ORIGINAL PAPER

Synthesis, characterization, and antibacterial and anticancerscreening of {M2+–Co3+–M2+} and {Co3+–M2+} (M is Zn, Cd, Hg)heterometallic complexes

Nagendra K. Kaushik • Anurag Mishra •

Afsar Ali • J. S. Adhikari • Akhilesh K. Verma •

Rajeev Gupta

Received: 18 April 2012 / Accepted: 1 September 2012 / Published online: 23 September 2012

� SBIC 2012

Abstract The cobalt(III) complexes Et4N[Co(L1)2] and

[Co(L2)3] [H2L1 is 2,6-bis(N-(2-pyridyl)carbamoyl)pyri-

dine and HL2 is 2-(N-(2-pyridyl)carbamoyl)pyridine] were

used as the building blocks for preparing a series of {M2?–

Co3?–M2?} (where M is Zn, Cd, or Hg) and {Co3?–M2?}

(where M is Zn or Cd) heterometallic complexes. All het-

erometallic complexes were characterized using a host of

spectroscopic methods (IR, NMR, and UV/vis spectroscopy

and mass spectrometry), elemental analysis, and conduc-

tivity measurements. One of the representative compounds,

{Hg2?–Co3?–Hg2?}, was characterized crystallographi-

cally, and it was revealed that two Hg(II) ions are coordi-

nated within the clefts created by the building block

Et4N[Co(L1)2]. The results of screening for anticancer

activity against the human brain tumor U87 cell line and

antibacterial activity against a range of resistant (Pseudo-

monas aeruginosa and Proteus vulgaris) as well as standard

(Staphylococcus aureus SA 96, P. aeruginosa MTCC 1688,

Klebsiella planticola MTCC 2272, and Escherichia coli T7)

bacterial strains indicate promising activities. Notably, the

observed activity was found to vary with the type of

building block and the secondary metal ion present in

the heterometallic complex. Treatment-induced cell death

[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-

mide, MTT and macrocolony assay), growth inhibition,

cytogenetic damage, cell cycle delay, and apoptosis were

studied as the parameters for cellular response.

Keywords Heterometallic complexes � Antibacterial

activity � Cytotoxic activity � DNA cell cycle analysis �Apoptosis

Introduction

Medicinal inorganic chemistry is continuously receiving

interest in the field of biomedical applications [1, 2]. This

interest is largely due to the potential application of metal-

containing compounds as antibacterial, antiviral, anti-

fungal, antimalarial, and antitumor agents [3–12]. The

recent advances rely on the fact that the concepts of

rational drug design are also applicable to metal-based drug

molecules. For a successful metal-based drug molecule,

precise knowledge of the thermodynamics and kinetics of

ligand substitution, stability, and redox reactions under

biologically relevant conditions is essential [13, 14]. In

addition, the choice of coordinated ligands and the metal’s

coordination geometry provides an invaluable ability to

fine-tune the chemical reactivity of metal complexes [15].

This information allows control of pharmacological prop-

erties, including cell uptake, distribution, binding, and

metabolism.

The identification and recognition of several metal

complexes as new anticancer and antimicrobial agents has

been related to their structural diversity and the properties

arising from binding/interaction with the biomolecular

N. K. Kaushik and A. Mishra have contributed equally.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00775-012-0937-5) contains supplementarymaterial, which is available to authorized users.

N. K. Kaushik � A. Mishra � A. Ali � A. K. Verma �R. Gupta (&)

Department of Chemistry, University of Delhi,

Delhi 110 007, India

e-mail: rgupta@chemistry.du.ac.in

J. S. Adhikari

Institute of Nuclear Medicine and Allied Sciences,

Lucknow Road, Timarpur, Delhi 110 054, India

123

J Biol Inorg Chem (2012) 17:1217–1230

DOI 10.1007/s00775-012-0937-5

cellular targets. In these endeavors, copper, zinc, and cobalt

complexes have been used in the treatment of many dis-

eases, including cancer [16–26], whereas chromium, man-

ganese, and iron complexes have been used for their

antibacterial activity [27]. The interaction of zinc, cad-

mium, and mercury with biomolecules is one of the most

studied fields in medicinal inorganic chemistry [28, 29].

Cadmium and mercury, in contrast, are toxic metals; how-

ever, they are widely used in many industrial processes

[30, 31]. The toxicity of cadmium is associated with the fact

that it often competes with zinc for a variety of important

binding sites in the cell, including gene regulation sites.

Heterometallic complexes and networks are currently

attracting a great deal of interest owing to the presence of

two different metals which not only contribute to fasci-

nating structures but also result in important applications

[32–40]. These materials have had important applications

in selective gas sorption [41], sensing [42–44], and catal-

ysis [45, 46]. Heterometallic complexes have been scarcely

developed for biomedical applications and only a few

examples have been reported [47–49]. We have recently

developed a strategy to selectively place two different

metals in close proximity to one another and were able to

prepare several {M2?–Co3?–M2?} and {Co3?–M2?} het-

erometallic complexes and networks (where M is Zn, Cd,

Hg) [50–55]. Interestingly, these heterometallic complexes

and networks were synthesized starting with Co3? com-

plexes of pyridine–amide ligands as the building blocks

(complexes 1 and 10, Fig. 1). Importantly, of several such

building block molecules, complexes 1 and 10 were also

shown to have high antimicrobial activity [56]. We rea-

soned that the presence of empty clefts in these molecules

enabled them to interact with the biologically important

metal ions required for the functioning of a bacterium cell.

In this article, we describe the synthesis and characteriza-

tion of a few {M2?–Co3?–M2?} and {Co3?–M2?} het-

erometallic complexes and report anticancer and

antibacterial studies. After in vitro screening on various

human cells, we performed genotoxicity analyses on

human brain cancer cells by studying the formation of

micronuclei. To the best of our knowledge, for the first

time, several heterometallic complexes have been used for

a comprehensive anticancer and antibacterial study.

Materials and methods

Chemicals

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-

mide (MTT), bisbenzimidazole derivative Hoechst 33342

bisbenzimide(2-[4-ethoxyphenyl]-5-[4-methyl-1-piperazin-

ylpiperazinyl]-2,5-bi-1H-benzimidazole)trihydrochloride),

Hanks’s balanced salt solution, Dulbecco’s modified

phosphate-buffered saline (PBS), Dulbecco’s modified

Eagle’s medium (DMEM), fetal calf serum, N-(2-

hydroxyethyl)piperazine-N-2-ethanesulfonic acid buffer,

propidium iodide (PI), ribonuclease A, and trypsin were

obtained from Sigma (USA). All other chemicals used

were of analytical grade from BDH, Glaxo Laboratories

(Qualigens), SRL, and Merck (India). Solvents were puri-

fied and dried according to the standard literature [57].

Bacterial culture and human cell lines

The clinically isolated resistant strains of Pseudomonas

aeruginosa and Proteus vulgaris and the standard strains of

Escherichia coli (DH5a and T7), Staphylococcus aureus

(SA 96), P. aeruginosa (MTCC 1688), and Klebsiella

planticola (MTCC 2272) used in this study were grown in

nutrient broth and subcultured as specified by the Microbial

Type Culture Collection and Gene Bank (MTCC, India).

Human cerebral glioma U87 and normal human embryonic

kidney (HEK) cells used in this study were grown as

monolayer cultures in DMEM with 10 % fetal calf serum

for both cell lines and antibiotics, i.e., penicillin (100 U/

ml), streptomycin (50 lg/ml), and nystatin (2 lg/ml).

Stock cultures were passaged every third day after har-

vesting the cells with 0.05 % trypsin and seeding

8 9 103 cells per square centimeter in tissue culture flasks

to maintain the cells in the exponential phase. All experi-

ments were performed in exponentially growing cells.

Syntheses

The complexes Et4N[Co(L1)2] (1) and [Co(L2)3] (10) were

prepared as reported earlier [50, 56]. The heterometallic

complexes 2, 5, 7, and 8 were synthesized according to our

recent reports [50, 51].

[Co(L1)2(Zn)2(NO3)3(DMF)] (3)

To a solution of Zn(NO3)2�6H2O (0.090 g, 0.30 mmol) in

dimethylformamide (DMF; 5 ml) was added a solution of

complex 1 (0.100 g, 0.12 mmol) dissolved in 5 ml DMF.

The resulting deep-red solution was stirred for 1 h at room

temperature. The reaction mixture was filtered, followed by

the removal of solvent under reduced pressure. The crude

product was isolated after washing with diethyl ether. The

product thus obtained was redissolved in DMF and layered

with diethyl ether. This afforded a crystalline product that

was filtered, washed with diethyl ether, and air-dried.

Yield: 0.10 g, 75 %. Elemental analysis: found (calculated)

(%) for C37H31N14O15CoZn2 (including one H2O): C,

40.58 (40.35); H, 2.45 (2.84); N, 17.87 (17.80). IR

1218 J Biol Inorg Chem (2012) 17:1217–1230

123

spectrum (KBr, 1/cm): 1,637 m(C=ODMF), 1,603 m(C=O),

and 1,385 m(NO3-). kmax/nm (DMF, e, dm3/mol/cm): 650

(90), 540 (sh, 145), 365 (sh, 4,310), 300 nm (sh, 10,580).

Mass spectrometry (MS; CH3CN): m/z = 1,083.98 [Co

(L1)2(Zn)2(NO3)3(DMF)] (calculated mass 1,083.42).

Molecular conductivity (approximately 1 mM, CH3CN,

25 �C): KM = 40 S cm2/mol (the range for a 1:1 electro-

lyte in CH3CN is 120–160). 1H NMR spectrum [d6-dime-

thyl sulfoxide (DMSO), 300 MHz, d, ppm]: 7.85 (d, 4H;

br, H2), 6.93 (m, 4H; br, H3), 7.49 (m, 4H; br, H4), 7.67 (d,

4H; H5), 7.0 (d, 4H; H9), 8.06 (t, 2H; H10), 2.89 (CH3,

DMF), 2.73 (CH3, DMF), 8.04 (CHO, DMF). 13C NMR

spectrum (d6-DMSO, 75 MHz, d, ppm): 147.68 (C2);

123.62 (C3); 124.10 (C4); 122.74 (C5); 147.68 (C6); 167.30

(C7); 158.53 (C8); 140.13 (C9); 138.38 (C10).

Et4N[Co(L1)2(Cd)2(Cl)4] (4)

To a solution of CdCl2 (0.057 g, 0.25 mmol) in dry DMF

(5 ml) was added a solution of complex 1 (0.100 g,

0.12 mmol) dissolved in 5 ml dry DMF. The deep-yellow

solution was stirred for 1 h at room temperature. The

reaction mixture was filtered, followed by the removal of

solvent under reduced pressure. The crude product was

obtained after trituration with diethyl ether. The crude

product thus obtained was dissolved in CH3CN and sub-

jected to vapor diffusion of diethyl ether. This afforded a

highly crystalline product within 48 h, which was filtered,

washed with diethyl ether, and dried under a vacuum. Yield:

0.09 g, 64 %. Elemental analysis: found (calculated) (%) for

C42H42N11O4CoCd2Cl4: C, 42.12 (42.38); H, 3.77 (3.56); N,

12.82 (12.94). IR spectrum (KBr, 1/cm, selected peaks):

1,600 m(C=O). kmax/nm (DMF, e, dm3/mol/cm): 660 (75), 520

(sh, 250), 460 (sh, 440), 300 nm (sh, 5130). MS (CH3CN):

m/z = 1,098.87 [Co(L1)2(Cd)2(Cl)3(DMF)] (calculated

mass 1,098.88). Molecular conductivity (approximately

1 mM, CH3CN, 25 �C): KM = 50 S cm2/mol. 1H NMR

spectrum (d6-DMSO, 300 MHz, d, ppm): 7.79 (d, 4H; br,

H2), 6.85 (m, 4H; br, H3), 7.40 (m, 4H; br, H4), 7.62 (d, 4H;

H5), 6.99 (d, 4H; H9), 8.04 (t, 2H; H10), 2.89 (CH3, DMF),

2.73 (CH3, DMF), 7.95 (CHO, DMF). 13C NMR spectrum

(d6-DMSO, 75 MHz, d, ppm): 147.38 (C2), 123.43 (C3),

138.11 (C4), 119.16 (C5), 156.78 (C6), 168.05 (C7), 162.44

(C8), 158.54 (C9), 138.43 (C10).

NN N

OO

Co

NN N

OO

N

NM

Cl

Cl

N

NM

Cl

Cl

M = Cd (4)M = Hg (6)

NN N

OO

Co

NN N

OO

N

NM

N

NM

S

S

NN N

OO

Co

NN N

OO

N

NZn

X

DMF

N

NZn

X

X

NN

O

Co

NN

O

N

N

N

NO

N

MX

X

X = Cl (2)X = NO3 (3)

M = Cd; S = DMF (5)M = Hg; S = DMSO (7)

M = Zn; X = Cl (8)M = Cd; X = NO3 (9)

NN

O

Co

NN

O

NN

N

NO

N

10

-

-

NN N

OO

Co

NN N

OO

N

NN

N

1

NO3

O3N

O3N

Fig. 1 Building blocks and

{M2?–Co3?–M2?} and {Co3?–

M2?} heterometallic complexes

(M is Zn, Cd, or Hg) used

in this study. DMFdimethylformamide, DMSOdimethyl sulfoxide

J Biol Inorg Chem (2012) 17:1217–1230 1219

123

Et4N[Co(L1)2(Hg)2(Cl)4] (6)

To a solution of HgCl2 (0.068 g, 0.25 mmol) in dry

CH3CN (8 ml) was added a solution of complex 1

(0.100 g, 0.12 mmol) dissolved in 5 ml dry CH3CN. The

resulting deep-yellow solution was stirred for 1 h at room

temperature. After the reaction mixture had been strirred,

the filtrate was subjected to diethyl ether diffusion. This

afforded a deep-yellow crystalline compound within

2–3 days. The product was filtered, washed with diethyl

ether, and dried under a vacuum. Yield: 0.12 g, 72 %.

Elemental analysis: found (calculated) (%) for C42H44N11

O5CoHg2Cl4 (including one H2O): C, 36.25 (36.43);

H, 3.07 (3.20); N, 11.08 (11.13). IR spectrum (KBr,

1/cm, selected peaks): 1,597 m(C=O). kmax/nm (DMF, e,dm3/mol/cm): 660 (180), 540 (sh, 240), 480 (sh, 1,980).

MS (CH3CN): m/z = 1,275.02 [Co(L1)2(Hg)2(Cl)3(DMF)]

(calculated mass 1,275.02). Molecular conductivity (appr-

oximately 1 mM, CH3CN, 25 �C): KM = 75 S cm2/mol.1H NMR spectrum (d6-DMSO, 400 MHz, d, ppm): 7.73 (d,

4H; H2), 6.82 (m, 4H; br, H3), 7.35 (m, 4H; H4), 7.70

(d, 4H; H5), 7.10 (d, 4H; H9), 7.99 (t, 2H; H10), 2.90 (CH3,

DMF), 2.75 (CH3, DMF), 7.97 (CHO, DMF). 13C NMR

spectrum (d6-DMSO, 100 MHz, d, ppm): 148.28 (C2),

123.05 (C3), 138.22 (C4), 118.78 (C5), 156.67 (C6), 167.49

(C7), 163.78 (C8), 153.43 (C9), 138.43 (C10).

[Co(L2)3Cd(NO3)2] (9)

To a solution of Cd(NO3)2�4H2O (0.068 g, 0.22 mmol) in

CH3CN (10 ml) was added a solution of complex 10

(0.100 g, 0.15 mmol) dissolved in 5 ml CH3CN. After the

reaction mixture had been strirred for 1 h, the filtrate was

subjected to vapor diffusion of diethyl ether. A highly

crystalline product resulted within 1 day and was filtered

and dried under a vacuum. Yield: 0.12 g, 88 %. Elemental

analysis: found (calculated) (%) for C33H26N11O10CoCd

(including one H2O): C, 43.42 (43.65); H, 2.87 (2.89); N,

12.59 (12.38). IR spectrum (KBr, 1/cm): 1,623, 1,598,

1,561 m(C=O); 1,379 m(NO3-). kmax/nm (DMF, e, dm3/mol/

cm-1): 520 (240), 309 nm (20,500). MS (CH3CN): m/z =

891.02 [Co(L2)3Cd(NO3)2] (calculated mass 891.01).

Molecular conductivity (approximately 1 mM, CH3CN,

25 �C): KM = 35 S cm2/mol. 1H NMR spectrum (d6-

DMSO, 300 MHz, d, ppm): 9.80, 9.55, 8.58, 8.40, 8.22,

7.78, 7.70, 7.62, 7.50, 7.20–7.30, 7.10–7.20, 6.70–6.80,

5.96, 5.76. 13C NMR spectrum (d6-DMSO, 75 MHz, d,

ppm): 169.82, 168.78, 168.40, 160.27, 159.64, 156.96,

156.39, 155.45, 151.02, 150.86, 150.19, 148.79, 147.87,

147.47, 141.18, 139.79, 136.41 (two signals are merged),

135.31, 136.15, 127.01, 125.30 (two signals are merged),

124.44, 123.84, 123.67, 123.44 (three signals are

merged), 122.44, 120.59, 119.38, 118.99.

Physical measurements

The conductivity measurements were done in organic sol-

vents using a model PT-825 digital conductivity bridge

from Popular Traders (India). The elemental analysis data

were obtained using an Elementar Analysensysteme Vario

EL-III instrument. The NMR spectral measurements were

done using either a Bruker Avance (300 MHz) or a JEOL

(400 MHz) instrument. The IR spectra (either as a KBr

pellet or as a mull in mineral oil) were recorded using a

PerkinElmer model 2000 Fourier transform IR spectrom-

eter. The absorption spectra were recorded using a Perk-

inElmer Lambda-25 spectrophotometer. The mass spectra

were obtained with a liquid chromatography–time-of flight

(KC-455) mass spectrometer from Waters.

Crystallography

The single crystals suitable for the X-ray diffraction studies

for complex 6 were grown by the slow diffusion of diethyl

ether into a DMF solution of the compound at room tem-

perature. The intensity data for complex 6 were collected

with an Oxford Diffraction CCD diffractometer with an

Xcalibur sapphire diffraction measurement device at

293(2) K using graphite-monochromated Mo Ka radiation

(k = 0.71073 A) [58]. A multiscan absorption correction

was applied [58]. The structure was solved by the direct

method using SIR-97 and refined by the full-matrix least-

squares method on F2 (SHELXL-97). All calculations were

performed using the WinGX crystallographic software

package [59]. The non-hydrogen atoms were refined

anisotropically, whereas the hydrogen atoms were placed

into the geometrically calculated positions using a riding

model. The crystal structure of complex 6 contains solvent-

accessible voids of 515 A3 due to the crystal packing

effect. The crystallographic data collection and structure

solution parameters are compiled in Table 1.

Crystallographic data (without structure factors) for the

structure reported in this article have been deposited with

the Cambridge Crystallographic Data Centre (CCDC) as

supplementary publication no. CCDC-874991. Copies of

the data can be obtained free of charge from the CCDC (12

Union Road, Cambridge CB2 1EZ, UK; Tel.: ?44-1223-

336408; Fax: ?44-1223-336003; e-mail: deposit@ccdc.

cam.ac.uk; website: http://www.ccdc.cam.ac.uk)

In vitro antibacterial activity (microbroth dilution

assay)

All complexes were screened [56, 60] in vitro against

clinically isolated resistant strains of P. aeruginosa and

P. vulgaris and standard strains of E. coli (DH5a and T7),

S. aureus (SA 96), P. aeruginosa (MTCC 1688), and

1220 J Biol Inorg Chem (2012) 17:1217–1230

123

K. planticola (MTCC 2272). Various methods are available

for the evaluation of the antibacterial activity of different

types of drugs. However, the most widely used method,

which consists in adding the drug in known concentrations

to cultures of the test organisms, was employed. Different

concentrations of the test compounds in 200 ll of culture

medium were prepared in 96-well flat-bottomed micro-

culture plates (Nunc, Nunclon) by the double-dilution

method. The wells were prepared in triplicate for each

concentration. Each well was inoculated with 10.0 ll of

bacterial suspension containing 107 cells per milliliter. The

plates were incubated at 37 �C for 16 h and the optical

density (OD) of the suspension was measured at 600 nm to

assess the inhibition of the cell growth caused by treatment

with the compounds. All tests were repeated three times.

In vitro cell growth inhibition assay (MTT assay)

Cells were seeded in 96-well plates at a concentration of

1 9 104 cells per well in 200 ll of complete medium and

were incubated for 24, 48, and 72 h at 37 �C in a 5 % CO2

atmosphere to allow cell adhesion to occur [61, 62]. Stock

solutions (2 mg/ml) of the compounds made in PBS were

filter-sterilized, then further diluted up to 0.45 lg/ml

incomplete medium for treatment against HEK and U87

cell lines. A 100-ll solution of the compound was added to

a 100-ll solution of fresh medium in wells to give final

concentrations of 0.45–1,000 lg/ml. All assays were per-

formed in two independent sets of quadruplicate tests. A

control group containing no drug was run in each assay.

Following 24, 48, and 72 h exposure of cells to the drug,

each well was carefully rinsed with 200 ll PBS. Cytotox-

icity was assessed using MTT. For this, 20 ll of MTT

solution (5 mg/ml double-distilled water) and 200 ll of

fresh, complete medium were added to each well, and the

plates were incubated for 4 h. Following incubation, the

medium was removed and the purple formazan precipitate

in each well was sterilized in 200 ll DMSO. Absorbance

was measured using a Tecan microplate reader (Molecular

Devices) at 570 nm and the results were expressed as the

percent viability, which is directly proportional to number

of metabolically active cells:

% Viability ¼ OD in sample well=OD in control well

� 100:

Clonogenic survival assay

After they had been harvested with 0.05 % trypsin,

150–400 (depending on the treatment) cells were plated

10–14 h before treatment with various concentrations of

drugs in DMEM at 37 �C. Cultured cells were treated with

various doses (62–1,000 lg/ml) of the compounds. After

the treatment, cells were incubated in the dark under a

humidified, 5 % CO2 atmosphere at 37 �C for 8–10 days to

allow colony formation. Colonies were fixed with methanol

and stained with 1 % crystal violet. Colonies of more than

50 cells were counted and the surviving fractions were

calculated [63]. Clonogenic survival curves were con-

structed from three independent experiments by least-

squares regression fitting averaged survival levels.

Cell cycle perturbations

Flow cytometry measurements of cellular DNA content after

24 h of treatment were performed with cells fixed with

ethanol (70 %) using the intercalating DNA fluorochrome PI

as described earlier [64–66]. Briefly, the cells (0.5–1 9 106)

were washed in PBS after the removal of ethanol and treat-

ment with ribonuclease A (200 lg/ml) for 30 min at 37 �C.

Subsequently, the cells were stained with PI (50 lg/ml) in

PBS. Measurements were made with a laser-based (488 nm)

flow cytometer (FACSCalibur; Becton, Dickinson, USA)

and data were acquired using Cell Quest (Becton, Dickinson,

USA). Cell cycle analysis was performed using the program

Modfit (Becton, Dickinson, USA).

Formation of micronuclei

Air-dried slides containing cells fixed with acetic acid–

methanol (1:3 v/v) were stained with Hoechst 33342

Table 1 Crystallographic data collection and structural refinement

parameters for complex 6

6

Molecular formula C42H42Cl4CoHg2N11O4

Formula weight 1,366.78

T (K) 298(2)

Crystal system Triclinic

Space group P-1

a (A) 12.3355(7)

b (A) 14.8451(9)

c (A) 15.2833(9)

a (�) 89.256(5)

b (�) 75.544(5)

c (�) 84.589(5)

V (A3) 2,697.9(3)

Z 2

d (g/cm3) 1.683

F(000) 1,316

Goodness of fit (F2) 0.968

R1, wR1

[I [ 2r(I)]

R1 = 0.0528,

wR1 = 0.1135

R1, wR2

(all data)

R2 = 0.0787,

wR2 = 0.1237

J Biol Inorg Chem (2012) 17:1217–1230 1221

123

(10 lg/ml in 0.1 M PBS, 0.45 M disodium phosphate

buffer containing 0.05 % Tween 20 detergent). Slides were

examined under a fluorescence microscope using a UV

excitation filter. Fluorescent nuclei were visualized using a

blue emission filter. Cells containing micronuclei were

counted from more 1,000 cells by employing the criteria of

Countrymen and Heddle [67]. The fraction of cells con-

taining micronuclei, called the M-fraction (%), was cal-

culated as follows:

M-fraction ð%Þ ¼ Nm=Nt � 100

where Nm is the number of cells with micronuclei and Nt is

the total number of cells analyzed. Since the formation of

micronuclei is linked to cell proliferation, the frequencies

of micronuclei were normalized with respect to the cell

numbers.

Detection of apoptotic cells

Morphologically, marked condensation and margination of

chromatin, fragmentation of nuclei, and cell shrinkage

characterize apoptotic cells, and a good correlation

between these morphological changes and DNA fragmen-

tation (ladder) as hallmarks has been demonstrated [65,

66]. The percentage of cells undergoing apoptosis was

determined microscopically and by flow cytometry using

PI-labeled cells. At least 1,000 cells were counted and the

percentage of apoptotic cells was determined from slides

prepared as described in ‘‘Formation of micronuclei.’’ In

flow-cytometric DNA analysis, the presence of a hypo-

diploid (sub-G0/G1) population (with PI-stained cells, as

described for cell cycle analysis) is indicative of an apop-

totic cell population. Measurements of cellular DNA con-

tent were made from PI-stained, ethanol-fixed cells as

described in ‘‘Cell cycle perturbations.’’ Analysis of the

hypodiploid population was performed using the program

Modfit (Becton, Dickinson, USA).

Results and discussion

Chemistry

The chemistry applied uses three classes of coordination

compounds for pharmacological purposes. The first class of

compounds includes the building block molecules 1 and

10. Compounds 1 and 10 were synthesized using either a

tridentate or a bidentate pyridine–amide-based ligand,

respectively [50, 56]. Complexes 1 and 10 have four and

three uncoordinated pyridine rings, respectively; these

converge to create molecular clefts. These molecular clefts

were used to bind the secondary metal ions to generate

the trimetallic complexes {Zn2?–Co3?–Zn2?} (2 and 3),

{Cd2?–Co3?–Cd2?} (4 and 5), and {Hg2?–Co3?–Hg2?} (6

and 7) as well as the bimetallic complexes {Co3?–Zn2?}

(8) and {Co3?–Cd2?} (9) (Fig. 1). Building blocks 1 and

10, trimetallic complexes 2, 5, and 7, and bimetallic

complex 8 were characterized crystallographically in our

earlier studies [50, 51].

The Fourier transform IR spectra of new complexes 3

and 9 show a strong stretch at approximately 1,385/cm due

to vibrations arising from the coordinated NO3- anion [68].

The other class of new compounds, 4 and 6, has the Et4N?

cation, which was supported by the observation of the mC–H

stretches. Conductivity measurements [69] of freshly pre-

pared solutions of complexes 3, 4, and 6 suggest a non-

conducting nature; however, with time, the conductivity

increases, suggesting partial dissociation of the weakly

coordinating anion. A similar observation was made for

complexes 5 and 7 [51]. Complexes 3, 4, and 6 were also

characterized by their high–resolution mass spectra. For

complex 3, the molecular ion peak was observed at

1,083.98 and fits well with the proposed composition of

[Co(L)2(Zn)2(NO3)3(DMF)] (calculated mass 1,083.42).

Both complex 4 and complex 6 displayed the molecular ion

peak at 1,098.87 (calculated 1,098.88) and 1,275.02 (cal-

culated 1,275.02), respectively, for the general composition

[(1)2(Cd/Hg)2(Cl)3(DMF)]. This observation suggests that

one of the chloride ions was replaced by the solvent DMF

during the mass spectral analysis. Importantly, conductiv-

ity measurements also indicated solvolysis of both of these

complexes. Thus, the solution studies indicate that both

complex 4 and complex 6 have a solution-based structure

as observed for complexes 2, 3, 5, and 7 where one of the

coordination sites is occupied by the solvent molecule.

Complex 9 displayed the molecular ion peak at 891.02

(calculated 891.01), corresponding to the composition

[(10)Cd(NO3)3].

All new complexes were also characterized by 1H and13C NMR spectroscopy owing to their diamagnetic state

(Fig. S1). The 1H NMR spectra of complexes 3, 4, and 6

are very similar to the spectrum of parent complex 1 as

well as the spectra of the other trimetallic complexes 2, 5,

and 7 [50, 51]. This is not unexpected as coordination of a

diamagnetic metal center may not have significantly altered

the chemical environment of the protons or carbons.

However, a few proton signals became quite broad, and we

tentatively assign them as the pyridine hydrogen closer to

the secondary metal center. The 1H NMR spectrum of

complex 9 was found to be highly complex owing to the

meridional (mer) configuration of three ligands around the

metal. Typically, a mer isomer has three identical groups

(coordinated Npyridine or Namide groups in complex 9;

Fig. 1) in a plane bisecting the molecule [70]. As a con-

sequence, the three coordinated ligands, not related through

symmetry, are expected to be NMR-inequivalent. The

1222 J Biol Inorg Chem (2012) 17:1217–1230

123

1H NMR spectrum shows this to be the case with threefold

signals (Fig. S1). For example, the deprotonated ligand is

expected to display eight proton signals due to two

chemically different pyridine rings; complex 9, however,

shows 24 proton signals due to three chemically non-

equivalent coordinated ligands. The 13C NMR spectrum of

complex 9 displays 33 signals due to three nonequivalent

ligands each having 11 unique carbon centers. Similar

NMR spectral results were observed for structurally char-

acterized complexes 8 and 10 [50, 56].

Although there was no ambiguity about the composition

of complexes 3 and 9 as their chemical analogues with a

coordinated chloride ion (i.e., complexes 2 and 8) have

been structurally characterized [50], there was doubt about

the composition of complexes 4 and 6 because their

microanalysis data supported the presence of coordinated

chloride ions, whereas the solution studies (conductivity

and mass spectral analysis in DMF) pointed towards the

replacement of one chloride ion by DMF. Thus, to ascer-

tain the solid-state structure, one of the representative

complexes, compound 6, was characterized crystallo-

graphically. This compound crystallized in the triclinic cell

system with the P-1 space group. The molecular structure

of this compound is shown in Fig. 2, and selected bond

distances and angles are presented in Table 2. As can be

seen from the molecular structure, two Hg(II) ions are

symmetrically placed within the molecular clefts created

by the building block molecule. Every Hg(II) ion is four-

coordinate, with the geometry distorted from a perfect

tetrahedron. The tetrahedral distortion parameter, s4 [71], is

0.865 and 0.850 for Hg1 and Hg2 ions, respectively. This

parameter has an ideal value of 1 for a perfect tetrahedral

geometry and 0 for a perfect square-planar geometry [71].

Every Hg(II) atom is coordinated by two Npyridine atoms

originating from the building block and the remaining two

coordination sites are occupied by the chloride ions. The

average Hg–Npyridine and Hg–Cl distances are 2.302 and

2.666 A, respectively. These distances are within the range

reported for similar examples in the literature [51, 72–74].

The geometry around the central Co3? ion is compressed

octahedral as noted for its precursor building block mole-

cule 1 [50] and other examples in the literature [51, 52, 73,

75]. The average Co–Npyridine distance (1.865 A) is

somewhat shorter than the average Co–Namide distance

(1.975 A) [50, 75]. Notably, these distances are compara-

ble to the distances for building block 1 and other hetero-

bimetallic complexes [50, 51, 75]. Two axial pyridine rings

are trans to each other and make an angle of more than

176� with the Co3? ion as also noted for complex 1

[50, 75]. The distorted N4 basal plane composed of

Namide groups perfectly houses the Co3? ion.

Fig. 2 Molecular structure of complex 6 with partial numbering

scheme. Thermal ellipsoids are drawn at the 50 % probability level,

and the cation and hydrogen atoms have been omitted for clarity

Table 2 Selected bond

distances (A) and bond angles

(�) for complex 6

Bond distance Bond angle

Co(1)–N(1) 1.969(5) N(6)–Co(1)–N(5) 176.2(2)

Co(1)–N(2) 1.969(5) N(3)–Co(1)–N(1) 89.6(2)

Co(1)–N(3) 1.988(5) N(3)–Co(1)–N(2) 92.4(2)

Co(1)–N(4) 1.974(5) N(1)–Co(1)–N(2) 162.6(2)

Co(1)–N(5) 1.857(5) N(3)–Co(1)–N(4) 162.4(2)

Co(1)–N(6) 1.872(5) N(1)–Co(1)–N(4) 93.6(2)

Hg(1)–N(7) 2.321(6) N(2)–Co(1)–N(4) 89.7(2)

Hg(1)–N(8) 2.291(6) N(8)–Hg(1)–N(7) 121.3(2)

Hg(2)–N(9) 2.262(5) N(9)–Hg(2)–N(10) 121.40(19)

Hg(2)–N(10) 2.332(5) Cl(1)–Hg(1)–Cl(2) 116.69(11)

Hg(1)–Cl(1) 2.429(2) Cl(3)–Hg(2)–Cl(4) 118.81(9)

Hg(1)–Cl(2) 2.440(3)

Hg(2)–Cl(3) 2.446(3)

Hg(2)–Cl(4) 2.439(2)

J Biol Inorg Chem (2012) 17:1217–1230 1223

123

The negative charge on the complex anion is balanced

by the Et4N? cation. Notably, the Et4N? group takes part

in weak interactions with the complex anion via various

C–H���Cl and C–H���C hydrogen-bonding interactions

(Fig. S2). The Oamide atom O3 of one molecule forms a

hydrogen bond with H27–C27 of the pyridine ring from

other molecule to form a one-dimensional chain. The het-

eroatom separation for O3 and C27 is 3.236 A. One such

chain is attached to another neighboring chain through the

Et4N? cation. The Oamide atoms O2 and O4 of two different

molecules in a chain form hydrogen bonds with the methyl

and methylene protons attached to the C42 and C37 carbon

atoms of the Et4N? cation. The chloride ion Cl1 interacts

weakly with H41–C41 of the Et4N? cation. The hetero-

atom separations for C42���O2, C37���O4, and C41���Cl1 are

3.376, 3.127, and 3.828 A, respectively. These interactions

result in the generation of a two-dimensional sheet

where complex anions and Et4N? groups are arranged

alternatively

Pharmacology

Antibacterial activity

In this study, all complexes were tested against pathogenic

clinically isolated resistant strains of P. aeruginosa and

P. vulgaris and standard strains of S. aureus (SA 96),

P. aeruginosa (MTCC 1688), K. planticola (MTCC 2272),

and E. coli (DH5a and T7) employing the microbroth

dilution method [56, 60] (Table 3). Gentamicin was used

as a reference drug for these studies. Complexes 2 and 4–7

show good activity against the resistant strain of P. vulgaris

and a standard strain of E. coli (T7), with minimum

inhibitory concentrations ranging from 15.6 to 62.5 lg/ml.

Complex 7 was four times more effective than complex 5

against the P. vulgaris strain and three times more effective

against E. coli (T7). Taken together, the data show com-

plex 7 was the most potent compound in this series against

P. vulgaris and the standard strain of E. coli (T7). Complex

4 was effective against the standard strain of P. aeruginosa,

with a minimum inhibitory concentration of 31.25 lg/ml.

Complex 4 was four times more effective than complexes 2

and 6 and three times more effective than complex 7

against the standard strain of P. aeruginosa. In the litera-

ture, a few coordination complexes have been reported

to have moderate activity against E. coli, S. aureus,

P. aeruginosa, Bacillus subtilis, and Candida albicans

microbes [76–78]. Other complexes appeared to have a

broad spectrum as they showed mild to moderate activity

towards most of the strains. Furthermore, complexes 2–9

also exhibited strong inhibitory characteristics against

clinically isolated resistant strains of E. coli and P. vul-

garis. We postulate that the effectiveness of these com-

plexes could be due to either easy diffusion through the cell

Table 3 Minimum inhibitory concentrations of substituted pyrazino[1,2-a]indoles on resistant and standard bacterial strains obtained by the

microbroth dilution method

Compound Minimum inhibitory concentration (lg/ml)

Standard strain Resistant strains

Escherichiacoli (DH5a)

Staphylococcusaureus (SA 96)

Pseudomonasaeruginosa (MTCC 1688)

Klebsiella planticola(MTCC 2272)

Escherichiacoli (T7)

Proteusvulgaris

Pseudomonasaeruginosa

2 250 250 125 125 250 31.25 125

3 500 – 500 500 125 – 500

4 125 250 31.25 – 62.5 62.5 125

5 250 250 125 500 31.25 250 250

6 500 125 500 500 31.25 62.5 500

7 250 62.5 250 500 31.25 15.6 250

8 125 125 125 250 125 62.5 500

9 – – 500 – – – 500

ZnCl2 500 – 250 500 500 500 250

Zn(NO3)2 125 – 250 250 1,000 500 250

Cd(NO3)2 125 – 250 – 250 125 125

CdCl2 125 – 62.5 – 250 62.5 125

Hg(NO3) 125 – 125 – 62.5 125 125

HgCl2 62.5 – 62.5 500 31.25 125 62.5

Gentamicin 250 15.6 7.8 250 7.8 3.9 125

All drugs were tested on bacteria at 107 cells per milliliter

Dash no activity

1224 J Biol Inorg Chem (2012) 17:1217–1230

123

membrane and/or an additional effect of labile sites present

on the secondary metal ions. The results suggest that these

complexes should be explored further as specific antibac-

terial drugs owing to their good activity against all exper-

imental strains.

Inhibitory effects of complexes on the proliferation of U87

brain cancer and normal HEK cells

The MTT cell proliferation assay has been widely accepted

as a reliable way to measure the cell proliferation rate and

conversely when metabolic events lead to apoptosis or

necrosis. The data obtained by the MTT assay show that

complexes 1–9 have inhibitory effects on the growth of

U87 brain cancer cells in a dose-dependent manner [61,

62]. All complexes effectively inhibited the growth of U87

cells, with their IC50 values ranging from 4.7 to 126 lg/ml.

Complexes 1, 2, 4, 6, and 7 were found to inhibit cell

growth in the range from 4.7 to 17 lg/ml after 24 h

treatment. All metal complexes were found to be less

cytotoxic to normal HEK cells than gentamicin (Table 4).

The data obtained suggest that the complexes are less

cytotoxic to normal HEK cells at moderate concentrations

(250–0.45 lg/ml). IC50 values of the heterometallic com-

plexes for HEK cells were in the range from 62.5 to

250 lg/ml. Metal salts—MCl2 or M(NO3)2 salts—were

more toxic to HEK cells than the heterometallic com-

plexes, and their IC50 values ranged from 19 to 64.1 lg/ml

(Table 5). These results indicate that the parent complexes

1 and 10 bind the secondary metal ions quite strongly and

do not release them under our experimental conditions. The

importance of such work lies in the possibility that

the next-generation metal complexes might be more effi-

cacious as antibacterial and anticancer agents. However,

further investigations relating to the structure and activity

of the complexes as well as their stability under biological

conditions will be helpful and may result in the design of

more potent antibacterial and anticancer agents for thera-

peutic use.

Inhibitory effects of complexes on the colony-forming

capacity of U87 cells

A dose-dependent decrease in the survival fraction of cells

was found by the macrocolony assay (clonogenicity) [63]

in the U87 cell line, where the extent of cell death was

marginally higher at higher concentrations (Fig. 3). The

effect of complex-induced cell death was studied as a

function of the concentration of the complex (62.5–1,000

lg/ml). Treatment increased cell death of the U87 cells in a

concentration-dependent manner (Fig. 3), suggesting inhi-

bition of the repair processes and or increased damage

fixation. The survival fractions of U87 cells treated with

complexes 1, 4, 6, and 8 were significantly higher than

Table 4 IC50 and IC90 values evaluated from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on U87 glioma and

normal human embryonic kidney (HEK) cells treated with complexes after 24 h exposure

Compound U87 cells HEK cells

IC50 (lg/ml) IC90 (lg/ml) IC50 (lg/ml) IC90 (lg/ml)

1 4.7 (±0.6, ±1) 34 (±5.4, ±1) 30 (±0.6, ±0.4) 483 (±28, ±1)

2 17 (±2.5, ±4) 121 (±10.4, ±1.9) 61 (±1.4, ±1) 416 (±28.8, ±1)

3 118 (±7.6, ±12.1) 533 (±57, ±10) 121.6 (±2.8, ±2) [1,000

4 17 (±2.5, ± 4) 128 (±2.8, ±0.53) 65 (±4.3, ±3) 483 (±28, ±1)

5 120 (±10, ± 15.8) 246 (±5.7, ±1) 60.7 (±1.2, ±0.8) 250

6 9.2 (±1, ± 2) 118 (±7.6, ±1.4) 18.4 (±2.7, ±1.9) 550 (±50, ±1.73)

7 7 (±1.6, ± 2) 34 (±5.4, ±1) 30 (±0.7, ±0.5) 283 (±28.8, ±1)

8 126 (±2.8, ± 4.5) 433 (±57.7, ±10.6) 61.6 (±1.44, ±1) 283 (±28.8, ±1)

9 61.6 (±1.4, ± 2.2) 283 (±28.8, ±5.3) 61.6 (±1.44, ±1) 500

Gentamicin NA NA 8.5 (±1.2, ±0.8) 60.8 (±1.4, ±0.5)

Values are the mean (± standard deviation, ± standard error) of three observations from three independent experiments

NA not applicable

Table 5 IC50 values evaluated from MTT assay on normal HEK

cells treated with metal salts for 24 h

Compound IC50 (lg/ml)

ZnCl2 64.1 (±5.2, ±4.3)

Zn(NO3)2 30 (±0.7, ±0.6)

Cd(NO3)2 19 (±2.5, ±2.1)

CdCl2 19 (±2.5, ±2.1)

Hg(NO3)2 28.75 (±3.3, ±2.7)

HgCl2 30 (±0.7, ±0.6)

Gentamicin 8.5 (±1.2, ±0.8)

Values are the mean (± standard deviation, ± standard error) of three

observations from three independent experiments

J Biol Inorg Chem (2012) 17:1217–1230 1225

123

those of other treated cells. This suggests that these

complexes inhibited the cell growth and colony-forming

capacity of the U87 cancer cells. Metal complexes 3, 5, 7,

and 9 were moderately active compared with complexes 1,

4, 6, and 8. In the literature, a few heterometallic com-

plexes have been reported to be moderately active against

U373MG brain cancer cells [79, 80].

Effect of complexes on DNA cell cycle perturbations

To investigate the role of cell cycle perturbations after

treatment, cell cycle distributions were analyzed by cyto-

fluorimetric measurements of cellular DNA contents after a

24 h interval [64–66] (Tables 6, 7). All complexes resulted

in cells having a hypodiploid DNA content (sub-G1

material) that is characteristic of apoptosis and reflects

fragmented DNA. Treatment of U87 cells with the com-

plexes at a concentration of 62.5 lg/ml for 24 h induced

apoptosis effects in 12 % (1), 10 % (2), 11 % (4), 11 %

(7), and 14 % (9) of the sub-G1 DNA peak. The hetero-

metallic complexes also transiently delayed cell cycle

progression, with a significant accumulation in G2–M

phase after 24 h post-treatment incubation (Fig. S2). This

delay was further increased after the concentration of the

metal complexes had been increased. Although the G2–M

block was not transient in the U87 cells, at higher con-

centration of complex 4, a profound decrease in G2–M

phase cells was observed in U87 cells, with a concomitant

increase in cell death. This is most likely due to apoptosis

as indicated by the appearance of cell population with

hypodiploid DNA content. We further conducted micro-

nucleus assay for the confirmation of apoptosis and cyto-

genetic damage by the metal complexes.

Cytogenetic damage

Mitotic death (linked to cytogenetic damage) and inter-

phase death (apoptosis) together account for the cytotox-

icity of many physicochemical agents, although the relative

contributions of the two death processes differ depend-

ing on the type of damaging agent. To investigate the

modifications of cytogenetic damage by the heterometallic

Table 6 DNA cell cycle analysis on U87 cells treated with com-

plexes at 62.5 lg/ml

Complex G0/G1 S G2/M Apoptosis

1 50 38 12 12

2 47 40 13 10

3 41 48 11 7

4 40 52 8 11

5 41 48 11 8

6 50 38 12 7

7 50 38 12 11

8 45 41 14 8

9 42 42 16 14

Control 45 43 12 3

The results are shown as the percent frequency of DNA

Fig. 3 Effects of heterometallic

complexes on the exponentially

growing human glioma cell line

U87 studied by macrocolony

assay. Data presented are mean

values from at least three

independent observations

1226 J Biol Inorg Chem (2012) 17:1217–1230

123

complexes, we studied treatment-induced formation of

micronuclei in gliomas (Figs. 4, 5). Since cell proliferation

influences treatment-induced expression of micronuclei,

the data from kinetic studies up to 24 h after treatment

were analyzed. Complexes 1, 2, 4, and 6–9 showed a sig-

nificant frequency of micronuclei. The frequency of cells

with micronuclei was in the range from 22 to 38 %. The

frequency of micronuclei of complexes 1, 2, 4, and 7–9 was

fourfold to fivefold more than for the untreated control

group. Importantly, more than 20–25 % of cells were found

with one or more micronuclei in these treated groups at

both 31.25 and 62.5 lg/ml.

Complex induced apoptosis in U87 cells

The apoptotic mode of cell death in the U87 cell line was

confirmed by microscopy and flow cytometry. The changes

in the cell morphology were characterized by typical

chromatin condensation and nuclear fragmentation [62, 65,

66] with disruption of plasma membrane integrity and the

appearance of a cell population with hypodiploid DNA

content. Flow-cytometric analysis of the hypodiploid sub-

G0/G1 population showed that the metal complexes

induced a significant level of apoptosis in gliomas. Com-

plexes 1, 2, 4, and 6–9 showed a significant frequency of

apoptosis. The frequency of apoptosis of cells treated with

complexes 1, 4 and 9 is 13–18 %, which is two to three

times more than that of the untreated control group. Cells

treated with complexes 1 and 4 showed a significant

increase in both the frequency of apoptosis and the

Table 7 DNA cell cycle analysis on U87 cells treated with com-

plexes at 31.25 lg/ml

Complex G0/G1 S G2/M Apoptosis

1 46 41 13 12

2 45 41 14 8

3 37 45 18 6

4 46 42 12 10

5 43 40 17 7

6 42 43 15 6

7 41 40 19 9

8 49 38 13 7

9 42 41 17 8

Control 45 43 12 3

The results are shown as the percent frequency of DNA

Fig. 4 Fluorescence images of cells stained with Hoechst 33342

treated with heterometallic complexes at two different concentrations

(31.25 and 62.5 lg/ml) as observed from in vitro micronucleus assay.

The cells shown with one or more micronuclei were scored for the

frequency of micronuclei (a–h). The untreated control experiment is

shown in i

J Biol Inorg Chem (2012) 17:1217–1230 1227

123

frequency of micronuclei. Moreover, the treatment signif-

icantly increased apoptosis in a concentration-dependent

manner in the U87 cell line (Tables 6, 7). The frequency of

apoptosis increased by nearly 50 % following treatment

with the metal complexes at 24 h. The nonapoptotic pop-

ulation consisted predominantly of G1 and S phase cells

and to a lesser extent of G2–M phase cells (Tables 6, 7).

Further, these results were supported by the morphological

analysis of apoptosis (Figs. 4, 5).

Conclusions

This report has shown the use of Co3? complexes as the

building blocks for the preparation of a series of hetero-

metallic complexes. All new heterometallic complexes

were thoroughly characterized by various physical meth-

ods, and one of the representative complexes was investi-

gated crystallographically. The {M2?–Co3?–M2?} and

{Co3?–M2?} (M is Zn, Cd, Hg) heterometallic complexes

showed an unusually broad range of activity against both

human U87 gliomas and bacterial strains. These complexes

are more efficacious on U87 cancer cells and less toxic to

normal human HEK cells. The anticancer studies clearly

demonstrate that treatment with heterometallic complexes

sensitizes the U87 cells by increasing both mitotic (linked

to cytogenetic damage) and interphase (apoptosis) death.

The increased cell death due to metal complexes was also

accompanied by a significant increase in the formation of

micronuclei in the U87 cell line. The results suggest that

the bound metal complexes may inhibit the rejoining of

strand breaks created during excision repair. Further

Fig. 5 Comparison of

formation of micronuclei (MNi)and apoptosis in human gliomas

exposed in vitro in G1/S/G2

phase to various complexes at

a 31.25 lg/ml and b 62.5 lg/ml

measured either in

mononucleated cells in cultures

or in binucleated cells in

cultures. The extent of genetic

damage induced by the

heterometallic complexes,

particularly at doses that inhibit

nuclear division, is evident from

the assay estimates. The data

represent the mean ± the

standard error of three replicate

cultures. C control

1228 J Biol Inorg Chem (2012) 17:1217–1230

123

studies are in progress to understand the mechanism by

which these heterometallic complexes induce the formation

of micronuclei and apoptosis in U87 gliomas. Importantly,

these heterometallic complexes may be regarded as lead

molecules for a new class of anticancer agents with easy

further modification.

Acknowledgments R.G. gratefully acknowledges financial support

from the Department of Science & Technology (DST), Government

of India. The authors thank the CIF-USIC of the University of Delhi

for instrumental facilities. N.K.K. thanks the ACBR for laboratory

facilities.

References

1. Jung Y, Lippard SJ (2007) Chem Rev 107:1387

2. Guo Z, Sadler PJ (1999) Angew Chem Int Ed 38:1512

3. Fricker SP (2007) Dalton Trans 4903

4. Hartinger CG, Dyson PJ (2009) Chem Soc Rev 38:391

5. Schatzschneider U (2010) Eur J Inorg Chem 1451

6. Fry NL, Mascharak PK (2011) Acc Chem Res 44:289

7. Iakovidou Z, Papageorgiou A, Demertzis MA, Mioglou E,

Mourelatos D, Kotsis A, Yadav PN, Kovala-Demertzi D (2001)

Anticancer Drugs 12:65

8. Patole J, Dutta S, Padhye S, Sinn E (2001) Inorg Chim Acta

318:207

9. Maurer RI, Blower PJ, Dilworth JR, Reynolds CA, Zheng Y,

Mullen GED (2002) J Med Chem 45:1420

10. Cowly AR, Dilworth JR, Donnely PS, Labisbal E, Sousa A

(2002) J Am Chem Soc 124:5270

11. Ferrari MB, Bisceglie F, Pelosi G, Sassi M, Tarasconi P, Cornia

M, Capacchi S, Albertini R, Pinelli S (2002) J Inorg Biochem

90:113

12. Jouad EM, Thanh XD, Bouet G, Bonneau S, Khan MA (2002)

Anticancer Res 22:1713

13. Barry NPE, Sadler PJ (2012) Chem Soc Rev 41:3264

14. Gray HB (2003) Proc Natl Acad Sci USA 100:3563

15. Abrams MJ, Murrer BA (1993) Science 261:725

16. Sorenson JRJ (1984) Chem Br 16:1110

17. Crouch RK, Kensler TW, Oberlew LW, Sorenson JRJ (1986) In:

Karlin KD, Zubieta J(eds) Biochemical and inorganic copper

chemistry, vol 1. Adenine, New York, p 139

18. Cvek B, Milacic V, Taraba J, Dou QP (2008) J Med Chem

51:6256

19. Anderson RF, Denny WA, Ware DC, Wilson WR (1996) Br J

Cancer 74:S48

20. Teicher BA, Holden SA (1987) Radiat Res 109:58

21. Teicher BA, Abrams M, Rosbe K, Herman T (1990) Cancer Res

50:6971

22. Ware DC, Siim BG, Robinson KG, Denny WA, Brothers PJ,

Clark GR (1991) Inorg Chem 30:3750

23. Ware DC, Palmer BD, Wilson WR, Denny WA (1993) J Med

Chem 36:1839

24. Wilson WR, Moselen JW, Cliffe S, Denny WA, Ware DC (1994)

Int J Radiat Oncol Biol Phys 29:323

25. Ware DC, Palmer HR, Brothers PJ, Rickard CEF, Wilson WR,

Denny WA (1997) J Inorg Biochem 68:215

26. Blower PJ, Dilworth JR, Maurer RI, Mullen GD, Reynolds CA,

Zheng Y (2001) J Inorg Biochem 85:15

27. Singh DP, Kumar R, Singh J (2009) Eur J Med Chem 44:1731

28. Frau0sto da Silva JJR, Williams RJP (1991) The biological

chemistry of the elements. Oxford University Press, Oxford

29. Parkin G (2004) Chem Rev 104:699

30. Waalkes MP (2000) J Inorg Biochem 79:241

31. Waalkes MP (2003) Mutat Res 533:107

32. Stork JR, Thoi VS, Cohen SM (2007) Inorg Chem 46:11213

33. Garibay SJ, Stork JR, Wang Z, Cohen SM, Telfer S (2007) Chem

Commun 4881

34. Halper SR, Do L, Stork JR, Cohen SM (2006) J Am Chem Soc

128:15255

35. Halper SR, Cohen SM (2005) Inorg Chem 44:486

36. Kitagawa S, Kitaura R, Noro S-I (2004) Angew Chem Int Ed

43:2334

37. Kitagawa S, Noro S-I, Nakamura T (2006) Chem Commun 701

38. Caskey SR, Matzger AJ (2008) Inorg Chem 47:7942

39. Wang Y, Bredenkotter B, Rieger B, Volkmer D (2007) Dalton

Trans 689

40. Ren P, Shi W, Cheng P (2008) Cryst Growth Des 8:1097

41. Murray LJ, Dinca M, Long JR (2009) Chem Soc Rev 38:1294

42. Sun YQ, Zhang J, Yang GY (2006) Chem Commun 4700

43. Zhao B, Chen XY, Chen Z, Shi W, Cheng P, Yan SP (2009)

Chem Commun 3113

44. Zhao XQ, Zhao B, Shi W, Cheng P (2009) Cryst Eng Commun

11:1261

45. Lee J, Farha OK, Roberts J, Scheidt KA, Nguyen ST, Hupp JT

(2009) Chem Soc Rev 38:1450

46. Ma L, Abney C, Lin W (2009) Chem Soc Rev 38:1248

47. Huxford RC, Rocca JD, Lin W (2010) Curr Opin Chem Biol

14:262

48. Rocca JD, Liu D, Lin W (2011) Acc Chem Res 44:957

49. Pu F, Liu X, Xu B, Ren J, Qu X (2012) Chem Eur J 18:4322

50. Mishra A, Ali A, Upreti S, Gupta R (2008) Inorg Chem

47:154

51. Mishra A, Ali A, Upreti S, Whittingham MS, Gupta R (2009)

Inorg Chem 48:5234

52. Singh AP, Gupta R (2010) Eur J Inorg Chem 4546

53. Kumar G, Singh AP, Gupta R (2010) Eur J Inorg Chem 5103

54. Singh AP, Ali A, Gupta R (2010) Dalton Trans 39:8135

55. Singh AP, Kumar G, Gupta R (2011) Dalton Trans 40:12454

56. Mishra A, Kaushik NK, Verma AK, Gupta R (2008) Eur J Med

Chem 43:2189

57. Perrin DD, Armarego WLF, Perrin DR (1980) Purification of

laboratory chemicals. Pergamon, Oxford

58. Oxford Diffraction (2009) CrysAlisPro, version 1.171.33.49b.

Oxford Diffraction, Abingdon

59. Farrugia LJ (2003) WinGX version 1.64, an integrated system of

windows programs for the solution, refinement and analysis of

single-crystal X-ray diffraction data. University of Glasgow,

Glasgow

60. Singh AP, Kaushik NK, Verma AK, Hundal G, Gupta R (2009)

Eur J Med Chem 44:1607

61. Mosmann T (1983) J Immunol Methods 65:55

62. Zheng L-W, Wu L-L, Zhao B-X, Dong W-L, Miao J-Y (2009)

Bioorg Med Chem 17:1957

63. Hoffman RM (1991) J Clin Lab Anal 5:133

64. Wang J-J, Shen Y-K, Hu W-P, Hsieh M-C, Lin F-L, Hsu M-K,

Hsu M-H (2006) J Med Chem 49:1442

65. Singh S, Dwarakanath BS, Mathew TL (2004) J Photochem

Photobiol B Biol 77:45

66. Yanagihara K, Nii M, Nuot K, Kamiya P, Tauchi T, Sawada T,

Seito T (1995) Int J Radiat Biol 77:677

67. Countryman PI, Heddle JA (1976) Mutation Res 41:321

68. Nakamoto K (1986) Infrared and Raman spectra of inorganic and

coordination compounds. Wiley, New York

69. Geary WJ (1971) Coord Chem Rev 7:81

70. Jensen AW, O’Brien BA (2001) J Chem Ed 78:954

71. Yang L, Powell DR, Houser RP (2007) Dalton Trans 955

72. Poojary MD, Manohar H (1984) Inorg Chim Acta 93:153

J Biol Inorg Chem (2012) 17:1217–1230 1229

123

73. Burchell TJ, Eisler DJ, Puddephatt RJ (2004) Inorg Chem

43:5550

74. Henry M, Hosseini MW (2004) New J Chem 28:897

75. Bricks JL, Reck G, Rurack K, Schlz B, Spieless M (2003) Su-

pramol Chem 15:189

76. Husain A, Nami SAA, Siddiqi KS (2010) J Mol Struct 970:117

77. Tabassum S, Zaki M, Arjmand F, Ahmad I (2012) J Photochem

Photobiol B Biol 114:108

78. Aliyu AO, Adamu H, Maikajes DB (2012) Glob J Sci Front Res

Chem 12:20

79. Tabassum S, Khan RA, Arjmand F, Aziz M, Juvekar AS, Zingde

SM (2011) Carbohydr Res 346:2886

80. Narla RK, Chen C-L, Dong Y, Uckun FM (2001) Clin Cancer

Res 7:2124

1230 J Biol Inorg Chem (2012) 17:1217–1230

123