Coordination-Driven Self-Assembly of Ionic Irregular Hexagonal Metallamacrocycles via an Organometallic Clip and Their Cytotoxicity Potency

Article abstract of DOI:10.1021/acs.inorgchem.7b01561

Two new irregular hexagons (6 and 7) were synthesized from a pyrazine motif containing an organometallic acceptor clip [bearing platinum(II) centers] and different neutral donor ligands (4,4′-bipyridine or pyrazine) using a coordination-driven self-assemb

Full text of DOI:10.1021/acs.inorgchem.7b01561

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pubs.acs.org/IC
Coordination-Driven Self-Assembly of Ionic Irregular Hexagonal
Metallamacrocycles via an Organometallic Clip and Their
Cytotoxicity Potency
†
†
†
‡
‡
Sourav Bhowmick, Achintya Jana, Khushwant Singh, Prerak Gupta, Ankit Gangrade,
,‡
,†
Biman B. Mandal,* and Neeladri Das*
†
Department of Chemistry, Indian Institute of Technology Patna, Bihta 801103, Bihar, India
‡
Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India
S Supporting Information
*
ABSTRACT: Two new irregular hexagons (6 and 7) were synthesized from a pyrazine motif containing an organometallic
acceptor clip [bearing platinum(II) centers] and different neutral donor ligands (4,4′-bipyridine or pyrazine) using a
coordination-driven self-assembly protocol. The two-dimensional supramolecules were characterized by multinuclear NMR, mass
spectrometry, and elemental analyses. Additionally, one of the macrocycles (6) was characterized by single-crystal X-ray analyses.
Macrocycles are unique examples of [2 + 2] self-assembled ensembles that are hexagonal but irregular in shape. These hexagon
frameworks require the assembly of only four tectons/subunits. The cytotoxicity of platinum(II)-based macrocycles was studied
using various cell lines such as A549 (human lung carcinoma), KB (human oral cancer), MCF7 (human breast cancer), and
HaCaT (human skin keratinocyte) cell lines, and the results were compared with those of cisplatin. The smaller macrocycle (7)
exhibited a higher cytotoxic effect against all cell types, and its sensitivity was found to be comparable with that of cisplatin for
A549 and MCF7 cells. Cell cycle analysis and live propidium iodide staining suggest that the macrocycles 6 and 7 induced a loss
of membrane integrity that ultimately might lead to necrotic cell death.
INTRODUCTION
motivated research interest in developing organometallic
complexes as drugs for such medical treatment and
investigating their potential in anticancer activity. Organo-
■
The strategy of utilizing metal−ligand coordination to yield
discrete molecular species has emerged as a well-established
protocol in supramolecular chemistry. Herein, the term
ensemble” has been frequently used to describe a molecular
entity that is usually in nanoscale dimensions. The phrase
molecular subunit” is used to describe organic or metal-bearing
organometallic) smaller molecules that bind with one another
via coordination bonds in a chemical reaction that is popularly
referred to as a “self-assembly” process. The phrase “self-
assembly” refers to a chemical reaction that utilizes the
molecular information present in the “molecular subunit” to
yield a molecular “ensemble” of predefined shape and size.
These reactions do not require any human intervention because
the information (such as the bite angle) contained in the
molecular subunit determines the geometrical shape of the
resultant supramolecular framework.
It is well-known that inorganic complexes such as cisplatin,
carboplatin, and others are widely used drugs for the treatment
of various types of cancers. However, there are certain
7
1
metallic complexes have been reported to be more potent than
their nonorganometallic/inorganic counterparts because the
“
7a,8
2
former are more inert kinetically relative to the latter.
In
recent years, the effectiveness of organometallic compounds as
“
(
potential anticancer drugs has been documented in a review
9
article. As far as the metal center is concerned, the use of
10
11
12
ruthenium, palladium, and platinum in anticancer organo-
metallic metallodrugs is more common. In this context, discrete
supramolecular species bearing multiple metal centers are new
additions in the library of compounds with anticancer activity
and coordination-driven self-assembly is a facile synthetic
strategy for the design of such discrete frameworks. Two-
dimensional metal-containing macrocycles (metallamacro-
cycles) and three-dimensional metallacages have been reported
3
1a,b,4
1
a,b,4a−c
9
to have biomedical applications. Their interaction with
biomolecular species such as DNA or cancerous cells has
13
been studied in the past. Among the different types of self-
5
limitations of cisplatin and related complexes that include, but
are not limited, to high toxicity, undesired side effects, shorter
half-life, and a lower spectrum of effectiveness as well as a
Special Issue: Self-Assembled Cages and Macrocycles
Received: June 19, 2017
6
tendency of tumor cells to acquire resistance. This has
©
XXXX American Chemical Society
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DOI: 10.1021/acs.inorgchem.7b01561
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Scheme 1. Synthesis of the Pyrazine-Based Organoplatinum Clip 5
assembled architectures whose biological properties have been
studied, a majority of these ensembles have ruthenium(III) as
the metal center. Literature reports describing the study of
the anticancer activity of discrete self-assembled ensembles
with 2 equiv of trans-PtI (PEt ) in the presence of CuI
2
3 2
(catalyst), triethylamine (base), and toluene (solvent) at room
temperature for 16 h (Scheme 1). 5 is a new organometallic clip
that was obtained in 68% yield as a yellowish semisolid and is
stable in air/moisture with high solubility in common organic
solvents. All new molecules (2−5) were characterized by
1
4
(
metallomacrocycles or metallocages) containing other metals
such as palladium(II) and platinum(II) are relatively less
1
13
common, although cisplatin contains platinum(II).
Fourier transform infrared (FT-IR) and NMR ( H and C)
spectroscopy, mass spectrometry, and elemental analyses. In
addition, the molecular structure of 4 was confirmed by single-
crystal X-ray analysis. In FT-IR spectra of 2 and 4, the presence
Herein, in a continuation of our research interest to develop
new metallosupramolecular architectures, we report the design
of a new organometallic diplatinum molecular “clip” as an
acceptor tecton that was used as a building block for the
synthesis of two nanodimensional molecular hexagonal-shaped
macrocycles. Subsequently, for the first time, cytotoxic efficacies
of platinum(II)-based macrocycles were studied (using these
macrocycles) against four different cancer cell lines, and the
results obtained were compared with those of cisplatin.
of strong bands at 2218 and 2212 cm− [ν(CC )],
1
str.
respectively, indicates the presence of an internal ethynyl
functional group in these products. In H NMR spectra of 2
1
and 4 (Supporting Information S2 and S3), the sharp singlets at
8
.65 and 8.66 ppm, respectively, were assigned to the aromatic
protons of the pyrazine ring present in these molecules. Four
1
sets of signals were observed in the H NMR spectra because of
RESULT AND DISCUSSION
four different kinds of chemically inequivalent protons in 2 and
■
4
(Supporting Information S2 and S3). Additionally, in the case
Synthesis and Characterization of the Pyrazine-Based
Organometallic Clip 5. A new pyrazine-based organometallic
clip 5, has been synthesized via multistep reactions using
commercially available 2,6-dichloropyrazine (Scheme 1) as the
synthon. First, 2,6-bis[(trimethylsilyl)ethynyl]pyrazine was
synthesized using a well-known Sonogashira cross-coupling
reaction, followed by deprotection of the trimethylsilyl group,
which yielded 2,6-diethynylpyrazine (1). Subsequently, using a
coupling reaction between 1 and 1,3-diiodobenzene in the
presence of cuprous iodide/palladium(0) catalysts and triethyl-
amine (base) at 60 °C for 16 h, 2,6-bis[(3-iodophenyl)-
ethynyl]pyrazine (2) was obtained in reasonably high yields
of 4, the sharp singlet at 3.12 ppm was due to an ethynyl
proton. All of the characteristic peaks corresponding to the
pyrazine, phenyl, and ethynyl units of both 2 and 4 were
1
3
1
observed in the C{ H} NMR spectrum (Supporting
Information S2 and S3). The new pyrazine-based organo-
metallic clip 5 has been characterized by FT-IR, multinuclear
1
13
31
NMR ( H, C, and P) spectroscopy, mass spectrometry, and
elemental analyses. In the FT-IR spectrum of 5, the presence of
two different ethynyl functional groups was evident because of
−1
1
the presence of intense peaks at 2210 and 2113 cm . In the H
NMR spectrum of 5 (Supporting Information S4), a sharp
singlet at 8.64 ppm corresponds to the aromatic protons of
(
>80%). In subsequent steps, 2,6-bis[(3-ethynylphenyl)-
ethynyl]pyrazine (4) was synthesized by the cross-coupling
reaction of 2 with (trimethylsilyl)acetylene in tetrahydrofuran
1
pyrazine. Four sets of signals were observed in the H NMR
because of the four different chemically inequivalent protons of
the phenyl ring (Supporting Information S4). As expected, the
(
THF) as the solvent and using the same catalyst and base as
those utilized in a previous step. Subsequently, deprotection
with tetra-n-butylammonium fluoride (TBAF; THF as the
solvent) yielded the desired intermediate (4). The pyrazine-
based organometallic clip 5 was finally synthesized by reacting 4
methylene and methyl protons of the PEt groups appear as
3
multiplets in the ranges of 2.26−2.19 and 1.22−1.14 ppm,
respectively. In the 31P NMR spectrum of 5 (Supporting
Information S4), one sharp singlet observed at 8.87 ppm,
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Figure 1. ORTEP representation of 4. Thermal ellipsoids were drawn at the 50% probability level. Hydrogen atoms were omitted for clarity.
Scheme 2. Design of Irregular Hexagonal Macrocycles 6 and 7
accompanied by concomitant 195Pt satellites (1JPPt = 1147 Hz),
suggests that all phosphorus nuclei attached to platinum(II)
centers are chemically equivalent in 5.
constructing discrete metal-containing macrocyclic species
derived from pyrazine molecules, we describe the use of a
new organometallic tecton (5) as a building block to yield two
hexagonal ensembles via a coordination-driven self-assembly
reaction. Usually the construction of discrete hexagonal
macrocyclic species requires the self-assembly of a relatively
larger number of donor and acceptor units. However, our
strategy involves the utilization of a relatively lesser number of
donor/acceptor units [2 + 2] to obtain a hexagonal entity that
otherwise requires a [6 + 6] or [3 + 3] combination of
complementary tectons. Herein, we have synthesized two
hexagonal-shaped macrocycles in which two ditopic acceptor
tectons self-assemble with two linear ditopic donor tectons to
yield a [2 + 2] assembly. The diplatinum acceptor organo-
metallic clip (5) was first reacted with 2 equiv of silver nitrate,
and the dinitrate derivative (generated in situ) was reacted with
a suitable linear ditopic donor tecton (4,4′-bipyridine or
pyrazine) in a 1:1 stoichiometric ratio (Scheme 2). This
resulted in the exclusive formation of the hexagonal macro-
cycles 6 and 7 via a [2 + 2] coordination-driven self-assembly
(Scheme 2). The crude product thus obtained (after solvent
evaporation) was washed with n-pentane and subsequently
X-ray Crystallographic Analysis of 4. Single crystals of 4
suitable for X-ray diffraction) were obtained by the slow
(
evaporation of its dichloromethane solution at ambient
temperature. Compound 4 crystallizes in the triclinic space
group P1. Crystal structure analysis reveals no unusual bond
̅
lengths or angles within the molecule. The molecular structure
with the numbering scheme is shown in Figure 1. Both pendent
1,3-diethynylbenzene moieties are in the same plane as the
central pyrazine ring. However, the orientations of the two
terminal ethynyl moieties of 4 are trans to each other. Detailed
crystallographic analysis revealed that both nitrogen atoms (N1
and N2) of the pyrazine ring of a given molecule 4 have
hydrogen-bonding interactions with the neighboring molecule.
The hydrogen-bond parameters found in 4 are tabulated in
Supporting Information S8. The intermolecular hydrogen-
bonding interactions are shown in Supporting Information S8.
Application of the Organometallic Clip 5 as a
Molecular Tecton for the Self-Assembly of Irregular
Hexagonal Macrocycles. Continuing our research interest in
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Figure 2. (a) 31P{ H} and (b) H NMR spectra of the macrocycle 6.
1
1
recrystallized from chloroform/methanol to obtain the desired
macrocycles as a yellowish microcrystalline solid in reasonable
yield (>90%). In both cases, the product obtained was soluble
in organic solvents.
The formation of macrocycles was subsequently confirmed
by analytical techniques such as multinuclear NMR spectros-
copy, electrospray ionization time-of-flight mass spectrometry
(ESI-TOF-MS), and elemental analyses. In addition, the
molecular structure of 6 was unambiguously determined by
3
1
1
X-ray crystallography analysis. P{ H} NMR analyses of 6 and
are consistent with the formation of a single, highly
symmetrical species due to the appearance of sharp singlets
7
Figure 3. ESI-MS data of the macrocycle 6.
195
respectively at 15.88 and 20.13 ppm with concomitant Pt
1
1
satellite peaks (6, J = 1164 Hz; 7, J = 1237 Hz). These
PPt
PPt
consecutive loss of nitrate counteranions from the expected [2
peaks are shifted relative to that of 5 (Δδ = 7.29 and 11.54
− 2+
+
−
2] macrocycle at m/z 1406.43 ([6 − 2NO ] ), 916.95 ([6
3
ppm) for products 6 and 7, respectively (Figure 2 and
− 3+ − 4+
3NO ] ), and 672.22 ([6 − 4NO ] ). Similarly, for
1
3
3
Supporting Information S5−S8). The H NMR spectra of
compound 7, peaks were observed at m/z 1330.42 ([7 −
macrocycles 6 and 7 also confirmed the formation of pure and
highly symmetrical structures. A significant shift in the position
of the peaks corresponding to the reacting donor and acceptor
− 2+
− 3+
2
4
NO ] ), 866.28 ([7 − 3NO ] ), and 634.21 ([7 −
− 4+
3
3
NO ] ) in the ESI-TOF-MS spectrum, confirming the
3
formation of the [2 + 2] macrocyclic architecture (Supporting
Information S9).
X-ray Crystallographic Analysis of 6. There are a handful
reports in the literature where the crystal structures of ionic
platina metallacycles [synthesized from platinum(II) acceptor
1
tectons was observed upon coordination. The H NMR spectra
of products 6 and 7 clearly suggested the incorporation of a
donor tecton (4,4′-bipyridine in the case of 6 and pyrazine in
the case of 7) and an acceptor pyrazine-based clip (5) in the
respective final product (Figure 2 and Supporting Information
15
tectons and bipyridine donors] are reported. After repeated
trials, X-ray-quality single crystals of 6 were obtained by the
slow vapor diffusion of diethyl ether into its solution in a
solvent mixture (CH Cl /CH OH) at room temperature.
1
S5−S8). In H NMR spectrum of 6, two doublet peaks
appeared at 8.89−8.88 and 8.66−8.65 ppm, and these were
assigned to the protons of the 4,4′-bipyridine units. The signals
at 8.67, 7.64, 7.45−7.44, and 7.32−7.31 ppm correspond to the
pyrazine and phenyl protons of the acceptor unit (Figure 2) in
2
2
3
Figure 4 shows the molecular structure of 6, which crystallizes
in the triclinic space group P1. The ball-and-stick presentation
̅
6
. The presence of the PEt3 groups attached to the
of the ionic irregular hexagon 6 is shown in Figure 4. The
molecular structure of 6 consists of two molecular clips that are
bridged by two 4,4′-bipyridine units through nitrogen atoms
platinum(II) centers was evident from the signals due to the
1
ethyl protons in the H NMR spectrum in the range of 1.85−
1.17 ppm (Figure 2), and this confirmed the incorporation of
and forms a highly twisted Pt molecular irregular hexagon, as
4
the acceptor tecton in this self-assembled product (6).
shown in Figure 4B. The geometry around each platinum is
close to square-planar, with the cis angles in the range of 86−
95° (Supporting Information S9 and S10). There are two
nitrate anions that are crystallographically disordered. They are
situated outside the hexagon pockets of 6. The important
crystallographic parameters for 6 are presented in Supporting
1
Similarly, in the case of 7, all proton signals in H NMR
were assigned correctly (Supporting Information S7).
The [2 + 2] self-assembly involving two units of donor and
acceptor tectons were confirmed by ESI-MS analysis. The ESI-
TOF-MS spectrum of 6 (Figure 3) shows signals due the
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Figure 4. (A) Ball-and-stick representation of 6. (B) Ball-and-stick representation illustrating the inherent twist present in the molecular irregular
−
hexagon 6. The ethyl groups attached to the phosphorus atoms and all counteranions (NO ) are omitted for clarity.
3
Figure 5. In vitro cytotoxicity of synthesized compounds (6 and 7) and cisplatin against (A) A549, (B) MCF7, (C) KB, and (D) HaCaT cells after
4
8 h of treatment as determined by MTT assay. Data points represent mean ± standard deviation for at least three independent experiments (#, p <
0
.05; ##, p < 0.01).
Information S9. The distance between Pt(01) and Pt(03) is
more toxic for each cell line and exhibited a lesser IC50 value
compared to 6. A five-point dose-dependent curve was plotted
for each cell line (Figure 5), and the IC50 concentration was
calculated for both compounds (Table 1). The IC50
8
8
.3414 (0.0017) Å, and that between Pt(02) and Pt(04) is
.3937 (0.0017) Å. The length of 6 is 29.959 Å, as defined by
the distance between the centroids of the pyrazine rings of the
molecular clip. The distortion in the hexagon shape of the
metallacycle 6 is probably due to the steric effect exerted by the
adjacent triethylphosphine ligands. This is more clearly evident
from the CPK representation (space-filled model of 6 in
Supporting Information S11); it appears that this repulsive
interaction has been partially relieved in the entire molecule by
incorporating a dihedral twist (Φ ≈ 20.6°), and this is possible
because of the free rotation of the terminal ethynylbenzene
moieties of the molecular clip 6.
Table 1. IC50 Concentration (μM) of Compounds against
Different Cancer Cells
IC /μM
50
A549 cells
MCF7 cells
KB cells
HaCaT cells
cisplatin
25.0 ± 0.3
>30.0
20.0 ± 0.3
17.0 ± 0.3
05.0 ± 0.2
08.0 ± 0.4
20.0 ± 0.1
16.0 ± 0.4
12.0 ± 0.3
20.0 ± 0.1
16.0 ± 0.5
6
7
20.0 ± 0.2
Overall, the stoichiometry and composition of the [2 + 2]
macrocycle, as predicted by NMR spectroscopy and mass
spectrometry, were confirmed by X-ray analysis of 6.
In Vitro Cytotoxicity and Determination of the IC50
Value. The cytotoxic activity of both compounds were tested
against four cell lines, viz., A549 (human lung carcinoma cell
line), KB (human oral cancer cell line), MCF7 (human breast
cancer cell line), and HaCaT (human skin keratinocyte cell
line) and compared with that of cisplatin. 7 was found to be
concentration is defined as the compound concentration at
which 50% of the cells were viable. Both compounds and
cisplatin showed concentration-dependent cytotoxic effects. At
lower concentrations (∼1 μM), no cytotoxic effect was
observed for any compound. A549 cells were found to be
least sensitive for cisplatin (IC value of 25.0 ± 0.3 μM) and 6
50
(IC50 value of >30.0 μM).
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Figure 6. Live−dead cell viability assay for (A) A549, (B) MCF7, (C) KB, and (D) HaCaT cells. Cells were treated with a 10 μM concentration of
each compound for 48 h and stained with live−dead fluorescent dyes. Green color staining represents live cells, whereas red color fluorescence
indicates dead cells with damaged membranes. Scale bar: 200 μm.
1
9
Interestingly, 7 exhibited superior cytotoxic effects (IC50
values of 20.0 ± 0.2 and 05.0 ± 0.2 μM) when compared
with cisplatin (IC values of 25.0 ± 0.3 and 20.0 ± 0.3 μM) for
of cisplatin was in accordance with earlier reports. A549 cells
did not show cell cycle arrest in any phase (Supporting
Information S12, Figure S18A) when treated with our
synthesized compounds (6 and 7). 7 and 6 induced cell cycle
arrest in the G2 phase for MCF7 and KB cells, respectively
(Supportin Information S12, Figure S18B,C), which indicates
50
A549 and MCF7 cells, respectively (Figure 5A,B; p < 0.01).
Moreover, 6 showed a similar effect against MCF7 cells and
was more cytotoxic than cisplatin at higher concentration (30
μM; p < 0.05). Overall, 6 with two additional benzene rings
exhibited lower cytotoxic effects in comparison with 7. Such
variations in cytotoxic effects might be attributed to differences
in the hydrophobicity and molecular weight, leading to
2
0
the possibility of apoptosis in these two cell lines. A slight G1
arrest was observed for HaCaT cells after treatment with 6 and
7 (Supporting Information S12, Figure S18D). Cell cycle data
indicate that cytotoxic effects of our synthesized compounds
might be followed by apoptosis in certain cases where cell cycle
arrest is observed.
16
alteration in the cellular uptake. Differences in the cellular
uptake would allow variable access of compounds to the nuclear
DNA, which might lead to inconsistent cytotoxic effects.
In order to further investigate the effect of synthesized
compounds on the cell viability and attachment, cells were
stained with two fluorescent dyes (Calcein AM and Ethidium
homodimer-1) after 48 h with constant 10 μM concentration.
Live cells were found to be firmly attached in all cases, and a
variable number of dead cells were observed based on the IC50
concentration of each compound. A549 and KB cells were
found to be swollen after treatment with 7 and cisplatin (Figure
In general, cells may follow either apoptosis or necrosis after
treatment with any cytotoxic compound. Apoptosis is also
known as programmed cell death and characterized by cell
shrinkage, DNA fragmentation, and chromatin condensation.
Necrosis, on the other hand, is induced by physical cell damage
and characterized by early cell membrane damage and cellular
swelling. Cells following the necrosis pathway of cell death may
be identified by staining with propidium iodide (PI) dye. This
dye is cell-impermeable and enters the cell only if the cell
membrane is damaged. Hence, PI staining is a suitable way of
assessing whether cells are undergoing necrosis. In the
present study, we followed the same and all of the cells were
stained with PI 48 h after treatment with each respective
compound. Cells were administered to flow cytometry analysis
without fixing them in ethanol. Data were acquired following a
similar gating for all of the samples, and % PI-positive cells
(undergoing necrosis) are reported (Supporting Information
S13, Figure S19).
6
A,C). In contrast, no such morphological change was observed
21
for the other two cell lines (Figure 6B,D).
Flow Cytometry Analysis. MTT data indicated that the
synthesized compounds (6 and 7 show cytotoxic effects against
various human cell lines (A549, MCF7, KB, and HaCaT). We
further investigated the mechanism of action of these
compounds by analyzing their effects on the cell cycle and
membrane integrity. Cell cycle checkpoints ensure the normal
growth of cells. In the case of DNA damage, the cell cycle is
arrested in that particular phase until the damaged DNA is
In all cases, cells were more sensitive to cisplatin and a higher
percentage of PI-positive cells was observed after treatment
compared to other groups (p < 0.01). However, MTT data
indicated that the cytotoxic effects of 7 are comparable to those
of cisplatin for A549 and MCF7 cells. This suggests that only a
1
7
repaired. Platinum (Pt)-based cytotoxic drugs are known to
18
target the DNA replication process. In the present study,
cisplatin treatment induced S and G2 phase arrest for all four
cell types (Supporting Information S12, Figure S18). The effect
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fraction of macrocycle-7-treated cells are undergoing possible
necrotic cell death, while the remaining population may follow
the apoptotic pathway. 7 has induced membrane damage for a
higher population of cells (A549 cells, 7.12 ± 0.03%; KB cells,
7
.31 ± 0.41%) compared to 6-treated cells (A549 cells, 5.17 ±
.13%; KB cells, 5.17 ± 0.25%; Supporting Information S13,
0
Figure S19A,C; p < 0.01). This might be an indication toward
necrosis-induced cell death. These data are in accordance with
live−dead images, where 7-treated cells exhibited a swollen
morphology (a signature of necrotic cell death; Figure 6A,C).
Although MCF7 cells did not exhibit any significant difference
between the two synthesized compounds (6 and 7), a
percentage of PI-positive cells was elevated compared with
the control group (Supporting Information S13, Figure S19B; p
<
0.05). On the contrary, both compounds were ineffective
against HaCaT cells, and values were comparable with those of
the control group (Supporting Information S13, Figure S19D).
In conclusion, data suggested that platinum-containing macro-
cyclic compounds (6 and 7) induced the loss of membrane
integrity against cancer cells and might serve as a potential
target for anticancer therapy.
5
32.12. Found: m/z 532.90.
Synthesis of Compound 4. In a 100 mL round-bottomed Schlenk
flask, 2 (0.5 g, 0.94 mmol), tetrakis(triphenylphosphine)palladium
(0.22 g, 0.19 mmol), and 46 mg of cuprous(I) iodide (54 mg, 0.28
mmol) were added in 30 mL of dry THF. Then 5 mL of dry
triethylamine and (trimethylsilyl)acetylene (0.5 mL, 3.76 mmol) were
added to the reaction mixture. The suspension was stirred for 16 h at
room temperature in the absence of light. After removal of the solvent,
the residue was washed twice with water and the organic phase was
dried over sodium sulfate. The solvent was removed in a rotary
evaporator, and the residue was purified by column chromatography
on 100−200 mesh silica gel. The desired product 3 (0.34 g, 76%) was
isolated with 3% ethyl acetate in hexane.
Desilylation of 3 (0.34 g, 0.72 mmol) was carried out in a THF
solution (10 mL) using TBAF (0.46 g, 1.44 mmol) as a deprotecting
agent for 5 min. The solution was evaporated in a rotary evaporator
followed by washing of the residual product with water and extraction
with dichloromethane. The organic phase was dried by sodium sulfate,
and the solvent was removed. Finally, the desired product 4 was
obtained as a white solid by purification using column chromatography
CONCLUSION
■
In conclusion, we have reported two new cationic and
hexagonal (irregular) macrocycles (6 and 7) derived from a
pyrazine-based organometallic acceptor clip 5 using only four
tectons in highly efficient coordination-driven self-assembly
reactions. 6 and 7 were characterized by well-known
spectroscopic techniques, and one of the macrocycles (6) was
characterized by single-crystal XRD analysis. The macrocycles
were tested against A549 (human lung carcinoma cell line), KB
(
human oral cancer cell line), MCF7 (human breast cancer cell
line), and HaCaT (human skin keratinocyte cell line) and
compared with cisplatin. It was observed that the smaller
macrocycle (7) has superior cytotoxic effects against all cancer
cells and its IC concentration is lower relative to cisplatin for
on 100−200 mesh silica gel using 5% ethyl acetate in hexane.
1
Yield: 0.20 g, 84%. Mp: 145−147 °C. H NMR (CDCl
, 400 MHz):
3
δ 8.66 (s, 2H, Ar−H), 7.75−7.74 (m, 2H), 7.61−7.58 (m, 2H, Ar−H),
50
1
3
7
.54−7.52 (s, 2H, Ar−H), 7.38−7.34 (m, 2H, Ar−H). C NMR
A549 and MCF7 cells. This report is a unique example, wherein
self-assembled two-dimensional ensembles derived from a
platinum(II) acceptor clip have been studied for their potential
application in anticancer therapy.
(CDCl , 75 MHz): δ 145.7, 139.5, 135.6, 133.2, 132.3, 128.6, 122.8,
3
1
1
8
21.7, 92.4, 85.9, 82.3, 78.3. IR (ATR): 3290, 3209, 2212, 1504, 1473,
207, 1163, 1004, 894, 783, 677 cm . Anal. Calcd for C H N : C,
−1
2
4
12
2
7.79; H, 3.68; N, 8.53. Found: C, 87.86; H, 3.76; N, 8.63. MS (ESI).
+
Calcd (26.7%; M ): m/z 328.37. Found: m/z 329.10.
EXPERIMENTAL SECTION
General Details. All chemicals and anhydrous solvents used in this
work were purchased from commercial sources and used without
further purification. Triethylamine was freshly distilled prior to use. 1
Synthesis of Compound 5. 4 (0.1 g, 0.30 mmol) and trans-
diiodobis(triethylphosphine)platinum(II) (0.63 g, 0.91 mmol) were
placed in a 100 mL Schlenk flask in the glovebox. Subsequently, 30 mL
of dry toluene and 10 mL of freshly distilled triethylamine were added
under nitrogen. The solution was stirred for 20 min at room
temperature before CuI (9 mg, 0.05 mmol) was added in one portion.
The reaction mixture was stirred overnight at room temperature.
Triethylammonium iodide precipitated from the solution and was
removed by filtration. Toluene was evaporated in a rotary evaporator,
and the resulting yellow residue was purified by column chromatog-
raphy on 100−200 mesh silica gel by eluting with 3% ethyl acetate in
hexane first and then gradually increasing the polarity of the eluant to
■
2
2
was prepared by following the reported literature procedures. A549
(
human lung carcinoma cell line), KB (human oral cancer cell line),
MCF7 (human breast cancer cell line), and HaCaT (human skin
keratinocyte cell line) were purchased from National Centre for Cell
Science, Pune, India. All of the cells were cultured and maintained in
Dulbecco’s modified Eagle medium (Gibco, USA) containing a 1% (v/
v) antibiotic−antimycotic solution (Himedia, India) and 10% (v/v)
fetal bovine serum (FBS; Gibco, USA) in a humidified CO incubator
2
at 37 °C. Phosphate-buffered saline (PBS) and Ribonuclease A
8% ethyl acetate in hexane to isolate 5 as a yellow semisolid.
1
(
RNaseA) were purchased from Sigma-Aldrich, USA. FT-IR spectra
Yield: 0.3 g, 68%. H NMR (400 MHz, CDCl
3
): δ 8.64 (s, 2H, Ar−
were recorded in a PerkinElmer Spectrum 400 FT-IR spectropho-
H), 7.50 (s, 2H, Ar−H), 7.42−7.40 (d, J = 7.6 Hz, 2H, Ar−H), 7.31 (s,
2H, Ar−H), 7.24 (s, 2H, Ar−H), 2.26−2.19 (m, 24H, −CH ), 1.22−
, 75 MHz): δ 145.4, 139.5,
133.9, 131.6, 129.0, 128.3, 121.1, 99.1, 93.5, 91.9, 85.2, 16.6, 8.1.
1
31
tometer. H and P NMR spectra were recorded on Bruker 400/500
MHz spectrometers. Elemental analyses were carried out using an
Elementar Vario Micro Cube elemental analyzer. ESI-MS analysis was
performed using a Bruker Impact ESI-Q-TOF system. A Tecan infinite
Pro M 200 reader with i-control software was used for to record the
absorbance. A EVOS FL (Life Technologies, USA) fluorescence
microscope was used.
2
13
1.14 (m, 36H, −CH ). C NMR (CDCl
3 3
31
1
1
P{ H} NMR (163 MHz, CDCl
): δ 8.59 ( JPPt = 1165 Hz). IR
3
(ATR): 2957, 2927, 2872, 2210, 2113, 1587, 1506, 1454, 1408, 1217,
1145, 1031, 1004, 887, 767, 727, 680 cm . MS (ESI). Calcd (M +
1): m/z 1442.19. Found: m/z 1443.5.
−1
+
Synthesis of Compound 2. A solution of compounds 1,3-
diiodobenzene (1.03 g, 3.12 mmol) and palladium tetrakis-
General Procedure for the Synthesis of Macrocycles 6 and 7.
To the solution of compound 5 (100 mg, 0.07 mmol) in chloroform
G
DOI: 10.1021/acs.inorgchem.7b01561
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Forum Article
(
4 mL) was added AgNO (24 mg, 0.14 mmol) in one portion, and
In Vitro Cytotoxicity. The cytotoxic activities of both compounds
6 and 7 was tested against A549, MCF7, KB, and HaCaT cell lines at
varying concentrations ranging from 1 to 30 μM. Cells were seeded in
3
the reaction mixture was stirred overnight in the absence of light at
room temperature. The precipitated AgI was filtered over a bed of
Celite, and the filtrate was collected as a yellow solution. Subsequently,
a methanolic solution of the respective donor tecton (bipyridine and
pyrazine; 0.02 mmol, 0.5 mL) was added dropwise to the
aforementioned filtrate with continuous stirring. The reaction mixture
was stirred for 15 h at room temperature. Solvents were removed on a
rotary evaporator, and the product obtained was washed several times
with n-pentane to obtain a yellow solid, which was finally dried in a
vacuum. 6 was crystallized by slow vapor diffusion of diethyl ether in
its corresponding concentrated dichloromethane-methanol solution
and 7 was crystallized as a yellow microcrystalline solid by vapor
diffusion of diethyl ether in its corresponding concentrated methanol
4
a 96-well flat-bottom plate, keeping a similar cell density (∼10 ) in
each well, and allowed to adhere for 24 h. Cells were further treated
with predefined concentrations of compounds 6 and 7 and cisplatin.
After 48 h of treatment, the viability of the cells was quantified by
measuring their metabolic activity using MTT assay [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. A 10% (v/v)
MTT stock solution (5 mg/mL) prepared in FBS free culture media
was added to the compound-treated cells and incubated for 4 h to
allow formazan crystal formation. These crystals were further dissolved
in 200 μL of DMSO, and the absorbance was measured at 570 nm
using a microplate reader. The absorbance values of compound-treated
cells were normalized with the untreated control group, and a five-
point dose-dependent curve was plotted to calculate the IC50 value of
each compound (the IC50 value is the concentration of the compound
at which 50% of cells are viable).
solution.
1
Macrocycle 6. Yield: 30 mg, 92%. H NMR (400 MHz, CDCl ): δ
3
8
.89−8.88 (d, J = 6.52 Hz, 8H, Ar−H), 8.67 (s, 4H, Ar−H), 8.66−8.64
(
d, J = 6.72 Hz, 8H, Ar−H), 7.64 (s, 4H, Ar−H), 7.45−7.44 (m, 4H,
Ar−H), 7.32−7.31 (m, 8H, Ar−H), 1.85−1.81 (m, 48H, −CH −),
2
13
1
1
9
.26−1.17 (m, 72H, −CH ). C NMR (CDCl , 75 MHz): δ 153.3,
The viability of cells after 48 h of compound treatment (10 μM
concentration) was assessed by staining the cells with a live−dead
solution. Cells were incubated with a working live−dead solution (1
μL of 4 mM Calcein AM and 4 μL of 2 mM Ethidium homodimer-1 in
3
3
52.6, 150.4, 145.8, 144.8, 139.5, 135.5, 131.6, 128.3, 126.3, 121.1,
3.1, 85.9, 14.3, 7.7. 31P{ H} NMR (162 MHz, CDCl ): δ 15.88 ( J
1
1
3
PPt
=
1
1164 Hz). IR (ATR): 2970, 2931, 2870, 2214, 2121, 1705, 1635,
−1
581, 1504, 1458, 1327, 1157, 1033, 887, 756, 740 cm . Anal. Calcd
2
mL of PBS) for 10 min at 37 °C in a humidified CO incubator.
2
for C116H156N P Pt : C, 51.78; H, 5.84; N, 4.16. Found: C, 51.89; H,
5
8
8
4
Calcein AM enters into live cells, which, in turn, undergoes ester
hydrolysis by a cellular esterase enzyme and fluoresces green
(excitation = 495 nm; emission = 516 nm). On the other hand,
Ethidium homodimer-1 specifically enters into dead cells, and, upon
binding with DNA, gives red fluorescence (excitation = 528 nm;
emission = 617 nm). Stained cells were washed three times with sterile
PBS and imaged under a fluorescent microscope.
2+
.98; N, 4.23. MS (ESI). Calcd for [M − 2NO ] : m/z 1406.32.
3
3+
Found: m/z 1406.43. Calcd for [M − 3NO ] : m/z 916.88. Found:
3
4+
m/z 916.59. Calcd for [M − 4NO ] : m/z 672.16. Found: m/z
3
6
72.22.
1
Macrocycle 7. Yield: 28 mg, 91%. H NMR (400 MHz, CDCl ): δ
3
8
7
−
1
1
.65 (s, 4H, Ar−H), 8.60 (s, 8H, Ar−H), 7.44 (s, 4H, Ar−H), 7.40−
.38 (m, 8H, Ar−H), 7.25−7.24 (m, 4H, Ar−H), 1.99−1.93 (m, 48H,
1
3
CH ), 1.27−1.25 (m, 72H, −CH ). C NMR (CDCl , 75 MHz): δ
Flow Cytometry. Cell Cycle Analysis. Cells treated with
compound were tested for any cell cycle arrest using flow cytometry.
2
3
3
45.7, 145.4, 144.8, 134.9, 134.5, 132.3, 129.3, 128.7, 121.1, 93.5, 85.2,
4.3, 7.7. 31P{ H} NMR (162 MHz, CDCl ): δ 20.13 ( J = 1238
1
1
Approximately 2 × 10 cells were seeded in each well of standard 6-
5
3
PPt
Hz). IR (ATR): 2972, 2924, 2875, 2223, 2124, 1640, 1583, 1463,
well tissue culture plates, and cells were allowed to adhere for 24 h in a
humidified incubator. Further, cells were treated with IC50
concentration of each compound for 48 h. After compound exposure,
cells were harvested and a cell pellet was fixed in ice cold 70% ethanol
and stored at −20 °C for further analysis. Fixed cells were washed
twice with PBS and incubated with RNaseA [200 μg/mL (w/v)] for 1
h at room temperature. RNaseA treatment helps to avoid nonspecific
interaction of PI with nucleic acids. After RNaseA treatment, 40 μg/
mL PI was added to the samples, and they were incubated on ice for
−1
1
331, 1273, 1149, 1038, 885, 752, 738 cm . Anal. Calcd for
C104H148N P Pt : C, 50.53; H, 5.86; N, 4.29. Found: C, 50.70; H, 5.94;
8
8
4
2+
N, 5.92. MS (ESI). Calcd for [M − 2NO ] : m/z 1330.40. Found:
3
3+
1
330.42. Calcd for [M − 3NO ] : m/z 866.27. Found: m/z 866.28.
3
4+
Calcd for [M − 4NO ] : m/z 634.20. Found: m/z 634.21.
3
Crystallographic Data Collection and Refinement. Single-
crystal data for 4 and 6 were collected on a Bruker D8 Quest
diffractometer equipped with a PHOTON 100 CMOS detector, using
a graphite monochromator and Mo Kα (λ = 0.71073 Å) radiation
operating at 50 kV and 30 mA. The unit cell measurement, data
collection (φ and ω scan), integration, scaling and absorption
1
0 min. Cytometry acquisition was done on a BD Accuri C6 plus flow
cytometer with a blue laser (488 nm) on the FL2 channel. Data were
analyzed using a ModFit LT, version 2.0 (Verity Software House), to
calculate the percentage of cells present in each phase of the cell cycle.
Live PI Staining. In order to analyze the membrane integrity of
compound-treated cells, cells were stained with PI dye. PI is a cell-
impermeable dye and stains only dead cells of a damaged cellular
membrane. Cells were cultured and treated with a specific
concentration of each compound as described earlier. After treatment,
cells were harvested and centrifuged at 1000 rpm for 5 min. A cell
pellet was washed three times with sterile PBS and resuspended in the
same. Each cell pellet was further treated with PI [15 μg/mL (w/v)
final concentration] at 4 °C for 10 min and vortexed gently.
Cytometry data acquisition was performed using a BD Accuri C6 plus
flow cytometer with a blue laser (488 nm) on the FL2 channel.
Statistical Analysis. All of the biological experiments (including
MTT assay and flow cytometry analysis) were performed in triplicate.
Significant differences among different experimental groups at various
time points were assessed by performing one-way analysis of variance
2
3
corrections for the crystals were done using Bruker Apex II software.
The structures were solved using a direct method, followed by full
2
matrix least-squares refinements against F (all data in HKLF 4
24
format) using SHELXTL. A multiscan absorption correction, based
on equivalent reflections, was applied to the data. All of the atoms
except platinum and phosphorus atoms were refined isotropically.
There are two disordered nitrate anions, which were isotropically
refined. The contributions of the most disordered solvent molecules
−
and counteranions (NO3 ) were removed from the diffraction data
25
using the SQUEEZE routine of PLATON software, and then final
refinements were carried out.
Crystallographic data for “data collection and structure refinement
parameters” and crystallographic data for 4 and 6 are summarized in
Supporting Information S8.
CCDC contains the supplementary crystallographic data for this
paper with deposition numbers of CCDC 1518730 and 1518833 for
compounds 4 and 6, respectively.
Preparation of Stock Solutions. 1 mM stock solutions of both
compounds (6 and 7) were prepared in DMSO (Sigma-Aldrich, USA),
whereas a cisplatin stock solution was prepared in normal saline (0.9%
sodium chloride). For cell culture experiments, 0.1−3% (v/v) of a
stock solution was used.
(ANOVA) following Tukey’s test using Origin 8.0 software. The
significance level was analyzed at both 95% and 99% confidence levels.
p < 0.05 was considered to be significant and p < 0.01 highly
significant.
H
DOI: 10.1021/acs.inorgchem.7b01561
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Forum Article
(
i) Fiedler, D.; Leung, D. H.; Bergman, R. G.; Raymond, K. N.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.7b01561.
■
Selective Molecular Recognition, C−H Bond Activation, and Catalysis
in Nanoscale Reaction Vessels. Acc. Chem. Res. 2005, 38, 349.
(
*
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j) Spokoyny, A. M.; Kim, D.; Sumrein, A.; Mirkin, C. A. Infinite
coordination polymer nano- and microparticle structures. Chem. Soc.
Rev. 2009, 38, 1218. (k) Bhowmick, S.; Chakraborty, S.; Das, A.;
Rajamohanan, P. R.; Das, N. Pyrazine-Based Organometallic Complex:
Synthesis, Characterization, and Supramolecular Chemistry. Inorg.
Chem. 2015, 54, 2543. (l) Bhowmick, S.; Chakraborty, S.; Das, A.;
Nallapeta, S.; Das, N. Pyrazine Motif Containing Hexagonal
Macrocycles: Synthesis, Characterization, and Host−Guest Chemistry
with Nitro Aromatics. Inorg. Chem. 2015, 54, 8994. (m) Jana, A.;
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1
13
1
H and C{ H} NMR spectra of compounds 2 and 4,
1
31
1
H and P{ H} NMR spectra of compounds 5−7, ESI-
MS analysis of macrocycles 6 and 7, X-ray crystallog-
raphy analysis of compound 4 and macrocycle 6 (PDF)
Accession Codes
CCDC 1518730 and 1518833 contain the supplementary
crystallographic data for this paper. These data can be obtained
free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by
emailing data_request@ccdc.cam.ac.uk, or by contacting The
Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
(
2) (a) Chen, L.-J.; Jiang, B.; Yang, H.-B. Transformable
nanostructures of cholesteryl-containing rhomboidal metallacycles
through hierarchical self-assembl. Org. Chem. Front. 2016, 3, 579.
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and self-organization. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 4763.
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AUTHOR INFORMATION
Corresponding Authors
E-mail: biman.mandal@iitg.ernet.in or mandal.biman@gmail.
com (B.B.M.). Tel.: +91-361-258 2225.
E-mail: neeladri@iitp.ac.in or neeladri2002@yahoo.co.in
N.D.). Tel.: +91-9631624708.
(
■
*
catenane. Nat. Chem. 2014, 6, 978. (d) Jana, A.; Das, N. Self-Assembly
of [2 + 2] Platina Macrocycles Using a Flexible Organometallic Clip.
Chemistryselect 2017, 2, 4099. (e) Tian, J.; Zhou, T.-Y.; Zhang, S.-C.;
Aloni, S.; Altoe, M. V.; Xie, S.-H.; Wang, H.; Zhang, D.-W.; Zhao, X.;
Liu, Y.; Li, Z.-T. Three-dimensional periodic supramolecular organic
framework ion sponge in water and microcrystals. Nat. Commun. 2014,
5, 5574. (f) Li, S.-H.; Zhang, H.-Y.; Xu, X.; Liu, Y. Mechanically
selflocked chiral gemini-catenanes. Nat. Commun. 2015, 6, 7590.
*
(
ORCID
Biman B. Mandal: 0000-0003-3936-4621
Neeladri Das: 0000-0003-3476-1097
Notes
(g) Bhowmick, S.; Chakraborty, S.; Marri, S. R.; Behera, J. N.; Das, N.
Pyrazine-based donor tectons: synthesis, self-assembly and character-
ization. RSC Adv. 2016, 6, 8992. (h) Chakraborty, S.; Mondal, S.;
Bhowmick, S.; Ma, J.; Tan, H.; Neogi, S.; Das, N. Triptycene based
organometallic complexes: a new class of acceptor synthons for
supramolecular ensembles. Dalton Trans. 2014, 43, 13270.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(
3) (a) Lehn, J.-M. Supramolecular Chemistry: Concepts and
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. and K.S. thank UGC, New Delhi,
India, for a Research Fellowship. The authors also acknowledge
SAIF-Panjab University and SAIF-IIT Patna for analytical
facilities. B.B.M. gratefully acknowledges funding support
through the Department of Biotechnology and Department
of Science and Technology, Government of India.
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