Enhancing the photocytotoxic potential of curcumin on terpyridyl lanthanide(iii) complex formation

Article abstract of DOI:10.1039/c2dt32042h

Lanthanide(iii) complexes [Ln(R-tpy)(cur)(NO₃)₂] (Ln = La(iii) in 1, 2; Gd(iii) in 5, 6) and [Ln(R-tpy)(scur)(NO₃) ₂] (Ln = La(iii) in 3, 4; Gd(iii) in 7, 8), where R-tpy is 4′-phenyl-2,2′:6′,2′′-terpyridine (ph-tpy in 1, 3, 5, 7), 4′-(1-pyrenyl)-2,2′:6′,2′′-terpyridine (py-tpy in 2, 4, 6, 8), Hcur is curcumin (in 1, 2, 5, 6) and Hscur is diglucosylcurcumin (in 3, 4, 7, 8), were prepared and their DNA photocleavage activity and photocytotoxicity studied. Complexes [La(ph-tpy)(cur)(NO ₃)₂] (1) and [Gd(ph-tpy)(cur)(NO₃)₂] (5) were structurally characterized. The complexes in aqueous-DMF showed an absorption band near 430 nm and an emission band near 515 nm when excited at 420 nm. The complexes are moderate binders to calf-thymus DNA. They cleave plasmid supercoiled DNA to its nicked circular form in UV-A (365 nm) and visible light (454 nm) via¹O₂ and OH pathways. The complexes are remarkably photocytotoxic in HeLa cells in visible light (λ = 400-700 nm) and are non-toxic in the dark. FACScan analysis of the HeLa cells treated with 2 and 4 showed cell death via an apoptotic pathway. Nuclear localization of 1-4 is evidenced from confocal imaging on HeLa cells. The hydrolytic instability of curcumin gets significantly reduced upon binding to the lanthanide ions while retaining its photocytotoxic potential. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c2dt32042h

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Enhancing the photocytotoxic potential of curcumin on
terpyridyl lanthanide(III) complex formation†
Cite this: Dalton Trans., 2013, 42, 182
Akhtar Hussain,a Kumar Somyajit,b Bhabatosh Banik,a Samya Banerjee,a
Ganesh Nagaraju*b and Akhil R. Chakravarty*a
Lanthanide(III) complexes [Ln(R-tpy)(cur)(NO3)2] (Ln = La(III) in 1, 2; Gd(III) in 5, 6) and [Ln(R-tpy)(scur)-
(NO3)2] (Ln = La(III) in 3, 4; Gd(III) in 7, 8), where R-tpy is 4’-phenyl-2,2’:6’,2’’-terpyridine (ph-tpy in 1, 3, 5,
7), 4’-(1-pyrenyl)-2,2’:6’,2’’-terpyridine (py-tpy in 2, 4, 6, 8), Hcur is curcumin (in 1, 2, 5, 6) and Hscur is
diglucosylcurcumin (in 3, 4, 7, 8), were prepared and their DNA photocleavage activity and photocyto-
toxicity studied. Complexes [La(ph-tpy)(cur)(NO3)2] (1) and [Gd(ph-tpy)(cur)(NO3)2] (5) were structurally
characterized. The complexes in aqueous-DMF showed an absorption band near 430 nm and an emission
band near 515 nm when excited at 420 nm. The complexes are moderate binders to calf-thymus DNA.
They cleave plasmid supercoiled DNA to its nicked circular form in UV-A (365 nm) and visible light
(454 nm) via 1O2 and ˙OH pathways. The complexes are remarkably photocytotoxic in HeLa cells in
visible light (λ = 400–700 nm) and are non-toxic in the dark. FACScan analysis of the HeLa cells treated
with 2 and 4 showed cell death via an apoptotic pathway. Nuclear localization of 1–4 is evidenced from
confocal imaging on HeLa cells. The hydrolytic instability of curcumin gets significantly reduced upon
binding to the lanthanide ions while retaining its photocytotoxic potential.
Received 4th September 2012,
Accepted 15th October 2012
DOI: 10.1039/c2dt32042h
www.rsc.org/dalton
Curcumin, 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-
3,5-dione, has two aryl buten-2-one (feruloyl) chromophores
Introduction
Curcumin (Hcur), a polyphenolic pigment of root turmeric iso- joined by a methylene group. The aryl rings with two ortho-
lated from herb Curcuma longa L., is well known for its medi- methoxy and phenolic hydroxyl groups are symmetrically
cinal values as traditional Indian and Chinese herbal linked in conjugation through a β-diketone moiety (Fig. 1).
medicine.1 The therapeutic activity of curcumin is due to its The presence of this β-diketone moiety in its structure makes
ability to interact with a number of molecular targets in the curcumin susceptible to hydrolysis.6 Complexation of curcu-
body.2 Curcumin is also known to exhibit anticancer activity. min to an oxophilic metal ion via the β-diketone moiety makes
In its native form it has been subjected to extensive studies for curcumin hydrolytically stable.7,8 Curcumin has an electronic
the treatment of a variety of cancers and other diseases.3–5 The absorption band around 420 nm with a shoulder at around
cytotoxicity of curcumin stems from its ability to induce apop-
tosis in cancer cells without causing any major adverse
impacts on the normal cells. Curcumin is reported to interfere
with the activity of the transcription factor NF-κB which is a
protein complex involved in the transcription of DNA.6
The clinical application of curcumin is, however, limited by
its poor bioavailability and pharmacokinetic profiles due to
its structural instability under physiological conditions.6
aDepartment of Inorganic and Physical Chemistry, Indian Institute of Science,
Bangalore-560 012, India. E-mail: arc@ipc.iisc.ernet.in; Tel: +91-80-22932533
bDepartment of Biochemistry, Indian Institute of Science, Bangalore-560 012, India.
E-mail: nganesh@biochem.iisc.ernet.in; Tel: +91-80-22933055
†Electronic supplementary information (ESI) available: Reaction scheme,
ESI-MS, IR, electronic and 1H NMR spectra, packing diagrams, DNA binding,
MTT assay and FACS plots (Fig. S1–S34). CCDC 866033 and 900324 for the com-
plexes 1 and 5. For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/c2dt32042h
Fig. 1 Schematic drawing of the complexes 1–8 and the ligands used.
182 | Dalton Trans., 2013, 42, 182–195
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450 nm which is responsible for its bright yellow colour. It characterized from their physicochemical data and their
shows green emission which is sensitive to the nature of the visible light-induced DNA cleavage and anticancer activity in
solvent.9 While general studies on curcumin and its metal HeLa cells studied. Complexes [La(ph-tpy)(cur)(NO3)2] (1) and
complexes are reported in the literature, the photocytotoxic [Gd(ph-tpy)(cur)(NO3)2] (5) were structurally characterized by
potential of curcumin metal complexes remains virtually un- X-ray crystallography. Significant results of this work include
explored despite their rich photophysical and photochemical remarkable photocytotoxicity in visible light of the curcumin
properties.7,10,11
complexes of La(III) and Gd(III) while being essentially non-
The present work stems from our interest to design and toxic in the dark. Complexes 1–4 in HeLa cells showed nuclear
synthesize ternary lanthanide(III) metal complexes of curcu- localization making the nuclear DNA as the potential target of
min/glycosylated curcumin and polypyridyl bases to study the curcumin complexes. The use of tridentate terpyridine
their visible light-induced plasmid DNA cleavage activity and ligands, as compared to the bidentate phenanthroline bases,
photocytotoxicity in cancer cells. Ln(III) complexes with their leads to higher coordination number of the complexes thus
poor stereochemical preferences and high coordination decreasing the possibility of having any undesirable hydrolytic
numbers could be suitably designed using photoactive organic DNA damage. Lanthanide compounds are well known to
ligands to achieve oxidative DNA cleavage activity and photocy- display significant hydrolytic DNA cleavage activity.19
totoxicity.12,13 In addition, the redox stability of the lantha-
nide(III) complexes could make them suitable for cellular
applications in photodynamic therapy (PDT) in the absence of
Results and discussion
Synthesis and general aspects
any chemical nuclease activity in the presence of cellular
thiols.14–16 Curcumin as a photosensitizer with a visible band
in the 400–500 nm region and as a fluorophore with green
emission property could be suitably utilized for imaging the
cancer cells by fluorescence microscopy to study the cellular
localization of the complexes besides damaging the cancer
cells on photo-irradiation. We have designed the present com-
plexes based on our dual strategy of combining imaging with
therapy. An attempt has been made to increase the aqueous
solubility of the complexes by derivatization of the OH groups
of curcumin by glucose.17 The presence of a photoactive terpyr-
idine base (R-tpy) in the ternary structure is expected to
augment the DNA photocleavage activity and photodynamic
efficacy of the complexes.18
Herein, we report new Ln(III) complexes, viz. [Ln(R-tpy)(cur)-
(NO3)2] (Ln = La(III) in 1, 2; Gd(III) in 5, 6) and [Ln(R-tpy)(scur)-
(NO3)2] (Ln = La(III) in 3, 4; Gd(III) in 7, 8), where R-tpy is 4′-
phenyl-2,2′:6′,2′′-terpyridine (ph-tpy in 1, 3, 5, 7), 4′-(1-pyrenyl)-
2,2′:6′,2′′-terpyridine (py-tpy in 2, 4, 6, 8), cur is the monoanio-
nic curcumin (in 1, 2, 5, 6) and scur is the monoanionic diglu-
cosylcurcumin (in 3, 4, 7, 8) (Fig. 1). The complexes were
The precursor complexes [Ln(ph-tpy)(NO3)3] and [Ln(py-tpy)-
(NO3)3] (Ln = La, Gd) were prepared from the reaction of
respective ph-tpy and py-tpy ligands in dichloromethane with
an ethanol solution of the corresponding metal nitrate. The
ternary complexes 1–8 were prepared by reacting the precursor
complex with Hcur or Hscur in the presence of triethylamine
as a base (Fig. 1, Fig. S1, ESI†). The complexes were character-
ized from the analytical and mass spectral data. Selected phys-
icochemical data are given in Table 1. The curcumin
complexes gave a molar conductivity value of ∼90 S cm2 Mol−1
in aqueous DMF (1 : 10 v/v) suggesting their 1 : 1 electrolytic
behaviour.20 One nitrate ligand seemed to get dissociated from
the complex in the solution phase to form the 1 : 1 electrolytes.
This is further evidenced from the ESI-MS data in aqueous
methanol or aqueous acetonitrile showing the presence of a
prominent [M-(NO3)]+ peak corresponding to the loss of one
nitrate anion (Fig. S2–S9, ESI†). The complexes are thus formu-
lated as [Ln(R-tpy)(cur/scur)(NO3)2] in the solid state and as
[Ln(R-tpy)(cur/scur)(NO3)](NO3) in an aqueous phase (R = ph
Table 1 Selected physicochemical data and DNA binding parameters for the complexes 1–8
IR (cm−1
)
Kb (mol−1)d [s]
ΔTm e (°C)
a
b
c
Complex
ν(CvO)
ν(CvC)
ν(NO3)
μeff
λf (nm) [Φf
]
ΛM
1
2
3
4
5
6
7
8
1598
1590
1590
1595
1595
1595
1592
1592
1490
1492
1495
1502
1494
1495
1495
1501
1395
1398
1398
1402
1400
1397
1398
1400
—
—
—
—
7.94
8.02
7.99
7.98
515 [0.02]
515 [0.04]
500 [0.01]
500 [0.03]
515 [0.01]
515 [0.03]
500 [0.01]
500 [0.03]
98
92
88
91
86
90
95
85
(6.5 0.3) × 104 [0.2]
(2.4 0.4) × 106 [0.5]
(6.7 0.2) × 104 [0.3]
(2.7 0.1) × 106 [0.6]
(6.6 0.2) × 104 [0.2]
(2.3 0.3) × 106 [0.5]
(7.0 0.2) × 104 [0.3]
(2.8 0.2) × 106 [0.6]
2.1
4.6
2.3
4.9
2.2
4.7
2.3
4.9
a μeff in μB using DMSO-d6 solutions of the Gd(III) complexes at 25 °C. b Quantum yield is measured in H2O–DMSO (1 : 20 v/v). The quantum yield
value for the curcumin ligand is 0.04 in H2O–DMSO (1 : 20 v/v). c ΛM, molar conductivity in S cm2 mol−1 in 10% aqueous DMF at 25 °C. d Kb, the
calf thymus DNA binding constant [s, MvH equation fitting parameter]. The Kb value for curcumin alone is 8.6 × 103 M−1 under similar
experimental conditions. e Change in the calf thymus DNA melting temperature.
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Fig. 3 An ORTEP view of the complex in 1·H2O showing 50% probability
thermal ellipsoids and the atom numbering scheme for the metal and hetero-
atoms. The hydrogen atoms are not shown for clarity.
Fig. 2 Absorption spectra of the complexes 7 and 8 in 10% aqueous DMF are
shown by solid curves. Fluorescence spectra in 5% aqueous DMSO are shown by
dotted curves (λex = 420 nm).
or py).21 The IR spectra of the complexes in the solid state
showed three strong bands around 1595, 1490 and 1395 cm−1
−
corresponding to the CvO, CvC (β-diketonate) and NO3
stretching vibrations, respectively, indicating bidentate coordi-
−
nation mode of the β-diketonate (cur or scur) and NO3
ligands (Fig. S10–S17, ESI†).22 The room temperature H-NMR
1
spectra of the diamagnetic La(III) complexes 1–4 in DMSO-d6
showed characteristic spectral features of the metal-bound
polypyridyl ligand and the β-diketonate moiety (Fig. S18–S21,
ESI†). The large J value of ∼15 Hz for the olefinic protons at
1,2 and 5,6 positions of the coordinated cur or scur ligand
indicates the presence of a conjugated CvC bond.17 The room
temperature solution magnetic susceptibility measurements of
Fig. 4 An ORTEP view of the complex in 5·H2O showing 50% probability
thermal ellipsoids and the atom labelling scheme for the metal and hetero-
atoms. The hydrogen atoms are omitted for clarity.
X-ray crystallography
the paramagnetic Gd(III) complexes 5–8 in DMSO-d6 gave mag- Complexes 1 and 5 were structurally characterized by a single
netic moment values of ∼8.0 μB suggesting the presence of crystal X-ray diffraction method. The crystals belonged to the
ˉ
seven unpaired electrons.
triclinic space group P1. The ORTEP views of the complexes
The electronic absorption spectra of the complexes in 10% are shown in Fig. 3 and 4 (unit cell packing diagrams in
aqueous DMF showed a strong ligand-based π → π* transition Fig. S23 and S24, ESI†) and selected bond distances and
centered at ∼434 nm with a shoulder near 450 nm (Fig. 2, angles are given in Tables 2 and 3. The complexes are discrete
Fig. S22, ESI†).9 Other ligand centered bands due to R-tpy mononuclear species with the Ln(III) centre in a nine co-ordi-
ligands were observed in the UV region with the py-tpy com- nate LnO6N3 coordination geometry. The 4′-substituted
plexes showing the lowest energy band centered at 350 nm.18 phenyl-terpyridine (ph-tpy) ligand displays a tridentate mode
The curcumin complexes displayed an emission band of coordination. The monoanionic cur and two nitrate ligands
centered at 515 nm (excitation at 420 nm) in 5% aqueous bind Ln(III) in a bidentate chelating fashion. The pyridine
DMSO giving fluorescence quantum yield (ϕ) values in rings of the ph-tpy ligand lie in a planar conformation and the
the range of 0.01–0.04 (Fig. 2, Table 1, Fig. S22, ESI†). The cur- bidentate nitrate anions are disposed trans to one another.
cumin fluorescence is retained in the complexes and this The La–O and La–N distances in 1·H2O are in the range of
has enabled us to study the cellular localization of 1–4 in 2.400(4) to 2.636(5) Å and 2.631(5) to 2.665(5) Å, respectively
HeLa cancer cells by confocal microscopy. Similarly, the com- (Table 2). The Gd–O and Gd–N distances in 5·H2O are in the
plexes of the glycosylated curcumin (Hscur) ligand showed range of 2.307(4) to 2.520(5) Å and 2.544(5) to 2.575(5) Å,
an emission band centred at 500 nm in 5% aqueous respectively (Table 3). The phenyl ring of the ph-tpy ligand
DMSO (excitation at 420 nm) giving fluorescence quantum makes a dihedral angle of 15.87° and 13.6° with the middle
yield values in the range of 0.01–0.03 (Fig. 2, Fig. S22, ESI,† pyridine ring of the terpyridine moiety due to steric encum-
Table 1). Glycosylation of curcumin has resulted in a blue-shift brance among the aromatic hydrogen atoms in 1 and 5,
of the emission band in comparison to the curcumin respectively. The Ln–O bond distances involving the cur ligand
complexes.
are shorter than those involving the nitrate ligands thus
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Table 2 Selected bond distances (Å) and angles (°) for 1·H2O with estimated
standard deviations (e.s.d.) in the parentheses
La(1)–O(1)
La(1)–O(2)
La(1)–O(3)
La(1)–O(5)
La(1)–O(6)
La(1)–O(8)
La(1)–N(1)
La(1)–N(2)
2.400(4)
2.410(4)
2.636(5)
2.609(5)
2.617(5)
2.615(5)
2.665(5)
2.694(4)
O(5)–La(1)–O(8)
O(6)–La(1)–O(8)
N(1)–La(1)–O(1)
N(1)–La(1)–O(2)
N(1)–La(1)–O(3)
N(1)–La(1)–O(5)
N(1)–La(1)–O(6)
N(1)–La(1)–O(8)
N(2)–La(1)–O(1)
N(2)–La(1)–O(2)
N(2)–La(1)–O(3)
N(2)–La(1)–O(5)
N(2)–La(1)–O(6)
N(2)–La(1)–O(8)
N(3)–La(1)–O(1)
N(3)–La(1)–O(2)
N(3)–La(1)–O(3)
N(3)–La(1)–O(5)
N(3)–La(1)–O(6)
N(3)–La(1)–O(8)
N(1)–La(1)–N(3)
138.82(15)
48.33(14)
155.30(15)
84.19(14)
102.09(15)
68.85(16)
115.88(16)
70.14(16)
141.23(13)
144.85(13)
74.70(14)
88.13(15)
110.19(15)
75.12(14)
82.49(14)
154.10(14)
68.27(16)
115.14(16)
78.82(15)
89.40(16)
121.45(14)
Fig. 5 Absorption spectral traces of (a) curcumin and (b) complex 1 in DMSO–
phosphate buffer (1 : 1 v/v, pH = 7.2, 37 °C). The spectra were recorded at an
interval of 20 min (up to 4 h) after incubation of the sample at 37 °C.
La(1)–N(3)
2.631(5)
O(1)–La(1)–O(2)
O(1)–La(1)–O(3)
O(1)–La(1)–O(5)
O(1)–La(1)–O(6)
O(1)–La(1)–O(8)
O(2)–La(1)–O(3)
O(2)–La(1)–O(5)
O(2)–La(1)–O(6)
O(2)–La(1)–O(8)
O(3)–La(1)–O(5)
O(3)–La(1)–O(6)
O(3)–La(1)–O(8)
O(5)–La(1)–O(6)
72.55(13)
80.12(14)
96.66(16)
71.86(15)
120.01(15)
112.82(16)
75.44(16)
86.78(15)
97.00(15)
48.20(14)
139.07(15)
148.55(15)
161.23(15)
Solubility and stability
All the complexes showed good solubility in DMF and DMSO
and moderate solubility in methanol and acetonitrile. The gly-
cosylated curcumin complexes were soluble in water but
showed poor solubility in chlorinated solvents due to the pres-
ence of the carbohydrate moieties. The complexes were found
to be stable in the monocationic form due to dissociation of
one nitrate anion and the solution stability of the cationic
species was ascertained from the ESI-MS and the molar con-
ductivity data. Curcumin is known to be unstable in an
aqueous buffer and various degradation products of curcumin
are reported in the literature.25,26 To study the aqueous stabi-
lity of curcumin and its lanthanide complexes, time dependent
electronic absorption spectral measurements were carried out
in DMSO–phosphate buffer (1 : 1, pH = 7.2, 37 °C). Curcumin
was found to degrade in the aqueous buffer medium within
4 h, the incubation time used to carry out the MTT (3-(4,5-
dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) exper-
iments, while the lanthanide complexes did not show any
apparent degradation (Fig. 5). This observation indicates that
complexation of curcumin with the Ln(III) ion significantly
increases its hydrolytic stability. Binding of the anticancer
drug curcumin to Ln(III) thus provides a convenient way to
deliver the drug into the target site without its degradation in
the physiological medium and this is likely to enhance the
therapeutic efficacy of the drug.
Table 3 Selected bond distances (Å) and angles (°) for 5·H2O with estimated
standard deviations (e.s.d.) in the parentheses
Gd(1)–O(1)
Gd(1)–O(2)
Gd(1)–O(3)
Gd(1)–O(5)
Gd(1)–O(6)
Gd(1)–O(8)
Gd(1)–N(1)
Gd(1)–N(2)
2.307(4)
2.315(4)
2.520(5)
2.493(5)
2.519(5)
2.491(5)
2.575(5)
2.574(5)
O(5)–Gd(1)–O(8)
O(6)–Gd(1)–O(8)
N(1)–Gd(1)–O(1)
N(1)–Gd(1)–O(2)
N(1)–Gd(1)–O(3)
N(1)–Gd(1)–O(5)
N(1)–Gd(1)–O(6)
N(1)–Gd(1)–O(8)
N(2)–Gd(1)–O(1)
N(2)–Gd(1)–O(2)
N(2)–Gd(1)–O(3)
N(2)–Gd(1)–O(5)
N(2)–Gd(1)–O(6)
N(2)–Gd(1)–O(8)
N(3)–Gd(1)–O(1)
N(3)–Gd(1)–O(2)
N(3)–Gd(1)–O(3)
N(3)–Gd(1)–O(5)
N(3)–Gd(1)–O(6)
N(3)–Gd(1)–O(8)
N(1)–Gd(1)–N(3)
139.39(17)
50.23(17)
152.86(16)
79.44(15)
104.15(17)
69.71(17)
115.86(18)
69.79(18)
140.68(14)
142.60(14)
74.37(16)
90.18(17)
111.97(17)
74.30(16)
80.40(15)
153.39(15)
68.21(17)
117.75(17)
77.80(16)
88.90(17)
126.02(16)
Gd(1)–N(3)
2.544(5)
O(1)–Gd(1)–O(2)
O(1)–Gd(1)–O(3)
O(1)–Gd(1)–O(5)
O(1)–Gd(1)–O(6)
O(1)–Gd(1)–O(8)
O(2)–Gd(1)–O(3)
O(2)–Gd(1)–O(5)
O(2)–Gd(1)–O(6)
O(2)–Gd(1)–O(8)
O(3)–Gd(1)–O(5)
O(3)–Gd(1)–O(6)
O(3)–Gd(1)–O(8)
O(5)–Gd(1)–O(6)
75.59(14)
78.36(15)
93.62(17)
72.05(18)
122.23(17)
116.94(16)
75.51(17)
83.97(16)
94.23(16)
50.12(15)
137.95(16)
147.11(16)
157.49(17)
DNA binding
DNA being one of the most important targets of many anti-
cancer agents, we investigated the DNA-binding strengths of
the lanthanide complexes using spectroscopic and hydrodyn-
amic methods such as absorption titration, thermal DNA
melting and viscosity measurements. The electronic absorp-
tion spectral titrations were carried out to monitor the binding
interactions of the complexes 1–8 with calf thymus (ct) DNA
(Fig. 6, Fig. S27, ESI†). The intrinsic equilibrium DNA binding
constants (Kb) are given in Table 1. The Kb values in the range
of 104–106 M−1 follow the order: 8–6–4–2 > 7–5–3–1. The py-tpy
complexes have significantly higher Kb values than their ph-tpy
analogues because of the presence of a planar pyrenyl moiety
in py-tpy. Metal complexes of py-tpy are known to form
π-assembled metallo-supramolecular arrays indicating the
ability of this ligand to strongly π-stack in between the DNA
making the nitrate ligand labile in a solution phase. The struc-
tural properties observed in the present complexes are similar
to the previously reported structures for analogous lanthanide
complexes.23,24 One of the two OH groups of the cur ligand of
one molecule in the unit cell is hydrogen-bonded to one of the
bound oxygen atoms of one nitrate anion of a neighbouring
molecule in the crystal lattice (Fig. S25 and S26, ESI†). The
other OH group and the nearby methoxy group of the cur
ligand are hydrogen-bonded to a lattice water molecule.
Although several metal complexes of curcumin are reported in
the literature, structurally characterized metal complexes of
the curcumin ligand are virtually unknown in the
literature.10,11
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The ΔTm values of ∼5 °C for the py-tpy complexes suggest
partial intercalative mode of binding to DNA.30 Viscosity
measurements were done to examine the effect of the com-
plexes on the relative specific viscosity of the solution contain-
ing the ct-DNA (Fig. 7(b), Fig. S28, ESI†). Since the relative
specific viscosity (η/η0) of a solution containing DNA gives a
measure of the increase in the contour length of DNA associ-
ated with the separation of base pairs caused by intercalation,
a classical intercalator such as ethidium bromide (EB) results
in a significant increase in the viscosity of the DNA solution (η
and η0 are the respective specific viscosities of a DNA solution
in the presence and absence of the complex). In contrast, a
partial and/or non-intercalative binding could result in less
pronounced effects on the viscosity.31 The groove binder
Hoechst 33258, used as a reference compound, showed no
apparent change in the DNA solution viscosity. The results
indicate a two step binding process in which the Ln(III) com-
plexes possibly first interact with the DNA surface followed by
groove binding (for ph-tpy complexes) and/or partial intercala-
tion (for py-tpy complexes).32
Fig. 6 Absorption spectral traces of complex 2 in 5 mM Tris-HCl buffer (pH 7.2)
on increasing the quantity of calf thymus DNA. The inset shows the least-
squares fit of Δεaf/Δεbf vs. [DNA] for [La(ph-tpy)(cur)(NO3)2] (1, ▲), [La(py-tpy)-
■
(cur)(NO3)2] (2, ), [La(ph-tpy)(scur)(NO3)2] (3, ▼) and [La(py-tpy)(scur)(NO3)2]
●
(4, ).
DNA photocleavage
The photo-induced DNA cleavage activity of the complexes 1–8
was studied using supercoiled (SC) pUC19 DNA (30 μM, 0.2 μg)
in Tris-HCl/NaCl (50 mM, pH, 7.2) buffer by irradiating the
samples with a low power monochromatic UV-A light of
365 nm (6 W) and a visible light of 454 nm. Selected DNA
photocleavage data are given in Table 4. The ph-tpy complexes
of cur and scur ligands showed only moderate DNA cleavage
activity in the UV-A light (lanes 7, 9, 11 and 13 in Fig. 8,
Table 4). In contrast, the py-tpy complexes (1.0 μM) with
greater DNA binding strength and photosensitizing ability
showed efficient photo-induced DNA cleavage activity for an
exposure time of 1 h (lanes 8, 10, 12 and 14 in Fig. 8). Control
experiments performed with only SC DNA or with the metal
salts and R-tpy ligands did not show any apparent
Fig. 7 (a) Derivative plots of dA260/dT vs. T for the thermal denaturation of
180 μM calf thymus DNA alone and on addition of complexes 5–8. (b) Plots of
(η/η0)1/3 vs. [compound]/[DNA] showing the effect of increasing concentration
●
of the complexes [Gd(ph-tpy)(cur)(NO3)2] (5, ◀), [Gd(py-tpy)(cur)(NO3)2] (6, ),
[Gd(ph-tpy)(scur)(NO3)2] (7, ▼), [Gd(py-tpy)(scur)(NO3)2] (8, ▲), ethidium
■
bromide (EB, ) and Hoechst 33 258 (◆) on the relative solution viscosities of
ct-DNA at 37.0 ( 0.1) °C in 5 mM Tris-HCl buffer (pH 7.2) and 180 μM calf
thymus DNA.
Table 4 Selected photo-induced SC pUC19 DNA (0.2 μg, 30 μM) cleavage
dataa in blue light of 454 nm and UV-A light of 365 nm
base pairs.27 The fitting parameter s of the McGhee–von
Hippel (MvH) equation generally gives an estimate of the
number of DNA bases associated with the complex and its
value of <1.0 is typically interpreted as arising from the aggre-
gation of hydrophobic molecules on the surface of DNA.28 The
larger value of s for the py-tpy complexes than the ph-tpy ana-
logues suggests better DNA binding strength of the py-tpy com-
plexes than the ph-tpy analogues.29
Thermal DNA denaturation experiments were performed to
obtain insights into the binding of the complexes 1–8 to
ct-DNA (Fig. 7(a), Fig. S28, ESI,† Table 1). A positive shift in the
DNA melting temperature (ΔTm) was observed upon addition
[Complex]454
%NC454
(%NC365)
d
Conditions
([Complex]365)b/μM
t454 (t365)c/h
DNA control
—
2 (1)
2 (1)
2 (1)
2 (1)
2 (1)
2 (1)
2 (1)
2 (1)
2 (1)
2 (2)
1
2
3
4
5
6
7
8
10 (1)
10 (1)
10 (1)
10 (1)
10 (1)
10 (1)
10 (1)
10 (1)
58 (35)
90 (92)
50 (30)
70 (80)
52 (33)
86 (88)
48 (28)
72 (80)
a SC and NC are the supercoiled and nicked circular forms of pUC19
DNA, respectively. Light sources: UV-A lamp of 365 nm (6 W) and
of the complexes to the DNA. An increase in the DNA melting visible laser light of 454 nm (50 mW power). b Complex concentrations
c
for visible light and UV-A light (in parentheses) experiments. t454 and
temperature suggests an increase in the stability of the double
helix upon addition of the complex. The ΔTm values of ∼2 °C
t365 are the photo-exposure time for 454 nm and 365 nm light
experiments. d %NC454 and %NC365 are the quantity of NC DNA forms
for the ph-tpy complexes indicate their groove binding nature. from the 454 nm and 365 nm experiments, respectively.
186 | Dalton Trans., 2013, 42, 182–195
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Fig. 8 Gel electrophoresis diagram showing the cleavage of SC pUC19 DNA
(0.2 μg, 30 μM) by the complexes 1–8 in 50 mM Tris-HCl/NaCl buffer (pH, 7.2)
containing 5% DMF on irradiation with UV-A light of 365 nm (6 W) for 1 h: lane
1, DNA control; lane 2, DNA + Hcur (1.0 μM, dark); lane 3, DNA + Hscur (1.0 μM,
dark); lane 4, DNA + Hcur (1.0 μM); lane 5, DNA + Hscur (1.0 μM); lane 6, DNA +
4 (1.0 μM, dark); lanes 7–14, DNA + complexes 1–8 (1.0 μM); lane 15, DNA +
methyl green (200 μM); lane 16, DNA + methyl green (200 μM) + 4 [lanes 2, 3
and 6 are for controls in the dark; lanes 1, 4 and 5 are for controls in the light].
Fig. 9 Gel electrophoresis diagram showing the cleavage of SC pUC19 DNA
(0.2 μg, 30 μM) by the complexes 1–8 in 50 mM Tris-HCl/NaCl buffer (pH, 7.2)
containing 5% DMF on irradiation with visible light of 454 nm (60 mW) for 2 h:
lane 1, DNA control; lane 2, DNA + Hcur (10.0 μM, dark); lane 3, DNA + Hscur
(10.0 μM, dark); lane 4, DNA + Hcur (10.0 μM); lane 5, DNA + Hscur (10.0 μM);
lane 6, DNA + 6 (10.0 μM, dark); lanes 7–14, DNA + complexes 1–8 (10.0 μM);
lane 15, DNA + methyl green (200 μM); lane 16, DNA + methyl green (200 μM)
+ 6 [lanes 2, 3 and 6 are for controls in the dark; lanes 1, 4 and 5 are for controls
in the light].
Fig. 10 Bar diagram showing the cleavage of SC pUC19 DNA (0.2 μg, 30 μM)
by [Gd(py-tpy)(cur)(NO3)2] (6, light shade) and [Gd(py-tpy)(scur)(NO3)2] (8, dark
shade) in visible light of 454 nm (60 mW) in the presence of various additives in
Tris-HCl buffer containing 10% DMF. The complex concentration and exposure
time are 1.0 μM and 2 h, respectively. The additive concentrations/quantities
are: sodium azide, 0.5 mM; KI, 0.5 mM; TEMP, 0.5 mM; DABCO, 0.5 mM; D2O,
16 μl; DMSO, 4 μl; catalase, 4 units and SOD, 4 units.
photocleavage of DNA. The complexes were not cleavage active
in the dark thus ruling out the possibility of any hydrolytic
DNA damage (lane 6 in Fig. 8). The complexes having the
py-tpy ligand are found to be more active than the ph-tpy ana-
logues in the presence of a photoactive pyrenyl moiety. The
DNA groove binding nature of the complexes was studied
using a minor groove binder distamycin-A and a major groove
binder methyl green.33 Distamycin-A and methyl green alone
showed ∼15% cleavage of the SC DNA in UV-A light. Addition
of complex 2 to the distamycin-A bound SC DNA did not show
any inhibition in the DNA cleavage activity. Addition of methyl
green to complex 2, however, inhibited the DNA photocleavage
activity suggesting DNA major groove binding mode of the
py-tpy complexes (lanes 15 and 16 in Fig. 8).34
The ph-tpy complexes showed moderate DNA cleavage
(∼55%) activity in visible light of 454 nm wavelength (lanes 7,
9, 11 and 13 in Fig. 9, Table 4). The py-tpy complexes with
their high DNA binding strength and photosensitizing ability
showed efficient photocleavage activity with essentially com-
plete cleavage of SC DNA using a complex of 10.0 μM for an
exposure time of 2 h (lanes 8, 10, 12, 14 in Fig. 9). Control
experiments with only SC DNA did not show any apparent clea-
vage of DNA. The curcumin ligands alone showed ∼25% clea-
vage of SC DNA in the dark. This could be due to generation of
reactive oxygen species in an aqueous solution of curcumin.9
Curcumin ligands as photosensitizers showed ∼40% photo-
cleavage of SC DNA under same conditions forming reactive
oxygen species (ROS).9 The cleavage efficacy of curcumin is
remarkably enhanced possibly due to efficient intersystem
crossing (ISC) upon complexation to the heavy metal ions, viz.
La(III) and Gd(III).35
Mechanistic aspects
The mechanistic aspects of the DNA photocleavage reactions
were studied using the py-tpy complexes and different addi-
tives (Fig. 10, Fig. S29, ESI†). The reactions involving molecular
oxygen could proceed via two major pathways, viz. the type-II
process, forming singlet oxygen (1O2) or a photo-redox pathway
forming reactive hydroxyl radicals (˙OH).36 Addition of singlet
oxygen quenchers, viz. sodium azide, TEMP or DABCO, to SC
DNA showed partial inhibition of the DNA photocleavage
activity of the complex. Hydroxyl radical scavengers, viz. DMSO
or catalase, also showed partial inhibition in the DNA cleavage
activity. The results suggest the formation of both singlet
oxygen and hydroxyl radicals as ROS. The singlet oxygen for-
mation was also evidenced from the enhancement of the DNA
cleavage activity in D2O due to longer lifetime of 1O2 in this
medium than in water.37 While a photoactive pyrenyl and/or
curcumin moiety could generate the singlet oxygen species in
a type-II process in UV-A light, formation of hydroxyl radicals
could take place from an electron transfer to the oxygen mo-
lecule from the photo-excited ligand generating radical
cations.38
Photocytotoxicity in visible light
The photocytotoxic behaviour of 1–8 was studied in HeLa cervi-
cal cancer cells using MTT assay which is based on the ability
of the mitochondrial dehydrogenases of viable cells to cleave
the tetrazolium rings of MTT forming dark purple membrane
impermeable crystals of formazan that could be estimated at
595 nm after solubilising in DMSO (Table 5). The La(III)
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The py-tpy and ph-tpy ligands alone were found to be neither
phototoxic nor dark toxic (Fig. S30, ESI†).18 The curcumin
ligand alone is reported to be less toxic with IC50 values of
8.2 0.2 μM in the light and 85.4 0.6 μM in the dark.7 The
Hscur ligand is non-toxic in both light and dark with higher
IC50 values (Table 5, Fig. S31, ESI†).
Table 5 A comparison of the IC50 values of the lanthanide complexes along
with the reference compounds in HeLa cells
IC50/μM
IC50/μM
(dark)
Light
source
Compound
(light)a
[La(ph-tpy)(cur)(NO3)2] (1)
[La(py-tpy)(cur)(NO3)2] (2)
[La(ph-tpy)(scur)(NO3)2] (3)
[La(py-tpy)(scur)(NO3)2] (4)
[Gd(ph-tpy)(cur)(NO3)2] (5)
[Gd(py-tpy)(cur)(NO3)2] (6)
[Gd(ph-tpy)(scur)(NO3)2] (7)
[Gd(py-tpy)(scur)(NO3)2] (8)
Curcumind
7.7( 1.3)
4.6( 0.6)
40( 2)
>100b
>100b
>100b
>100b
>100b
>100b
>100b
>100b
85.4 0.6b
>41
Visiblec
Visiblec
Visiblec
Visiblec
Visiblec
Visiblec
Visiblec
Visiblec
Visiblec
Visiblef
UV-Ai
Complexation of curcumin and glycosylated curcumin to
Ln(III) retains their photocytotoxic potential. Complexes of the
glycosylated curcumin (Hscur) are much less active compared
to their curcumin analogues. This could be due to reduced
lipophilicity of the Hscur complexes than their curcumin ana-
logues.39 The present results are of importance since related
lanthanide complexes, viz. [Ln(dppz)2(NO3)3], [Ln(dppz)(acac)3]
and [Ln(pydppz)(acac)2(NO3)], are known to be active only in
the UV-A light of 365 nm.24,40–42 We have used cisplatin as a
non-PDT model complex with an IC50 value of 7.5 μM in the
dark in HeLa cells for an incubation time of 24 h.40 Photo-
frin®, the FDA approved PDT drug, is known to give an IC50
value of 4.3( 0.2) μM in 633 nm light (5 J cm−2 power) and
>41 μM in the dark in HeLa cells.43 Complex trans-
[Pt(N3)2(OH)2(NH3)(py)] is reported to give an IC50 value of
6.1( 0.5) μM in UV-A light of 365 nm and >244.3 μM in the
dark in HaCaT cancer cells.44 A comparison of the IC50 values
suggests that the present Ln(III) complexes of curcumin are
potent cytotoxic agents in visible light with low dark toxicity
thus making them suitable for further studies towards photo-
therapeutic applications (Table 5). The presence of a pyrenyl-
terpyridine base enhances the photocytotoxicity of the curcu-
min complexes.
18( 2)
6.5( 1.0)
4.5( 1.0)
60( 3)
23( 2)
8.2 0.2
4.28 0.20
0.34
0.46 0.05
0.16 0.01
0.04 0.01
68.7
Photofrin®e
[La(dppz)2(NO3)3]g
>100h
>100h
>3b,l
[La(dppz)(acac)3]j
UV-Ai
[La(pydppz)(acac)2(NO3)]k
[La(py-tpy)(acac)(NO3)2]m
Cisplating
UV-Ai
>200b
7.5h
UV-Ai
UV-Ai
a For 4 h incubation in the dark followed by exposure to light for 1–8.
b For
4 h
incubation in the dark. c Visible light photoreactor
(400–700 nm, 10 J cm−2). d Data from ref. 7. e Data from ref. 43. f Red
light of 633 nm (5 J cm−2). g Data from ref. 40. h Data correspond to
24 h incubation in the dark. i UV-A light (365 nm, 0.55 J cm−2). j Data
from ref. 24. k Data from ref. 42. l The concentration was limited to
3.0 μM due to limited solubility of the complex. m Data from ref. 24.
FACScan analysis
The mechanistic aspects of cell death were studied by FACScan
analysis using La(III) complexes 1 and 4.45 In order to ascertain
whether the photocytotoxic effect is by induction of apoptosis,
flow cytometric analysis was carried out using HeLa cells
treated with 1 and 4 at three different complex concentrations
(Fig. 12, Fig. S32, S33, ESI†). Cells treated with the complex in
the dark were exposed to visible light of 400–700 nm and then
Fig. 11 Cell viability plots showing the photocytotoxicity of (a) the complexes
1 (red squares in the light and black squares in the dark) and 2 (red circles in the
light and black circles in the dark) and (b) complexes 7 (red triangles in the light
and black triangles in the dark) and 8 (red squares in the light and black squares
in the dark) in HeLa cells on 4 h incubation in the dark followed by exposure to
visible light (400–700 nm, 10 J cm-2) for 1 h, as determined from the MTT assay.
complexes 1 and 2 of curcumin upon prior incubation for 4 h
in the dark and subsequent photo-exposure to visible light
(400–700 nm Luzchem photoreactor) for 1 h showed a dose-
dependent decrease in cell viability giving respective IC50
values of 7.7 1.3 and 4.6 0.6 μM (panel (a) in Fig. 11). The
La(III) complexes 3 and 4 of glycosylated curcumin under
similar experimental conditions exhibited only moderate
decrease in the cell viability with respective IC50 values of 40
2 and 18 2 μM (panel (b) in Fig. 11). The photocytotoxic be-
haviour of the Gd(III) complexes is similar to their La(III) ana-
logues (panels (c) and (d) in Fig. 11). The cells unexposed to
light but treated with the complexes gave an IC50 value of
>100 μM. The complexes are thus non-toxic in the dark but
become highly cytotoxic upon irradiation with visible light.
Fig. 12 Bar diagrams showing the FACS analysis of the HeLa cells treated with
(a) complex 2 and (b) complex 4 using different complex concentrations. The
graph is plotted over and above the % apoptosis observed in cells, treated with
DMSO (1% v/v), as the vehicle control.
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Fig. 13 A time-course collection of confocal microscopy images of the HeLa cells treated with the complexes 1–4 (10 μM) and propidium iodide (PI) after 2 and
4 h of incubation. Each panel consists of fluorescence (top left), bright field (top right), PI-staining (bottom left) and merged (bottom right) images respectively.
Scale bar = 10 μm.
stained with propidium iodide to observe any fragmented DNA extent, of the nucleus. This is indicative of the fact that the
which is indicated by an increase in the sub-G1/G0 population. complexes are taken up by the cells via diffusion only and not
It was observed that a concentration of 5 μM of 1 and 10 μM of via any receptor mediated pathway. No change in the nuclear
4 induced appreciable apoptosis in 24 h and the extent of morphology was observed in the confocal images of the cells,
apoptosis increased with increasing complex concentrations. indicating the fact that the complexes remain harmless in the
No significant induction of apoptosis by the complex was dark but cause significant damage to the cells upon photo-
observed when the sample was kept in the dark. This suggests exposure leading to apoptosis. In contrast, the lanthanide-
a non-toxic nature of the complex in the dark and cytotoxicity based MRI agents are useful only for tumour detection but not
arising only upon photo-activation.
for its selective damage.48–50
Cellular uptake
The cellular uptake of an anticancer drug is of importance for
its effectiveness against tumors.46 The fluorescence of curcu-
min and its complexes 1–4 was monitored by confocal fluor-
Experimental
Materials
escence microscopy in order to study the cellular uptake and All reagents and chemicals were purchased from commercial
their localization behavior in the HeLa cells (Fig. 13). It was sources (s.d. Fine Chemicals, India; Sigma-Aldrich, USA) and
observed that 1–4 get internalized into the cells within 2 h of used without further purifications. Solvents were purified by
incubation and the extent of uptake increases further on standard procedures.51 Curcumin (95% curcuminoid content,
increasing the incubation time to 4 h. In contrast, the fluor- 80% curcumin content) was purchased from Sigma-Aldrich
escence of the curcumin dye alone inside the cells decreased and purified into individual components by following a
significantly when incubation time was increased from 2 h to reported procedure.52 Supercoiled (SC) pUC19 DNA (caesium
4 h. This behaviour of curcumin could be attributed to its chloride purified) was purchased from Bangalore Genie
decomposition with time in the cellular medium.7,47 Complex (India). Tris-(hydroxymethyl)aminomethane-HCl (Tris-HCl)
2 showed the most significant cellular uptake among the com- buffer (pH = 7.2) was prepared using deionized and sonicated
plexes. Complexes 3 and 4 showed diminished fluorescence triple distilled water. Calf thymus (ct) DNA, agarose (molecular
intensity compared to that of 1 and 2 inside the cells. This biology grade), distamycin-A, methyl green, catalase, superox-
trend in cellular uptake behaviour gets reflected in the cyto- ide dismutase (SOD), 2,2,6,6-tetramethyl-4-piperidone (TEMP),
toxic potentials of the complexes. Incubation of the HeLa cells 4-diazabicyclo[2.2.2]octane (DABCO), ethidium bromide (EB),
with either compound showed uptake and retention of the propidium iodide and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
complexes in the nucleus as well as in the cytoplasm as a nyltetrazolium bromide (MTT) were purchased from Sigma
diffused green fluorescence all over the cell. This was also evi- (USA). The terpyridine derivatives, viz. 4′-phenyl-2,2′:6′,2′′-ter-
denced from co-staining of the cells using propidium iodide pyridine (ph-tpy) and 4′-(1-pyrenyl)-2,2′:6′,2′′-terpyridine (py-
(PI) which stains the nucleus of the HeLa cells. The co-staining tpy), were prepared following literature procedures.53,54 The
data clearly indicate the retention of the complexes in the glycosylated curcumin, viz. diglucosylcurcumin (Hscur), 1,7-bis-
nucleus and in the cytoplasm too. The intracellular localiz- (3-methoxy-4-β-D-glucopyranos-1-yloxophenyl)hepta-1,6-diene-
ation of the complexes did not show any granular appearance, 3,5-dione, was prepared according to a literature method
but largely a diffused staining of the cytoplasm and to some (Fig. S34, ESI†).17
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Measurements
To a methanol suspension (15 ml) of the precursor
complex, viz. [Ln(ph-tpy)(NO3)3] (0.32 g, La(III); 0.33 g, Gd(III),
0.5 mmol) or [Ln(py-tpy)(NO3)3] (0.38 g, La(III); 0.39 g, Gd(III),
0.5 mmol), was added a deaerated methanol solution (25 ml)
of the β-diketone ligand (0.54 g, Hcur; 1.04 g, Hscur,
1.5 mmol) which was neutralized with Et3N (0.15 g, 1.5 mmol)
prior to the reaction. The brick red solid obtained after 1 h of
stirring was isolated, washed with cold methanol followed by
diethyl ether and finally dried in a vacuum over P4O10 [yield:
∼70%]. The characterization data for the complexes are given
below.
The elemental analyses were done using a Thermo Finnigan
Flash EA 1112 CHNS analyzer. The infrared spectra were
recorded on a Bruker ALPHA FT-IR spectrometer. Electronic
spectra were recorded on a PerkinElmer Lambda 650 UV-VIS
spectrometer. Molar conductivity measurements were per-
formed using a Control Dynamics (India) conductivity meter.
Electrospray ionization mass spectral (ESI-MS) measurements
were carried out using a Bruker Daltonics Esquire 300 Plus ESI
1
Model instrument. H NMR spectra of the diamagnetic La(III)
complexes were recorded at room temperature on a Bruker
400 MHz NMR spectrometer. Room temperature fluorescence
quantum yield measurements were done using a PerkinElmer
LS 55 fluorescence spectrometer using coumarin-153 laser dye
as a reference with a known Φ value of 0.56 in acetonitrile.55
Samples were deaerated prior to spectral measurements. The
complexes and the reference were excited at 420 nm, maintain-
ing nearly equal absorbance and the emission spectra were
recorded from 450 to 600 nm. The integrated emission inten-
sity was calculated using Origin Pro 8.1 software and the
quantum yield was calculated using the equation: (ΦS/ΦR) =
[La(ph-tpy)(cur)(NO3)2] (1) (M
= 939): Anal. calc. for
C42H34N5O12La: C, 53.68; H, 3.65; N, 7.45. Found: C, 53.81; H,
3.48; N, 7.56%. ESI-MS in 10% aqueous MeOH: m/z 877 [M −
(NO3−)]+. IR data/cm−1: 3062 w, 1598 s, 1490 vs, 1430 s, 1395 s,
1278 s, 1214 m, 1156 m, 1121 m, 1026 w, 968 m, 840 w, 817 w,
755 m, 734 m, 682 w, 640 w, 630 w, 609 w, 578 w, 515 w,
453 m, 408 w (vs, very strong; s, strong; m, medium; w, weak).
UV-visible in 10% DMF [λmax/nm (ε/dm3 mol−1 cm−1)]: 454 sh
(36 200), 432 (41 300), 319 (10 100), 273 (22 600) (sh, shoulder).
1H-NMR in DMSO-d6 (δ, ppm): 8.78 (d, 2H, J = 4.8 Hz), 8.70 (s,
2H), 8.67 (d, 2H, J = 8.4 Hz), 8.05 (d, 2H, J = 7.8 Hz), 7.90 (d,
2H, J = 7.4 Hz), 7.60–7.50 (m, 5H), 7.50 (d, 2H, J = 15.3 Hz),
7.45 (m, 2H), 7.14 (d, 2H, J = 8.15 Hz), 6.83 (d, 2H, J = 7.4 Hz),
6.66 (d, 2H, J = 15.3 Hz), 6.01 (s, 1H), 3.85 (s, 6H) (s, singlet; d,
doublet; m, multiplet). Molar conductivity in 10% aqueous
DMF at 298 K [ΛM/S cm2 mol−1]: 98.
2
(AS/AR) × ((OD)S/(OD)R) × (nS /nR2), where ΦS and ΦR are the flu-
orescence quantum yields of the sample and the reference
respectively, AS and AR are the area under the fluorescence
spectra of the sample and the reference respectively, (OD)S and
(OD)R are the respective optical densities of the sample and
the reference solution at the wavelength of excitation, and nS
and nR are the refractive indices for the respective solvents
used for the sample and the reference.56,57 Fluorescence
microscopic investigations were carried out on a Leica DM IL
microscope with an integrated Leica DFC400 camera and IL50
image software. Confocal microscopy was done using a con-
focal scanning electron microscope (Leica, TCS SP5 DM6000).
Room temperature magnetic susceptibilities of the DMSO-d6
solutions of the Gd(III) complexes containing 1% TMS (v/v) as
the internal reference were obtained by a solution NMR
method with a Bruker AMX-400 NMR spectrometer.58 The
magnetic moments were calculated by the Evans method
using the equation: μeff = 0.0618(ΔfT/fM), where Δf is the
observed shift in frequency of the TMS signal, T is the temp-
erature (K), f is the operating frequency (MHz) of the NMR
spectrometer, and M is the molarity of the complex in the
solution.59
[La(py-tpy)(cur)(NO3)2] (2) (M = 1063): Anal. calc. for
C52H38N5O12La: C, 58.71; H, 3.60; N, 6.58. Found: C, 58.90; H,
3.72; N, 6.63%. ESI-MS in 10% aqueous MeOH: m/z 1001[M −
(NO3−)]+. IR data/cm−1: 3070 w, 1590 s, 1492 vs, 1398 s,
1280 m, 1155 m, 1224 m, 1026 w, 970 m, 843 w, 815 w, 755 w,
721 w, 680 w, 642 w, 607 w, 575 w, 544 w, 513 w, 451 m, 409
w. UV-visible in 10% DMF [λmax/nm (ε/dm3 mol−1 cm−1)]: 454
sh (39 300), 432 (44 600), 348 (38 300), 331 (30 500), 317
1
(25 700), 277 (48 400). H-NMR in DMSO-d6 (δ, ppm): 8.80 (d,
2H, J = 8.3 Hz), 8.75 (d, 2H, J = 4.6 Hz), 8.70 (s, 2H), 8.48–8.28
(m, 8H), 8.20 (t, 1H, J = 8.1 Hz), 8.10–8.05 (m, 4H), 7.52 (d, 2H,
J = 15.2 Hz), 7.42 (m, 2H), 7.11(d, 2H, J = 8.10 Hz), 6.85 (d, 2H,
J = 7.2 Hz), 6.63 (d, 2H, J = 15.2 Hz), 6.02 (s, 1H), 3.83 (s, 6H).
Molar conductivity in 10% aqueous DMF at 298 K [ΛM/S
cm2 mol−1]: 92.
[La(ph-tpy)(scur)(NO3)2] (3) (M = 1263): Anal. calc. for
C54H54N5O22La: C, 51.31; H, 4.31; N, 5.54. Found: C, 51.40; H,
4.22; N, 5.62%. ESI-MS in 10% aqueous MeOH: m/z 1201[M −
(NO3−)]+. IR data/cm−1: 3304 s, 2928 w, 1590 s, 1495 vs,
Syntheses of the complexes 1–8
The complexes were prepared by a general synthetic route. A 1460 m, 1398 s, 1356 m, 1252 s, 1221 m, 1148 w, 1127 w, 1070
methanol solution (15 ml) of Ln(NO3)3·6H2O (0.43 g for La(III) s, 1028 s, 894 w, 842 w, 811 w, 760 w, 665 m, 635 w, 620 w, 582
and 0.45 g for Gd(III), 1.0 mmol) was added dropwise to a w, 519 w, 454 w, 413 w. UV-visible in 10% DMF [λmax, nm
dichloromethane solution (15 ml) of the terpyridine ligand (ε/dm3 mol−1 cm−1)]: 446 sh (28 050), 417 (35 200), 322 sh
1
(ph-tpy, 0.31 g; py-tpy, 0.44 g; 1.0 mmol) with stirring for 1 h (21 700), 270 (22 000). H-NMR in DMSO-d6 (δ, ppm): 8.80 (d,
at 25 °C. The reaction mixture was cooled to 0 °C to obtain a 2H, J = 4.7 Hz), 8.75 (s, 2H), 8.71 (d, 2H, J = 4.3 Hz), 8.08 (d,
crystalline solid of [Ln(ph-tpy)(NO3)3] or [Ln(py-tpy)(NO3)3] as 2H, J = 7.7 Hz), 7.95 (d, 2H, J = 7.6 Hz), 7.64–7.55 (m, 5H), 7.53
the precursor complex which was filtered, washed with ice- (d, 2H, J = 15.3 Hz), 7.30–7.40 (m, 2H), 7.13 (d, 2H, J = 8.2 Hz),
cold ethanol followed by diethyl ether and finally dried in a 6.83 (d, 2H, J = 7.6 Hz), 6.65 (d, 2H, J = 15.3 Hz), 6.01 (s, 1H),
vacuum and used directly in the next step.
5.16 (d, 2H, J = 7.5 Hz), 5.08 (m, 2H), 4.60 (m, 2H), 4.50
190 | Dalton Trans., 2013, 42, 182–195
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(m, 2H), 4.33 (m, 2H), 3.87 (m, 4H), 3.76 (s, 6H). Molar
conductivity in 10% aqueous DMF at 298 K [ΛM/S cm2 mol−1]:
88.
Table 6 Selected crystallographic data and structure refinement parameters
for the complexes 1·H2O and 5·H2O
Empirical formula
Formula weight
C42H36LaN5O13
(1·H2O)
957.67
C42H36GdN5O13
(5·H2O)
976.01
[La(py-tpy)(scur)(NO3)2] (4) (M = 1387): Anal. calc. for
C64H58N5O22La: C, 55.38; H, 4.21; N, 5.05. Found: C, 55.45; H,
3.98; N, 5.12%. ESI-MS in 10% aqueous MeOH: m/z 1325 [M −
(NO3−)]+. IR data/cm−1: 3317 s, 2926 w, 1595 s, 1502 vs,
1461 m, 1402 m, 1253 s, 1150 w, 1125 w, 1066 m, 1028 s,
901 w, 845 w, 803 w, 755 w, 661 w, 620 w, 570 w, 545 w, 484 w,
454 w, 423 w. UV-visible in 10% DMF [λmax, nm (ε/dm3 mol−1
cm−1)]: 446 sh (30 800), 417 (42 200), 348 (38 000), 331 (31 200),
(g mol−1
)
Crystal system
Space group
a (Å)
b (Å)
c (Å)
α (°)
β (°)
Triclinic
P1
Triclinic
P1
ˉ
ˉ
10.7473(6)
12.5084(8)
15.0670(9)
86.953(5)
89.168(5)
79.358(5)
1987.8(2)
2
10.7275(6)
12.4582(8)
15.0207(9)
86.869(4)
89.236(4)
80.443(4)
1976.6(2)
2
1
γ (°)
316 (25 900), 278 (37 300). H-NMR in DMSO-d6 (δ, ppm): 8.81
V (Å3)
Z
(d, 2H, J = 8.5 Hz), 8.78 (d, 2H, J = 4.6 Hz), 8.76 (s, 2H),
8.49–8.25 (m, 8H), 8.15 (t, 1H, J = 8.2 Hz), 8.10–8.03 (m, 4H),
7.50 (d, 2H, J = 15.1 Hz), 7.42 (m, 2H), 7.10 (d, 2H, J = 8.0 Hz),
6.83 (d, 2H, J = 7.5 Hz), 6.63 (d, 2H, J = 15.1 Hz), 6.03 (s, 1H),
5.22 (d, 2H, J = 8.2 Hz), 5.10 (m, 2H), 4.55 (m, 2H), 4.46
(m, 2H), 4.29 (m, 2H), 3.85 (m, 4H), 3.74 (s, 6H). Molar conduc-
tivity in 10% aqueous DMF at 298 K [ΛM/S cm2 mol−1]: 91.
T (K)
293(2)
1.600
0.71073
1.150
293(2)
1.640
0.71073
1.753
ρcalc (g cm−3
λ/Å (Mo-Kα)
)
μ (cm−1
)
Data/restraints/
6983/0/550
8503/0/650
parameters
F(000)
968
1.034
0.0582/0.1213
0.0870/0.1430
0.595, −0.681
982
0.978
0.0526/0.0884
0.1476/0.1147
0.750, −1.473
Goodness-of-fit on F2
R(Fo)a, I > 2σ(I)/wR (Fo)b
R (all data)/wR (all data)
Largest diff. peak and
[Gd(ph-tpy)(cur)(NO3)2] (5) (M
= 958): Anal. calc. for
C42H34N5O12Gd: C, 52.66; H, 3.58; N, 7.31. Found: C, 52.48; H,
3.43; N, 7.25%. ESI-MS in 10% aqueous MeOH: m/z 896 [M −
(NO3−)]+. IR data/cm−1: 3060 w, 1595 s, 1494 vs, 1400 s, 1282 s,
1220 m, 1156 m, 1125 s, 1036 m, 1005 m, 975 m, 820 m, 780 w,
768 m, 727 w, 695 w, 654 w, 6312 w, 583 w, 550 w, 519 w,
458 m, 415 w. UV-visible in 10% DMF [λmax, nm (ε/dm3 mol−1
cm−1)]: 454 sh (34 400), 431(39 600), 319 (12 700), 276 (29 000).
μeff = 7.94 μB at 298 K. Molar conductivity in 10% aqueous
DMF at 298 K [ΛM/S cm2 mol−1]: 86.
[Gd(py-tpy)(cur)(NO3)2] (6) (M = 1082): Anal. calc. for
C52H38N5O12Gd: C, 57.72; H, 3.54; N, 6.47. Found: C, 57.78; H,
3.48; N, 6.53%. ESI-MS in 10% aqueous MeOH: m/z 1020 [M −
(NO3−)]+. IR data/cm−1: 3065 w, 1595 s, 1495 vs, 1397 s, 1282 s,
1220 s, 1157 s, 1118 s, 1026 w, 974 m, 843 m, 821 m, 765 w,
hole (e Å−3
)
a R = Σ||Fo| − |Fc||/Σ|Fo|. b wR = {Σ[w(Fo − Fc2)2]/Σ[w(Fo)2]}1/2; w =
2
[σ2(Fo)2 + (AP)2 + BP]−1, where P = (Fo + 2Fc2)/3, A = 0.0473, B = 0.0000
2
for complex 1·H2O; A = 0.0475, B = 0.0000 for complex 5·H2O.
(40 700), 348 (41 200), 330 (35 300), 277 (42 900), 267 (39 800).
μeff = 8.06 μB at 298 K. Molar conductivity in 10% aqueous
DMF at 298 K [ΛM/S cm2 mol−1]: 85.
X-ray crystallographic procedure
735 w, 610 w, 580 w, 560 w, 516 w, 454 m, 412 w. UV-visible in The crystal structures of [La(ph-tpy)(cur)(NO3)2]·H2O (1·H2O)
10% DMF [λmax, nm (ε/dm3 mol−1 cm−1)]: 452 sh (39 300), 429 and [Gd(ph-tpy)(cur)(NO3)2]·H2O (5·H2O) were obtained by a
(45 500), 348 (25 300), 330 (20 140), 278 (25 200), 264 (28 500). single crystal X-ray diffraction method. Selected crystallo-
μeff = 8.02 μB at 298 K. Molar conductivity in 10% aqueous graphic parameters are given in Table 6. Single crystals were
DMF at 298 K [ΛM/S cm2 mol−1]: 90.
grown by slow evaporation of a solution of the complex in a
[Gd(ph-tpy)(scur)(NO3)2] (7) (M = 1282): Anal. calc. for MeOH–MeCN mixture. The source of the lattice H2O could be
C54H54N5O22Gd: C, 50.58; H, 4.24; N, 5.46. Found: C, 50.69; H, the solvents used for crystallization. Crystal mounting was
4.32; N, 5.52%. ESI-MS in 10% aqueous MeOH: m/z 1220 [M − done on glass fibre with epoxy cement. All geometric and
(NO3−)]+. IR data/cm−1: 3310 s, 2929 w, 1592 s, 1495 vs, 1398 s, intensity data were collected at room temperature using an
1360 s, 1256 s, 1220 m, 1160 w, 1125 w, 1067 s, 1026 s, 896 w, automated Bruker SMART APEX CCD diffractometer equipped
840 w, 811 w, 790 w, 665 w, 623 w, 582 w, 530 w, 457 w, 420 w. with a fine focus 1.75 kW sealed tube Mo-Kα X-ray source (λ =
UV-visible in 10% DMF [λmax, nm (ε/dm3 mol−1 cm−1)]: 447 sh 0.71073 Å) with increasing ω (width of 0.3° per frame) at a
(25 700), 421 (35 060), 324 (19 500), 279 (32 030), 267 (35 600). scan speed of 10 s per frame for complex 1 and 5 s per frame
μeff = 7.99 μB at 298 K. Molar conductivity in 10% aqueous for complex 5. Intensity data, collected using ω-2θ scan mode,
DMF at 298 K [ΛM/S cm2 mol−1]: 95.
were corrected for Lorentz-polarization effects and for absorp-
[Gd(py-tpy)(scur)(NO3)2] (8) (M = 1406): Anal. calc. for tion.60 The structures were solved by a combination of Patter-
C64H58N5O22Gd: C, 54.66; H, 4.16; N, 4.98. Found: C, 54.79; H, son and Fourier techniques and refined by a full-matrix least-
4.22; N, 4.88%. ESI-MS in 10% aqueous MeOH: m/z 1344 [M − squares method using SHELX system of programs.61 All hydro-
(NO3−)]+. IR data/cm−1: 3330 s, 2924 m, 1592 s, 1501 vs, 1400 s, gen atoms belonging to the complexes were refined using a
1254 s, 1150 w, 1130 m, 1061 s, 1020 s, 845 s, 802 w, 760 w, riding model. All non-hydrogen atoms were refined anisotropi-
670 w, 615 w, 570 w, 542 w, 460 w, 420 w. UV-visible in 10% cally. Perspective views of the molecules were obtained by
DMF [λmax, nm (ε/dm3 mol−1 cm−1)]: 447 sh (29 500), 420 ORTEP.62
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DNA binding experiments
500 μM; catalase, 4 units; SOD, 4 units) prior to the addition of
the lanthanide complex. For the D2O experiment, this solvent
was used for dilution of the sample to 20 μl sample volume.
The samples after incubation in a dark room were added to
the loading buffer containing 0.25% bromophenol blue, 0.25%
xylene cyanol, 30% glycerol (3.0 μl) and the solution was
finally loaded on 1% agarose gel containing 1.0 μg ml−1 ethi-
dium bromide (EB). Electrophoresis was done in a dark room
for 2.0 h at 60 V in TAE (Tris-acetate EDTA) buffer. Bands were
visualized in UV light and photographed. The extent of SC
DNA cleavage was estimated from the intensities of the bands
using the UVITEC Gel Documentation System. Due corrections
were made for the low level of the NC form of DNA present in
the original SC DNA sample and for low affinity of EB binding
to SC compared to the NC form of DNA.65 The observed error
in measuring the gel band intensities was within 3–5%.
DNA binding experiments were carried out in Tris-HCl/NaCl
buffer (5 mM Tris-HCl, 5 mM NaCl, pH 7.2) using a DMF solu-
tion of the complexes 1–8. Calf thymus DNA (ca. 350 μM NP)
in this buffer medium gave a ratio of ca. 1.9 : 1 of UV absor-
bance at 260 and 280 nm indicating that the DNA is apparently
free from protein. The concentration of DNA was estimated
from its absorption intensity at 260 nm with a known molar
extinction coefficient value (ε) of 6600 dm3 mol−1 cm−1 63
.
Absorption titration experiments were done by varying the con-
centration of the ct-DNA while keeping the metal complex con-
centration constant. Due correction was made for the
absorbance of DNA itself. Each spectrum was recorded after
equilibration of the sample for 5 min. The intrinsic equili-
brium binding constant (Kb) and the fitting parameter (s) of
1–8 to DNA were obtained by the McGhee–von Hippel (MvH)
method using the expression of Bard et al.28,64
Cell culture
DNA melting experiments were carried out by monitoring
the absorbance of ct-DNA (180 μM) at 260 nm at various temp-
eratures, both in the absence and presence of the complexes.
Measurements were carried out using a PerkinElmer Lambda
650 UV/VIS spectrometer with a PTP-1 + 1 Peltier temperature
controller at an increase rate of 0.5 °C per min. Viscometric
titrations were performed with a Schott Gerate AVS 310 Auto-
mated Viscometer thermostated at 37 °C in a constant temp-
erature bath. The concentration of ct-DNA was 150 μM in NP
(nucleotide pair) and the flow times were measured using an
automated timer. Each sample was measured 3 times and an
average flow time was calculated. Plots of (η/η0)1/3 vs. [com-
pound]/[DNA] were made, where η is the viscosity of DNA in
the presence of the compound and η0 is that of DNA alone. Vis-
cosity values were calculated from the observed flow time of
DNA-containing solutions (t) corrected for that of the buffer
alone (t0), η = (t − t0)/t0. Due corrections were made for the vis-
cosity of DMF solvent present in the aqueous buffer solution
of DNA.
HeLa (human cervical carcinoma) cells were maintained in
Dulbeco’s modified eagle’s medium (DMEM) supplemented
with 10% FBS (fetal bovine serum), 100 IU ml−1 of penicillin,
100 μg ml−1 of streptomycin and 2 mM of Glutamax at 37 °C
in a humidified incubator at 5% CO2. The adherent cultures
were grown as monolayers and were passaged once in 4–5 days
by treatment with 0.25% Trypsin–EDTA.
Photocytotoxicity experiments
The photocytotoxicity of the complexes was studied using the
MTT assay which is based on the ability of mitochondrial
dehydrogenases in the viable cells to cleave the tetrazolium
rings of MTT forming dark blue membrane impermeable crys-
tals of formazan which could be readily estimated at 595 nm
on detergent solubilization.66 The level of the formazan
formed gave a measure of the number of viable cells. Approxi-
mately, 8000 cells of human cervical carcinoma (HeLa) were
plated in a 96 well culture plate in DMEM containing 10% FBS
and after 24 h of incubation at 37 °C in a CO2 incubator,
different concentrations of the lanthanide complexes dissolved
DNA cleavage experiments
The cleavage of supercoiled (SC) pUC19 DNA (30 μM, 0.2 μg, in 1% DMSO were added to the cells and incubation was con-
2686 base-pairs) in Tris-HCl/NaCl (50 mM, pH, 7.2) buffer was tinued for 4 h in the dark. The medium was subsequently
studied by agarose gel electrophoresis. For photo-induced DNA replaced with PBS and photo-irradiation was done in visible
cleavage studies, the reactions were carried out under illumi- light (400–700 nm) using a Luzchem Photoreactor (Model
nated conditions using UV-A light of 365 nm (6 W, Model LZC-1, Ontario, Canada) fitted with Sylvania make 8 fluor-
LF-206.LS UV lamp from Bangalore Genei) and in visible laser escent white tubes with a fluence rate of 2.4 mW cm−2 to
light of 454 nm (Spectra Physics continuous-wave (CW) Ar–Kr provide a total light dose of 10 J cm−2. Post irradiation, PBS
mixed-gas ion laser Model Stabilities 2018-RM of 60 mW laser was replaced with DMEM-FBS and incubation was continued
power). The concentration of the complexes in DMF or the for a further 24 h in the dark. After incubation, 20 μl of 5 mg
additives in buffer corresponded to the quantity in 2.0 μl stock ml−1 of MTT was added to each well and incubated for an
solution after dilution to the 20 μl final volume using Tris-HCl additional 3 h. After discarding the culture medium, 200 μl of
buffer. The solution path length in the sample vial was DMSO was added to dissolve the formazan crystals and the
∼5 mm. After light exposure, each sample was incubated for absorbance at 595 nm was measured using a BIORAD ELISA
1.0 h at 37 °C and analyzed for the nicked circular (NC) and/or plate reader. Cytotoxicity of the test compounds was measured
linear DNA forms from SC DNA using gel electrophoresis. as the percentage ratio of the absorbance of the treated cells to
Mechanistic studies were done using different additives (NaN3, the untreated controls. The IC50 values were determined by
500 μM; TEMP, 500 μM; DABCO, 500 μM; DMSO, 4 μl; KI, nonlinear regression analysis (GraphPad Prism 5).
192 | Dalton Trans., 2013, 42, 182–195
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FACScan analysis
complexes are essentially non-toxic in the dark but become
highly cytotoxic on photoactivation. Binding of curcumin to
the Ln(III) ion increases the hydrolytic stability and photocyto-
toxicity of curcumin. This is likely to increase the bioavailabil-
ity of curcumin in a metal bound form. The photoactivated
anticancer activity of lanthanide curcumin complexes is unpre-
cedented in the literature. This work opens up a new area of
research on the photochemotherapeutic applications of curcu-
min and its lanthanide complexes. Nuclear localization of the
complexes in HeLa cells could enable the complexes to bind
the chromosomal DNA and damage the nuclear DNA on
photoactivation. With a significant PDT effect, the curcumin
lanthanide complexes could serve the dual purpose of detect-
ing the tumour while remaining dormant inside the cell in the
dark until subjected to photoactivation causing apoptotic cel-
lular death selectively in the photo-exposed cells leaving the
unexposed cells unaffected. In contrast, the lanthanide-based
MRI agents are useful for only tumour detection but not for
any tumour damage.
Briefly, HeLa cells (0.5 × 106 cells) plated overnight were
treated with different concentrations of the complexes 2 and 4
in DMEM for 4 h, followed by exposure to visible light
(400–700 nm) for
1 h using a Luzchem Photoreactor
(400–700 nm). The cells were then cultured overnight, har-
vested and fixed using chilled 70% ethanol. The cells were
treated with 50 μg ml−1 RNaseA for 4 h at 42 °C, stained with
propidium iodide staining solution (20 μg ml−1 in PBS) for 2 h
at 4 °C. The cells were analyzed by flow cytometry (FACSCanto,
Beckton Dickenson) to determine the
population.67
%
apoptotic
Confocal microscopy
HeLa cells (4 × 104 cells mm−2), plated on cover slips, were
incubated with 10 μM of the complexes 1–4 at the time inter-
vals of 2 and 4 h in the dark, fixed with 4% formaldehyde for
10 min at 25 °C and washed with PBS. Cells were later permea-
bilized by incubating with 90% methanol for 10 min at room
temperature. This was followed by incubation with propidium
iodide staining solution for 30 min at room temperature in the
presence of 100 μg ml−1 RNaseA. The cells were washed free of Acknowledgements
excess propidium iodide and mounted. Images were acquired
using a confocal scanning microscope (Zeiss, LSM510 apocro-
mat) and analysed using LSM image browser software.68
We thank the Department of Science and Technology (DST),
Government of India, for financial support (SR/S5/MBD-02/
2007). Work in the laboratory of G. N. is supported by the
Department of Biotechnology (DBT), Government of India,
and DST. We are thankful to DST for a CCD diffractometer
facility and DBT for an imaging facility. A. R. C. thanks the
Conclusions
In conclusion, we have studied the photoactivated DNA clea- DST for the J. C. Bose national fellowship. A. H., K. S. and
vage and anticancer activity of new ternary lanthanide com- B. B. thank the Council of Scientific and Industrial Research
plexes having tridentate N,N,N-donor terpyridine and O,O- (CSIR), New Delhi, for research fellowships. We are thankful to
donor curcumin and glycosylated curcumin ligands. The Dr O. Joy, P. Pai, A. Kavya, Deepti and Minakshi for the FACS
crystal structures of complexes 1 and 5 having a terpyridine analysis and confocal microscopy data and S. Mukherjee for
base and the curcumin ligand show a nine-coordinate LnO6N3 his help in the crystallographic work.
coordination environment with two nitrate ligands bound to
the metal center. The La(III) and Gd(III) complexes show the
LnO4N3 core in a solution phase due to dissociation of one
nitrate ligand. The complexes show electronic absorption and
References
emission spectral bands in the visible region. The py-tpy com-
plexes which bind to DNA in an intercalative mode display
efficient UV-A (365 nm) and visible (454 nm) light-induced
plasmid DNA cleavage activity via mechanistic pathways that
involve formation of both singlet oxygen and hydroxyl radicals
in the presence of photoactive pyrenyl and curcumin moieties.
Our observation of the DNA photocleavage activity in visible
light is of significance since the lanthanide complexes of non-
macrocyclic organic molecules are known to be active only in
the UV light. The present complexes do not show any apparent
hydrolytic DNA cleavage activity thus making them suitable for
potential photochemotherapeutic applications.
The curcumin complexes of lanthanide ions display
remarkable photocytotoxicity in HeLa cancer cells giving IC50
values that are comparable to that of Photofrin. The glycosy-
lated curcumin complexes show better aqueous solubility but
moderate photocytotoxicity in visible light. These curcumin
1 (a) H. Hatcher, R. Planalp, J. Cho, F. M. Torti and
S. V. Torti, Cell. Mol. Life Sci., 2008, 65, 1631;
(b) V. S. Govindarajan and W. H. Stahl, Crit. Rev. Food Sci.
Nutr., 1980, 12, 199.
2 B. B. Aggarwal and K. B. Harikumar, Int. J. Biochem. Cell
Biol., 2009, 41, 40.
3 (a) M.-H. Teiten, S. Eifes, M. Dicato and M. Diederich,
Toxins, 2010, 2, 128; (b) B. B. Aggarwal, A. Kumar and
A. C. Bharti, Anticancer Res., 2003, 23, 363; (c) A. Goel,
A. B. Kunnumakkara and B. B. Aggarwal, Biochem. Pharma-
col., 2008, 75, 787; (d) G. Sa and T. Das, Cell Div., 2008, 3, 1;
(e) A. Shehzad, F. Wahid and Y. S. Lee, Arch. Pharm. Chem.
Life Sci., 2010, 343, 489.
4 (a) F. Yang, P. L. G. P. Lim, A. N. Begum, O. J. Ubeda,
M. R. Simmons, S. S. Ambegaokar, P. Chen, R. Kayed,
C. G. Glabe, S. A. Frautschy and G. M. Cole, J. Biol. Chem.,
2005, 280, 5892; (b) M. Garcia-Alloza, L. A. Borrelli,
This journal is © The Royal Society of Chemistry 2013
Dalton Trans., 2013, 42, 182–195 | 193

View Article Online
Paper
Dalton Transactions
A. Rozkalne, B. T. Hyman and B. J. Bacskai, J. Neurochem., 16 (a) B. Banik, P. K. Sasmal, S. Roy, R. Majumdar, R. R. Dighe
2007, 102, 1095.
5 (a) N. Dhillon, B. B. Aggarwal, R. A. Newman, R. A. Wolff,
A. B. Kunnumakkara, J. L. Abbruzzese, C. S. Ng,
and A. R. Chakravarty, Eur. J. Inorg. Chem., 2011, 1425;
(b) P. K. Sasmal, S. Saha, R. Majumdar, R. R. Dighe and
A. R. Chakravarty, Chem. Commun., 2009, 1703.
V. Badmaev and R. Kurzrock, Clin. Cancer Res., 2008, 14, 17 (a) E. Ferrari, S. Lazzari, G. Marverti, F. Pignedoli,
4491; (b) R. A. Sharma, S. A. Euden, S. L. Platton,
D. N. Cooke, A. Shafayat, H. R. Hewitt, T. H. Marczylo,
B. Morgan, D. Hemingway, S. M. Plummer,
M. Pirmohamed, A. J. Gescher and W. P. Steward, Clin.
F. Spagnolo and M. Saladini, Bioorg. Med. Chem., 2009, 17,
3043; (b) K. Mohri, Y. Watanabe, Y. Yoshida, M. Satoh,
K. Isobe, N. Sugimoto and Y. Tsuda, Chem. Pharm. Bull.,
2003, 51, 1268.
Cancer Res., 2004, 10, 6847; (c) C. H. Hsu and A. L. Cheng, 18 S. Roy, S. Saha, R. Majumdar, R. R. Dighe, E. D. Jemmis
Adv. Exp. Med. Biol., 2007, 595, 471. and A. R. Chakravarty, Dalton Trans., 2011, 40, 1233.
6 (a) B. B. Aggarwal, C. Sundaram, N. Malani and 19 (a) M. Komiyama, N. Takida and H. Shigekawa, Chem.
H. Ichikawa, Adv. Exp. Med. Biol., 2007, 595, 1;
(b) P. Anand, S. G. Thomas, A. B. Kunnumakkara,
C. Sundaram, K. B. Harikumar, B. Sung, S. T. Tharakan,
Commun., 1999, 1443; (b) S. J. Franklin, Curr. Opin. Chem.
Biol., 2001, 5, 201; (c) C. Liu, M. Wang, T. Zhang and
H. Sun, Coord. Chem. Rev., 2004, 248, 147.
K. Misra, I. K. Priyadarsini, K. N. Rajasekharan and 20 W. J. Geary, Coord. Chem. Rev., 1971, 7, 81.
B. B. Aggarwal, Biochem. Pharmacol., 2008, 76, 1590.
7 S. Banerjee, P. Prasad, A. Hussain, I. Khan, P. Kondaiah
and A. R. Chakravarty, Chem. Commun., 2012, 48, 7702.
8 (a) D. Pucci, T. Bellini, A. Crispini, I. D’Agnano,
P. F. Liguori, P. Garcia-Orduña, S. Pirillo, A. Valentini and
21 J. C. G. Bünzli, A. E. Merbach and R. M. Nielson, Inorg.
Chim. Acta, 1987, 139, 151.
22 K. Nakamoto, Infrared and Raman Spectra of Inorganic and
Coordination Compounds, John Wiley & Sons, New York,
3rd edn, 1978.
G. Zanchettab, Med. Chem. Commun., 2012, 3, 462; 23 Y. Fukuda, A. Nakao and K. Hayashi, J. Chem. Soc., Dalton
(b) F. Kuhlwein, K. Polborn and W. Beck, Z. Anorg. Allg.
Chem., 1997, 623, 1211.
9 (a) I. K. Priyadarsini, J. Photochem. Photobiol., C, 2009, 10,
Trans., 2002, 527.
24 A. Hussain, S. Gadadhar, T. K. Goswami, A. Karande and
A. R. Chakravarty, Eur. J. Med. Chem., 2012, 50, 319.
81; (b) T. A. Dahl, P. Bilski, K. J. Reszka and C. F. Chignell, 25 Y.-J. Wang, M.-H. Pan, A.-L. Cheng, L.-I. Lin, Y.-S. Ho,
Photochem. Photobiol., 1994, 59, 290.
C.-Y. Hsieh and J.-K. Lin, J. Pharm. Biomed. Anal., 1997, 15,
10 (a) M. Sagnou, D. Benaki, C. Triantis, T. Tsotakos,
1867.
V. Psycharis, C. P. Raptopoulou, I. Pirmettis, 26 L. Shen and H.-F. Ji, Trends Mol. Med., 2012, 18, 138.
M. Papadopoulos and M. Pelecanou, Inorg. Chem., 2011, 27 H. Krass, E. A. Plummer, J. M. Haider, P. R. Barker,
50, 1295; (b) A. Valentini, F. Conforti, A. Crispini, A. De
N. W. Alcock, Z. Pikramenou, M. J. Hannon and
Martino, R. Condello, C. Stellitano, G. Rotilio, M. Ghedini,
D. G. Kurth, Angew. Chem., Int. Ed., 2001, 40, 3862.
G. Federici, S. Bernardini and D. Pucci, J. Med. Chem., 28 J. D. McGhee and P. H. von Hippel, J. Mol. Biol., 1974, 86,
2009, 52, 484. 469.
11 (a) K. Krishnankutty and V. D. John, Synth. React. Inorg. 29 A. M. Angeles-Boza, P. M. Bradley, P. K.-L. Fu, S. E. Wicke,
Met.-Org., Nano-Met. Chem., 2003, 33, 343; (b) X.-Z. Zhao,
T. Jiang, L. Wang, H. Yang, S. Zhang and P. Zhou, J. Mol.
Struct., 2010, 984, 316.
J. Bacsa, K. R. Dunbar and C. Turro, Inorg. Chem., 2004, 43,
8510.
30 (a) Y. An, S.-D. Liu, S.-Y. Deng, L.-N. Ji and Z.-W. Mao,
J. Inorg. Biochem., 2006, 100, 1586; (b) L. E. Gunther and
A. S. Yong, J. Am. Chem. Soc., 1968, 90, 7323.
12 (a) E. J. New, D. Parker, D. G. Smith and J. W. Walton, Curr.
Opin. Chem. Biol., 2010, 14, 238; (b) C. P. Montgomery,
B. S. Murrey, E. J. New, R. Pal and D. Parker, Acc. Chem. 31 J. M. Veal and R. L. Rill, Biochemistry, 1991, 30, 1132.
Res., 2009, 42, 925.
13 D. Parker and J. A. G. Williams, J. Chem. Soc., Dalton Trans.,
1996, 3613.
14 (a) H. J. Nyst, I. B. Tan, F. A. Stewart and A. J. M. Balm,
Photodiagn. Photodyn. Ther., 2009, 6, 3; (b) M. Ethirajan,
32 P. P. Pellegrini and J. R. Aldrich-Wright, Dalton Trans.,
2003, 176.
33 T. Phillips, I. Haq, A. J. H. M. Meijer, H. Adams, I. Soutar,
L. Swanson, M. J. Sykes and J. A. Thomas, Biochemistry,
2004, 43, 13657.
Y. Chen, P. Joshi and R. K. Pandey, Chem. Soc. Rev., 2011, 34 (a) K. E. Erkkila, D. T. Odom and J. K. Barton, Chem. Rev.,
40, 340; (c) A. Bergamo and G. Sava, Dalton Trans., 2011,
40, 7817; (d) D. Crespy, K. Landfester, U. S. Schubert and
A. Schiller, Chem. Commun., 2010, 46, 6651.
1999, 99, 2777; (b) S. Delaney, M. Pascaly,
P. K. Bhattacharya, K. Han and J. K. Barton, Inorg. Chem.,
2002, 41, 1966.
15 (a) N. J. Farrer, L. Salassa and P. J. Sadler, Dalton Trans., 35 (a) S. Tobita, M. Arakawa and I. Tanaka, J. Phys. Chem.,
2009, 10690; (b) N. J. Farrer and P. J. Sadler, Bioinorg. Med.
Chem., 2011, 1; (c) U. Schatzschneider, Eur. J. Inorg. Chem.,
1984, 88, 2697; (b) S. Tobita, M. Arakawa and I. Tanaka,
J. Phys. Chem., 1985, 89, 5649.
2010, 1451; (d) A. D. Ostrowski and P. C. Ford, Dalton 36 K. Szacilowski, W. Macyk, A. Drzewiecka-Matuszek,
Trans., 2009, 10660.
M. Brindell and G. Stochel, Chem. Rev., 2005, 105, 2647.
194 | Dalton Trans., 2013, 42, 182–195
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Dalton Transactions
Paper
37 (a) A. U. Khan, J. Phys. Chem., 1976, 80, 2219; 49 (a) P. Caravan, Acc. Chem. Res., 2009, 42, 851;
(b) P. B. Merkel and D. R. Kearns, J. Am. Chem. Soc., 1972,
94, 1029.
38 (a) M. Tanaka, K. Ohkubo and S. Fukuzumi, J. Phys. Chem.
A, 2006, 110, 11214; (b) M. L. Cunningham, J. S. Johnson,
S. M. Giovanazzi and M. J. Peak, Photochem. Photobiol.,
1985, 42, 125.
(b) P. Caravan, Chem. Soc. Rev., 2006, 35, 512.
50 (a) D. Parker, R. S. Dickins, H. Puschmann, C. Crossland
and J. A. K. Howard, Chem. Rev., 2002, 102, 1977;
(b) M. Bottrill, L. Kwok and N. J. Long, Chem. Soc. Rev.,
2006, 35, 557; (c) P. Caravan, J. J. Ellison, T. J. McMurry and
R. B. Lauffer, Chem. Rev., 1999, 99, 2293.
39 (a) E. Ferrari, S. Lazzari, G. Marverti, F. Pignedoli, 51 D. D. Perrin, W. L. F. Armarego and D. R. Perrin, Purifi-
F. Spagnolo and M. Saladini, Bioorg. Med. Chem., 2009, 17,
3043; (b) K. Mohri, Y. Watanabe, Y. Yoshida, M. Satoh,
cation of Laboratory Chemicals, Pergamon Press, Oxford,
1980.
K. Isobe, N. Sugimoto and Y. Tsuda, Chem. Pharm. Bull., 52 O. Vajragupta, P. Boonchoon and L. J. Berliner, Free Radi-
2003, 51, 1268. cals Res., 2004, 38, 303.
40 A. Hussain, D. Lahiri, M. S. A. Begum, S. Saha, 53 S. A. Moya, R. Pastene, H. L. Bozec, P. J. Baricelli,
R. Majumdar, R. R. Dighe and A. R. Chakravarty, Inorg.
Chem., 2010, 49, 4036.
A. J. Pardey and J. Gimeno, Inorg. Chim. Acta, 2001,
312, 7.
41 A. Hussain, S. Saha, R. Majumdar, R. R. Dighe and 54 W. Leslie, R. A. Poole, P. R. Murray, L. J. Yellowlees,
A. R. Chakravarty, Indian J. Chem., Sect. A: Inorg., Bio-inorg.,
Phys., Theor. Anal. Chem., 2011, 50A, 519.
A. Beeby and J. A. G. Williams, Polyhedron, 2004, 23,
2769.
42 A. Hussain, S. Gadadhar, T. K. Goswami, A. A. Karande and 55 J. Guilford II, R. Jackson, C. Choi and W. R. Bergmark,
A. R. Chakravarty, Dalton Trans., 2011, 41, 885. J. Phys. Chem., 1985, 89, 294.
43 E. Delaey, F. Van Laar, D. De Vos, A. Kamuhabwa, P. Jacobs 56 J. R. Lakowicz, Principles of Fluorescence Spectroscopy,
and P. De Witte, J. Photochem. Photobiol., B, 2000, 55, 27. Kluwer Academic/Plenum Publishers, New York, 1999.
44 F. S. Mackay, J. A. Woods, P. Heringová, J. Kašpárková, 57 A. T. R. Williams, S. A. Winfield and J. N. Miller, Analyst,
A. M. Pizarro, S. A. Moggach, S. Parsons, V. Brabec and
1983, 108, 1067.
P. J. Sadler, Proc. Natl. Acad. Sci. U. S. A., 2007, 104, 20743.
58 D. F. Evans, J. Chem. Soc., 1961, 1987.
45 (a) M. Roy, B. V. S. K. Chakravarthi, C. Jayabaskaran, 59 D. F. Evans, J. Chem. Soc., 1959, 2003.
A. A. Karande and A. R. Chakravarty, Dalton Trans., 2011, 60 N. Walker and D. Stuart, Acta Crystallogr., Sect. A: Fundam.
40, 4855; (b) C. H. Ng, K. C. Kong, S. T. Von, P. Balraj,
Crystallogr., 1983, 39, 158.
P. Jensen, E. Thirthagiri, H. Hamada and M. Chikira, 61 G. M. Sheldrick, SHELX-97, Program for Crystal Structure
Dalton Trans., 2008, 447.
Solution and Refinement, University of Göttingen, Göttin-
46 (a) T. Storr, K. H. Thompson and C. Orvig, Chem. Soc. Rev.,
gen, Germany, 1997.
2006, 35, 534; (b) K. H. Thompson and C. Orvig, Dalton 62 C. K. Johnson, ORTEP-III, Report ORNL-5138, Oak Ridge
Trans., 2006, 761. National.
47 (a) R. A. Sharma, W. P. Steward and A. J. Gescher, Adv. Exp. 63 M. E. Reichmann, S. A. Rice, C. A. Thomas and P. Doty,
Med. Biol., 2007, 595, 453; (b) L. Shen and H. F. Ji, Trends J. Am. Chem. Soc., 1954, 76, 3047.
Mol. Med., 2012, 18, 138; (c) Y.-J. Wang, M.-H. Pan, 64 M. T. Carter, M. Rodriguez and A. J. Bard, J. Am. Chem.
A.-L. Cheng, L.-I. Lin, Y.-S. Ho, C.-Y. Hsieh and J.-K. Lin,
J. Pharm. Biomed. Anal., 1997, 15, 1867.
48 (a) E. J. Werner, A. Datta, C. J. Jocher and K. N. Raymond,
Soc., 1989, 111, 8901.
65 J. Bernadou, G. Pratviel, F. Bennis, M. Girardet and
B. Meunier, Biochemistry, 1989, 28, 7268.
Angew. Chem., Int. Ed., 2008, 47, 8568; (b) A. Datta and 66 T. Mosmann, J. Immunol. Methods, 1983, 65, 55.
K. N. Raymond, Acc. Chem. Res., 2009, 42, 938; 67 X. Qu and L. Qing, J. Biochem. Mol. Biol., 2004, 37, 445.
(c) J. L. Major and T. J. Meade, Acc. Chem. Res., 2009, 42, 68 J. L. McClintock and B. P. Ceresa, Invest. Opthalmol. Vis.
893.
Sci., 2010, 51, 3455.
This journal is © The Royal Society of Chemistry 2013
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