Targeted photocytotoxicity by copper(II) complexes having vitamin B6and photoactive acridine moieties

Article abstract of DOI:10.1016/j.ejmech.2016.07.003

Copper(II) pyridoxal Schiff base complexes [Cu(L¹/L²)(B)]ClO₄(1–4), where HL¹is 4-(((2-(1H-imidazol-4-yl)ethyl)imino)methyl)-5-(hydroxymethyl)-2-methylpyridin-3-ol (in 1 and 2), HL²is 2-(((2-(1H-imidazol-4-yl)ethyl)imino)methyl)phenol (in 3, 4), B is 11-(9-acridinyl)dipyrido[3,2-a:2′,3′-c]phenazine (acdppz in 1 and 3), dipyrido[3,2-a:2′,3′-c]phenazine (in 2) and 1,10-phenanthroline (in 4), were synthesized, characterized and their photocytotoxicity in visible light, intracellular localization, cellular uptake and DNA photocleavage activity were studied. Complex 4 was characterized by X-ray crystallography. Complexes 1 and 3 having acdppz as photosensitizer showed significant photocytotoxicity in visible light in HeLa and MCF7 cells giving IC₅₀value of <0.6?μM, while being relatively non-toxic in dark. The complexes were non-toxic to non-tumorigenic HPL1D cells both in light and dark conditions. Complex 1 showed significant localization in the cytoplasm of HeLa cells within 4?h of treatment, as evidenced from confocal microscopy. DCFDA assay on 1 suggested generation of intracellular reactive oxygen species in HeLa cells upon photo-exposure. Importantly, Annexin-V-FITC/PI assay indicated photo-induced apoptotic cell death.

Full text of DOI:10.1016/j.ejmech.2016.07.003

Accepted Manuscript
Targeted photocytotoxicity by copper(II) complexes having vitamin B6 and
photoactive acridine moieties
Nandini Mukherjee, Santosh Podder, Samya Banerjee, Shamik Majumdar, Dipankar
Nandi, Akhil R. Chakravarty
PII:
S0223-5234(16)30550-5
10.1016/j.ejmech.2016.07.003
EJMECH 8721
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 May 2016
Accepted Date: 3 July 2016
Please cite this article as: N. Mukherjee, S. Podder, S. Banerjee, S. Majumdar, D. Nandi, A.R.
Chakravarty, Targeted photocytotoxicity by copper(II) complexes having vitamin B6 and photoactive
acridine moieties, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.07.003.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
913 Targeted photocytotoxicity by copper(II) complexes
914 having vitamin B6 and photoactive acridine moieties
915 Nandini Mukherjee a, Santosh Podder b, Samya Banerjee a, Shamik
916 Majumdar b, Dipankar Nandi b,*, Akhil R. Chakravarty a,*
917
918
919
920
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
560 012, India
921
922
923
924
Graphical abstract: Ternary copper(II) complex of acridinyldipyridophenazine and vitamin
B6-derived Schiff base showed significant PDT effect in HeLa and MCF-7 cancer cells with
cytosolic localization, apoptotic cell death by photo-generated ROS and less toxicity in non-
tumorous HPL1D cells.
925
926
PICTOGRAM
927
46

ACCEPTED MANUSCRIPT
Ternary copper(II) complex of acridinyldipyridophenazine and vitamin B6-derived Schiff
base showed significant PDT effect in HeLa and MCF-7 cancer cells with cytosolic
localization, apoptotic cell death by photo-generated ROS and less toxicity in non-tumorous
HPL1D cells.

ACCEPTED MANUSCRIPT
1
2
Targeted photocytotoxicity by copper(II) complexes
having vitamin B6 and photoactive acridine moieties
3
4
Nandini Mukherjee a, Santosh Podder b, Samya Banerjee a, Shamik
Majumdar b, Dipankar Nandi b,*, Akhil R. Chakravarty a,*
5
6
7
8
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
560 012, India
9
A B S T R A C T
10
11
12
13
14
15
16
17
18
19
20
21
22
Copper(II) pyridoxal Schiff base complexes [Cu(L1/L2)(B)]ClO4 (1-4), where HL1 is 4-(((2-
(1H-imidazol-4-yl)ethyl)imino)methyl)-5-(hydroxymethyl)-2-methylpyridin-3-ol (in 1 and
2), HL2 is 2-(((2-(1H-imidazol-4-yl)ethyl)imino)methyl)phenol (in 3, 4), B is 11-(9-
acridinyl)dipyrido[3,2-a:2’,3’-c]phenazine (acdppz in 1 and 3), dipyrido[3,2-a:2’,3’-
c]phenazine (in 2 ) and 1,10-phenanthroline (in 4), were synthesized, characterized and their
photocytotoxicity in visible light, intracellular localization, cellular uptake and DNA
photocleavage activity were studied. Complex 4 was characterized by X-ray crystallography.
Complexes 1 and 3 having acdppz as photosensitizer showed significant photocytotoxicity in
visible light in HeLa and MCF7 cells giving IC50 value of <0.6 µM, while being relatively
non-toxic in dark. The complexes were non-toxic to non-tumorigenic HPL1D cells both in
light and dark conditions. Complex 1 showed significant localization in the cytoplasm of
HeLa cells within 4h of treatment, as evidenced from confocal microscopy. DCFDA assay on
1 suggested generation of intracellular reactive oxygen species in HeLa cells upon photo-
1

ACCEPTED MANUSCRIPT
23
24
25
26
27
28
29
30
31
exposure. Importantly, Annexin-V-FITC/PI assay indicated photo-induced apoptotic cell
death.
*Corresponding authors
E-mail adddress: arc@ipc.iisc.ernet.in (A.R. Chakravarty)
Tel.: +91-80-22932533
E-mail address: nandi@biochem.iisc.ernet.in (D. Nandi)
Tel: +91-80-22933051
Keywords:
Medicinal chemistry
32
33
34
35
36
Copper
Vitamin B6
Acridine
Photocytotoxicity
Tumour selectivity
Highlights:
37
38
39
40
41
42
43
44
45
•
•
•
•
•
The copper(II) acridine conjugates are remarkably PDT active in visible light.
The complexes are efficient photo-cleavers of plasmid DNA in UV-A and red light.
Flow cytometry of the active complex shows ROS generation upon light exposure.
Confocal microscopy shows cytosolic localization of complex 1 in HeLa cells.
The complex having vitamin-B6 moiety shows selectivity towards cancer cells.
Abbreviations: acdppz, 11-(9-acridinyl)dipyrido[3,2-a:2’,3’-c]phenazine; ct-DNA, calf
thymus DNA; CT, charge transfer; DABCO, 1,4-diazabicyclo[2.2.2]octane; DCFDA, 2’,7’-
dichlorofluorescein diacetate; DMEM, Dulbecco’s Modified Eagle’s Medium; DMF,
2

ACCEPTED MANUSCRIPT
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
dimethylformamide; DMSO, dimethyl sulfoxide; DNA, deoxyribonucleic acid; DPBS,
Dulbecco’s phosphate buffered saline; dppz, dipyrido[3,2-a:2’,3’-c]phenazine; EB, ethidium
bromide; EDTA, ethylenediaminetetraacetic acid; FBS, fetal bovine serum; FITC, fluorescein
isothiocyanate; HeLa, Henrietta Lacks; HOMO, highest occupied molecular orbital; HPL1D,
human peripheral lung epithelial 1D; LUMO, lowest unoccupied molecular orbital; MCF7,
Michigan Cancer Foundation 7; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide; MvH, McGhee-von Hippel; NC, nicked circular; ORTEP, Oak Ridge Thermal-
Ellipsoid Plot; PBS, phosphate buffered saline; PDT, photodynamic therapy; phen, 1,10-
phenanthroline; PI, propidium iodide; ROS, reactive oxygen species; SC, supercoiled; SOD,
superoxide dismutase; TBAP, tetrabutylammonium perchlorate; TEMP, 2,2,6,6-tetramethyl-
4-piperdone.
Introduction
Photodynamic therapy (PDT) as a non-invasive treatment modality of various types of
cancers has three essential components, viz., a photosensitizer (PS), visible light source and
tissue oxygen [1-5]. Upon photo-activation, the photosensitizer leads to generation of
cytotoxic reactive oxygen species (ROS) that causes the death of the photo-exposed cancer
cells, while the light unexposed normal cells remain unaffected [6,7]. The advent of PDT
started from the seminal discovery of the photodynamic action of acridine dye in light on
paramecium in 1900 by Oscar Raab from the school of von Tappeiner [8, 9]. Subsequently,
Dougherty et al. reported haematoporphyrin derivative (HpD) with improved PDT activity
that led to the development of Photofrin as the FDA approved PDT drug showing activity in
red light of 633 nm forming singlet oxygen as the ROS [10-12]. The macrocyclic porphyrin
and phthalocyanine based organic dyes are known to suffer from side effects like prolonged
skin photosensitivity, low tumour selectivity and hepatotoxicity causing jaundice due to
3

ACCEPTED MANUSCRIPT
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
oxidative degradation of the drug [13-15]. These drawbacks can be circumvented by
designing transition metal complexes of photoactive ligands as new generation PDT agents
[16-18].
The present work stems from our interest to use acridine as a photoactive moiety in a
transition metal complex having an additional tumour targeting moiety for better efficacy of
the PDT agent. Acridine alone and its various derivatives including acridine orange were
earlier used as potential anti-tumour agents but with limited success (Chart 1) [19-26].
Acridine as a strong DNA intercalator is known to generate singlet oxygen species on UV
light irradiation [23]. While UV light is not preferred in conventional PDT, acridine dye
appended to a ligand bound to a transition metal can be activated in visible light. Besides, the
emission property of acridine (λem ~525 nm) could be used for cellular imaging to study its
localization inside the cells. These properties of acridine make it a suitable choice as a
photosensitizer in PDT with suitable modification for its targeted delivery into the cancer
over normal cells [27]. We have designed a ternary complex consisting of: (i) a
dipyridophenazine (dppz) base with a pendant photoactive acridine moiety (acdppz), (ii)
copper(II) as a bio-essential redox active metal ion and (iii) a Schiff base ligand having a
vitamin B6 (VB6) moiety. VB6 is chosen for its tumour targeting properties [28,29]. VB6 is
known to be taken up in cells by facilitated diffusion through VB6 transporting membrane
carriers (VTC) [30-32]. Besides, VB6 dependent enzyme serine hydroxymethyltransferase
(SHMT) plays a major role in DNA biosynthesis [33,34]. The enhanced SHMT activity in
tumours is known for highly proliferative and mitotic cells [35,36]. Due to the higher uptake
of VB6 in cancer cells as compared to the normal cells, the ternary copper(II) complexes
having a VB6 Schiff base ligand are expected to show greater cancer cell selectivity.
Herein, we present the synthesis, characterization and anti-cancer properties of new ternary
copper(II) complexes, viz., [Cu(L1/L2)(B)]ClO4 (1-4) of 1,10-phenanthroline (phen in 4) and
4

ACCEPTED MANUSCRIPT
96
97
its derivatives (B: acdppz in 1 and 3; dppz in 2) and tridentate Schiff base ligands derived
from VB6 (HL1: in 1 and 2) or salicylaldehyde (HL2: in 3 and 4) (Fig 1). Complexes 1 and 3
have the acridine moiety conjugated to dppz for its singlet oxygen generation property on
photo-activation [23]. Complexes 2 and 4 without the acridine moiety are used as controls.
Both the Schiff base ligands have a histamine moiety because of its anti-tumorigenesis
activity [37]. To explore the role of VB6 in tumour cell selectivity and greater cellular
uptake, control complex 3 of salicylaldehyde Schiff base is prepared and its biological
properties were compared with that of complex 1. Complex 4 is structurally characterized by
X-ray crystallography to know the coordination mode of the ligands at the metal centre. The
photocytotoxicity, cellular uptake, DNA binding and light-induced DNA cleavage activity of
the complexes are investigated. Complex 1 is used for cellular imaging. The significant
results of this study include: (i) observation of remarkable PDT activity of the acridine
complex, (ii) generation of singlet oxygen as the ROS on photo-activation of the acridine
moiety, (iii) cytosolic localization and (iv) tumour targeting property of the complex having
the VB6 moiety.
98
99
100
101
102
103
104
105
106
107
108
109
110
111
2. Chemistry
112
113
114
115
116
117
118
119
120
The Schiff base ligands were synthesized by refluxing a dry methanol solution of pyridoxal
hydrochloride (neutralized with 1.0 eq. of triethylamine) or salicylaldehyde and histamine
dihydrochoride (neutralized with 2.0 eq. of triethylamine) for 3h [38]. The 1,10-
phenanthroline (phen) derivatives (acdppz, dppz) were prepared following literature
procedures [39,40]. 9-Chloroacridine was prepared by bubbling anhydrous HCl through
ethanol solution of acridine followed by cooling at 4°C. Next step was a solventless
nucleophilic substitution reaction of 9-chloroacridine and o-phenylenediamine in presence of
sulfur at 130 °C to yield 9-(3,4-diaminophenyl)acridine (DAA). DAA was then refluxed with
1,10-phenanthroline-5,6-dione in ethanol for 4h. The solution was cooled to room
5

ACCEPTED MANUSCRIPT
121
122
123
124
125
126
temperature to get a yellow solid of acdppz. Dppz was prepared from a reaction of o-
phenylenediamine and 1,10-phenanthroline-5,6-dione. Copper(II) complexes 1-4 were
prepared by a general method in two steps (Scheme S1, supporting information). Copper(II)
acetate in methanol was reacted with the corresponding phen ligand. This was followed by
addition of the appropriate Schiff base in its deprotonated form. The complexes were isolated
as their perchlorate salts on addition of excess NaClO4.
127
3. Pharmacology
128
129
130
131
132
133
134
135
136
137
138
139
The cytotoxicity of the complexes 1-4 was determined by MTT assay in two cancer cell lines
viz., HeLa and MCF7 and in non-tumorous HPL1D cells, in dark and on photo-exposure to
visible light (400-700 nm). The incorporation of the complexes inside the HeLa, MCF7 and
HPL1D cells was ascertained by flow cytometric analysis. Intracellular localization of
complex 1 in HeLa cells was studied by confocal microscopy, where the green emissive
property of acdppz ligand was used for imaging. The mode of cell death upon photo-exposure
was assessed by Annexin-V-FITC/PI assay in HeLa cells. DCFDA assay was performed in
HeLa cells to detect generation of any reactive oxygen species (ROS) upon photo-irradiation.
The calf-thymus DNA binding strengths of the complexes were evaluated by spectroscopic
analyses and viscometric titration experiments. Chemical nuclease and photonuclease activity
of the complexes 1-4 were studied by analyzing their ability to cleave plasmid supercoiled
(SC) DNA into the nicked circular (NC) form.
140
141
4. Results and discussion
4.1.1. Synthesis and general aspects
142
143
The copper(II) complexes 1 and 2 of tridentate vitamin B6 Schiff base ligand were prepared
by first reacting copper(II) acetate monohydrate in methanol with respective bidentate
6

ACCEPTED MANUSCRIPT
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
heterocyclic base (acdppz in CHCl3-CH3OH for 1, dppz in CH3OH for 2) followed by
addition of the monoanionic Schiff base ligand (HL1). Complex 3 was synthesized following
the same procedure where deprotonated salicylaldehyde Schiff base ligand (HL2) was used
instead of HL1. Complex 4 was synthesized using phen as the heterocyclic base and
salicylaldehyde Schiff base as the NNO donor ligand. All the complexes were isolated in
~75% yield as their perchlorate salts (Fig. 1). Complexes 2 and 3 were prepared to explore
the role of the acridinyl moiety in showing the photocytotoxic effect and vitamin B6 moiety
for cellular uptake. The structure of the complexes was elucidated by synthesizing and
characterizing complex 4 by X-ray crystallography. The complexes were characterized from
the elemental, mass spectral and other physicochemical data (Table 1). The ESI-MS spectra
of the complexes in CH3CN showed prominent molecular ion peak corresponding to [M-
(ClO4)]+ (Fig. S1-S4, supporting information). The complexes were one-electron
paramagnetic with µeff values of ~1.8 µB at 25°C. They were 1:1 electrolytes in DMF giving
molar conductance values of 66-78 S m2 M-1 at 25°C. The solid state IR spectra of the
complexes showed characteristic peak for the metal coordinated C=N stretching vibration at
~1620 cm-1. The complexes also showed peaks for the uncoordinated perchlorate anion at
~1080 cm-1 and ~620 cm-1 (Fig. S5, supporting information) [41]. The UV-Visible
absorption spectra of the copper(II) complexes showed a Laporte forbidden d-d band at ~690
nm with low molar extinction coefficient (ε) value (Fig. 2). Complex 2 showed intense bands
at 380 and 360 nm corresponding to the π-π* and n-π* transitions of the dppz moiety.
Complexes 1 and 3 were found to have marginally red shifted bands at 390 nm and 362 nm
[39,42]. This red shift is attributed to the presence of the planar acridine moiety. All the
complexes showed another ligand based spectral band at ~270 nm in the UV region.
Complexes 1 and 3 on excitation at 390 nm showed green fluorescence at 520 nm and 515
nm, respectively (Fig. 2). The fluorescence quantum yield (ΦF) of 1 and 3 are ~0.07 in
7

ACCEPTED MANUSCRIPT
169
170
171
172
173
174
175
176
177
178
DMSO with anthracene (in ethanol) and fluorescein (in 0.1 M NaOH) being used as
standards. The low value of ΦF could be due to the presence of paramagnetic copper(II) ion
bonded to the acridine-dppz ligand. The cyclic voltammetric experiments were done in DMF-
0.1 M TBAP. The complexes showed redox activity involving both the metal center and the
ligands (Table 1, Fig. S6, supporting information). Complexes 1-3 showed a ligand based
reduction near –1.0 V vs. SCE. Complex 4 did not show any significant reduction peak for
the ligand. The complexes showed a quasi-reversible response for the Cu(II)-Cu(I) couple
near 0.0 V vs. SCE (Fig. S7, supporting information) [43]. The presence of metal based redox
process could lead to some dark toxicity of the complexes in presence of cellular reducing
thiols like glutathione (GSH).
179
4.1.2. X-ray crystal structure
180
181
182
183
184
185
186
187
188
189
190
191
Complex 4 was crystallized as its perchlorate salt from CH3OH solution and structurally
characterized by single crystal X-ray diffraction method [44-46]. The crystals belong to the
monoclinic space group C2/c with 8 molecules in the unit cell. An ORTEP view of the
complex is shown in Fig. 3. The structure showed the presence of a monocationic copper(II)
complex with a lattice perchlorate anion. The metal has a distorted square-pyramidal
geometry with a CuN4O core where the ONN-donor Schiff base ligand displays tridentate
chelating mode of bonding. The imidazole nitrogen of the ligand is bonded at the axial site of
the metal center. The Cu(1)-O(1) bond distance is 1.900(6) Å and the Cu-N distances are in
the range of 1.959(7) to 2.284(7) Å. Selected crystallographic parameters are given in Table
S1 (vide supporting information). Selected bond distances and bond angles are listed in Table
S2 (vide supporting information). Important bonding parameters are presented in the caption
of Fig. 3. The unit cell packing diagram is shown in Fig. S8 (vide supporting information)
192
4.2. Computational study
8

ACCEPTED MANUSCRIPT
193
194
195
196
197
198
199
The energy optimized structures of the complexes were obtained from DFT calculations (Fig.
4; Fig. S9, supporting information) [47-49]. The HOMO of the complexes 1 and 3 was
located on the acridine moiety with LUMO being concentrated over the phenazine moiety of
the acdppz ligand. The energy optimized structure of complex 2 revealed that the Schiff base
ligand and the phenazine moiety constitute the HOMO and LUMO, respectively. Complex 4
was found to have contribution from the Schiff base and phenanthroline ligand in the
formation of HOMO and LUMO, respectively.
200
4.3. Stability of the complexes
201
202
203
204
The solution stability of the complexes was studied in DMSO-DPBS (1:1 v/v) by monitoring
the change in the UV-Visible absorption intensity within a time period of 6h (Fig. S10,
supporting information). The complexes were fairly stable up to 6h and showed no apparent
change in the absorption intensity within the given time period.
205
206
4.4. Cellular incorporation studies
207
208
209
210
211
212
213
214
215
216
Cellular uptake of the fluorescent complex 1 was studied in HeLa, MCF7 and HPL1D cells
by FACS analysis. The cells were incubated with three different concentrations of the
complex for 4h in the dark. For HeLa and MCF7, 100% of the cells showed positive signal
for the uptake of the complex in all the three doses used (Fig. 5; Fig. S11, supporting
information). In contrast, a dose dependent increase in the fluorescence signal was observed
for HPL1D with 100% cell positivity at only the highest dose used (Fig 5; Fig S12,
supporting information). This indicates higher uptake of the complex in cancer cells as
compared to the normal ones. Complete incorporation of the complex in all the cell lines was
observed within 4h of incubation and this helped us to standardize the incubation time of the
complexes for other in-vitro studies prior to light irradiation.
9

ACCEPTED MANUSCRIPT
217
4.5. Photocytotoxicity studies
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
The photocytotoxic behaviour of the complexes 1-4 and the ligands was studied in three
different cell lines, viz., HeLa (human cervical cancer), MCF7 (human breast cancer) and
HPL1D (non-transformed human epithelial lung cells) by MTT assay (Fig. 6; Fig. S13-S16,
supporting information). The IC50 values are listed in Table 2 along with few other selected
compounds [28,43,50]. HPL1D was chosen as a normal control cell line to study the targeting
property of the VB6 moiety in the complexes. The cells were treated with different
concentrations of the complexes and the ligands. Incubation was done at 37°C for 4h in the
dark. They were subsequently subjected to photo-irradiation with visible light (400-700 nm)
for 1h while keeping appropriate dark controls. The complexes showed remarkable
photocytotoxicity in the cancer cells with IC50 values ranging from 0.36-8.0 µM in visible
light, while being comparatively less toxic in the dark. The IC50 values of the complexes were
found to be >40 µM in HPL1D cells in light or dark suggesting their less toxic behaviour in
normal cells. The extent of photocytotoxicity of the complexes followed the order: 1 > 3 > 2
> 4 for HeLa and 1 ≈ 3 >2 > 4 for MCF7. Complex 1 was found to be the most PDT active
among the complexes with ~40 fold lower IC50 value in light (IC50 = 0.36 µM) with respect to
the dark value (IC50 = 14 µM) in HeLa cells. The PDT effect of complex 1 is comparable to
that of Photofrin® [50]. Complex 3 having the acdppz ligand also showed significant
photocytotoxicity in HeLa cells with an IC50 value of 0.85 µM. Complexes 1 and 3 exhibited
similar PDT activity in MCF7 cells and the IC50 values showed ~14 fold increase when the
treated cells were exposed to light. Complex 2 displayed only ~2 fold increase in the
cytotoxicity upon light exposure in the cancer cells. The enhanced photocytotoxicity of
complexes 1 and 3 over complex 2 is due to the presence of photoactive acridine moiety
which is known to generate singlet oxygen species as the ROS upon photo-irradiation via a
Type-II process [51]. Complex 2 having only dppz as the photosensitizer is less effective and
10

ACCEPTED MANUSCRIPT
242
243
244
245
246
247
may follow a photo-redox pathway to generate ROS. Complex 4, devoid of any photoactive
moiety, did not show any significant change in the IC50 value on photoirradiation in any of
the cancer cell lines used. The dark toxicity of the complexes could be attributed to the
reduction of Cu(II) to Cu(I) by intracellular reducing agents like GSH and other thiols
[52,53]. All the ligands were tested for their cytotoxicity and found to be essentially nontoxic
in all the three cell lines with IC50 values >50 µM (in the dark and in visible light).
248
249
250
251
252
253
254
To examine the effect of VB6 Schiff base in cellular uptake, HeLa cells were treated
with vitamin B6 prior to the addition of the complexes 1 and 3. The IC50 value of complex 1
in light showed considerable increase to a value of ~0.90 µM from ~0.36 µM, whereas for
complex 3 the change was not significant (0.93 µM from ~0.85 µM) (Fig. 6). This suggests
greater uptake of vitamin B6 conjugated complex 1 as compared to its salicylaldehyde
analogue 3 [54]. Further cellular studies were carried out in HeLa cells using the most
effective complex 1 and the results are presented in the following sections.
255
4.6. Detection of intracellular ROS
256
257
258
259
260
261
262
263
264
265
DCFDA assay was performed in HeLa cells to examine the involvement of intracellular
reactive oxygen species (ROS) in photocytotoxicity of the complexes. DCFDA (2’,7’-
dichlorofluorescein diacetate) permeates through the cell membrane and gets converted to the
non-fluorescent DCF by intracellular esterases [55]. ROS oxidizes DCF to a fluorescent
species (emission maximum at 528 nm) thus enabling detection of ROS by FACS analysis.
HeLa cells were treated with complex 1 with varying concentrations both in presence and
absence of light (400-700 nm). Higher fluorescence was observed in light treated cells in a
dose dependent manner (Fig 7). The shift in fluorescence signal was not significant when the
cells were kept in the dark suggesting the generation of ROS only upon light activation. The
ROS formed on photo-activation possibly leads to cell death [56].
11

ACCEPTED MANUSCRIPT
266
4.7. Mode of cell death
267
268
269
270
271
272
273
274
275
The cells in the early stages of apoptosis undergo various morphological changes including
flipping of phosphatidylserine from inner portion of the cell membrane to the outer leaflet.
Annexin-V protein is known to bind this enzyme. To estimate the percentage of cells
undergoing early apoptosis, Annexin-V-FITC/PI assay was performed in HeLa cells where
the cells were treated with complex 1 for 4h with subsequent photo-irradiation for 1h. After
12h of incubation, flow cytometric analysis was done on the cells. Complex 1 induced ~13%
early apoptosis upon light exposure whereas ~9% of cells were in the late stage of apoptosis.
The percentage of cells in necrotic mode (PI stained) was considerably less as compared to
that in the apoptotic mode (Fig. 8).
276
4.8. Cellular localization
277
278
279
280
281
282
283
Confocal microscopy was used to monitor the localization of the green fluorescent complex 1
in the HeLa cells. The cells were treated with the complex in dark and after 4h incubation the
microscopic images were taken. Propidium iodide (PI) was chosen as the nuclear staining dye
since its excitation and emission maxima fall in a different range (535 nm and 617 nm
respectively) as compared to that of complex 1 (390 nm and 520 nm respectively). The
complex was found to localize primarily in the cytosol with minimal accumulation in the
nucleus (Fig. 9).
284
285
286
287
288
289
4.9. DNA binding and cleavage
DNA binding propensity of the complexes 1-4 was examined using different methods. The
data are given in Table 1. UV-Visible absorption titration showed that with increasing
addition of ct-DNA, absorption intensity of the charge transfer (CT) bands of the complexes
decrease (Fig. S17, supporting information). This spectral change was recorded to determine
the intrinsic equilibrium DNA binding constant (Kb) of the complexes. Kb values of the
12

ACCEPTED MANUSCRIPT
290
291
292
293
294
295
296
297
298
299
300
301
302
complexes 1-4 are in the range of 4.8 x 104 to 1.2 x 105 M-1. The complexes showed their
DNA binding strengths in the following order: 2 > 3 > 1 > 4. The DNA binding of the
complexes was also studied by viscometric titration data. Intercalation of a moiety between
base pairs generally leads to an increase in the contour length of DNA resulting in an
enhancement of its specific viscosity [57]. The relative specific viscosity (η/η0) of ct-DNA
was found to increase upon addition of the complexes giving the same order: 2 > 3 > 1 > 4
(Fig. 10). Ethidium bromide (EB) was used as a standard DNA intercalator and Hoechst dye
as a groove binder [58]. Complex 2 having an extended planar aromatic dppz ligand showed
significant intercalative binding to ct-DNA while complex 4 exhibited groove binding.
Although acridine is known to be a good DNA intercalator, complexes 1 and 3 with acdppz
ligand showed only partial intercalative binding to DNA. This might be due to the steric bulk
of acdppz with the acridine moiety being covalently linked to dppz in a nearly perpendicular
orientation (Fig. 4).
303
304
305
306
307
308
309
310
311
312
313
With the knowledge from confocal microscopy that complex 1 localizing
predominantly in the cytosol with minimal localization in the nucleus, mitochondrial DNA
could be a potential target of the complex [59]. Moreover, generation of ROS from the
acridine moiety in the active complex intrigued us to elucidate its nature upon light treatment.
We thus studied the DNA photocleavage activity of the complexes using supercoiled (SC)
pUC19 DNA. Complexes 1-3 (15 µM) were found to cleave ~70% of SC DNA to its NC
form in UV-A light of 365 nm. Complex 4 lacking any photo-active moiety did not show any
significant photocleavage of SC DNA. The choice of UV-A light was based on the CT band
at ~362 nm. The controls like DNA alone in light and the complexes in the dark did not show
any DNA photocleavage activity. The complexes did not show any DNA cleavage activity in
argon atmosphere indicating the necessity of O2 for ROS generation.
13

ACCEPTED MANUSCRIPT
314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
Complex 1 was also studied for mechanistic details of the cleavage process in UV-A
light of 365 nm and red light of 705 nm (Fig. 11, Fig. S18- S20, supporting information). Red
light was chosen because of the presence of the copper based d-d band near 690 nm. Several
external additives were used to detect the ROS involved in the cleavage process. Complex 1
(15 µM) in UV-A light (365 nm) showed cleavage of ~70% of SC DNA which reduced to
only ~35% in presence of the singlet oxygen quenchers like NaN3, TEMP and DABCO.
Addition of hydroxyl radical scavengers, viz., DMSO, KI and catalase gave ~40% NC DNA.
The cleavage activity of complex 1 was enhanced considerably (83%) in presence of D2O
which is known to stabilize 1O2 [60]. SOD was found to partially inhibit DNA photocleavage
activity (~50%) suggesting initial formation of superoxide radical anion which rapidly
•
degrades to OH radicals. Complex 1 (50 µM) also showed considerable cleavage activity
upon red light exposure (~63%). In presence of hydroxyl radical scavengers the extent of SC
DNA cleavage reduced to ~38%, whereas it was ~13% upon addition of singlet oxygen
quenchers. In D2O, the SC DNA cleavage efficiency of 1 was enhanced to 76% (Fig S18,
1
•
supporting information). The mechanistic data suggest formation of both O2 and OH in
presence of two different photoactive moieties, viz. acridine and dppz. To explore the role of
1
acridine in O2 generation, mechanistic study was performed using non-acridine complex 2.
•
The DNA photocleavage activity of 2 was found to decrease upon addition of OH radical
1
scavengers (~25% from ~78%) in UV-A light (Fig S19, supporting information). The O2
quenchers had no effect on its DNA photocleavage activity (~61%). It is thus inferred that the
acridine moiety is responsible for the generation of singlet oxygen species via type-II
pathway, while the dppz moiety generates the hydroxyl radical from a photo-redox pathway
enabled by the favourable potential of the Cu(II)-Cu(I) redox couple.
337
338
The chemical nuclease activity of the complexes was studied in the dark by using
glutathione (GSH) as a reducing agent and H2O2 as an oxidizing agent [61]. The complexes
14

ACCEPTED MANUSCRIPT
339
340
341
342
343
344
345
346
1-4 were found to be cleavage inactive in presence of H2O2. Complexes 1-3 (15 µM),
however, showed significant cleavage of SC DNA in presence of GSH (Fig. S20, supporting
information). This behaviour could be explained due to the presence of the Cu(II)-Cu(I)
redox couple at ~0.0 V which leads to facile reduction of Cu(II) to Cu(I) by GSH. The
cleavage efficiency of complex 4 was found to be less (~38%) in an identical condition. This
might be due to its inefficient binding to DNA. The dark cellular toxicity arising from the
chemical nuclease activity is expected to be low considering cytosolic localization of the
complexes.
347
5. Conclusions
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
Utilizing the singlet oxygen generation property of acridine, a ternary copper(II) complex (1)
of vitamin B6 Schiff base and acridinyldipyridophenazine (acdppz) ligand was designed for
targeted PDT. The complex was synthesized, characterized and its photocytotoxicity and
DNA photocleavage activity studied in both UV-A and visible light. Both 1 and its analogue
3 exhibited remarkable photocytotoxicity in HeLa and MCF7 cells with IC50 values in the
nanomolar range. They were relatively less toxic in the dark. Interestingly, complexes 1-4 did
not show any significant photo-cytotoxicity (>40 µM) in normal HPL1D cells. Complex 1
showed higher uptake than complex 3 possibly due to the presence of vitamin B6 derivative
in 1 that is likely to provide VTC mediated cellular uptake. Incorporation of acridine into the
complex enhanced singlet oxygen generation as observed from the DNA photo-cleavage
study. The visible light-induced cell death mainly proceeded through apoptotic pathway as
evidenced from the Annexin-V-FITC/PI assay. Confocal microscopy revealed primarily
cytosolic localization of complex 1 as compared to the nucleus. The results are of
significance as acridine and its organic derivatives alone are less effective in PDT and
generally devoid of any targeted PDT. The acridine-conjugated copper(II) complexes are
15

ACCEPTED MANUSCRIPT
363
364
likely to provide new impetus in PDT for their remarkable targeted photocytotoxicity and
tumour imaging properties.
6. Experimental Section
6.1. Materials
365
366
367
368
369
370
371
372
373
374
375
The reagents and chemicals were purchased from commercial sources (s.d. Fine Chemicals,
India; Sigma-Aldrich, USA) and used without further purification. The solvents were distilled
and dried following the literature procedures [62]. Supercoiled pUC19 DNA (cesium chloride
purified) was purchased from Bangalore Genei (India). MTT, DCFDA, propidium iodide,
Dulbecco’s modified eagles medium, Dulbecco’s phosphate buffered saline, fetal bovine
serum (FBS), Annexin-V/ PI kit, calf thymus (ct) DNA, agarose (molecular biology grade),
KI, NaN3, SOD, catalase, TEMP, DABCO, bromophenol blue, xylene cyanol, EB and
Hoechst 33342 were obtained from Sigma-Aldrich, USA. Ligands dppz and acdppz were
prepared following literature procedures [39,40].
376
6.2. Instrumentation
377
378
379
380
381
382
383
384
385
The elemental analyses were done using Thermo Finnigun Flash EA 1112 CHN analyser.
The infrared, electronic and fluorescence spectra were obtained using Bruker Alpha, Perkin-
Elmer Spectrum 650 and Perkin-Elmer LS 55 spectrophotometer, respectively. Molar
conductivity was measured with a Control Dynamics (India) conductivity meter.
Electrochemical measurements were made at 298K using EG&G PAR model 253 Versastat
potentiostat/galvanostat and a three electrode setup with glassy carbon as working, platinum
wire as auxiliary and saturated calomel electrode as a reference electrode (SCE).
Tetrabutylammonium perchlorate (TBAP, 0.1M) in DMF was used as a supporting
electrolyte. TBAP being a perchlorate salt was handled with care. Magnetic susceptibility of
16

ACCEPTED MANUSCRIPT
386
387
388
389
390
391
392
the complexes was measured using Sherwood scientific magnetic susceptibility balance at
298 K. Electronspray ionization mass spectral (ESI-MS) data were obtained using Agilent
1
6538 Ultra high definition (UHD) accurate Mass-Q-TOF (LC-HRMS) instrument. H NMR
spectral measurements for the ligands were done using Bruker 400 MHz NMR spectrometer.
Fluorescence assisted cell sorting experiments were done using FACS Verse and Canto
instrument (BD Biosciences). Confocal microscopic images were obtained using Leica
microscope (TCS-SP5, Germany) having oil immersion lens with magnification of 63X.
393
6.3. Synthesis of the complexes 1-4
394
395
396
397
398
399
400
401
402
403
404
405
The complexes 1-4 were prepared following a general synthetic procedure in which
copper(II) acetate monohydrate (0.20 g, 1 mmol) was dissolved in methanol (10 mL) and a
methanol solution (15 mL) of the phenanthroline base (0.20 g phen for 4, 0.28 g dppz for 2
and 0.46 g acdppz for 1, 3; 1 mmol) was added to it. The reaction mixture was stirred for 15
min at room temperature. Schiff base ligand HL1 (0.26 g, 1 mmol, for 1 and 2) or HL2 (0.21 g,
1 mmol, for 3 and 4) was dissolved in methanol (10 mL) and deprotonated by addition of one
eq. of triethylamine (0.10 g, 1 mmol). The solution containing monoanionic Schiff base was
added to the reaction mixture and stirred at room temperature for 1 h. To this was added
excess of sodium perchlorate and the complexes were precipitated as their perchlorate salts.
The precipitate was filtered off and washed thoroughly with chilled methanol and diethyl
ether and dried in vacuum over P4O10. Caution! The perchlorate salts were handled in small
quantities and with care.
406
6.3.1. [Cu(acdppz)(L1)](ClO4) (1)
407
408
409
Yield: 71%. Anal. Calcd for C44H32ClCuN9O6: C, 59.93; H, 3.66; N, 14.30. Found: C,
59.61; H, 3.35; N, 14.12. ESI-MS in MeCN (m/z): 781.1988 [M ̶
(ClO4)]+ . ΛM in DMF: 68 S
m2 M-1. UV-Vis in 10% aqueous DMF [λmax/nm (ε/M-1cm-1)]: 690 (200), 388 (23,380), 362
17

ACCEPTED MANUSCRIPT
410
411
412
(21,305), 267 (64,195). FT-IR (cm-1): 3390w, 3250w, 3061w, 1620vs (C=N), 1556s, 1482w,
1408s, 1357w, 1076s, 820m, 758m, 731m, 620s, 423m (vs, very strong; s, strong; m,
medium; w, weak). µeff = 1.84 µB at 298K.
413
6.3.2. [Cu(dppz)(L1)](ClO4) (2)
414
415
416
417
418
Yield: 78%. Anal. Calcd for C31H25ClCuN8O6: C, 52.84; H, 3.58; N, 15.90. Found: C,
52.45; H, 3.28; N, 15.72. ESI-MS in MeCN (m/z): 604.1441 [M ̶
(ClO4)]+. ΛM in DMF: 78 S
m2 M-1. UV-Vis in 10% aqueous DMF [λmax/nm (ε/M-1cm-1)]: 690 (180), 380 (18,224), 360
(18,510), 271 (60,615). FT-IR (cm-1): 3380w, 3254w, 3076w, 1628vs (C=N), 1562s, 1481w,
1408s, 1352w, 1079s, 819w, 744w, 719w, 614s, 423w. µeff = 1.8 µB at 298K.
419
6.3.3. [Cu(acdppz)(L2)](ClO4) (3)
420
421
422
423
424
Yield: 75%. Anal. Calcd for C43H29ClCuN8O5: C, 61.72; H, 3.49; N, 13.39. Found: C,
61.41; H, 3.22, N, 13.17. ESI-MS in MeCN (m/z): 736.1764 [M ̶
(ClO4)]+. ΛM in DMF: 67 S
m2 M-1. UV-Vis in 10% aqueous DMF [λmax/nm (ε/M-1cm-1)]: 690 (210), 388 (24,870), 362
(25,370), 271 (65,900). FT-IR (cm-1): 3357w, 3067w, 1620s (C=N), 1560s, 1487m, 1412s,
1356s, 1083vs, 820m, 759s, 730s, 620s, 423w. µeff = 1.85 µB at 298K.
425
6.3.4. [Cu(phen)(L2)](ClO4) (4)
426
427
428
429
430
Yield: 70%. Anal. Calcd for C24H20ClCuN5O5: C, 51.71; H, 3.62; N, 12.56. Found: C,
51.44; H, 3.29; N, 12.35. ESI-MS in MeCN (m/z): 458.1183 [M ̶
(ClO4)]+ . ΛM in DMF: 66 S
m2 M-1. UV-Vis in 10% aqueous DMF [λmax/nm (ε/M-1cm-1)]: 730 (200), 272 (60,650). FT-IR
(cm-1): 3375w, 3065w, 1620vs (C=N), 1570m, 1476m, 1405s, 1360s, 1082s, 848s, 727vs,
654m, 620m, 457m. µeff = 1.82 µB at 298K.
431
6.4. Solubility and stability
18

ACCEPTED MANUSCRIPT
432
433
434
435
436
The complexes 1-4 were soluble in DMSO and DMF, moderately soluble in acetonitrile,
methanol, chloroform and dichloromethane. They were insoluble in hydrocarbon solvents.
The complexes were stable both in solid and solution phases. The solution phase stability of
complex 1 was studied by monitoring its UV-visible spectra over a time period of 6h in
DMSO-phosphate buffer (1:1 v/v) and it showed no apparent degradation.
437
6.5. X-ray crystallographic procedure
438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
453
454
The crystal structure of complex 4 was obtained by single crystal X-ray diffraction method.
The crystals were obtained on slow evaporation of a methanol solution of the complex.
Crystal mounting was done on glass fiber with epoxy cement. All geometric and intensity
data were collected at room temperature using an automated Bruker SMART APEX CCD
diffractometer equipped with a fine focus 1.75 kW sealed tube Mo-Kα X-ray source ( λ =
0.71073 Å) with increasing ω (width of 0.3° frame) at a scan speed of 5 sec per frame.
Intensity data were collected using ω-2θ mode and corrected for Lorentz-polarization effect
and for absorption [44]. The structure was solved using Patterson and Fourier techniques and
refined by full matrix least squares method using SHELX system of programs [45]. The
hydrogen atoms belonging to the complex were corrected by using riding model. All non-
hydrogen atoms were refined anisotropically. The view of the molecule was obtained by
ORTEP [46]. Selected crystal data are: C24H20ClCuN5O5, Fw/ g M-1 = 557.44, space group
C2/c, a = 21.201(3), b = 15.123(3), c = 15.661(3) Å,
α = 90º, β = 112.351(7)º, γ = 90º, V
= 4644.3(14) Å3, Z = 8, ρcalcd = 1.594 g cm−3, T = 293(2)K, reflections collected/unique:
55592/4207, F(000) = 2280, Goodness-of-fit = 1.257, R(F0) [wR(F0)] = 0.1227 [0.2623]
for 2762 reflections [I > 2σ(I)] and 329 parameters; R (all data)[wR (all data)] = 0.1704
[0.2823]. The CCDC deposition number is 1445849.
455
6.6. Computational study
19

ACCEPTED MANUSCRIPT
456
457
458
459
The calculations were done in Gaussian09 program [47-49]. The geometries of the complexes
1-4 were optimized by density functional theory (DFT) using B3LYP level of theory and
LanL2DZ basis set. The calculated coordinates are given in Tables S3-S6 (vide supporting
information).
460
6.7. Cell culture
461
462
463
HeLa, MCF7 and HPL1D cells were grown in 100 mm polymer culture dishes (SPL Life
sciences, Korea) in DMEM supplemented with 10% FBS, 100 U penicillin/mL, 100 µg
streptomycin/mL and incubated in a humidified 5% CO2 incubator (Sanyo, UK) at 37 °C.
464
465
6.8. Cellular experiments
6.8.1. Cell viability assay
466
467
468
469
470
471
472
473
474
475
476
477
478
The photocytotoxic behavior of the complexes in two cancer cell lines viz. HeLa, MCF7 and
in normal HPL1D cells was studied by MTT assay [63]. For each individual cell line,
approximately 8000 cells were seeded in two 96 well plates in 100 µL media per well. The
cells were grown for 24h in at 37 °C in a CO2 incubator. The complexes were dissolved in
1% DMSO and were added to the cells at different concentrations. Incubation was continued
for another 4h and then the media of one plate was replaced with DPBS and photoirradiation
was given for 1h in broad-band visible light (400-700nm) using a Luzchem Photoreactor
(Model LZC-1, Ontario, Canada; light fluence rate: 2.4 mW cm-2; light dose: 10 J cm-2). This
was followed by removal of DPBS and addition of fresh media. The media of the other plate
was discarded and replaced with fresh media. Both the plates were incubated for a further
period of 20h in the dark which was followed by addition of 25 µL MTT (4 mg mL-1) in each
well and additional 3h incubation in dark. Finally the culture media was discarded from each
well of the plates and 200 µL DMSO was added to each well to solubilize the purple
20

ACCEPTED MANUSCRIPT
479
480
481
482
formazan crystals. The absorbance at 540 nm was measured using a Molecular Devices
Spectra Max M5 plate reader. The cytotoxicity of the complexes was measured as the
percentage ratio of absorbance of treated cells to the untreated controls. The IC50 values were
determined by using nonlinear regression analysis (Graph Pad Prism 6).
483
6.8.2. Cellular incorporation assay
484
485
486
In 6 wells plates, 3 x 105 cells per well were plated and grown for 24 h. Cells were treated
with the different doses (low, 1 ꢀM; medium, 2 ꢀM; high, 3 ꢀM) of complex 1 and incubated
for 4h. Cells were then trypsinized, washed with DPBS and analyzed by flow cytometry.
487
6.8.3. Detection of intracellular ROS by DCFDA assay
488
489
490
491
492
493
494
To ascertain the involvement of reactive oxygen species (ROS) in the cellular activity of the
complexes, generation of ROS was determined using 2’7’-dichlorofluorescein diacetate
(DCFDA) assay [64]. Cells were incubated with complex 1 (low, 0.5 ꢀM; medium, 1 ꢀM;
high, 3 ꢀM) for 4h followed by photoexposure (400-700nm) for 1h in PBS. Cells were then
trypsinized and 10 µM of DCFDA was added to 0.25 x106 cells. The cells were incubated at
37°C for 30 min in dark. The cells were washed with DPBS and acquired on a FACS Verse
flow cytometer (Biosciences, USA).
495
6.8.4. Annexin-V/FITC assay
496
497
498
499
500
501
Annexin-V and PI staining were performed according to the manufacturer’s instructions
(eBioscience, USA). Briefly, HeLa cells (0.25 x 106) were incubated with complex 1
(high dose, 3 ꢀM) in 10% DMEM for 4h followed by 1h of photoexposure (400-700 nm)
and cultured for 10h. Then Annexin-V was added to the cells and incubated at room
temperature for 10 min in the dark. The cells were then washed and propidium iodide
(PI) was added. The fluorescence of the cells was acquired immediately using FACS
21

ACCEPTED MANUSCRIPT
502
503
504
505
506
Verse flow cytometer. On the basis of forward scatter height and forward scatter area,
single cells were selected. This gate was applied on the forward area versus side scatter
area plot to select on live singe cells. On application of these two gates, 10,000 events
were recorded. For apoptosis studies, only the single cells were gated. WinMDI (USA)
software was used to analyse the results.
507
6.8.5. Confocal microscopy
508
509
510
511
512
513
514
515
The emissive property of complex 1 was used in studying its internal localization in HeLa
cells using a confocal microscope (Leica,TCS-SP5, Germany). Cells were grown on glass
cover slips in a 12 well plate at a seeding density of 2 x 105 cells/1.5 mL media for 24h
followed by addition of 1 (15 ꢀM) and further incubation for 4h in the dark. The media was
then discarded from each well and cells were fixed and permeabilized with chilled methanol
for 3 min at -20°C. Methanol was removed and cells were washed thoroughly with DPBS.
Nuclear staining was done by incubating the cells with PI (0.25 mg mL-1) for 3 min. Finally,
the cover slips were mounted on slides and visualized under a confocal microscope.
516
6.9. DNA binding experiments
517
518
519
520
521
522
523
524
525
Binding of the complexes 1-4 to ct-DNA was studied by spectral and viscometric
measurements. Tris-HCl/NaCl buffer (5 mM Tris-HCl, 5 mM NaCl, pH=7.2) was used for
UV-visible absorption and viscometric titrations using ct-DNA of 180 µM and 150 µM
respectively. DMF was used to dissolve the complexes. Purity of ct-DNA was checked from
the ratio of absorbance at 260 and 280 nm, which was found to be approximately 1.9: 1. The
concentration of DNA was measured from its absorption intensity at 260 nm with its known
molar extinction coefficient (ε) of 6600 M-1 cm-1. UV-Vis absorption titration experiments
were performed by changing the concentration of ct-DNA while keeping constant complex
concentration (30 µM). Due corrections were made for absorbance of the complexes and ct-
22

ACCEPTED MANUSCRIPT
526
527
528
529
530
531
532
533
534
535
DNA itself. Each spectrum was recorded only after 5 min of ct-DNA addition to allow
equilibration of the sample. The intrinsic binding constant (Kb) of the complexes to ct-DNA
was calculated using Mcghee-von Hippel (MvH) equation [65,66]. The non-linear regression
2
analysis equation was: (εa - εf)/(εb - εf) = (b - (b2 - 2Kb Ct[DNA]t/s)1/2)/2KbCt, where b = 1+
KbCt + Kb[DNA]t/2s, εa is the molar extinction coefficient of the CT band at a given DNA
concentration, εf is the molar extinction coefficient of DNA unbound complex, εb is molar
extinction coefficient of complex when fully bound to DNA, Ct is the total complex
concentration, [DNA]t is the DNA concentration, Kb is the equilibrium binding constant and s
is the MvH fitting parameter. The graph plotting and non-linear least square analyses were
done using Origin Lab, version 8.
536
537
538
539
540
541
542
543
544
The viscosity measurements were carried out using a Schott Gerate AVS 310
Automated Viscometer connected to a constant temperature bath at 37°C. A 100 µL of
complex solution (200 µM) in DMF was added each time to 150 µM stock solution of DNA
in Tris-HCl buffer and the average flow time (t) was measured after homogenizing the
solution. The average flow time of only buffer solution (t0) was also measured with an
automated timer. The viscosity values were determined from the average flow time of DNA-
containing solutions (t), duly corrected for that of the buffer alone (t0), η= (t-t0)/t0. The
relative specific viscosity of DNA (η/η0)1/3 was plotted against [complex]/ [DNA], where η is
the specific viscosity of DNA in presence of the complex and η0 is that of DNA alone.
545
6.10. DNA cleavage experiments
546
547
548
549
DNA cleavage experiments were performed using supercoiled pUC19 DNA (1 µL, 30 µM,
2686 base-pairs) in 50 mM Tris-HCl buffer containing 50 mM NaCl solution and DMF
solution of the complexes. Chemical nuclease activity was studied using GSH as a reducing
agent and H2O2 as an oxidizing agent. Photocleavage activity of the complexes was studied
23

ACCEPTED MANUSCRIPT
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
using UV-A light (365 nm) and diode laser of red light (705 nm, 38 mW, Model: LQC705-
38E from Newport Corporation with LD module, continuous wave (CW) elliptical beam).
Mechanistic studies were carried out by using different additives (NaN3: 5 mM, TEMP: 5
mM, DABCO: 5mM, DMSO: 4 µL, KI: 5 mM, Catalase: 4 units, SOD: 4 units), which were
added to SC DNA prior to the addition of the complexes and light exposure. Each sample
was incubated in the dark for 2h at 37 °C before 30 min of UV-A light exposure and for 1h
before 2h of red-light exposure. The samples were then loaded with a buffer containing 25%
bromophenol blue, 25% xylene cyanol, 30% glycerol (3µL) and finally in 1% agarose gel
containing 0.5µg/mL ethidium bromide (EB). The gel electrophoresis was carried out in a
dark chamber for ~2h at 60V in TAE (Tris Acetate EDTA) buffer. The bands were visualized
by UV-light and photographed. The intensities of the bands, which gave the measure of
extent of SC DNA cleavage, were determined using UVITECH Gel Documentation System.
Due corrections were made for low percentage of NC DNA present in the supplied SC DNA
and for the low binding affinity of EB towards SC DNA as compared to the its NC and linear
forms [67]. The error in quantification of band intensities was ~5%. Appropriate controls
were kept for all the experiments.
566
567
568
569
570
571
572
573
574
Acknowledgements
We thank the Department of Science and Technology (DST), Government of India, for
financial support (SR/S5/MBD-02/2007, EMR/2015/000742). A.R.C. thanks DST for J.C.
Bose national fellowship. A part of this study was performed at the Bioimaging and flow
cytometry facility, Division of Biological Sciences, IISc. This facility is supported by the
Department of Biotechnology (DBT)-IISc partnership program. We are thankful to Dr.
Sanjoy Mukherjee for the DFT study. N.M. thanks the University Grants Commission
(UGC), New Delhi, for a research fellowship. The authors thank the Alexander von
Humboldt Foundation for donation of an elctroanalytical system.
24

ACCEPTED MANUSCRIPT
575
576
577
578
579
580
581
582
583
Supplementary data
The supplementary data contain synthetic scheme (Scheme S1), ESI-MS (Fig. S1-S4), IR
spectra (Fig. S5), cyclic voltammograms (Fig. S6, S7), unit cell packing (Fig. S8), optimized
structure (Fig. S9), stability studies (Fig. S10), cellular uptake (Fig. S11,S12), MTT plots
(Fig. S13-S16), DNA binding plot (Fig. S17) and DNA cleavage gel and bar diagrams (Fig.
S18-S20), crystallographic parameters (Tables S1, S2) and coordinates of the optimized
structures (Tables S3-S6). Supplementary data associated with this article can be found, in
the online version, at doi:xxxxxxxxxxxxxxxx.
References
584
585
586
587
588
589
590
591
592
593
594
595
596
597
598
599
600
[1] W.M. Sharman, C.M. Allen, J.E.V. Lier, Drug Discov. Today 4 (1999) 507-517.
[2] R. Bonnett, Photodynamic Action, Chemical Aspects of Photodynamic Therapy,
vol. 1, Gordon & Breach, London, U.K, 2000, pp. 57-88.
[3] D.E.J.G.J. Dolmans, D. Fukumura, R.K. Jain, Nat. Rev. Cancer 3 (2003) 380-387.
[4] S.B. Brown, E.A. Brown, I. Walker, Lancet Oncol. 5 (2004) 497-508.
[5] J.F. Lovell, T.W.B. Liu, J. Chen, G. Zheng, Chem. Rev. 110 (2010) 2839-2857.
[6] N.L. Oleinick, R.L. Morris, I. Belichenko, Photochem. Photobiol. Sci. (2001) 1-21.
[7] K. Szacilowski, W. Macyk, A. Drzewiecka-Matuszek, M. Brindell, G. Stochel, Chem.
Rev. 105 (2005) 2647-2694.
[8] O. Raab, Z. Biol. 39(1900) 524-546.
[9] H.V. Tappeiner, O. Raab, Munch. Med. Wochenschr. 47(1900) 5.
[10] X.Y. He, R.A. Sikes, S. Thomsen, W.K. Chung, S.L. Jacques, Photochem. Photobiol.
59 (1994) 437-468.
[11] I.J. Macdonald, T J. Dougherty, J. Porphyrins Phthalocyanins 5 (2001) 105-129.
[12] K. Takahira, M. Sano, H. Arai, H. Hanai, Lasers Med. Sci. 19 (2004) 89-94.
[13] H. Kato, T. Horai, K. Furuse , M. Fukuoka, S. Suzuki, Y. Hiki, Y. Ito, S. Mimura, Y.
Tenjin, H. Hisazumi, Y. Hayata, Jpn. J. Cancer Res. 4 (1993) 1209-1214.
25

ACCEPTED MANUSCRIPT
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
619
620
621
622
623
624
625
626
627
[14] M. Ochsner, J. Photochem. Photobiol. B. Biol. 32 (1996) 3-9.
[15] U.O. Nseyo, B. Shumaker, E.A. Klein, K. Sutherland, J. Urol. 160 (1998) 39-44.
[16] (a) J.M. Hope, J.J. Wilson, S.J. Lippard, Dalton Trans. 42 (2013) 3176-3180;
(b) Y. Zhao, N.J. Farrer, H. Li, J.S. Butler, R.J. McQuitty, A. Habtemariam, F. Wang,
P.J. Sadler, Angew. Chem. Int. Ed. 52 (2013) 13633-13637.
[17] X. Wang, Z. Guo, Chem. Soc. Rev. 42 (2013) 202-224.
[18] B.A. Albani, B. Peña, N.A. Leed, N.A.B.G. . Paula, C. Pavani, M. S. Baptista,
K.R. Dunbar, C. Turro, J. Am. Chem. Soc. 136 (2014) 17095-17101.
[19] H.-J. Xiang, L. An, W.-W. Tang, S.-P. Yang, J.-G. Liu, Chem. Commun. 51 (2015)
2555-2558.
[20] (a) E.T. Martins, H. Baruah, J. Kramarczyk, G. Saluta, C.S. Day, G.L. Kucera, U.
Bierbach, J. Med. Chem. 44 (2001) 4492-4496;
(b) C.G. Barry, E.C. Turney, C.S. Day, G. Saluta, G.L. Kucera, U. Bierbach, Inorg.
Chem. 41 (2002) 7159-7169;
(c) K. Cheung-Ong, K.T. Song, Z. Ma, D. Shabtai, A.Y. Lee, D. Gallo, L.E. Heisler, G.W.
Brown, U. Bierbach, G. Giaever, C. Nislow, Chem. Biol. 7 (2012) 1892-1901.
[21] P. Yang, Q. Yang, X. Qian, L. Tong, X. Li, J. Photochem. Photobiol. B: Biol.
84 (2006) 221-226.
[22] K. Kusuzaki, H. Murata, T. Matsubara, H. Satonaka, T. Wakabayashi, A. Mastsumine,
A. Uchida, In Vivo 21 (2007) 205-214.
[23] S. Banerjee, P. Prasad, I. Khan, A. Hussain, P. Kondaiah, A.R. Chakravarty, Z. Anorg.
Allg. Chem. 640 (2014) 1195–1204.
[24] L. Cizeková, A. Grolmusová, Z. Ipóthová, Z. Barbieriková, V. Brezová, L. Hunáková, J.
Imrich, L. Janovec, I. Dovinová, H. Paulíková, Bioorg. Med. Chem. 22 (2014) 4684-4693.
[25] G. Cholewiñski, K. Dzierzbicka, A.M. Koodziejczyk, Pharmacological Reports 63
(2011) 305-336.
[26] V. Murray, J.K. Chen, A.M. Galea, Anti-Cancer Agents Med. Chem. 14 (2014) 695-705.
26

ACCEPTED MANUSCRIPT
628
[27] F. Schmitt, L. Juillerat-Jeanneret, Anti-Cancer Agents Med. Chem. 12 (2012) 500-525.
629
630
631
632
633
634
635
636
637
638
639
640
641
642
643
644
645
646
647
648
649
650
651
652
653
[28] (a) S. Banerjee, A. Dixit, R.N. Shridharan, A.A. Karande, A.R. Chakravarty, Chem.
Commun. 50(2014) 5590-5592;
(b) S. Banerjee, A. Dixit, A.A. Karande, A.R. Chakravarty, Dalton. Trans. 45 (2016) 783-
796.
[29] R. Spector, C.E. Johanson, J. Neurochem. 103 (2007) 425-438.
[30] S. Pandey, P. Garg, K.T. Lim, J. Kim, Y.H. Choung, Y.J. Choi, P.H. Choung, C.S.
Cho, J.H. Chung, Biomaterials 34 (2013) 3716-3728.
[31] S. Pandey, P. Garg, S. Lee, H.W. Choung, Y.H. Choung, P.H. Choung, J.H. Chung,
Biomaterials 35 (2014) 9332-9342.
[32] J. Stolz, M. Vielreicher, J. Biol. Chem. 278 (2003) 18990-18996.
[32] H. Hellman, S. Mooney, Molecules 15 (2010) 442-459.
[33] S. Mooney, J. E. Leuendorf, C. Hendrickson, H. Hellmann, Molecules 14 (2009) 329-
351.
[34] G. Giardina, P. Brunotti, A. Fiascarelli, A. Cicalini, M. G. S. Costa, A. M. Buckle, M.
L. D. Salvo, A. Giorgi, M. Marani, A. Paone, S. Rinaldo, A. Paiardini, R. Contestabile, F.
Cutruzzola, FEBS J. 282 (2015) 1225-1241.
[35] J.F. Gregory, Nutr. Rev. 50 (1992) 295-297.
[36] J.V. Skelly, S.B. Renwick, K.J. Chave, P.G. Sanders, K. Snell, U. Baumann, Acta.
Crystallogr. D. 54 (1998) 1030-1031.
[37] B. Ruffell, L.M . Coussens. Nat. Med. 17 (2011) 43-44.
[38] T. Mukherjee, J.C. Pessoa, A. Kumar, A.R. Sarkar, Inorg. Chem. 50 (2011) 4349-
4361.
[39] M. Mariappan, M. Suenaga, A. Mukhopadhyay, P. Raghavaiah, B.G. Maiya, Inorg.
Chim. Acta 376 (2011) 340-349.
[40] (a) J.E. Dickeson, L.A. Summers, Aust. J. Chem. 23 (1970) 1023-1027;
27