Photocytotoxic oxovanadium(IV) complexes of ferrocenyl-terpyridine and acetylacetonate derivatives

Article abstract of DOI:10.1016/j.ejmech.2015.01.003

Oxovanadium(IV) complexes [VO(Fc-tpy)(acac)](C10₄) (1), [VO(Fc-tpy)(nap-acac)](C10₄) (2), [VO(Fc-tpy)(py-acac)](C10₄) (3) and [VO(Ph-tpy)(py-acac)](C10₄) (4) of 4'-ferrocenyl-2,2':6',2"-terpyridine (Fc-tpy) and 4'-phenyl-2,2':6',2"-terpyridine (Ph-tpy) having monoanionic acetylacetonate (acac), naph-thylacetylacetonate (nap-acac) or pyre ny lace ty lace to nate (py-acac) ligand were prepared, characterized and their photocytotoxicity in visible light studied. The ferrocenyl complexes 1-3 showed an intense charge transfer band near 585 nm in DMFand displayed Fc'/Fc and V(1V)/V(111) redox couples near 0.66 V and -0.95 V vs. SCE in DMF-0.1 M TBAP. The complexes as avid binders to calf thymus DNA showed significant photocleavage of plasmid DNA in green light (568 nm) forming "OH radicals. The complexes that are photocytotoxic in HeLa and MCF-7 cancer cells in visible light (400 700 nm) with low dark toxicity remain nontoxic in normal fibroblast 3T3 cells. ICP-MS and fluorescence microscopic studies show significant cellular uptake of the complexes. Photo-irradiation of the complexes causes apoptotic cell death by ROS as evidenced from the DCFDA assay.

Full text of DOI:10.1016/j.ejmech.2015.01.003

European Journal of Medicinal Chemistry 92 (2015) 332e341
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Photocytotoxic oxovanadium(IV) complexes of ferrocenyl-terpyridine
and acetylacetonate derivatives
,
*
Babu Balaji a, Babita Balakrishnan b, Sravanakumar Perumalla a, Anjali A. Karande b
,
,
*
Akhil R. Chakravarty a
a Department of Inorganic and Physical Chemistry, Indian Institute of Science, Sir C.V. Raman Avenue, Bangalore 560 012, India
b Department of Biochemistry, Indian Institute of Science, Sir C.V. Raman Avenue, Bangalore 560 012, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Oxovanadium(IV) complexes [VO(Fc-tpy)(acac)](ClO4) (1), [VO(Fc-tpy)(nap-acac)](ClO4) (2), [VO(Fc-
tpy)(py-acac)](ClO4) (3) and [VO(Ph-tpy)(py-acac)](ClO4) (4) of 4ʹ-ferrocenyl-2,2ʹ:6ʹ,2ʺ-terpyridine (Fc-
tpy) and 4ʹ-phenyl-2,2ʹ:6ʹ,2ʺ-terpyridine (Ph-tpy) having monoanionic acetylacetonate (acac), naph-
thylacetylacetonate (nap-acac) or pyrenylacetylacetonate (py-acac) ligand were prepared, characterized
and their photocytotoxicity in visible light studied. The ferrocenyl complexes 1e3 showed an intense
charge transfer band near 585 nm in DMF and displayed Fcþ/Fc and V(IV)/V(III) redox couples near 0.66 V
and ꢀ0.95 V vs. SCE in DMF-0.1 M TBAP. The complexes as avid binders to calf thymus DNA showed
Received 1 August 2014
Received in revised form
8 November 2014
Accepted 3 January 2015
Available online 3 January 2015
Keywords:
Medicinal chemistry
Vanadium
ꢁ
significant photocleavage of plasmid DNA in green light (568 nm) forming OH radicals. The complexes
that are photocytotoxic in HeLa and MCF-7 cancer cells in visible light (400e700 nm) with low dark
toxicity remain nontoxic in normal fibroblast 3T3 cells. ICP-MS and fluorescence microscopic studies
show significant cellular uptake of the complexes. Photo-irradiation of the complexes causes apoptotic
cell death by ROS as evidenced from the DCFDA assay.
Ferrocene
Photocytotoxicity
Cellular imaging
Apoptosis
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
other chemotherapeutic treatments is its selectivity to target only
the photo-exposed cancerous cells leaving unexposed normal cells
Photodynamic therapy (PDT) which has emerged as a non-
invasive treatment modality for various types of cancers using
the FDA approved hematoporphyrin drug Photofrin® involves
administration of the photosensitizer (PS) to the body, followed by
its activation by light at the cancer site with formation of singlet
oxygen killing the tumour cells [1e5]. The advantage of PDT over
unaffected thus minimizing toxic side effects. Photofrin and its
analogues suffer from prolonged skin photosensitivity and hepa-
totoxicity due to formation of bilirubin on oxidative degradation of
the porphyrin core [6,7]. Non-porphyrinic transition metal com-
plexes are shown to be efficient photocytotoxic agents with low
dark toxicity [8e15]. The metal complexes could be suitably
designed to show novel PDT activity following different reaction
pathways, producing hydroxyl radicals or singlet oxygen to cause
cellular damage [16,17].
Abbreviations: acac, acetylacetonate; ct-DNA, calf thymus DNA; DCFDA, 20,70-
dichlorofluorescein diacetate; DMEM, Dulbecco's Modified Eagle's Medium; DMF,
dimethylformamide; DMSO, dimethyl sulfoxide; DNA, deoxyribonucleic acid;
DPPH, 2,2-diphenyl-1-picrylhydrazyl; EB, ethidium bromide; EDTA, ethylenedi-
aminetetraacetate; EPR, electron paramagnetic resonance; FBS, fetal bovine serum;
Fc, ferrocenyl; Fc-tpy, 4ʹ-ferrocenyl-2,2ʹ:6ʹ,2ʺ-terpyridine; ICP-MS, inductively
coupled plasma mass spectrometry; MLCT, metal-to-ligand charge transfer; MTT, 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MvH, McGhee-von
Hippel; NAC, N-acetylcysteine; nap-acac, naphthylacetylacetonate; PBS, phosphate
buffered saline; PDT, photodynamic therapy; Ph-tpy, 4ʹ-phenyl-2,2ʹ:6ʹ,2ʺ-terpyr-
idine; PI, propidium iodide; py-acac, pyrenylacetylacetonate; ROS, reactive oxygen
species; TBAP, tetrabutylammonium perchlorate.
We have recently shown that ferrocenyl conjugates of transition
metals significantly enhance their photo-chemotherapeutic po-
tential compared to the one lacking the ferrocenyl unit [18e20].
Ferrocenyl derivatives with their non-toxic, redox active and lipo-
philic properties have been used to prepare novel antitumor,
antimalarial and antifungal agents [21e26]. Mechanistic studies
have shown that the anticancer activity of the ferrocenium cation is
ꢁ
due to generation of reactive oxygen species (ROS) including OH
radicals and these radicals are responsible for DNA degradation and
strand-breakage [27]. We have also shown in a recent report that
* Corresponding authors.
E-mail addresses: anjali@biochem.iisc.ernet.in (A.A. Karande), arc@ipc.iisc.ernet.
in (A.R. Chakravarty).
ferrocene-conjugated
oxovanadium(IV)
complexes
of
http://dx.doi.org/10.1016/j.ejmech.2015.01.003
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

B. Balaji et al. / European Journal of Medicinal Chemistry 92 (2015) 332e341
333
curcuminoids are photocytotoxic in cancer cells [28]. Curcumin has
a dione moiety which in its enolized form resembles the metal
binding properties of acetylacetonates [29]. The present work
stems from our interests to extend this chemistry further by using
different acetylacetonate derivatives in a ternary structure in which
the oxovanadium(IV) moiety is bound to a tridentate ferroce-
nylterpyridine ligand and a bidentate acetylacetonate derivative.
Curcumin with its poor bioavailability is known to be susceptible to
hydrolytic degradation in the physiological medium [30]. In
contrast, the acac ligands are expected to be of higher stability in a
biological medium. The acac derivatives bearing fluorophoric
pendants like naphthyl or pyrenyl moiety could be used to study
the cellular localization of the complexes by fluorescence micro-
scopy. The 40-ferrocenyl-2,20:60,200-terpyridine (Fc-tpy) ligand with
its intense charge transfer band is used as a photosensitizer.
Herein, we report the synthesis, characterization, photo-
cytotoxicity and cellular imaging of ferrocenyl-terpyridine (Fc-tpy)
oxovanadium(IV) complexes of acetylacetone derivatives, viz.
[VO(Fc-tpy)(acac)](ClO4) (1), [VO(Fc-tpy)(nap-acac)](ClO4) (2),
[VO(Fc-tpy)(py-acac)](ClO4) (3) and [VO(Ph-tpy)(py-acac)](ClO4)
(4), where acac is acetylacetonate, nap-acac is naph-
thylacetylacetonate and py-acac is pyrenylacetylacetonate (Scheme
1). Complex 4 having the ferrocenyl moiety replaced by a phenyl
group was used as a control. Significant results of this study include
remarkable photocytotoxicity of the py-acac complexes and cyto-
solic localization of complex 3 as evidenced from the fluorescence
microscopy imaging study. The pyrenyl complex shows similar
photocytotoxicity as observed for its reported curcumin analogue
[28].
product. The metal precursor vanadyl perchlorate was prepared by
reacting vanadyl sulphate with calcium perchlorate. Oxovanadiu-
m(IV) complexes 1e4 were prepared by a general synthetic method
in two steps. First, the acetylacetonate derivative after neutralizing
with dilute NaOH was reacted with vanadyl perchlorate for 30 min.
Corresponding terpyridine ligand in CHCl3eMeOH was then added
and stirred for further 30 min. The complex was isolated as its
perchlorate salt.
2.2. Pharmacology
Photocytotoxicity of the oxovanadium(IV) complexes 1e4 in
HeLa and MCF-7 cells was evaluated by MTT assay in dark and
visible light (400e700 nm). The cellular uptake of the complexes
was evaluated by ICP-MS. The cellular localization studies were
carried out by fluorescence microscopy using the blue fluorescence
of the naphthyl and pyrenyl groups present in the complexes. The
mechanism of cell death on photo-irradiation was assessed by
Hoechst staining. The generation of reactive oxygen species (ROS)
in the cells on photo-irradiation was evaluated by DCFDA assay. The
oxovanadium(IV) complexes were also evaluated for their calf
thymus DNA binding strengths and plasmid DNA photocleavage
activities. The DNA binding studies were done by spectroscopic
methods, viz. electronic absorption titration, DNA melting and
viscosity measurements. The DNA cleavage activity of the com-
plexes was studied using plasmid supercoiled (SC) form of DNA.
The extent of DNA cleavage forming nicked circular (NC) DNA was
quantified by gel electrophoresis.
2.3. Synthesis and general aspects
2. Results and discussion
Complexes [VO(Fc-tpy)(L)](ClO4) (1e3) of acetylacetone de-
rivatives (L: acac in 1; nap-acac in 2; py-acac in 3) and the ferro-
cenyl terpyridine ligand (Fc-tpy) were prepared in good yield.
Complex 4 was prepared using Ph-tpy instead of Fc-tpy and used as
a control species. Complexes 1e4 were characterized from the
analytical, mass spectral and other physicochemical data (Table 1).
The molar conductance value of ~82 S m2 Mꢀ1 of the complexes in
DMF at 25 ꢂC suggests their 1:1 electrolytic behaviour. The ESI mass
spectra of the complexes in MeCN showed a single peak corre-
sponding to the molecular ion peak (m/z) as [VO(Fc-tpy)(L)]þ. The
isotopic distribution pattern of the complexes is in accordance with
the calculated one. The IR spectra of the complexes in the solid state
showed C]O and C]C bands that are shifted to lower wave-
numbers compared to the free ligands due to binding to the
metal. The complexes showed band for V]O and ClOꢀ4 within
958e963 cmꢀ1 and 1090-1096 cmꢀ1, respectively [33]. The 3d1-
VO2þ complexes are one-electron paramagnetic giving a magnetic
moment value of ~1.64 mB. The complexes showed broad 1H NMR
spectra due to their paramagnetic nature. The complexes showed
eight line pattern in the ESR spectra due to hyperfine coupling of
the unpaired electron with the 51V nucleus (I ¼ 7/2), confirming the
3d1-VO(IV) oxidation state. Complexes 1e3 showed an intense
band at ~585 nm which is assignable to the metal to ligand charge
transfer transition (MLCT) (Fig. 1) [34]. This band is absent in the
spectrum of the phenyl analogue 4 indicating the involvement of
the ferrocenyl moiety for this charge transfer transition. Complex 4
showed only a weak ded transition at 774 nm. The ded band in 1e3
was found to be masked by the MLCT band. The complexes 2e4
showed fluorescence property (Fig.1). Complex 2 having a naphthyl
moiety showed emission bands at 406 and 430 nm, while the
pyrenyl complexes 3 and 4 showed emission bands at 387 and
409 nm, respectively. Cyclic voltammetry of the redox active
complexes in DMF-0.1 M TBAP showed quasi-reversible Fcþ/Fc
redox couple near 0.62 V (Fc ¼ ferrocenyl unit) and the V(IV)/V(III)
2.1. Chemistry
The terpyridine ligands (Fc-tpy and Ph-tpy) were prepared by
reacting corresponding aldehyde with 2-acetylpyridine and NaOH
with subsequent condensation of the intermediate species with
ammonium acetate in refluxing ethanol [31]. The 1-
naphthylacetylacetone (Hnap-acac) and pyrenylacetylacetone
(Hpy-acac) were prepared by following a literature procedure [32].
1-Acetylnaphthalene or 1-acetylpyrene was refluxed with sodium
sand in dry ethyl acetate for 4 h and poured into cold water. The
aqueous layer was acidified with dilute HCl to precipitate out the
Scheme 1. Schematic drawings of the complexes 1e4 and ligands used.

334
B. Balaji et al. / European Journal of Medicinal Chemistry 92 (2015) 332e341
Table 1
Selected physicochemical and DNA binding data for the complexes 1e4.
Complex
1
2
3
4
IR/cmꢀ1 (in KBr)
n
n
(V]O)
(ClO4)
958
1096
581 (2608)
0.62 (170)
958
1095
583 (3014)
0.63 (155)
960
1096
586 (3536)
0.62 (165)
961
1095
777 (90)
e
lmax/nm (ε/dm3 Mꢀ1 cmꢀ1
)
a
Ef/V (D
Ep/mV)b
FcþeFcc
Ep [V]d
ꢀ0.93
ꢀ0.98
ꢀ1.01
ꢀ0.97
LMe/S m2 Mꢀ1
84
86
79
74
f
giso
Kb
1.99
1.98
1.98
1.98
(4.5 0.5) ꢃ 104
(3.2 0.3) ꢃ 105
(9.6 0.4) ꢃ 105
(3.8 0.3) ꢃ 106
g
h
D
Tm (ꢂC)
1.1
2.6
4.9
4.4
a
Visible electronic spectral band in DMFeTris HCl buffer (1:1 v/v).
b
Redox data in DMF-0.1 M TBAP. The potentials are vs. SCE. Scan rate ¼ 50 mV sꢀ1. Ef ¼ (Epa þ Epc)/2,
Ep ¼ (Epa ꢀ Epc), where Epa and Epc are the anodic and cathodic peak
D
potentials, respectively.
c
Fcþ/Fc redox couple.
d
Cathodic peak for the VIVeVIII redox couple.
e
Molar conductance in DMF at 25 ꢂC.
f
From room-temperature EPR spectra in DMF solution with DPPH as an internal standard.
Intrinsic equilibrium DNA binding constant from the UVevisible experiment.
Change in the calf thymus DNA melting temperature.
g
h
Fig. 1. The electronic spectra of [VO(Fc-tpy)(py-acac)](ClO4) (3) (ddd) and [VO(Ph-
tpy)(py-acac)](ClO4) (4) (- - -) in DMFeTriseHCl buffer (1:1 v/v) with the inset showing
the emission spectrum of 2e4 in DMF.
couple as an irreversible reduction in the range from ꢀ0.91
to ꢀ0.97 V. Complex 4 showed only the V(IV)eV(III) reduction peak
at ꢀ0.98 V.
In order to gain more detailed insight into the photophysical
properties of the complexes, density functional theory (DFT) and its
time-dependent version (TDDFT) were applied and the details are
Fig. 2. Frontier orbital diagram of complexes 1e4 showing a) HOMO, HOMO-1, LUMO,
LUMOþ1 and (b) the HOMOeLUMO energy gap of the ligand (Fc-tpy) and the com-
plexes 1e4.
presented as Supporting Information [35e37]. The calculated ab-
sorption band positions for 1e3 at 600 nm are responsible for the
experimentally observed intense band at ~585 nm. This absorption
is associated with charge transfer from the HOMO to LUMO orbital
for 1 and 2, while it is HOMOe1 to LUMO orbital of 3 (Fig. 2). No
such band was observed for the phenyl analogue 4. The HOMO of 1
and 2 and HOMOe1 of 3 have predominant contribution from the
ferrocenyl moiety (~97%). The LUMO has a major contribution from
the terpyridine (tpy) moiety (~83%) and some V]O character (~9%).
The metal-to-ligand charge-transfer (MLCT) is from the ferrocenyl
to the terpyridine moiety in the complex. The TDDFT data are in
agreement with the experimental results.
indicating the solution stability of the complexes. The inertness of
ferrocene moiety in complexes 1e3 in dark is established by
monitoring the cyclic voltammograms of FceFcþ couple in com-
plexes with time. The cyclic voltammograms remained the same
over the period of 4 h indicating ferrocenyl moiety not undergoing
any oxidation when kept in dark. The 51V NMR of complex 3 in dark
with time showed no peaks in spectra due to paramagnetic V(IV)
moiety in 3. This suggests that the complex not undergoing any
oxidation to give diamagnetic V(V) species which is 51V NMR active.
2.4. Stability of the complexes
2.5. Photocytotoxicity
The stability of the complexes was studied in 1% DMSO-PBS
buffer (pH ¼ 7.2) using UV-Visible spectroscopic technique. There
was no change in the spectral features of the complexes in 24 h
The photocytotoxicity of the complexes 1e4 in cervical HeLa
cancer and breast MCF-7 cancer cells and in normal fibroblast 3T3

B. Balaji et al. / European Journal of Medicinal Chemistry 92 (2015) 332e341
335
Fig. 3. MTT assay showing photocytotoxicity of 3 and 4 in HeLa (a, b) and MCF-7 (c, d) cancer cells and in normal 3T3 cells (e, f) on 4 h incubation in dark (with black symbol) and on
photo-irradiation with visible light of 400e700 nm (shown as open circle), as determined from the MTT assay.
cells was studied in dark and visible light (400e700 nm) by MTT
assay (Fig. 3). To optimize the incubation time of the complexes,
MTT assay was done in HeLa cells by varying the incubation time to
2, 4, 6 and 8 h using complex 3. Cellular uptake of complex 3 into
the cancer cells was studied by FACS analysis which showed that 3
was taken up significantly within 4 h of incubation with the HeLa
cells and remained the same after 6 or 8 h. It was also observed that
increasing the incubation time resulted in an increase in the dark
toxicity and a 4 h incubation showed better photocytotoxicity.
Based on these results, a 4 h incubation period was chosen before
photo-irradiating the samples with visible light of 400e700 nm
(10 J cmꢀ2) to obtain the IC50 values of the complexes. The com-
plexes were found to be significantly photocytotoxic compared to
their activity in dark. The IC50 values for the complexes are given in
Table 2 along with few related compounds [38e42]. The ferrocenyl
complex
3
showed better photocytotoxicity than its phenyl
M in HeLa and MCF-7
analogue 4 with IC50 values of 5.4 and 2.1
m
cells, respectively. This difference could be attributed to the pres-
ence of an MLCT band in complex 3 in the visible region (590 nm)
which is absent in complex 4. We have thus observed an improved
photocytotoxicity on incorporating the ferrocenyl organometallic
moiety. The complexes in general showed better activity in MCF-7
cells than in HeLa cells. In order to see the selectivity of the com-
plexes from cancerous to the normal cells, we did MTT assay using
normal fibroblast 3T3 cell line. Interestingly, the complexes showed
10 times less photocytotoxicity in normal 3T3 cells compared to the
Table 2
IC50 values of 1e4 with other relevant compounds in HeLa and MCF-7 cancer cells and in normal 3T3 fibroblast cell.
Compound
HeLa
IC50
MCF-7
IC50
M) darka
3T3
IC50
(m
M) darka
IC50
(
m
M) lightb
(
m
IC50
(m
M) lightb
(
m
M) darka
IC50 (m
M) lightb
1
2
3
4
40.2 ( 1.1)
45.1 ( 1.2)
37.6 ( 1.2)
40.1 ( 1.1)
>100
35.5 ( 1.0)
34.7 ( 1.9)
54.2 ( 1.1)
>100
24.4 ( 1.1)
15.5 ( 1.0)
5.4 ( 0.5)
19.9 ( 1.1)
39.3 ( 1.8)
13.4 ( 1.1)
17.8 ( 1.2)
2.4 ( 0.3)
12.0
41.2 ( 1.0)
48.7 ( 1.1)
38.5 ( 1.1)
42.4 ( 1.1)
>100
5.6 ( 0.9)
3.3 ( 0.6)
2.1 ( 0.3)
15.5 ( 1.2)
32.4 ( 1.3)
e
>100
>100
>100
>100
>100
e
66.8 ( 1.1)
60.5 ( 1.0)
54.5 ( 1.0)
96.7 ( 2.1)
>100
Fc-tpy ligand
[VO(Fc-tpy)(dppz)](ClO4)2
[VO(Fc-pic)(Curc)](ClO4)d
[VO(Fc-tpy)(Curc)](ClO4)e
[VOCl(dppz)2]Clf
Photofrin®g
c
e
e
e
e
e
e
e
e
46.4 ( 1.5)
22.5 ( 1.1)
>41
4.28
2.0 ( 0.2)h
Cisplatin
71.3 ( 2.9)i
68.7 ( 3.4)i
2.0 ( 0.3)j
a
The IC50 values for complexes 1e4 and ligands correspond to 4 h incubation in the dark without any photoexposure. The IC50 values for ligands in all the above three cell
lines are given as Supplementary data.
b
The IC50 values of complexes 1e4 and ligands correspond to 4 h incubation in the dark followed by photoexposure to visible light (400ꢀ700 nm, 10 J cmꢀ2) for 1 h.
c
The IC50 values are from Ref. [18].
d
The IC50 values are from Ref. [29].
The IC50 values are from Ref. [28].
The IC50 values are from Ref. [38].
e
f
g
The Photofrin® IC50 values (633 nm excitation; fluence rate: 5 J cmꢀ2) are taken from Ref. [39] (converted to M using the approximate molecular weight of Photofrin®,
m
600 g Mꢀ1).
h
i
The Photofrin® IC50 values (2 h exposure; white bulb; fluence rate: 5.5 ꢃ 10ꢀ2 mW cmꢀ2) are taken from Ref. [40].
The IC50 values for 4 h treatment are taken from Ref. [41].
j
The IC50 values for 96 h treatment are taken from Ref. [42].

336
B. Balaji et al. / European Journal of Medicinal Chemistry 92 (2015) 332e341
Fig. 4. Cellular vanadium content as determined by ICP-MS in HeLa and MCF-7 cancer
cells upon incubation with the complexes 1e4 (30
M) at 37 ꢂC for 4 h.
m
cancerous ones (HeLa, MCF-7) and remained inactive in dark. The
complexes are thus active against only the cancerous cells leaving
the normal cells unaffected. When photo-irradiation was done in
the presence of N-acetyl cysteine as a ROS scavenger, the activity of
the complexes decreased significantly in both the cells indicating
possible involvement of ROS in the cellular damage.
Fig. 5. a) DCFDA assay used for ROS generation in HeLa cells by complex 2 (10 mM) on
exposure to visible light (400e700 nm). b) A comparison of the DCF derived fluores-
cence intensity of the cells treated with complexes 1e4 in dark and light.
2.6. Cellular uptake from ICP-MS
The observed difference in the photocytotoxic activity of the
complexes among the cancer cells could be related to their cellular
uptake. The vanadium content associated with the cancer cells
oxidation forms 20,7’-dichlorofluorescein (DCF) showing an emis-
sion maximum at 528 nm. The assay was done by fluorescence-
activated cell sorting (FACS) method in which the cancer cells on
upon incubation for 4 h in dark with complexes 1e4 (30 mM) at
treatment with the complexes (10 mM) showed a significant posi-
37 ꢂC was measured by ICP-MS (Fig. 4). The DMSO (1% in DMEM
media) treated cells were used as a control. The uptake of the
complexes follows the order: 3 > 4 > 1 > 2 in both the cell lines with
better uptake in MCF-7 compared to HeLa cells. The ferrocenyl
tive shift in the fluorescence intensity due to formation of DCF in
the presence of ROS generated on photo-irradiation of the com-
plexes (Fig. 5). To verify further the ROS mediated cytotoxicity, the
cancer cells were treated with complex 2 and N-acetyl-L-cysteine
complex 3 (1.83 0.07
in MCF-7) showed better cellular uptake than its phenyl analogue 4
(0.60 0.04
g/106 cells, 1.89 0.05 g/106 cells) (Table 3). This
m m
g/106 cells in HeLa, 2.39 0.07 g/106 cells
(NAC) as ROS quencher followed by photo-irradiation with visible
light [43]. The fluorescence intensity was found to reduce in pres-
ence of NAC. The results indicate that the complexes generate ROS
in the cellular medium only upon photo-irradiation but not in the
dark. The positive control cells (H2O2 treated cells) showed a sig-
nificant shift compared to the control cells. A comparison of the DCF
derived fluorescence intensity of the cells treated with complexes
1e4, the production of ROS follows the order: 3 > 4 > 2 > 1. This
trend could be related to the higher cellular uptake of complex 3
than the other complexes.
m
m
could be due to higher lipophilic nature of the ferrocenyl moiety
present in complex 3.
2.7. Measurement of intracellular ROS
Generation of any reactive oxygen species (ROS) by the com-
plexes (10
mM) was examined by dichlorofluorescein diacetate
(DCFDA) assay. DCFDA is a cell permeable fluorogenic probe that on
2.8. Fluorescence microscopy
Table 3
Uptake of the oxovanadium(IV) complexes (1e4) by HeLa and MCF-7 cells after 4 h
incubation by ICP-MS.
Fluorescence microscopic study was done to visualize the
localization of the complexes in the cells using the emission
property of the pyrenyl and naphthyl moieties. The ICP-MS analysis
showed that the complexes were taken up effectively into the cells.
HeLa cells, on treatment with complex 3 for different time intervals
(2 and 4 h), showed preferential uptake and it localized predomi-
nantly in the cytosol as seen from the blue fluorescence of 3 in
panels (e) and (h) of Fig. 6. MCF-7 cells treated with complex 3
showed the same trend of cytosolic localization. It is evident that
there is not much difference in the fluorescence intensity of the
cells treated with 3 in 2 and 4 h time point. This is in agreement
Compound
Vanadium contenta
HeLa cells
(m
g/106 cells)
MCF-7 cells
Cells alone
0.09 0.04
0.59 0.30
0.43 0.10
1.83 0.07
0.60 0.04
0.10 0.01
1.44 0.04
1.29 0.06
2.39 0.07
1.89 0.05
1
2
3
4
a
Total accumulation of vanadium in HeLa and MCF-7 cells for complexes 1e4
after 4 h at 37 ꢂC.

B. Balaji et al. / European Journal of Medicinal Chemistry 92 (2015) 332e341
337
Fig. 6. A time-course collection of fluorescence microscopic images of untreated control cells (panels: a-c and jel), [VO(Fc-tpy)(py-acac)](ClO4) (3) (10
mM) in HeLa and MCF-7 cells.
Panels (d), (g), (m) and (p) show the blue emission of 3. Panels (b), (e), (h), (k), (n) and (q) correspond to the red emission of propidium iodide (PI) dye. Panels (c), (f), (i), (l), (o) and
(r) display the merged images of the first two panels. Scale bar ¼ 20
mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version
of this article.)
showed nuclear fragmentation, chromatin condensation indicating
apoptotic cell death (Fig. 7, panels (d, f)). But nuclei of the cells
when kept in dark for 4 h with complexes 3 and 4 were found to be
healthy (Fig. 7, panels (a, c, e)). The results suggest that cell death
occurred only in light with no apparent cell death in the dark.
2.9. Mechanistic aspects
To identify the possibility of formation of any hydroxyl radical as
ROS by the complexes on photo-irradiation, EPR spin trapping was
done using DMPO as a spin trap. Photo-irradiation of complex 3
(100
mM) in 10% DMSO-Tris HCl buffer with visible light in the
presence of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) (100 mM)
for 30 min gave a four line EPR spectrum which corresponds to
DMPO-OH adduct with a hyperfine splitting of aN ¼ aH ¼ 14.9 Gauss
[44]. No such EPR signal was observed when complex 4 was treated
with DMPO in dark. Addition of superoxide dismutase (SOD) prior
to irradiation led scavenging of the ROS. No DMPO-OH adduct
formation was noticed in the presence of catalase. The reduction of
DMPO-OH formation by SOD is possibly due to formation of un-
stable DMPO-Oe2 ꢁ spin adduct with reduced life time [45]. The effect
of catalase addition indicates formation of H2O2 in the photo-
chemical reaction. The results suggest oxidation of the ferrocenyl
moiety to ferrocenium ion on light activation and the ferrocenium
ion which being unstable in a buffer medium degrades to form the
hydroxyl radicals [46,47]. The involvement of the Fc unit under-
going oxidation on photo-irradiation was evidenced from the
spectral studies showing the absorption maximum shifting to
590 nm with an increase of the intensity of that band with time
[20]. A similar spectral change was noticed when complex 3 was
oxidized chemically by cerric(IV) ammonium nitrate. Complex 4
lacking the ferrocenyl moiety did not show any such spectral
change.
Fig. 7. Propidium Iodide (PI) staining of the HeLa cells treated with complexes 3 and 4
for 4 h incubation in dark followed by light exposure to visible light (400e700 nm,
10 J cmꢀ2) for 1 h. Panels (a), (c) and (e) are the fluorescence images of the control,
complexes 3 and 4 treated cells (PI stained) in dark respectively. Panels (b), (d) and (f)
are the fluorescence images of the control and complexes 3, 4 treated cells in light
respectively. The PDT effect is shown by arrows in panels (d) and (f). The scale bar is
20 mm.
with the cellular uptake results of complex 3 by FACS. Staining with
PI which specifically stains the nucleus revealed minor localization
of the complex within the nucleus (panels (f), (i), (o) and (r)). Cells
treated with the complexes 2 and 4 showed similar cellular local-
ization as observed for complex 3.
2.10. DNA binding and photocleavage
Since the complexes showed partial localization within the
nucleus of the cells where DNA could be the possible target for
observed cytotoxicity, we studied the DNA binding and photo-
cleavage properties of the complexes. The ct-DNA binding studies
were carried out by UV-visible absorption titration, DNA melting
To see the PDT effect, complexes 3 and 4 (10 mM) were added to
the HeLa cells and incubated for 4 h in dark followed by exposure to
visible light of 400e700 nm for 1 h (Fig. 7). Fluorescence images of
the cells incubated with the complex on visible light exposure

338
B. Balaji et al. / European Journal of Medicinal Chemistry 92 (2015) 332e341
Table 4
Selected SC pUC19 DNA (0.5
and viscometric methods. Selected DNA binding data are given in
Table 1. UVeVis absorption titration method was used to determine
the intrinsic DNA binding constant (Kb) of the complexes by
monitoring the change in the absorption intensity of the ligand-
centred band of the complexes 1e4 at ~280 nm. The complexes
showed significant hypochromicity. The Kb values were in the range
of 104e106 Mꢀ1 following the order: 3 > 4 > 2 > 1. The binding data
suggest partial intercalative binding mode of the complexes 3 and 4
having planar aromatic moieties, while complexes 1 and 2 have
surface and/or partial groove binding to ct-DNA. Thermal DNA
denaturation experiments showed a positive shift in the DNA
m
g) cleavage data for complexes 1e4.
%NCb form %NCb form
Reaction conditionsa
l
¼ 568 nm ¼ 785 nm
l
DNA control
2
3
DNA þ [VO(Fc-tpy)(acac)](ClO4) (1) 56
46
62
DNA þ [VO(Fc-tpy)(nap-
acac)](ClO4) (2)
70c
DNA þ [VO(Fc-tpy)(py-acac)](ClO4) 88d
76
70
(3)
DNA þ [VO(Ph-tpy)(py-acac)](ClO4) 26e
(4)
a
melting temperature (DTm) upon addition of the complexes to the
In Tris-buffer medium (pH ¼ 7.2).
b
NC ¼ nicked circular form of pUC19 DNA.
l
, laser wavelength. Photo-exposure
ct-DNA. Again, the Tm values suggest primarily groove binding
D
time (t) ¼ 2 h. Concentration of complexes 1e4 was 40
mM for 568 nm and 50 mM for
nature of 1 and 2 and partial intercalative mode of binding of the
pyrenyl complexes to ct-DNA. Viscosity measurements were car-
ried out to examine the effect of the complexes on the specific
relative viscosity of the ct-DNA which gave a measure on the in-
crease in its contour length associated with the separation of the
DNA base pairs caused by intercalation. The viscosity data also
indicate groove binding nature of 1 and 2 and partial intercalation
of 3 and 4 to the ct-DNA.
785 nm experiment.
c
The ligand Hnap-acac alone gave 5% NC form at this wavelength.
The ligand Hpy-acac alone gave 8% NC form at this wavelength.
No visible band was observed at this wavelength for this complex.
d
e
3. Conclusions
Ferrocenyl-terpyridyl oxovanadium(IV) complexes of acetyla-
cetonate derivatives having pendant fluorophores are designed,
synthesized, characterized and their DNA photocleavage and visible
light-induced cytotoxic activity studied. The ferrocenyl complexes
show significant DNA photocleavage activity compared to its
phenyl analogue. Mechanistic study revealed formation of hydroxyl
radicals as the reactive oxygen species. The complexes show high
cellular uptake. They are remarkably PDT active in MCF-7 cells
compared to HeLa cells on photo-excitation of their MLCT band. The
positive effect of the ferrocenyl moiety is evidenced from a four-
fold increase in photocytotoxicity of the ferrocenyl complex 3
compared to its phenyl analogue 4. The IC50 value of complex 3 in
light in MCF-7 cells is similar to that of Photofrin®. The ferrocenyl
complex 3 showed ~4-fold increase in cellular uptake compared to
its phenyl analogue 4 and this could be due to the lipophilic nature
of the ferrocenyl moiety. The fluorescence property of the naphthyl
and pyrenyl moieties in the complexes is used for cellular imaging
which showed localization of the complex predominantly in the
cytosol and partially in the nucleus of the HeLa and MCF-7 cells. The
photocytotoxicity of the acetylacetonate derivatives compares well
with their reported curcumin analogues. The light-induced cell
death by the complexes proceeds via an apoptotic pathway. The
results are of importance toward developing ferrocenyl conjugates
of transition metals as potential metal-based photocytotoxic
agents.
We have studied the DNA photocleavage activity of the com-
plexes using supercoiled pUC19 DNA in Tris-HCl/NaCl (50 mM,
pH ¼ 7.2) buffer using green and red light of 568 and 785 nm
wavelength that were chosen based on the MLCT band of the
complexes 1e3. The ferrocenyl complexes (40 mM) showed signif-
icant cleavage of SC DNA giving ~80% nicked circular (NC) DNA in
red light (Fig. 8, Table 4). Complex 4 lacking the ferrocenyl moiety is
less active in light of 568 nm. Control experiments using only DNA
in light or the complexes in dark did not show any significant DNA
cleavage activity. The complexes in red light of 785 nm showed
efficient photocleaveage of DNA giving ~70% NC DNA formation.
Mechanistic studies were carried out using complex 3 in the
presence of various additives to explore any formation of reactive
oxygen species. The complex did not show any DNA photocleavage
activity under an argon atmosphere indicating possible involve-
ment of ROS like singlet oxygen and/or hydroxyl radicals. Singlet
oxygen quenchers, viz. sodium azide and TEMP did not show any
inhibition of the DNA photocleavage activity and addition of D2O in
which singlet oxygen has longer life time did not show any
enhancement in the DNA photocleavage activity, thus ruling out
any singlet oxygen formation [48]. The hydroxyl radical scavengers,
viz. DMSO, KI and catalase exhibited significant inhibitory effect on
the DNA photocleavage activity suggesting the formation of hy-
droxyl radical (ꢁOH) as the ROS. Superoxide dismutase (SOD)
showed only partial inhibitory effect and this could be due to
ꢁ
spontaneous dismutation of superoxide radical anion to the OH
radicals.
4. Experimental
4.1. Materials and methods
The reagents and chemicals were procured from commercial
sources (s.d. Fine Chemicals, India; SigmaeAldrich, USA) and used
without further purifications. Solvents were purified by standard
procedures prior to use [49]. Synthesis of the complexes was per-
formed by standard Schlenk technique under nitrogen atmosphere.
Supercoiled (SC) pUC19 DNA (cesium chloride purified) was pur-
chased from Bangalore Genie (India). Ferrocenecarboxaldehyde,
acetyl naphthalene, pyrene, sodium perchlorate, 5,5-dimethyl-1-
pyrroline 1-oxide (DMPO), calf thymus (ct) DNA, agarose (molec-
ular biology grade), catalase, superoxide dismutase (SOD), 2,2,6,6-
tetramethyl-4-piperidone (TEMP), MTT (3-(4,5-dimethylthiazole-
2-yl)-2,5-diphenyltetrazolium bromide) and ethidium bromide
Fig. 8. Cleavage of SC pUC19 DNA (0.2
m
g, 30
m
M) by ligands (Hnap-acac, Hpy-acac)
and complexes 1e4 (30 M) and ligands (30
m
mM) in 50 mmol Tris-HCl/NaCl buffer
(pH ¼ 7.2) containing 10% DMF on photo-irradiation with light of 568 nm and 785 nm
wavelengths (2 h exposure): lane 1, DNA control; lane 2, DNA þ Hpy-acac; lane 3,
DNA þ Hnap-acac; lanes 4e7, DNA þ 1e4 (40 M, 568 nm); lanes 8e11, DNA þ 1e4
m
(EB)
were
purchased
from
SigmaeAldrich
(USA).
(50
mM, 785 nm), respectively. SC and NC are the supercoiled and nicked circular forms
of the DNA.
Tris(hydroxymethyl)aminomethane-HCl (TriseHCl) buffer solution

B. Balaji et al. / European Journal of Medicinal Chemistry 92 (2015) 332e341
339
was prepared using deionized and sonicated triple-distilled water.
The ligands 4'-ferrocenyl-2,2':60,200-terpyridine (Fc-tpy) and phe-
nylterpyridine (Ph-tpy), acetylacetone derivatives viz., Hnap-acac,
Hpy-acac were prepared according the literature procedures
[31,32].
1290w, 1255w, 1223w, 1143w, 1095w, 1095vs (ClOꢀ4 ), 1032m, 958s
(V]O), 911w, 878w, 788m, 751w, 674w, 623m, 573w, 514w, 474w.
meff ¼ 1.68 mB at 298 K.
4.3.3. [VO(Fc-tpy)(py-acac)](ClO4) (3)
Yield: 72%. Anal. Calcd for C45H32N3O7ClFeV: C, 62.20; H, 3.71; N,
4.84. Found: C, 62.42; H, 3.78; N, 4.72. ESI-MS (m/z) in MeCN: 769
4.2. General methods
[M-(ClO4)]þ. LM in DMF: 79 S m2
M
ꢀ1. UVeVis in DMFeTris HCl
The elemental analysis was done using a Thermo Finnigan
FLASH EA 1112 CHNS analyzer. The electronic, infrared and emis-
sion spectra were recorded on PerkineElmer Lambda 650, Per-
kineElmer spectrum one 55 and Horiba Jobin Yvon max4
spectrometer, respectively. Molar conductivity measurements were
carried out using a Control Dynamics (India) conductivity meter.
Cyclic voltammetric measurements were made at room tempera-
ture on an EG&G PAR 253 VersaStat potentiostat/galvanostat using
a three electrode configuration consisting of a glassy carbon
working, platinum wire auxiliary and saturated calomel reference
electrode (SCE) with 0.1 M tetrabutylammonium perchlorate as a
supporting electrolyte. Electrospray ionization (ESI) mass spectral
measurements were made using Bruker Daltonics make Esquire
300 Plus ESI model. Magnetic measurements were done in the solid
state at 25 ꢂC using a magnetic susceptibility balance, Sherwood
Scientific, Cambridge, UK.
buffer (1:1 v/v) [lmax, nm (ε, dm3 Mꢀ1 cmꢀ1)]: 586 (3536), 396
(9465), 368sh (16220), 346 (20160), 286 (35430) (sh, shoulder). FT-
IR (cmꢀ1): 3441br, 3074w, 1610s, 1563s, 1536sm, 1517w, 1499m,
1451m, 1460w, 1435m, 1388m, 1372m, 1324w, 1267w, 1255w,
1096vs (ClOꢀ4 ), 1031m, 963s (V]O), 856w, 848w, 791m, 822w,
771wm 714w, 670w, 625m, 579w, 542w, 517w, 478w. meff ¼ 1.62 mB
at 298 K.
4.3.4. [VO(Ph-tpy)(py-acac)](ClO4) (4)
Yield: 74%. Anal. Calcd for C41H28N3O7ClV: C, 64.70; H, 3.71; N,
5.52. Found: C, 64.58; H, 3.78; N, 5.40. ESI-MS (m/z) in MeCN: 661
ꢀ1
M . UVeVis in DMFeTris HCl
[M-(ClO4)]þ. LM in DMF: 74 S m2
buffer (1:1 v/v) [lmax, nm (ε, dm3 Mꢀ1 cmꢀ1)]: 777 (90), 392sh
(7896), 358 (11820), 280 (47850). FT-IR (cmꢀ1): 3451br, 3072w,
1608s, 1561s, 1526sm, 1491m, 1441m, 1434m, 1372m, 1324w,
1263w, 1255w, 1095vs (ClOꢀ4 ), 1032m, 961s (V]O), 851w, 791m,
822w, 710w, 672w, 622m, 532w, 482w. meff ¼ 1.65 mB at 298 K.
CAUTION: Perchlorate salts being potentially explosive, only small
quantity of the sample was used with due precautions.
4.4. Solubility
4.3. Synthesis of the complexes 1e4
The complexes were soluble in acetonitrile, chloroform,
dichloromethane, dimethyl sulfoxide (DMSO), dimethylformamide
(DMF) and 1% DMSO-DMEM media. They were sparingly soluble in
water.
VOSO4 (0.16 g, 1.0 mmol) was dissolved in 1.0 ml of ethanol and
to this was added BaCl2.2H2O (0.244 g, 1.0 mmol) taken in 1.0 ml of
ethanol and the mixture was stirred for 1.0 h at 25 ꢂC. The mixture
was centrifuged to remove precipitated barium sulphate. The clear
solution was separated and to this was added the acetylacetone
derivative (1.0 mmol; Hacac, 0.01 g; Hnap-acac, 0.21 g; Hpy-acac,
0.27 g), previously neutralized with NaOH (0.04 g in 1.0 ml wa-
ter). The solution was stirred for 30 min. To this solution was added
Fc-tpy or Ph-tpy dissolved in CHCl3:MeOH (1:4 v/v, 5.0 ml) to get a
dark blue solution on stirring for an additional 30 min. The complex
was precipitated as its perchlorate salt by adding an excess of
aqueous NaClO4. The precipitate was isolated and washed with cold
methanol and diethyl ether followed by drying in vacuum. The
ferrocenyl complexes 1e3 were navy blue coloured amorphous
solids, while the phenyl complex 4 was golden yellow coloured
amorphous solid.
4.5. Computational methodology
All calculations were performed using GAUSSIAN09 program
suite and the geometries of the complexes were optimized at
B3LYP/6-31g (d,p) level of theory [35e37]. Details are given as
Supporting Information. Time-dependent density functional theory
(TD-DFT) calculations were carried out to investigate the optical
properties of the complexes in DMF. The lowest 40 transitions up to
400 nm were taken into account in the calculations of the ab-
sorption spectra. Molecular orbital (MO) compositions were
calculated using the Multiwfn program [50].
4.6. Cell culture
4.3.1. [VO(Fc-tpy)(acac)](ClO4) (1)
Yield: 69%. Anal. Calcd for C30H26N3O7ClFeV: C, 52.77; H, 3.84; N,
6.15. Found: C, 52.50; H, 3.78; N, 6.10ꢀ. 1ESI-MS (m/z) in MeCN: 583
HeLa (human cervical carcinoma), MCF-7 (human breast
adenocarcinoma), 3T3 (standard normal fibroblast) cells were
maintained in Dulbecco's Modified Eagle's Medium (DMEM) sup-
plemented with 10% fetal bovine serum (FBS), 100 IU mlꢀ1 of
penicillin, 100 mg mlꢀ1 of streptomycin and 2 mM of Glutamax at
37 ꢂC in a humidified incubator at 5% CO2. The adherent cultures
were grown as monolayer and were passaged once in 4e5 days by
exposure to 0.25% Trypsin-EDTA.
[M-(ClO4)]þ. LM in DMF: 84 S m2
M . UVeVis in DMFeTris HCl
buffer (1:1 v/v) [lmax, nm (ε, dm3 Mꢀ1 cmꢀ1)]: 581 (2608), 427
(1025), 334 (10080), 287 (15175). FT-IR (cmꢀ1): 3424br, 3082w,
1610s, 1564s, 1516s, 1477s, 1435m, 1373s, 1290w, 1250m, 1096vs
(ClOꢀ4 ), 1041m, 1026m, 958s (V]O), 902w, 830w, 794m, 766w,
732m, 700w, 676w, 660w, 623m, 570w, 555w, 515w, 478w (br,
broad; vs, very strong; s, strong; m, medium; w, weak). meff ¼ 1.65
mB at 298 K.
4.7. Cellular experiments
4.3.2. [VO(Fc-tpy)(nap-acac)](ClO4) (2)
4.7.1. Cytotoxicity of the complexes
Yield: 79%. Anal. Calcd for C39H30N3O7ClFeV: C, 58.93; H, 3.80; N,
5.29. Found: C, 58.82; H, 3.71; N, 5.20. ESI-MS (m/z) in MeCN: 695
The cytotoxicity of the complexes in HeLa and MCF-7 cells was
assessed by MTT assay [51]. About 1 ꢃ 104 cells were seeded into
ꢀ1
[M-(ClO4)]þ. LM in DMF: 86 S m2
M
. UVeVis in DMFeTris HCl
96-well plates in 100 mL media per well. The cells were allowed to
buffer (1:1 v/v) [lmax, nm (ε, dm3 Mꢀ1 cmꢀ1)]: 583 (3014), 425
(1285), 330 (13175), 293 (17930). FT-IR (cmꢀ1): 3420br, 3074w,
2922w, 1709m, 1612s, 1556s, 1516s, 1480s, 1460w, 1436m, 1376s,
grow for 24 h in a CO2 incubator at 37 ꢂC. Different concentrations
of the complexes dissolved in 1% DMSO were added to the cells.
Incubation was continued for a further period of 4 h at 37 ꢂC in the

340
B. Balaji et al. / European Journal of Medicinal Chemistry 92 (2015) 332e341
CO2 incubator. After incubation, the medium was replaced with PBS
and photo-irradiation was performed for 1 h in visible light of
400e700 nm using Luzchem Photoreactor (Model LZC-1, Ontario,
Canada; light fluence rate: 2.4 mW cmꢀ2; light dose ¼ 10 J cmꢀ2).
For experiment using NAC as a ROS scavenger, photo-irradiation
was done in presence of 0.5 mM NAC containing PBS. Post irradi-
ation, PBS was replaced with 10% DMEM and cells were cultured for
exposed to visible light for 1 h and images were captured.
4.8. EPR spin trap with DMPO
EPR spectra were recorded at room temperature using a Bruker
ESP 300 EPR spectrometer at 9.5 GHz (X-band) employing 100 kHz
field modulation. EPR measurements for the oxovanadium(IV)
complexes were done in 4 mM DMF solution at 25 ꢂC. For spin-
a further period of 20 h in dark followed by addition of 25
4 mg mlꢀ1 of MTT to each well and incubated for an additional 3 h.
The culture medium was discarded and a 200 L volume of DMSO
mL of
ꢁ
trapping OH, the sample solution containing complex 4 (100
mL,
m
1 mM), DMPO (100
mL, 0.5 M) and 800
mL phosphate buffer was
was added to dissolve the purple formazan crystals. The absorbance
at 540 nm was determined using an ELISA microplate reader
(BioRad, Hercules, CA, USA). Cytotoxicity of the test compounds
was measured as the percentage ratio of the absorbance of the
treated cells to the untreated controls. The IC50 values were
determined by nonlinear regression analysis (GraphPad Prism 5).
photo-irradiated with visible light for 30 min. The solution was
then transferred to an EPR quartz capillary tube to record the
spectra at 25 ꢂC. The g values were estimated using solid DPPH as
an internal standard (g ¼ 2.0036).
4.9. DNA binding and cleavage experiments
4.7.2. Cellular uptake by ICP-MS
The ct-DNA binding experiments were done in TriseHCl/NaCl
buffer (5 mM TriseHCl, 5 mM NaCl, pH ¼ 7.2) or phosphate buffer
(pH ¼ 7.4) using DMF solution of the complexes 1e4 following
reported procedures [53,54]. DNA photocleavage studies were
carried out using DMF solutions of the complexes and supercoiled
pUC19 DNA in 50 mM TriseHCl buffer containing 50 mM NaCl. The
DNA photocleavage studies were carried out in visible light of
568 nm of 50 mW laser power using a Spectra Physics Water-
Cooled Mixed-Gas Ion Laser Stabilite® 2018-RM (continuous-wave
(CW) beam diameter at 1/e2 ¼ 1.8 mm 10% and beam divergence
with full angle ¼ 0.7 mrad 10%) and in near IR 785 nm from a
diode laser (100 mW laser power, Model LQC785-100C from
Spectra Physics with LD module) [55]. Appropriate controls were
used for all the experiments and suitable inhibitors were used for
mechanistic investigations, as described above.
Uptake of the oxovanadium(IV) complexes was determined by
measuring the cellular vanadium content using ICP-MS. HeLa and
MCF-7 cells (~0.5 ꢃ 106 cells) were grown in 6 wells plate and
incubated at 37 ꢂC under 5% CO2 atmosphere for overnight. The
culture medium was removed and replaced with a medium con-
taining the complex (30 mM). After incubation for 4 h, the medium
was removed, washed twice with 3 ml PBS to remove any excess
complex from the extracellular media, trypsinized and digested in
concentrated nitric acid (65% HNO3) at 70 ꢂC for 2 h. Dilution was
done with Milli-Q water to the final volume of 10 ml and analysed
for ICP-MS. The instrument was calibrated for vanadium using
standard solutions containing 1, 10, 100 and 1000 ppb vanadium.
4.7.3. Measurement of intracellular ROS from DCFDA assay
Cell permeable DCFDA when oxidized by cellular ROS is known
to generate fluorescent DCF having an emission maximum at
528 nm [52]. To determine the intracellular ROS, ~0.5 ꢃ 106 HeLa
Acknowledgments
cells were incubated with the complexes (10 mM) for 4 h in dark.
We thank the Department of Science and Technology (DST),
Government of India, and the Council of Scientific and Industrial
Research (CSIR), New Delhi, for financial support (CSIR No.
01(2559)/12/EMR-II and DST No. SR/S5/MBD-02/2007). We are
thankful to the Alexander von Humboldt Foundation, Germany, for
donation of an electroanalytical system. A.R.C. thanks DST for J.C.
Bose national fellowship. We thank Prof. S.V. Bhat and Mrs. S.K.
Bhagyashree for assistance in the EPR experiments and Dr. R.
Chakrabarti for the ICP-MS facility. We thank the Department of
Biotechnology (DBT) for the FACS facility. S.P. thanks SERC of our
institute for the computational facility.
The media was replaced with PBS followed by photo-irradiation for
1 h in serum free conditions. The cells were harvested by trypsi-
nization and a single cell suspension was made. The cells were
washed with PBS to remove any extracellular complex and treated
with 1
mM DCFDA solution in DMSO and incubated in dark for
30 min at room temperature. The intracellular fluorescence of DCF
was monitored by flow cytometry in the FL-1 channel. Control
experiments were performed for (a) untreated cells, in order to
determine the contribution from auto-fluorescence of cells and (b)
cells treated with DCFDA only. Hydrogen peroxide treated cells
were taken as a positive control.
Appendix A. Supplementary data
4.7.4. Cellular uptake from fluorescence microscopy
The emissive property of the complexes 2 and 3 was utilized to
study the cellular localization of the complexes using fluorescence
microscopy. HeLa cells (~3 ꢃ 104) were grown on glass cover slips in
each 6 well plate for 24 h. The cells were subsequently treated with
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.01.003.
References
the complexes (10
mM) for 2 and 4 h in dark. Cells were then
washed with PBS to remove any extracellular complex and fixed
with 4% formaldehyde for 10 min at room temperature. This was
followed by incubation with propidium iodide (PI) staining solution
[1] M. Ethirajan, Y. Chen, P. Joshi, R.K. Pandey, Chem. Soc. Rev. 40 (2011)
340e362.
[2] J.P. Celli, B.Q. Spring, I. Rizvi, C.L. Evans, K.S. Samkoe, S. Verma, B.W. Pogue,
T. Hasan, Chem. Rev. 110 (2010) 2795e2838.
[3] P. Agostinis, K. Berg, K.A. Cengel, T.H. Foster, A.W. Girotti, S.O. Gollnick,
S.M. Hahn, M.R. Hamblin, A. Juzeniene, D. Kessel, M. Korbelik, J. Moan, P. Mroz,
D. Nowis, J. Piette, B.C. Wilson, J. Golab, CA Cancer J. Clin. 61 (2011) 250e281.
[4] R. Bonnett, Chemical Aspects of Photodynamic Therapy, Gordon & Breach,
London, 2000.
[5] P. Couleaud, V. Morosini, C. Frochot, S. Richeter, L. Raehm, J.-O. Durand,
Nanoscale 2 (2010) 1083e1095.
[6] M.J. Ochsner, Photochem. Photobiol. B 32 (1996) 3e9.
[7] S.I. Moriwaki, J. Misawa, Y. Yoshinari, I. Yamada, M. Takigawa, Y. Tokura,
Photodermatol. Photoimmunol. Photomed. 17 (2001) 241e243.
(50 m m
g mlꢀ1 RNase A, 20 g mlꢀ1 PI in PBS) for 1 h at 42 ꢂC. The cells
were washed free of excess PI, mounted in 90% glycerol solution
containing Mowiol, an anti-fade reagent, and sealed. Images were
acquired using Apotome.2 fluorescence microscope (Carl Zeiss,
Germany) using an oil immersion lens at 63ꢃ magnification. The
images were analyzed using the AxioVision Rel 4.9.1 (Carl Zeiss,
Germany) software. Similarly, to view the PDT effect, cells treated
with complexes 3 and 4 were plated on glass cover-slip and

B. Balaji et al. / European Journal of Medicinal Chemistry 92 (2015) 332e341
341
[8] U. Schatzschneider, Eur. J. Inorg. Chem. (2010) 1451e1467.
[32] D.F. Evans, J. Chem. Soc. (1961) 1987e1993.
[9] X. Jia, F.F. Yang, J. Li, J.Y. Liu, J.P. Xue, J. Med. Chem. 56 (2013) 5797e5805.
[10] N.J. Farrer, L. Salassa, P.J. Sadler, Dalton Trans. (2009) 10690e10701.
[11] E. Ranyuk, N. Cauchon, K. Klarskov, B. Guerin, J.E. van Lier, J. Med. Chem. 56
[33] P.K. Sasmal, R. Majumdar, R.R. Dighe, A.R. Chakravarty, Dalton Trans. 39
(2010) 7104e7113.
[34] K. Hutchison, J.C. Morris, T.A. Nile, J.L. Walsh, Inorg. Chem. 38 (1999)
2516e2523.
ꢀ
(2013) 1520e1534.
[12] M.A. Sgambellone, A. David, R.N. Garner, K.R. Dunbar, C. Turro, J. Am. Chem.
Soc. 135 (2013) 11274e11282.
[35] M.J. Frisch, et al., Gaussian 03, Revision B.4, Gaussian Inc., Pittsburgh, PA, 2003
(vide Supplementary data for full reference).
[13] P. Prasad, I. Khan, P. Kondaiah, A.R. Chakravarty, Chem. Eur. J. 19 (2013)
17445e17455.
[36] A.D. Becke, J. Chem. Phys. 98 (1993) 5648e5652.
[37] C. Lee, W. Yang, R.G. Parr, Phys. Rev. B 37 (1988) 785e789.
[38] P.K. Sasmal, S. Saha, R. Majumdar, S. De, R.R. Dighe, A.R. Chakravarty, Dalton
Trans. 39 (2010) 2147e2158.
[14] N.L. Fry, P.K. Mascharak, Acc. Chem. Res. 44 (2011) 289e298.
[15] A.R. Chakravarty, M. Roy, Prog. Inorg. Chem. 57 (2012) 119e202.
[16] C.J. Burrows, J.G. Muller, Chem. Rev. 98 (1998) 1109e1151.
[17] K. Szaciłowski, W. Macyk, A. Drzewiecka-Matuszek, M. Brindell, G. Stochel,
Chem. Rev. 105 (2005) 2647e2694.
[39] E. Delaey, F. Van Larr, D. De Vos, A. Kamuhabwa, P. Jacobs, P. De Witte,
J. Photochem. Photobiol. B 55 (2000) 27e36.
ꢀ
[40] F. Menard, V. Sol, C. Ringot, R. Granet, S. Alves, C.L. Morvan, Y. Queneau,
[18] B. Balaji, B. Banik, P.K. Sasmal, B. Maity, R. Majumdar, R.R. Dighe,
A.R. Chakravarty, Eur. J. Inorg. Chem. (2012) 126e135.
[19] T.K. Goswami, S. Gadadhar, M. Roy, M. Nethaji, A.A. Karande, A.R. Chakravarty,
Organometallics 31 (2012) 3010e3021.
N. Ono, P. Krausz, Bioorg. Med. Chem. 17 (2009) 7647e7657.
[41] S. Saha, D. Mallick, R. Majumdar, M. Roy, R.R. Dighe, E.D. Jemmis,
A.R. Chakravarty, Inorg. Chem. 50 (2011) 2975e2987.
[42] I. Ott, K. Schmidt, B. Kircher, P. Schumacher, T. Wiglenda, R. Gust, J. Med.
Chem. 48 (2005) 622e629.
[43] S.Y. Sun, Cancer Biol. Ther. 9 (2010) 109e110.
[44] G.M. Pieper, C.C. Felix, B. Kalyanaraman, M. Turk, A.M. Roza, Free Radic. Biol.
Med. 19 (1995) 219e225.
[20] B. Maity, M. Roy, B. Banik, R. Majumdar, R.R. Dighe, A.R. Chakravarty, Or-
ganometallics 29 (2010) 3632e3641.
ꢁ
[21] E. Hillard, A. Vessieres, L. Thouin, G. Jaouen, C. Amatore, Angew. Chem. Int. Ed.
45 (2006) 285e290.
[22] C. Ornelas, New J. Chem. 35 (2011) 1973e1985.
[23] G. Gasser, I. Ott, N. Metzler-Nolte, J. Med. Chem. 54 (2011) 3e25.
[24] J. Manosroi, K. Rueanto, K. Boonpisuttinant, W. Manosroi, C. Biot, H. Akazawa,
T. Akihisa, W. Issarangporn, A. Manosroi, J. Med. Chem. 53 (2010) 3937e3943.
[25] F. Dubar, G. Anquetin, B. Pradines, D. Dive, J. Khalife, C. Biot, J. Med. Chem. 52
(2009) 7954e7957.
[45] M. Hoebeke, H.J. Schuitmakerz, L.E. Janninkz, T.M.A.R. Dubbelman, A. Jakobs,
A.V. Vorst, Photochem. Photobiol. 66 (1997) 502e508.
[46] B. Maity, B.V.S.K. Chakravarthi, M. Roy, A.A. Karande, A.R. Chakravarty, Eur. J.
Inorg. Chem. (2011) 1379e1386.
[47] G. Tabbì, C. Cassino, G. Cavigiolio, D. Colangelo, A. Ghiglia, I. Viano, D. Osella,
J. Med. Chem. 45 (2002) 5786e5796.
[26] D. Dive, C. Biot, ChemMedChem 3 (2008) 383e391.
[27] D. Osella, M. Ferrali, P. Zanello, F. Laschi, M. Fontani, C. Nervi, G. Cavigiolio,
Inorg. Chim. Acta 306 (2000) 42e48.
[48] P.B. Merkel, D.R. Kearns, J. Am. Chem. Soc. 94 (1972) 1029e1030.
[49] D.D. Perrin, W.L.F. Armarego, D.R. Perrin, Purification of Laboratory Chemicals,
Pergamon Press, Oxford, 1980.
[28] B. Balaji, B. Balakrishnan, S. Perumalla, A.A. Karande, A.R. Chakravarty, Eur. J.
Med. Chem. 85 (2014) 458e467.
[50] T. Lu, F. Chen, J. Comput. Chem. 33 (2012) 580e592.
[51] T. Mosmann, J. Immunol. Methods 65 (1983) 55e63.
[52] A. Gomes, E. Fernandes, J.L.F.C. Lima, J. Biochem. Biophys. Methods 65 (2005)
45e80.
[53] J.D. McGhee, P.H. von Hippel, J. Mol. Biol. 86 (1974) 469e489.
[54] M.T. Carter, M. Rodriguez, A.J. Bard, J. Am. Chem. Soc. 111 (1989) 8901e8911.
[55] P.K. Sasmal, S. Saha, R. Majumdar, R.R. Dighe, A.R. Chakravarty, Inorg. Chem.
49 (2010) 849e859.
[29] B. Balaji, K. Somyajit, B. Banik, G. Nagaraju, A.R. Chakravarty, Inorg. Chim. Acta
400 (2013) 142e150.
ꢀ
[30] M.A. Tomren, M. Masson, T. Loftsson, H.H. Tønnesen, Int. J. Pharm. 338 (2007)
27e34.
ꢀ~
[31] E.C. Constable, A.J. Edwards, R. Martínez-Manez, P.R. Raithby,
A.M.W.C. Thompson, J. Chem. Soc. Dalton Trans. (1994) 645e650.