Versatile coordination ability of thioamide ligand in Ru(II) complexes: Synthesis, computational studies,: In vitro anticancer activity and apoptosis induction

Article abstract of DOI:10.1039/c7nj01828b

The reaction of a (2-hydroxy-5-nitrophenyl)(pyrrolidin-1-yl) methanethione ligand (L), with an equimolar amount of [RuHCl(CO)(PPh₃)₃] afforded two structurally different ruthenium(ii) complexes of formulas [Ru(CO)(Cl)(PPh₃)₂(O^S)] (1) and [Ru(CO)(PPh₃)₂(N^O^S)] (2) by varying the experimental reactions conditions. Interestingly, the ligand behaves in a monobasic bidendate OS fashion in reflux conditions vis C-N bond cleavage, while it acts as a dibasic tridendate NOS donor at room temperature towards the ruthenium(ii) precursor. The synthesised ruthenium complexes 1 and 2 are fully characterized by means of analytical (elemental analysis), spectral [FT-IR, UV-vis, NMR (¹H, ¹³C and ³¹P)] and fluorescence methods, respectively. The solid state molecular structures of the ligand and the complexes were studied by using single crystal X-ray crystallography, which confirms the different coordination mode of the ligand with the ruthenium ion and reveals the presence of distorted octahedral geometry. Notably, in complex 1, the two triphenylphosphine co-ligands occupy trans positions, whereas in complex 2 they are mutually cis disposed. The intercontacts in the crystal structures are examined using Hirshfeld surface analysis. The stability of the complexes is also studied by time-dependent UV-visible and ESI-mass spectral methods. Moreover, the cytotoxic effect of the complexes is examined on cancerous cell lines such as HeLa and MCF-7 along with a cell viability assay against non-cancerous NIH-3T3 cells under in vitro conditions and the results showed that the complexes exhibit significant anticancer activity. Furthermore, the morphological changes and apoptosis induction have been studied by AO-EB dual staining methods, Hoechst 33258 and flow cytometry techniques.

Full text of DOI:10.1039/c7nj01828b

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Versatile coordination ability of thioamide ligand
in Ru(II) complexes: synthesis, computational
studies, in vitro anticancer activity and apoptosis
induction†
Cite this:DOI: 10.1039/c7nj01828b
a
b
Ramasamy Raj Kumar,
Rengan Ramesh *a and Jan Grzegorz Małecki
The reaction of a (2-hydroxy-5-nitrophenyl)(pyrrolidin-1-yl) methanethione ligand (L), with an equimolar
amount of [RuHCl(CO)(PPh3)3] afforded two structurally different ruthenium(II) complexes of formulas
[Ru(CO)(Cl)(PPh3)2(O^S)] (1) and [Ru(CO)(PPh3)2(N^O^S)] (2) by varying the experimental reactions
conditions. Interestingly, the ligand behaves in a monobasic bidendate OS fashion in reflux conditions vis
C–N bond cleavage, while it acts as a dibasic tridendate NOS donor at room temperature towards the
ruthenium(II) precursor. The synthesised ruthenium complexes 1 and 2 are fully characterized by means
of analytical (elemental analysis), spectral [FT-IR, UV-vis, NMR (1H, 13C and 31P)] and fluorescence
methods, respectively. The solid state molecular structures of the ligand and the complexes were
studied by using single crystal X-ray crystallography, which confirms the different coordination mode of
the ligand with the ruthenium ion and reveals the presence of distorted octahedral geometry. Notably,
in complex 1, the two triphenylphosphine co-ligands occupy trans positions, whereas in complex 2 they
are mutually cis disposed. The intercontacts in the crystal structures are examined using Hirshfeld surface
analysis. The stability of the complexes is also studied by time-dependent UV-visible and ESI-mass spectral
methods. Moreover, the cytotoxic effect of the complexes is examined on cancerous cell lines such as
HeLa and MCF-7 along with a cell viability assay against non-cancerous NIH-3T3 cells under in vitro
conditions and the results showed that the complexes exhibit significant anticancer activity. Furthermore,
the morphological changes and apoptosis induction have been studied by AO-EB dual staining methods,
Hoechst 33258 and flow cytometry techniques.
Received 25th May 2017,
Accepted 20th July 2017
DOI: 10.1039/c7nj01828b
rsc.li/njc
easily be modified by variation of the parent aldehydes or
ketone for synthesis. These ligands are vital structural motifs
Introduction
In recent years, there has been a surge in the development of widely used as key intermediates and versatile building blocks for
thioamide-based ligands designed for biologically active transition the construction of biologically important sulphur-containing
metal complexes. Thioamides are sulphur analogues of amides heterocycles.1,2 They are of considerable pharmacological interest
and are a starting material for the preparation of thiazoles, since a number of derivatives have shown a broad spectrum of
amidenes and amidrazones which coordinate strongly with metal chemotherapeutic properties. In particular, heterocyclic thio-
ions. The chemical interest arises from the ability of thioa- amides have antibacterial, antineoplastic, antimalarial, antiviral
mides to adopt various coordination modes, leading to enor- and antitumour activities.3–5 Although uncomplexed thioamides
mous structural diversity of their metal complexes. They can show interesting biological activity, metal complexes of thioamides
show a marked increase in activity.6,7 Currently, considerable effort
is being directed towards the synthesis and structural characteriza-
a Centre for Organometallic Chemistry, School of Chemistry,
tion of metal complexes of aromatic thioamides,8–11 due to their
Bharathidasan University, Tiruchirappalli-620 024, Tamil Nadu, India.
highly interesting chemical and biological properties.12,13
E-mail: ramesh_bdu@yahoo.com, rramesh@bdu.ac.in; Fax: +91-431-2407045,
+91-431-2407020; Tel: +91-431-2407053
The amide bond is a crucial backbone in organic chemistry
and constitutes the key functional group in peptides, polymers,
and many natural products and pharmaceuticals.14 The signifi-
cant increase in the interest in the reactivity of transition metals
leading to the cleavage of C–X bonds (where X = O, S, N)15 can
be attributed to how this nature can lead to new chemical
b Department of Crystallography, Institute of Chemistry, University of Silesia,
40-006 Katowice, Poland
† Electronic supplementary information (ESI) available: 1H, 13C and 31P NMR spectra
for the ligand and complexes (1–2), ESI-Mass, stability studies and MTT assay for the
complexes. CCDC 1472340, 1473794 and 1518538. For ESI and crystallographic data in
CIF or other electronic format see DOI: 10.1039/c7nj01828b
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transformations with practical implications for several indus- Results and discussion
trial processes. Dehydrodesulfurization,16 deoxygenation,17 and
Synthesis of the complexes
denitrogenation18 of crude oil, catalytic degradation of
Reaction of the (2-hydroxy-5-nitrophenyl)(pyrrolidin-1-yl) methan-
ethione ligand (L) with an equimolar amount of [RuHCl(CO)-
(PPh3)3] resulted in two different entities, namely [Ru(CO)(Cl)-
(PPh3)2(O^S)] (1) and [Ru(CO)(PPh3)2(N^O^S)] (2), with different
structural features by varying the reaction conditions. The ligand
behaves as a monobasic bidendate (O, S) which undergoes C–N
bond cleavage resulting in the formation of a new ruthenium(II)
benzothialdehyde complex (1) in benzene medium under
reflux conditions. However, the same ligand acts as a dibasic
tridendate (OSN) in complex (2) under stirring in benzene at
room temperature. Coordination was immediate, as a change of
colour from yellow to red was observed. Both the ruthenium(II)
complexes have been isolated as red coloured, air stable solids
and are non-hygroscopic in nature. The complexes were isolated
as red solid in 72–80% yield (Scheme 1). The new ruthenium
complexes are highly soluble in polar organic solvents such
as chloroform, dichloromethane, acetonitrile, DMF and DMSO.
They are found to be diamagnetic, characteristic of the low-spin
d6 ruthenium(II) complexes. The identities of the complexes
were confirmed by using elemental analysis, NMR, mass and
X-ray analysis. The analytical data of the two ruthenium
complexes are in good agreement with the molecular formula
proposed.
We take into account that when the starting precursor
is a ruthenium hydride complex [RuH(Cl)(CO)(PPh3)3], the
acid–base reaction conditions have an impact on the reaction
products. At boiling temperature, the strongly stressed coordi-
nated ligand is vulnerable to nucleophilic attack and the
C–N bond is broken. Moreover, the negative potential in the
studied complex wraps the sulphur and nitrogen atoms (cf.
Fig. S1 in the ESI†) and the potentials on the halogen atoms
are considerably lower than the ones on the S or N atoms.
This shows that the C–N bond in the coordinated ligand is
susceptible to substitution. The CQS bond distance in the free
ligand and in complex 1 is 1.67 and 1.63 Å respectively, whereas
the C–S bond distance in complex 2 is 1.83 Å, indicating a
partial double bond character in CQS bonds and a weakening
of the C–S bond after coordination by the ligand to the metal
centre.
halocarbons,19 and catalysis and extrusion of nitrogen and
ammonia from organic compounds20 are the most prominent
examples among a plethora of reactions which involve C–X bond
cleavage. Although some of these processes are well understood,
reactions leading to the cleavage of the C–N bonds are particularly
challenging and information on them remains scarce.21 There
have been few reports on transition-metal-mediated cleavage of
aliphatic C–N bonds.22–25 Rupture and repair of the porphyrin
inner core: carbon–nitrogen bond breaking and formation
in ruthenium complexes of an N,N0-bridged porphyrin have
been described.26 The C–N bond cleavage of anilines by a
(Salen)ruthenium(VI) nitrido complex has been reported.27
Amide C–N bond cleavage and formation of nitride promoted
by a niobium(II) cluster has been reported by Tayebani et al.28
Facile C–N cleavage in a series of bridged lactams has been
reported.29 The activation of the relatively inert C–N bonds of
anilines under mild conditions has been reported.30 Catalytic
C–C bond formation reactions proceeding via cleavage of C–N bonds
in aniline derivatives through catalysis by the ruthenium complex
[RuH2(CO)(PPh3)3] have also been reported in the literature.31
Recently, solvent-assisted formation of ruthenium(II)/copper(I)
complexes containing thiourea derivatives has been reported
from in vitro biological perspectives in which the ligand under-
went C–N cleavage during complex formation.32
Cisplatin and its analogues are still one of the world’s best-
selling anticancer drugs. The limitation of using cisplatin
is that it leads to side effects and dose-limiting nephrotoxicity
and the development of drug resistance prevents its potential
efficacy.33 Nevertheless, this limited effectiveness due to severe
side effects and acquired resistance due to prolonged treatment
has spurred investigators to find alternative metal-based
drugs. Among the different metal complexes generating interest,
ruthenium complexes have shown great potential and remain the
subject of extensive drug discovery efforts.34,35 In this respect,
ruthenium complexes are better suited because of their variable
oxidation states, low toxicity, selectivity for cancer cells, ability to
penetrate tumor cells reasonably well, ability to bind effectively to
DNA and ability to mimic iron in binding to biomolecules.36
To the best of our knowledge, reports on thioamide ligands
undergoing amide C–N bond cleavage are scarce in the literature.
Therefore, in this investigation, we have taken a ruthenium unit
along with
a (2-hydroxy-5-nitrophenyl)(pyrrolidin-1-yl) methan-
ethione ligand. In an effort to substantiate this speculation, the
chemistry of structurally diverse ruthenium(II) complexes is
described in this paper along with synthesis, crystal structures and
in vitro anticancer activities. To get an insight into the geometry and
the electronic structure of these complexes, density functional theory
(DFT) studies have also been included. Based on X-ray diffraction,
Hirshfeld surface analysis and the 2D Finger plots have been
analysed and visualized. In addition, the in vitro anticancer activity
of the complexes against human cancer cell lines by MTT assay and
the mechanism of cell death were investigated using AO-EB, Hoechst
33258 staining methods and flow cytometry analysis.
Scheme 1 Synthetic route for the ruthenium(II) complexes 1 and 2. The
coordinating atoms are shown in distinct colours.
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Spectroscopic characterization of the complexes
FT-IR spectra. The IR spectrum of the free ligand displayed
characteristic stretching bands at 3172 and 765 cmꢀ1 due to its
–OH and CQS functional groups.37 The –OH stretching vibra-
tions are not observed in the complexes, which indicates that the
ligand underwent tautomerization and subsequent coordination
to ruthenium metal via the phenolic oxygen upon complexation.
The band observed in the region of 1315–1316 cmꢀ1 due to (nC–O
)
further confirms the coordination through the phenolic oxygen.38
A strong band in the region of 1953 to 1954 cmꢀ1 is due to the
terminally coordinated carbonyl group and is observed at a
slightly higher frequency than in the precursor ruthenium
complexes. In addition, a set of four bands at 540, 695, 745
and 1434 cmꢀ1 in the spectra of the complexes was attributed to
ruthenium bound triphenylphosphine groups.39 A band is observed
at 744 cmꢀ1 in complex 1 which corresponds to the coordination of
thione sulphur to the ruthenium(II) ion and a Ru–Cl stretching
mode appears in the region of 425 cmꢀ1 respectively.40 Moreover,
complex 2 displays new bands at 695 cmꢀ1 and 518 cmꢀ1
corresponding to nC–S (thiol sulphur) and nRu–N stretching modes
respectively. The coordination of thiol sulphur to ruthenium is
further supported by the lengthening of the C–S bond, which
is evidenced by the X-ray structure. Based on the IR spectral
data it is clearly inferred that the ligand is coordinated to the
ruthenium(II) ion in a bidendate mode via the phenolic oxygen
and thione sulphur in complex 1 whereas the ligand binds to
ruthenium(II) ion in a tridentate mode through the pyrrolidine
nitrogen, phenolic oxygen and thiolate sulphur in complex 2.
The experimental and calculated infrared spectra of the complexes
in KBr pellets are given in Fig. S2 (ESI†).
Fig. 1 Frontier molecular orbitals of the ligand (A) and complexes (B–C),
and their energy gaps.
To get an insight into the electronic structures of the ruthenium
complexes, calculations with the DFT method at the B3LYP/6-
31G**/DZVP (the DZVP basis was used for the ruthenium(II) ion)
level were carried out. Contour plots of the molecular orbitals of
the ligand and complexes were generated using Gauss view 9.0
and the frontier molecular orbitals of A–C were determined and
are depicted in Fig. 1. Moreover, analysis of the frontier mole-
cular orbitals is useful for understanding the spectroscopic
properties such as electronic absorption and emission spectra.
So, the density of states (DOS) and overlap population density of
states (OPDOS) in terms of Mu¨llikan population analysis were
calculated and the pictorial representation of the MO composi-
tions and their contributions to the chemical bonding are
presented in Fig. S3 and S4 (ESI†). Based on the calculated
electronic structures of the complexes, the absorption spectra of
the complexes 1 and 2, and the lowest energy, broad and low
intensity absorption bands on the UV-vis spectra, the complexes
have a metal-to-ligand charge transfer character.
Further, the composition of the frontier HOMOs and LUMO/
L+1 indicates that the MLCT transitions are accompanied by
charge transfer from the chloride to L and intraligand transitions.
A similar character is indicated by the second absorption band
with a maximum at 395 nm but in this band the charge transfer
between triphenylphosphine and L plays a crucial role because
the transitions between lower occupied molecular orbitals and
the LUMO were calculated in the energy range. The higher
energy bands are attributed to transitions of ligand–ligand
charge transfer type. The results of the experimental absorption
measurements resemble the theoretically calculated results.
The experimental and calculated UV-vis spectra of complexes
1 and 2 with the calculated electronic transitions are shown in
Fig. S5 (ESI†).
Electronic spectra. The electronic absorption spectra of the
complexes in dry CHCl3 showed three to four bands in the
region of 240–600 nm. The ground state of ruthenium(II) in an
1
octahedral environment is A1g, arising from the t62g configuration,
and the excited states corresponding to the t52g eg1 configurations are
3
1
1
3T1g, T2g, T1g, and T2g. Hence, four bands corresponding to the
1A1g - 3T1g, 1A1g - 3T2g, 1A1g - 1T1g and 1A1g - 1T2g transitions
are possible in order of increasing energy. The bands around
1
1
600–555 nm and 395–414 nm are assigned to A1g - T1g and
charge transfer transitions respectively. The charge transfer bands
observed in the complexes due to M - L transition are possible in
the visible region. Moreover, the presence of carbonyl, triphenyl-
phosphine and heterocyclic bases as ligands, which are capable of
producing a strong ligand field increasing eꢁg orbital, places these
levels relatively high in energy. Therefore, the lowest charge transfer
bands due to excitation of an electron from the metal t2g level to an
unfilled molecular orbital derived from the p* ligands level
should appear in a relatively higher energy region compared to
those due to t2g ! egꢁ transitions.41,42 The high intensity bands
in the region of 240–270 nm were characterized by the ligand-
centered transitions and have been designated as p–p* and n–p*
transitions for the electrons localized on the thioamide ligand. The
pattern of the electronic spectra of the complexes indicated the
NMR spectra. Evidence of chelation can also be obtained from
the NMR spectra of the ligand and the ruthenium complexes. The
1H NMR spectra of the free ligand and complexes were recorded
presence of an octahedral environment around the ruthenium(II) in CDCl3 solution and the spectra are depicted in Fig. S6 and S8
ion, similar to other formerly reported ruthenium(II) complexes.41 (ESI†). In the 1H NMR spectra, the signal due to the phenolic –OH
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and four methylene groups (pyrrolidine ring) of the free ligand (L)
appear at 9.8 and 2.0–3.9 ppm, respectively. The –OH groups
are absent in the spectra of the complexes, supporting the
deprotonation and coordination via the phenolic oxygen to
the ruthenium(II) ion. This fact confirms the deprotonation of
the ligand which is also confirmed by the IR data. In the NMR
spectrum of the free ligand, four methylene groups (pyrrolidine
ring) on the thioamide-N atoms appeared separately, suggesting
their non-equivalence and slow-rotation of the thioamide C–N
bond on the NMR time scale.43,44 In complex 1, the (–HCQS)
proton appears as a singlet in the region of 8.0 ppm; also,
methylene groups in the pyrrolidine ring signal disappear in
the aliphatic region of 2.0–3.9 ppm suggesting the cleavage
of the amide C–N bond. The overlapping multiplets observed
in the region around d 7.1–7.6 ppm are due to the presence of
aromatic protons and triphenylphosphine groups. Interestingly,
the methylene protons of the pyrrolidine ring are observed in the
region of 2.7–3.8 ppm suggesting that the pyrolidine ring is
involved in coordination via nitrogen to the ruthenium ion in
complex 2. The 13C NMR spectra of the ligand and ruthenium
complexes were recorded in CDCl3 solutions and the spectra are
shown in Fig. S7 and S9 (ESI†). The 13C NMR spectra clearly
showed that the chemical shift of the thioketone carbon moves
upfield from 189 ppm in the ligand to 158–170 ppm in the
ruthenium complexes, indicating the reduced C–S bond order
upon complexation. The aromatic carbons of the free ligand and
triphenylphosphines in the complexes show signals in the region
of 117–140 ppm. The 13C NMR spectra of the complexes showed
peaks at 186 and 190 ppm corresponding to the terminal
carbonyl group. The methylene and methine carbons in complex 2
show signals at 24–53 ppm. The coordination of the triphenyl-
phosphines as co-ligands and their configuration in the ruthenium
complexes 1–2 are confirmed from the 31P NMR of single crystals of
Fig. 2 ORTEP view of the ligand with 30% probability level.
Fig. 3 ORTEP diagram of complex 1 in CH3OH at the 30% probability
the ruthenium complexes and the spectra are shown in Fig. S10 level, with hydrogen atoms being omitted for clarity.
(ESI†). The appearance of a singlet at d 27.2 and 43.3 ppm for
complex 1 and 2 suggests the presence of two magnetically
equivalent triphenylphosphines. The NMR spectra of the com-
plexes 1 and 2 therefore confirm the coordination mode of the
ligand to the Ru(II) ion and the results are consistent with the
molecular structures revealed by X-ray diffraction studies.
X-ray crystallographic studies. As part of the structural
characterization, the molecular structure of the ligand (L) and
the complexes 1 and 2 have been determined by single crystal
X-ray analysis. The single crystals of the ligand and complexes
were obtained from the slow evaporation method using methanol
and dichloromethane–acetonitrile solvents. Perspective views of the
molecular structures with the atom numbering scheme are depicted
in Fig. 2–4. The summary of the data collection and refinement
parameters are given in Table S1 (ESI†). Whereas selected bond
lengths and bond angles are gathered in Table S2 (ESI†).
In complex 1, the facile cleavage of the C–N bond during
reaction of L with the ruthenium(II) precursor is unexpected
and seems to be metal-induced amide bond cleavage as shown
in Scheme 1. [Ru(CO)(Cl)(PPh3)2(O^S)] (1) crystallizes in the
monoclinic space group P21/n and the structure of the complex
Fig. 4 ORTEP diagram of complex 2 at the 30% probability level, with
hydrogen atoms being omitted for clarity.
is shown in Fig. 3. The crystal structure also reveals the
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presence of two solvent molecules (CH3OH) per complex. The
ligand coordinates in a bidendate manner to the Ru(II) via the
O, S donors forming one six membered chelate ring. One
carbonyl group (trans to the phenolic oxygen) and one chloride
ion form an OSCOCl square plane and the phosphorous atoms
of the two triphenylphosphine ligands occupy the two axial
sites. However, the presence of a strong p-acidic character of
CO in the complex has probably forced the bulky PPh3 to take
up mutually trans positions for steric reasons. Ruthenium is
therefore sitting in an OSCOClP2 coordination environment,
which is distorted octahedral in nature as reflected in all the
bond parameters around ruthenium. The bite angles around the
Ru(II) ion, O(2)–Ru(1)–S(1), O(2)–Ru(1)–P(1), and S(1)–Ru(1)–P(1),
are equal to 90.62(5)1, 91.77(5)1 and 89.93(3)1, respectively, and
the bond lengths are 2.2916(7) Å for Ru(1)–S(1), 2.0914(16) Å
for Ru(1)–O(2), 2.4133(6) Å for Ru(1)–Cl(1) and 1.637(3) Å for
C(8)–S(1). The two triphenylphosphine ligands are mutually
trans disposed with Ru(1)–P(1) and Ru(1)–P(2) distances of
2.4045(7) and 2.4268(7) Å, respectively. The two Ru–P bond
Fig. 5 Hirshfeld surfaces for the ligand (L) and complexes 1 and 2 mapped
with di, de, and dnorm (A–C).
lengths are similar and comparable with those found in Ru(1)–O(2) and 1.835(3) Å for C(2)–S(1). The two triphenylpho-
other reported ruthenium complexes containing PPh3.45 The sphine ligands are mutually cis disposed with Ru(1)–P(1) and
Ru(1)–O(2) distance equal to 2.0914(16) Å is shorter than Ru(1)–P(2) distances of 2.4084(7) and 2.3627(7) Å, respectively.
Ru(1)–S(1) 2.2916(7) Å. The thione form is confirmed by the The two Ru–P bond lengths are similar and comparable with
bond length of C(8)–S(1) being 1.637(3) Å, which is very close to those found in other reported ruthenium complexes containing
a formal CQS bond length (1.676(2) Å). In the complexes, the PPh3.48 In addition, the Ru–N bond length equal to 2.259(2) Å is
CQS bond distances are no longer than that of the free ligand comparable with other reported ruthenium complexes.49 The thiol
but the phenolic C–O bond length is slightly reduced upon form is confirmed by the bond length of C(2)–S(1) 1.835(3) Å which
coordination of the ligand to the ruthenium. The bond lengths is very close to other ruthenium complexes. The bond lengths
and bond angles are in good agreement with reported data on and bond angles are in good agreement with reported data on
related ruthenium(II) complexes.46,47
related octahedral ruthenium(II) complexes.50
In the free ligand, the amide C–N bond distance is equal to
We analysed the intermolecular interactions in the crystal
1.310(3) Å, which is elongated up to 1.520(3) Å after coordination structures of the free ligand and ruthenium complexes using
to ruthenium(II). Furthermore, the geometry of the thioamide Hirshfeld surface analysis. This approach is a graphical tool for
part of the ligand changes significantly during the complexation. visualizing and understanding intermolecular interactions.51
The S–C–N angle decreases from 122.56(18)1 in the free ligand to In Fig. 5 are presented the Hirshfeld surfaces on which are
103.03(15)1 in the complex 2. The data indicate a strong weak- mapped the internal distances (di), i.e. the distances from the
ness of the amide bond (activation). So under mild reaction surface to the nearest nucleus inside, outside surface (de) and
conditions (room temperature) complex 2 can be isolated.
normalized contact distance (dnorm), defined in terms of de, di
[Ru(CO)(PPh3)2(N^O^S)] (2) crystallized in the monoclinic and the vdW radii of the atoms.
space group P21/n and the structure of the complex is given in
The share of each molecular interaction is shown in Fig. 6
Fig. 4. The thioamide ligand behaves in a tridentate manner and hydrogen bonds for the ligand and complexes are gathered
and coordinates to the Ru(II) via the pyrrolidine nitrogen N, in Table S3 (ESI†). In the structures of L and complexes 1 and 2, a
phenolic O and thiolate S donors forming one four and one six dominant role is played by the HꢂꢂꢂH interactions with distances
membered chelate ring. In addition, one carbonyl group (trans to around (white colour on the dnorm surface) and longer (blue colour)
the phenolic oxygen) and pyrrolidine nitrogen also coordinate to than the van der Waals radii separation. The OꢂꢂꢂH and HꢂꢂꢂO
the Ru(II) ion to form a NOSCO square plane and the phosphor- contacts, i.e. hydrogen bonding, comprise 30.5% of the total
ous atoms of the two triphenylphosphine ligands occupy the cis Hirshfeld surface area of the L molecule. The SꢂꢂꢂH and vice versa
position for better P interaction.45 Hence, ruthenium is placed interactions, which create the infinite H-bond chain (cf. Fig. S11
in a NOSCOP2 coordination environment, which is distorted in (ESI†), participate in the surface area with 14.6% and display
octahedral in nature as reflected in all the bond parameters the wings on the 2D fingerplot at top left and bottom right. The
around ruthenium. This complex displays a significantly dis- top wing (di o de) corresponds to the O–H donor, whereas that
torted octahedral geometry imposed on by its four and six at the bottom right (de o di) corresponds to the surface around
membered chelate rings. The bite angles around the Ru(II) the S acceptor.
ion are O(2)–Ru(1)–S(1) 88.01(5)1, O(2)–Ru(1)–P(1) 84.60(5)1,
The Hꢂ ꢂ ꢂH interactions play a major role covering 55% and
O(2)–Ru(1)–P(2) 94.32(5)1 and N(1)–Ru(1)–S(1) 68.82(6)1 with 59% of the total Hirshfeld surface areas in the case of 1 and 2
bond lengths of 2.3976(7) Å for Ru(1)–S(1), 2.1090(16) Å for respectively. The Cꢂ ꢂ ꢂH, i.e. C–Hꢂ ꢂ ꢂp, interactions are present in
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Fig. 7 UV-vis absorption spectrum of complexes 1(A) and 2(B) in aqueous
PBS buffer solution, at pH 7.4, to a final concentration of 1 ꢃ 10ꢀ3
M
recorded after different time intervals.
(80%) (71%) and HOMO - L+1 (71%), indicating that the
transition has MLCT character.
Stability studies. A reasonable stability in aqueous media
plays an important role for the development of anticancer drug
candidates. The solution chemistry of the complexes 1 and 2
are studied by time-dependent UV-visible absorption spectro-
Fig. 6 2D fingerprint plots and percentage contributions to the Hirshfeld
surface area for the ligand and complexes 1 and 2(A–C). Percentages are
given on the histogram only for the major atom-type/atom-type contacts.
both ruthenium complexes and participate in the area with scopy. The complexes are first dissolved in DMSO, and the
about 21% and 22% share, and the Cꢂ ꢂ ꢂC that is pꢂ ꢂ ꢂp inter- concentrated DMSO solutions were diluted using aqueous PBS
action participates only in the structure of complex 1 in the buffer, at pH 7.4, to a final concentration of 1 ꢃ 10ꢀ3 M. The
surface, contributing 1.6%. However, in the same compound, a UV-visible absorption spectra of these solutions are recorded
similar contribution comes from Nꢂ ꢂ ꢂO contact (1.7%), which for different time intervals over 24 h at room temperature and
in this case means the N–Oꢂ ꢂ ꢂp interaction. The parameters of shown in Fig. 7. In addition, the stability of the complexes in
these intermolecular interactions in the structures of the com- DMSO was also monitored at various time intervals and the
plexes are summarized in Table S4 (ESI†). The analysis shows spectra are shown in Fig. S14 (ESI†). The spectral profiles for
that in the case of 1 the p-stacking, Nꢂ ꢂ ꢂO/N–Oꢂ ꢂ ꢂp and Hꢂ ꢂ ꢂCl complexes 1 and 2 did not change, which clearly indicates
interactions affect, to a certain extent, the structural stabilization the stability nature of the complexes in DMSO and aqueous
as compared to complex 2. The TGA curves show that the medium. These absorption bands remain unchanged over 24 h,
complex 1 is stable upto 200 1C and the decomposition of 2 and also no new absorption bands were observed during the
starts at 160 1C as shown in Fig. S12 (ESI†). The lower stability of experimental period showing the substantial stability of the
2 is connected on one hand with the presence of the pyrrolidine complexes in DMSO and PBS buffer solutions.
moiety in the ligand which causes stress in the coordinated
ligand. On the other hand the mentioned above intermolecular plexes was also acquired from ESI-MS spectral studies. Hence, we
interactions in 1 play a stabilizing role. have recorded mass spectra for the complexes 1 and 2 and they
Furthermore, information about the composition of the com-
Emission properties. The ruthenium(II) complexes containing are shown in Fig. S15 (ESI†). Mass spectrometric measurements
strong chromophoric groups often show, in addition to intense were carried out under positive ion ESI conditions with different
MLCT transitions, interesting luminescence properties; such cone voltages, using acetonitrile as the solvent and the mobile
properties have also been explored in this type of complex. The phase. The ESI spectra of complexes 1 and 2 exhibit base peaks at
luminescence studies of the complexes were carried out in m/z 871.0 and m/z 905.2, respectively.
degassed chloroform solution at room temperature and are
illustrated in Fig. S13 (ESI†). When an excitation wavelength
In vitro anticancer activity
corresponding to the lowest energy absorption B400 nm was
Cancer cell growth inhibition (MTT assay). The antiprolifera-
used, no emission was observed. However, when an excitation tive activities of the ruthenium complexes were measured against
wavelength of 560 nm was used, the two ruthenium complexes HeLa and MCF-7 along with NIH 3T3 cell lines by using the MTT
showed red emission falling within 615–630 nm. It is likely that [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] assay
the emission originates from the lowest energy metal-to-ligand (colorimetric assay) and the widely used clinical drug, cisplatin, was
charge transfer (MLCT) state, probably involving dp(Ru)–p*(ligand) included as a positive control. The ability of the ruthenium(II)
transitions, similar to the MLCT observed in Ru(II) bipyridyl complexes to arrest the proliferation of cancer cells was investigated
complexes.52 The complexes are luminescent in nature at room after exposure for 48 h. The cytotoxicity results were specified by
temperature and possess light emitting properties. This assign- means of cell viability curves and expressed with values in the
ment is also supported by the analysis of the frontier orbitals of the studied concentration range from 0.1–100 mM. The activities
complexes. The dichloromethane solutions of the compounds corresponding to inhibition of cancer cell growth at the maximum
excited at 560 nm reveal emission with a maximum at 613 nm. level, indicated as IC50 values linked to inhibition of cancer cell
The excitation wavelength corresponds to the Hꢀ1 - LUMO growth at the 50% level, are noted. It is to be noted that the
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Table 1 Cytotoxicity (IC50, mM) of the ligand and complexes 1 and 2 (n. e.:
no effect), selectivity index (SI) and calculated partition coefficients (log P)
IC50 values (mM)
Complexes HeLa
SI
MCF-7
SI
NIH-3T3
log P
1
2
L
45.6 ꢄ 0.2 5.40 43.6 ꢄ 0.5 5.65 246.60 ꢄ 0.8 ꢀ0.82
60.7 ꢄ 0.6 4.11 57.3 ꢄ 1.2 4.35 249.48 ꢄ 0.4 ꢀ1.23
n.e.
—
n.e.
—
n.e.
—
—
Cisplatin
21.3 ꢄ 0.2 11.38 19.5 ꢄ 0.6 12.43 242.52 ꢄ 0.6
Fig. 8 AO and EB dual staining of MCF-7 cells with the control and treated
with complex 1 (50 mM) showing the changes in nuclear morphology.
precursor and the ligand did not show any inhibition of the cell
growth even up to 300 mM which clearly indicates that chelation
of the ligand with a metal ion is important for the observed
cytotoxicity properties of the complexes.
morphology, AO and EB dual staining of the MCF-7 cell line
treated with complex 1 (50 mM) was carried out and the results
are shown in Fig. 8.
The resulting 50% growth inhibitory concentration (IC50
)
values are summarized in Table 1 and graphically depicted in
Fig. S16 (ESI†). From the experimental results, the IC50 values
show that complex 1 (bidendate mode) exhibits a slightly
higher inhibitory effect than complex 2 (tridentate) towards
the tested cancer cell lines indicating that the leaving group,
and as consequence the aquation rate, plays an important role
in the anticancer activity. Another factor that has been con-
sidered as important in anticancer activity of metal complexes
is their hydrophobicity. So, we have investigated the hydropho-
bicity of the present ruthenium complexes, which can be
related to their cytotoxicity. The calculated log P values are
ꢀ0.82 and ꢀ1.23 for complex 1 and 2 respectively. Complex 1
has the highest log P value and therefore enhances the hydro-
phobicity to facilitate the crossing of the cell membrane, which
of leads to cell death as a possible mechanism. The results of
MTT assays revealed that the complexes showed notable activity
against both the HeLa and MCF-7 cell lines. Out of the two
different cell lines studied, the proliferation of the MCF-7 cell
line was arrested to a larger extent than HeLa cells by the
complexes under investigation. Though the above mentioned
complexes are active against the cell lines under in vitro cyto-
toxicity experiments and show slightly less cytotoxic activity
than cisplatin, the cytotoxicity of the complexes (1–2) is much
better than similar ruthenium complexes containing hydrazone
ligands.53–55 The IC50 value of the complexes against NIH-3T3
(non-cancerous cells) is found to be above 240 mM and did not
show any damage to normal cells. The experimental results of
the cell viability tests suggest that the ruthenium(II) complexes
have the ability to arrest the proliferation of cancer cells without
causing any damage to the normal cells.
The experiment was based on the discrimination of live cells
from dead cells on the support of morphological changes. Acridine
orange passes through the plasma membrane and stains the DNA
of live cells with the appearance of green fluorescence. On the
other hand, EB is excluded from the cells having an intact plasma
membrane and stains the DNA of dead cells, showing orange
fluorescence. After treatment of the MCF-7 cells with complex 1 for
24 h and irradiation with visible light significant gross cytological
changes of reddish-orange emission are observed with the help
of a confocal microscope. The controls, which were incubated
in the dark, showed only predominant green emission. These
morphological changes indicated that the cells were committed
to cell death, mostly via an apoptosis pathway.
Additionally, when complex 1 treated MCF-7 cells were
stained with Hoechst 33258, apoptotic features such as nuclear
shrinkage and chromatin condensation were also observed
(Fig. 9). Hence the results of AO–EB and Hoechst staining
assays suggest that complex 1 induces apoptosis in MCF-7 cells.
Flow cytometry analysis. In our present inspection, apoptosis
was detected by a flow cytometric technique using the Annexin V
protocol, with the aid of an Annexin V-FITC apoptosis detection Kit
to perform double-staining with propidium iodide and Annexin
V-FITC. Hence, we have measured in the various cell populations
staining with fluorescein isothiocyanate (FITC labelled Annexin V
(green fluorescence) and dye exclusion of the non-vital dye
propidium iodide (PI) (red fluorescence negative). The test is easy
to perform and discriminates between intact cells (FITC/PIꢀ),
and apoptotic (FITC+/PIꢀ) and necrotic cells (FITC+/PI+).
The selectivity index (SI) for each compound is expressed as
the IC50 of the pure compound in a normal cell line/the IC50 of the
pure compound in a cancer cell line. The calculated selectivity
index for complex 1 and 2 is 5.40, 5.65, 4.11 and 4.35 against HeLa
and MCF-7 cell lines, respectively. In the present study, complex 1
demonstrated strong selectivity towards the tested cancer cells.
Apoptotic event: AO–EB and Hoechst staining assays. In
order to ascertain the morphological changes the Acridine
Orange and Ethidium Bromide (AO and EB) dual staining
technique is commonly used. To investigate the mechanistic
Fig. 9 Hoechst 33258 staining of MCF-7 cells with the control and treated
aspects of cell death and to determine the changes of nuclear with complex 1 (50 mM) showing the changes in nuclear morphology.
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visualized showing that hydrogen bonding is the dominating
interaction for the construction of this architecture. In vitro
cytotoxicity results showed that the two ruthenium complexes
exhibit significant anticancer activity against HeLa and MCF-7 cell
lines without affecting the normal NIH-3T3 cells. Furthermore,
the mode of cell death assessed by AO-EB, Hoechst 33258 staining
techniques and flow cytometry analyses revealed that the com-
plexes induce apoptosis in the cancer cell lines. On the basis of
these results, the ruthenium complexes may be suitable candidates
for further evaluation in in vivo experiments.
Experimental section
Materials and instrumentation
All reagents and solvents were of high purity grade and were
purchased from commercial sources and used as received.
Commercial RuCl3ꢂ3H2O was purchased from Loba-Chemie Pvt.
Ltd. The solvents were purified and dried according to standard
procedures.56 The starting material [RuH(Cl)(CO)(PPh3)3] was
prepared according to a reported method.57 5-Nitro salicylalde-
hyde, pyrrolidine and sulphur were obtained from Aldrich.
Microanalytical (C, H, N) data were obtained with a Perkin-
Elmer model 240C elemental analyser. Melting points were
recorded in the Boetius micro heating table and were uncor-
rected. The infrared spectra of the complexes were recorded in
KBr pellets with a Nicolet iS5 spectrophotometer in the range
4000–400 cmꢀ1. (1H, 13C) NMR spectra were recorded with a
Bruker 400 MHz using CDCl3 as a solvent and tetramethylsilane
as an internal reference. The 31P NMR spectra were measured
with a Bruker instrument by using CDCl3 as a solvent and
o-phosphoric acid as an external standard. The mass spectra of
the complexes 1 and 2 were recorded using a Q-TOF mass
spectrometer. Electronic spectra were recorded on a Varian
Cary 300 Bio UV-vis spectrophotometer using cuvettes of
1 cm path length. Emission spectral intensity measurements
were carried out by using a Jasco FP-6500 spectrofluorimeter.
Thermal analysis measurements were made in the temperature
range of 20–900 1C, in an inert atmosphere, at a scanning rate
of 10 1C minꢀ1 using a Perkin Elmer Pyris 1 thermogravimetric
Analyser. 3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) was purchased from Sigma-Aldrich and used
as received. HeLa and MCF-7 cells were received from National
centre for cell Science (NCCS), Pune, India. The Annexin V-FITC
Kit (APOAF-20TST) from Sigma Aldrich was utilized as per the
instructions from the manufacturer. All other chemicals and
reagents used for the biological studies were of high quality and
biological grade.
Fig. 10 Flow cytometric results after the exposure of MCF-7 cells to
complex 1 at (25–200 mM) for 48 h respectively. Propidium iodide and
annexin positive (Q2) and propidium iodide positive (Q4).
The MCF-7 cells were treated with various concentrations
(25–200 mM) of complex 1 with a 48 h incubation period. The
flow cytometry results are shown in Fig. 10.
The Annexin V+/PI+ (Q2) population represented cells under-
going apoptosis and increased by 16.6%, 24.7%, 33.4% and
42.6% for 25 mM, 50 mM, 100 mM and 200 mM concentrations of
the investigated ruthenium complex. This observation is in good
agreement with the results of fluorescence double staining methods.
From these results, we can infer the apoptosis-inducing
potential of the complex with different potencies, in accordance
with their in vitro cytotoxicities.
Conclusions
In this work, two structurally different new organometallic
ruthenium complexes [Ru(CO)(Cl)(PPh3)2(O^S)] (1) and [Ru(CO)-
(PPh3)2(N^O^S)] (2) were isolated from the reaction of a (2-hydroxy-
5-nitrophenyl)(pyrrolidin-1-yl) methanethione ligand (L) with
[RuHCl(CO)(PPh3)3] under varying experimental conditions. The
stoichiometric reaction afforded two ruthenium complexes having
distinct ligation, which were characterized using various analytical,
spectral and single crystal X-ray crystallographic techniques. It
is very interesting to note that one of the obtained complexes, 1,
has the ligand coordinated in the OS coordination mode
governed by amide C–N bond cleavage, whereas in complex 2,
the same ligand coordinated in NOS dibasic tridentate fashion.
The starting Ru–H precursor can attack the thioamide as a
Preparation of the (2-hydroxy-5-nitrophenyl)(pyrrolidin-1-yl)
methanethione ligand
reducing agent during the reaction under reflux conditions via Ligands of this class were prepared by a modified literature
amide C–N bond cleavage. The two ruthenium complexes are procedure.58 A mixture of sulphur (0.5 g, 1 mmol), pyrrolidine
emissive and possess a light emitting property. Using X-ray (1.109 g, 1 mmol), 5-nitrosalicylaldehyde (1.25 g, 0.66 mmol),
structural analysis, supported by a detailed Hirshfeld surface and acetic acid (0.3 mL) in 20 cm3 of N,N-dimethylformamide
analysis, intermolecular contacts have been analysed and (DMF) was heated to 100 1C over 1 h with stirring. The mixture
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was stirred for an additional 3 h at this temperature and cooled 560 (906), 392 (3022), 271 (7312), 241 (10 143). 1H-NMR
to room temperature. The resulting mixture was poured with (400 MHz, CDCl3): 7.1–7.6 (m, 33H, aromatic), 2.8–2.9 (s, 4H),
stirring into 300 cm3 of water cooled in an ice bath and the 3.3–3.7 (s, 3H) ppm. 13C NMR (100 MHz, CDCl3): 24.5, 26.4, 29.6,
stirring was continued until the oily product had solidified. The 45.5, 53.4 (ꢀCH2 and –CH), 117.7, 123.6, 125.9, 127.6, 127.7,
solids were filtered, washed with water, dried in air, and 128.1, 128.5, 128.6, 128.7, 128.8, 130.3, 131.6, 132, 132.1, 132.2,
dissolved in 600 cm3 of hot methanol. The remaining solids 132.3, 132.4, 133.8, 133.9, 134.5, 140.1 (Ar-CPPh3 + Ar-CLigand),
(mainly unreacted sulphur) were removed by filtration and the 158.8 (C–S), 190.6 (CRO) ppm. 31P NMR (162 MHz, CDCl3)
red solution was treated with charcoal. The filtered solution 43.32 (s, PPh3). ESI-MS: calculated for C48H42N2O4P2RuS is 905.9;
was evaporated to 30 cm3 under reduced pressure to give a faint found 905.2.
yellow precipitate, which was recrystallized from warm diethyl
ether. Yield = 72%.
X-ray crystallography
Single crystals of the ligand L and complexes 1 and 2 suitable
for X-ray diffraction studies were grown by slow evaporation of
Analytical and spectral data for the ligand
(2-Hydroxy-5-nitrophenyl)(pyrrolidin-1-yl)methanethione (L). methanol and a dichloromethane–acetonitrile mixture at room
Color: Pale yellow; yield 72%, M.P.: 148 1C. Anal. calc. for temperature. A single crystal of suitable size was mounted
C11H12N2O3S (252.29): C, 52.36, H, 4.79, N, 11.10, S, 12.70)%; in turn on an Gemini A Ultra Oxford Diffraction automatic
found C, 52.29, H, 4.69, N, 11.08, S, 12.72)%. Selected IR bands diffractometer equipped with a CCD detector, and used for data
(KBr, n in cmꢀ1) = 3172 (–OH), 765 (CQS). UV-vis (lmax/nm) 240, collection. X-ray intensity data were collected with graphite
1
292, 371. H NMR (400 MHz, CDCl3): 9.8 (br, 1H, OH), 7.1–8.1 monochromated Mo Ka radiation (l = 0.71073 Å) at a tempera-
(3H, Ar-H), 3.7–4.0 (4H, a-CH2), 2.0–2.2 (4H, b-CH2) ppm. ture of 295(2) K, with the o scan mode. Ewald sphere reflec-
13C NMR (100 MHz, CDCl3): 189.5 (CQS), 160.6 (C–OH), 139.8, tions were collected up to 2y = 50.101. Lorentz, polarization and
126.5, 125.7, 122.1, 119.2 (Ar-C), 55, 53.9 (a-CH2), 26.5, 24.34 empirical absorption corrections using spherical harmonics
(b-CH2) ppm.
implemented in the SCALE3, ABSPACK scaling algorithm59
were applied. The structures were solved by the direct method.
All the non-hydrogen atoms were refined anisotropically using
Synthesis of the ruthenium(II) complexes
All the reactions were carried out under anhydrous conditions the full-matrix, least-squares technique on F2. The Olex2,60
and the ruthenium complexes were prepared by the following SHELXS, and SHELXL61 programs were used for all the calcula-
general procedure: To a benzene solution of [RuHCl(CO)(PPh3)3] tions. Atomic scattering factors were those incorporated in the
was added the appropriate (2-hydroxy-5-nitrophenyl)(pyrrolidin- computer programs.
1-yl) methanethione ligand. The resulting mixture was kept at
room temperature and under reflux conditions for 5 h. Coordi-
Theoretical calculations
nation was immediate, as a change of colour from yellow into The calculations were carried out using the Gaussian09 program.62
red was observed. The reaction mixture changed colour gradually The DFT/B3LYP63,64 method was used for the geometry optimiza-
and the reactions were monitored by TLC. The solution was tion and electronic structure determination. The calculations were
concentrated to about 3 mL and petroleum ether (60–80 1C) performed using the 6-31G** functions for carbon, nitrogen,
was added. The obtained solid residue was filtered and washed sulfur, chloride, oxygen and phosphorus atoms and 6-31G for
with hexane. On evaporation of the resulting solution, pure hydrogen. The DZVP basis set65 with f functions with exponents
compounds were obtained and were dried under vacuum to 1.94722036 and 0.748930908 was used to describe the ruthe-
give reasonable yields (72–80%).
nium atom. The 6-31G** and 6-31G basis sets were taken from
[Ru(CO)(Cl)(PPh3)2(O^S)](1). Color: Red. Yield: 72%. M.P.: the Gaussian09 program. The PCM solvent model was used in
202 1C. Anal. calc. For C44H34ClNO4P2RuS (871.28 g molꢀ1): the Gaussian calculations with dichloromethane as the solvent.
C, 60.65; H, 3.93; N, 1.60; S, 3.68%. Found: C, 60.25; H, 3.66; Frequency calculation was carried out, verifying whether the
N, 1.50; S, 3.58%. FT-IR (KBr, cmꢀ1) 1316 (C–O), 744 (CQS), 1954 optimized molecular structure corresponds to energy minimum,
(Ru–CO). UV-vis CHCl3, lmax/nm; e/dm3 molꢀ1 cmꢀ1: 555 (936), thus only positive frequencies were expected. The CIS(D) (single
1
395 (3159), 270 (7642), 242 (10 524). H-NMR (400 MHz, CDCl3): excitation configuration interaction with doubles correction)
8.0 (s, 1H, HCQS), 7.1–7.6 (m, 33H, aromatic) ppm. 13C NMR method was used to describe the first singlet and triplet excited
(100 MHz, CDCl3): 126.2, 127.6, 128.1, 128.5, 128.6, 129.6, 130.2, states of the ruthenium complex.66 The contribution of a group
130.8, 131, 131.2, 131.6, 131.9, 132.1, 132.3, 132.9, 134.4, 134.5, to a molecular orbital was calculated using Mu¨lliken population
134.7, 134.8, 137.4 (Ar-CPPh3 + Ar-CLigand), 170.2 (CQS), 186.9 analysis. GaussSum 3.067 was used to calculate group contribu-
(CRO) ppm. 31P NMR (162 MHz, CDCl3) 27.26 ppm (s, PPh3). tions to the molecular orbitals and prepare the partial density of
ESI-MS: calculated for C44H34ClNO4P2RuS is 871.2; found 871.0. states (DOS) and overlap partial density of states (OPDOS)
[Ru(CO)(PPh3)2(N^O^S)](2). Color: Red. Yield: 80%. M.P.: diagram. These spectra were created by convoluting the mole-
165 1C. Anal. calc. For C48H42N2O4P2RuS (905.90 g molꢀ1): cular orbital information with GAUSSIAN curves of unit height
C, 63.63; H, 4.67; N, 3.09; S, 3.53%. Found: C, 63.25; H, 4.36; and FWHM of 0.3 eV. The electrostatic potential (ESP) surfaces
N, 3.15; S, 3.45%. FT-IR (KBr, cmꢀ1) 1315 (C–O), 695 (C–S), 1953 were plotted by using gOpenMol v2.31 program. The Hirshfeld
(Ru–CO), 518 (Ru–N). UV-vis CHCl3, lmax/nm; e/dm3 molꢀ1 cmꢀ1
:
surfaces analysis were performed using the crystal geometries
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that were used as input to the TONTO68 integrated with the The results are given as the mean values obtained from three
Crystal explorer program.69
independent experiments.
Cytotoxicity: maintenance of cell lines
Fluorescent double staining experiment
The tests were done in monolayer cells detached with trypsin-
ethylenediaminetetraacetic acid (EDTA) to make single cell
suspensions and viable cells were counted using a hemo-
cytometer and diluted with medium containing 5% FBS to give
a final density of 1 ꢃ 105 cells per mL. One hundred microlitres
per well of cell suspension were seeded into 96-well plates at a
plating density of 10 000 cell per well and incubated to allow for
cell attachment at 37 1C, under conditions of 5% CO2, 95% air
and 100% relative humidity. After 24 h, the cells were treated
with serial concentrations of the test complexes. The complexes
were initially dissolved in neat DMSO and an aliquot sample
solution was diluted to twice the desired final maximum test
concentration with a serum free medium. An additional four
serial dilutions were made to provide a total of five sample
concentrations. Aliquots of 100 mL of these different sample
dilutions were added to the appropriate wells already containing
100 mL of medium, resulting in the required final sample con-
centrations. Following sample addition, the plates were incubated
for an additional 48 h at 37 1C, 5% CO2, 95% air and 100% relative
humidity. The medium without complexes served as a control and
triplicates were maintained for all concentrations.
Acridine Orange and Ethidium Bromide (AO and EB) staining
was performed as follows: The MCF-7 cells were treated with
complex 1 at 50 mM and incubated for 24 hours in a CO2
incubator at 37 1C. The cells were removed by trypsination and
collected by centrifugation including the non adherent cells. The
cell pellet was re-suspended in medium and the cell suspensions
(25 mL) were transferred to glass slides. Dual fluorescent staining
solution (1 mL) containing 100 mg mLꢀ1 AO and 100 mg mLꢀ1
EB (AO/EB, Sigma) was added to each suspension and then
covered with a coverslip. The morphology of apoptotic cells was
examined and the cells were counted within 20 min using a
fluorescence microscope.
Hoechst 33258 staining method
Hoechst 33258 staining was done using a method described
earlier but with slight modifications. 5 ꢃ 105 MCF-7 cells were
treated with IC50 concentrations of complex 1 for 24 h in a
6-well culture plate and fixed with 4% paraformaldehyde
followed by permeabilization with 0.1% Triton X-100. The cells
were then stained with 50 mg mLꢀ1 Hoechst 33258 for 30 min
at room temperature. The cells undergoing apoptosis, repre-
sented by the morphological changes of apoptotic nuclei, were
observed and imaged using an epifluorescence microscope
(Carl Zeiss, Germany).
MTT assay
3-[4,5-Dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT)
is a yellow water soluble tetrazolium salt. A mitochondrial enzyme
in living cells, succinate-dehydrogenase, cleaves the tetrazolium
ring, converting the MTT to insoluble purple formazan. There-
fore, the amount of formazan produced is directly proportional
to the number of viable cells. After 48 h of incubation, 15 mL of
MTT (5 mg mLꢀ1) in phosphate buffered saline (PBS) was
added to each well and incubated at 37 1C for 4 h. The medium
with MTT was then flicked off and the formed formazan
crystals were solubilized in 100 mL of DMSO and then the
absorbance was measured at 570 nm using a micro plate reader.
The % cell inhibition was determined using the following
formula.
Apoptosis detection-flow cytometry analysis
MCF-7 cells were grown in 6-well plates and defined at four
different concentrations of complex 1 with a 48 h incubation
period. The Annexin V-FITC kit uses annexin V conjugated with
fluorescein isothiocyanate (FITC) to label phosphatidylserine
sites on the membrane surface of apoptotic cells. Briefly, the
cells were trypsinised and washed with Annexin binding buffer
and incubated with Annexin V FITC and PI for 30 minutes and
immediately analysed using a FACS Aria-II flow cytometer. The
forward and side scattering parameters were adjusted to keep
the background fluorescence to a minimum. The results were
carefully analysed by using DIVA software and the percentage of
positive cells was calculated.
% Cell Inhibition = 100 ꢀ Abs (sample)/Abs (control) ꢃ 100.
A nonlinear regression graph was plotted between % cell
inhibition and Log concentration and IC50 was determined
using GraphPad Prism software.
Acknowledgements
Partition coefficient determination
The hydrophobicity values of complexes 1 and 2 were measured R. R. K. thanks the DST-INSPIRE Program for the financial
by using the ‘‘Shake flask’’ method in octanol–water phase assistance as a Senior Research Fellow (IF130075). We thank
partitions as reported earlier.70 The complexes were dissolved UGC-SAP and DST-India (FIST programme) for the use of the
in a mixture of water and n-octanol followed by shaking for Bruker 400 MHz NMR UV-visible and Fluorescence spectral
1 hour. The mixture was allowed to settle over a period of facilities at the School of Chemistry, Bharathidasan University.
30 minutes and the resulting two phases were collected separately The GAUSSIAN09 calculations have been carried out using
without cross contamination of one solvent layer into another. resources provided by the Wroclaw Centre for Networking
The concentration of the complexes in each phase was deter- and Supercomputing, WCSS, Wroclaw, Poland (http://wcss.pl),
mined by UV-vis absorption spectroscopy at room temperature. Grant Number 18.
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27 W.-L. Man, J. Xie, Y. Pan, W. W. Y. Lam, H.-K. Kwong, K.-W.
Ip, S.-M. Yiu, K.-C. Lau and T.-C. Lau, J. Am. Chem. Soc.,
2013, 135, 5533–5536.
28 M. Tayebani, K. Feghali, S. Gambarotta and C. Bensimon,
Organometallics, 1997, 16, 5084–5088.
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