Dinuclear CuI complexes of pyridyl-diazadiphosphetidines and aminobis(phosphonite) ligands: Synthesis, structural studies and antiproliferative activity towards human cervical, colon carcinoma and breast cancer cells

Article abstract of DOI:10.1039/c4dt00832d

The copper(i) complexes containing phosphorus donor ligands such as diazadiphosphetidine, cis-{(o-OCH₂C₅H₄N)P(μ- N^tBu)}₂ (1) and aminobis(phosphonite), C₆H ₅N{P(OC₆H₃(OMe-o)(C₃H ₅-p))₂}₂ (2, PNP), have been synthesized. Treatment of 1 with copper iodide afforded the 1D coordination polymer [{Cu(μ-I)}₂{(o-OCH₂C₅H₄N)P(μ- N^tBu)}₂]_n (3). Treatment of 3 with 2,2′-bipyridine (bpy) and 1,10-phenanthroline (phen) produced mixed-ligand complexes [(L)₂Cu₂{(o-OCH₂C₅H ₄N)P(μ-N^tBu)}₂][I]₂ (4 L = bpy; 5 L = phen) in good yields. The reaction of 2 with copper iodide yielded a rare tetranuclear copper complex [(CuI)₂C₆H ₅N(PR₂)₂]₂ (6), which on subsequent treatment with various pyridyl ligands produced binuclear complexes [{Cu(μ-I)(py)}₂(μ-PNP)] (7), [Cu₂(μ-I)(bpy) ₂(μ-PNP)]I (8), [Cu₂(μ-I)I(bpy)(μ-PNP)] (9), [Cu₂(phen)(bpy)(μ-PNP)](OTf)₂ (10), [Cu ₂(μ-I)I(phen)(μ-PNP)] (11) and [Cu₂(μ-I)(phen) ₂(μ-PNP)]I (12), in an almost quantitative yield. The new copper(i) complexes (4, 5 and 7-12) were tested for anti-cancer activity against three human tumor cell lines. Compounds 5, 10 and 12 showed in vitro antitumor activity 5-7 fold higher than cisplatin, the most used anticancer drug. These three most potent compounds (5, 10 and 12) were chosen for detailed study to understand their mechanism of action. The copper(i) compounds studied in the present investigation were found to inhibit tumor cell growth by arresting cells at the S-phase of the cell cycle. The characteristic nuclear morphology of treated cells showed signs of DNA damage. The experimental evidence clearly indicated that these compounds initiated apoptosis, which is mediated through the p53 pathway. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4dt00832d

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Dinuclear CuI complexes of pyridyl-
diazadiphosphetidines and aminobis(phosphonite)
ligands: synthesis, structural studies and
Cite this: Dalton Trans., 2014, 43,
11339
antiproliferative activity towards human cervical,
colon carcinoma and breast cancer cells†
Aijaz Rashid,‡a Guddekoppa S. Ananthnag,‡b Susmita Naik,b Joel T. Mague,c
Dulal Panda*a and Maravanji S. Balakrishna*b
The copper(I) complexes containing phosphorus donor ligands such as diazadiphosphetidine, cis-
{(o-OCH2C5H4N)P(µ-NtBu)}2 (1) and aminobis(phosphonite), C6H5N{P(OC6H3(OMe-o)(C3H5-p))2}2
(2, PNP), have been synthesized. Treatment of 1 with copper iodide afforded the 1D coordination polymer
[{Cu(µ-I)}2{(o-OCH2C5H4N)P(µ-NtBu)}2]n (3). Treatment of 3 with 2,2’-bipyridine (bpy) and 1,10-phen-
anthroline (phen) produced mixed-ligand complexes [(L)2Cu2{(o-OCH2C5H4N)P(µ-NtBu)}2][I]2 (4 L = bpy;
5 L = phen) in good yields. The reaction of 2 with copper iodide yielded a rare tetranuclear copper
complex [(CuI)2C6H5N(PR2)2]2 (6), which on subsequent treatment with various pyridyl ligands produced
binuclear complexes [{Cu(μ-I)(py)}2(μ-PNP)] (7), [Cu2(μ-I)(bpy)2(μ-PNP)]I (8), [Cu2(μ-I)I(bpy)(μ-PNP)] (9),
[Cu2(phen)(bpy)(μ-PNP)](OTf)2 (10), [Cu2(μ-I)I(phen)(μ-PNP)] (11) and [Cu2(μ-I)(phen)2(μ-PNP)]I (12), in an
almost quantitative yield. The new copper(I) complexes (4, 5 and 7–12) were tested for anti-cancer activity
against three human tumor cell lines. Compounds 5, 10 and 12 showed in vitro antitumor activity 5–7 fold
higher than cisplatin, the most used anticancer drug. These three most potent compounds (5, 10 and 12)
were chosen for detailed study to understand their mechanism of action. The copper(I) compounds
studied in the present investigation were found to inhibit tumor cell growth by arresting cells at the
S-phase of the cell cycle. The characteristic nuclear morphology of treated cells showed signs of DNA
damage. The experimental evidence clearly indicated that these compounds initiated apoptosis, which is
mediated through the p53 pathway.
Received 19th March 2014,
Accepted 19th May 2014
DOI: 10.1039/c4dt00832d
www.rsc.org/dalton
present severe toxic and undesirable side effects such as
nephrotoxicity and neurotoxicity, and often the treatment
Introduction
Ever since the active cancer drug cisplatin was discovered,1 failure was also due to the development of resistance to these
a variety of transition metal complexes were looked upon as complexes.9–12 To make chemotherapy more effective with
potential anticancer reagents.2–4 Even today, platinum based minimum side-effects, it is necessary to design metallodrugs
drugs such as cisplatin and carboplatin are highly active which are less toxic and highly active in smaller dosages,
towards testicular and ovarian cancer, bladder carcinoma and especially against cell lines that have acquired high resistance
non-small-cell lung cancer.5–8 However, these compounds towards cisplatin.13,14 In this context, modification of the
ligand structure and employment of other platinum metals
were given importance to overcome the toxic side effects.15 The
aDepartment of Biosciences and Bioengineering, Indian Institute of Technology
focus was on group 11 metals for cancer therapy owing to the
Bombay, Powai, Mumbai 400076, India
bPhosphorus Laboratory, Department of Chemistry, Indian Institute of Technology
Bombay, Powai, Mumbai 400076, India
long known utility of gold complexes in the treatment of arthritis,
turberculosis and, in the 1970s, p388 leukemia.16 Saddler and
others have extensively studied antitumor properties of gold(I)
as well as ruthenium(II) complexes containing phosphines,
N-heterocyclic carbenes and nitrogen based ligands.8,17–23
Copper complexes are considered to be important anti-
cancer agents because of their bio-friendly nature as copper
cDepartment of Chemistry, Tulane University, New Orleans, LA 70118, USA.
E-mail: krishna@chem.iitb.ac.in, panda@iitb.ac.in
†Electronic supplementary information (ESI) available. CCDC 977645–977647.
For ESI and crystallographic data in CIF or other electronic format see DOI:
10.1039/c4dt00832d
‡These authors contributed equally to this work.
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plays a significant role in biological systems. Copper is an {(o-OCH2C5H4N)P(μ-NtBu)}2 (1) in good yield. Compound 1 is a
essential trace metal for living organisms and plays a major light yellow viscous liquid, sensitive to air and moisture. The
role in several enzymatic activities, in particular redox reac- 31P{1H} NMR spectrum of 1 displayed a single resonance at
tions which have an influence on the antioxidant system of the 133.5 ppm and the mass spectrum showed the molecular
organism.24 The medicinal properties of copper complexes in ion peak at m/z 420.2. The aminobis(phosphonite), C6H5N-
anti-inflammatory, antiarthritic, antiulcer and anticonvulsant {P(OC6H3(OMe-o)(C3H5-p))2}2 (2), was prepared according to
diseases including anticancer properties25–31 are also well the literature procedure.48
documented. Due to the selective permeability of cancer cell
The treatment of 1 with two equivalents of copper(I) iodide
membranes to copper complexes and their strict regulation on in a 1 : 1 mixture of dichloromethane and acetonitrile resulted
intracellular concentration encouraged the synthesis of copper- in the formation of the copper(I) coordination polymer [{Cu(µ-
based compounds as probable antiproliferative agents with I)}2{(o-OCH2C5H4N)P(µ-NtBu)}2]n (3). Addition of two equi-
relatively less severe side effects than the present standard valents of 2,2′-bipyridine or 1,10-phenanthroline to the sus-
anticancer drugs.32,33 Several copper(I) complexes containing pension of 3 in dichloromethane at room temperature yielded
either phosphorus(III) based compounds or pyridyl-type cationic dinuclear complexes [(bpy)2Cu2{(o-OCH2C5H4N)P(µ-
ligands have been screened for anticancer activity with promis- NtBu)}2][I]2 (4) and [(phen)2Cu2{(o-OCH2C5H4N)P(µ-NtBu)}2][I]2
ing results.34–43 Recently, we have studied the antitumor (5) as yellow crystalline solids (Scheme 1). The 31P{1H} NMR
activity of gold(I) and copper(I) complexes and also metal-free spectra of 4 and 5 showed single resonances at 109.8 and
thio and selenol compounds which inhibit the growth of 110.7 ppm, respectively.
cancer cells by inducing apoptosis.44–46 In the present study,
The reaction of aminobis(phosphonite), C6H5N{P-
several mixed-ligand copper(I) complexes of diazadiphos- (OC6H3(OMe-o)(C3H5-p))2}2 (2) (hereafter referred to as ‘PNP’),
phetidine or cyclodiphosphazane appended with pyridyl func- with one or two equivalents of copper(I) iodide in acetonitrile
tionalities and aminobis(diphosphonite) ligands along with resulted in the formation of a rare tetranuclear copper
2,2′-bipyridine or 1,10-phenanthroline ligands have been syn- complex [(CuI)2(μ-PNP)]2 (6) irrespective of the stoichiometry
thesized and screened for in vitro anticancer activities against of the reactants and the reaction conditions.49 The reactions of
human cervical cells (HeLa), breast cancer cells and human 6 with pyridyl ligands such as pyridine, 2,2′-bipyridine and
colorectal carcinoma cells along with cisplatin, the most 1,10-phenanthroline led to the formation of several mixed-
widely used antitumor drug47 for comparison. Among them, ligand complexes. Attempts to prepare mixed-ligand complexes
the three most potent compounds (5, 10 and 12) were chosen in a one-pot reaction led to the isolation of stable homoleptic
for detailed study to understand their mechanism of action. amine complexes. In order to prepare mixed-ligand complexes,
The copper(I) compounds studied in the present investigation complex 6 was initially isolated and subsequently reacted with
were found to inhibit tumor cell growth by arresting cells at various pyridyl ligands.
the S phase of the cell cycle.
The reaction of 6 with an excess of pyridine resulted in
the formation of a white crystalline dinuclear complex,
[{Cu(μ-I)(py)}2(μ-PNP)] (7), whereas the reaction of 6 with 2,2′-
bipyridine in 1 : 4 and 1 : 2 molar ratios afforded dinuclear
complexes, [Cu2(μ-I)(bpy)2(μ-PNP)]I (8) and [Cu2(μ-I)I(bpy)-
Results and discussion
Synthesis of ligands and copper(I) complexes
The reaction of two equivalents of pyridine-2-methanol with (μ-PNP)] (9), respectively, as bright yellow crystalline solids. In
cis-{ClP(μ-NtBu)}2 in the presence of triethylamine yielded cis- complex 9, one of the Cu–I bond was cleaved by 2,2′-bipyridine
Scheme 1 Synthesis and copper complexes of cis-{(o-OCH2C5H4N)P(μ-NtBu)}2 (1).
11340 | Dalton Trans., 2014, 43, 11339–11351
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to give both tri- and tetra-coordinated copper centers, whereas four-membered {(o-OCH2C5H4N)P(µ-NtBu)}2 and {Cu(µ-I)}2
in complex 8, one of the bridging iodides was completely dis- units arranged alternatively in a zig-zag fashion to form a one
placed to produce a cationic complex with iodide as the dimensional coordination polymer.45,51,52 The copper centers
counter anion.50 Similar reactions of 6 with 1,10-phenanthro- are in a distorted tetrahedral environment consisting of two
line in 1 : 2 and 1 : 4 molar ratios gave [Cu2(μ-I)I(phen)(μ-PNP)] bridging iodides, a phosphorus atom from the cyclodiphos-
(11) and [Cu2(μ-I)(phen)2(μ-PNP)]I (12), respectively. The reac- phazane ring and a nitrogen atom of the pyridyl group. The
tion of 9 with two equivalents of AgOTf in acetonitrile followed Cu⋯Cu distance in 3 [3.2653(4) Å] is noticeably greater than
by one equivalent of 1,10-phenanthroline afforded [Cu2(phen)- the sum of the van der Waals radii for the copper(I) ion, thus
(bpy)(μ-PNP)](OTf)2 (10) as a pale yellow crystalline solid overruling any metallophilic interactions. The four-membered
(Scheme 2). The 31P{1H} NMR spectra of 7–9, 11 and 12 [Cu(µ-I)]2 units are puckered [torsion angle is 13.78(1)°], but
showed broad single resonances around 103 ppm, whereas the the diazadiphosphetidine rings are almost planar. The mean
31P{1H} NMR spectrum of 10 showed a broad single resonance Cu–I distances in 3 [2.6448 Å] is in good agreement with the
at 111.7 ppm. The molecular structures of 3, 9 and 11 were same observed in analogous complexes [1,3-C6H4{OP(µ-
confirmed by single crystal X-ray diffraction studies.
NtBu)2PN(H)tBu}2{Cu2(µ-I)2}2]n,51 [{Cu2(μ-I)2}2{(OC6H4OMe-o)-
P(μ-NtBu)}2]n,52 [{Cu2(μ-I)2CH3CN}4({(NC4H8NMe)P(μ-NtBu)}2)8]45
and [Cu(µ-I)(dppp)]2).53
Molecular structures of 3, 9 and 11
The crystals of 9 and 11 suitable for X-ray diffraction
studies were grown from acetonitrile solution. The unit cell of
the complex 9 contains two independent molecules with very
similar bonding parameters. In both the complexes 9 and 11
the Cu1 and Cu3 have distorted tetrahedral geometry being co-
ordinated to one phosphorus atom, one iodine atom and two
pyridyl nitrogen atoms, whereas Cu2 and Cu4 exhibit a tri-
gonal planar environment coordinated by one phosphorus and
The perspective views of molecular structures of 3, 9 and 11 are
shown in Fig. 1–3. The selected bond lengths [Å] and bond
angles [°] are given in Tables 1 and 2. Crystallographic data and
the details of the structure determinations are given in Table 3.
The yellow crystal of 3 suitable for single crystal X-ray diffr-
action study was obtained by slow diffusion of copper(I) iodide
solution in acetonitrile into a dichloromethane solution of 1 at
room temperature. The core structure of 3 consists of cyclic
Scheme 2 Reaction of 6 with pyridyl ligands.
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Fig. 1 The molecular structure of [{Cu(µ-I)}2{(o-OCH2C5H4N)P(µ-NtBu)}2]n (3). All hydrogen atoms and lattice solvents are omitted for clarity.
Thermal ellipsoids are drawn at the 50% probability level.
Fig. 2 (a) Molecular structure of [Cu2(μ-I)I(C10H8N2){C6H5N{P(OC6H3(OMe-o)(C3H5-p))2}2}] (9) showing the atom numbering schemes. (b) Inter-
molecular π–π stacking in 9. All hydrogen atoms are omitted for clarity. Thermal ellipsoids are drawn at the 50% probability level.
Fig. 3 (a) Molecular structure of [Cu2(μ-I)I(C12H8N2){C6H5N{P(OC6H3(OMe-o)(C3H5-p))2}2}] (11) showing the atom numbering schemes. (b) Core
structure of 11. All hydrogen atoms and lattice solvents are omitted for clarity. Thermal ellipsoids are drawn at the 50% probability level.
two iodine atoms. The two Cu–P distances differ significantly Cu2–I1 = 2.5651(5) Å (11)]. The terminal Cu–I distances [Cu2–
[Cu1–P1 = 2.1516(1) Å; Cu2–P2 = 2.1921(1) Å (9) and Cu1–P1 = I2 = 2.5230(6) Å (9) and Cu2–I2 = 2.5196(5) Å (11)] are shorter
2.1769(1) Å; Cu2–P2 = 2.1913(1) Å (11)], and similar is the case than the bridging Cu–I distances. The shorter Cu–P distance
for Cu–I distances to the bridging iodide [Cu1–I1 = 2.5760(6) Å in case of four-coordinated copper bonded to 2,2′-bipyridine/
and Cu2–I1 = 2.5860(5) Å (9) and Cu1–I1 = 2.6433(5) Å and phenanthroline is possibly due to the π–π stacking between
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Complexes 9 and 11 both show intramolecular π–π interactions
between one of the phenyl groups and the 2,2′-bipyridine and
the 1,10-phenanthroline moiety, respectively. In addition, the
complex 9 shows a non-covalent intermolecular π–π interaction
between the two 2,2′-bipyridine moieties having parallel dis-
placed π–π stacking alignment with the minimum distance
between the two 2,2′-bipyridine rings being 3.59 Å (Fig. 2b).54
The complex 11 does not show the analogous intermolecular
π–π interaction between the two 1,10-phenanthroline groups.
Table 1 Selected bond distances [Å] and bond angles [°] for complex 3
Bond length [Å]
Bond angle [°]
Cu1–I1
Cu1–I2
Cu1–P1
P1–O1
P2–N1
P1–N2
2.6384(4)
2.6512(4)
2.2166(6)
1.633(2)
1.697(2)
1.703(2)
I1–Cu1–P1
I1–Cu1–N3
I2–Cu1–P1
Cu1–I1–Cu2
I1–Cu1–I2
I2–Cu2–P2
I2–Cu2–N4
130.67(2)
102.84(5)
114.90(2)
76.30(1)
101.63(1)
122.16(2)
103.11(5)
Antiproliferative properties of copper complexes
Table 2 Selected bond distances [Å] and bond angles [°] for 9 and 11
11 11
1.685(4) 1.685(3) P1–N1–P2 120.1(2) 119.51(18)
The copper(I) complexes containing phosphorus based ligands
and pyridyl ligands have shown good to moderate antiproli-
ferative activity towards various cancer cell lines.25,55,56
Recently we reported the antitumor activity of copper(I) com-
plexes containing monocoordinated cyclodiphosphazanes and
chelating pyridyl ligands which have shown superior activity
towards cervical and breast cancer cell lines compared to cis-
platin.44 In the present investigation we wanted to examine the
anticancer properties of two distinct but related aminopho-
sphine systems: (a) cyclodiphosphazane appended with pyridyl
functionalities to form the large-bite bis(bidentate) system,
and (b) short-bite symmetric bis(phosphino)amine ligands, to
assess their anticancer properties. It is anticipated that these
chelating ligands would readily form copper(I) ionic complexes
and probably show better cytotoxicity. The antiproliferative
activity of these two series of copper complexes against the
human cervical cancer (HeLa) cell line was checked and
different compounds showed varied levels of inhibition as pre-
sented in Table 4. Under similar conditions, 10 μM cisplatin
9
9
P1–N1
P2–N1
1.694(3) 1.689(3) N1–P1–Cu1 122.75(1) 121.09(11)
2.1517(1) 2.1769(1) N1–P2–Cu2 116.62(1) 115.12(11)
Cu1–P1
Cu2–P2
Cu1–I1
Cu2–I1
Cu2–I2
Cu1–N2
Cu1–N3
2.1921(1) 2.1913(1) P1–Cu1–I1
2.5759(5) 2.6433(5) P2–Cu2–I1
2.5859(6) 2.5650(5) P2–Cu2–I2
114.71(3) 122.54(3)
119.12(4) 121.70(3)
128.8(4) 127.93(3)
2.5230(6) 2.5196(5) Cu1–I1–Cu2 68.59(2) 64.33(2)
2.049(3) 2.062(3) I1–Cu2–I2 111.83(2) 110.21(2)
2.056(4) 2.078(3) N2–Cu1–N3 80.32(14) 81.25(13)
Cu1–Cu2 2.909(4) 2.7735(6)
one of the phenoxy group on P1 and the 2,2′-bipyridyl/phen-
anthroline ligand. The Cu–N bond lengths (Cu1–N2 = 2.048(3) Å
and Cu1–N3 = 2.056(3) Å) differ slightly. The distance between
two copper centers in the complex 9 is 2.9085(7) Å greater than
the sum of the van der Waals radii indicating no metal–metal
interaction between the two copper atoms. The complex 11
shows the presence of the cuprophillic interaction between the
two copper metals as the Cu–Cu distance is 2.7735(6) Å. The
I2–Cu2–I1 and Cu1–I1–Cu2 angles in complexes 9 and 11 are
111.83(2)°, 68.590(1)° and 110.21(2)°, 64.33(2)°, respectively.
inhibited the proliferation of HeLa cells by 44
these results, the three most potent compounds (5, 10 and 12)
were chosen for detailed study to understand their mechanism
7%. From
Table 3 Crystallographic data for 3, 9 and 11
3
9
11
Formula
Formula weight
Crystal system
Space group
a [Å]
b [Å]
c [Å]
C20H30Cu2I2N4O2P2·CH3CN
C56H57Cu2I2N3O8P2
1342.87
C58H57Cu2I2N3O8P2·CH3CN
842.35
Monoclinic
P21/n (no. 14)
10.4251(7)
22.1496(14)
12.9548(8)
90
97.561(1)
90
2965.4(3)
4
1407.99
Monoclinic
P21/n (no. 14)
12.4841(3)
17.0323(4)
27.1967(7)
90
94.464(2)
90
5765.4(2)
4
Triclinic
ˉ
P1 (no. 2)
10.9180(9)
23.2001(18)
24.1557(19)
65.123(1)
81.611(1)
86.293(1)
5491.5(8)
4
α [°]
β [°]
γ [°]
V [Å3]
Z
ρcalc [g cm−3
]
1.887
3.652
1640
1.624
2.013
2688
1.624
1.923
2828
µ (Mo-Kα) [mm−1
F(000)
]
T (K)
2θ range [°]
Total no. of reflns
No. of indep. reflns
Rint
R
wR
100
100
1.9–28.3
95 613
19 471
0.061
0.0433
0.1062
1.03
150
2.2–28.0
51 118
7145
3.2–25.0
41 807
10 122
0.059
0.0370
0.0931
1.03
0.037
0.0203
0.0467
1.07
S
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Table 4 Effects of different copper complexes on the proliferation of
Table 5 Half maximal inhibitory concentrations (IC50) of 5, 10, 12
HeLa cells after 24 h
IC50 a (μM)
MCF-7
IC50 (μM)
HeLa
IC50 (μM)
HCT116
% Inhibitiona at 5 µM
% Inhibition at 10 µM
Compounds
Compounds
5
10
12
1.6 0.3
2.4 0.6
1.7 0.3
1.6 0.1
2.5 0.2
2.3 0.5
3.9 0.5
1.9 0.1
4
5
7
8
9
10
11
12
38
87
27
25
13
86
33
92
22
2
2
4
4
3
2
3
2
3
60
97
36
36
23
93
41
96
44
2
3
2
3
3
2
3
4
7
2
0.1
a IC50 data are average of three independent experiments for each cell
types. SD stands for standard deviation of mean.
Cisplatin
tumor cells are much stronger than cisplatin. The structural
activity correlation of compounds 4, 5, 10 and 12 suggested
that the complexes containing 1,10-phenanthroline ligands
showed more potency as compared to those containing 2,2′-
bipyridine ligands. The better π-stacking ability of 1,10-phen-
a % of inhibition of cell proliferation is average of three independently
performed experiments. SD stands for standard deviation of mean.
of action. The antiproliferative activity of 5, 10 and 12 against anthroline ligands58 seems to add more effectiveness to the
the human cervical cancer cell line (HeLa), breast cancer cell copper complex than 2,2′-bipyridine ligands (Fig. 4, 5 and
line (MCF-7) and human colon carcinoma cell line (HCT116) Tables 4, 5).
was determined (Fig. 4). The compounds 5, 10 and 12, in
The antiproliferative activity of these compounds is character-
culture, inhibited the proliferation of cells in a concentration istic of metal–ligand complexes as individual ligands and copper
dependent manner. Half maximal inhibitory concentration iodide fail to show any cytotoxicity. For example, ligand 1 at 6 μM
(IC50) of 5 was found to be 1.6 0.3, 1.6 0.1 and 2.3 0.5 μM showed 3.5 1% inhibition of cell growth, whereas the ligand 2
in MCF-7, HeLa and HCT116 cell lines, respectively. Similarly, and copper iodide did not show any cytotoxicity even at 6 μM
compounds 10 and 12 inhibited proliferation of MCF-7, HeLa concentration. To determine the effect of the copper complexes
and HCT116 cell lines with IC50 values of 2.4 0.6, 2.5 0.2, on cell cycle progression of the MCF-7 cell line, flow-cytometric
3.9 0.5 μM and 1.7 0.3, 2 0.1, 1.9 0.1 μM, respectively analysis was performed after 48 h treatment with compounds.
(Fig. 4 and Table 5). Under similar conditions, cisplatin was The compounds block cell cycle progression at the S phase of
reported to inhibit the proliferation of HeLa, HCT116 and the cell cycle (Fig. 6). For example, in the case of the control 17%
MCF-7 cells with IC50 of 8, 16 and 15 μM, respectively.57 Thus, cells were in the S phase of the cell cycle and the number of cells
the antiproliferative efficacies of 5, 10, and 12 against cultured in the S phase increased to 44%, 48% and 52% in the presence
Fig. 4 The effects of 5, 10 and 12 on human cervical cancer (HeLa), human colon carcinoma (HCT116) and breast cancer (MCF-7) cells after 24, 24
and 48 h.
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Fig. 5 Structural activity correlation of complexes 4, 5, 10 and 12.
Fig. 6 Effects of compounds 5 (6A) and 10 (upper panels 6B) and 12 (lower panels 6B) on cell cycle of MCF-7 cells.
of 5 at 1.5, 3 and 6 µM, respectively (Fig. 6 and Table 6). FACS
analysis for Annexin V and propidium iodide staining was
Table 6 Effects of 5, 10 and 12 on MCF-7 cell cycle progression
used to confirm apoptosis induction by these complexes. The
compounds 5, 10 and 12 increased the number of cells in early
and late stages of apoptosis (Fig. 7 and Table 7).
The apoptosis-causing ability of the compounds was
further confirmed by performing DNA fragmentation assays.
When a cell undergoes apoptosis, endonucleases cleave the
internucleosomal linker DNA that results in fragmentation of
the genome.59 The fragmented genome gives a laddering
pattern on running the gel which is a characteristic signature
Compound 5
Compound 10
Compound 12
% of cells in different phases of cell cycle
Conc. (µM)
G1
S
G2
G1
S
G2
G1
S
G2
0
1.5
3
55
48
43
43
17
44
48
52
28
8
9
59
63
52
39
11
23
41
58
30
14
7
59
61
53
46
11
34
46
50
30
5
1
6
5
3
4
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ing the DNA damage as shown in Fig. 9 and S1.† The damage
was more pronounced at higher concentrations.
In comparison with the control, treatment with compounds
5 and 12 at 1.5, 3 and 6 µM concentrations increased nuclear
localization of p53 from 1.8% in the control to 25, 33 and 42%
and 32, 40, and 47%, respectively (Fig. 9, S1† and Table 8).
Since p21 is the downstream protein of p53 and is indispensi-
ble for p53 mediated G1/S arrest,64,65 p21 localization to the
nucleus will give an insight into the effect of these compounds
on the progression of the cell cycle. In the control, 2% of the
cells showed nuclear localization of p21, while treatment with
5 and 12 at 1.5, 3, and 6 µM concentrations increased its
nuclear localization to 29, 35, and 45% and 27, 42, and 48%,
respectively (Fig. 9 and S1† and Table 8).
Fig. 7 Apoptosis determination by FACS analysis of MCF-7 cells.
In summary, we have identified a few copper complexes as
potent anticancer agents. The complexes caused strong
S-phase arrest which can be due to the DNA damage. The
pathway of apoptosis and S-phase arrest seems to follow
the p53 and p21 route. The results are in agreement with the
general mechanism followed by G1/S blocking agents of the
cell cycle. The stability of complexes under in vitro conditions
and in aqueous medium for more than 16 h indicates the
absence of any covalent interactions with DNA through substi-
tution or hydrolysis. Therefore, non-covalent interactions such
as hydrogen-bonding or π–π interactions may be responsible
for antiproliferative activities. Recent computational studies58
have shown that the planar heteroaromatic diimines interact
with DNA base pairs by aromatic π–π stacking. The superior
activity of 1,10-phenanthroline containing compounds is prob-
ably due to better π–π stacking interaction compared to 2,2′-
bipyridine containing complexes. Further efforts to definitively
unravel the uncertainties about the actual interactions would
be warranted.
Table 7 The distribution of cells in different states of apoptosis in the
presence of copper complexes, 5, 10 and 12
Q1 (% dead
Cells)
Q2 (% late
apoptosis)
Q3 (% living
cells)
Q4 (% earlier
apoptosis)
Sample
Control
0.4
0.6
2.8
0.9
0.9
23.5
30.1
16.9
98.6
19.1
18.1
33.6
0.5
56.8
49.1
48.4
5 (8 μM)
10 (8 μM)
12 (8 μM)
of apoptosis.60 In comparison with the control, compounds 5,
10 and 12 caused DNA fragmentation comparable to etopo-
side, a known DNA damaging agent (Fig. 8).61
Since the role of tumor suppressor p53 is well known in
apoptosis,62,63 we were interested in knowing whether apopto-
sis by these compounds is mediated through the p53 pathway
or not. Although the structures of 10 and 12 are quite similar,
compound 12 was considered for this study as it was found to
be a more potent cytotoxic agent. The vehicle-treated MCF 7
cells showed compact and circular DNA morphology. In con-
trast, treatment with 5 and 12 induced disorganization of the
nucleus with a granular and hollow appearance clearly indicat-
Conclusions
Di- and tetranuclear copper(I) complexes containing aminobis-
(phosphonites) and cyclodiphosphazane ligands were pre-
pared and reacted with various pyridyl ligands to form binuc-
lear mixed-ligand complexes. The mixed-ligand copper(I)
complexes have shown highly promising antiproliferative
activities towards various human cancer cell lines. Copper(I)
compounds were found to suppress tumor cell growth by
arresting cells at the S-phase of the cell cycle. The nuclear mor-
phology of treated cells clearly showed DNA damage. The
experimental evidence clearly indicated that these compounds
initiated apoptosis, which is mediated through the p53
pathway. Copper being an essential cofactor in several
enzymes and physiological processes, and also not being
designated as a carcinogenic element, may be less toxic than
other transition metals such as platinum, ruthenium and
palladium. Further, the antiproliferative activities and apopto-
sis induction of the copper complexes presented in this paper
are several-fold higher than the cisplatin, in remarkably low
concentrations.
Fig. 8 DNA laddering experiment suggesting fragmentation of
genomic DNA.
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Fig. 9 Compound 5 mediated apoptosis through p53 and p21 pathway. There is prominent nuclear translocation of p53 and its downstream protein
p21.
1
CDCl3 (or DMSO-d6) as an internal lock; chemical shifts of H
and 13C{1H} NMR spectra are reported in ppm downfield from
TMS, used as an internal standard. The chemical shifts of
31P{1H} NMR spectra are referred to 85% H3PO4 as an external
standard. The microanalyses were performed using a Carlo
Erba Model 1112 elemental analyzer. Mass spectra were
recorded using Waters Q-Tof micro. The melting points were
observed in capillary tubes and are uncorrected.
Table 8 Nuclear translocation of p53 and p21 in the presence of
copper complexes 5 and 12
% cell p53 nuclear
localization
% cell p21 nuclear
localization
Sample
Control
1.75
25
33
42
32
40
47
2
29
35
45
27
42
48
5 (1.5 μM)
5 (3 μM)
5 (6 μM)
12 (1.5 μM)
12 (3 μM)
12 (6 μM)
Synthesis of cis-{(o-OCH2C5H4N)P(µ-NtBu)}2 (1)
A mixture of pyridine-2-methanol (1.0 g, 9.16 mmol) and tri-
ethylamine (1.3 mL, 0.943 g, 9.32 mmol) in diethyl ether
(30 mL) was added to cis-{ClP(μ-NtBu)}2 (1.26 g, 4.58 mmol)
also in diethyl ether (30 mL) at 0 °C. The reaction mixture was
allowed to stir for 6 h at room temperature. The triethyl-
amine hydrochloride salt thus formed was filtered off and the
solvent was removed under reduced pressure to obtain cis-
{(o-OCH2C5H4N)P(µ-NtBu)}2 (1) as a pale yellow viscous liquid.
Experimental section
General procedures
All manipulations were performed using standard vacuum-line
and Schlenk techniques under a nitrogen atmosphere unless
otherwise stated. All the solvents were purified by conventional
methods and distilled prior to use. The compounds cis-{ClP-
(μ-NtBu)}2 66 and C6H5N{P(OC6H3(OMe-o)(C3H5-p))2}2 (2)48 were
prepared according to the published procedures. CuI, AgOTf,
2,2′-bipyridine and 1,10-phenanthroline were purchased from
Aldrich Chemicals and used without further purification.
Other chemicals were obtained from commercial sources and
purified prior to use.
1
Yield: 91% (1.75 g). H NMR (400 MHz, CDCl3): δ 8.54 (m, 2H,
3
3
py), 7.68 (t, JHH = 8 Hz, 2H, py), 7.55 (d, JHH = 8 Hz, 2H, py),
3
7.16 (t, JHH = 6 Hz, 2H, py), 5.15 (s, 4H, CH2), 1.24 (s, 18H,
tBu). 13C{1H} NMR (100 MHz, CDCl3): 159.3, 148.9, 136.6,
122.1, 121.2, 64.7, 51.4 (t, 2JPC = 12.5 Hz), 31.1 (t, 3JPC = 9.6 Hz).
31P{1H} NMR (162 MHz, CDCl3): δ 133.5 (s). HRMS: Calcd for
C20H31N4O2P2 [M + H]: 421.1922; Found: 421.1907.
Instrumentation
Synthesis of [{Cu(µ-I)}2{(o-OCH2C5H4N)P(µ-NtBu)}2]n (3)
The NMR spectra were recorded at the following frequencies: An acetonitrile (10 mL) solution of CuI (0.028 g, 0.147 mmol)
400 MHz (1H), 100 MHz (13C), 162 MHz (31P) using either was added dropwise to
a well-stirred solution of cis-
Varian VXR 400 or Bruker AV 400 spectrometers. 13C and 31P {(o-OCH2C5H4N)P(µ-NtBu)}2 (1) (0.031 g, 0.074 mmol) in
NMR spectra were acquired using broad band decoupling. The dichloromethane (10 mL) at room temperature and stirring
spectra were recorded in CDCl3 (or DMSO-d6) solutions with was continued further for 4 h. The solvent was removed under
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vacuum and the residue obtained was washed with 2 × 5 mL of and stored at −20 °C for a day to give a white crystalline solid.
petroleum ether to give an analytically pure sample of 3 as a Yield: 82% (0.0278 g). Mp: >230 °C. 1H NMR (400 MHz,
3
yellow solid. Yield: 80% (0.047 g). Mp: >270 °C. 1H NMR CDCl3): δ 8.14 (br s, Pyr-4-H, 2H), 7.88 (d, JHH = 8 Hz, Pyr-2-6-
3
(400 MHz, DMSO-d6): δ 8.78 (m, py, 2H), 7.66–7.57 (m, py, 6H), H, 4H), 7.52 (t, JHH = 8 Hz, Pyr-3-5-H, 4H), 6.36–7.39 (m, ArH,
3
t
5.20 (d, CH2, JPH = 16 Hz, 4H), 1.26 (s, Bu, 18H). 31P{1H} 17H), 6.01–5.91 (m. CH, 4H), 5.12–5.06 (m, CH2, 8H), 3.63 (s,
NMR (162 MHz, CDCl3): δ 113.5 (br s). Anal. Calcd for OCH3, 12H), 3.34 (d, CH2, 8H, JHH = 8 Hz). 31P{1H} NMR
3
C20H30N4I2O2P2Cu2·CH3CN: C, 31.37; H, 3.95; N, 8.31%. (162 MHz, CDCl3):
δ
104.7 (br s). Anal. Calcd for
Found: C, 31.49; H, 3.82; N, 8.42%.
C56H59N3P2O8I2Cu2·CH2Cl2: C, 47.88; H, 4.30; N, 2.94%.
Found: C, 47.58; H, 4.54; N, 3.12%.
Synthesis of [(bpy)2Cu2{(o-OCH2C5H4N)P(µ-NtBu)}2][I]2 (4)
To a suspension of 3 (0.048 g, 0.06 mmol) in dichloromethane Synthesis of [Cu2(µ-I)I(bpy){C6H5N{P(OC6H3(OMe-o)-
(5 mL) was added dropwise a solution of 2,2′-bipyridine (C3H5-p))2}2}] (8)
(0.019 g, 0.12 mmol) in the same solvent (5 mL) at room temp-
erature. The reaction mixture was stirred for further 2 h, con-
To a solution of 6 (0.03 g, 0.0126 mmol) in acetonitrile (5 mL)
was added dropwise a solution of 2,2′-bipyridine (0.008 g,
centrated to 2 mL and layered with petroleum ether (3 mL).
0.0504 mmol) in the same solvent (5 mL) and the reaction
The turbid yellow solution was stored at room temperature for
mixture was stirred for 4 h. The resulting yellow solution was
24 h to obtain 4 as a yellow crystalline solid. Yield: 82%
concentrated and stored at −10 °C for 24 h to give an analyti-
(0.054 g) Mp: 210–212 °C (dec). 1H NMR (400 MHz, CDCl3):
cally pure product as bright yellow crystals. Yield: 89%
3
δ 8.89 (s br, 4H), 8.53 (d, JHH = 4 Hz, 4H), 7.90 (s br, 4H),
1
(0.0337 g). Mp: 119–121 °C. H NMR (400 MHz, CDCl3): δ 8.11
3
t
7.67–7.16 (m, 12H), 5.35 (d, JPH = 8.2 Hz, 4H), 1.29 (s, Bu,
18H). 31P{1H} NMR (162 MHz, CDCl3): δ 109.8 (br s). Anal.
Calcd for C40H46N8P2O2Cu2I2: C, 43.14; H, 4.16; N, 10.06%.
Found: C. 43.62; H, 4.05; N, 10.01%.
(br s, Pyr-3-3′-H, 2H), 7.75 (br s, Pyr-6-6′-H, 2H), 7.52–6.47 (m,
ArH, 21H), 5.95–5.87 (m, CH, 4H), 5.13–5.08 (m, CH2, 8H),
3.64 (br s, OCH3, 12H), 3.28 (br s, CH2, 8H). 31P{1H} NMR
(162 MHz, CDCl3):
δ
103.7 (br s). Anal. Calcd for
C66H69N5P2O8I2Cu2: C, 52.74; H, 4.63; N, 4.66%. Found: C,
53.10; H, 4.67; N, 4.38%.
Synthesis of [(phen)2Cu2{(o-OCH2C5H4N)P(µ-NtBu)}2][I]2 (5)
This was synthesized by a procedure similar to that of 4, using
3 (0.1 g, 0.125 mmol) and 1,10-phenanthroline (0.05 g,
0.25 mmol). Yield: 79% (0.118 g). Mp: 232–234 °C (dec).
1H NMR (400 MHz, CDCl3): δ 9.32 (s, br, 4H), 8.48–7.14 (m,
20H), 5.41 (d, 3JPH = 8 Hz, 4H), 1.36 (s, tBu, 18H). 31P{1H} NMR
Synthesis of [Cu2(µ-I)I(bpy){C6H5N{P(OC6H3(OMe-o)-
(C3H5-p))2}2}] (9)
This was synthesized by a procedure similar to that for 8 using
2,2′-bipyridine (0.004 g, 0.0256 mmol) and
6 (0.03 g,
(162 MHz, CDCl3):
δ
110.7 (br s). Anal. Calcd for
0.0126 mmol). Yield: 85% (0.0288 g). Mp: 166–169 °C. 1H NMR
(400 MHz, CDCl3): δ 8.01 (br d, Pyr-3-3′-H, 2H), 7.72 (br d, Pyr-
6-6′-H, 2H), 7.56 (br d, Pyr-4-4′-H, 2H), 7.46 (br t, Pyr-5-5′-H,
2H), 7.32–6.50 (m, ArH, 17H), 5.97–5.87 (m, CH, 4H),
5.29–5.07 (m, CH2, 8H), 3.64 (br s, OCH3, 12H), 3.28 (br s,
CH2, 8H). 31P{1H} NMR (162 MHz, CDCl3): δ 103.5 (br s). Anal.
Calcd for C56H57N3P2O8I2Cu2: C, 50.08; H, 4.28; N, 3.13%.
Found: C, 49.78; H, 4.14; N, 3.57%.
C44H46N8I2O2P2Cu2: C, 45.49; H, 3.99; N, 9.65%. Found:
C. 45.07; H, 4.60; N, 9.52%.
Synthesis of [(CuI)4{PhN{P(OC6H4(OMe-o)(C3H5-p))2}2}2] (6)
A solution of cuprous iodide (0.014 g, 0.074 mmol) in aceto-
nitrile (5 mL) was added to
a solution of 2 (0.03 g,
0.037 mmol) in acetonitrile (5 mL). After stirring for 4 h, the
solvent was evaporated under vacuum to obtain an oily residue
which on washing several times with petroleum ether and
drying under vacuum gave a white crystalline solid. Yield: 89%
Synthesis of [Cu2(phen)(bpy){C6H5N{P(OC6H3(OMe-o)-
(C3H5-p))2}2}](OTf)2 (10)
(0.0391 g). Mp: 123–125 °C. 1H NMR (400 MHz, CDCl3):
3
δ 7.74–7.30 (m, C6H5, 5H), 6.73 (d, C6H3, 4H, JHH = 8.0 Hz), To a solution of 9 (0.027 g, 0.02 mmol) in acetonitrile (5 mL)
6.58 (s, C6H3, 4H), 6.52 (d, C6H3, 4H, JHH = 6.4 Hz), 5.97–5.90 was added AgOTf (0.0052 g, 0.02 mmol) and the reaction
3
(m, CH, 4H), 5.10–5.04 (m, CH2, 8H), 3.63 (s, OCH3, 12H), 3.30 mixture was stirred for 1 h. The AgI formed was filtered
3
(d, CH2, 8H, JHH = 8 Hz). 31P{1H} NMR (162 MHz, CDCl3): through a frit with celite and a solution of 1,10-phenanthroline
δ 86.3 (br s). Anal. Calcd for C92H98N2I4O16P4Cu4: C, 46.56; H, (0.004 g, 0.02 mmol) in acetonitrile was added dropwise. The
4.16; N, 1.18%. Found: C, 46.23; H, 4.01; N, 1.19%.
resulting yellow solution was stirred for another 2 h, the
solvent removed under vacuo, and the residue obtained dis-
solved in 1 mL of dichloromethane and diluted with 3 mL of
petroleum ether to give a yellow precipitate. The precipitate
Synthesis of [{Cu(µ-I)(py)}2{C6H5N{P(OC6H3(OMe-o)-
(C3H5-p))2}2}] (7)
Pyridine (5 mL) was added to compound
6
(0.03 g, was separated, and dried under vacuo to obtain an analytically
1
0.0126 mmol) dropwise and allowed to stir at room temp- pure product. Yield: 91% (0.0281 g). Mp: 176–178 °C. H NMR
erature for 4 h. The resulting yellow solution was evaporated (400 MHz, CDCl3): δ 8.70–7.10 (m, ArH, 25H), 5.97–5.90 (m,
to dryness under vacuum; the residue was dissolved in CH, 4H), 5.11–5.06 (m, CH2, 8H), 3.48 (br s, OCH3, 12H), 3.22
dichloromethane (2 mL), layered with petroleum ether (3 mL) (br s, CH2, 8H). 31P{1H} NMR (162 MHz, CDCl3): δ 112.3 (br s).
11348 | Dalton Trans., 2014, 43, 11339–11351
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Anal. Calcd for C69H65NF3I5O11P2SCu2: C, 53.63; H, 4.24; N, three independent experiments performed in a 96 well plate
4.53, S, 2.08%. Found: C, 53.39; H, 3.96; N, 4.29, S, 1.91%.
on different days for each cell line.
Synthesis of [Cu2(µ-I)I(phen){C6H5N{P(OC6H3(OMe-o)-
(C3H5-p))2}2}] (11)
p53 and p21 staining for determining p53 mediated apoptotic
pathway
This was prepared by a procedure similar to that for 8 using
1,10-phenanthroline (0.005 g, 0.0256 mmol) and 6 (0.03 g, MCF-7 cells were incubated with either vehicle or different
1
0.0126 mmol). Yield: 87% (0.03 g). Mp: 130–132 °C. H NMR concentrations of 5 and 12 for 24 h. Cells were collected by
(400 MHz, CDCl3): δ 8.48 (br s, Pyr-3-3′-H, 2H), 7.93 (br d, Pyr- centrifugation, fixed with 3.7% formaldehyde and then
4-7-H, 2H), 7.73 (br d, Pyr-2-9-H, 2H), 7.41 (s, Pyr-5-6-H, 2H), immunostained using antibody against p53 and p21 (1° 1 : 300,
7.35–7.18 (m, ArH, 17H), 5.97–5.90 (m, CH, 4H), 5.11–5.06 (m, 2° 1 : 300). Secondary antibody was alexa linked anti-mouse anti-
CH2, 8H), 3.62 (br s, OCH3, 12H), 3.22 (br s, CH2, 8H). 31P{1H} body generated in rabbit. The DNA was stained with Hoechst
NMR (162 MHz, CDCl3): δ 103.6 (br s). Anal. Calcd for 33258. The images were captured at 40× using a Nikon micro-
C58H57N3P2O8I2Cu2: C, 50.96; H, 4.20; N, 3.07%. Found: scope and the processing was done by an image pro plus Soft-
C, 51.18; H, 4.29; N, 3.15%.
ware (Media Cybernetics, Silver Spring, MD, USA). The cells
following apoptosis through a p53 dependent manner showed
red nuclear staining due to the translocation of p53 and p21
into the nucleus.67
Synthesis of [Cu2(µ-I)(phen)2{C6H5N{P(OC6H3(OMe-o)-
(C3H5-p))2}2}]I (12)
This was prepared by a procedure analogous to 8 using
1,10-phenanthroline (0.01 g, 0.0504 mmol) and 6 (0.03 g,
0.0126 mmol). Yield: 79% (0.0308 g). Mp: >160 °C (dec).
Annexin V/propidium iodide (PI) staining for apoptosis
1H NMR (400 MHz, CDCl3): δ 8.46 (br s, Pyr-3-3′-H, 2H), 7.91 Samples for Annexin V/propidium iodide (PI) staining were
(br d, Pyr-4-7-H, 2H), 7.72 (br d, Pyr-2-9-H, 2H), 7.40 (s, Pyr-5-6- prepared as described recently.67,69,70 MCF-7 cells were incu-
H, 2H), 7.34–7.16 (m, ArH, 17H), 5.99–5.89 (m, CH, 4H), bated with either vehicle or 8 µM concentrations of 5, 10 and
5.13–5.07 (m, CH2, 8H), 3.62 (br s, OCH3, 12H), 3.22 (br s, 12 for 24 h. The cells were dislodged by trypsin-EDTA treat-
CH2, 8H). 31P{1H} NMR (162 MHz, CDCl3): δ 103.6 (br s). Anal. ment, collected by cyto-spinning at 2400 rpm for 10 min at
Calcd for C70H65N5I2O8P2Cu2: C, 54.34; H, 4.23; N, 4.53%. 25 °C, washed twice with PBS and resuspended in 1× binding
Found: C, 54.37; H, 4.03; N, 4.93%.
buffer. Then the cells were incubated with 5 µL of FITC-Annex-
inV and 5 µL of propidium iodide for 15 minutes at room
temperature in the dark. Annexin and PI content of the live
Antiproliferative studies
Materials. Sulforhodamine B, Hoechst 33258, mouse anti- cells was quantified by flow cytometry (FACS Aria Beckton
p53 IgG, mouse anti-p21 IgG antibodies and apoptosis detec- Dickenson) and analyzed using the Modfit LT program (Verity
tion kit (Annexin V-propidium iodide) were purchased from Softwares, USA).
Santa Cruz Biotechnology, CA, USA. All other reagents were of
analytical grade and obtained from Sigma, MO, USA and mL−1) were incubated without or with different (1.5 µM, 3 µM
Himedia, Mumbai, India. and 6 µM) concentrations of 5, 10 and 12. DNA were stained
Cell cycle analysis using FACS. MCF-7 cells (∼1 × 106 cells
Cell culture. MCF-7, HeLa and HCT116 cells were grown in with 50 μg mL−1 propidium iodide and quantified by flow cyto-
MEM (Eagle’s minimum essential medium) (Himedia, metry.44,67 The data were analyzed by Modfit LT program
Mumbai, India). MEM contains 10% fetal bovine serum, 2.2 g (Verity Softwares, USA).
L−1 sodium bicarbonate and 1% antibiotic–antimycotic solu-
tion composed of streptomycin, amphoterecin B and penicil-
lin. Cells were grown at 37 °C in a humidified atmosphere of
DNA fragmentation assay for determination of apoptosis
5% carbon dioxide and 95% air. Cell lines were maintained in MCF-7 cells were grown in 25 mL culture flasks, once the cell
a 25 mL cell culture flask (Nunc) at 37 °C.
density reached 1 × 106 cells mL−1, the cells were incubated
Cell proliferation assay. Cytotoxicity of different copper with 5, 10 and 12 at 8 μM and etoposide at 30 μM for 48 h.
complexes on HeLa cell line proliferation at 5 µM and 10 µM After compound treatment, the cells were dislodged and col-
concentrations was determined by sulforhodamine B assay67,68 lected by centrifugation.71 The pellets were washed with PBS,
Briefly, cells were seeded in 96 well cell culture plates dissolved in lysis buffer (50 mM Tris, pH 8.0/10 mM ethylene-
(Himedia, Mumbai, India) for 24 h. Then, the medium was diaminetetraacetic acid–0.5% SL-sarcosine–0.5 mg mL−1 of
removed and fresh media containing 0.1% DMSO or different proteinase K), and incubated at 50 °C for 1 h in a heating
concentrations of each compound were added into the wells. block. After heat treatment, 5 µL of RNase (1 mg mL−1) was
The antiproliferative activity of 5, 10 and 12 against human added to the mixture and it was incubated at 50 °C for 1 h.
cervical cancer (HeLa), human colon carcinoma (HCT116) and Finally, the mixture was incubated at 65 °C for 2 min, the
breast cancer (MCF-7) cells was determined after 24, 24 and temperature was lowered to 25 °C and the sample was run on a
48 h, respectively. The extent of inhibition of cell proliferation 1.8% agarose gel. The DNA bands were analyzed using UV gel
was determined by the SRB assay.67,68 Data are the average of doc.
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X-Ray crystallography
5 J. W. Ho, Recent Pat. Anti-Cancer Drug Discovery, 2006, 1,
129–134.
Crystals of each of the compounds 3 and 9 suitable for X-ray
crystal analysis were mounted on a Cryoloop with a drop of
Paratone oil and placed in the cold nitrogen stream of the
Kryoflex attachment of the Bruker APEX CCD diffractometer,
whereas crystals of 11 were mounted on a CCD Oxford Diffrac-
tion XCALIBUR-S diffractometer equipped with an Oxford
Instruments low-temperature attachment. Data were collected
at 100(2) K for 3 and 9 and 150(2) K for 11 using graphite-
monochromated Mo Kα radiation (λα = 0.71073 Å). The data
were collected by the standard ω-2θ scan techniques (for 11)
or APEX2 programme suite (for 3 and 9)72 and were scaled and
reduced using the SAINT software.73 Multiple measurements
of equivalent reflections provided the basis for an empirical
absorption correction as well as a correction for any crystal
deterioration during the data collection (SADABS74). The struc-
tures 3 and 9 were solved by the Patterson method, whereas 11
was solved by direct methods and refined by full-matrix least-
squares procedures using the SHELXTL program package.75
Hydrogen atoms attached to carbon were placed in calculated
positions and included as riding contributions with isotropic
displacement parameters tied to those of the attached non-
hydrogen atoms. The isotropic thermal parameters of the
hydrogen atoms were fixed at 1.2 times that of the corres-
ponding carbon for phenyl hydrogen and 1.5 times for
C(CH3)3. In the final refinement, the hydrogen atoms were
riding with the carbon atom to which they were bonded.
Crystallographic data (excluding structure factors) for the
structures reported in this paper have been deposited with the
Cambridge Crystallographic Data Centre as supplementary
publication no. CCDC 977645 (compound 3), 977646 (com-
pound 9), 977647 (compound 11).
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The work is supported by a grant SR/S1/IC-17/2010 from
Department of Science and Technology (DST), New Delhi, and
a grant from Department of Atomic energy, Government of
India. GSA thanks CSIR, New Delhi, AR and SN thanks UGC,
New Delhi for Senior Research Fellowships. DP thanks
DAE-SRC fellowship. We also thank the Department of Chem-
istry Instrumentation Facilities, IIT Bombay, for spectral and
analytical data. The Chemistry Department of Tulane Univer-
sity is thankful for support of the X-ray crystallography
laboratory.
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