Pyrazine based Pt(II) bis-alkynyl organometallic complexes: Synthesis, characterization, and cytotoxic effect on A549 human lung carcinoma cells

Article abstract of DOI:10.1002/aoc.3824

A facile and efficient synthesis of a series of five new pyrazine-based organometallic complexes is being reported. The complexes (2–6) have been fully characterized. Molecules 2, 3 and 6 were structurally characterized by single crystal X-ray crystallographic analyses. Additionally, anticancer properties of these organometallic complexes have been studied against A549 human lung carcinoma cells. Biological studies suggest that complex 5 (with pendant pyridine moieties) exhibited maximal growth inhibitory cytotoxic effect even at lower concentration (nearly 0.5–1?μM) whereas the other four compounds (2, 3, 4 and 6) were non-toxic below 1?μM concentration. Two complexes (4 and 6) were effective only at higher doses (approx 30?μM) while the remaining two organometallic complexes (2 and 3) were able to inhibit 50% cell growth at slightly lower concentration (nearly 10?μM). As far as IC₅₀ is concerned, 5 exhibited a minimum value ranging between 3–5?μM which is comparable with cisplatin under similar conditions.

Full text of DOI:10.1002/aoc.3824

Received: 16 January 2017
DOI: 10.1002/aoc.3824
Revised: 3 March 2017
Accepted: 4 March 2017
F U L L PA P E R
Pyrazine based Pt(II) bis‐alkynyl organometallic complexes:
Synthesis, characterization, and cytotoxic effect on A549 human
lung carcinoma cells
Sourav Bhowmick1 | Achintya Jana1 | Subba R. Marri2 | Prerak Gupta3 | J.N. Behera2 |
Biman B. Mandal3 | Neeladri Das1
1 Department of Chemistry, Indian Institute
of Technology Patna, Patna 801 106, Bihar,
A facile and efficient synthesis of a series of five new pyrazine‐based organometal-
lic complexes is being reported. The complexes (2–6) have been fully characterized.
Molecules 2, 3 and 6 were structurally characterized by single crystal X‐ray crystal-
lographic analyses. Additionally, anticancer properties of these organometallic com-
India
2 School of Chemical Sciences, National
Institute of Science Education and Research
(NISER), Bhubaneswar 751005, India
3 Department of Biosciences and
Bioengineering, Indian Institute of
Technology Guwahati, Guwahati 781039,
Assam, India
plexes have been studied against A549 human lung carcinoma cells. Biological
studies suggest that complex 5 (with pendant pyridine moieties) exhibited maximal
growth inhibitory cytotoxic effect even at lower concentration (nearly 0.5–1 μM)
whereas the other four compounds (2, 3, 4 and 6) were non‐toxic below 1 μM
concentration. Two complexes (4 and 6) were effective only at higher doses (approx
30 μM) while the remaining two organometallic complexes (2 and 3) were able to
inhibit 50% cell growth at slightly lower concentration (nearly 10 μM). As far as
IC50 is concerned, 5 exhibited a minimum value ranging between 3–5 μM which
is comparable with cisplatin under similar conditions.
Correspondence
Neeladri Das, Department of Chemistry,
Indian Institute of Technology Patna, Patna
801 106, Bihar, India.
Email: neeladri@iitp.ac.in;
neeladri2002@yahoo.co.in
Biman B. Mandal, Department of
Biosciences and Bioengineering, Indian
Institute of Technology Guwahati, Guwahati,
India.
KEYWORDS
A549 human lung carcinoma cells, anticancer, cytotoxic effect, organometallic complex, pyrazine, X‐ray
crystallography
Email: biman.mandal@iitg.ernet.in; mandal.
biman@gmail.com
1 | INTRODUCTION
not limited to undesirable harmful side‐effects and antitu-
mor activity with a relatively narrow spectrum.[4] In this
Research in the development of new organometallic
complexes bearing metal alkynyl linkages has gained
much importance since the last decade.[1] The interest in
such π‐conjugated metal–organic units is attributed to their
potential use as smart materials with applications in the field
of electronics, optics, sensors and design of luminescent
devices.[2] More importantly, several organometallic
complexes have been screened as potential candidates for
use in treatment of (lungs) cancer.[3] The need to develop
new anticancer compounds stems from the fact that widely
used platinum based drugs (cisplatin, carboplatin and
oxaliplatin) have several drawbacks that include but are
context, it may be mentioned that polynuclear platinum
complexes may be considered as alternative drugs to
cisplatin (and its derivatives) with the objective to widen
the spectrum of platinum based anticancer drugs concomi-
tantly reducing the other side effects.[5]
As far as organometallic complexes with multiple plati-
num centers are concerned, several organic motifs such as
anthracene, phenanthrene, carbazole, fluorene, truxene,
iptycene, oxadiazole, and others have been incorporated.[6]
Recently, we have reported Pt(II) organometallic complexes
derived from 2,6‐dichloropyrazine as a precursor unit.[7] This
current research interest, i.e., development of pyrazine unit
Appl Organometal Chem. 2017;e3824.
wileyonlinelibrary.com/journal/aoc
Copyright © 2017 John Wiley & Sons, Ltd.
1 of 11
https://doi.org/10.1002/aoc.3824

2 of 11
BHOWMICK ET AL.
containing Pt(II) organometallic complexes, has prompted us
to further explore synthesis of newer π‐conjugated Pt(II)‐
ethynyl molecules with a central pyrazine moiety. In this con-
text and to the best of our knowledge, pyrazine‐containing
and π‐conjugated Pt(II)‐alkynyl complexes are quite rare
and their biological activities have not been studied to date.
Herein, we report the synthesis and characterization of
five new organometallic complexes (2–6) that contain a pyr-
azine ring in the center and have two Pt(II)‐alkynyl branches
protruding on either side. All five complexes have been char-
and 6 have been structurally characterized by using single
crystal X‐ray crystallography analysis.
In the FTIR spectrum of 2–6, the presence of an ethynyl
functional group was evident because of the presence of an
intense peak in the region 2080–2100 cm−1. The 31P{1H}
NMR spectrum of each complex (2–6) exhibits one sharp
singlet signal at δ 11.30, 11.96, 11.26, 11.47 and
11.50 ppm with accompanying 195Pt satellites of 1JPPt = 1182,
1177, 1193, 1174 and 1175 Hz, respectively (Figure 1 and
supporting information S2‐6). The appearance of one sharp
signal in the 31P{1H} spectrum is indicative of all phosphorus
nuclei (attached to platinum(II) centers) being chemically
equivalent. It also suggests that complexes (2–6) have an
overall symmetrical structure. The respective 1H NMR
spectra of 2–6 (recorded in CDCl3) display a sharp singlet
in the range of 8.10–8.12 ppm and this corresponds to the
two chemically equivalent aromatic protons present in the
pyrazine ring. As expected, the methylene and methyl
protons of the PEt3 groups appear as multiplets in the range
of 2.21–1.17 ppm. In case of 2, the three sets of signals in
the range of 7.29 to 7.27 ppm, 7.23 to 7.19 and 7.14 to
7.12 ppm are attributed to the phenyl protons in this molecule
(supporting information S2). For 3, the three sets of signals in
the range of 7.58 to 7.56 ppm, 7.47 to 7.45 and 7.41 to
7.31 ppm are ascribed to the biphenyl protons in this product
(supporting information S3). In complex 4, two sets of sig-
nals corresponding to the protons of the terminal phenyl rings
are observed in the range of 7.23 to 7.22 and 6.77 to
6.75 ppm. Additionally, the methoxy protons in the case of
4 are observed as a sharp singlet at 3.78 ppm (supporting
1
acterized by FTIR, NMR [31P{1H} and H], ESI‐MS and
CHN analyses. Additionally, the structure of three complexes
(2, 3 and 6) has been unambiguously determined using single
crystal X‐Ray analysis. Anticancer activity of all five organo-
metallic complexes was tested using human lung carcinoma
cells (A549) and their cytotoxicity has been compared with
cisplatin under similar conditions.
2 | RESULT AND DISCUSSION
2.1 | Synthesis and characterization of the
pyrazine containing organometallic complexes
2,6‐dichloropyrazine has been used as the synthon for the
synthesis of all five organometallic platinum(II) bis‐alkynyl
complexes 2–6. 2,6‐diethynylpyrazine obtained in the first
step (Scheme 1) was reacted with two equivalents of trans‐
PtI2(PEt3)2 in the presence of CuI (catalytic amount) and
triethylamine (base) to yield the organometallic linker (1).7
1
Subsequently, using
a
dehydrohalogenation protocol,
information S4). The H spectrum of complexes 5 and 6 are
reaction of 1 with various terminal alkynyl derivatives (at
ambient temperature) yielded the respective organometallic
complexes 2–6 in high yields and these have been character-
ized by Fourier transform infrared (FTIR), multinuclear
NMR (1H and 31P{1H}), ESI‐MS spectroscopy and elemen-
tal analysis. Additionally, organometallic complexes 2, 3
somewhat similar with the appearance of two sets of signals
in the aromatic region – signals in the range of 8.40 to
8.39 ppm and 7.10 to 7.08 ppm in complex 5 due to the
pyridyl protons (Figure 1) and peaks in the range
7.49–7.46 ppm and 7.30–7.28 ppm due to phenyl protons
in complex 6 (supporting information S6).
SCHEME 1 Synthesis of pyrazine based
organometallic complexes 2–6

BHOWMICK ET AL.
3 of 11
1
FIGURE 1 (a) 31P{1H} NMR and (b) H NMR spectra of complex 5
FIGURE 2 Asymmetric unit of 2 (ORTEP plot with 30% probability) hydrogen atoms are omitted for clarity (color code: C = gray; N = blue;
P = pink and Pt = orange)
2.2 | X‐ray crystallography analysis of
organometallic complex C48H72N2P4Pt2(2)
X‐ray quality crystals of 2 were obtained by slow evaporation
from a solution of ethylacetate and hexane. Complex 2
crystallized with the space group of Pbca. In the asymmetric
unit (Figure 2), the two platinum atoms have the same coor-
dination environment (square‐planar geometry and bonded
to two carbon and two phosphorus atoms). The distance
between the two platinum atoms is 10.9 Å. The angle
between the Pt‐pyrazine‐Pt is around 133.5°. Two weak
non‐classical interactions (C‐H…N and C‐H…π) were also
observed as depicted in Figure 3. One of the nitrogen atoms
present in the pyrazine ring has a weak hydrogen bond inter-
action (3.0 Å) with a phenyl C‐H unit(s) present in a neigh-
boring molecule. This interaction gets extended along the
ab‐plane throughout the lattice thereby generating a 1D–
polymer as shown in Figure 3(a). Additionally, C‐H…π
(3.2 Å) interactions between the methyl C‐H unit(s) and aro-
matic rings (both pyrazine and phenyl) present in adjacent
molecules are also observed. These C‐H…π interactions also
get extended through the entire lattice along the c‐axis as
FIGURE 3 (a) non‐classical hydrogen bond interactions (fragmented
bonds) of the complex 2 viewed along the c‐axis. Some parts of the
molecules omitted for clarity. (b) View of C‐H….π interactions in the
solid state structure shown with the fragmented bonds

4 of 11
BHOWMICK ET AL.
shown in the Figure 3(b). The selected bond lengths and
bond angles are given in the supporting formation S10‐12.
2.3 | X‐ray crystallography analysis of
organometallic complex C60H80N2P4Pt2(3)
Crystals of 3 were obtained also by slow evaporation from a
solution of ethylacetate and hexane. The complex 3 crystal-
lized with a P‐1space group. The asymmetric unit of 3 is
shown in Figure 4. In the asymmetric unit, the two Pt(II)
atoms have the same coordination environment and a
square‐planar geometry. The distance between the two Pt
atoms is 10.6 Å. The Pt‐pyrazine‐Pt angle was observed to
be 127.7°. The solid state structure of 3 is similar to that of
2 except that in 3, an extra phenyl group is present. As a
result, the nature of weak interactions is somewhat different
in 2 and 3. The solid state crystal structure of 3 contains
two different types of non‐classical hydrogen (C‐H….N and
C‐H….π) bond interactions as shown in Figure 5. Nitrogen
centers of the pyrazine unit display weak hydrogen bond
interactions with an ethyl C‐H hydrogen (C‐H….N = 3.0 Å)
present in adjacent molecules. These interactions extend in
the lattice along the a‐axis to form a 1D–polymer as shown in
Figure 5(a). Additionally, C‐H….π (3.2 Å) interactions
between the methyl C‐H and the aromatic rings (both pyr-
azine and phenyl) present in adjacent molecules are also
observed. These are extended along the a‐axis through the
entire crystal lattice as shown in Figure 5(b). The selected
bond distances and bond angles are given in supporting infor-
mation S12‐14.
FIGURE 5 (a) non‐classical hydrogen bond interactions (fragmented
bonds) in the crystal lattice of 3 as viewed along the a‐axis. (b) View of
C‐H….π interactions of 3 in the solid sate structure shown with the
dotted lines. Some part of the atoms in the molecules have been removed
The crystal lattice of 6 also consists of various non‐classical
weak interactions (C_H….N and C‐H….π) as shown in
Figure 7. One of the nitrogen atoms on the pyrazine moiety
shows hydrogen bonding interactions with the methyl C‐H
of an adjacent molecule thereby generating a 1D–polymeric
chain in the ac‐plane [Figure 7(a)]. C‐H….π interactions
(3.2 Å) between methyl C‐H and aromatic rings (both phenyl
and pyrazine ring) present in a neighboring molecule are
also observed as shown in the Figure 7(b). These
interactions are extended in the crystal structure along the
b‐axis. Additionally, the nitrile nitrogen atoms also display
weak interactions (3.0 Å) with ethyl C‐H hydrogen atoms
present in phosphines of an adjacent molecule. The
selected bond lengths and bond angles are given in
supporting information S15.
2.4 | X‐ray crystallography analysis of
organometallic complex C50H70N4P4Pt2 (6)
Slow evaporation of an ethylacetate/hexane solution of 6
yielded good quality and transparent single crystals of 6 for
X‐Ray diffraction. The complex 6 crystallized in the P2/c
space group. The asymmetric unit of this complex is shown
in Figure 6 with two Pt(II) atoms having square‐planar
geometry. The angle between the Pt‐pyrazine‐Pt is 114.9°.
The distance between the two Platinum centers is 10.05 Å.
FIGURE 4 Asymmetric unit of 3
(ORTEP plot with 35% probability).
Hydrogen atoms are omitted for clarity (color
code: C = gray; N = blue; P = pink and
Pt = green)

BHOWMICK ET AL.
5 of 11
FIGURE 6 Asymmetric unit of 6. The
ellipsoids are shown at 35% probability level.
Hydrogen atoms are omitted for clarity (color
code: C = gray; N = blue; P = pink and
Pt = green)
of intraligand transitions (π ! π*) and [dπ(Pt) ! π*(C ≡ C)
2‐arene] involving the peripheral alkynyl‐arene moieties that
are absent in case of ligand 1.
2.6 | In vitro cytotoxicity
The use of platinum complexes is quite common as a thera-
peutic agent against tumorigenic diseases.[9] However, their
clinical implementation is limited owing to emergence of
resistance and side effects. It is well known that cisplatin is
a Pt(II) containing anticancer drug that has two chloride
and two NH3 ligands attached to the square planar Pt center.
This molecule, upon being administered in saline water,
undergoes hydrolysis leading to the substitution of chloride
ligands by water. In the resulting intermediate aqua complex
cis‐[PtCl(NH3)2(H2O)]+, the labile water molecules can be
easily displaced by purine/pyrimidine bases of DNA.[10]
However, in complexes (2–6) reported in this manuscript,
the Pt center is not bound to easily displaceable (labile)
groups such as chloride or water. Therefore we were curious
to study the cytotoxicity of these complexes (2–6) that don't
have suitable leaving groups and hence cannot coordinate to
DNA, unlike cisplatin. Thus, the pyrazine based organome-
tallic complexes reported herein were investigated for their
potential cytotoxicity. The dose dependent cytotoxic effect
of organometallic compounds 2–6 was tested with A549
human lung carcinoma cells. The experimental compound
concentration was adopted based on the prior studies ranging
between 1 and 30 μM.[11,12] A cisplatin stock solution was
prepared in normal saline owing to its inactivation in
DMSO.[12] All the compounds showed a dose dependent
cytotoxic effect (Figure 9). However, cisplatin exhibited a
similar inhibitory effect at the following four different con-
centrations: 30 μM, 10 μM, 5 μM and 1 μM (nearly 50% cell
viability). This might be attributed to emergence of drug
resistance developed after treatment.[13] Complex 5 exhibited
maximal growth inhibitory effect even at lower concentration
(below 3 μM) whereas other compounds viz. 2, 3, 4, and 6
were non‐toxic below 1 μM concentration (p < 0.05, which
means that the cytotoxic activity of compound 5 was
FIGURE 7 (a) non‐classical hydrogen bond interactions between the
C‐H….N in the solid state structure of 6. (b) View of C‐H…π interactions
in ac‐plane
2.5 | Photophysical properties of
organometallic complexes 1–6
A methanolic solution (2 μM) of the dinuclear platinum(II)
organometallic complexes (1–6) was utilized to record their
respective UV–Vis absorption spectrum in solution state.
Complex 1 exhibits a strong band with a maximum centered
at 369 nm whereas in case of 2–6, two intense absorption
bands were observed beyond 300 nm. The first one appears
in the range of 315–325 nm and the second one was observed
in the range 365–375 nm with extinction coefficients of the
order of 104 dm 3mol−1 cm−1 (Figure 8 supporting
Information S7‐9). In the case of iodoplatinum(II) precursor
1, the absorption band centered at 369 nm is assigned to
metal‐to‐ethynyl metal to ligand charge transfer (MLCT)
transition such as [dπ(Pt) ! π*(C ≡ C)2‐pyra‐2,6] as well
as intraligand [π ! π*(C ≡ C)2‐pyra‐2,6] transition involving
of the central alkynyl core.[8] Similarly, for dinuclear
complexes 2–6, the band in the region of 365–375 may be
ascribed to [dπ(Pt) ! π*(C ≡ C)2‐pyra‐2,6] MLCT and
[π ! π*(C ≡ C)2‐pyra‐2,6]. In these complexes (2–6), the
additional band in the range of 315–325 nm is due to mixing

6 of 11
BHOWMICK ET AL.
FIGURE 8 UV–Vis absorption spectrum of linker 1 and 2
TABLE 1 IC50 value of each compound with A549 cells calculated
after 48 h treatment
Compound
IC50 (μM)
2
10–30
10–30
>30
3
4
5
6
3–5
10–30
3–10
Cisplatin
10 μM). Complex 5 (with pendant pyridine moieties)
exhibited a minimum IC50 value i.e. ~5 μM. Cisplatin
however exhibited almost similar inhibitory effect at higher
concentrations (3–30 μM), we considered a minimum
inhibitory concentration i.e. 3–10 μM as IC50 for this drug.
This is in accordance with prior reports.[14,15] Thus, the cyto-
toxic effect of 5 is comparable to that of cisplatin whereas
others (2,3, 4 and 6) are less potent relative to cisplatin.
To further assess the cell viability in the presence of
different organometallic compounds, we stained the drug
treated cells with Live‐Dead solution.[16] For fluorescent
imaging experiments, IC50 treated groups for each compound
was used (Figure 10). It is evident from the fluorescent
images that untreated cells exhibited well spread native like
morphology with abundant presence of live cells (green
color). On the contrary, the drug treated groups exhibited
round appearance with simultaneous occurrence of dead cells
(red color). A comparatively lesser number of live cells were
observed in the drug treated wells relative to untreated group.
Also, all the dead cells could not be imaged since non‐
adherent dead cells were washed off during the repeated
washing steps while staining. These results suggest that 5
exhibits most efficient A549 cell growth inhibition at
minimum concentration comparable with widely used drug
cisplatin.
FIGURE
9
In vitro cytotoxicity of various organometallic
compounds and cisplatin with A549 cells post 48 h treatment. O.D.
(optical density) of the solution is a direct measure of cell viability. A
higher O.D. Value indicates higher number of metabolically active
viable cells. *p < 0.05 and **p < 0.01
significantly higher as compared to other compounds at 95%
confidence level). Cells exhibited well spread morphology at
1 μM and lower concentrations indicating healthy cells. On
the other hand, at 30 μM concentration, rounded cell
morphology was observed for all organometallic compounds
suggesting cytotoxic effect of these compounds at higher
concentrations.
Furthermore, an IC50 value of different organometallic
compounds was obtained by calculating the percentage cell
viability. O.D. values of drug treated groups were normalized
with the control untreated group. The IC50 value of a drug is
defined as the minimum inhibitory concentration at which
50% of cells are viable as compared to the untreated control.
IC50 values for molecules 2–6 and cisplatin are listed in
Table 1. Complexes 4 and 6 were effective only at higher
doses (~30 μM). Complexes 2 and 3 were able to inhibit
50% cell growth at slightly lower concentrations (nearly

BHOWMICK ET AL.
7 of 11
FIGURE 10 Live‐dead cell viability assay of A549 cells after 48 h treatment with IC50 concentration of each compound. Green fluorescence is
depicting the live cells while red fluorescence is indicative of dead cells. (scale bar 400 μm)
organometallic complexes as therapeutic agents for other
malignant cells is currently underway in our laboratories.
3 | CONCLUSION
In conclusion, we describe herein the synthesis and character-
ization of five new organometallic complexes (2–6) contain-
ing two Pt(II) phosphine‐alkynyl units attached to a central
pyrazine ring at its C2 and C6 positions. Each platinum cen-
ter is further covalently linked to various aromatic or hetero
aromatic units as peripheral units. The cytotoxic potency of
these five organoplatinum complexes was studied with
A549 human lung carcinoma cells and results obtained were
compared with the effect due to cisplatin. Our experimental
data suggest that 5 (compound bearing terminal pyridine
units), showed better growth inhibitory effects than other
complexes (2, 3, 4 and 6). In fact, among the five
complexes, 5 exhibits the most efficient A549 cell growth
inhibition at minimum concentration and this result is
comparable with that of cisplatin. This result is interesting
because unlike cisplatin, 5 does not have a leaving group(s)
attached to Pt(II) centers. The cytotoxic behaviour of these
complexes may be attributed to their non‐covalent interaction
with DNA. These results will form the basis of further studies
on exploring the mechanism of cytotoxic activity of such
organometallic complexes containing multiple Pt(II) centres.
In depth analysis of results and exploration of these
3.1 | Experimental section
3.1.1 | General details
All chemicals and anhydrous solvents used in this work were
purchased from commercial sources and used without further
purification. A549 (human lung carcinoma cells) were
procured from NCCS (National Centre For Cell Science),
Pune, India. MTT (3‐(4, 5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyl
tetrazolium bromide, Calcein AM and Ethidium homodimer‐1
were purchased from Sigma‐Aldrich (St. Louis, MO, USA),
whereas Dulbecco's modified eagle medium (DMEM) and
Fetal bovine serum (FBS) were purchased from GIbco
(Camarillo, CA, USA). Antibiotic‐antimycotic solution was
purchased from Himedia, India. Linker 1 was prepared by
following the reported literature by Bhowmick et al.[7] FTIR
spectra were recorded in a PerkinElmer Spectrum 400 FTIR
spectrophotometer. 1H and 31P{1H} NMR spectra were
recorded on a Bruker 400 MHz spectrometer. Elemental
analysis was carried out using a Thermo Scientific Flash
2000 organic elemental analyzer. ESI‐MS spectra of the

8 of 11
BHOWMICK ET AL.
compounds were recorded using a Bruker UltrafleXtreme™
ESI mass spectrometer. UV–Vis spectra were recorded using
a Shimadzu UV‐2550 spectrophotometer.
to isolate complex 4 as a yellow solid. Yield: 70 mg, 69%.
Mp: 128–130 °C. H NMR (400 MHz, CDCl3): δ 8.10 (s,
1
2H, Ar − H), 7.23–7.22 (d, 4H, J = 1.72 Hz, Ar − H),
6.77–6.75 (dd, 4H, J = 1.84 Hz, 1.88 Hz, Ar − H), 3.78 (s,
6H, −OCH3), 2.21–2.14 (m, 24H, −CH2), 1.24–1.16 (m,
36H, −CH3). 31P{1H} NMR (163 MHz, CDCl3): δ 11.26
(1JPPt = 1193 Hz). IR (ATR): 2966, 2877, 2355, 2086,
1741, 1545, 1491, 1454, 1379, 1274, 1232, 1170, 1147,
3.1.2 | General procedure for the synthesis of
compound 2–6
The organometallic complex 1 (100 mg, 0.080 mmol) and the
respective ethynyl compound (0.161 mmol) were charged in
a 25 mL Schlenk flask in the glove box. Subsequently,
10 ml anhydrous THF, CuI (3 mg, 0.016 mmol) and 6 ml
freshly distilled triethylamine were added under nitrogen.
The reaction mixture was stirred for overnight at room
temperature, after which the solvent was removed using a
rotary evaporator. The resulting yellow residue was dissolved
in dichloromethane, washed with water and dried over
anhydrous Na2SO4. The filtrate obtained was evaporated
and the crude product thus obtained was purified by column
chromatography on basic alumina with respective eluent
(Ethyl acetate and Hexane).
1026, 827, 763, 729 cm−1
.
Anal. Calcd for
C50H76N2O2P4Pt2: C, 48.00; H, 6.12; N, 2.24. Found: C,
48.07; H, 6.18; N, 2.29. MS (ESI) m/z (%): 1251.42 (100)
(M+).
Organometallic complex 5
The organometallic complex 5 was purified by column
chromatography on basic alumina with 10% ethyl acetate in
hexane to isolate complex 5 as a reddish semisolid. Yield:
65 mg, 67%.1H NMR (400 MHz, CDCl3): δ 8.40–8.39 (d,
4H, J = 4.16 Hz, Ar − H), 8.12 (s, 2H, Ar − H), 7.10–7.08
(d, 4H, J = 5.60 Hz, Ar − H), 2.21–2.11 (m, 24H, −CH2),
1.29–1.12 (m, 36H, −CH3). 31P{1H} NMR (163 MHz,
CDCl3): δ 11.47 (1JPPt = 1174 Hz). IR (ATR): 2967, 2935,
2349, 2092, 1586, 1489, 1448, 1384, 1219, 1208, 1151,
1031, 873, 817, 761, 732 cm−1.MS (ESI) m/z (%): 1193.38
(61.8) (M+).
Organometallic complex 2
The organometallic complex 2 was purified by column chro-
matography on basic alumina with 6% ethyl acetate in hexane
to isolate complex 2 as an off white solid. Yield: 70 mg, 73%.
Mp: 158–160 °C.1H NMR (400 MHz, CDCl3): δ 8.11 (s, 2H,
Ar − H), 7.29–7.27 (m, 4H, Ar − H), 7.23–7.19 (m, 4H,
Ar − H), 7.14–7.12 (m, 2H, Ar − H), 2.21–2.14 (m, 24H,
−CH2), 1.25–1.17 (m, 36H, −CH3). 31P{1H} NMR
(163 MHz, CDCl3): δ 11.30 (1JPPt = 1182 Hz). IR (ATR):
2872, 2360, 2092, 1485, 1454, 1430, 1384, 1223, 1151,
1032, 762, 736 cm−1. Anal. Calcd for C48H72N2P4Pt2: C,
48.40; H, 6.09; N, 2.35. Found: C, 48.45; H, 6.11; N, 2.39.
MS (ESI) m/z (%): 1191.40 (64) (M+).
Organometallic complex 6
The organometallic complex 6 was purified by column
chromatography on basic alumina with 10% ethyl acetate in
hexane to isolate complex 6 as a yellowish solid. Yield:
75 mg, 76%. Mp: 192–195 °C.1H NMR (400 MHz, CDCl3):
δ 8.12 (s, 2H, Ar − H), 7.49–7.46 (d, 4H, J = 8.32 Hz,
Ar − H), 7.30–7.28 (d, 4H, J = 8.32 Hz, Ar − H),
2.19–2.12 (m, 24H, −CH2), 1.24–1.12 (m, 36H, −CH3).
31P{1H} NMR (163 MHz, CDCl3): δ 11.50 (1JPPt = 1175 Hz).
IR (ATR): 2966, 2872, 2355, 2218, 2092, 1590, 1490, 1454,
1384, 1222, 1151, 1029, 854, 827, 761, 732 cm−1. Anal.
Calcd for C50H70N4P4Pt2: C, 48.38; H, 5.68; N, 4.51. Found:
C, 48.48; H, 5.76; N, 4.62. MS (ESI) m/z (%): 1241.39 (66.6)
(M+).
Organometallic complex 3
The organometallic complex 3 was purified by column chro-
matography on basic alumina with 5% ethyl acetate in hexane
to isolate complex 3 as an off white solid. Yield: 85 mg, 78%.
1
Mp: 170–174 °C. H NMR (400 MHz, CDCl3): δ 8.12 (s,
2H, Ar − H), 7.58–7.56 (d, 4H, J = 9.88 Hz, Ar − H),
7.47–7.45 (d, 4H, J = 10.89 Hz, Ar − H), 7.41–7.31 (m,
10H, Ar − H), 2.21–2.17 (m, 24H, −CH2), 1.28–1.17 (m,
36H, −CH3). 31P{1H} NMR (163 MHz, CDCl3): δ 11.96
(1JPPt = 1177 Hz). IR (ATR): 2972, 2878, 2081, 1597,
Single‐crystal structure determination
A suitable single crystal of each compound was carefully
selected under a polarizing microscope and mounted at the
tip of the thin glass fiber using cyanoacrylate (super glue)
adhesive. Single crystal structure determination by X‐ray
diffraction was performed on a Bruker Smart Apex II
diffractometer equipped with an Oxford Cryostream low‐
temperature device and a fine‐focus sealed‐tube X‐ray source
(Mo‐Kα radiation, λ = 0.71073 Å, graphite monochromated)
operating at 50 kV and 30 mA. The structure was solved by
direct methods using SHELXS‐97,[17] which readily revealed
all the heavy atom positions (Pt, P) and enabled us to locate
1485, 1454, 1302, 1222, 1145, 1032, 873, 839, 750 cm−1
.
Anal. Calcd for C60H80N2P4Pt2: C, 53.65; H, 6.00; N, 2.09.
Found: C, 53.74; H, 6.06; N, 2.12. MS (ESI) m/z (%):
1343.46 (100) (M+).
Organometallic complex 4
The organometallic complex 4 was purified by column chro-
matography on basic alumina with 4% ethyl acetate in hexane

BHOWMICK ET AL.
9 of 11
the other non‐hydrogen (C and N) positions from the differ-
ence Fourier maps. An empirical absorption correction based
on symmetry equivalent reflections was applied using
SADABS.[18] The hydrogen positions of carbon atoms were
found in the difference Fourier maps. For the final refine-
ment, the hydrogen atoms of three compounds were placed
geometrically and held in the riding mode. The last cycles
of refinement included atomic positions, anisotropic thermal
parameters for all the non‐hydrogen atoms, and isotropic
thermal parameters for all the hydrogen atoms. Full ‐matrix
least‐squares structure refinement against ∣F2∣ was carried out
using the SHELXTL‐PLUS[19] package of programs. In the
complex 2, few of the atoms were found to have thermal disor-
der and thus the disordered atoms were refined isotropically.
Details of the structure determination and final refinements
for three compounds are listed in supporting information S10.
Effect of organometallic compounds and cisplatin was
further tested by monitoring the cell viability and attachment.
After 48 h drug treatment, cells were washed with sterile
phosphate buffered saline (PBS, pH 7.4) and incubated with
working live‐dead solution (1 μL of 4 mM Calcein AM and
4 μl of 2 mM Ethidium homodimer‐1 in 2 mL PBS, Sigma
Aldrich, USA) for 10 min. Calcein AM is a cell permeable
dye which reacts with live cells and fluoresces green
(Excitation: 495 nm, Emission: 516 nm) whereas Ethidium
homodimer‐1 reacts with dead cells and fluoresces red
(Excitation: 528 nm, Emission: 617 nm). Post incubation,
cells were washed with PBS and their morphology was
visualized under fluorescence microscope (EVOS FL, Life
Technologies, Camarillo, CA, USA).
3.2 | Statistical analysis
In vitro cytotoxic assessment was performed in triplicates for
each concentration of organometallic compounds under
study. One way analysis of variance (ANOVA) was
performed using Origin 8.0 software following Tukey's test
to examine the significant difference among different groups.
*p < 0.05 was considered as significant and **p < 0.01 as
highly significant.
3.1.3 | In vitro cytotoxicity
A549 cells (human lung carcinoma cells) were procured from
NCCS (National Centre For Cell Science). Culture of A549
cells was maintained in Dulbecco's Modified Eagle's
Medium (DMEM) added with 10% Fetal Bovine Serum
(FBS) and 1% antibiotic‐antimycotic solution (Himedia,
India) in a humidified incubator at 5% CO2, 37 °C. 1 mM
stock solution of all compounds was prepared in DMSO
(Dimethyl sulfoxide, Sigma‐Aldrich, USA). The final
concentration of DMSO in culture media was kept between
0.01 to 3% (v/v). On the other hand, cisplatin was dissolved
in normal saline (0.9% sodium chloride) and 1 mM stock
solution was used for cell treatment. Cytocompatibility of
all organometallic compounds and cisplatin was assessed by
measuring the cellular metabolic activity of A549 using
MTT dye (3‐(4, 5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyl
tetrazazolium bromide, Sigma‐Aldrich, St. Louis, MO,
USA). Reduction of tetrazolium MTT dye in to blue colored
formazan crystals by NAD(P)H dependent oxidoreductase
provide an indirect measurement of viable cells present in
the culture.[20] Equal numbers of A549 cells (~104) were cul-
tured in 96‐well tissue culture plate. Cells were allowed to
adhere and grow for 24 h. Cells were then treated with
predefined concentration of each compound and cisplatin
for 48 h. Post treatment, cells were added with 10% (v/v)
MTT stock solution (5 mg/ml) prepared in serum free media
and incubated for 4 h. It led to formation of blue formazan
crystals which were further solubilized in 200 μL DMSO
(Dimethyl sulfoxide, SRL, Kolkata, India). Absorbance was
read at 570 nm using microtiter plate reader (Tecan infinite
Pro M 200 reader with i‐control software). A dose dependent
curve was produced to calculate IC50 value defined as 50%
growth inhibition in presence of drug compared to untreated
control.
4 | ASSOCIATED CONTENT
Author contributions
N.D. conceived the research and supervised the experimental
work related to synthesis and characterization of organome-
tallic molecules reported herein. S.B. synthesized all
compounds reported in this manuscript and performed the
UV–Vis. Spectroscopy. A. J. assisted S.B. in his experiments.
J.N.B. and S.R.M collected XRD data and refined structure
of 2, 3 and 6. P.G. carried out the experiments related to eval-
uation of biological activity of 2–6 and cisplatin reported
herein and these biological studies were supervised by B.B.
M. All authors have contributed to interpretation of results,
compiling the manuscript and have approved the final
manuscript.
NOTES
The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
N.D. thanks the Indian Institute of Technology (IIT) Patna for
financial support. S.B. thanks IIT Patna for an Institute
Research Fellowship. A.J. thanks UGC, New Delhi for a
Research Fellowship. The authors also acknowledge SAIF‐
Panjab University for analytical facilities. B.B.M. greatly

10 of 11
BHOWMICK ET AL.
U. Rothlisberger, P. J. Dyson, C. A. Davey, Nat. Commun. 2014, 5,
3462. k) M. Frik, A. Martínez, B. T. Elie, O. Gonzalo, D. R. D.
Mingo, M. Sanau, R. S. Delgado, T. Sadhukha, S. Prabha, J. W.
Ramos, I. Marzo, M. Contel, J. Med. Chem. 2014, 57, 9995. l) P.
V. Simpson, L. A. Watson, A. Barlow, G. Wang, M. P. Cifuentes,
M. G. Humphrey, Angew. Chem. Int. Ed. 2016, 55, 2387. m) A.
Colombo, C. Dragonetti, D. Marinotto, S. Righetto, G. Griffini,
S. Turri, H. Akdas‐Kilig, J.‐L. Fillaut, A. Amar, A. Boucekkine,
C. Katan, Dalton Trans. 2016, 45, 11052.
acknowledges funding support through Department of
Biotechnology (DBT) and Department of Science and
Technology (DST), Government of India.
REFERENCES
[1] a) T. D. Cook, S. N. Natoli, P. E. Fanwick, T. Ren, Organometallics
2015, 34, 686. b) E. S.‐H. Lam, W. H. Lam, V. W.‐W. Yam, Inorg.
Chem. 2015, 54, 3624. c) S. Li, J. Huang, F. Zhou, T. R. Cook, X.
Yan, Y. Ye, B. Zhu, B. Zheng, P. J. Stang, J. Am. Chem. Soc. 2014,
136, 5908. d) W. Wang, B. Sun, X.‐Q. Wang, Y.‐Y. Ren, L.‐J.
Chen, J. Ma, Y. Zhang, X. Li, Y. Yu, H. Tan, H.‐B. Yang,
Chem.‐Eur. J. 2015, 21, 6286. e) S. Bhowmick, S. Chakraborty,
A. Das, S. Nallapeta, N. Das, Inorg. Chem. 2015, 54, 8994. f) T.
Rena, Chem. Commun. 2016, 52, 3271. g) S. F. Tyler, S. N. Natoli,
B. Vlaisavljevich, P. E. Fanwick, T. Ren, Inorg. Chem. 2015, 54,
10058. h) M. C.‐L. Yeung, V. W.‐W. Yam, Chem. Sci. 2013, 4,
2928. i) T. D. Cook, P. E. Fanwick, T. Ren, Organometallics
2014, 33, 4621.
[4] a) L. Kelland, Nat. Rev. Cancer 2007, 7, 573. b) Y. Jung, S. J.
Lippard, Chem. Rev. 2007, 107, 1387. c) R. C. Todd, S. J. Lippard,
Metallomics 2009, 1, 280. d) X. Yao, K. Panichpisal, N. Kurtzman,
K. Nugent, Am. J. Med. Sci. 2007, 334, 115. e) S. R. McWhinney,
R. M. Goldberg, H. L. McLeod, Mol. Cancer Ther. 2009, 8, 10. f)
A. S. Abu‐Surrah, M. Kettunen, Curr. Med. Chem. 2006, 13, 1337.
g) X. Y. Wang, Z. J. Guo, Dalton Trans. 2008, 1521. h) A. Rabik,
M. E. Dolan, Cancer Treat. Rev. 2007, 33, 9. i) E. Wong, C. M.
Giandomenico, Chem. Rev. 1999, 99, 2451.
[5] a) N. P. Farrell, Chem. Soc. Rev. 2015, 44, 8773. b) X. Wang, Z.
Guo, Chem. Soc. Rev. 2013, 42, 202. c) G. N. Kaluerovic´, R.
Paschke, Curr. Med. Chem. 2011, 18, 4738. d) W. K. C. Lo, G.
S. Huff, D. Preston, D. A. McMorran, G. I. Giles, K. C. Gordon,
J. D. Crowley, Inorg. Chem. 2015, 54, 6671. e) N. P. E. Barry, P.
J. Sadler, Chem. Commun. 2013, 49, 5106. f) K. S. Lovejoy, S. J.
Lippard, Dalton Trans. 2009, 48, 10651. g) G. Sava, G. Jaouen,
E. A. Hillard, A. Bergamob, Dalton Trans. 2012, 41, 8226. h) K.
Mitra, U. Basu, I. Khan, B. Maity, P. Kondaiah, A. R. Chakravarty,
Dalton Trans. 2014, 43, 751. i) D. N. Kolishetti, S. J. Lippard, O.
C. Farokhzad, Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 1850. j)
F. S. Mackay, J. A. Woods, P. Heringova, J. Kasparkova, A. M.
Pizzaro, S. A. Moggach, S. Parsons, V. Brabec, P. J. Sadler, Proc.
Natl. Acad. Sci. U. S. A. 2007, 104, 20743. k) J. S. Butler, J. A.
Woods, N. J. Farrer, M. E. Newton, P. J. Sadler, J. Am. Chem.
Soc. 2012, 134, 16508.
[2] a) O. S. Wenger, Chem. Rev. 2013, 113, 3686. b) Y. Tanaka, K. M.‐
C. Wong, V. W.‐W. Yam, Chem. – Eur. J. 2013, 19, 390. c) M. C.‐
L. Yeung, V. W.‐W. Yam, Chem. Soc. Rev. 2015, 44, 4192. d) J.
Xiang, T.‐K. Wang, Q. Zhao, W. Huang, C.‐L. Ho, W.‐Y. Wong,
J. Mater. Chem. C 2016, 4, 921. e) W.‐Y. Wong, C.‐L. Ho, Acc.
Chem. Res. 2010, 43, 1246. f) G.‐J. Zhou, W.‐Y. Wong, Chem.
Soc. Rev. 2011, 40, 2541. g) C. Y.‐S. Chung, V. W.‐W. Yam, Chem.
– Eur. J. 2014, 20, 13016. h) K. Costuas, S. Rigaut, Dalton Trans.
2011, 40, 5643. i) A. K.‐W. Chan, K. M.‐C. Wong, V. W.‐W. Yam,
J. Am. Chem. Soc. 2015, 137, 6920. j) T. D. Cook, S. N. Natoli, P.
E. Fanwick, T. Ren, Organometallics 2016, 35, 1329. k) V. K.‐M.
Au, D. Wu, V. W.‐W. Yam, J. Am. Chem. Soc. 2015, 137, 4654.
l) E. Rossi, A. Colombo, C. Dragonetti, D. Roberto, R. Ugo, A.
Valore, L. Falciola, P. Brulatti, M. Cocchi, J. A. G. Williams,
J. Mater. Chem. 2012, 22, 10650. m) J. Boixel, V. Guerchais, H.
L. Bozec, D. Jacquemin, A. Amar, A. Boucekkine, A. Colombo,
C. Dragonetti, D. Marinotto, D. Roberto, S. Righetto, R. D.
Angelis, J. Am. Chem. Soc. 2014, 136, 5367. n) D. Wei, M. S.
Kodikara, M. Morshedi, G. J. Moxey, H. Wang, G. Wang, C.
Quintana, C. Zhang, R. Stranger, M. P. Cifuentes, M. G.
Humphrey, ChemPlusChem 2016, 81, 621.
[6] a) Y. Li, D. P.‐K. Tsang, C. K.‐M. Chan, K. M.‐C. Wong, M.‐Y.
Chan, V. W.‐W. Yam, Chem. – Eur. J. 2014, 20, 13710. b) S. Y.‐
L. Leung, A. Y.‐Y. Tam, C.‐H. Tao, H. S. Chow, V. W.‐W. Yam,
J. Am. Chem. Soc. 2012, 134, 1047. c) S. Bhowmick, S.
Chakraborty, S. R. Marri, J. N. Behera, N. Das, RSC Adv. 2016,
6, 8992. d) S. Chakraborty, S. Mondal, S. Bhowmick, J. Ma, H.
Tan, S. Neogi, N. Das, Dalton Trans. 2014, 43, 13270. e) S.‐H.
Lee, C. T.‐L. Chan, K. M.‐C. Wong, W. H. Lam, W.‐M. Kwok,
V. W.‐W. Yam, Dalton Trans. 2014, 43, 17624. f) E. S.‐H. Lam,
D. P.‐K. Tsang, W. H. Lam, A. Y.‐Y. Tam, M.‐Y. Chan, W.‐T.
Wong, V. W.‐W. Yam, Chem. – Eur. J. 2013, 19, 6385. g) S. Y.‐
L. Leung, W. H. Lam, V. W.‐W. Yam, Proc. Natl. Acad. Sci.
2013, 110, 7986. h) S. Chakraborty, S. Bhowmick, J. Ma, H. Tan,
N. Das, Inorg. Chem. Front. 2015, 2, 290. i) H.‐L. Wong, C.‐H.
Tao, N. Zhu, V. W.‐W. Yam, Inorg. Chem. 2011, 50, 471.
[3] a) A. K. Singh, D. S. Pandey, Q. Xua, P. Braunstein, Coord. Chem.
Rev. 2014, 270, 31. b) Z. Liu, I. Romero‐Canelón, A.
Habtemariam, G. J. Clarkson, P. J. Sadler, Organometallics 2014,
33, 5324. c) K. B. G. Singh, M. P. Grant, B. W. Harper, A. M.
Krause‐Heuer, M. Manohar, N. Orkey, J. R. Aldrich‐Wright, Curr.
Top. Med. Chem. 2011, 11, 521. d) F. Navas, S. Perfahl, C. Garino,
L. Salassa, O. Novakova, C. Navarro‐Ranninger, P. J. Bednarski, J.
Malina, A. G. Quiroga, J. Inorg. Biochem. 2015, 153, 211. e) P.
Elumalai, Y. J. Jeong, D. W. Park, D. H. Kim, H. Kim, S. C. Kang,
K.‐W. Chi, Dalton Trans. 2016, 45, 6667. f) S. Mokesch, M. S.
Novak, A. Roller, M. A. Jakupec, W. Kandioller, B. K. Keppler,
Organometallics 2015, 34, 848. g) J. Ruiz, C. Vicente, C. Haro,
D. Bautista, Inorg. Chem. 2013, 52, 974. h) Z. Liu, P. J. Sadler,
Acc. Chem. Res. 2014, 47, 1174. i) S. Mukhopadhyay, R. K. Gupta,
R. P. Paitandi, N. K. Rana, G. Sharma, B. Koch, L. K. Rana, M. S.
Hundal, D. S. Pandey, Organometallics 2015, 34, 4491. j) Z.
Adhireksan, G. E. Davey, P. Campomanes, M. Groessl, C. M.
Clavel, H. Yu, A. A. Nazarov, C. H. F. Yeo, W. H. Ang, P. Droge,
[7] a) S. Bhowmick, S. Chakraborty, A. Das, P. R. Rajamohanan, N.
Das, Inorg. Chem. 2015, 54, 2543. b) A. Jana, S. Bhowmick, S.
Kaur, H. K. Kashyap, N. Das, Dalton Trans. 2017, 46, 1986.
[8] a) H. Masai, K. Sonogashira, N. Hagihara, Bull. Chem. Soc. Jpn.
1971, 44, 2226. b) L. Saksteder, E. Baralt, B. A. DeGraff, C. M.
Lukehart, J. N. Demas, Inorg. Chem. 1991, 30, 2468.
[9] S. Dasari, P. B. Tchounwou, Eur. J. Pharmacol. 2014, 740, 364.
[10] C. Bauer, T. Peleg‐Shulman, D. Gibson, A. H. J. Wang, Europ. J.
Biochem. 1998, 256, 253.

BHOWMICK ET AL.
11 of 11
[11] H. Pruchnik, T. Lis, M. Latocha, A. Zielińska, F. P. Pruchnik,
J. Inorg. Biochem. 2016, 156, 14.
[19] G. M. Sheldrick, SHELXTL‐PLUS Program for Crystal Structure
Solution and Refinement; University of Gottingen: Gottingen,
Germany.
[12] B. J. Pages, Y. Zhang, F. Li, J. Sakoff, J. Gilbert, J. R. Aldrich‐
Wright, Eur. J. Inorg. Chem. 2015, 2015, 4167.
[20] S. K. Nandi, B. Kundu, A. Mahato, N. L. Thakur, S. N. Joardar, B.
B. Mandal, Integr. Biol. 2015, 7, 250.
[13] M. D. Hall, K. A. Telma, K. E. Chang, T. D. Lee, J. P. Madigan, J.
R. Lloyd, I. S. Goldlust, J. D. Hoeschele, M. M. Gottesman,
Cancer Res. 2014, 74, 3913.
SUPPORTING INFORMATION
Additional Supporting Information may be found online in
the supporting information tab for this article.
[14] B. D. Lopez‐Ayllon, V. Moncho‐Amor, A. Abarrategi, I. Ibañez de
Cáceres, J. Castro‐Carpeño, C. Belda‐Iniesta, R. Perona, L. Sastre,
Cancer med. 2014, 3, 1099.
[15] R. Zhang, Y. Li, Z. Wang, L. Chen, X. Dong, X. Nie, Tumour Biol.
2015, 36, 8585.
How to cite this article: Bhowmick S, Jana A, Marri
SR, et al. Pyrazine based Pt(II) bis‐alkynyl
organometallic complexes: Synthesis, characterization,
and cytotoxic effect on A549 human lung carcinoma
cells. Appl Organometal Chem. 2017:e3824. https://
doi.org/10.1002/aoc.3824
[16] S. Dhar, S. J. Lippard, Proc. Natl. Acad. Sci. 2009, 106, 22199.
[17] G. M. Sheldrick, SHELXL‐97; University of Gottingen: Gottingen,
Germany, 1997.
[18] SADABS; Bruker AXS, Inc: Madison, WI, 2001.