In vivo and in vitro evaluation of highly specific thiolate carrier group copper(II) and zinc(II) complexes on Ehrlich ascites carcinoma tumor model

Article abstract of DOI:10.1016/j.ejmech.2010.09.004

A new series of copper(II) and zinc(II) complexes have been designed and synthesized using a new type of Schiff bases derived from the reaction of 3-(3-phenyl-allylidene)-pentane-2,4-dione with para substituted aniline and benzene-1,2-dithiol. Their struc

Full text of DOI:10.1016/j.ejmech.2010.09.004

European Journal of Medicinal Chemistry 45 (2010) 5438e5451
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
In vivo and in vitro evaluation of highly specific thiolate carrier group copper(II)
and zinc(II) complexes on Ehrlich ascites carcinoma tumor model
,
a
b
c
d
N. Raman a , R. Jeyamurugan , R. Senthilkumar , B. Rajkapoor , Scott G. Franzblau
*
a Research Department of Chemistry, VHNSN College, Virudhunagar-626 001, Tamil Nadu, India
b Department of Pharmaceutical Chemistry, Swami Vivekanandha College of Pharmacy, Elayampalayam, Tiruchengodu-637 205, Tamil Nadu, India
c Department of Pharmacology, Sebha University, Sebha, Libya
d Institute for Tuberculosis Research, College of Pharmacy, University of Illinois, Chicago, IL 60612-723, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of copper(II) and zinc(II) complexes have been designed and synthesized using a new type
of Schiff bases derived from the reaction of 3-(3-phenyl-allylidene)-pentane-2,4-dione with para
substituted aniline and benzene-1,2-dithiol. Their structures have been established by analytical and
Received 10 June 2010
Received in revised form
30 August 2010
Accepted 1 September 2010
Available online 15 September 2010
spectral data. The higher 3 and low A values together with positive reduction potentials for these copper
k
complexes suggest that they can mimic the functional properties of naturally occurring proteins. In vivo
and in vitro antitumor functions of the complexes against Ehrlich ascites carcinoma tumor model have
been investigated. The minimum inhibitory concentration of the complexes has also been investigated
against Mycobacterium tuberculosis strain H37Rv. These complexes exhibit significant antitumor, cyto-
toxic and antituberculosis activity.
Keywords:
DNA binding
Nuclease activity
Antitumor activity
Benzene dithiolate complexes
Ehrlich ascites carcinoma
Human cancer cell lines
H37Rv strain
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
side effects. These include renal impairment, neurotoxicity and
ototoxicity (loss of balance/hearing), which are only partially
The better understanding of both the chemical properties and
the pharmacological action of cisplatin has guided the develop-
ment of analogs having always cis geometry but differing from
cisplatin either for the ammine carrier ligands or for the leaving
chlorides. In general, modification of the chloride leaving groups of
cisplatin results in compounds with different pharmacokinetic
properties, whereas modification of the carrier ligands alters the
efficacy and/or the spectrum of activity of the resulting complex.
Thousands of platinum compounds have been synthesized and
screened for their antitumor activity, but little success has been
achieved so far in finding novel platinum-based drugs active
towards cisplatin resistant/refractory tumors [1,2]. Pt(II) has
a strong thermodynamic preference for binding to S-donor ligands.
For that reason, one would predict that platinum compounds
would perhaps never reach DNA, with many cellular platinophiles
(S-donor ligands, such as glutathione, methionine) as competing
ligands in the cytosol [3,4]. Despite the success of cisplatin,
however, it lacks selectivity for tumor tissue, which leads to severe
reversible when the treatment is stopped. With long-term or high-
dose therapy, severe anemia may develop [5,6]. To address these
problems, modified versions of cisplatin, leading to second and
third generation platinum-based drugs have been synthesized over
the past 30 years. Several platinum complexes are currently in
clinical trials, but some of these new complexes have not yet
demonstrated such significant advantages over cisplatin.
With the exception of platinum(II) compounds, there is rela-
tively little mechanistic information on how metal anti-cancer
drugs function, but it is clear that metal ions can work by a variety
of different routes. Non-platinum active compounds are likely to
have mechanism of action, biodistribution and toxicity which are
different from those of platinum drugs and might be effective
against human cancers that are poor chemosensitive or have
become resistant to conventional platinum drugs. Among the
different therapeutic strategies to eradicate cancer cells through
DNA damage, the view of using transition metal complexes, capable
of oxidative or hydrolytic DNA cleavage as anti-cancer drugs, is
a challenging topic in bioinorganic chemistry [7,8].
Inthelastfew decades, interestinthestudyofmetalethiolateshas
ensued as a result of the following factors [9e15]: (i) Thiolates reduce
* Corresponding author. Tel.: þ91 092451 65958; fax: þ91 4562 281338.
E-mail address: drn_raman@yahoo.co.in (N. Raman).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.09.004

N. Raman et al. / European Journal of Medicinal Chemistry 45 (2010) 5438e5451
5439
the toxic effect of soft heavy metals such as Cd, Hg and Pb. (ii) Thiolate
donors are present in the coordination sphere of metal ions in active
sites of metalloproteins [16]; e.g, Fe(II) inpeptide deformylase [17], Co
(II) in the active site of nitrile hydratase [18], [NiFe]-hydrogenase [19]
and metallothioneins containing Zn, Hg, Cd, and Cu [20]. (iii) In the
application of certain metalethiolates in medicine, such as Au(I)-
thiolates for the treatment of arthritis and triphenylphosphinegold(I)
compounds as antitumor agents [21]. Recently technetiume and
rheniumethiolates have gained importance in medical radio-
therapeutic applications [22]. (iv) Metalethiolates are known to be
involved in radioactive protective efficacy and protection against
alkylating reagents. These metal complexes contribute to a greater
capacity to scavenge the superoxide radicals produced on exposure to
ionizing radiation [23,24].
structural and electronic properties of drugeDNA complexes and
their mechanism of binding is the key step in elucidating the prin-
ciples of their anti-cancer activity. In vivo and in vitro antitumor
functions of synthesized complexes against Ehrlich ascites carci-
noma (EAC) tumor model were investigated. The antitumor activity
was assessed by hematological parameters, median survival time
and cell viability with trypan blue dye exclusion assay. In vitro
antitumor activity was performed by MTT assay against human
cervical cancer cell lines (HeLa), Human laryngeal epithelial cancer
(Hep2), Human liver cancer (HepG2) and Human breast cancer
This inspires for developing new non-platinum anti-cancer
agents which include the incorporation of carrier groups that can
target tumor cells with high specificity. Hence, we have a strategy
to modify the previously reported [25] cisplatin based copper(II)
and zinc(II) complexes using benzene-1,2-dithiol. One of the main
reasons for this adaptation is that the soft metals are strongly
bonded with soft bases like thiolate. This type of adaptation may
arise due to the slower intracellular hydrolysis of thiolate ligands
compared to the monodentate chloro ligands of cisplatin. This
amendment may reduce the toxicity in intracellular metabolism
compared to cisplatin. Bearing these facts in mind, it is planned to
synthesize and characterize new copper(II) and zinc(II) complexes
of the above type and to investigate the in vivo and in vitro anti-
tumor activity of the synthesized complexes against Ehrlich ascites
carcinoma (EAC) tumor model.
2. Chemistry
The starting Knoevenagel condensate Schiff base ligands were
prepared as described in the literature procedure [25], by the
condensation of 3-(3-phenyl-allylidene)-pentane-2,4-dione with
para substituted aniline(X); X ¼ eNO2 (L1), eH (L2), eOH (L3)
and eOCH3 (L4). They were refluxed with copper(II)/zinc(II) chlo-
ride to form [MLCl2] type complexes. The labile chlorine atom was
replaced by benzene-1,2-dithiolate ligand (bdt) to form [ML(bdt)]
type complexes. Synthetic route for the preparation of complexes is
depicted in Scheme 1. The synthesized ligands and their complexes
were stable to air and moisture. The ligands were soluble in
common organic solvents but their complexes were soluble only in
DMF and DMSO. The synthesized ligands were characterized by the
usual spectral and analytical techniques. The complexes were
found to be 1:1 electrolytic nature in 10ꢀ3 M DMF solution,
implying the non-coordination of chloride anion to the central
metal ion. The absence of counter (chloride) ion was confirmed
from Volhard’s test. The elemental analysis results for the metal
complexes were also agreed with the calculated values showing
that the complexes have 1:1 metal/ligand ratio. The analytical data,
conductivity and magnetic moment of the complexes are given
along with their synthetic procedures.
3. Pharmacology
The DNA binding study of the synthesized complexes with CT
DNAwas carried out byelectronic absorption spectroscopy, viscosity
measurements, cyclic voltammetry and differential pulse voltam-
metry. Gel electrophoresis experiments using pUC19 circular
plasmid DNA were performed with the ligands and their complexes
in the presence and absence of H2O2 as an oxidant. The nuclease
activity of the complexes was also investigated in the presence of
a free radical scavenger, dimethylsulfoxide (DMSO) and singlet
oxygen quencher, azide ion (NaN3). A detailed understanding of the
Scheme 1. Synthetic route for the preparation of complexes.

5440
N. Raman et al. / European Journal of Medicinal Chemistry 45 (2010) 5438e5451
(MCF-7). The complexes were also investigated against Mycobacte-
rium tuberculosis strain H37Rv. The minimal inhibitory concentra-
tion (MIC) of the complexes was determined.
in the ligands. The downfield shift of this signal in the complexes (d
162.0e165.0 ppm) indicated the coordination of azomethine
nitrogen to metal. A new peak appeared in all the complexes in the
region of d 132e135 ppm indicating the presence of thiolate carbon
4. Results and discussion
4.1. Chemistry
atom. The solvent DMSO-d6 carbon peak appeared at d 39.7 ppm.
4.1.4. Mass spectra
The mass spectrum of L3 ligand showed M þ 1 peak at m/z 397
(15.6%) corresponding to [C26H24N2O2]þ ion. Moreover, the spec-
trum exhibited the fragments at m/z 107, 77 and 66 corresponding
to [C6H5NO]þ, [C6H5]þ and [C5H6]þ respectively. The mass spectrum
of [CuL3(bdt)] showed peaks at m/z 598 and 599 with 19.8 and
15.7% abundances, respectively. The peak at m/z 598 may represent
the molecular ion peak of the complex and the other peaks are
isotopic species. The strongest peaks (base peak) at m/z 397 and
283 represent the stable species C26H24N2O2 and C18H10N4
respectively. Also, the spectrum exhibited the fragments at m/z 107,
77 and 66 corresponding to [C6H5NO]þ, [C6H5]þ and [C5H6]þ
respectively. The m/z of all the fragments of ligands and their
complexes confirmed the stoichiometry of the complexes is being
of the type [ML(bdt)]. It was further supported by the mass spectra
of all the complexes. The observed peaks were in good agreement
4.1.1. Infrared spectra
In IR spectra, the n(C]N) band presented in the ligands was
shifted to lower frequency by ca.30 cmꢀ1 on complexation [26]. The
free eOH group of the ligand L3 vibrated at ca. 3435 cmꢀ1 [26] did
not show any significant shift on complex formation. The absence
of n
(eSH) band, which appeared around 2550e2600 cmꢀ1 as strong
band, indicated the evidence of coordination of thiolate ligand
through metal ion. Coordination of thiolate ligand with metal ion
was also proven by the appearance of a n(CeS) band in the region
1050e1150 cmꢀ1 as strong vibration. It was further supported by
the appearance of a new band in the region 380e410 cmꢀ1 for all
the complexes, assigned to the MeS band vibration and another
new band observed in the region 434e452 cmꢀ1 for all the
complexes, attributed to the MeN vibration [27].
4.1.2. Electronic spectra and magnetic moments
The geometry of the metal complexes had been deduced from
electronic spectra and magnetic moment data of the complexes.
The free ligands exhibited two intense bands at 46256e41854 and
28965e27635 cmꢀ1 which are due to
p /
p* and n / p* transi-
tions respectively. In all metal complexes, these absorption bands
were observed at 44345e40816 and 29876e35428 cmꢀ1. It indi-
cates the coordination of the ligands with metal ions. The electronic
spectra of Cu(II) complexes showed two broad, unusually high
intensity (
3
¼ 1800e3650 Mꢀ1 cmꢀ1) shoulder bands in the visible
region, around 16949e17035 and 14684e14815 cmꢀ1, which are
2
2
assigned to B1g
/ /
2A1g and B1g 2Eg transitions respectively.
The electronic spectral data suggest a square-planar geometry
around the Cu(II) ion. The observed magnetic moment of the Cu(II)
complexes (1.84e1.92 B.M) at room temperature indicates the non-
coupled mononuclear complexes of magnetically diluted d9 system
with S ¼ 1/2 spin-state square-planar structure. The monomeric
nature of the complexes was further supported by the microana-
lytical and FAB mass spectral data. The electronic absorption
spectra of the diamagnetic Zn(II) complexes showed bands at
38759e37453, 24213e24331 and 23866e23041 cmꢀ1 which were
assigned to intra-ligand charge transfer transitions [28].
4.1.3. NMR spectra of zinc complexes
The NMR spectra of ligands and their diamagnetic Zn(II)
complexes were recorded in DMSO-d6. In 1H NMR, a set of
mutiplets in the range of d 6.8e7.8 ppm, observed for all the ligands
and their Zn(II) complexes are assigned to aromatic region. The
phenolic eOH proton for L3 ligand and its zinc complexes was
observed as a singlet ca.
d 11.8 and 11.4 ppm respectively. It is
suggesting that phenolic eOH group is not taking part in the
complexation. The absence of eSH proton in all the complexes in
the region wd 7.8 ppm suggested the deprotonation of eSH group.
This result indicates that the thiolate group involves in the coor-
dination with metal ion upon complexation. 1H NMR spectra of
aliphatic methyl protons exhibited at
d 2.1e2.6 ppm for all the
Schiff base ligands and their Zn(II) complexes. The peaks at
d
6.8e7.1 ppm were assigned to phenyl protons.
The 13C NMR spectra of the ligands and their zinc complexes
were recorded in DMSO-d6. The aromatic carbon peaks were
observed at 120.0e130.0 ppm. The ligands showed signal at
156.0e159.0 ppm indicating the presence of azomethineecarbon
d
d
Fig. 1. EPR spectra of [CuL1(bdt)] at 300 K (a) and 77 K (b).

N. Raman et al. / European Journal of Medicinal Chemistry 45 (2010) 5438e5451
5441
Table 1
The spin Hamiltonian parameters of Cu(II) complexes in DMSO at 300 K and 77 K.
Complexes
g-tensor
A ꢁ 10ꢀ4 (cmꢀ1
)
gk/A
G
k
g
k
gt
giso
A
k
At
Aiso
[CuL1(bdt)]
[CuL2(bdt)]
[CuL3(bdt)]
[CuL4(bdt)]
2.18
2.23
2.26
2.21
2.07
2.12
2.09
2.05
2.15
2.11
2.16
2.13
125.2
125.8
120.6
108.9
18.5
21.3
23.7
20.2
63.9
74.5
68.3
80.5
182
186
194
202
4.12
4.16
4.20
4.10
with their empirical formulae as indicated from microanalytical
data. Thus, the mass spectral data reinforced the conclusion drawn
from the analytical and conductance values.
4.1.5. EPR spectral study
The EPR spectra of all of the copper(II) complexes were recorded
in DMSO at 300 and 77 K (Fig. 1). The spin Hamiltonian parameters
calculated for the complexes are given in Table 1. The g values are
k
greater than the corresponding gt values and therefore the
unpaired electron occupies the dx2ꢀy2 molecular orbital [29]. All the
spectra exhibited a typical four-line pattern. The spectral data were
consistent with typical monomers but with distorted tetrahedral
copper(II) geometry [30]. It is known that as the tetrahedral
distortion increases, the g will increase with a decrease in A [31].
k
k
For blue copper proteins the A values are in the 15e90 ꢁ 10ꢀ4 cmꢀ1
k
region due to deviation from planarity, towards a suppressed
tetrahedron. The observed A values (w110 ꢁ 10ꢀ4 cmꢀ1) revealed
k
the large deviation of the complexes from the regular square-
planar geometry. The A values of these complexes are on the
k
borderline between naturally occurring blue copper proteins and
typical planar structures. Often the g /A quotient is empirically
k
k
treated as an index of tetrahedral distortion [30]. The g /A values
k
k
are expected to be in the 105e135 and 150e250 range respectively
for square-planar and tetrahedral distorted copper(II) complexes.
In the present case the g /A values are between 182 and 202; it is
k
k
assumed that they have geometry distorted from planarity towards
tetrahedral. It is also reflected in the unusual gain in intensity in the
visible spectra. According to Hathaway [32,33], the G values within
the range 4.1e4.2 for all copper complexes indicate negligible
exchange interaction of CueCu in the complexes.
Based on the above analytical and spectral data, the structure of
complexes is shown in Fig. 2.
Fig. 2. Structure of complexes.
The intercalative binding of colored drugs to DNA helix has been
characterized by large changes in the absorbance (hypochromism)
and appreciable shifts in wavelength (red shift) [39]. The observed
hypochromism in the present case is very large compared to that
observed for potential intercalators [40]. Since the present
complexes do not contain any fused aromatic ring to facilitate
intercalation, it is clear that the weak MeS bonds in the complex
facilitate ligand displacement on adding DNA and that the inter-
action of DNA with metal(II) through sugarephosphate oxygen, N7
of purines etc., is stabilized. However, all the complexes do not
undergo complete ligand displacement even if R value reaches 25. It
appears that all the complexes bind to DNA through its axial
position [38]. This leads to labilization of the complexes followed by
displacement of thiolato ligands by DNA. Moreover, the planarity of
the CuN2S2 chromophore of the complexes encourages its partial
intercalation between the base pairs of DNA and stabilizes the
complex against ligand displacement.
4.2. Pharmacology
4.2.1. DNA binding studies
4.2.1.1. Electronic spectral studies. Electronic absorption spectra are
initially employed to study the binding of complexes and DNA.
Binding of complex to DNA through intercalation usually results in
hypochromism and red shift (bathochromism), due to the inter-
calative mode involving a strong stacking interaction between
aromatic chromophore and the base pairs of DNA. The extent of the
hypochromism in the visible MLCT band is commonly consistent
with the strength of intercalative interaction [34e36].
The electronic spectra of all the copper complexes exhibited
a single ded band around 610 nm as well as a more intense S
(s) / Cu(II) ligand to metal charge transfer (LMCT) band around
410 nm and 430 nm [37,38]. On the addition of calf thymus DNA
a considerable decrease in absorptivity of the LMCT band was
observed for all the complexes and the change in the absorbance for
[CuL1(bdt)] as a function of added DNA is shown in Fig. 3. There is
a considerable decrease in the absorbance only upto R ¼ 20
(R ¼ [NP]/[Copper complex]).
Also absorption spectral titrations of all the present complexes
with ATP were carried out to understand their susceptibility
towards ligand displacement. At the [ATP]:[complex ] ratio of 1:1
for all the complexes complete displacement occurs suggesting the
formation of a 1:1 Cu2þ:ATP complex obviously involving N(7) and

5442
N. Raman et al. / European Journal of Medicinal Chemistry 45 (2010) 5438e5451
The cyclic voltammogram of the complexes in buffer (Table 3)
reveals a non-Nernstian behavior of the Cu(II)/Cu(I) couple, as may
be judged from the limiting peak potential separation ( Ep
Epꢂ,
D
D
value extrapolated to zero scan rate) of 62e78 mV (59 mV for a one-
electron diffusion controlled reversible process). Redox behavior of
[CuL1(bdt)] in DMF versus Ag/AgCl with TBAP as supporting elec-
trolyte at different scan rate is shown in Fig. 4. Linearity of the ipc
versus square root of scan rate plot (passing through origin) and the
values of ipa/ipc (z1) suggest that the redox tends to become fairly
reversible. The redox potentials of the present complexes follow
the trends [CuL4(bdt)] > [CuL3(bdt)] > [CuL2(bdt)] > [CuL1(bdt)]
illustrating the ability of chelate rings to stabilize the Cu(I) state.
The later are expected to illustrate the nuclease activity of copper
complexes, as their Cu(II) and Cu(I) forms are involved in the
proposed cleavage mechanism (Scheme 2).
A typical CV behavior of synthesized complexes in the presence
of excess DNA is shown in Fig. 5. A large decrease in peak currents of
almost all the complexes on adding DNA is consistent with the
displacement of the thio ligand in the complexes, revealed by
spectral results. The shift in E1/2 for complexes on binding to
Cu2þeDNA suggests that both Cu(II) and Cu(I) forms bind to DNA
but to different extents. Analogous to the treatment of the associ-
ation of small molecules with micelles [42] and DNA [43], the ratio
Fig. 3. Charge transfer spectrum of [CuL1(bdt)] in the absence (—) and the presence
(d) of increasing amount of CT DNA; R ¼ [NP]/[complex].
of the equilibrium constants, K2þ/K for the binding of the Cu(II) and
þ
phosphate oxygens of ATP [41]. So it is clear that similar donors
present in DNA displace the thiolate ligands bound to metal.
In order to compare the binding strength of the complexes with
CT DNA, the intrinsic binding constants Kb were obtained by
monitoring the changes in absorbance for the complexes with
increasing concentration of DNA. Kb was obtained from the ratio of
Cu(I) forms of complexes to DNA can be estimated from the net shift
in E1/2, assuming reversible electron transfer. For a Nernstian elec-
tron transfer in a system in which both the oxidized and reduced
forms associate with a third species such as DNA in solution, Scheme
2 can be applied. Here CuLnþeDNA represents the CuLnþ complex
bound to Cu2þeDNA. Thus for the one-electron process,
slope to the intercept from the plots of [DNA]/(3a
ꢀ
3f) versus [DNA].
The Kb values of copper and zinc complexes are shown in Table 2.
The high Kb value for all complexes is due to the labialization of
weak MeS bonds and flexible versatile ligands in complexes which
greatly facilitate groove binding/stacking with the base pairs. The
binding strength of the synthesized complexes with DNA is shown
as in the following order: eNO2 > eOH > eOCH3 > eH.
Eꢂ0 ꢀ Eꢂ0 ¼ 0:059logðK =K2þÞ
þ
b
f
where Efꢂ’ and Ebꢂ’ are the formal potentials of the, Cu(II)/Cu(I)
couple in the free and bound forms, respectively. Thus from the
shift in E1/2 values, K2þ/K ratios were calculated.
þ
For all the synthesized complexes K2þ was higher than K . This
þ
suggests that B-DNA with small C-DNA character tends to stabilize
the Cu(II) over the Cu(I) state of the complex obviously by elec-
trostatic interaction. The DNA strand which may be considered as
a local ‘solvent’ environment as far as bound metal complex is
concerned differs from the bulk medium in dielectric constant and
charge distribution and so has a substantial effect on the electron
transfer thermodynamics [44]. Further, as DNA has the hydrophilic
coat/hydrophobic core structure [45], the interplay between elec-
trostatic and hydrophobic (intercalative) interactions can be
important in the overall binding of the charged species possessing
a planar aromatic moiety [46].
4.2.1.2. Electrochemical behavior. The application of electro-
chemical methods to the study of metallointercalation and coor-
dination of metal ions and chelates to DNA provides a useful
complement to the previously used methods of investigation such
as UVeVis spectroscopy. As further exploring the binding of the
present complexes with DNA, cyclic and differential pulse vol-
tammetric studies were carried out both in the presence and
absence of DNA.
Table 2
Charge transfer properties of interaction of synthesized complexes with CT DNA.
In addition to changes in the formal potential, the voltammetric
current decreased upon the addition of Cu2þeDNA as shown in
Table 3. The decrease in current in CV experiments may be attrib-
uted to the diffusion of copper complexes bound to the large, slowly
diffusing DNA molecule. The change in current upon Cu2þeDNA
addition can be explained in terms of the diffusion of an equilib-
rium mixture of free and Cu2þeDNA-bound metal complex to the
SI. No
Complexes
lmax (nm)
Dl (nm)
%H
M
Free
Bound
1
2
3
4
5
6
7
8
[CuL1(bdt)]
[CuL2(bdt]
[CuL3(bdt)]
[CuL4(bdt)]
[ZnL1(bdt)]
[ZnL2(bdt)]
[ZnL3(bdt)]
[ZnL4(bdt)]
413.0
434.0
394.0
420.4
408.5
428.3
400.7
425.6
417.1
429.6
403.3
419.7
413.4
425.6
409.7
421.8
416.0
438.0
396.4
423.2
411.6
430.7
403.3
427.8
419.5
431.8
404.8
420.9
415.0
426.3
410.8
423.9
3.0
4.0
2.4
2.8
3.1
2.7
2.6
2.2
2.4
2.2
1.5
1.2
2.4
0.7
1.1
2.1
19
19
12
13
17
16
12
12
10
09
05
07
11
12
09
09
2.4
2.2
1.2
1.1
2.0
2.0
1.9
1.9
1.8
1.8
1.0
0.9
1.7
1.0
1.0
1.3
electrode surface. The slope of the ipc
ꢀ
n1/2 plot decreased with
increase in the ratio R (¼[NP]/[Cu complex]), indicating a reduction
in the apparent diffusion coefficient of copper complexes as [NP] is
increased. This decrease in the diffusion coefficient value with
increase in R reveals the quantitative binding of the complex to
DNA. The ratio of anodic to cathodic peak current, ipa/ipc, was less
than unity under all experimental conditions, indicating that the Cu
(II) complex species is stable on the time scale of the CV
measurement. It was further supported by the differential pulse
voltammogram experiment which is shown in Fig. 6.

N. Raman et al. / European Journal of Medicinal Chemistry 45 (2010) 5438e5451
5443
Table 3
Electrochemical parameters of interaction of copper complexes with CT DNA.
SI. No
Complexes
蜐Ep (V)
E1/2 (V)
Free
Decrease of ipc (%)
ipa/ipc
Free
K /K2þ
þ
Free
Bound
Bound
Bound
1
2
3
4
[CuL1(bdt)]
[CuL2(bdt)]
[CuL3(bdt)]
[CuL4(bdt)]
0.727
0.645
0.567
0.529
0.597
0.432
0.322
0.553
0.493
0.470
0.492
0.383
0.558
0.380
0.439
0.568
41
34
27
19
0.59
0.79
0.72
0.76
0.77
0.54
0.61
0.61
0.07
0.18
0.34
0.52
Zn2þ ꢀ DNA#Zn2þ þ DNA
Zn2þ ꢀ 2eꢀ#Zn0
The dissociation constant (Kd) of the Zn(II)eDNA complex was
obtained using the following equation:
These results demonstrate that rather straightforward electro-
chemical methods can be employed to characterize the inter-
calative interaction between a metal complexes or electro active
species and DNA. The electrochemical oxidation and reduction of
selected bound species on DNA can also be carried out and favor-
able circumstances may allow strand scission in the DNA.
The electrochemical behavior of all the zinc complexes in the
absence of DNA showed only cathodic potential corresponding to
Zn(II/0). The cathodic peak appeared in the positive potential of
0.812e0.924 V range corresponding to the two electron reduction
of Zn(II) complexes. Addition of CT DNA to the zinc complexes
solution resulted in a shift of cathodic peak potentials to more
negative values and a decrease of the cathodic currents. The shift of
the cathodic potential of the complexes in the presence of DNA to
more negative values indicates a binding interaction between the
complexes and DNA that makes the complexes less readily reduc-
ible. The drop of the voltammetric currents in the presence of CT
DNA can be attributed to diffusion of metal complexes bound to
higher and slowly diffusing DNA molecule. The decreased extents
of the peak currents observed for the complexes upon addition of
DNA may indicate copper complexes possess more DNA-binding
affinity than zinc complexes. Typical voltammetric data of zinc
complexes at different DNA concentrations are summarized in
Table 4.
ꢀ
ꢁ
Kd
½DNAꢃ
ip2
¼
i2po ꢀ ip2 #i2po ꢀ ½DNAꢃ
where Kd is dissociation constant of the complex Zn(II)eDNA, i2po
and i2p are reduction current of Zn(II) in the absence and presence of
DNA respectively. The low dissociation constant values (Table 4) of
Zn(II) ions were indispensable for structural stability of complexes
Zn(II)eDNA which participate in the replication, degradation and
translation of genetic material of all species.
4.2.1.3. Viscosity measurements. Under appropriate conditions
intercalation causes a significant increase in the viscosity of DNA
solutions due to the increase in the separation of base pairs at
intercalation sites and hence an increase in overall DNA contours
length. By contrast, ligands that bind exclusively in the DNA
grooves (e.g. netropsin, distamycin), under the same conditions,
typically cause less pronounced changes (positive or negative) or
no changes in DNA solution viscosity [44]. As a means for further
exploring the binding of the copper complexes, viscosity
measurements were carried out on DNA pretreated with copper(II)
chloride by varying the concentration of the added complexes. The
Differential pulse voltammogram of the Zn(II) complexes
observed a positive potential shift along with significant decreasing
of current intensity during the addition of increasing amounts of
DNA. It indicates that zinc ions stabilize the duplex (GC pairs) by
intercalating way. Hence, the complexes of the electroactive species
(Zn(II)) with DNA, the electrochemical reduction reaction can be
divided into two steps:
values of relative specific viscosity (h/h
o), where ho is the specific
viscosity contribution of DNA in the absence of complex, were
plotted against 1/R (¼[complex]/[NP]) (Fig. 7a and b). The synthe-
sized complexes slightly decreased the specific viscosity of DNA
possibly due to a number of factors including a change in confor-
mation, flexibility or solvation of DNA molecules. However, in view
of the tendency of DNA towards particulate formation, it is most
likely that extensive aggregation of DNA on binding to the
complexes [47] would sharply reduce the number of independently
moving DNA molecules in solution and hence the viscous drag that
comes from molecules diffusing into each other. Thus, a similar
decrease in
h/
ho for other complexes has been ascribed to DNA
aggregation [47]. In contrast to all the complexes exhibit two
distinct modes of binding; the relative specific viscosity increases
slowly up to 1/R ¼ 0.25 and then steadily but the extent of increase
is lesser than that for the known intercalator, ethidium bromide
Fig. 4. Redox behavior of [CuL1(bdt)] in DMF versus Ag/AgCl with TBAP as supporting
electrolyte at scan rate (a) 150, (b) 125, (c) 100, (d) 75, (e) 50, (f) 25, and (g) 10 mV sꢀ1
.
Scheme 2. Redox behavior of copper complex in the presence of DNA.

5444
N. Raman et al. / European Journal of Medicinal Chemistry 45 (2010) 5438e5451
Table 4
Electrochemical parameters of interaction of zinc complexes with CT DNA.
SI. No Complexes
Ep (V)
Free
ipc
(
m
A)
Kd ꢁ 10ꢀ10 (mol Lꢀ1
)
Bound Free Bound
1
2
3
4
[ZnL1(dppz)]Cl2 0.924 0.932 0.63 0.40
[ZnL2(dppz)]Cl2 0.884 0.893 0.59 0.41
[ZnL3(dppz)]Cl2 0.841 0.848 0.60 0.52
[ZnL4(dppz)]Cl2 0.812 0.823 0.47 0.38
3.2
2.3
2.1
1.6
the reaction between the metal complex and oxidant may be
explained as follows:
Cu(II)L þ eꢀ / Cu(I)L
Cu(I)L þ O2 / Cu(II)L þ O2ꢀ
2Oꢀ2 þ 2Hþ / H2O2 þ O2
Fig. 5. Redox behavior of [CuL1(bdt)] both in the absence (—) and the presence (d) of
different concentration of DNA using cyclic voltammogram. Supporting electrolyte,
50 mM NaCl, 5 mM TriseHCl, pH ¼ 7.1. Scan rate 100 mV sꢀ1
.
(EB) [48] illustrating the partial stacking interaction of complexes
with DNA as discussed above. Spectral and electrochemical
evidences also support such an intercalative mode of interaction.
4.2.2. DNA cleavage studies
4.2.2.1. Chemical nuclease activity. Gel electrophoresis experiments
using pUC19 circular plasmid DNA were performed with the ligands
and their complexes in the presence and absence of H2O2 as an
oxidant. At micro-molar concentrations, for 2 h incubation periods,
the ligands exhibited no significant cleavage activity in the absence
or presence of oxidant (H2O2). The nuclease activity was greatly
enhanced by the incorporation of metal ion in the respective ligands.
The nuclease activity of the complexes was also investigated in the
presence of a free radical scavenger, dimethylsulfoxide (DMSO) and
singlet oxygen quencher, azide ion (NaN3). From Fig. 8a and b, it is
evident that the complexes cleave DNA more efficiently in the
presence of an oxidant. This may be attributed to the formation of
hydroxyl free radicals. The production of a hydroxyl radical due to
Fig. 7. (a). Ratio of the specific viscosity of DNA in the presence of copper complex to
that of free CT DNA vs. 1/R (¼[Copper complex]/[NP]) in the presence of [CuL1(bdt)]
(A), [CuL2(bdt)] (:), [CuL3(bdt)] (C) and [CuL4(bdt)] (-). (b). Ratio of the specific
viscosity of DNA in the presence of zinc complex to that of free CT DNA vs. 1/R (¼[Zinc
complex]/[NP]) in the presence of [ZnL1(bdt)] (:), [ZnL2(bdt)] (A), [ZnL3(bdt)] (C)
and [ZnL4(bdt)] (-).
Fig. 6. Redox behavior of [CuL1(bdt)] both in the absence (—) and the presence (d) of
different concentration of DNA using differential pulse voltammogram. Supporting
electrolyte, 50 mM NaCl, 5 mM TriseHCl, pH ¼ 7.1. Scan rate 100 mV sꢀ1
.

N. Raman et al. / European Journal of Medicinal Chemistry 45 (2010) 5438e5451
5445
radical scavenger or in the presence of singlet oxygen quencher. It
means that some DNA was cleaved by other than an oxidative
mechanism, possibly by a discernable hydrolytic path.
4.2.2.2. Hydrolytic cleavage and ligation of the DNA linearized by
zinc(II) complexes. Although the cleavage reaction through zinc
complexes does not require additional external agents, we carefully
investigated the possibility that diffusible OH radical, singlet
oxygen (1O2) or superoxide anion radical were involved in this
reaction. The cleavage efficiency does not change in the presence of
DMSO as potential OH radical scavengers, NaN3 as potential 1O2
inhibitors, or SOD as a superoxide anion radical inhibitor [50e52].
In conclusion, the results presented here rule out the involvement
of any oxidation inhibitors in the strand cleavage, and this reaction
most probably occurs through a hydrolytic mechanism. Further
experiments support this assumption. It is well known that in DNA
hydrolytic cleavage 30-OH and 50-OPO3 (50-OH and 30-OPO3) frag-
ments remain intact and that these fragments can be enzymatically
ligated and end-labeled. Fig. 9a and b shows that the linear DNA
fragments cleaved by zinc complex can be relegated by T4 ligase
just like the linear DNA mediated by EcoR1. Hence, this result
implied that the process of DNA cleavage by the complex occurs via
a hydrolytic path.
Fig. 8. (a). Gel electrophoresis diagram showing the cleavage of SC pUC19 DNA (0.2 mg)
by the synthesized complexes (50 mM) in the presence of MPA (5 mM): lane 1, DNA
control; lane 2, DNA þ L1 (50
m
M); lane 3, DNA þ [CuL1(bdt)] þ H2O2; lane 4,
DNA þ [CuL1(bdt)] þ DMSO (4
m
L) þ H2O2; lane 5, DNA þ [CuL1(bdt)] þ distamycin
(50
m
M) þ H2O2; lane 6, DNA þ [CuL1(bdt)] þ SOD (1U) þ H2O2; lane 7, DNA þ L2; lane
4.2.2.3. Photonuclease activity. Photo-induced DNA cleavage exper-
iments were carried out in UV and visible light using the ligands and
8, DNA þ [CuL2(bdt)] þ H2O2. (b). Gel electrophoresis diagram showing the cleavage of
SC pUC19 DNA (0.2
(5 mM): lane 1, DNA
DNA
[CuL2(bdt)]
mg) by the synthesized complexes (50 mM) in the presence of MPA
þ
[CuL2(bdt)]
DMSO (4 L)
þ
distamycin (50
H2O2; lane 3, DNA
m
M)
þ
H2O2; lane 2,
þ
their copper complexes (50 and 100
mM) and SC pUC19 DNA (0.2 mg,
þ
þ
m
þ
L3; lane 4,
33.3 M) in the presence and absence of various inhibitors.
m
DNA þ [CuL3(bdt)] þ H2O2; lane 5, DNA þ L4; lane 6, DNA þ [CuL4(bdt)] þ H2O2; lane 7,
DNA þ [CuL4(bdt)] þ DMSO (4
m
L) þ H2O2; lane 8, DNA þ [CuL4(bdt)] þ NaN3.
Cu(I)L þ H2O2 / Cu(II)L þ OHꢀ þ OHꢄ
Oꢀ2 þ H2O2 / O2 þ OHꢀ þ OHꢄ
These OH free radicals participate in the oxidation of the
deoxyribose moiety, followed by hydrolytic cleavage of the sugar
phosphate backbone [49]. The more pronounced nuclease activity of
these adducts in the presence of oxidant as compared to the parent
copper complexes may be due to the increased production of
hydroxyl radicals. Even in the absence of oxidant, the mixed ligand
complexes exhibit significant DNA cleavage activity when compared
to the parent complexes. This may be due to the ability of the mixed
ligand complexes to associate with DNA, facilitated by hydrophobic
interaction leading to fair nucleolytic cleavage. It is clear that the
order of cleavage activity in the presence of oxidant (H2O2) follows
the order [CuL1(bdt)] > [CuL3(bdt)] > [CuL4(bdt)] > [CuL2(bdt)]. To
determine the groove selectivity of the complexes, control experi-
ments were performed using minor groove binder distamycin. The
addition of distamycin did not inhibit the cleavage for the
complexes. This result suggests major groove binding for the
complexes.
The nuclease activity of the complexes had also been investi-
gated in the presence of a free radical scavenger (dimethylsulf-
oxide) and a singlet oxygen quencher (azide ions). To this end,
standard scavengers of reactive oxygen intermediates were
included in the electrophoresis process (Fig. 8a and b). Azide
inhibited to some extent, indicating the involvement of singlet
oxygen in the cleavage activity. The hydroxyl radical scavenger,
dimethylsulfoxide diminished the nuclease activity of the
compound, which is indicative of the involvement of the hydroxyl
radical in the cleavage process. Nuclease activity of the complexes
was not completely diminished either in the presence of free
Fig. 9. (a). Gel electrophoresis diagram for ligation of SC pUC19 DNA linearized by zinc
complexes: lane 1, DNA markers; lane 2, DNA
þ
[ZnL1(bdt)]; lane 3,
DNA þ [ZnL1(bdt)] þ T4 DNA ligase; lane 4, DNA þ [ZnL1(bdt)] þ EcoR1; lane 5,
DNA þ [ZnL1(bdt)] þ EcoR1 þ T4 DNA ligase. (b). Gel electrophoresis diagram for
ligation of SC pUC19 DNA linearized by zinc complexes: lane 1, DNA þ [ZnL2(bdt)]; lane
2, DNA
DNA
þ
[ZnL2(bdt)]
þ
T4 DNA ligase; lane 3, DNA
þ
[ZnL3(bdt)]; lane 4,
[ZnL4(bdt)]; lane 6,
þ
[ZnL3(bdt)]
þ
T4 DNA ligase; lane 5, DNA
þ
DNA þ [ZnL4(bdt)] þ T4 DNA ligase.

5446
N. Raman et al. / European Journal of Medicinal Chemistry 45 (2010) 5438e5451
Table 6
Effect of Cu(II) and Zn(II) complexes treatment on the survival of tumor-bearing
mice.
Treatment
MST
Increase in life span
Tumor control
5-FU
14.56 ꢅ 0.56
34.83 ꢅ 0.87*
30.17 ꢅ 0.60*
32 ꢅ 0.58*
e
140.21
108.06
120.69
112.62
106.89
121.86
101.17
105.72
102.07
[CuL1(bdt)]
[CuL2(bdt)]
[CuL3(bdt)]
[CuL4(bdt)]
[ZnL1(bdt)]
[ZnL2(bdt)]
[ZnL3(bdt)]
[ZnL4(bdt)]
30.83 ꢅ 0.75*
30 ꢅ 0.58*
32.17 ꢅ 0.95*
29.17 ꢅ 0.60*
29.83 ꢅ 0.79*
29.3 ꢅ 1.20*
N ¼ 6; d of drug treatment ¼ 9, *P < 0.01 versus tumor control. Data were analyzed
by one-way ANOVA followed by Dunnett’s test.
oxygen from its stable triplet to the cytotoxic singlet state. More-
over, it suggests the formation of singlet oxygen as the respective
species in a type-II process in the metal assisted photo-excitation
process involving ligand nep* and p* transitions (Fig.10a and b).
pe
4.2.3. Antitumor activity
4.2.3.1. Effect of complexes on hematological parameters.
Hematological parameters of tumor-bearing mice on day 14
showed significant changes when compared with the normal mice
(Table 5). The total white blood cell (WBC) count, proteins and
packed cell volume (PCV) were found to increase with a reduction
in the hemoglobin content of red blood cell (RBC). The differential
count of WBC showed that the percentage of neutrophils increased
(P < 0.001) while that of lymphocytes decreased (P < 0.001). At the
same time interval, complexes (100 mg/kg/day, p.o.) treatment
could change these altered parameters to near normal. The reliable
criteria for judging the value of any anti-cancer drug are prolon-
gation of lifespan and decrease of WBC from blood [54,55]. The
results of the present study showed an antitumor effect of
complexes against EAC in Swiss albino mice.
The analysis of the hematological parameters showed minimum
toxic effect in mice treated with complexes. After 14 days of
transplantation, complexes were able to reverse the changes in the
hematological parameters consequent to tumor inoculation. The
present study revealed that the complexes were cytotoxic towards
EAC.
Fig. 10. (a). Gel electrophoresis diagram showing the photocleavage of pUC19 DNA by
the synthesized complexes in DMFeTris buffer medium and in the presence of various
reagents on irradiation with UV light at 360 nm: lane 1, DNA control (60 min); lane 2,
DNA
þ
L1 (60 min); lane 3, DNA
þ
[CuL1(bdt)]
þ
D2O (60 min); lane 4,
DNA þ [CuL1(bdt)] (60 min); lane 5, DNA þ [CuL1(bdt)] þ DMSO (60 min); lane 6,
DNA þ [CuL1(bdt)] þ SOD (60 min); lane 7, DNA þ [CuL1(bdt)] þ NaN3 (60 min); lane 8,
DNA þ [CuL2(bdt)] (60 min). (b). Gel electrophoresis diagram showing the photo-
cleavage of pUC19 DNA by the synthesized complexes in DMFeTris buffer medium and
in the presence of various reagents on irradiation with UV light at 360 nm: lane 1,
DNA þ [CuL2(bdt)] þ D2O (60 min); lane 2, DNA þ [CuL2(bdt)] þ DMSO (60 min); lane
3, DNA þ [CuL2(bdt)] þ NaN3 (60 min); lane 4, DNA þ [CuL2(bdt)] þ SOD (60 min); lane
5, DNA þ L3 (60 min); lane 6, DNA þ [CuL3(bdt)] (60 min); lane 7, DNA þ L4 (60 min);
lane 8, DNA þ [CuL4(bdt)] (60 min).
The DNA cleavage of ligand alone was inactive. The result indi-
cated the importance of the metal in the complexes for observing
the photo-induced DNA cleavage activity. All the complexes cleaved
the pUC19 DNA from its SC to NC form even in the absence of
inhibitors on irradiation with UV light at 360 nm. In the presence of
singlet oxygen quencher like sodium azide inhibited the cleavage.
An enhancement of photocleavage of DNA was observed in D2O
solvent in which 1O2 had longer life time [53]. Hydroxyl radical
scavenger DMSO or KI did not show any significant inhibition in the
DNA cleavage activity. The results are indicative of the presence of
a type-II in which the photo-excited complexes activate molecular
4.2.3.2. Effect of Cu(II) and Zn(II) complexes on survival time. The
effect of complexes on the survival of tumor-bearing mice is shown
Table 5
Effect of Cu(II) and Zn(II) complexes on hematological parameters of EAC tumor-bearing mice.
Design of treatment Hb (gm %)
RBC 106 Cells/CU.MM WBC 103 Cells/CU.MM Total protein mg% PCV (%)
Differential count (%)
Lymphocytes Neutrophils Monocytes
Normal
12.92 ꢅ 0.15
5.78 ꢅ 0.4a
4.9 ꢅ 0.12
6.78 ꢅ 0.14
5.73 ꢅ 0.26
16.86 ꢅ 0.55
25.98 ꢅ 0.56a
17.02 ꢅ 0.26d
16.98 ꢅ 0.51d
17.1 ꢅ 0.45d
66.4 ꢅ 1.36
32.6 ꢅ 1.6
1 ꢅ 0.45
Tumor control
[CuL1(bdt)]
[CuL2(bdt)]
[CuL3(bdt)]
[CuL4(bdt)]
[ZnL1(bdt)]
[ZnL2(bdt)]
[ZnL3(bdt)]
[ZnL4(bdt)]
2.46 ꢅ 0.14a
4.26 ꢅ 0.19d
3.44 ꢅ 0.15a,e
18.74 ꢅ 0.47a
15.54 ꢅ 0.3a,d
14.96 ꢅ 0.54a,d
12.16 ꢅ 0.63a,d
11.4 ꢅ 0.42a,d
11.86 ꢅ 0.32a,d
12.54 ꢅ 0.36a,d
11.78 ꢅ 0.32a,d
11.04 ꢅ 0.72a,d
12.23 ꢅ 0.34a
7.44 ꢅ 0.17b,d
7.62 ꢅ 0.27a,d
8.56 ꢅ 0.17a,d
7.96 ꢅ 0.41a,d
7.44 ꢅ 0.24b,d
8.14 ꢅ 0.14a,d
6.76 ꢅ 0.23d
7.06 ꢅ 0.24c,d
26.2 ꢅ 1.16a 72.8 ꢅ 1.07a 1 ꢅ 0.45
64.2 ꢅ 0.86d 35.2 ꢅ 1.24d 0.6 ꢅ 0.4
12.48 ꢅ 0.21d
12.38 ꢅ 0.13d
68 ꢅ 1.0d
31.6 ꢅ 1.03d 0.4 ꢅ 0.24
11.04 ꢅ 0.16a,d 3.94 ꢅ 0.13b,d
63.2 ꢅ 1.28d 36.2 ꢅ 1.46d 0.6 ꢅ 0.4
13 ꢅ 0.14d
4.84 ꢅ 0.15d
4.6 ꢅ 0.21d
19.98 ꢅ 0.54b,d 61.4 ꢅ 1.21d 38 ꢅ 1.23d
0.6 ꢅ 0.24
12.48 ꢅ 0.29d
11.1 ꢅ 0.43a,d
11.78 ꢅ 0.27d
12.28 ꢅ 0.23d
18.96 ꢅ 0.43d
17.2 ꢅ 0.45d
18.94 ꢅ 0.71d
18.04 ꢅ 0.47d
67.2 ꢅ 1.93d 32.4 ꢅ 2.11d 0.4 ꢅ 0.4
65.6 ꢅ 1.72d 33.8 ꢅ 1.83d 0.6 ꢅ 0.4
64.8 ꢅ 1.28d 34.6 ꢅ 1.63d 0.6 ꢅ 0.4
4.2 ꢅ 0.12d
4.04 ꢅ 0.21c,d
3.92 ꢅ 0.11b,d
64.6 ꢅ 1.36d 35 ꢅ 1.14d
0.4 ꢅ 0.24
a
P < 0.001.
P < 0.01.
P < 0.05 versus Normal.
P < 0.001.
b
c
d
e
P < 0.01 versus Tumor control. Data were analyzed by using one-way ANOVA followed by TukeyeKramer multiple comparison test.

N. Raman et al. / European Journal of Medicinal Chemistry 45 (2010) 5438e5451
5447
Table 7
Table 9
In vitro cytotoxic activity of Cu(II) and Zn(II) complexes in EAC cell
Minimum inhibitory concentration of complexes against
line.
Mycobacterium tuberculosis.
Treatment compounds
IC50
(
m
g/mL)
Complex
MIC (
2.9
>100
9.9
12.5
3.8
m
g/mL)
[CuL1(bdt)]
[CuL2(bdt)]
[CuL3(bdt)]
[CuL4(bdt)]
[ZnL1(bdt)]
[ZnL2(bdt)]
[ZnL3(bdt)]
[ZnL4(bdt)]
114.47
190.55
183.38
108.18
170.59
182.35
175.40
186.56
[CuL1(bdt)]
[CuL2(bdt)]
[CuL3(bdt)]
[CuL4(bdt)]
[ZnL1(bdt)]
[ZnL2(bdt)]
[ZnL3(bdt)]
[ZnL4(bdt)]
Rifampicin
>100
19.2
35.2
0.1
Average of 3 determinations, 3 replicates IC50, Drug concentration
inhibiting 50% cellular growth following 3 h of drug exposure.
cells were treated for 72
complexes and cisplatin (0.1e100
h
with various concentrations of
g/mL), relative cell survival
in Table 6. The MST of the treated groups was compared with those
of control groups by the following calculation:
m
progressively decreased in a dose-dependent manner. The IC50 of
the synthesized complexes were determined for all cell lines.
Among the complexes, [CuL3(bdt)] showed low IC50 against HeLa
and MCF-7 cell lines. However, all the complexes showed higher
IC50 when compared to the standard cisplatin.
T ꢀ C
Increase in life span ¼
ꢁ 100
C
where number of days treated animals survived and
T
¼
C ¼ number of days control animals survived.
The basic criteria for any anti-cancer drug should prolong the
life span of the tumor bearing animals. The effect of [CuL2(bdt)] and
[ZnL1(bdt)] complexes on the survival of tumor-bearing mice
showed at dose of 100 mg/mL a life span values 120.69 and 121.86%
respectively. These complexes were judged to be significant anti-
tumor activity against tumor-bearing mice. The other complexes
were also showed significant increase in the life span of the tumor-
bearing mice which were almost comparable with that of 5-FU.
These results clearly demonstrated the antitumor effect of
complexes against EAC.
4.2.4. Antimycobacterial assay
The activity of the complexes against M. tuberculosis virulent
strain H37Rv was determined. The minimum inhibitory concen-
tration (MIC) against M. tuberculosis was determined and results
are presented in Table 9. The lowest MIC values of eNO2 group
containing complexes were more active against H37Rv strain than
the other complexes. The highest MIC was found for the unsub-
stituted group containing Cu(II) and Zn(II) complexes. On the other
hand, the least active complexes were the unsubstituted Cu(II) and
Zn(II) complexes, which exhibited MIC > 100 mg/mL. Although the
complexes have not shown better antitubercular activity than the
other substituted (eNO2, eOH and eOCH3) complexes, in general
all of the complexes exhibited good activity and, except unsub-
stituted group complexes, all of them were less active than the
standards.
4.2.3.3. Effect of Cu(II) and Zn(II) complexes on cytotoxicity in
vitro. The in vitro cytotoxicity studies were performed by trypan
blue dye exclusion method. The IC50 values of all the synthesized
complexes were given in Table 7. Among the all of the complexes
tested, [CuL4(bdt)] and [CuL1(bdt)] have lowest IC50 values of 108.18
and 114.47 mg/mL when compared with other complexes. From the
5. Conclusions
result, it is inferred that [CuL4(bdt)] and [CuL1(bdt)] complexes
have higher cytotoxicity and anti-cancer effects on cancer cell line
than other complexes.
Few new copper(II) and zinc(II) complexes of the type [ML(bdt)],
[L ¼ Schiff base derived from the condensation of 3-(3-phenyl-
allylidene)-pentane-2,4-dione and para substituted aniline(X);
X ¼ eNO2 (L1), eH (L2), eOH (L3) and eOCH3 (L4); bdt ¼ benzene-
1,2-dithiol] have been synthesized and structurally characterized.
They have adopted square-planar geometry around the central
metal ion. Mechanistic investigations show a major groove binding
for the synthesized complexes with DNA. Chemical nuclease
activity of the synthesized copper complexes in the presence of an
oxidizing agent, hydrogen peroxide via mechanistic pathway
involves the formation of hydroxyl radical as the reactive species.
Pathways involving singlet oxygen in the DNA photocleavage
reactions have been proposed from the observation of the complete
inhibition of the cleavage in the presence of sodium azide and
enhancement of cleavage in D2O. Hydroxyl radical scavengers like
DMSO do not show any significant effect in the DNA cleavage
activity. This result suggests the formation of singlet oxygen as the
reactive species in a type-II process. The hydrolytic cleavage of DNA
by the zinc complexes is supported by the evidence from free
radical quenching and T4 ligase ligation. The antitumor and in vitro
cytotoxic activities of synthesized complexes have been examined.
The results suggest that all the complexes produce a potent anti-
tumor and cytotoxic effect against EAC. All of the complexes exhibit
good antimycobacterial activity except unsubstituted group
complexes.
4.2.3.4. Effect of Cu(II) and Zn(II) complexes on human cancer cell
lines by MTT assay. In vitro anti-cancer activity was assessed by MTT
assay against human cervical cancer cell lines (HeLa), Human
laryngeal epithelial cancer (Hep2), Human liver cancer (HepG2)
and Human breast cancer (MCF-7). IC50 values of the complexes
were displayed in Table 8. Cisplatin was used as standard. When the
Table 8
In vitro cytotoxic effect of Cu(II) and Zn(II) complexes against human cancer cell
lines.
Compounds
IC50 (mg/mL)
HeLa
Hep2
HepG2
MCF-7
[CuL1(bdt)]
[CuL2(bdt)]
[CuL3(bdt)]
[CuL4(bdt)]
[ZnL1(bdt)]
[ZnL2(bdt)]
[ZnL3(bdt)]
[ZnL4(bdt)]
Cisplatin
17
34
31
14
36
30
42
44
33
52
58
26
54
34
46
52
0.25
2
14
9
7
18
6
8
3
16
24
22
26
0.65
14
21
24
31
0.72
0.53
Average of 3 determinations, 3 replicates IC50, Drug concentration inhibiting 50%
cellular growth following 72 h of drug exposure.

5448
N. Raman et al. / European Journal of Medicinal Chemistry 45 (2010) 5438e5451
6. Experimental protocols
[CuL3(bdt)]. Yield: 38%. IR (KBr pellet, cmꢀ1): 1621
1570 (eHC]C); 1068 (eCeS ring str.; bdt); 3432 (eOH); 430
n(eC]N);
n
n
n
6.1. Materials and methods
(MeN); 382 (MeS). MS m/z (%): 599 [M þ 1]þ. Anal. Calc. for
C32H26N2O2CuS2: Cu, 10.6; C, 64.3; H, 4.4; N, 4.7; S, 10.7; Found: Cu,
10.4; C, 64.0; H, 4.2; N, 4.5; S, 10.4 (%). LM 10ꢀ3
(ohmꢀ1 cm2 molꢀ1) ¼ 3.3. lmax (cmꢀ1) in DMF, 38023, 24876,
23866, 16978, 14815. meff (BM): 1.84.
All reagents and chemicals were procured from Merck products.
Solvents used for electrochemical and spectroscopic studies were
purified by standard procedures [56]. DNA was purchased from
Bangalore Genei (India). Agarose (molecular biology grade),
ethidium bromide (EB) were obtained from Sigma (USA). Tris
(hydroxymethyl)aminomethaneeHCl (TriseHCl) buffer solution
was prepared using deionized, sonicated triply distilled water.
[CuL4(bdt)]. Yield: 49%. IR (KBr pellet, cmꢀ1): 1615
1587 (eHC]C); 1283, 1085, (eCeOeCe); 1095 (eCeS ring str.;
n(eC]N);
n
n
n
bdt); 429 (MeN); 397 (MeS). MS m/z (%): 629 [M þ 1]þ. Anal. Calc.
for C34H32N2O2CuS2: Cu, 10.1; C, 65.0; H, 5.1; N, 4.5; S, 10.2; Found:
Cu, 9.8; C, 64.7; H, 5.0; N, 4.2; S, 10.0 (%). LM 10ꢀ3
(ohmꢀ1 cm2 molꢀ1) ¼ 7.3. lmax (cmꢀ1) in DMF, 37453, 24331, 23585,
16949, 14793, meff (BM): 1.89.
6.2. Physical measurement
Carbon, hydrogen and nitrogen analysis of the complexes were
carried out on a CHN analyzer Calrlo Erba 1108, Heraeus. The
infrared spectra (KBr discs) of the samples were recorded on
a PerkineElmer 783 series FTIR spectrophotometer. The electronic
absorption spectra in the 200e1100 nm were obtained on a Shi-
madzu UV-1601 spectrophotometer. 1H and 13C NMR spectra
(300 MHz) of the ligands and their zinc complexes were recorded
on a Bruker Avance DRX 300 FT-NMR spectrometer using CDCl3 and
DMSO-d6 as solvents respectively. Tetramethylsilane was used as
internal standard. Fast atomic bombardment mass spectra
(FAB-MS) were obtained using a VGZAB-HS spectrometer in
a 3-nitrobenzylalcohol matrix. The X-band EPR spectra of the
complexes were recorded at RT (300 K) and LNT (77 K) using DPPH
as the g-marker. Molar conductance of 10ꢀ3 M solutions of the
complexes in N,N0-dimethylformamide (DMF) were measured at
room temperature with a Deepvision Model-601 digital direct
reading deluxe conductivity meter. Magnetic susceptibility
measurements were carried out by employing the Gouy method at
room temperature on powder sample of the complexes.
CuSO4$5H20 was used as calibrant. Electrochemical measurements
were performed on a CHI620C electrochemical analyzer with three
electrode system of a glassy electrode as the working electrode,
a platinum wire as auxiliary electrode and Ag/AgCl as the reference
electrode. Solutions were deoxygenated by purging with N2 prior to
measurements. The metal contents of the complexes were deter-
mined according to the literature method [57]. The purity of ligands
and their complexes were evaluated by column and thin layer
chromatography.
[ZnL1(bdt)]. Yield: 47%. IR (KBr pellet, cmꢀ1): 1610
1580 (eHC]C); 1096 (eCeS ring str.; bdt); 1478, 1321, 861
(eCeN str; eNO2); 431 (MeN); 379 (MeS). 1H NMR (
): (aromatic)
6.8e7.2 (m); (eCH3, 6H), 2.3 (s). 13C NMR (
): 125.0e127.6 (C1 to C6
n(eC]N);
n
n
n
d
d
and C13); 136.0 (C7); 129 (C8); 154.6 (C9); 18.8 (C10); 152.3 (C11);
121.8 (C12); 146.5 (C14); 125.8, 129.7,132.7 (C15, C16, C17, bdt). MS m/z
(%): 661 [M þ 1]þ. Anal. Calc. for C32H26N4O4ZnS2: Zn, 9.9; C, 58.2;
H, 4.0; N, 8.5; S, 9.7; Found: Zn, 9.7; C, 57.9; H, 4.0; N, 8.2; S, 9.4 (%).
LM 10ꢀ3 (ohmꢀ1 cm2 molꢀ1) ¼ 9.9. lmax (cmꢀ1) in DMF, 38810,
24412, 23068. meff (BM): diamagnetic.
[ZnL2(bdt)]. Yield: 42%. IR (KBr pellet, cmꢀ1): 1616
1585 (eHC]C); 1089 (eCeS ring str.; bdt); 427 (MeN); 372
(MeS).1H NMR ( ): (aromatic) 6.8e7.2 (m); (eCH3, 6H), 2.6 (s). 13
NMR ( ): 125.5e128.0 (C1 to C6 and C13); 137.0 (C7); 129.1 (C8);
n(eC]N);
n
n
d
C
d
152.0 (C9); 17.5 (C10); 148.4 (C11); 121.0 (C12); 130.0 (C13); 126.8,
129.7, 133.7 (C15, C16, C17, bdt). MS m/z (%): 569 [M þ 1]þ. Anal. Calc.
for C32H26N2ZnS2: Zn, 11.5; C, 67.7; H, 4.6; N, 4.9; S, 11.3; Found: Zn,
11.2; C, 67.4; H, 4.5; N, 4.7; S, 11.0 (%). LM 10ꢀ3
(ohmꢀ1 cm2 molꢀ1) ¼ 5.7 lmax(cmꢀ1) in DMF, 38365, 24395, 23318.
meff (BM): diamagnetic.
[ZnL3(bdt)]. Yield: 48%. IR (KBr pellet, cmꢀ1): 1619
1574 (eHC]C); 1085 (eCeS ring str.; bdt); 3432 (eOH); 435
(MeN); 391 (MeS). 1H NMR (
): (aromatic) 6.8e7.4 (m); (eOH,
1H), 11.7; (eCH3, 6H), 2.3 (s). 13C NMR (
): 125.1e129.0 (C1 to C6);
n(eC]N);
n
n
n
d
n
d
137.0 (C7); 129.0 (C8); 152.2 (C9); 14.5 (C10); 13.0 (C11); 123.6 (C12);
120.3 (C13); 152.4 (C14); 127.5,129.9,134.5 (C15, C16, C17, bdt). MS m/z
(%): 601 [M þ 1]þ. Anal. Calc. for C32H26N2O2ZnS2: Zn, 10.9; C, 64.1;
H, 4.4; N, 4.7; S, 10.7; Found: Zn, 10.7; C, 63.8; H, 4.4; N, 4.3; S, 10.4
(%). LM 10ꢀ3 (ohmꢀ1 cm2 molꢀ1) ¼ 4.7. lmax (cmꢀ1) in DMF, 38046,
24895, 23871. meff (BM): diamagnetic.
6.3. Synthesis of complexes
[ZnL4(bdt)]. Yield: 53%. IR (KBr pellet, cmꢀ1): 1621
1587 (eHC]C); 1283, 1085 (eCeOeCe); 1105 (eCeS ring str.;
bdt); 439 (MeN); 398 (MeS).1H NMR (
): (aromatic) 7.0e7.4 (m);
(eOCH3, 6H), 3.6(s); (eCH3, 6H), 2.3 (s). 13C NMR (
): 125.0e128.0
n(eC]N);
To a stirred ethanol solution of the Schiff base(s) (5 mmol),
a solution of copper(II)/zinc(II)chloride (5 mmol) in ethanol was
added dropwise. After the reaction for 1 h at 70 ꢂC, a solution of bdt
(5 mmol) in ethanol was added. The reaction solution was refluxed
for 2 h. After cooling the reaction mixture to an ambient temper-
ature, the formed solid was filtered, washed with diethyl ether and
finally dried in vacuum.
n
n
n
d
d
(C1 to C6); 137.0 (C7); 128.6 (C8); 150.2 (C9); 16.8 (C10); 141.6 (C11);
123.2 (C12); 116.3 (C13); 154.5 (C14); 59.4 (C15); 127.7, 130.0, 134.8
(C15, C16, C17, bdt). MS m/z (%): 631 [M þ 1]þ. Anal. Calc. for
C34H32N2O2ZnS2: Zn, 10.4; C, 64.8; H, 5.1; N, 4.5; S, 10.2; Found: Zn,
10.0; C, 64.2; H, 5.0; N, 4.2; S, 10.0 (%). LM 10ꢀ3
(ohmꢀ1 cm2 molꢀ1) ¼ 8.3. lmax (cmꢀ1) in DMF, 37458, 24335,
23582. meff (BM): diamagnetic.
[CuL1(bdt)]. Yield: 42%. IR (KBr pellet, cmꢀ1): 1612
1576 (eHC]C); 1058 (eCeS ring str.; bdt); 1476, 1322, 861 n
n(eC]N);
n
n
(eCeN str; eNO2); 432 (MeN); 386 (MeS). MS m/z (%): 659
[M þ 1]þ. Anal. Calc. for C32H26N4O4CuS2: Cu, 9.7; C, 58.4; H, 4.0; N,
8.5; S, 9.7; Found: Cu, 9.5; C, 58.0; H, 4.0; N, 8.2; S, 9.5 (%). LM 10ꢀ3
(ohmꢀ1 cm2 molꢀ1) ¼ 5.0 lmax in DMF (cmꢀ1), 38759, 24213, 23041,
17035, 14684. meff (BM): 1.94.
6.4. DNA binding studies
[CuL2(bdt)]. Yield: 51%. IR (KBr pellet, cmꢀ1): 1619
(eHC]C); 1076
n
(C]N); 1584
All the experiments involving the interaction of the complexes
with Calf thymus (CT) DNA were carried out in TriseHCl buffer
(50 mM TriseHCl, pH 7.2) containing 5% DMF at room temperature.
A solution of CT DNA in the buffer gave a ratio of UV absorbance at
260 and 280 nm of about 1.89:1, indicating the CT DNA sufficiently
free from protein. The CT DNA concentration per nucleotide was
n
n(CeS ring str.; bdt); 441 (MeN); 394 (MeS). MS
m/z (%): 567 [M þ 1]þ. Anal. Calc. for C32H26N2CuS2: Cu,11.2; C, 67.9;
H, 4.6; N, 5.0; S, 11.3; Found: Cu, 11.0; C, 67.5; H, 4.5; N, 4.7 (%).
LM 10ꢀ3 (ohmꢀ1 cm2 molꢀ1) ¼ 7.0 lmax (cmꢀ1) in DMF, 38314,
24390, 23310, 16992, 14771. meff (BM): 1.9.

N. Raman et al. / European Journal of Medicinal Chemistry 45 (2010) 5438e5451
5449
determined by absorption spectroscopy using the molar absorption
DMSO (4
experiment, the solvent was used for the dilution of the sample to
18
L. The sample after incubation for 1 h at 37 ꢂC in a dark chamber
was added to the loading buffer containing 0.25% bromophenol
blue, 0.25% xylene cyanol, 30% glycerol (3 L) and the solution was
finally loaded on 0.8% agarose gel containing 1 g/mL ethidium
mL) prior to the addition of the each complex. For the D2O
coefficient of 6600 Mꢀ1 cmꢀ1 at 260 nm [58].
m
6.4.1. Absorption spectroscopic studies
Absorption titration experiments were performed by main-
m
taining the metal complex concentration as constant at 50
mM
m
while varying the concentration of the CT DNA within 40e400
m
M.
bromide. Electrophoresis was carried out in a dark chamber for 3 h
at 50 V in TriseacetateeEDTA buffer. Bands were visualized by UV
light and photographed.
While measuring the absorption spectrum, equal quantity of CT
DNA was added to both the complex solution and the reference
solution to eliminate the absorbance of CT DNA itself. From the
absorption data, the intrinsic binding constant Kb was determined
6.6. Hydrolytic DNA cleavage experiment
from the plot of [DNA]/(3a
equation:
ꢀ
3f) vs. [DNA] using the following
The 25
mL total volume of mixture of 77
mM pUC19 DNA with
zinc complexes of 50
m
M in 50 mM TriseHCl buffer (pH 7.2) con-
ꢀ
ꢁ
ꢀ
ꢁ
h
ꢀ
ꢁiꢀ1
taining 50 mM NaCl was incubated for 30 min at 37 ꢂC. To eliminate
the effect of the oxidative species which results from the oxygen
dissolved in the solution on the hydrolysis reactions, the incubation
of all samples was performed under anaerobic conditions such as
deoxygenated and highly purified water, isolation of air, and in
nitrogen atmosphere. The hydrolytic reactions were quenched by
½DNAꢃ= 3a
ꢀ
3f ¼ ½DNAꢃ= 3b
ꢀ
3f þ Kb 3b
ꢀ
3f
where [DNA] is the concentration of CT DNA in base pairs. The
apparent absorption coefficients 3a, 3f and 3b correspond to Aobsd/
[M], the extinction coefficient for the free metal(II) complex and
extinction coefficient for the metal(II) complex in the fully bound
form, respectively [59]. Kb is given by the ratio of slope to the
intercept.
the addition of 4 mL TBE (89 mM Tris, 89 mM boron hydroxide,
2 mM EDTA) sample buffer containing xylene cyanol and bromo-
phenol blue. The electrophoresis of DNA cleavage products was
performed on 0.8% agarose gel. The gels were run at 50 V for 45 min
in 0.01 M pH 7.2 NaH2PO4 and Na2HPO4 buffer. To perform simple
kinetic analysis, the electrophoresis bands were visualized by
staining in an ethidium bromide solution and photographed on UV
transilluminator at 360 nm.
6.4.2. Electrochemical methods
Cyclic voltammetry and Differential pulse voltammogram
studies were performed on a CHI620C electrochemical analyzer
with three electrode system of glassy carbon as the working elec-
trode, a platinum wire as auxiliary electrode and Ag/AgCl as the
reference electrode. Solutions were deoxygenated by purging with
N2 prior to measurements. The freshly polished glassy electrode
6.7. Ligation experiment on the DNA by zinc complexes
was modified by transferring a droplet of 2
m
L of 2.5 ꢁ 10ꢀ3 M of CT
First, the linear DNA was isolated from the pUC 19 DNA cleavage
products provided by zinc complexes. Then in each parallel
DNA solution on to the surface, followed by air drying. Then the
electrode was rinsed with distilled water. Thus, a CT DNA-modified
glassy carbon electrode was obtained.
experiment, the 25
m
L total volume of mixture containing 2
mL
ligation buffer, 1 L 0.1 M ATP, 4
m
mL 40% PEG, and 3 Weiss units T4
DNA ligase reacted with the linear DNA for 24 h at room temper-
ature. The ligation products were monitored by the electrophoresis
and visualized by staining in an ethidium bromide solution.
6.4.3. Viscosity measurements
Viscosity experiments were carried on an Ostwald viscometer,
immersed in a thermostated water-bath maintained at a constant
temperature at 30.0 ꢅ 0.1 ꢂC. CT DNA samples of approximately
0.5 mM were prepared by sonicating in order minimize complex-
ities arising from CT DNA flexibility [60]. Flow time was measured
with a digital stopwatch three times for each sample and an
6.8. Antitumor activity
6.8.1. Animals
average flow time was calculated. Data were presented as (
versus the concentration of the metal(II) complexes, where
h
/
h0 1/3
is the
)
Adult Swiss female albino mice (20e25 g) were procured from
Animal house, Vivekananda College of Pharmacy, Trichengodu,
Tamil Nadu, India and used throughout the study. They were
housed in microlon boxes in controlled environment (temperature
25 ꢅ 2 ꢂC and 12 h dark/light cycle) with standard laboratory diet
and water ad libitum. The experiments were performed in accor-
dance with guidelines established by the European community for
the care and use of laboratory animals, and were approved by the
Institutional Animal Ethics Committee (IAEC) of Vivekananda
College of Pharmacy, Tamil Nadu, India.
h
viscosity of CT DNA solution in the presence of complex, and h0 is
the viscosity of CT DNA solution in the absence of complex.
Viscosity values were calculated after correcting the flow time of
buffer alone (t0),
h
¼ (t e t0)/t0 [61].
6.5. Nuclease activity
The extent of cleavage of super coiled (SC) pUC19 DNA (33.3 mM,
0.2
m
g) to its nicked circular (NC) form was determined by agarose
6.8.2. Cells
gel electrophoresis in 50 mM TriseHCl buffer (pH 7.2) containing
50 mM NaCl. For photo-induced DNA cleavage studies, the reac-
tions were carried out under illuminated conditions using UV
sources at 360 nm. After exposure to light, each sample was incu-
bated for 1 h at 37 ꢂC and analyzed for the photo-cleaved products
using gel electrophoresis as discussed below. The inhibition reac-
tions for the “chemical nuclease” reactions were carried out under
dark condition by adding reagent distamycin (50 mM)/DMSO (4 mL),
prior to the addition of the each complex and the oxidizing agent
hydrogen peroxide. The inhibition reactions for the photo-induced
EAC cells were obtained through the courtesy of Amala Cancer
Research Centre, Trissur, India. They were maintained by weekly
intraperitoneal inoculation of 106 cells/mouse [62].
6.8.3. Effect of Cu(II) and Zn(II) complexes on survival time [63]
Animals were inoculated with 1 ꢁ106 cells/mouse on day ‘0’ and
treatment with synthesized complexes started 24 h after inocula-
tion, at a dose of 100 mg/kg/day, p.o. The control group was treated
with the same volume of 0.9% sodium chloride solution. All the
treatments were given for nine days. The median survival time
(MST) of each group, consisting of 10 mice was noted. The
DNA were carried out at 360 nm using reagents NaN3 (100 mM)/

5450
N. Raman et al. / European Journal of Medicinal Chemistry 45 (2010) 5438e5451
antitumor efficacy of complexes was compared with that of 5-
fluorouracil (Dabur Pharmaceutical Ltd, India; 5-FU, 20 mg/kg/day,
i.p. for 9 days).
well. If the dye turned pink, indicating bacterial growth, the dye
was then added to all remaining wells in the plate. The results were
read on the following day and Minimum Inhibitory Concentration
(MIC) values of the complexes were calculated. Rifampicin was
used as positive control.
6.8.4. Effect of Cu(II) and Zn(II) complexes on hematological
parameters [63]
In order to detect the influence of complexes on the hemato-
logical status of EAC-bearing mice, a comparison was made among
three groups (n ¼ 5) of mice on the 14th day after inoculation. The
groups comprised of (1) tumor-bearing mice (2) tumor-bearing
mice treated with complexes (100 mg/kg/day, p.o. for the first 9
days) and (3) control mice (normal). Blood was drawn from each
mouse by the retro orbital plexus method and the white blood cell
count (WBC), red blood cell count (RBC), hemoglobin, protein and
packed cell volume (PCV) were determined [64e66].
Acknowledgements
The authors express their heartfelt thanks to the Department of
Science and Technology, New Delhi for financial assistance. They
also express their gratitude to the College Managing Board, VHNSN
College, Virudhunagar for providing research facilities.
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mg/mL), and amphotericin-B
(5
m
g/mL) under a humidified atmosphere of 5% CO2 at 37 ꢂC until
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