incubated again for
2 h, then 80 lL of lyses buffer was added to each wellof the plate, which was covered in
aluminum foil to prevent the oxidation of the dye and placed on a shaker overnight.
AbsorbancewasmeasuredonanELISA readerat562 nmwavelength toascertain the%
inhibition of the test and also to compare with the DMSO control.
Results and discussion
The reaction leading to synthesis of ANA and its metal complexes is given in
Scheme 2. The ligand ANA was prepared by the self-condensation of nicotinamide
in the presence of ammonium sulfamate followed by acid-catalyzed hydrolysis. The
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R. Konakanchi et al.
Scheme 2 The synthesis of ANA and its metal complexes M = Ni, Pd, Co and Cu, X = Cl
ligand and Pd(II) complex were also characterized by 1H and 13C NMR (Fig. 4a–d).
Analytical data show that all the complexes have 1:2:2 (metal:ANA:chloride)
stoichiometry (Table 1). The structure of the ligand was elucidated by the
recrystallization of ANA in ethanol. The coordination chemistry of ANA was
explored by the reaction of ANA with corresponding metal salts. Each synthetic
step involved here is straightforward and provided good to moderate yield of the
desired product in pure form.
IR and far-IR spectral studies
The IR spectral data of the ligand (ANA) and its metal complexes are given in
Table 2. The mN–H of NH2 of ANA occurs at 3396 cm-1, the peak at 1667 cm-1
was assigned to mC=O of CHO group and the peak at 1575 cm-1 corresponds to the
mC=N (py) stretching frequency of the heteroatomic ring. The IR spectra of
complexes were similar to the ligand (ANA) with minor shifts in the position of the
peaks. In all the spectra of the complexes, mC=N (py) peak shifted significantly to the
lower wave number [28, 29], suggesting that the pyridine nitrogen of ANA is
coordinated to the metal center. In the far-IR region, the new peaks at
472–421 cm-1 were assigned to mM–N, and at 362, 303, 296, and 290 cm-1 were
assigned to mM-Cl.
Electronic spectra
Generally, electronic absorption studies provide quick and reliable information
about the geometry of metal complexes. These data can serve as an effective tool to
differentiate the tetrahedral, square-planar or octahedral geometries of the transition
metal complexes. The electronic spectral bands of the ligand (L) and its complexes
are listed in Table 3. The UV–Vis spectrum of ANA shows two bands at 38,461 and
28,653 cm-1, corresponding, respectively, to the p ? p* and n ? p* transitions.
However, the Cu(II) complex shows a band around 24,096 cm-1 and a shoulder at
2
2
2
2
12,195 cm-1, which may be due to B1g ? B2g and B1g ? Eg transitions,
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Synthesis, characterization, biological screening and…
Fig. 4
[Pd(ANA)2Cl2] complex
a
1H NMR and b 13C NMR of 2-aminonicotinaldehyde (ANA), c 1H NMR and d 13C NMR of
123

R. Konakanchi et al.
Fig. 4 continued
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Synthesis, characterization, biological screening and…
123

R. Konakanchi et al.
Table 2 IR and far-IR spectral
data (cm-1
Compound
mN–H
mC=O
mC=N(py)
mM–N
mM–Cl
)
Ligand (ANA)
[Ni(ANA)2Cl2]
[Pd(ANA)2Cl2]
[Co(ANA)2Cl2]
[Cu(ANA)2Cl2]
3396
3390
3389
3385
3391
1667
1658
1653
1654
1660
1575
1542
1550
1512
1542
–
–
472
446
451
421
362
303
296
290
Table 3 Electronic spectral data of the ligand and its metal complexes
Compound
ANA
Band position (cm–1
)
Transition
Geometry
28,653
38,461
9500
n ? p*
p ? p*
3T1(F) ? 3A2(F)
3T1(F) ? 3T1(P)
1A1g ? 1Eg
[Ni(ANA)2Cl2]
[Pd(ANA)2Cl2]
[Co(ANA)2Cl2]
[Cu(ANA)2Cl2]
Tetrahedral
19,157
22,540
31,750
9000
Square planar
Tetrahedral
Charge transfer
4A2(F) ? 4T1(F)
4A2(F) ? 4T1(P)
2B1g ? 2Eg
20,400
12,195
24,096
Square planar
2B1g ? 2B2g
respectively, characteristic of square-planar geometry [30]. The UV–Vis spectrum
of the Co(II) complex usually shows three bands in the regions 3000–5000,
6000–10,000 and 15,000–21,000 cm-1
,
which may be assigned to
4A2(F) ? T2(F), A2(F) ? T1(F) and A2(F) ? T1(P) transitions, respectively.
However, in the present Co(II) complex, the latter two bands, observed at around
9000 and 20,400 cm-1, are characteristic of tetrahedral geometry [31]. The Pd(II)
complex exhibits two bands around 22,540 and 31,750 cm-1, which may be
4
4
4
4
4
1
1
assigned to A1g ? Eg and charge transfer transitions, respectively, characteristic
of square-planar geometry [32]. The Ni(II) complex is found to be paramagnetic and
its absorption spectrum showed two bands around 9500 and 19,157 cm-1, which
3
3
could be assigned to T1(F) ? 3A2(F) and T1(F) ? 3T1(P) transitions, respec-
tively, which were orderly with reported tetrahedral geometry [33].
Square-planar Cu(II) complexes show magnetic moment values in the range of
1.8–2.1 BM. These values are somewhat greater than the spin-only value of 1.73
BM and this is due to the spin–orbit coupling followed by lowering of symmetry
[34]. The magnetic moment of the Cu(II) complex is observed as 2.01 BM which
also supports square-planar geometry around the metal ion [35]. The magnetic
moment of the Co(II) complex is 3.87 BM which suggests the paramagnetic
tetrahedral geometry of the complex molecule, while the Ni(II) complex is also
paramagnetic and the magnetic moment at room temperature is found to be 2.91
BM, well in agreement with tetrahedral geometry [36]. The Pd(II) complex is
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Synthesis, characterization, biological screening and…
diamagnetic in nature, and, based on this, square-planar geometry has been
proposed [37].
To study the thermodynamic stability of the complexes in aqueous medium, a
series of parts were prepared with suspensions of 10 mg of the complexes in 20 mL
of water in each part. In UV–Vis spectra of the filtrate of each part at various time
intervals (60 s, 1 h, 5 h, 10 h and 12 h), the spectral patterns were found to be
invariant with the time of suspension, which indicates that the complexes were
thermodynamically stable in aqueous medium. The same trend was observed in the
buffer medium in which biological studies were made.
Cyclic and differential pulse voltammograms
The cyclic (CV) and differential pulse (DPV) voltammograms of all the metal
complexes were recorded in CH3CN (containing 0.1 M TBAP) (Fig. 5). The values
obtained were calibrated with respect to the Fc?/Fc couple. [Cu(ANA)2Cl2] showed
‘
a reversible (ipc/ipa = 0.9–1.0) and diffusion-controlled [ipc/v = constant in the
scan rate (v) range 50–500 mV/s] one-electron transfer (DEp = 60–70 mV;
DEp = 65 3 mV for Fc?/Fc couple) couple at -181 mV. On the other hand,
[Co(ANA)2Cl2] showed an irreversible peak at 967 mV, whereas the Ni(II) complex
of ANA was observed to be electrochemically silent, as expected. The irreversible
oxidative peak observed at this potential cannot be attributed to the oxidation to
Co(II) to Co(III) because the standard reduction potential of Co(III)/Co(II) is ?1.81.
Hence, this voltammetric irreversible oxidative peak is assigned to the removal of
an electron from the lone pair of electrons available on one of the two NH2 groups
of the otherwise chemically equivalent ligand molecules.
NMR spectral studies
1H NMR spectra of ANA were recorded in DMSO-d6, with signals at d 9.84 ppm (s,
1H), 8.24 ppm (d, J = 8.0 Hz, 1H), 8.00 ppm (d, J = 8.0 Hz, 1H), 7.55 ppm (s,
2H, –NH2), 6.72–6.75 ppm (m, 1H). The 13C NMR spectrum of ANA exhibits
Fig. 5 Cyclic and differential
pulse voltamograms of
a [Co(ANA)2Cl2] and
b [Cu(ANA)2Cl2] in CH3CN
123

R. Konakanchi et al.
signals from 112.69 to 194.12 ppm. The signal at 194.12 ppm is assigned to
aldehyde carbon, the peaks at 155.36 ppm and 112.69 ppm are due to quaternary
carbons and the peaks at 113.44, 145.02 and 158.70 ppm are for the remaining
carbons. These peaks in the spectra of the complex appeared with positive
coordination-induced shifts, indicating that the pyridine N of ANA is involved in
bonding with the metal ion.
Crystal structure of ANA
The single-crystal data of ANA are listed in Table 4. The crystallized in the
monoclinic system, with space group P21/c with Z = 8. In the crystal structure, an
asymmetric unit consists of two molecules of ANA, labeled separately as A and B
with the atom-numbering shown in Fig. 6. The classical intramolecular hydrogen
bonding of N–HÁÁÁO (N2–H2aÁÁÁO1 and N4–H2aÁÁÁO2) and intermolecular hydrogen
bonding of N–HÁÁÁN (N2–H2b…..N1 and N4–H4bÁÁÁN3) leads to the formation of
dimeric units of molecules of B and herring bone stacking of molecules of A along
the b-axis (Fig. 7a, b). Furthermore, the non-classical inter-molecular hydrogen
bonding of C–H…..N/O between dimeric units of molecules (C3–H3ÁÁÁO2 and C10–
H10ÁÁÁN3) as a result of glide plane gives rise to a different type of herring bone
stacking pattern of molecules expanded on two dimensions (Fig. 7c). In addition to
this hydrogen bonding, the aldehyde hydrogen H12, aromatic ring carbon hydrogens
H4 and H9 are also involved in trifurcated hydrogen bonding with O1, which
connects the molecules together in the crystal structure Fig. 8. The aromatic rings
˚
are further stabilized by weak displaced p–p interaction (3.955A). The overall
intermolecular non-covalent interactions and packing diagram of the molecules
extended in two dimensions are shown in Fig. 9. In a nutshell, the three-dimensional
network of ANA consists of classical and non-classical hydrogen bonding networks
for its 3D architecture (Fig. 10). The H-binding data of ANA are listed in Table 5.
Electron spin resonance
The electron spin resonance spectrum of the Cu(II) complex was recorded in
benzene solution at room temperature in the solid state (Fig. 11). It shows a broad
band centered at g = 2.21, without a resolved hyperfine structure with the values
gk = 2.144 and g? = 2.087. The gk [ g? [ 2.0023, showing that the unpaired
2
electron in the ground state of Cu(II) predominantly lies in the d2X – orbital and
Y
also refere to a square-planer geometry around the Cu(II) ions [38]. The observed gk
value of the metal complex is less than 2.3, which is in agreement with the covalent
character of the metal ligand bond [39]. The geometric parameter G is calculated
using the equation:
gk À 2:0023
G ¼
:
g? À 2:0023
According to Hathaway and Billing [40, 41], if the value of G is greater than 4, this
indicates that the exchange interaction between the Cu(II) centers in the solid state
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Synthesis, characterization, biological screening and…
Table 4 Crystallographic data
Compound
Ligand (ANA)
of ANA
Empirical formula
Formula weight
C6H6N2O
122.13
˚
Wavelength (A)
0.71073
Monoclinic
P21/c
Crystal system
Space group
˚
a (A)
22.8474 (14)
3.9552 (2)
13.1652 (8)
90
˚
b (A)
˚
c (A)
a (°)
b (°)
c (°)
94.465 (4)
90
Volume (3A
˚
)
1186.1 (1)
8
Z
-3
Dcalc. (M
g m
)
1.368
Temperature (K)
Absorption coefficient (mm-1
296
)
0.097
F (000)
Crystal size (mm3)
512
0.23 9 0.20 9 0.18
0.89–25.99
12,978/2321
25.99, 100%
0.1902
h range for data collection (°)
Reflections collected/unique
Completeness to h
Independent reflections (Rint
Max./min. transmission
Data/parameters
Goodness-of-fit on F2
Final R indices[I [ 2r(I)]
wR2 = 0.1071b,c
)
0.9827/0.9779
2321/212
0.881
P
P
(|FO| -|FC|)/ |FO|
P
a
R1
=
R1 = 0.0497a
P
b
wR2 = { [w (F2O – FC2 )2]/
[w(F2O)2]}1/2
c
R indices(all data)
wR2 = 0.1152b,c
R1 = 0.0733a
w =
1/[r2(F2O) ? (aP)2 ? bP] with
P = [F2o ? 2F2c]/3
Extinction coefficient
Largest diff. peak and hole (eA-3
0.044 (4)
a = 0.0333 and b = 0.000 for
ANA
˚
)
0.235 and -0.235
is negligible, whereas when its value is less than 4, considerable exchange inter-
actions exist in the solid complex. The value G = 1.672 for the exchange inter-
action parameter for the Cu(II) complex is less than 4, indicating that there is a spin
exchange interaction between Cu(II) ions in the solid state.
Antibacterial activity
The in vitro antibacterial evaluation results reveal that the ligand ANA does not
show any activity against the strains, the complexes [Cu(ANA)2Cl2] and
[Co(ANA)2Cl2] exhibited good activity against S. aureus (12.5 lg/mL) and
123

R. Konakanchi et al.
Fig. 6 ORTEP diagram of ligand up to 50% probability showing intramolecular N–HÁÁÁO hydrogen
bonds (with atom numbering)
moderate activity against three strains, B. subtilis (25 lg/mL), E. coli (25 lg/mL)
and K. pneumoniae (25 lgmL). The complexes [Ni(ANA)2Cl2] and [Pd(ANA)2Cl2]
exhibited poor activity against S. aureus, B. subtilis, E. coli and K. pneumoniae
compared to the standard drug streptomycin. ANA does not show any activity,
whereas the activity of the [Cu(ANA)2Cl2] and [Co(ANA)2Cl2] complexes against
four strains may be attributed to the coordination of ANA with metals. The MIC of
the complexes are presented in Table 6. In conclusion, [Co(ANA)2Cl2] and
[Cu(ANA)2Cl2] complexes showed good activity against S. aureus and moderate
activity against B. subtilis, E. coli and K. pneumoniae in comparision with the
[Ni(ANA)2Cl2] and [Pd(ANA)2Cl2] complexes.
Antifungal activity
ANA and its metal complexes were investigated for in vitro antifungal activity
against the fungal strains Aspergillus niger and Pencillium notatum, and compared
with the standard drug Ketoconazole with MIC values. The results of the in vitro
antifungal activity in MIC of the tested compounds are presented in Table 7 in
which it can be seen that the complexes [Cu(ANA)2Cl2] and [Co(ANA)2Cl2]
exhibited very good activity against Aspergillus niger and Pencillium notatum, with
IC50 values (6.25 and 6.25 lg/mL) and (6.25 and 12.5 lg/mL), respectively. The
complexes [Ni(ANA)2Cl2] and [Pd(ANA)2Cl2] exhibited moderate to poor activity
against the strains, with IC50 values (50 and 50 lg/mL) and (50 lg/mL). The ligand
ANA had no activity, but its complexes [Cu(ANA)2Cl2] and [Co(ANA)2Cl2]
showed good activity.
The metal ion and ligand association exerts a synergistic effect on the activity of
the free ligand [42, 43]. The metal ion–ligand interaction in the complexes leads to
the activity of changing the electron distribution in the ligand. In conclusion,
[Co(ANA)2Cl2] and [Cu(ANA)2Cl2] complexes showed potent activity against
Aspergillus niger and Pencillium notatum, in comparison with the [Ni(ANA)2Cl2]
and [Pd(ANA)2Cl2] complexes.
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Synthesis, characterization, biological screening and…
123

R. Konakanchi et al.
bFig. 7 a Intermolecular interactions of the ligand ANA, intermolecular hydrogen bonding depicting
dimer formation of B and herring bone pattern formation A. b Herring bone stacking pattern of molecule
A along b axis. c Herring bone pattern of dimeric units of molecules B
Fig. 8 Intermolecular non-classical hydrogen bonding between molecules A and B
Antioxidant activity
The IC50 values of the ligand and its metal complexes are given in Table 8. It can be
seen that, by increasing the concentration of the compounds, the scavenging activity
also increases. The DPPH assay of the metal complexes (1–4) was calculated as
percentage inhibition (Fig. 12). From the results, it is shown that the scavenging
effect of the free ligand is significantly less compared to the metal complexes [25].
The antioxidant activity of the complexes [Ni(ANA)2Cl2] and [Cu(ANA)2Cl2] show
better activity with IC50 values at 12.39 1.26 and 14.82 1.27 lM, respectively.
Analogues [Pd(ANA)2Cl2] and [Co(ANA)2Cl2] complexes showed moderate
activity with IC50 values, 16.27 1.19 and 19.49 1.30 lM, respectively.
According to IC50 values, the scavenging activity order of the compounds follows:
½Ni(ANA)2Cl2Š [ ½Cu(ANA)2Cl2Š [ ½Pd(ANA)2Cl2Š [ ½Co(ANA)2Cl2Š [ ANA:
In conclusion, the complexes [Ni(ANA)2Cl2] and [Cu(ANA)2Cl2] complexes have
shown good anti-oxidant activity compared to the [Pd(ANA)2Cl2] and
[Co(ANA)2Cl2] complexes.
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Synthesis, characterization, biological screening and…
Fig. 9 Two-dimensional stacking diagram depicting the intermolecular H-bonding
Anticancer activity
The ligand and its metal complexes were evaluated against HeLa, MCF-7 and HEK-
293T using MTT assay, with cis-platin is used as standard drug. The relationship
between the surviving fraction and drug concentration was plotted to obtain the
survival curves of HeLa, MCF-7 and HEK 293T (Fig. 13a–c). The IC50 values
calculated for the synthesized compounds against the tested cell lines are listed in
Table 9. The results show that the [Ni(ANA)2Cl2] complex exhibited good activity
against the three cancer cell lines, with IC50 values 26.40 0.29, 22.86 0.29 and
34.23 0.27 lM, respectively, compared with the standard drug cis-platin. The
[Pd(ANA)2Cl2] complex showed good activity against the HeLa cell line with IC50
values 27.48 0.20 lM, the [Co(ANA)2Cl2] complex showed good activity
against the MCF-7 cell line with IC50 values 27.48 0.12 lM and [Cu(ANA)2
Cl2] complex showed significant activity against the two cell lines, HeLa and MCF-
7, with IC50 values 29.04 0.37 and 31.26 0.18 lM, respectively. The metal
complexes were also screened for cytotoxic activity against HEK-293. IC50 values
of the [Pd(ANA)2Cl2], [Co(ANA)2Cl2] and [Cu(ANA)2Cl2] complexes were
123

R. Konakanchi et al.
Fig. 10 3D-crystal structure of ANA
Table 5 Hydrogen-bond parameters of ANA
Compound D–H…A
ANA
D–H
DÁÁÁA
HÁÁÁÁA
\D–HÁÁÁÁA Symmetry code
N2–H2aÁÁÁO1 1.00 (2) 2.745 (2) 2.02 (2) 127 (2)
N4–H4aÁÁÁO2 0.97 (2) 2.781 (2) 2.06 (3) 130 (2)
x, y, z
x, y, z
C3–H3ÁÁÁO2
C4–H4ÁÁÁO1
0.91 (2) 3.662 (2) 2.79 (2) 158 (2)
x, y - 1, z ? 1
0.98 (2) 3.538 (3) 2.61 (2) 154 (2)
x, - y ? 1/2, z ? 1/2
x, - y ? 1/2, z ? 1/2
x, - y ? 1/2 ? 1, z - 1/2
-x,- y ? 1/2 ? 1, z - 1/2
-x ? 1, - y ? 1,-z
C
10–H10ÁÁÁN4 0.96 (2) 3.666 (2) 2.88 (2) 138 (1)
C9–H9ÁÁÁO1
C
0.94 (2) 3.472 (2) 2.57 (2) 159 (2)
12–H12ÁÁÁO1 1.01 (2) 3.558 (3) 2.59 (2) 159 (2)
N4H4bÁÁÁN3
0.92 (2) 3.073 (2) 2.14 (2) 176 (2)
N2–H2bÁÁÁN1 0.84 (2) 3.082 (2) 2.25 (2) 167 (2)
-x, y ? 1/2,
- z = 1/2 ? 1
72.32 0.21, 40.58 0.20 and 44.74 0.35 lM, respectively. The results are
presented in Table 9. In conclusion, the [Ni(ANA)2Cl2] complex showed good
activity against HeLa, the MCF-7. [Pd(ANA)2Cl2] complex against HeLa and the
[Co(ANA)2Cl2] complex against MCF-7.
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Synthesis, characterization, biological screening and…
Fig. 11 ESR spectrum of the Cu(II) complex
Table 6 Antibacterial activity
MIC (lg/mL)
of the compounds MIC (lg/mL)
Compound
S. aureus B. subtilis K. pneumoniae E. coli
ANA
[100
[100
100
50
[100
[100
[100
25
[100
50
[Ni(ANA)2Cl2] 50
[Pd(ANA)2Cl2] 100
[Co(ANA)2Cl2] 12.5
[Cu(ANA)2Cl2] 12.5
50
25
50
25
25
25
Streptomycin
6.25
12.5
12.5
6.25
Molecular docking studies
Our aim was to explain the interactions of metal complexes with protein receptors,
and we have chosen a computer-aided experimental method, molecular docking.
Chem Draw Ultra 12.0 has been used to draw the 2D structures of the metal
complexes and the structures have been fully optimized with the small hf/3–21 g*
[44], basic set by using Guassian 09 [45]. The crystallographic 3D structure of the
EGFR protein receptor was retrieved from RSC PDB (an information portal for
123

R. Konakanchi et al.
Table 7 Antifungal activity of
the compounds (MIC (lg/mL)
MIC (lg/mL)
Compound
Asperegillus niger Pencillium notatum
ANA
[100
50
[100
50
[Ni(ANA)2Cl2]
[Pd(ANA)2Cl2]
[Co(ANA)2Cl2]
[Cu(ANA)2Cl2]
50
[100
12.5
6.25
3.125
6.25
6.25
Standard(Ketoconazole) 3.125
Table 8 IC50 (lM) values of
DPPH scavenging activity of
ligand and its metal complexes
Sample no.
Compound codes
IC50 (lM)
1
2
3
4
5
6
[Ni(ANA)2Cl2]
[Pd(ANA)2Cl2]
[Co(ANA)2Cl2]
[Cu(ANA)2Cl2]
ANA
12.39 1.26
16.27 1.19
19.49 1.30
14.82 1.27
20.60 1.38
6.53 1.19
Order of reactivity: 1 [ 4 [
2 [ 3 [ 5
(Standard) Ascorbic Acid
Fig. 12 Radical scavenging activity of ligand and metal complexes in terms of IC50 value (50%
b
c
inhibition): Letters over columns:a p \ 0.05, p \ 0.01, p \ 0.001 and p [ 0.05 as non-significant. It
was determined by unifactorial analysis of variance (ANOVA) followed by Dunnett’s multiple
comparison test using Graphpad Prism Software v.5.3
biological macromolecular structures), PDB ID: 4HJO. It is an attractive target in
cancer disease. EGFR plays a major role in the causing of cancer including squa-
mous-cell carcinoma of the lung, anal cancers, and epithelia tumors of the head and
neck [46] in the human body. Mutations involving EGFR lead to its constant
123

Synthesis, characterization, biological screening and…
Fig. 13 Survival curves of HeLa, MCF-7 and HEK293 T cell lines. a Anticancer activity (HeLa),
b anticancer activity (MCF-7), c anticancer activity (HEK293)
123

R. Konakanchi et al.
Table 9 IC50 values of anticancer activity of compounds (lM)
Sample no.
Compound codes
IC50 values (lM)
HeLa
MCF-7
HEK 293T
1
2
3
4
5
6
[Ni(ANA)2Cl2]
[Pd(ANA)2Cl2]
[Co(ANA)2Cl2]
[Cu(ANA)2Cl2]
ANA
26.40 0.29
27.48 0.20
34.23 0.31
29.04 0.37
56.26 0.49
10.14 0.17
22.86 0.29
40.580 0.30
27.48 0.12
31.26 0.18
46.86 0.28
4.77 0.16
34.23 0.27
72.32 0.21
40.58 0.20
44.74 0.35
51.03 0.41
52.56 0.39
Standard (Cis-platin)
Table 10 Binding energies of
metal complexes with EGFR
protein receptor (4HJO)
Sample no.
Compound
Binding energy (K cal/mol)
1
2
3
4
[Co(ANA)2Cl2]
-4.16
-4.64
-4.88
-5.46
[Ni(ANA)2Cl2]
[Cu(ANA)2Cl2]
[Pd(ANA)2Cl2]
activation, and it develops uncontrolled cell division [47]. The extracted protein
structures were prepared by removing water molecules and minimizing the energy
of the protein receptor with UCSF chimera 1.10.1 software used for docking. The
metal complexes [Co(ANA)2Cl2], [Ni(ANA)2Cl2], [Cu(ANA)2Cl2] and [Pd(ANA)2
Cl2] were bound to the receptor with minimum binding energies -4.16, -4.64,
-4.88 and -5.46 K cal/mol, respectively. The docking results were compared with
the experimental IC50 values in Table 10. The best conformations of ligands
forming a cluster into the binding pocket of the receptor are shown in Fig. 14a–d.
From these results, we can conclude that, in the protein EGFR complex, all the
interactions of the [Co(ANA)2Cl2] complex are hydrophobic interactions with
amino acids, Ser167, Tyr126, Ser267, Glu171, His346, Phe214, Phe270, Phe163,
Val170, and Ala264. In the [Ni(ANA)2Cl2]-protein receptor complex, all the similar
interactions were found with the amino acids, Phe699, Asp813, Ala698, Phe699,
Lys721, Asp813, Asn818, Lys851, Pro853, and Arg817. In the [Cu(ANA)2Cl2]
protein receptor complex, the same kind of interactions were found with amino
acids, Arg817, Asn818, Asp813, Asp831, Ala698, and Pro853. In addition to these
interactions, p–p stacking interactions were observed.
123

Synthesis, characterization, biological screening and…
Fig. 14 a–d Shows the binding process and interactions of metal complexes of [Co(ANA)2Cl2],
[Ni(ANA)2Cl2], [Cu(ANA)2Cl2] and [Pd(ANA)2Cl2] to the binding sites of the EGFR protein receptor
123

R. Konakanchi et al.
Conclusions
We have, for the first time, demonstrated the coordination behavior of 2-aminoni-
cotinaldehyde with transition metals Ni(II), Pd(II), Co(II) and Cu(II). The ligand
ANA and its metal complexes have been synthesized and characterized by using
various spectroscopic techniques. ANA acts as a mono-dentate ligand towards the
metal ions through the N atom of the pyridine ring, orienting the aldehyde and
amine potential functional groups of the ligand in the metal complexes. Based on
these studies, the structures of the Pd(II) and Cu(II) complexes may be tentatively
proposed as having square-planar geometry, whereas the Ni(II) and Co(II)
complexes show tetrahedral geometry with the [M(N)2(Cl)2] coordination sphere.
The biological activity may be specific due to metal–ligand orientations facilitating
hydrophobic interactions and p–p stacking in the protein receptor complex. The
metal complexes Co(II) and Cu(II) were potent antibacterial and antifungal agents.
The Ni(II) complex showed the highest scavenging activity and displayed good
anticancer activity against the HeLa, MCF-7 and HEK 293 human cancer cell lines,
compared with other metal complexes.
Supplementary material
CCDC 801685 contains the supplementary crystallographic data of ligand ANA.
The data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/
retrieving.html, or from the Cambridge Crystallographic Data Centre, 12 Union
Road, Cambridge, CB2 1EZ, UK; fax: (?44) 1223-336-033; or e-mail:
deposit@ccdc.cam.ac.uk.
Acknowledgements I am very grateful to Dr. Munirathinam, IISC Bangalore, for providing the single-
crystal XRD facility. I am grateful to the Ministry of Human Resource Development (MHRD), India for
awarding a research fellowship to me.
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