Photocytotoxic ferrocene-appended (l-tyrosine)copper(II) complexes of phenanthroline bases

Article abstract of DOI:10.1016/j.poly.2012.06.018

Copper(II) complexes of ferrocene(Fc)-conjugated reduced Schiff base of l-tyrosine (Fc-TyrH), viz., [Cu(Fc-Tyr)(L)](ClO₄), where L is 1,10-phenanthroline (phen, 1), dipyrido[3,2-d:2′,3′-f]quinoxaline (dpq, 2), dipyrido[3,2-a:2′,3′-c]phenazine (

Full text of DOI:10.1016/j.poly.2012.06.018

Polyhedron 52 (2013) 1287–1298
Contents lists available at SciVerse ScienceDirect
Polyhedron
journal homepage: www.elsevier.com/locate/poly
Photocytotoxic ferrocene-appended (L-tyrosine)copper(II) complexes
of phenanthroline bases
a,
Tridib K. Goswami a, Sudarshan Gadadhar b, Anjali A. Karande b, , Akhil R. Chakravarty
⇑
⇑
a Department of Inorganic and Physical Chemistry, Indian Institute of Science, Sir C.V. Raman Avenue, Bangalore 560012, India
b Department of Biochemistry, Indian Institute of Science, Sir C.V. Raman Avenue, Bangalore 560012, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 18 June 2012
Copper(II) complexes of ferrocene(Fc)-conjugated reduced Schiff base of
L
-tyrosine (Fc-TyrH), viz., [Cu(Fc-
Tyr)(L)](ClO4), where L is 1,10-phenanthroline (phen, 1), dipyrido[3,2-d:20,30-f]quinoxaline (dpq, 2),
dipyrido[3,2-a:20,30-c]phenazine (dppz, 3) and 2-(naphthalen-1-yl)-1H-imidazo[4,5-f][1,10]phenanthro-
line (nip, 4), were prepared and tested for their photocytotoxicity in cancer cells. [Cu(Fc-Phe)(phen)](-
Dedicated to Alfred Werner on the 100th
Anniversary of his Nobel prize in chemistry
in 1913.
ClO4) (5) of
L-phenylalanine and [Cu(Ph-Tyr)(L)(ClO4)] of the reduced Schiff base Ph-TyrH derived from
benzaldehyde and
L-tyrosine having phen (6) and dppz (7), and [Cu(Ph-Phe)(phen)(ClO4)] (8) using L-
phenylalanine were prepared and used as controls. Complexes 5 and 6 were structurally characterized
by X-ray crystallography. A copper(II)-based d–d band near 600 nm and a ferrocenyl band at ꢀ450 nm
were observed in DMF–Tris–HCl buffer (1:4 v/v) in respective complexes. The complexes are photoclea-
vers of pUC19 DNA in visible light forming ꢁOH radicals. They are cytotoxic in HeLa (human cervical can-
cer) and MCF-7 (human breast cancer) cells showing an enhancement of cytotoxicity in visible light.
Fluorescence imaging shows nuclear localization of the complexes.
Keywords:
Ferrocene
Copper
Crystal structure
DNA Photocleavage
Photocytotoxicity
Cellular imaging
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
against cisplatin resistant cell line A2780 [29]. Among the metal-
based PDT agents, 3d and 4d metal complexes are reported to show
Metal complexes have been extensively studied for their vari-
ous therapeutic applications [1–5]. Among them, cisplatin and its
analogues are found to show anticancer activity against different
cancer cell lines [1,6–9]. The dark toxicity and poor selectivity
are generally the major issues for the failure and limitations of
the platinum(II) based chemotherapeutic drugs. This has led to
the discovery of platinum(IV) prodrugs which can be activated in
the cancer cells by chemical or photochemical means [10–14].
Cytotoxicity resulting from photoactivation of the prodrugs which
are otherwise non-toxic to the cells in dark is an attractive possibil-
ity to circumvent the toxicity associated with the chemotherapeu-
tic drugs [15–21]. Photodynamic therapy (PDT) has received
significant current attentions due to its non-invasive nature, re-
duced side effects and for its selective action on the tumour leaving
unexposed healthy cells unaffected [15–18]. The porphyrin-based
PDT agents show skin sensitivity and hepato-toxicity [22,23]. This
has prompted search for metal-based PDT agents as photosensitiz-
ers [24–28]. Sadler and co-workers reported a Pt(IV)-azido prodrug
which on photoexcitation generates the active species which is ac-
tive against a number of cell lines in in-vitro studies, significantly
photocytotoxicity in visible light [21,24–35]. Unlike the organic
PDT agents, the redox active metal complexes are known to show
generation of hydroxyl radicals as the reactive oxygen species
(ROS) by a photo-redox pathway [36,37].
The present work stems from our interests to develop the
chemistry of ferrocene-conjugated copper(II) complexes as poten-
tial photocytotoxic agents in visible light [38–40]. The utility of fer-
rocene-appended breast cancer drug tamoxifen, commonly known
as ferrocifen, is well known against both hormone dependent and
independent cases while tamoxifen is inactive for the hormone
independent ones [41]. This augmentation in the activity of ferroc-
ifen is attributed to the reversible redox chemistry of ferrocene
[42,43]. Again, the anti-malarial activity of chloroquine gets en-
hanced on attaching the ferrocene moiety to the parent drug
[44]. Besides the ferrocene-conjugates, several half-sandwich
arene-metal complexes are known to show prominent anticancer
activity, particularly for metastatic cancer cells [45–47]. The chem-
istry of organometallic PDT agents showing photocytotoxicity in
visible light is relatively new. While organometallic complexes of
Fe and W are known to show DNA cleavage activity in high power
UV-A light, the ferrocene-conjugated copper(II) and oxovana-
dium(IV) complexes display photodynamic effect in HeLa and
MCF-7 cell lines in visible light [38–40,48–52]. Porphyrin-conju-
gated (arene)ruthenium(II) complexes are reported to show PDT
⇑
Corresponding authors. Fax: +91 80 23601552.
E-mail addresses: anjali@biochem.iisc.ernet.in (A.A. Karande), arc@ipc.iisc.erne-
t.in (A.R. Chakravarty).
0277-5387/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.poly.2012.06.018

1288
T.K. Goswami et al. / Polyhedron 52 (2013) 1287–1298
activity due to the presence of a porphyrin moiety which acts as a
photosensitizer [53,54].
(molecular biology grade), distamycin, methyl green, KI, catalase,
NaN3, -histidine, SOD (superoxide dismutase), 2,2,6,6-tetra-
L
Herein, we present the synthesis, characterization, DNA bind-
ing, photo-enhanced DNA cleavage activity and photocytotoxic
properties of four ferrocene-appended reduced Schiff base (Fc-
methyl-4-piperidone (TEMP), acridine orange (AO), ethidium bro-
mide (EB), bromophenol blue, xylene cyanol, Dulbecco’s Modified
Eagle’s Medium (DMEM), propidium iodide (PI) and MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were
purchased from Sigma (USA). Dipyrido[3,2-d:20,30-f]quinoxaline
(dpq) and dipyrido[3,2-a:20,30-c]phenazine (dppz) were prepared
by reported procedures using 1,10-phenanthroline-5,6-dione as a
precursor reacted with ethylenediamine and 1,2-phenylenedi-
amine, respectively [56,57]. 2-(Naphthalen-1-yl)-1H-imidazo[4,5-
f][1,10]phenanthroline (nip) was prepared following literature
procedure using 1,10-phenanthroline-5,6-dione and 1-naphthal-
dehyde [58]. Fc-TyrH was prepared according to a literature proce-
dure [59].
The elemental analysis was carried out using a Thermo Finnigan
Flash EA 1112 CHN analyzer. The infrared, UV–Vis and emission
spectra were recorded on PerkinElmer Lambda 35, PerkinElmer
Spectrum one 55 and PerkinElmer LS 55 spectrophotometer,
respectively. Molar conductivity measurements were performed
using a Control Dynamics (India) conductivity meter. Room tem-
perature solution state magnetic susceptibilities of the copper(II)
complexes in DMSO-d6 containing 1% TMS (v/v) as the internal ref-
erence were obtained by Evans method using Bruker AMX-400
NMR spectrometer [60,61]. The magnetic moments were calcu-
TyrH) copper(II) complexes of L-tyrosine and phenanthroline bases,
viz., [Cu(Fc-Tyr)(L)](ClO4), where L is 1,10-phenanthroline (phen in
1), dipyrido[3,2-d:20,30-f]quinoxaline (dpq in 2), dipyrido[3,2-
a:20,30-c]phenazine (dppz in 3) and 2-(naphthalen-1-yl)-1H-imi-
dazo[4,5-f][1,10]phenanthroline (nip in 4) (Fig. 1). The (1,10-phe-
nanthroline)copper(II) complex of the reduced Schiff base
derived from L-phenylalanine and ferrocenecarboxaldehyde, viz.,
[Cu(Fc-Phe)(phen)](ClO4) (5) is prepared, structually characterized
and evaluated for its biological potential to explore the effect of
the ferrocene moiety in this series of complexes. Complexes
[Cu(Ph-Tyr)(L)](ClO4)], where L is phen (in 6) and dppz (in 7) and
Ph-TyrH is the reduced Schiff base of benzaldehyde and
and [Cu(Ph-Phe)(phen)(ClO4)] (8), where Ph-Phe is the reduced
Schiff base of -phenylalanine and benzaldehyde are prepared
L-tyrosine,
L
and used as control species to study the visible light-induced
cytotoxicity in HeLa and MFC-7 cancer cell lines (Fig. 1). Significant
results of this study are the enhanced activity in the DNA photoc-
leavage and photocytotoxicity of the ferrocene-appended com-
plexes compared to the controls which lack the ferrocenyl
moiety. Complex 4 with a pendant naphthyl fluorophore is used
to study the cellular uptake. The fluorescence microscopic images
showed significant nuclear localization of complex 4 in HeLa cells.
lated using the equation: leff = 0.0618(DfT/fM), where Df is the ob-
served shift in frequency of the TMS signal, T is the temperature
(K), f is the operating frequency (MHz) of the NMR spectrometer,
and M is the molarity of the complex in the solution. Cyclic voltam-
metric measurements were carried out at 25 °C on a EG&G PAR
Model 253 VersaStat potentiostat/galvanostat using a three elec-
trode set-up with a glassy carbon working, platinum wire auxiliary
and a saturated calomel reference (SCE) electrode. Tetrabutylam-
monium perchlorate (TBAP) (0.1 M) was used as a supporting elec-
trolyte in DMF. The electrochemical data were uncorrected for
junction potentials. 1H NMR spectra were recorded at room tem-
perature on a Bruker 400 MHz NMR spectrometer. Electrospray
ionization mass spectral measurements were done using Esquire
3000 plus ESI (Bruker Daltonics) Spectrometer. Fluorescence
microscopic investigations were carried out on fluorescence micro-
scope (Carl Zeiss, Germany) using an oil immersion lens.
2. Experimental
2.1. Materials and measurements
All the reagents and chemicals were obtained from commercial
sources (s.d. Fine Chemicals, India; Sigma–Aldrich, USA) and used
as such. Solvents used were purified by reported procedures [55].
Supercoiled pUC19 DNA (cesium chloride purified) was purchased
from Bangalore Genie (India). Tris-(hydroxymethyl)aminometh-
ane–HCl (Tris–HCl) buffer solution was prepared using deionized
and sonicated triple distilled water. Calf thymus (ct) DNA, agarose
2.2. Synthesis
2.2.1. Preparation of [Cu(Fc-Tyr)(L)](ClO4) (L = phen, 1; dpq, 2; dppz,
3; nip, 4)
Complexes 1–4 were prepared by a general synthetic procedure
in which a 0.2 g (1.0 mmol) quantity of copper(II) acetate.hydrate
in 15 mL aqueous methanol (1:1 v/v) was reacted with the hetero-
cyclic base (L: phen, 0.19 g; dpq, 0.23 g; dppz, 0.29 g; nip, 0.35 g,
1.0 mmol) while stirring at room temperature for 0.5 h followed
by addition of solid Fc-TyrH (1.0 mmol, 0.38 g) in small portions
with continuous stirring. The reaction mixture was stirred for
1.5 h, and the product was isolated as a green solid in ꢀ70% yield
on addition of a methanol solution of NaClO4 (1.0 mmol, 0.12 g).
The solid was isolated, washed with water and cold methanol fol-
lowed by drying in vacuum over P4O10 (Yield: 0.53 g, 74% for 1;
0.55 g, 71% for 2; 0.56 g, 68% for 3; 0.61 g, 69% for 4).
Anal. Calc. for C32H28N3O7ClFeCu (1): C, 53.28; H, 3.91; N, 5.82.
Found: C, 53.09; H, 3.88; N, 5.91%. Selected IR data (cmꢂ1): 3220br,
2155w, 2076w, 2015w, 1630vs (COOasym), 1517s, 1430s, 1370m
(COOsym), 1253m, 1096vs (ClO4ꢂ), 916w, 842s, 720s, 621s, 555w,
484s, 429w. (vs, very strong; s, strong; m, medium; w, weak; br,
broad). ESI-MS in MeOH: m/z 621 [Mꢂ(ClO4ꢂ)]+. UV–Vis in DMF–
Fig. 1. Schematic drawings of the complexes [Cu(Fc-Tyr)(L)](ClO4) (L = phen, 1;
dpq, 2; dppz, 3; nip, 4), [Cu(Fc-Phe)(phen)](ClO4) (5), [Cu(Ph-Tyr)(L)(ClO4)]
(L = phen, 6; dppz, 7) and [Cu(Ph-Phe)(phen)(ClO4)] (8) and the heterocyclic bases
used.
Tris–HCl buffer (1:4 v/v) [kmax/nm (
e
/Mꢂ1 cmꢂ1)]: 590 (119), 440
eff = 1.80 lB at
(297), 272 (28125), 295sh (9075) (sh, shoulder).
l

T.K. Goswami et al. / Polyhedron 52 (2013) 1287–1298
1289
298 K in DMSO-d6 containing 1% TMS (v/v). KM = 79 S m2 Mꢂ1 in
DMF at 25 °C.
2.2.4. Preparation of Ph-TyrH and Ph-PheH
-Tyrosine or -phenylalanine (0.36 g of L-tyrosine or 0.33 g of L-
L
L
Anal. Calc. for C34H28N5O7ClFeCu (2): C, 52.80; H, 3.65; N, 9.05.
Found: C, 52.62; H, 3.65; N, 9.19%. Selected IR data (cmꢂ1): 3235br,
3085w, 2995w, 2080w, 2012w, 1636vs (COOasym), 1512m, 1485w,
1447w, 1383s (COOsym), 1238m, 1086vs (ClO4ꢂ), 920w, 814s, 726s,
620s, 556w, 485s, 436m. ESI-MS in MeOH: m/z 673 [Mꢂ(ClO4ꢂ)]+.
phenylalanine, 2.0 mmol) was dissolved in dry methanol (15 mL)
by addition of one equivalent of NaOH (0.08 g, 2.0 mmol) with
continuous stirring. Benzaldehyde (0.2 mL, 2.0 mmol) was added
drop-wise to the above solution. The mixture was refluxed for
an hour, cooled and then treated with an excess of solid NaBH4
in an ice-bath with constant stirring. After stirring for ꢀ15–
20 min the solvent was removed to get a mass which was dis-
solved in water and treated with dilute HCl to maintain a pH of
ꢀ5–6. A white solid precipitated out was isolated, thoroughly
washed with water and cold methanol and finally dried in vacuum
over P2O5. Yield: 0.43 g (ꢀ78%) for Ph-TyrH and 0.41 g (ꢀ80%) for
Ph-PheH.
UV–Vis in DMF–Tris–HCl buffer (1:4 v/v) [kmax/nm (e
/Mꢂ1 cmꢂ1)]:
595 (123), 452 (298), 337 (4050), 323 (66650), 298sh (13925), 258
(41650). leff = 1.81 lB at 298 K in DMSO-d6 containing 1% TMS (v/
v). KM = 82 S m2 Mꢂ1 in DMF at 25 °C.
Anal. Calc. for C38H30N5O7ClFeCu (3): C, 55.42; H, 3.67; N, 8.50.
Found: C, 55.18; H, 3.38; N, 8.20%. Selected IR data (cmꢂ1): 3450br,
3240w, 2925w, 2015w, 1638vs (COOasym), 1497s, 1419s, 1355s
(COOsym), 1235 m, 1075vs (ClO4ꢂ), 817s, 764s, 726s, 620s, 487m,
424m. ESI-MS in MeOH: m/z 723 [Mꢂ(ClO4ꢂ)]+. UV–Vis in DMF–
Anal. Calc. for C16H17NO3 (Ph-TyrH): C, 70.83; H, 6.32; N, 5.16.
Found: C, 70.55; H, 6.60; N, 5.30%. ESI-MS in MeOH: m/z 294
[M+Na+]. Selected IR data (cmꢂ1): 1580vs (COOasym), 1511s,
1437m, 1390vs (COO)sym, 1332m, 1250s, 1104m, 1058w, 1032w,
995w, 964w, 920w, 820s, 777m, 746s, 695s, 634m, 568m, 535s,
487s. 1H NMR (D2O, ppm): d HPh (7.22, m, 5H), Ha (3.61, d, 1H,
Tris–HCl buffer (1:4 v/v) [kmax/nm (e
/Mꢂ1 cmꢂ1)]: 585 (129), 440
(473), 377 (10625), 360 (10750), 275 (48825).
leff = 1.78 lB at
298 K in DMSO-d6 containing 1% TMS (v/v). KM = 84 S m2 Mꢂ1 in
DMF at 25 °C.
2
Anal. Calc. for C43H34N5O7ClFeCu (4): C, 58.19; H, 3.86; N, 7.89.
Found: C, 58.01; H, 4.01; N, 7.99%. Selected IR data (cmꢂ1): 3221br,
3082w, 2925w, 2327w, 2075w, 2012w, 1614vs (COOasym), 1512s,
1449m, 1362s (COOsym), 1236m, 1089vs (ClO4ꢂ), 916w, 807s,
776s, 725s, 661w, 621s, 556w, 484s, 429m. ESI-MS in MeOH: m/z
787 [Mꢂ(ClO4ꢂ)]+. UV–Vis in DMF–Tris–HCl buffer (1:4 v/v)
2JHH = 12 Hz), Ha’ (3.43, d, 1H, JHH = 12 Hz), Hb (3.15, m, 1H), Hc
2
(2.65, m, 2H), Hd (6.86, d, 2H, JHH = 8 Hz), He (6.52, d, 2H,
2JHH = 8 Hz) (vide Supplementary data for hydrogen atom
labelling).
Anal. Calc. for C16H17NO2 (Ph-PheH): C, 75.27; H, 6.71; N, 5.49.
Found: C, 75.09; H, 6.82; N, 5.41%. ESI-MS in MeOH: m/z 256
[M+H+], 278 [M+Na+]. Selected IR data (cmꢂ1): 1593vs (COOasym),
1495m, 1433s, 1385vs (COO)sym, 1330m, 1204m, 1082m, 1029m,
971w, 916w, 865m, 804w, 745s, 694vs, 616w, 582m, 529s, 483s,
432w. 1H NMR (D2O, ppm): d HPh,Ph’ (7.18, m, 10H), Ha (3.60, d,
[kmax/nm (e
/Mꢂ1 cmꢂ1)]: 615 (125), 455 (503), 301 (22225), 260
(29650).
leff = 1.77 lB at 298 K in DMSO-d6 containing 1% TMS
(v/v). KM = 79 S m2 Mꢂ1 in DMF at 25 °C.
2
2
1H, JHH = 12.4 Hz), Ha’ (3.42, d, 1H, JHH = 12.8 Hz), Hb (3.19, m,
1H), Hc (2.75, m, 2H) (vide Supplementary data for hydrogen atom
labelling).
2.2.2. Preparation of Fc-PheH
L-Phenylalanine (0.36 g, 1.0 mmol) was dissolved in dry metha-
nol (10 mL) by addition of one equivalent NaOH (0.04 g, 1.0 mmol)
with continuous stirring. This solution is added drop-wise to a
methanol solution of ferrocenecarboxaldehyde (0.21 g, 1 mmol).
The mixture was refluxed for 2 h, cooled in an ice bath and treated
with 5 eq. of NaBH4 for another ꢀ30 min with continuous stirring.
Removal of solvent resulted in a sticky mass which was dissolved
in water and pH was maintained to 5–6 with dilute HCl. A brown
precipitate thus appeared was isolated and thoroughly washed
with water, methanol and diethyl ether and finally dried in vac-
uum over P4O10. Yield: 0.31 g (ꢀ85%).
2.2.5. Preparation of [Cu(Ph-Tyr)(L)(ClO4)] (L = phen, 6; dppz, 7)
Complexes 6 and 7 were prepared following a similar procedure
as described for complexes 1–4. The dark blue methanolic solution
of the complex on slow evaporation gave a dark blue solid which
was washed with samll portions of cold methanol, water and final-
ly dried in vacuo over P4O10 (Yield: 0.43 g, ꢀ70% for 6, 0.52 g, ꢀ73%
for 7).
Anal. Calc. for C28H24N3O7ClCu (6): C, 54.82; H, 3.94; N, 6.85.
Found: C, 54.64; H, 3.69; N, 6.99%. Selected IR data (cmꢂ1):
3120br, 3010w, 2927w, 2335w, 1637vs (COOasym), 1515s,
1430s, 1378m (COOsym), 1348m, 1273m, 1228m, 1090–1052vs
(ClO4ꢂ), 970s, 872w, 835s, 760s, 712s, 619s, 583m, 546w, 511w,
453w, 430w. ESI-MS in MeOH: m/z 513 [Mꢂ(ClO4ꢂ)]+. UV–Vis in
Anal. Calc. for C20H21NO2Fe (Fc-PheH): C, 66.13; H, 5.83; N, 3.86.
Found: C, 66.02; H, 5.89; N, 3.79%. ESI-MS in MeOH: m/z 386
[M+Na+]. Selected IR data (cmꢂ1): 1583vs (COOasym), 1495m,
1405vs (COO)sym, 1106w, 1028w, 998m, 856s, 746w, 699m,
551w, 478s, 444w, 415m. 1H NMR (D2O, ppm): d HPh (7.17, m,
DMF–Tris–HCl buffer (1:4 v/v) [kmax/nm (e
/Mꢂ1 cmꢂ1)]: 610 (85),
5H), Hcpo (4.10, s, 2H), Hcpm (4.05, s, 2H) Hcp’ (4.02, s, 5H), Ha
272 (24475), 295sh (9000). leff = 1.77 lB at 298 K in DMSO-d6 con-
2
2
(3.32, d, 1H, JHH = 12.8 Hz), Ha’ (3.17, d, 1H, JHH = 12.4 Hz), Hb
(3.24, m, 1H), Hc (2.77, m, 2H) (vide Supplementary data for hydro-
gen atom labelling).
taining 1% TMS (v/v). KM = 66 S m2 Mꢂ1 in DMF at 25 °C.
Anal. Calc. for C34H26N5O7ClCu (7): C, 57.07; H, 3.66; N, 9.79.
Found: C, 56.91; H, 3.68; N, 10.06%. Selected IR data (KBr phase,
cmꢂ1): 3225br, 3085w, 2997w, 2357w, 1643vs (COOasym),
1497s, 1420m, 1357vs (COOsym), 1232w, 1093–1055vs (ClO4ꢂ),
923w, 817m, 762s, 728s, 620s, 577w, 425s. ESI-MS in MeOH: m/z
615 [Mꢂ(ClO4ꢂ)]+. UV–Vis in DMF–Tris–HCl buffer (1:4 v/v)
2.2.3. Preparation of [Cu(Fc-Phe)(phen)](ClO4) (5)
Complex 5 was prepared by following the same procedure as
described for complexes 1–4. Yield: 0.56 g (ꢀ79%).
[kmax/nm (e
/Mꢂ1 cmꢂ1)]: 580 (124), 377 (12425), 360 (12725),
Anal. Calc. for C32H18N3O6ClFeCu (5): C, 54.48; H, 4.00; N, 5.96.
Found: C, 54.19; H, 3.81; N, 6.03%. Selected IR data (cmꢂ1): 3245br,
3080w, 2937w, 2320w, 2038w, 1980w, 1630vs (COOasym), 1521s,
1495w, 1430s, 1368s (COOsym), 1236w, 1205w, 1097vs (ClO4ꢂ),
1001m, 917m, 845s, 746m, 719s, 653w, 621s, 563m, 481s. ESI-
MS in MeOH: m/z 605 [Mꢂ(ClO4ꢂ)]+. UV–Vis in DMF–Tris–HCl buf-
275 (52715). leff = 1.80 lB at 298 K in DMSO-d6 containing 1%
TMS (v/v). KM = 68 S m2 Mꢂ1 in DMF at 25 °C.
2.2.6. Preparation of [Cu(Ph-Phe)(phen)(ClO4)] (8)
Complex 8 was prepared in good yield by the same procedure as
described for 6 and 7. Yield: 0.45 g (ꢀ75%).
fer (1:4 v/v) [kmax/nm (
(24360).
(v/v). KM = 82 S m2 Mꢂ1 in DMF at 25 °C.
e
/Mꢂ1 cmꢂ1)]: 605 (125), 440 (315), 273
Anal. Calc. for C28H24N3O6ClCu (8): C, 56.28; H, 4.05; N, 7.03.
Found: C, 55.99; H, 4.01; N, 7.22%. Selected IR data (cmꢂ1):
3070br, 2933w, 2357w, 2179w, 1978w, 1610vs (COOasym),
leff = 1.79 lB at 298 K in DMSO-d6 containing 1% TMS

1290
T.K. Goswami et al. / Polyhedron 52 (2013) 1287–1298
1521s, 1495m, 1440s, 1382s, 1384s (COOsym), 1219w, 1093–
1051vs (ClO4ꢂ), 967w, 926 m, 849s, 754s, 718s, 699s, 617s,
553m, 494m, 427m. ESI-MS in MeOH: m/z 497 [Mꢂ(ClO4ꢂ)]+.
molecules were obtained by ORTEP [64,65]. Selected crystallo-
graphic data are given in Table 1.
UV–Vis in DMF–Tris–HCl buffer (1:4 v/v) [kmax/nm (e
/Mꢂ1 cmꢂ1)]:
2.5. DNA binding and cleavage experiments
615 (130), 275 (41875). leff = 1.80 lB at 298 K in DMSO-d6 contain-
ing 1% TMS (v/v). KM = 65 S m2 Mꢂ1 in DMF at 25 °C.
The DNA binding and cleavage experiments were carried out
using calf thymus DNA and supercoiled (SC) pUC19 DNA by follow-
ing reported standard procedures [66]. For UV–Vis absorption
titration, Tris–HCl buffer of pH 7.2 was used and the concentration
2.3. Solubility and stability
The complexes were soluble in MeOH, DMF, DMSO, MeCN and
in the aqueous mixtures of these solvents; less soluble in CHCl3
and CH2Cl2, and insoluble in hydrocarbon solvents. They were sta-
ble in both solid and solution phases.
of ct-DNA was 235
were carried out in phosphate buffer (pH 6.8) using ct-DNA of
200 M and complexes of 20 M by varying the temperature from
lM. DNA thermal denaturation experiments
l
l
40 to 90 °C. The ratio of the complex and DNA concentration was
1:10. The DNA viscometric titrations were done in Tris–HCl buffer
(pH 7.2) using ct-DNA of 160 lM. The SC pUC19 DNA cleavage
2.4. X-ray crystallographic procedures
activity of the complexes 1–6 was studied in visible light of 454,
568 and 647 nm wavelengths using a Spectra Physics Water-
Cooled Mixed-Gas Ion Laser StabiliteÒ 2018-RM (continuous-wave
(CW) beam diameter at 1/e2 = 1.8 mm 10% and beam divergence
with full angle = 0.7 mrad 10%) and in the dark using external
additives like glutathione (GSH) and hydrogen peroxide. Various
singlet oxygen quenchers and radical scavengers were used for
chemical nuclease and DNA photocleavage mechanistic studies to
detect the formation of any reactive oxygen species (ROS).
The crystal structures of [Cu(Fc-Phe)(phen)](PF6) (5a) and
[Cu(Ph-Tyr)(phen)(ClO4)] (6) were obtained by single crystal X-
ray diffraction method. Crystals of 5a were obtained by slow evap-
oration of an aqueous methanol solution of 5 in the presence of
NH4PF6. Crystals of 6 were obtained from the methanol solution
of the complex on slow evaporation of the solvent. Crystal mount-
ing was done on glass fibres with epoxy cement. All geometric and
intensity data were collected at room temperature using an auto-
mated Bruker SMART APEX CCD diffractometer equipped with a
fine focus 1.75 kW sealed tube Mo
(k = 0.71073 Å) with increasing (width of 0.3° per frame) at a
scan speed of 5 s per frame. Intensity data, collected using an
2h scan mode, were corrected for Lorentz-polarization effects and
for absorption [62]. The structure solution was done by the combi-
nation of Patterson and Fourier techniques and refined by full-ma-
trix least-squares method using SHELX system of programs [63]. All
hydrogen atoms belonging to the complex were in their calculated
positions and refined using a riding model. All non-hydrogen
atoms were refined anisotropically. The perspective views of the
K
a
X-ray source
2.6. Cell culture
x
x
-
HeLa (human cervical carcinoma) and MCF-7 (human breast
adenocarcinoma) cells were maintained in Dulbecco’s Modified Ea-
gle’s Medium (DMEM), supplemented with 10% foetal bovine ser-
um (FBS), 100 IU mlꢂ1 of penicillin, 100 g mlꢂ1 of streptomycin
l
and 2 mM Glutamax at 37 °C in a humidified incubator at 5%
CO2. The adherent cultures were grown as monolayer and were
passaged once in 4–5 days by trypsinizing with 0.25% Trypsin–
EDTA.
Table 1
2.7. Cell viability assay
Selected crystallographic data for the complexes [Cu(Fc-Phe)(phen)](PF6) (5a) and
[Cu(Ph-Tyr)(phen)(ClO4)] (6).
HeLa and MCF-7 cells after treatment were subjected to MTT as-
say. The photocytotoxicity of the complexes was based on the abil-
ity of mitochondrial dehydrogenases in the viable cells to cleave
the tetrazolium rings of MTT to form dark blue membrane imper-
meable crystals of formazan that were measured at 540 nm giving
an estimate of the number of viable cells [67]. Approximately,
15 ꢃ 103 cells of HeLa or 2 ꢃ 104 cells of MCF-7 were plated in a
96-well culture plate in DMEM supplemented with 10% foetal bo-
vine serum (10% DMEM) and cultured overnight. Different concen-
trations of the complexes were added to the cells, and incubation
was continued for 4 h in the dark. After incubation, the medium
was replaced with 50 mM phosphate buffer, pH 7.4 containing
150 mM NaCl (PBS) and photo-irradiation was done for 1 h in vis-
ible light of 400–700 nm using Luzchem Photoreactor (Model LZC-
[Cu(Fc-
Phe)(phen)](PF6)
[Cu(Ph-
Tyr)(phen)(ClO4)]
Empirical formula
Formula weight (g Mꢂ1
Crystal system
Space group
Unit cell dimensions
a (Å)
C
32H28CuF6FeN3O2P
C30H24ClCuN3O7
613.49
Triclinic
P1
)
750.93
Monoclinic
C2
20.5815(13)
11.0487(5)
15.7115(9)
90.00
121.622(5)
90.00
3042.3(3)
4
293(2)
1.639
0.71073
1.301
5298/ 1/ 415
1524
7.9670(5)
9.0097(6)
9.5968(6)
82.528(2)
77.484(2)
78.595(3)
656.47(7)
1
296(2)
1.552
0.71073
0.987
4085/ 3/ 363
315
1.026
0.0317 [0.0858]
0.0331 [0.0867]
0.591, ꢂ0.329
b (Å)
c (Å)
a
(°)
b (°)
c
(°)
V (Å3)
Z
T (K)
1, Ontario, Canada; light fluence rate: 2.4 mW cmꢂ2
; light
qcalc (g cmꢂ3
)
dose = 10 J cmꢂ2). PBS was replaced with 10% DMEM after irradia-
tion. Incubation was continued for a further period of 20 h in dark
k (Å) (Mo K
a
)
l
(cmꢂ1
)
l
L of 4 mg mlꢂ1 of MTT to each well and
Data/restraints/parameters
followed by addition of 25
incubated for an additional 3 h. The culture medium was dis-
carded, and a 200 L volume of DMSO was added to dissolve the
F(000)
Goodness-of-fit (GOF) on F2
0.970
R (Fo)a, I > 2
r
(I) [wR (Fo)b]
0.0404 [0.0980]
0.0531 [0.1031]
0.406, ꢂ0.355
l
formazan crystals. The absorbance at 540 nm was determined
using an ELISA microplate reader (BioRad, Hercules, CA, USA).
The cytotoxicity of the test compounds was measured as the per-
centage ratio of the absorbance of the treated cells over the un-
treated controls. The IC50 values were determined by nonlinear
regression analysis (GraphPad Prism).
R (all data) [wR (all data)]
Largest diff. peak and hole
(e Åꢂ3
)
P
P
a
R = ||Fo| ꢂ |Fc||/ |Fo|.
P
P
b
2
½
wR = { [w(Fo ꢂ Fc2)2]/
[w(Fo)2]}
;
w = [r
2(Fo)2 + (AP)2 + BP]ꢂ1
,
where
P = (Fo2 + 2Fc2)/3, A = 0.0519; B = 0.0000 for 5a and A = 0.0626; B = 0.0489 for 6.

T.K. Goswami et al. / Polyhedron 52 (2013) 1287–1298
1291
2.8. Nuclear staining experiment
by various spectroscopic and analytical methods. Selected physico-
chemical data are given in Table 2. The complexes are stable in a
solution phase as evidenced from their ESI-MS spectra showing
essentially the molecular ion peak as [Mꢂ(ClO4)]+ in methanol.
Complexes 6–8 show mass spectral peaks that correspond to the
species showing loss of perchlorate ligand in methanol, while the
complexes have a metal-bound perchlorate ligand in the solid
state. The IR spectra of the complexes display characteristic
stretching bands at ꢀ1630 cmꢂ1 and ꢀ1360 cmꢂ1 due to COO of
the amino acid. The complexes also show characteristic stretching
bands for ClO4ꢂ at ꢀ1080 cmꢂ1. There is, however, broadening and
splitting of the ClO4ꢂ band observed in the IR specꢂtra of complexes
6–8 indicating the presence of metal bound ClO4 ligand [70,71].
The IR spectral data reveal the structural differences of the two
types of complexes having or not having the ferrocenyl moiety.
The complexes 1–5 are 1:1 electrolytic in nature with molar con-
ductance value of ꢀ80 S m2 Mꢂ1 in DMF at 25 °C [72]. Complexeꢂs
6–8 are also 1:1 electrolytic with the loss of the coordinated ClO4
in the solution state giving molar conductance value of
ꢀ65 S m2 Mꢂ1 in DMF at 25 °C. Magnetic moment values of
ꢀ1.8 lB at 25 °C suggest the presence of a one-electron paramag-
netic 3d9-Cu(II) center in all the complexes. The UV–Vis spectra
of 1–8, recorded in DMF–Tris–HCl buffer (1:4 v/v), display a broad
and weak copper-centered d–d band in the range of 580–615 nm
(Fig. 2). A moderately intense ferrocene-centered band is observed
near 440 nm in 1–5 [73]. The ligand-centered electronic transitions
are observed in the UV region. Complex 4 exhibits an emission
spectral band at 418 nm on excitation at 300 nm in DMF at 25 °C
with a quantum yield (/) value of 0.11 (Fig. 2). The emissive prop-
erty of this complex is used for cellular imaging. Complexes 1–5
show quasi-reversible cyclic voltammetric response at ꢀ0.5 V vs.
SCE in DMF-0.1 M TBAP due to the Fc+–Fc couple (Table 2). There
is a significant positive shift of ꢀ100 mV of the Fe(III)–Fe(II) poten-
tial in 1–5 compared to that of ferrocene (0.43 V). The complexes
also show a quasi-reversible cyclic voltammetric response near
ꢂ0.1 V due to the Cu(II)-Cu(I) couple. Complexes 6–8 show the
Cu(II)–Cu(I) quasi-reversible response near ꢂ0.12 V vs. SCE. The li-
gand reduction peaks appear near ꢂ1.1 and ꢂ1.7 V. The minor dif-
ference in the Cu(II)/Cu(I) redox values observed between two
different types of complexes is probably due their structural simi-
larities in the solution phase.
HeLa cells cultured on cover slips were photo-irradiated with
visible light of 400–700 nm (light fluence rate = 2.4 mW cmꢂ2
;
light dose = 10 J cmꢂ2) following 4 h of incubation in the dark in
the presence of 5 M of complex 3. The cells were then allowed
to recover for 2 h, washed three times with PBS and stained with
acridine orange/ethidium bromide (AO/EB) mixture (1:1, 10 M)
l
l
for 15 min and observed at 20ꢃ magnification with a fluorescence
microscope (Carl Zeiss). The images were analyzed using the ‘‘Im-
age J’’ image browser [68].
2.9. Fluorescence microscopy
HeLa cells (4 ꢃ 104 cells/mm2), plated on cover slips, were incu-
bated with 5 lM of the nip complex 4 for different time intervals
from 1 to 4 h in dark, fixed with 4% paraformaldehyde for 10 min
at room temperature and washed with PBS. This was followed by
incubation with PI staining solution (50 lg/ml RNase A, 20 lg/ml
PI in PBS) for 1 h at 42 °C. The cells were washed free of excess
PI and mounted in 90% glycerol solution containing Mowiol, an
anti-fade reagent, and sealed. Images were acquired using Apo-
tome.2 fluorescence microscope (Carl Zeiss, Germany) using an
oil immersion lens at 63ꢃ magnification. The images were ana-
lyzed using the AxioVision Rel 4.8.2 (Carl Zeiss, Germany) software
[69].
3. Results and discussion
3.1. Synthesis and general aspects
Copper(II) complexes [Cu(Fc-Tyr)(L)](ClO4) (1–4) having ferro-
cene-conjugated L-tyrosine reduced Schiff base and N,N-donor het-
erocyclic bases (L: phen, 1; dpq, 2; dppz, 3; nip, 4) were prepared in
good yield from the reaction of ferrocenylmethyltyrosine (Fc-TyrH)
with copper(II) acetate monohydrate and the respective phenan-
throline base in methanol. Complex of ferrocenylmethylphenylala-
nine and 1,10-phenanthroline, viz., [Cu(Fc-Phe)(phen)](ClO4) (5)
was prepared by reacting the ligands with copper(II) acetate
monohydrate and structurally characterized by X-ray crystallogra-
phy. To explore the effect of the ferrocenyl moiety on the overall
DNA cleavage activity and photocytotoxicity of 1–5, three control
complexes, viz., [Cu(Ph-Tyr)(L)(ClO4)] with L as phen (in 6) and
dppz (in 7), where Ph-TyrH is the reduced Schiff base derived from
3.2. Crystal structure
benzaldehyde and
where Ph-Phe is the reduced Schiff base of benzaldehyde and
phenylalanine were prepared. The complexes were characterized
L
-tyrosine and [Cu(Ph-phe)(phen)(ClO4)] (8),
Complex 5 as its hexfluorophosphate salt (5a) and complex 6
were structurally characterized by single crystal X-ray diffraction
method. Complex 5a crystallized in the C2 space group of the
L
-
Table 2
Selected physicochemical data for the complexes [Cu(Fc-Tyr)(L)](ClO4) (L = phen, 1; dpq, 2; dppz, 3; nip, 4), [Cu(Fc-Phe)(phen)](ClO4) (5), [Cu(Ph-Tyr)(L)(ClO4)] (L = phen, 6; dppz,
7) and [Cu(Ph-Phe)(phen)(ClO4)] (8).
a
Complex
IR/cmꢂ1
[m
(ClO4ꢂ)]
kmax/nm (
e
/Mꢂ1 cmꢂ1
)
Ef/V (D
Ep/mV)b
KMc/S m2 Mꢂ1
leff /
d lB
Fc+–Fc
Cu(II)-Cu(I)
1
2
3
4
5
6
7
8
1096
1086
1075
1089
1097
1071
1074
1072
440 (297), 590 (119)
452 (298), 595 (123)
440 (473), 585 (129)
455 (503), 615 (125)
440 (315), 605 (125)
610 (85)
0.49 (98)
0.49 (95)
0.50(80)
0.50 (110)
0.49 (110)
–
ꢂ0.13 (410)
ꢂ0.12 (470)
ꢂ0.11 (490)
ꢂ0.16 (400)
ꢂ0.15 (300)
ꢂ0.14 (380)
ꢂ0.12 (457)
ꢂ0.13 (270)
79
82
84
80
82
66
68
65
1.80
1.81
1.78
1.79
1.79
1.77
1.80
1.80
580 (124)
615 (130)
–
–
a
In DMF–Tris–HCl buffer (1:4 v/v). The bands at ꢀ450 and 600 nm are ferrocene-based and Cu(II)-based, respectively.
b
Fc+–Fc and Cu(II)–Cu(I) couple in DMF-0.1 M TBAP, Ef = 0.5(Epa + Epc),
D
Ep = (Epa ꢂ Epc), where Epa and Epc are the anodic and cathodic peak potentials, respectively. The
potentials are vs. SCE. Scan rate = 50 mV sꢂ1
.
c
Molar conductivity in DMF.
Magnetic moment at 298 K using DMSO-d6 solution of the complexes.
d

1292
T.K. Goswami et al. / Polyhedron 52 (2013) 1287–1298
Fig. 2. The electronic spectra of [Cu(Fc-Tyr)(L)](ClO4) [L = phen in 1 (—); dppz in 3
(– ꢅ –)] and [Cu(Ph-Tyr)(L)(ClO4)] [L = phen in 6 (ꢅ ꢅ ꢅ); dppz in 7 (- - -)] in DMF–Tris–
HCl buffer (1:4 v/v). The arrows indicate the wavelengths of light used for the
photo-induced DNA cleavage studies. The inset shows the emission spectrum of
[Cu(Fc-Tyr)(nip)](ClO4) (4, ꢅ ꢅ ꢅ) in DMF.
Fig. 4. ORTEP view of [Cu(Ph-Tyr)(phen)(ClO4)] (6) showing 50% probability
thermal ellipsoids and the atom numbering scheme for the metal and hetero atoms.
Table 3
Selected bond distances (Å) and bond angles (°) of [Cu(Fc-Phe)(phen)](PF6) (5a) and
[Cu(Ph-Tyr)(phen)(ClO4)] (6).
monoclinic crystal system with four molecules in the unit cell.
Complex 6 crystallized in the P1 space group of the triclinic crystal
system with one molecule in the unit cell. The ORTEP views of the
complexes are shown in Figs. 3 and 4. Selected bond distances and
angles data are given in Table 3. Complex 5a is a heterobimetallic
species having the Cu(II) and Fe(II) centers. The X-ray structure
shows a square-planar geometry of Cu(II) in a CuN3O coordination
[Cu(Fc-Phe)(phen)](PF6) (5a)
[Cu(Ph-Tyr)(phen)(ClO4)] (6)
Cu1–O1
Cu1–N1
Cu1–N2
Cu1–N3
O1–Cu1–N1
O1–Cu1–N2
N1–Cu1–N2
O1–Cu1–N3
N1–Cu1–N3
N2–Cu1–N3
1.923(3)
1.987(4)
2.007(4)
2.009(4)
92.82(16)
170.94(17)
81.93(15)
85.71(12)
175.23(17)
100.10(17)
1.648a
1.958(3)
1.968(3)
1.986(3)
2.022(3)
176.08(15)
93.11(13)
83.10(14)
83.25(12)
100.28(12)
168.56(12)
–
environment (s = 0.071) [74]. This geometry is favored due to the
steric requirements of the reduced Schiff base ligand where both
sides of the square plane are blocked by the phenyl and ferrocenyl
groups. Both the Fc-Phe and phenanthroline ligands coordinate in a
bidentate fashion. The Cu–N and Cu–O bond distances are 2.009(4)
and 1.923(3) Å, respectively, for the Fc-Phe ligand. The Cu–N bond
distances are 1.987(4) and 2.007(4) Å for the heterocyclic base. The
cyclopentadienyl (Cp) rings of the ferrocenyl moiety are in a nearly
1
Fe(1)–C0
Fe(1)–C0
1.655a
–
2
a
1
2
C0 and C0 are two centroids of the Cp rings in the ferrocenyl moiety com-
prising of atoms C(23) to C(27) and C(28) to C(32), respectively.
eclipsed conformation with a dihedral angle between the
g
5-C5H5
and
g
5-C5H4 rings of 2.38°. The average Fe–C distance is 2.038 Å.
respectively. The Ph-Tyr ligand shows bidentate N,O-coordination
to the Cu(II) centre. The phenanthroline base coordinates in a
bidentate fashion with two Cu-N bond distances within 1.968(3)
The chiral carbon of L-phenylalanine in Fc-Phe ligand retains its
‘‘S’’-configuration.
The structure of 6 consists of a discrete complex having Cu(II) as
to 2.022(3) Å. The chiral carbon of L-tyrosine has an ‘‘S’’-configura-
the metal center. The metal exhibits an axially elongated distorted
square-pyramidal CuN3O2 geometry (s = 0.125) with the perchlo-
rate ion occupying the elongated fifth coordination site. The equa-
tion. The IR and X-ray crystal structural data show major structural
differences for the ferrocenyl-conjugates from their non-ferrocenyl
analogues in the solid state.
torial and axial Cu–O bond distances are 1.958(3) and 2.555(4) Å,
3.3. DNA binding properties
The affinity of the complexes to bind to calf-thymus DNA was
studied using spectral, DNA melting and viscometric methods. Se-
lected DNA binding data are presented in Table 4. UV–Vis absorp-
tion 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-centered band of the com-
plexes 1–8 at ꢀ270 nm. Significant hypochromicity with minor
bathochromic shift suggests primarily groove binding nature of
the complexes to ct-DNA in Tris–HCl buffer medium. Classical
DNA intercalators that
p-stack between two DNA base pairs are
known to cause much larger hypochromicity with bathochromic
shift of the spectral bands [75]. The Kb values of the complexes
range within (5.0 0.2) ꢃ 104 to (4.2 0.4) ꢃ 105 Mꢂ1 giving the
DNA binding affinity order as: 3 ꢄ 7 > 4 ꢄ 2 > 1 ꢄ 5 > 6 ꢄ 8
[76,77]. Complex 3 having an extended aromatic phenazine ring
shows highest ct-DNA binding strength compared to its dpq and
Fig. 3. ORTEP view of the cationic complex in [Cu(Fc-Phe)(phen)](PF6) (5a) showing
50% probability thermal ellipsoids and the atom numbering scheme for the metal
and hetero atoms.

T.K. Goswami et al. / Polyhedron 52 (2013) 1287–1298
1293
phen analogues. The phenanthroline complexes 1, 5 and 8 showed
similar DNA binding propensities.
not show any significant chemical nuclease activity in the concen-
tration range used for our experiments [80]. The chemical nuclease
activity follows the order: 3 (Fc-Tyr-dppz) ꢄ 7 (Ph-Tyr-dppz) > 2
(Fc-Tyr-dpq) ꢄ 4 (Fc-Tyr-nip) > 1 (Fc-Tyr-phen) ꢄ 5 (Fc-Phe-
phen) > 6 (Ph-Tyr-phen) ꢄ 8 (Ph-Phe-phen). The nuclease activity
is found to generally correlate with the duplex DNA binding order
of the complexes.
The mechanistic aspects of the chemical nuclease activity was
evaluated using additives, viz., hydroxyl radical scavengers (cata-
lase, DMSO, KI) and singlet oxygen quenchers (NaN3 and TEMP).
Addition of hydroxyl radical scavengers is found to inhibit the
DNA cleavage activity of the complexes, while singlet oxygen
quenchers show no apparent effect in the presence of GSH or
H2O2. The results indicate the involvement of hydroxyl radicals
as the DNA cleaving agents. DNA major groove binder methyl
green and DNA minor groove binder distamycin-A were used to
study the groove binding preferences of the complexes. The chem-
ical nuclease activity of 1 and 2 gets significantly inhibited in the
The DNA melting temperature which gives a measure of the du-
plex stability can be used to study the binding interactions of the
complexes to DNA. The DNA duplex on melting unwinds to give
single stranded DNA resulting in an increase in the absorbance at
260 nm as the bases separate out from each other. An increase in
the DNA melting temperature indicates the stability of double he-
lix. A DNA intercalator like ethidium bromide (EB) generally in-
creases the stability of the double helix rendering thermal
denaturation more difficult [78]. A significant stabilization of the
ct-DNA was observed on treatment with complex 3 compared to
the untreated sample giving a
D
Tm value of 4.5 °C. The
DTm values
range within 1.0–4.5 °C for 1–8 with EB giving a
D
Tm value of
11.2 °C indicating primarily groove binding nature of the com-
plexes with partial intercalation for complex 3 (Fig. 5a).
Viscometric titration experiments were performed to investi-
gate the change in the relative specific viscosity of the ct-DNA on
interacting with the complexes. It is known that an increase in
the contour length of DNA results in an increase in its relative vis-
cosity. Classical intercalator like EB is known to show significant
increase in the relative viscosity of the DNA solution [79]. In con-
trast, groove binding or partially intercalating molecules cause lit-
tle or no effect on the relative viscosity of the DNA solution.
Hoechst dye, which is a DNA groove binder, shows only minor
change in the viscosity of ct-DNA. The plots of relative viscosity
presence of distamycin (100
in the presence of methyl green (100
l
M), while no apparent effect is there
M) indicating minor groove
l
binding preference of these complexes. Complex 3 showed inhibi-
tion in the presence of methyl green and not with distamycin sug-
gesting its major groove binding preference.
3.4.2. DNA photocleavage activity
The ability of the complexes to cleave DNA when irradiated
with visible laser radiation was studied using supercoiled (SC)
(g/g0) g and g0 are the specific
1/3 vs. [complex]/[DNA] ratio, where
viscosity of DNA in the presence and absence of the complex,
respectively, for 1–5, ethidium bromide and Hoechst dye indicate
primarily surface aggregation and/or groove binding nature of
the complexes (Fig. 5b). Complex 3 with an extended phenazine
ring in the dppz ligand shows significant increase in the ct-DNA
viscosity.
pUC19 DNA (30 lM, 0.2 lg) in Tris–HCl/NaCl (50 mM, pH, 7.2) buf-
fer (Figs. 7 and 8). Monochromatic visible light of wavelengths 454,
568 and 647 nm of 50 mW laser power were used from a tunable
continuous-wave (CW) Ar–Kr mixed-gas ion laser to carry out
the photo-induced DNA cleavage reactions. The wavelengths were
chosen based on the presence of two metal-centered electronic
spectral bands of the complexes near 450 and 600 nm (Fig. 2). Con-
trol experiments using DNA alone showed no apparent photoclea-
3.4. DNA cleavage study
vage of DNA in visible light. The phen complexes 1and 5 (20 lM)
3.4.1. Chemical nuclease activity
showed moderate DNA cleavage activity when irradiated with blue
The chemical nuclease activity of the complexes 1–8 (10
was studied in the presence of external reagents, viz., glutathione
l
M)
light of 454 nm giving ꢀ50% cleavage of SC DNA to its NC form.
Complexes 2–4 (20 lM) nicked >80% of SC DNA in blue light. Con-
(GSH, 1 mM) as a reducing agent and hydrogen peroxide (H2O2,
trol complexes 6–8 having the Ph-Tyr or Ph-Phe ligand showed
much lower DNA photocleavage activity than their ferrocenyl ana-
logues. The cleavage activity using 454 nm laser light follows the
order: 3 [(Fc-Tyr)-Cu-(dppz)] > 4 [(Fc-Tyr)-Cu-(nip)] ꢄ 2 [(Fc-Tyr)-
Cu-(dpq)] > 7 [(Ph-Tyr)-Cu-(dppz)] > 1 [(Fc-Tyr)-Cu-(phen)] ꢄ 5
[(Fc-Phe)-Cu-(phen)] > 6 [(Ph-Tyr)-Cu-(phen)] ꢄ 8 [(Ph-Phe)-Cu-
(phen)] (Fig. 7).
200
30
l
M) as an oxidizing agent using SC pUC19 DNA (0.2
lg,
lM) in 50 mM Tris–HCl/50 mM NaCl buffer of pH 7.2 (Fig. 6).
GSH and H2O2 were chosen because of the presence of Fe(III)–Fe(II)
and Cu(II)–Cu(I) redox couples in the complexes 1–5. Complexes
1–5 showed significant chemical nuclease activity in the presence
of both H2O2 and GSH. Complexes 1 and 3 showed moderately
higher chemical nuclease activity in the presence of the oxidizing
agent compared to their non-ferrocenyl analogues 6 and 7 due to
the involvement of the redox active ferrocenyl moiety. Control
experiments with the ligands, the copper(II) salt, H2O2 or GSH
alone did not show any significant cleavage of SC DNA under sim-
ilar experimental conditions. Although ferrocenylated lysine con-
jugate is reported to cleave DNA in the presence of reducing
agents at high complex concentration, Fc-TyrH or Fc-PheH did
The complexes showed significant photo-induced DNA cleavage
activity in green light of 568 nm and red light of 647 nm. A 25 lM
dppz complex 3 in green light gave ꢀ85% cleavage of SC DNA. The
dpq complex 2 and nip complex 4 cleaved >70% of the SC DNA in
green light. The phen complexes 1 and 5 (25 lM) displayed lower
DNA cleavage activity probably due to photo-inactive nature of
phen. The complexes also displayed significant DNA cleavage activ-
ity in red light of 647 nm. Complexes 2–4 (25 lM) cleaved >70% of
Table 4
DNA binding data for [Cu(Fc-Tyr)(L)](ClO4) (L = phen, 1; dpq, 2; dppz, 3; nip, 4), [Cu(Fc-Phe)(phen)](ClO4) (5), [Cu(Ph-Tyr)(L)(ClO4)] (L = phen, 6; dppz, 7) and [Cu(Ph-
Phe)(phen)(ClO4)] (8).
1
2
3
4
5
6
7
8
Kba/Mꢂ1
(7.6 0.5) ꢃ 104
(2.3 0.5) ꢃ 105
(4.2 0.4) ꢃ 105
(2.1 0.6) ꢃ 105
(7.4 0.3) ꢃ 104
(5.1 0.4) ꢃ 104
(4.0 0.3) ꢃ 105
(5.0 0.2) ꢃ 104
sb
0.09
1.4
0.12
2.4
0.16
4.5
0.11
2.7
0.09
1.3
0.04
1.0
0.12
3.5
0.05
1.1
c
D
Tm /°C
a
b
c
Intrinsic equilibrium DNA binding constant from the UV–Vis experiment.
Binding site size.
Change in the calf thymus DNA melting temperature.

1294
T.K. Goswami et al. / Polyhedron 52 (2013) 1287–1298
Fig. 5. (a) DNA melting temperature plot using ct-DNA (200 lM NP) in the absence and presence of 20 lM ethidium bromide (EB) and complexes 1, 3, 6 and 7 in 5 mM
phosphate buffer (pH 6.8); (b) The effect of increasing concentration of EB (.), Hoechst dye (J), 1 (j), 2 (d), 3 (N), 4 (") and 5 (ꢀ) on the relative viscosity of ct-DNA at
37.0( 0.1) °C in 5 mM Tris–HCl buffer (pH 7.2, [ct-DNA] = 160 M).
l
Fig. 6. Gel electrophoresis diagram showing the chemical nuclease activity of the complexes 1–8 (10
lM) using SC pUC19 DNA (0.2 lg, 30 lM b.p.) in the presence of 1.0 mM
glutathione (GSH) as a reducing (lanes 2–9) and 200 M H2O2 as an oxidizing agent (lanes 10–17) for an incubation time of 2 h: lane-1, DNA control; lane-2, DNA + 1; lane-3,
l
DNA + 2; lane-4, DNA + 3; lane-5, DNA + 4; lane-6, DNA + 5; lane-7, DNA + 6; lane-8, DNA + 7; lane-9, DNA + 8; lane-10, DNA + 1; lane-11, DNA + 2; lane-12, DNA + 3; lane-13,
DNA + 4; lane-14, DNA + 5; lane-15, DNA + 6; lane-16, DNA + 7; lane-17, DNA + 8.
the SC DNA in red light. The role of the ferrocenyl moiety in the
DNA photo-cleavage reactions is evidenced from the greater activ-
ity of the ferrocenyl complexes 1, 3 and 5 than their phenyl ana-
logues 6–8. Complexes 1 and 3 showed remarkable DNA cleavage
activity in green and red light. The cleavage activity in green and
red light follows a similar order as observed for blue light (Table 5).
Control experiments using copper(II) perchlorate and the ligands
alone did not show any significant DNA photocleavage activity un-
der similar reaction conditions. Ferrocene derivatives like 4-ferr-
ocenylbutanoate are known to generate radical species under
high energy flash photolysis conditions due to their photo-oxida-
tion, but the ferrocenylated amino acid ligands used in the present
study did not show any apparent DNA photocleavage activity when
irradiated with low energy light within the PDT spectral window
[81].
Complexes 6–8 lacking the ferrocenyl moiety showed signifi-
cant DNA hydrolytic cleavage activity in dark. Complex 6 showed
ꢀ23% cleavage of SC DNA in dark without any external agents
where as its ferrocenyl analogue showed only ꢀ9% cleavage. The
ꢂ
labile axial ClO4 ligand in 6–8 undergoes dissociation in aqueous
buffer and the resulting species could bind the phosphodiester
bond causing hydrolytic damage of DNA. In contrast, the Fc-Tyr
complexes are structurally rigid due to steric protection of two ax-
ial sites. The complexes did not show any significant DNA photoc-
leavage activity under argon suggesting possible involvement of
the reactive oxygen species (ROS) in the DNA cleavage reactions.
Fig. 7. Bar diagram showing visible light-induced DNA cleavage activity of the
complexes 1–8 using SC pUC19 DNA (0.2
and laser power of 50 mW. [Complex] = 20
wavelengths. Colour code: white, 454 nm light; light gray, 568 nm light; dark gray,
647 nm light; black, sample unexposed to light.
l
g, 30
l
M b.p.) for an exposure time of 2 h
M for other
Fig. 8. Bar diagram showing the mechanistic data for the visible light-induced DNA
lM for 454 nm and 25
l
cleavage activity of [Cu(Fc-Tyr)(dppz)](ClO4) (3) at 454 nm (20
(25 M) using SC pUC19 DNA (0.2 g, 30 M b.p.) for an exposure time of 2 h.
Colour code: light gray, 454 nm; black, 647 nm.
lM) and 647 nm
l
l
l

T.K. Goswami et al. / Polyhedron 52 (2013) 1287–1298
1295
Table 5
Selected DNA (SC pUC19, 0.5 lg) cleavage data for [Cu(Fc-Tyr)(L)](ClO4) (L = phen, 1;
dpq, 2; dppz, 3; nip, 4), [Cu(Fc-Phe)(phen)](ClO4) (5), [Cu(Ph-Tyr)(L)(ClO4)] (L = phen,
6; dppz, 7) and [Cu(Ph-Phe)(phen)(ClO4)] (8) in visible light.
Reaction
conditions
%NC form
(k = 454 nm)
%NC form
(k = 568 nm)
%NC form
(k = 647 nm)
a
DNA control
DNA + 1
DNA + 2
DNA + 3
DNA + 4
DNA + 5
DNA + 6
DNA + 7
DNA + 8
4
2
3
52
82
92
81
48
26
63
23
42
73
85
71
37
28
70
26
45
77
82
73
40
30
65
29
a
In Tris-buffer medium (pH 7.2). k, Laser wavelength. Photo-exposure time
(t) = 2 h. Concentration of the complexes 1–6 was 20 lM for the 454 nm and 25 lM
for the 568 and 647 nm experiments.
Fig. 9. Cell viability plots for the cytotoxic effect of the dppz complexes 3 (j) and 7
(d) in HeLa cells in dark (solid symbols) and in the presence of visible light (hollow
symbols, 400–700 nm, 10 J cmꢂ2).
The ROS could form following either type-I and/or type-II pathway.
The ascertain this, mechanistic aspects of the DNA photocleavage
reactions were studied in visible light of 454 and 647 nm wave-
lengths using the dppz complex 3 and different additives (Fig. 8).
Singlet oxygen quenchers, viz., NaN3, TEMP or L-histidine did not
show any significant inhibition in the DNA cleavage activity thus
eliminating the involvement of a type-II process. The hydroxyl rad-
ical scavengers, viz., DMSO, KI and catalase showed significant
inhibition in the DNA photocleavage suggesting the formation of
hydroxyl radical via a photo-redox pathway [36–40]. Partial inhibi-
tion in the DNA photocleavage activity was observed on addition of
superoxide dismutase (SOD) indicating formation of superoxide
radical anion as an intermediate species [82].
3.5. Cytotoxicity study
Fig. 10. Cell viability plots for the cytotoxic effect of the dppz complexes 3 (j) and
7 (d) in MCF-7 cells in dark (solid symbols) and in the presence of visible light
(hollow symbols, 400–700 nm, 10 J cmꢂ2).
Cellular toxicity of the complexes 1–6 against HeLa (human cer-
vical carcinoma) and MCF-7 (human breast adenocarcinoma) can-
cer cell lines in dark and visible light was evaluated from the MTT
assay (Figs. 9 and 10). A dose dependent anti-proliferative activity
of the complexes was observed against both HeLa and MCF-7 cells
in dark as expected due to reduction of copper(II) to copper(I) by
thiols like GSH and cysteine and subsequent generation of radicals.
Photo-irradiation of the samples with visible light of 400–700 nm
resulted in an enhanced cytotoxicity of the complexes. The ferroce-
nyl complexes were found to be more photo-cytotoxic in both the
cell lines compared to the non-ferrocenyl analogues probably due
to the involvement of the ferrocene moiety. An enhancement in
the cytotoxicity of the complexes in HeLa cells was observed when
exposed to visible light compared to the samples in dark. The dppz
complex 3 was most toxic to HeLa cells with an IC50 value of
to be relatively non-toxic compared to the other complexes and
this could be due to its reduced uptake or quick efflux from the
cancer cells.
3.6. Acridine orange/ethidium bromide dual staining
The mechanistic aspects of the cell death were obtained from
acridine orange/ethidium bromide (AO/EB) dual staining of the
HeLa cells treated with complex 3 (Fig. 11). EB being unable to en-
ter the cells with intact cell membrane, stains only the nucleus of
late apoptotic cells with intense red colour. This gives an idea of
the cell death based on the changes in nuclear morphology upon
PDT [37,84]. The cells treated with complex 3 in dark showed no
apparent change in the nuclear morphology and no apparent EB
0.9 lM when exposed to visible light. The IC50 values of the com-
plexes along with Photofrin and cisplatin are given in Table 6
[37,83]. The observed photocytotoxicity of the complexes is com-
parable to that of Photofrin. Cisplatin lacking any photoactive moi-
ety showed an IC50 value of ꢀ70
l
M in both dark and light under
staining. In contrast, the cells treated with 5 lM of complex 3
similar experimental conditions. Photo exposure of the cells in ab-
sence of the complex showed no apparent reduction in the cell via-
bility. A similar photo-enhanced anti-proliferative behaviour of the
complexes was seen on the breast cancer cell line (MCF-7) on
exposure to visible light. Again, the dppz complex 3 was most cyto-
and photo-irradiated with visible light displayed significant in-
crease in the apoptotic nuclear morphology wherein the nuclei
had condensed significantly with intense EB staining and no AO
staining (Fig. 11, panels (c) and (d)). Cells cultured in the dark
and the cells with DMSO control showed negligible nuclear con-
densation and no EB staining (Fig. 11, panels (a) and (b)). The re-
sults indicate that the photo-irradiated cells are in the late stage
of apoptosis, which enables EB to enter the cells and stain the
nucleus.
toxic towards MCF-7 cells giving an IC50 of 0.76 lM in visible light.
The activities of the complexes are similar in both the cell lines
used. The ligands or the metal salt alone were significantly non-
toxic both in dark and visible light. The dpq complex 2 was found

1296
T.K. Goswami et al. / Polyhedron 52 (2013) 1287–1298
Table 6
A comparison of the IC50 values of [Cu(Fc-Tyr)(L)](ClO4) (L = phen, 1; dpq, 2; dppz, 3; nip, 4), [Cu(Fc-Phe)(phen)](ClO4) (5), [Cu(Ph-Tyr)(L)(ClO4)] (L = phen, 5; dppz, 6) and [Cu(Ph-
Phe)(phen)(ClO4)] (8) with PhotofrinÒ and cisplatin in HeLa and MCF-7 cancer cells.
Compound
HeLa
IC50
MCF-7
IC50
(l
M) darka
IC50
(
l
M) visible lightb
(
l
M) darka
IC50 (l
M) visible lightb
1
2
3
4
5
6
7
8
9.33 ( 0.28)
11.11 ( 0.39)
2.31 ( 0.19)
4.07 ( 0.16)
10.06 ( 0.50)
13.84 ( 0.73)
4.25 ( 0.31)
11.53 ( 0.48)
>41
4.63 ( 0.15)
6.81 ( 0.27)
0.90 ( 0.03)
1.49 ( 0.11)
4.17 ( 0.22)
9.56 ( 0.31)
2.08 ( 0.20)
5.68 ( 0.19)
4.3 ( 0.2)
9.29 ( 0.22)
11.37 ( 0.55)
2.70 ( 0.30)
5.73 ( 0.31)
11.30 ( 0.41)
8.50 ( 0.33)
3.62 ( 0.23)
12.90 ( 0.45)
–
5.02 ( 0.17)
6.23 ( 0.27)
0.76 ( 0.03)
2.53 ( 0.17)
5.47 ( 0.33)
4.91 ( 0.18)
1.62 ( 0.05)
6.94 ( 0.32)
–
PhotofrinÒc
Cisplatind
71.3( 2.9)
68.7( 3.4)
–
–
a
b
c
The IC50 values correspond to 24 h incubation in dark.
The IC50 values correspond to 4 h incubation in dark followed by photo-exposure to visible light (400–700 nm, 10 J cmꢂ2).
The IC50 values (633 nm excitation; fluence rate: 5 J cmꢂ2) of PhotofrinÒ are taken from reference No. 83 (converted to
l
M using the approximate molecular weight of
PhotofrinÒ = 600 g Mꢂ1).
d
The IC50 values are taken from reference No. 37.
3.7. Cellular localization
cating that the complex primarily localizes in the nucleus at that
time (panel (i) in Fig. 12). The cells were stained for the nucleus
with propidium iodide (PI) which stains the nucleic acids and in
the presence of RNase degrades the cellular RNA (panels (b), (f)
and (j) in Fig. 12). The cells are treated with RNase-A which de-
grades the cellular RNA to avoid PI staining of the RNA in the cyto-
plasm. On merging the blue fluorescence of 4 and red fluorescence
of PI, the panels (g) and (k) in Fig. 12 turned pink in colour at the
position of the nucleus indicating nuclear localization of complex
4. The bright field images confirm that the fluorescence images
are from the cellular organelles and are not artifacts (Fig. 12, panels
(d), (h) and (l)). Further, to confirm whether the fluorescence is due
to sticking of the compound on the cell or nuclear membrane, opti-
cal sectioning of the cells was done to analyze the cells in 3D. The
The delivery of a therapeutic agent to the targeted cellular
organelles is the key to the success of such compounds [85–87].
Incorporation of a fluorescent naphthyl moiety in complex 4 was
primarily aimed to study the uptake and localization of the com-
plex in the cellular organelles in HeLa cells from microscopic
images. The cells were treated with complex 4 for different time
intervals and observed by fluorescence microscopy (Fig. 12). It
was observed that at 1.0 h time point the complex is primarily in
the cytoplasm with minor accumulation in the nucleus (panel
(a), Fig. 12). After 2.0 h the complex moves from the cytoplasm
and predominantly localizes in the nucleus of the HeLa cells with
reduced blue fluorescence from the cytoplasm (panel (e) in
Fig. 12). At 4 h the intensity of fluorescence further increases indi-
cells, sectioned along the z-axis, at intervals of 1 lm, clearly
Fig. 11. Acridine orange (white arrow)/ethidium bromide (red arrow) (AO/EB) dual staining of HeLa cells treated with complex 3 (5 lM) to study the nuclear morphology.
Panels (a) and (b) correspond to the cells treated with only DMSO in dark and light, respectively; panel (c) corresponds to the cells treated with complex 3 in dark and panel
(d) corresponds to the cells treated with complex 3 and irradiated with visible light (400–700 nm, 10 J cmꢂ2). The scale bar in the panels corresponds to 20
lM. (Colour
online.)

T.K. Goswami et al. / Polyhedron 52 (2013) 1287–1298
1297
Fig. 12. A time-course collection of fluorescence microscopic images of HeLa cells treated with complex 4 (5
lM) and propidium iodide (PI). Panels (a), (e) and (i) correspond
to the blue emission of complex 4 and the respective images were taken after 1, 2 and 4 h. Panels (b), (f) and (j) correspond to the red emission of PI. Panels (c), (g) and (k) are
the merged images of the first two panels. Panels (d), (h) and (l) are the bright field images. The scale bar in panel (a) corresponds to 20 M.
l
showed blue fluorescence in the whole nucleus and not an artifact
due to deposition of the complex on cell or nuclear membrane.
We are thankful to the DST for a CCD difractometer facility, Alexan-
der von Humboldt Foundation, Germany, for donation of an elec-
troanalytical system. ARC thanks DST for J. C. Bose national
fellowship. TKG and SG thank CSIR, New Delhi, for research
fellowships.
4. Conclusions
Ternary copper(II) complexes containing ferrocene-conjugated
L-tyrosine reduced Schiff base and phenanthroline bases were syn-
Appendix A. Supplementary data
thesized, characterized and their photo-induced DNA cleavage
activity, cytotoxicity and cellular localization studied. The com-
plexes are designed to serve the dual purpose as DNA binder and
photosensitizer. The effect of the ferrocenyl moiety is observed
from the cytotoxicity of the ferrocenyl complexes. The ferrocenyl
complexes showed less undesirable hydrolytic cleavage of DNA
than the Ph-Tyr complexes. The complexes exhibit efficient groove
binding propensity to ct-DNA with partial intercalative binding
mode of the dppz complexes. The complexes show moderate
chemical nuclease activity in the presence of both reducing
(GSH) and oxidizing (H2O2) agents involving both the metal cen-
ters. The complexes displaying significant photo-induced DNA
cleavage activity in blue, green and red light are remarkably
photo-cytotoxic towards cervical and breast cancer cells. Signifi-
cant photodynamic effect on nuclear morphology is observed
when HeLa cells are treated with complex 3 and subsequently
photo-irradiated in comparison to the non-irradiated samples.
The fluorescence emission of the nip complex 4 bearing a naphthyl
moiety is successfully used for cellular imaging which shows nu-
clear localization of the complex in the HeLa cells. The results
showing remarkable photocytotoxicity of the ferrocene-conjugates
having copper(II) centre are of significance in the virtually unex-
plored chemistry of bioorganometallics in PDT.
CCDC 878189 and 878190 contains the supplementary crystal-
lographic data for complexes 5a and 6. These data can be obtained
free of charge via http://www.ccdc.cam.ac.uk/conts/retriev-
ing.html, or from the Cambridge Crystallographic Data Centre, 12
Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223 336 033; or
e-mail: deposit@ccdc.cam.ac.uk. Supplementary data associated
with this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.poly.2012.06.018.
References
[1] N.J. Wheate, S. Walker, G.E. Craig, R. Oun, Dalton Trans. 39 (2010) 8113.
[2] H.-K. Liu, P.J. Sadler, Acc. Chem. Res. 44 (2011) 349.
[3] A. Bergamo, G. Sava, Dalton Trans. 40 (2011) 7817.
[4] T.W. Hambley, Science 318 (2007) 1392.
[5] A. Bergamo, C. Gaiddon, J.H.M. Schellens, J.H. Beijnen, G. Sava, J. Inorg.
Biochem. 106 (2012) 90.
[6] L. Kelland, Nat. Rev. Cancer 7 (2007) 573.
[7] D. Wang, S.J. Lippard, Nat. Rev. Drug Disc. 4 (2005) 307.
[8] K.S. Lovejoy, S.J. Lippard, Dalton Trans. (2009) 10651.
[9] L. Ronconi, D. Fregona, Dalton Trans. (2009) 10670.
[10] S. Dhar, N. Kolishetti, S.J. Lippard, O.C. Farokhzad, Proc. Natl. Acad. Sci. U. S. A.
108 (2011) 1850.
[11] S. Dhar, W.L. Daniel, D.A. Giljohann, C.A. Mirkin, S.J. Lippard, J. Am. Chem. Soc.
131 (2009) 14652.
[12] F.S. Mackay, N.J. Farrer, L. Salassa, H.-C. Tai, R.J. Deeth, S.A. Moggach, P.A.
Wood, S. Parsons, P.J. Sadler, Dalton Trans. (2009) 2315.
[13] N.J. Farrer, J.A. Woods, L. Salassa, Y. Zhao, K.S. Robinson, G. Clarkson, F.S.
Mackay, P.J. Sadler, Angew. Chem. Int. Ed. 49 (2010) 8905.
[14] S.J. Berners-Price, Angew. Chem. Int. Ed. 50 (2011) 804.
[15] R. Bonnett, Chemical Aspects of Photodynamic Therapy, Gordon & Breach,
London, U.K., 2000.
Acknowledgements
We thank the Department of Science and Technology (DST),
Government of India, for financial support (SR/S5/MBD-02/2007).

1298
T.K. Goswami et al. / Polyhedron 52 (2013) 1287–1298
[16] M. Ethirajan, Y. Chen, P. Joshi, R.K. Pandey, Chem. Soc. Rev. 40 (2011) 340.
[17] A.M. Bugaj, Photochem. Photobiol. Sci. 10 (2011) 1097.
[18] M. Korbelik, Photochem. Photobiol. Sci. 10 (2011) 664.
[19] N.L. Fry, P.K. Mascharak, Acc. Chem. Res. 44 (2011) 289.
[20] D. Crespy, K. Landfester, U.S. Schubert, A. Schiller, Chem. Commun. 46 (2010)
6651.
[52] P.K. Sasmal, S. Saha, R. Majumdar, R.R. Dighe, A.R. Chakravarty, Inorg. Chem. 49
(2010) 849.
[53] F. Schmitt, P. Govindaswamy, G. Süss-Fink, W.H. Ang, P.J. Dyson, L. Juillerat-
Jeanneret, B. Therrien, J. Med. Chem. 51 (2008) 1811.
[54] F. Schmitt, P. Govindaswamy, O. Zava, G. Süss-Fink, L. Juillerat-Jeanneret, B.
Therrien, J. Biol. Inorg. Chem. 14 (2009) 101.
[21] G.-J. Chen, X. Qiao, J.-L. Tian, J.-Y. Xu, W. Gu, X. Liu, S.-P. Yan, Dalton Trans. 39
(2010) 10637.
[55] D.D. Perrin, W.L.F. Armarego, D.R. Perrin, Purification of Laboratory Chemicals,
Pergamon Press, Oxford, 1980.
[22] M. Ochsner, J. Photochem. Photobiol., B 32 (1996) 3.
[23] S.I. Moriwaki, J. Misawa, Y. Yoshinari, I. Yamada, M. Takigawa, Y. Tokura,
Photodermatol. Photoimmunol. Photomed. 17 (2001) 241.
[24] N.J. Farrer, L. Salassa, P.J. Sadler, Dalton Trans. (2009) 10690.
[25] A.D. Ostrowski, P.C. Ford, Dalton Trans. (2009) 10660.
[26] H.T. Chifotides, K.R. Dunbar, Acc. Chem. Res. 38 (2005) 146.
[27] U. Basu, I. Khan, A. Hussain, P. Kondaiah, A.R. Chakravarty, Angew. Chem. Int.
Ed. 51 (2012) 2658.
[28] U. Schatzschneider, Eur. J. Inorg. Chem. (2010) 1451.
[29] F.S. Mackay, J.A. Woods, P. Heringová, J. Kašpárková, A.M. Pizarro, S.A.
Moggach, S. Parsons, V. Brabec, P.J. Sadler, Proc. Natl. Acad. Sci. U.S.A. 104
(2007) 20743.
[56] J.E. Dickeson, L.A. Summers, Aust. J. Chem. 23 (1970) 1023.
[57] E. Amouyal, A. Homsi, J.-C. Chambron, J.-P. Sauvage, J. Chem. Soc., Dalton Trans.
(1990) 1841.
[58] S.S. Bhat, A.A. Kumbhar, H. Heptullah, A.A. Khan, V.V. Gobre, S.P. Gejji, V.G.
Puranik, Inorg. Chem. 50 (2011) 545.
[59] S.C. Sahoo, M. Ray, Dalton Trans. (2009) 3230.
[60] D.F. Evans, J. Chem. Soc. (1959) 2003.
[61] D.F. Evans, T.A. James, J. Chem. Soc., Dalton Trans. (1979) 723.
[62] N. Walker, D. Stuart, Acta Crystallogr., Sect. A 39 (1983) 158.
[63] G.M. Sheldrick, Acta Cryst. A64 (2008) 112.
[64] C.K. Johnson, ORTEP-III, Report ORNL–5138; Oak Ridge National Laboratory,
Oak Ridge, TN, 1976.
[30] T. Gianferrara, I. Bratsos, E. Alessio, Dalton Trans. (2009) 7588.
[31] A.A. Holder, D.F. Zigler, M.T. Tarrago-Trani, B. Storrie, K.J. Brewer, Inorg. Chem.
46 (2007) 4760.
[65] L.J. Farrugia, J. Appl. Cryst. 30 (1997) 565.
[66] T.K. Goswami, B.V.S.K. Chakravarthi, M. Roy, A.A. Karande, A.R. Chakravarty,
Inorg. Chem. 50 (2011) 8452.
[32] G.S. Smith, B. Therrien, Dalton Trans. 40 (2011) 10793.
[33] R.N. Garner, J.C. Gallucci, K.R. Dunbar, C. Turro, Inorg. Chem. 50 (2011) 9213.
[34] L.E. Joyce, J.D. Aguirre, A.M. Angeles-Boza, A. Chouai, P.K.-L. Fu, K.R. Dunbar, C.
Turro, Inorg. Chem. 49 (2010) 5371.
[35] A.M. Angeles-Boza, P.M. Bradley, P.K.-L. Fu, M. Shatruk, M.G. Hilfiger, K.R.
Dunbar, C. Turro, Inorg. Chem. 44 (2005) 7262.
[67] T. Mosmann, J. Immunol. Methods. 65 (1983) 55.
[68] J.E. Coligan, A.M. Kruisbeck, D.H. Margulies, E.M. Shevach, W. Strober, in: R.
Coico (Ed.), Related Isolation Procedures and Functional Assay, Current
Protocols in Immunology, John Wiley & Sons, Inc., New York, 1995. p 3.17.1.
[69] J.L. McClintock, B.P. Ceresa, Invest. Opthalmol. Vis. Sci. 51 (2010) 3455.
[70] B.J. Hathaway, A.E. Underhill, J. Chem. Soc. (1961) 3091.
[71] A.K. Patra, M. Nethaji, A.R. Chakravarty, Dalton Trans. (2005) 2798.
[72] W.J. Geary, Coord. Chem. Rev. 7 (1971) 81.
[36] B. Maity, M. Roy, B. Banik, R. Majumdar, R.R. Dighe, A.R. Chakravarty,
Organometallics 29 (2010) 3632.
[37] S. Saha, D. Mallick, R. Majumdar, M. Roy, R.R. Dighe, E.D. Jemmis, A.R.
Chakravarty, Inorg. Chem. 50 (2011) 2975.
[38] B. Maity, M. Roy, S. Saha, A.R. Chakravarty, Organometallics 28 (2009) 1495.
[39] B. Maity, S. Gadadhar, T.K. Goswami, A.A. Karande, A.R. Chakravarty, Dalton
Trans. 40 (2011) 11904.
[73] Y.S. Sohn, D.N. Hendrickson, H.B. Gray, J. Am. Chem. Soc. 933 (1971) 3603.
[74] A.W. Addison, T.N. Rao, J.V. Reedijk, G.C. Verschoor, J. Chem. Soc., Dalton Trans.
(1984) 1349.
[75] A.M. Pyle, J.P. Rehmann, R. Meshoyrer, C.V. Kumar, N.J. Turro, J.K. Barton, J. Am.
Chem. Soc. 111 (1989) 3051.
[40] T.K. Goswami, M. Roy, M. Nethaji, A.R. Chakravarty, Organometallics 28 (2009)
1992.
[76] A. Hussain, D. Lahiri, M.S.A. Begum, S. Saha, R. Majumdar, R.R. Dighe, A.R.
Chakravarty, Inorg. Chem. 49 (2010) 4036.
[41] A. Nguyen, S. Top, A. Vessieres, P. Pigeon, M. Huche, E.A. Hillard, G. Aouen, J.
Organomet. Chem 692 (2007) 1219.
[77] T.K. Goswami, S. Gadadhar, M. Roy, M. Nethaji, A.A. Karande, A.R. Chakravarty,
Organometallics 31 (2012) 3010.
[42] E. Hillard, A. Vessiéres, L. Thouin, G. Jaouen, C. Amatore, Angew. Chem. Int. Ed.
45 (2006) 285.
[78] M. Cory, D.D. Mckee, J. Kagan, D.W. Henry, J.A. Miller, J. Am. Chem. Soc. 107
(1985) 2528.
[43] G. Gasser, I. Ott, N. Metzler-Nolte, J. Med. Chem. 54 (2011) 3.
[44] D. Dive, C. Biot, ChemMedChem 3 (2008) 383.
[45] C.G. Hartinger, P.J. Dyson, Chem. Soc. Rev. 38 (2009) 391.
[46] P.J. Dyson, G. Sava, Dalton Trans. (2006) 1929.
[47] S. Chatterjee, S. Kundu, A. Bhattacharyya, C.G. Hartinger, P.J. Dyson, J. Biol.
Inorg. Chem. 13 (2008) 1149.
[48] B. Balaji, B. Banik, P.K. Sasmal, B. Maity, R. Majumdar, R.R. Dighe, A.R.
Chakravarty, Eur. J. Inorg. Chem. (2012) 126.
[49] B. Banik, P.K. Sasmal, S. Roy, R. Majumdar, R.R. Dighe, A.R. Chakravarty, Eur. J.
Inorg. Chem. (2011) 1425.
[50] P.K. Sasmal, S. Saha, R. Majumdar, R.R. Dighe, A.R. Chakravarty, Chem.
Commun. (2009) 1703.
[51] P.K. Sasmal, R. Majumdar, R.R. Dighe, A.R. Chakravarty, Dalton Trans. 39 (2010)
7104.
[79] K.E. Reinert, Biochim. Biophys. Acta 319 (1973) 135.
[80] P.J. Higgins, A.M. Gellett, Bioorg. Med. Chem. Lett. 19 (2009) 1614.
[81] S.R. Logan, Can. J. Chem. 69 (1991) 540.
[82] G. Tabbì, C. Cassino, G. Cavigiolio, D. Colangelo, A. Ghiglia, I. Viano, D. Osella, J.
Med. Chem. 45 (2002) 5787.
[83] E. Delaey, F. Van Larr, D. De Vos, A. Kamuhabwa, P. Jacobs, P. De Witte, J.
Photochem. Photobiol. B 55 (2000) 27.
[84] N.J. Wheate, C.R. Brodie, J.G. Collins, S. Kemp, J.R. Aldrich-Wright, Mini Rev.
Med. Chem. 7 (2007) 627.
[85] T. Storr, K.H. Thompson, C. Orvig, Chem. Soc. Rev. 35 (2006) 534.
[86] K.H. Thompson, C. Orvig, Dalton Trans. (2006) 761.
[87] N. Fozia, A. Wüstholz, R. Kinscherf, N. Metzler-Nolte, Angew. Chem. Int. Ed. 44
(2005) 2429.