Synthesis, structural characterization and in vitro biological screening of some homoleptic copper(II) complexes with substituted guanidines

Article abstract of DOI:10.1016/j.ejmech.2011.11.029

A series of homoleptic copper(II) complexes (1a-8a) with N,N′,N″-trisubstituted guanidines, [Cu(II){PhCONHC(NHR)NPh} ₂] (where R = phenyl (1a), n-butyl (2a), sec-butyl (3a), cyclohexyl (4a), 1-naphthyl (5a), 2,4-dichlorophenyl (6a), 3,4-dichlor

Full text of DOI:10.1016/j.ejmech.2011.11.029

European Journal of Medicinal Chemistry 48 (2012) 26e35
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, structural characterization and in vitro biological screening of some
homoleptic copper(II) complexes with substituted guanidines
,
c
a
Ghulam Murtaza a, Muhammad Khawar Rauf a, Amin Badshah a , Masahiro Ebihara , Muhammad Said ,
*
Marcel Gielen b, Dick de Vos d, Erum Dilshad e, Bushra Mirza e
a Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
b Vrije Universiteit Brussel, Faculty of Engineering, HNMR Unit, 1050 Brussels, Belgium
c Department of Chemistry, Faculty of Engineering, Gifu University Yanagido, Gifu 501-1193, Japan
d Pharmachemie B.V., P.O. Box 552, 2003 RN, Haarlem, The Netherlands
e Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan
a r t i c l e i n f o
a b s t r a c t
A series of homoleptic copper(II) complexes (1ae8a) with N,N0,N00-trisubstituted guanidines, [Cu(II)
{PhCONHC(NHR)NPh}2] (where R ¼ phenyl (1a), n-butyl (2a), sec-butyl (3a), cyclohexyl (4a), 1-naphthyl
(5a), 2,4-dichlorophenyl (6a), 3,4-dichlorophenyl (7a), and 3,5-dichlorophenyl (8a)) have been synthe-
sized and characterized by elemental analyses, FT-IR, UVevisible, 1H and 13C NMR spectroscopy, and
single crystal X-ray diffraction analysis. The X-ray crystal structures revealed that the complexes 2a and
4a are mononuclear in the solid state and that the geometry around the copper atom is nearly square
planar. In both the cases, N,N0,N00-trisubstituted guanidine ligands have been coordinated to the Cu(II)
through the oxygen and nitrogen atoms. The synthesized guanidines and their complexes were initially
screened for their anti-microbial activities, and Brine Shrimps Lethality assay. The complexes were also
screened for in vitro cytotoxicity activity in human cell lines carcinomas A498, EVSAT, H226, IGROV, M19,
MCF-7 and WIDR. The results show a moderate level of cytotoxicity against these seven human cancer
cell lines as compared with standard chemotherapeutic drugs.
Article history:
Received 8 June 2011
Received in revised form
14 November 2011
Accepted 15 November 2011
Available online 1 December 2011
Keywords:
Copper(II) complexes
N,N0,N00-trisubstituted guanidines
Cytotoxicity
Crystal structures
Human cell lines
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
a physiological feature of many tumor tissues and cells as was
demonstrated in many types of human cancers including breast,
One of the fundamental goals in medicinal chemistry is the
development of new anticancer and anti-microbial therapeutic
agents. Cancer treatment using metal-based drugs is one of the
very effective strategies as the metal ions are capable of binding to
nucleic acids stereo-specifically with varying strength [1e3].
Copper being an essential metal for animals and non-toxic in
controlled quantity is implicated in many diseases [4]. The toxicity
of metal atoms is due to those reactions of the redox-active metal
ions which generate reactive oxygen species (ROS) that damage
DNA and other biomolecules, probably via Haber-Weiss or Fenton-
like reactions [5]. The accumulation of copper is observed to be
prostate, colon, lung and brain [6e8]. However, there are several
metal-organic compounds that actively and specifically inhibit the
chymotrypsin like activity of the proteasome in vitro and in human
tumor-cell cultures [9]. Copper(II) complexes have been extensively
utilized in DNA cleavage for the generation of activated oxygen
species by redox cycling properties between Cu(II) and Cu(I),
presumably a hydroxyl radical, which can react at several nucleic
acid sites to break it [10e12]. Copper(II) complexes with N and O
donating ligands have also been studied for their nuclease prop-
erties [13,14]. The copper accumulates in tumors due to the selec-
tive permeability of the cancer cell membrane to copper
compounds. For these reasons, a number of copper complexes have
been screened for their anticancer activity and some of them were
found active both in vivo and in vitro [15e17].
Abbreviations: DMSO, Dimethylsulphoxide; DMF, Dimethylformamide; TLC,
Thin layer chromatography; SRB, Sulphorhodamine B; FCS, Fetal Calf Serum; PBS,
Fetal Bovine Serum; RPMI, Roswell Park Memorial Institute; TCA, Trichloroacetic
acid.
* Corresponding author.
E-mail addresses: mkhawarrauf@yahoo.co.uk (M.K. Rauf), aminbadshah@yahoo.
com (A. Badshah).
Guanidine functionalities are found in a variety of natural
compounds, either in cyclic form, or as terminal groups of pendent
substituents [18]. Some of the naturally occurring guanidines were
screened for their nuclease activity and exhibit cytotoxic properties
[19]. The guanidine group acts as an inhibitor of urokinase that
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.11.029

G. Murtaza et al. / European Journal of Medicinal Chemistry 48 (2012) 26e35
27
plays a vital role in tumor metastasis and is implicated in a large
number of malignancies, including breast, lungs, bladder, stomach,
cervix, kidney, and brain cancers [20]. Various guanidine
compounds have also been synthesized and tested for their anti-
tumor activity [21,22]. The studies had revealed that polyaromatic
guanidinium derivatives lengthen the DNA helix forming stronger
complexes which confer cytotoxic activities [23].
Though the anticancer activity of different platinum and palla-
dium metals appear to be promising, these metals are unnatural to
the human body because there is no effective mechanism for their
rejection as for the other metals like copper and iron. It is, therefore,
an endeavor to find out the cytotoxic effects of copper(II)
complexes. Herein, we report the synthesis of a series of copper(II)
complexes of novel substituted acylguanidines with a view to
design anticancer drugs that are more potent as well as friendly to
the human body. As some reports demonstrated that copper
complexes of guanidines also exhibit antibacterial and antifungal
activities [24] that is enhanced by complexation with copper [15],
so we are also presenting the bactericidal and fungicidal activities
for the synthesized compounds.
In the complexes, significant changes were observed in the C]O
band which shifted toward smaller value in all complexes showing
single bond behavior due to bonding with the metal atom. The
disappearance of the weak NeH band was observed, which
provides supporting information about the complex formation. The
sharp NeH band appeared in the same region with some shift
towards higher value. MeO and MeN bands are also observed in
the range 443e488 and 272e278 cmꢀ1 respectively in all the
complexes, which are within the specific regions for CueO and
CueN bond [27].
1H and 13C NMR spectra of all the guanidines were taken in
chloroform but the complexes being paramagnetic in nature could
not be analyzed by NMR. In 1H NMR compound 2e4 have one NeH
proton resonating up-field in the range 4.73e4.96 ppm and another
one downfield in the range 12.12e12.13 ppm. The downfield proton
was identified as the PheNH proton that appeared downfield due
to the attachment of electron withdrawing aromatic groups on
nitrogen atoms. The other reason may be the involvement of
Hebonding with the carbonyl oxygen. In the remaining compounds
(1 and 5e8) both NeH protons appeared downfield in the range
10.30e12.15 ppm due to the presence of aromatic groups on both
nitrogen atoms. Moreover the signals of the NH protons are often
broad because the 14Ne1H coupling is only partly eliminated by the
quadrupole relaxation of 14N, due to which eNHeCH2e show
spinespin decoupling effect and not couple with each other. The
methylene protons appear as a broad singlet (pyramid like) due to
“methylene enveloping” and keto-enol tautomerism existing in
these guanidine structures, which also justify the broadening of NH
proton and the appearance of NHeCH2e methylene protons as
a broad pyramid like singlet [28]. Aromatic protons were not well
resolved and appeared as complex patterns in their specific region.
In 13C NMR, the carbonyl carbon appeared at 176.7e178.4 ppm in all
compounds, while the CN3 carbon was present at 156.0e158.7 ppm
which is the specific region for the guanidine moiety [29].
2. Results and discussion
2.1. Chemistry
A series of guanidine ligands (1e8), N,N0-diphenyl-N"-benzoyl-
guanidine (1), N-phenyl-N0-(n-butyl)-N00-benzoylguanidine (2),
N-phenyl-N0-(sec-butyl)-N00-benzoylguanidine (3), N-phenyl-N0-
cyclohexyl-N"-benzoylguanidine (4), N-phenyl-N0-(1-naphthyl)-
N00-benzoylguanidine (5), N-phenyl-N0-(2,4-dichlorophenyl)-N00-
benzoylguanidine (6), N-phenyl-N0-(3,4-dichlorophenyl)-N00-ben-
zoylguanidine (7) and N-phenyl-N0-(3,5-dichlorophenyl)-N00-ben-
zoylguanidine (8) were synthesized from N-benzoyl-N0-
phenylthiourea by using a standard catalytic guanylation reaction
method [25a]. By reacting these ligands with copper(II) acetate,
a series of homoleptic complexes (1ae8a), bis(N,N0-diphenyl-
N"-benzoylguanidinato)copper(II) (1a), bis(N-phenyl-N0-(n-butyl)-
N00-benzoylguanidinato)copper(II) (2a), bis(N-phenyl-N0-(sec-
butyl)-N00-benzoylguanidinato)copper(II) (3a), bis(N-phenyl-N0-
cyclohexyl-N"-benzoylguanidinato)copper(II) (4a), bis(N-phenyl-
N0-(1-naphthyl)-N00-benzoylguanidinato)copper(II) (5a), bis(N-
phenyl-N0-(2,4-dichlorophenyl)-N00-benzoylguanidinato)copper(II)
All the synthesized complexes were characterized by elemental
analysis using a CHNS analyzer and atomic absorption spectroscopy
was used to find the concentration of copper. The results indicated
that the ligand to metal ratio in complexes is 1:2 which was further
confirmed by the crystal structures of complexes 2a and 4a.
2.3. Magnetic susceptibility
(6a),
bis(N-phenyl-N0-(3,4-dichlorophenyl)-N00-benzoylguanidi-
Magnetic susceptibility of the synthesized complexes was
determined by the magnetic susceptibility balance model Auto
MSB. All the complexes were paramagnetic with magnetic
nato)copper(II) (7a) and bis(N-phenyl-N0-(3,5-dichlorophenyl)-N00-
benzoylguanidinato)copper(II) (8a) were also synthesized. The
purity of the ligands and complexes was checked by TLC with
Merck Kieselgel GF 254 Plates, elemental analysis, 1H- and 13C-NMR
(Bruker ARX, 300 MHz spectrometer).
moments (m) in the range of 1.60e1.67 BM, which is characteristic
for the 3 d9 electronic configuration of Cu(II) complexes.
2.4. Structural studies
2.2. Synthesis and spectroscopic studies
Compound 1, Fig. 1, is a typical N,N0,N00-trisubstituted guanidine
with normal geometric parameters [30]. In the asymmetric unit,
there are two independent molecules with slightly different
conformations. The CeO bond distance indicates that the bond has
full double bond character while the CeN bond lengths indicate
partial double bond character (Table 2). The guanidine moiety and
carbonyl group are coplanar due to intramolecular NeH.O
The entire polysubstituted guanidines and their Cu(II)
complexes were synthesized in good yield, which were character-
ized by different analytical techniques. In FT-IR spectra of ligands
two peaks, a sharp and other weak, were observed for NeH bonds
in the range 3246e3393 cmꢀ1, except for 1 which have electroni-
cally similar environment for both NeH. The weak NeH band at
longer wavelength may be attributed to the involvement of
hydrogen bonding which was also confirmed by the crystal struc-
ture of compounds 1. The C]N stretching frequency in all the
compounds was observed in the range 1544e1586 cmꢀ1 which is
intermediate between single and double bond and shows the
conjugation between all the three nitrogen atoms of the guanidine
moiety [26]. A sharp peak for C]O was also observed in the range
1601e1656 cmꢀ1 for all the guanidines.
hydrogen bonding, forming
a six-membered ring commonly
observed in this class of compounds (Table 3).
The compounds 2a and 4a, are the copper(II) complexes with
the anionic bidentate chelating ligands, 2 and 4, respectively, as
shown in Figs. 2 and 3. Each complex has a square-planar geometry
in which the metal atom lies at an inversion center. Some selected
geometric parameters are listed in Table 2. As shown in Table 2, 2a
and 4a have very similar geometric parameters. The shorter C1eN1

28
G. Murtaza et al. / European Journal of Medicinal Chemistry 48 (2012) 26e35
Fig. 1. Molecular diagram of (1); displacement ellipsoids are drawn at the 50% probability level.
and longer C1eO1 distance as compared to the corresponding ones
in 1 indicate that the CeO has more single bond character and the
CeN more double bond character than those in 1. The plane of the
guanidinate moiety (O1/C1/N1/C2/N2/N3) is slightly tilted from the
coordination plane of 2a (CuN2O2), 11.02(11)ꢁ. It is also observed in
the structure of 4a.
Table 1
Crystallographic data for 1, 2a and 4a.
1
2a
4a
Formula
C20 H17 N3
O
C36 H40 Cu
N6 O2
652.28
Tetragonal
I 41/a
34.330(14)
C40 H44 Cu
N6 O2
704.37
Monoclinic
P21/n
10.043(3)
10.584(4)
16.180(6)
93.978(4)
1715.6(10)
2
Table 2
FW
315.37
Monoclinic
P21/n
13.850(4)
13.582(3)
18.892(5)
113.890(3)
3249.2(15)
8
Selected bond lengths (Å), angles (ꢁ) and torsion angles (ꢁ) for 1, 2a and 4a.
Crystal system
Space group
a (Å)
b (Å)
c (Å)
1
O(1)eC(1)
N(1)eC(1)
N(1)eC(2)
N(2)eC(2)
1.247 (17)
1.359 (18)
1.339 (17)
1.338 (18)
1.357 (18)
127.20(12)
119.38(12)
124.28(13)
118.51(13)
117.19(12)
O(2)eC(21)
N(4)eC(21)
N(4)eC(22)
N(5)eC(22)
1.247 (18)
1.359 (18)
1.334 (18)
1.344 (19)
1.355 (19)
127.36(13)
118.94(12)
123.95(13)
118.94(13)
117.09(13)
5.631(2)
b
(ꢁ)
V (Å3)
Z
6637(5)
8
N(3)eC(2)
N(6)eC(22)
O(1)eC(1)eN(1)
C(1)eN(1)eC(2)
N(1)eC(2)eN(2)
N(1)eC(2)eN(3)
N(2)eC(2)eN(3)
2a
O(2)eC(21)eN(4)
C(21)eN(4)-C(22)
N(4)eC(22)eN(5)
N(4)eC(22)eN(6)
N(5)eC(22)eN(6)
T (ꢁC)
ꢀ150
ꢀ150
ꢀ150
DC Mg/m3
1.289
1.306
1.363
m
(mmꢀ1
)
0.082
1328
0.699
2744
0.45 ꢂ 0.36
ꢂ 0.30
3.36 to 27.47
0.682
742
F(000)
Crystal size (mm3)
0.35 ꢂ 0.31
0.30 ꢂ 0.25
ꢂ 0.22
ꢂ 0.20
Cu(1)eO(1)
O(1)eC(1)
N(1)eC(2)
N(3)eC(2)
O(1)eCu(1)eO(1*)
O(1)eCu(1)eN(1)
C(1)eO(1)eCu(1)
C(9)eN(2)eCu(1)
4a
1.912 (17)
1.277(3)
1.362(3)
1.355(3)
180.0
90.14(7)
126.20(14)
120.26(13)
Cu(1)eN(2)
N(1)eC(1)
N(2)eC(2)
1.966 (18)
1.315(3)
1.339(3)
q
range (ꢁ)
3.22 to 27.48
3.01 to 27.48
Index ranges
ꢀ16<¼h<¼17 ꢀ41<¼h<¼44 ꢀ13<¼h<¼11
ꢀ17<¼k<¼11 ꢀ44<¼k<¼40 ꢀ9<¼k<¼13
ꢀ22<¼l<¼24 ꢀ4<¼l<¼7
ꢀ21<¼l<¼20
14,006
3903 (0.0280)
99.46%
N(1)eCu(1)eN(1*)
O(1)eCu(1)eN(1*)
C(2)eN(1)eCu(1)
180.0
89.86(7)
123.69(15)
Reflections collected
Indept. Reflect. [R(int)]
Completeness to
26,248
26,475
3793 (0.0450)
99.6%
7416 (0.0325)
99.5%
q
¼ 25.00ꢁ
Max. & min. transmission
0.9822
& 0.9720
0.8176
0.787 & 0.710
Cu(1)eO(1)
O(1)eC(1)
N(1)eC(2)
N(3)eC(2)
O(1)eCu(1)eO(1*)
O(1)eCu(1)eN(1)
C(1)eO(1)eCu(1)
C(9)eN(2)eCu(1)
1.909 (14)
1.277(2)
1.363(2)
1.360(2)
180.0
90.32(6)
126.17(12)
120.11(12)
Cu(1)eN(2)
N(1)eC(1)
N(2)eC(2)
1.964 (16)
1.310(2)
1.331(2)
& 0.7437
3793/0/206
1.27
0.052
0.103
Data/restraints/parameters 7416/0/449
3903/0/231
1.11
0.040
0.098
0.796, ꢀ0.479
Goodness-of-fit on F2
R1 [I > 2 (I)]
1.15
0.054
0.111
0.253, ꢀ0.190
s
N(1)eCu(1)eN(1*)
O(1)eCu(1)eN(1*)
C(2)eN(2)eCu(1)
180.0
89.68(6)
124.01(12)
wR2 [all data]
Largest peak and
0.326, ꢀ0.434
hole (eÅꢀ3
)

G. Murtaza et al. / European Journal of Medicinal Chemistry 48 (2012) 26e35
29
Table 3
2.6.2. In vitro cytotoxicity screening
ꢁ
The intramolecular hydrogen bonds for 1, distance, Å; angle,
.
As brine shrimps assay (a prescreening test for antitumor
activities) showed good results, these compounds were further
evaluated for their in vitro cytotoxicity in human cell lines.
Thus far little is known in a systematic way about the in vitro
cytotoxicity of copper complexes. Mostly some compounds have
been studied, large series of compounds have not been tested
[15e17,34e36]. It has been suggested that copper complexes may
function as a topoisomerase II inhibitor [17], anyhow DNA binding
has been mentioned [36]. In the present series of copper complexes
of guanidines ID50 values were in the high range of those of cisplatin.
Aryl substituted compounds are the most active, in particular C6H5
substituted. There is no consistent similar trend in activity as to the
different cell lines. This indicates that there is room for structural
variation affecting different cell lines in a different way. When
extrapolated to the human clinical situation this observation might
be of importance. It is not yet clear which factors, such as size or
electronic properties of the substituent are relevant to the in vitro
cytotoxic activity. ID50 values in Table 7 indicate that 1a is more
active than the standard drug etoposide (ETO) against lungs carci-
noma cells (H226), whereas, 7a have shown a comparable activity
against the same. In short, data collected so far indicate promising
in vitro cytotoxicity. They constitute a highly useful reference basis
for future further investigations.
Compound
D
H
A
DH(Å)
N2 H2N O1 0.870(2) 1.877(19) 2.581 (16) 136.7(17)
N5 H6N O2 0.880(2) 1.820(2) 2.551 (17) 139.2(18)
HA(Å)
DA(Å)
DHA(ꢁ)
1
2.5. UVevisible spectroscopy
Fig. 4(a) and 4(b) present the overlay of UVevisible spectra of
3a, taken in DMSO at different intervals of time i.e. for fresh solution
(day one), after three, five and seven days. The spectra were
recorded for two wavelength ranges with different concentrations,
264e400 nm (1.6 ꢂ 10ꢀ5 M) and 400e800 nm (1.6 ꢂ 10ꢀ4 M). The
absorption spectra of complex 3a exhibit bands in the regions
270e280, 290e295(weak shoulder), 525e710 (broad-band) and
735e750 nm. In UV-region, one peak appears at 275 nm, that may
be attributed to the pep* transition of benzene rings of the ligands.
The appearance of the broad peaks in the 525e710 nm regions
corresponds to the ded electronic transition of the metal d-orbitals.
In DMSO solution, a unique weak absorption at 745 nm is observed
[31]. Keeping in view all this, it is perquisite that the copper(II)
complexes (1ae8a) are also stable in test solution medium as well
as in solid state.
2.6.3. In vitro antibacterial activity
2.6. Pharmacology
Antibacterial screening of the synthesized compounds was
carried out by using four bacterial strains two Gram positive
Staphylococcus aureus (ATCC 6538), Micrococcus luteus (ATCC 10240)
and two Gram negative, Escherichia coli (ATCC 15224), Salmonella
setubal (ATCC 19196). The zones of inhibition values in Table 5
represent the mean value of the three readings with standard
deviation [STDEV]. Zones of more than 12 mm are characteristic for
significant activities, 10e12 mm, for good activities, 7e9 mm low,
and below 7 mm, non-significant activities.
2.6.1. Brine shrimps lethality assay
The cytotoxicity of the synthesized compounds was evaluated
by the brine shrimp lethality method as reported earlier [32]. LD50
was calculated which showed that most of the compounds had
good cytotoxic activities. Compound 2 is the most active of all the
compounds with LD50 1.7
mg/ml, while the rest of the compounds
showed activities with LD50 ranging from 20 to 779
mg/ml. Results,
given in Table 4, indicate that the guanidines (1e8) show good
cytotoxic activities; similar results were observed with the copper
complexes (1ae8a). The active compounds showed concentration-
dependent toxicities, as reported earlier [33].
The results revealed that compound 2 shows good to low
activity against M. luteus, S. setubal and S. aureus and that
compound 7a shows some activity against M. luteus and S. aureus.
Fig. 2. Molecular diagram of (2a); displacement ellipsoids are drawn at the 50% probability level.

30
G. Murtaza et al. / European Journal of Medicinal Chemistry 48 (2012) 26e35
Fig. 3. Molecular diagram of (4a); displacement ellipsoids are drawn at the 50% probability level.
None of the compounds showed activity against E. coli. Previously
the activity was shown by the guanidine ligands to some extent
[37,38], while Cu enhanced the antibacterial activity of the guani-
dine ligands [39].
2.6.4. Antifungal activity
All the synthesized compounds were subjected to antifungal
assays by using the Agar tube dilution method [40] against four
fungal strains i.e. Mucor species, Aspergillus niger, Aspergillu flavus,
and Fusarium solani. The terbinafine was used as standard drug and
the results are summarized in the Table 6. The activity was inves-
tigated on the basis of percent growth inhibition; inhibition more
than 70% was considered a significant activity, 60e70% good,
50e60% moderate and below 50% non-significant.
The complex 5a showed good activity against Mucor, A. niger,
and F. solani while compound 7, 8 and 1a showed good activities
Table 4
Brine shrimps assay for guanidine ligands (1, 2, 5e8) and complexes (1ae3a,
5ae8a).
Test compounds
Percentage mortality of brine shrimps
at concentrations
LD50 (mg/ml)
1000 ppm
100 ppm
10 ppm
1
2
5
6
7
8
1a
2a
3a
5a
6a
7a
8a
80
100
40
100
100
100
67
65
70
57
10
100
14
40
80
30
40
15
50
10
20
47
20
0
100
0
0
25
0
20
0
5
0
7
301.73
1.701
360.38
96.03
20.30
149.72
310.22
535.35
111.82
779.44
146.74
70.74
Fig. 4. (a) UVevisible spectra of 3a (264e400 nm, 1.6 ꢂ 10ꢀ5 M), taken in DMSO for
fresh solution (day one), after three, five and seven days. (b) UVevisible spectra of 3a
(400e800 nm, 1.6 ꢂ 10ꢀ4 M), taken in DMSO for fresh solution (day one), after three,
five and seven days.
100
100
100
17
0
183.28

G. Murtaza et al. / European Journal of Medicinal Chemistry 48 (2012) 26e35
31
Table 5
4. Experimental
In vitro antibacterial activity of synthesized guanidines (1, 2, 5e8), complexes
(1ae3a, 5ae8a) and standard drugs.
4.1. Materials and methods
Test compounds
% zone of inhibition of samples (mm)
All experiments were carried out under the specified conditions
of temperature. Solvents were distilled from the drying agents and
degassed before use.
M. luteus
E. coli
S. setubal
S. aureus
1
2
e
e
e
e
e
10.8 ꢃ 0.163
7.4 ꢃ 0.081
12.5 ꢃ 0.81
1 mg/ml
1 mg/ml
0.4 mg/ml
NMR spectra were recorded on a Bruker ARX, 300 MHz spec-
trometer. 1H NMR (300.13 MHz): internal standard solvent CDCl3
(7.28 ppm from TMS): internal standard TMS; 13C NMR
(75.47 MHz): internal standard solvent CDCl3 (77.0 ppm from TMS):
internal standard TMS; the splitting of proton resonances in the
reported 1H NMR spectra are defined as s ¼ singlet, d ¼ doublet,
t ¼ triplet, q ¼ quartet and m ¼ complex pattern; coupling
constants are reported in Hz. FTeIR spectra were recorded as KBr
5
6
7
8
1a
2a
3a
5a
6a
7a
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
pellets on
a Bio-Rad Excalibur FTeIR Model FTS 3000 MX
11 ꢃ 0.408
0.8 mg/ml
e
12 ꢃ 0.20
0.6 mg/ml
7.7 ꢃ 0.20
1 mg/ml
37 ꢃ 0.57
49.3 ꢃ 0.33
e
(400e4000 cmꢀ1) and on ATR with Perkin Elmer System 2000
(200e500 cmꢀ1). The elemental analyses were performed using
a LECO-932 CHNS analyzer while the metal concentrations were
determined on an Atomic Absorption Spectrophotometer Perkin
Elmer 2380. Magnetic susceptibilities of the complexes (1ae8a)
were recorded with a Magnetic Susceptibility Balance Auto MSB.
Absorption spectra were measured on a UVeVisible spectrometer;
Shimadzu 1800 at constant temperature of 25 ꢃ 1 ꢁC. The melting
points were determined on a Bio Cote SMP10- UK and are uncor-
rected. All solvents were purified by distillation, dried under an
atmosphere of nitrogen and stored over molecular sieves 4 Å.
Benzoic acid, potassium thiocyanate, thionyl chloride, aniline,
triethylamine, mercuric chloride, n-butylamine, sec-butylamine,
cyclohexylamine, 1-naphthylamine, 2,4-dichloroaniline, 3,4-
dichloroaniline, 3,5-dichloroaniline and copper(II) acetate were
purchased from SigmaeAldrich and used as received without
further purification.
8a
e
e
Roxithromycin
Cefixime
DMSO
36 ꢃ 0.577
46.33 ꢃ 0.33
e
30.3 ꢃ 1.02
56 ꢃ 2.30
e
19.7 ꢃ 0.88
44.6 ꢃ 0.70
e
ꢃ - mean standard deviation of triplicate, (ꢀ)- no zone of inhibition observed,
Roxithromycin and Cefixime -as positive control, DMSO-as negative control.
against F. solani and compound 1 against the Mucor species. The rest
of the compounds did not show significant activities. The overall
results show that guanidine ligands had good/moderate antifungal
activities with little enhancement when these guanidine were
coordinated with copper. According to some previous studies
guanidines have shown similar results [15,41].
3. Conclusion
4.2. Synthesis of N,N0,N00-trisubstituted guanidine ligands (1e8)
In the present study of guanidine ligands and their complexes it
has been shown that guanidine ligands exhibit anti-microbial and
cytotoxic activities and that almost similar results are shown by
their Cu complexes with a little enhancement. The cytotoxicity data
obtained show an interesting variable pattern among the different
human cell lines. They constitute a valuable starting point for future
investigations. ID50 values indicate that 1a and 7a have shown
promising results against lungs carcinoma cells (H226). UVeVisible
studies in DMSO solutions indicate that the copper(II) complexes
(1ae8a) are also stable in in test solution medium as well.
The guanidine ligands were synthesized from N-benzoyl-N0-
phenylthiourea, by using a standard guanylation reaction method
[25a]. N-benzoyl-N0-phenylthiourea was mixed in a 1:1 M ratio
with the desired substituted aniline in DMF in which triethylamine
was already added in 2 M ratio. The reaction mixture was main-
tained at temperatures below 5 ꢁC by applying an ice bath. Mercuric
chloride (1:1) was added to the reaction mixture and vigorously
stirred for overnight. Progress of reaction was monitored by TLC
until all the thiourea was consumed. Chloroform (20 ml) was added
and the suspension was filtered through a sintered glass funnel to
remove the residue (HgS). The solvents were evaporated under
reduced pressure and the residue was dissolved in 20 ml of CH2Cl2,
washed with water (4 ꢂ 30 ml) and the organic phase was dried
over anhydrous MgSO4. The solvent was evaporated and the
residue was recrystallized from ethanol to get the crystals of the
compounds (1e8) (see Scheme 1)
Table 6
Antifungal activity of guanidine ligands (1, 2, 5e8) and complexes (1ae3a, 5ae8a).
Test compounds
Compounds showing %inhibition against
fungal strains
A. Niger
F. Solani
Mucor
A. Flavus
1
2
5
6
7
8
1a
2a
3a
5a
6a
7a
43.90
ꢀ4.87
37.19
36.58
24.39
40.24
ꢀ2.43
0
42.36
51.3
55.5
9.72
63.88
64.58
61.8
75
62.5
52.7
54.1
53.5
54.8
100
64.28
39.28
61.30
44.04
35.11
41.07
ꢀ4.16
40.47
20.83
76.19
ꢀ4.16
1.78
41.8
34.6
ꢀ2.0
18.3
36.7
16.3
39.7
ꢀ3.0
16
36
35
46.8
62.2
100
49
4.2.1. N,N0-diphenyl-N00-benzoylguanidine (1)
Quantities used were 5.6 ml (40 mmol) triethylamine, 2.0 ml
(20 mmol) aniline 5.12 g (20 mmol) of N-benzoyl-N0-phenylthio-
urea and 5.44 g (20 mmol) mercuric chloride; Yield 4.54 g, 72%;
yellowish white solid; m.p. 96e98 ꢁC; FTeIR (KBr, cmꢀ1) 3389,
3157, 3054, 1603, 1562, 1526, 1491, 1446, 1351, 1022, 901, 750, 693,
511, 480; 1H NMR (300 MHz, CDCl3, 25 ꢁC)
d 7.29e7.80 (m, 13H,
26.8
62.19
52.43
9.75
30.48
100
ArH), 8.25 (d, 2H, J ¼ 7.2 Hz, ArHc¼c’), 10.30 (br, s, 2H, ReNH); 13C
NMR (75.47 MHz, CDCl3, 25 ꢁC)
d 124.3 (4C), 126.1 (2C), 128.0 (2C),
8a
43.45
100
84
129.3 (2C), 129.5 (4C), 131.5, 136.4, 138.1 (2C) (AromaticeC), 156.6
(C, CN3), 178.3 (C, C]O); Anal. Calcd. for C20N3H17O (315.37): C,
76.17; H, 5.43; N, 13.32; Found: C, 76.23; H, 5.67; N, 13.40.
Terbinafine
Linear length in -ve control
41
72

32
G. Murtaza et al. / European Journal of Medicinal Chemistry 48 (2012) 26e35
Table 7
Cytotoxicity of some selected copper(II) complexes of guanidines1a, 2a, 3a, 5a, 7a, and 8a, and of some reference compounds like doxorubicin (DOX), cisplatin (CPT), 5-
flourouracil (5-FU), methotrexate (MTX), etoposide (ETO) and taxol (TAX). ID50 values (ng/ml) of test compounds in vitro using SRB as cell viability test.
Test compounds
Human cell line
A498 (Renal-F)
EVSA-T (Breast)
H226 (Lungs)
IGROV (Ovarian)
M19MEL (Melanoma -Skin)
MCF-7 (Breast)
WIDR (Colon)
1a
2a
3a
5a
7a
8a
DOX
CPT
5-FU
MTX
ETO
TAX
4254
41062
25000
23911
10454
22351
90
2253
143
37
7578
12002
10870
6365
3334
7507
8
422
475
5
317
2998
18845
16617
12854
7046
13190
199
3269
340
2287
3934
<3.2
2955
29840
27083
24709
8480
24133
60
169
297
7
580
1959
4779
3428
2901
1196
2413
16
558
442
23
505
7765
15593
16971
8544
6080
9893
10
699
750
18
2594
<3.2
8359
7048
11152
14672
8394
10786
11
967
225
< 3.2
150
<3.2
1314
<3.2
<3.2
<3.2
< 3.2
In separate experiments an inter-experimental CV of 1e11% and an intra-experimental CV of 2e4% were obtained.
4.2.2. N-phenyl-N0-(n-butyl)-N00-benzoylguanidine (2)
129.1 (2C), 130.1 (2C), 131.2, 136.2, 138.8 (AromaticeC), 158.2 (C,CN3),
177.5 (C, C]O); Anal. Calcd. for C18N3H21O (295.38): C, 73.19; H, 7.17;
N, 14.23; Found: C, 73.30; H, 7.05; N, 14.43.
Quantities used were 5.6 ml (40 mmol) triethylamine, 2.0 ml
(20 mmol) n-butylamine, 5.12 g (20 mmol) N-benzoyl-N0-phenyl-
thiourea and 5.44 g (20 mmol) mercuric chloride. Yield 4.96 g, 84%;
yellowish white solid; m.p. 88e90 ꢁC; FTeIR (KBr, cmꢀ1) 3288, 3062,
2959, 2926, 2866, 1620,1544, 1500,1495, 1394,1352,1256, 1144, 1069,
1023, 907, 812, 752, 698, 617, 565, 499; 1H NMR (300 MHz, CDCl3,
4.2.4. N-phenyl-N0-cyclohexyl-N00-benzoylguanidine (4)
Quantities used were 5.6 ml (40 mmol) triethylamine, 2.3 ml
(20 mmol) cyclohexylamine, 5.12 g (20 mmol) N-benzoyl-N0-
phenylthiourea and 5.44 g (20 mmol) mercuric chloride; Yield
25 ꢁC)
d
0.97 (t, 3H, J ¼ 7.2 Hz, CH3eCH2eCH2eCH2e), 1.40 (tq, 2H,
J ¼ 7.2, 7.5 Hz, CH3eCH2eCH2eCH2e), 1.61 (tt, 2H, J ¼ 7.2, 7.5 Hz,
CH3eCH2eCH2eCH2e) 3.55 (br, s, 2H, NHeCH2eCH2e), 4.96 (br, s,1H,
ReNH), 7.28e7.49 (m, 8H, ArH), 8.29 (d, 2H, J ¼ 6.9 Hz, ArHc¼c’), 12.12
5.27 g, 82%; colourless solid; m.p. 120e123 ꢁC; FTeIR (KBr, cmꢀ1
)
3326, 3235, 3060, 2930, 2853, 1623, 1586, 1564, 1496, 1448, 1254,
1142, 1026, 901, 837, 760, 691, 580, 493; 1H NMR (300 MHz, CDCl3,
(br, s,1H, PheNH); 13C NMR (75.47 MHz, CDCl3, 25 ꢁC)
d
13.8 (CH3), 20.1
25 ꢁC)
d
1.23e2.09 (m, 11H, cyclohexyleH), 4.86 (br, s, 1H, ReNH)
(CH3eCH2), 31.8 (CH2eCH2eCH2), 41.2 (NHeCH2), 125.5 (2C), 126.8,
127.9 (2C), 129.1 (2C), 130.0 (2C), 131.2, 136.2, 138.6 (AromaticeC),
158.7 (C, CN3), 177.5 (C, C]O); Anal. Calcd. for C18N3H21O (295.38): C,
73.19; H, 7.17; N, 14.23; Found: C, 73.23; H, 7.23; N, 14.31.
7.28e7.49 (m, 10H, ArH), 8.25 (d, 2H, J ¼ 7.2 Hz, ArHc¼c’), 12.12 (br, s,
13
1H, PheNH); C NMR (75.47 MHz, CDCl3, 25 ꢁC)
d 24.8 (Ce3 and
Ce5 in cyclohexyl), 25.6 (Ce4 in cyclohexyl), 33.1 (Ce2 and Ce6 in
cyclohexyl), 50.2 (Ce1 in cyclohexyl), 125.2 (2C), 126.6, 127.9 (2C),
129.1 (2C), 130.0 (2C), 131.5, 136.3, 138.7 (AromaticeC), 157.8 (C,
CN3),177.5 (C, C]O); Anal. Calcd. for C20N3H23O (321.4): C, 74.70; H,
7.21; N, 13.07; Found: C, 74.34; H, 7.40; N, 12.96.
4.2.3. N-phenyl-N0-(sec-butyl)-N00-benzoylguanidine (3)
Quantities used were 5.6 ml (40 mmol) triethylamine, 2.1 ml
(20 mmol) sec-butylamine, 5.12 g (20 mmol) N-benzoyl-N0-phenyl-
thiourea and 5.44 g (20 mmol) mercuric chloride; Yield 4.67 g, 79%;
yellowish white solid; m.p. 61e63 ꢁC; FTeIR (KBr, cmꢀ1) 3246, 2963,
4.2.5. N-phenyl-N0-(1-naphthyl)-N00-benzoylguanidine (5)
Quantities used were, 5.6 ml (40 mmol) triethylamine, 2.86 g
(20 mmol) 1-naphthylamine, 5.12 g (20 mmol) N-benzoyl-N0-
phenylthiourea and 5.44 g (20 mmol) mercuric chloride; Yield
2930, 2872, 1656, 1576, 1496, 1377, 1225, 1135, 1067, 1020, 894, 746,
1
716, 679, 589, 490; H NMR (300 MHz, CDCl3, 25 ꢁC)
d 0.98 (t, 3H,
J ¼ 7.2 Hz, terminal CH3),1.23 (d, 3H J ¼ 6.6 Hz, branched CH3),1.57 (tt,
2H, J ¼ 7.2, 7.2 Hz, CH2), 4.37 (br, s, 1H, CH), 4.73 (br, s, 1H, ReNH),
7.17e7.92 (m, 8H, ArH), 8.29 (d, 2H, J ¼ 6.9 Hz, ArHc¼c’),12.13 (br, s,1H,
5.48 g, 75%; light brown solid; m.p. 132e134 ꢁC; FTeIR (KBr, cmꢀ1
)
3351, 3052, 1614, 1561, 1449, 1352, 1240, 777, 751, 692, 616; 1H NMR
(300 MHz, CDCl3, 25 ꢁC)
7.18e8.17 (m, 15H, ArH), 8.31 (d, 2H,
J ¼ 6.0 Hz, ArHc¼c’), 10.54 (br, s, 1H, ReNH), 12.15 (br, s, 1H, PheNH);
13C NMR (75.47 MHz, CDCl3, 25 ꢁC)
120.1, 122.5, 123.4, 124.2, 125.2
d
PheNH);13C NMR (75.47 MHz, CDCl3, 25ꢁC)
d 10.5 terminalCH3), 20.6
(branched CH3), 29.6 (CH2), 48.7 (CH), 125.4 (2C), 126.7, 127.9 (2C),
d
(2C), 125.8, 127.1, 127.5, 128.1 (2C), 128.6, 129.0 (2C), 129.4 (2C),
131.5, 131.9, 134.7, 137.1, 138.3 (AromaticeC), 157.7 (C, CN3), 178.6 (C,
C]O); Anal. Calcd. for C24N3H19O (365.43): C, 78.88; H, 5.24; N,
11.50; Found: C, 78.57; H, 4.93; N, 11.75.
4.2.6. N-phenyl-N0-(2,4-dichlorophenyl)-N00-benzoylguanidine (6)
Quantities used were 5.6 ml (40 mmol) triethylamine, 3.3 g
(20 mmol) 2,4-dichloroaniline, 5.12 g (20 mmol) N-benzoyl-N0-
phenylthiourea and 5.44 g (20 mmol) mercuric chloride; Yield
5.84 g, 76%; colourless solid; m.p. 156e158 ꢁC; FTeIR (KBr, cmꢀ1
)
3379, 3166, 3058, 1601, 1561, 1519, 1362, 1292, 1192, 811, 745, 699,
588, 487; 1H NMR (300 MHz, CDCl3, 25 ꢁC)
7.04e8.40 (m, 11H,
ArH), 8.21 (d, 2H, J ¼ 9.0 Hz, ArHc¼c’), 10.88 (br, s, 1H, ReNH), 12.13
(br, s, 1H, PheNH); 13C NMR (75.47 MHz, CDCl3, 25 ꢁC)
122.3,
d
d
123.9, 125.6, 125.8 (2C), 127.1, 127.4, 127.6 (2C), 128.9, 129.2 (2C),
130.1 (2C), 131.8, 135.2, 137.8, 140.7 (AromaticeC), 156.0 (C, CN3),
178.5 (C, C]O); Anal. Calcd. for C20N3H15OCl2 (384.26): C, 62.51; H,
3.93; N, 10.94; Found: C, 62.43; H, 3.71; N, 10.99.
Scheme 1. Synthesis of N,N0,N00-trisubstituted guanidines (1e8) and their copper(II)
complexes (1ae8a).

G. Murtaza et al. / European Journal of Medicinal Chemistry 48 (2012) 26e35
33
4.2.7. N-phenyl-N0-(3,4-dichlorophenyl)-N00-benzoylguanidine (7)
Quantities used were 5.6 ml (40 mmol) triethylamine, 3.3 g
(20 mmol) 3,4-dichloroaniline, 5.12 g (20 mmol) N-benzoyl-N0-
phenylthiourea and 5.44 g (20 mmol) mercuric chloride; Yield
5.69 g, 74%; yellow solid; m.p. 99e100 ꢁC; FTeIR (KBr, cmꢀ1) 3393,
3162, 3062, 1603, 1561, 1516, 1356, 1281, 1194, 1129, 1024, 905, 745,
66.29; H, 6.18; N, 12.88; Cu, 9.74; Found: C, 66.15; H, 6.16; N, 12.98;
Cu, 9.43; meff, 1.62 BM.
4.3.4. Bis(N-phenyl-N0-cyclohexyl-N00-benzoylguanidinato)
copper(II) (4a)
Quantities used were 1.28 g (4 mmol) N-phenyl-N0-cyclohexyl-
N"-benzoylguanidine (4) and 0.4 g (2 mmol) Cu(CH3COO)2.H2O;
695, 588, 490; 1H NMR (300 MHz, CDCl3, 25 ꢁC)
ArH), 8.36 (d, 2H, J ¼ 8.1 Hz, ArHc¼c’), 10.53 (br, s, 1H, ReNH), 11.98
(br, s, 1H, PheNH); 13C NMR (75.47 MHz, CDCl3, 25 ꢁC)
121.5, 125.5
d 7.13e8.24 (m,11H,
Yield 1.1 g, 78%; blue solid; m.p. 225e226 ꢁC; FTeIR (KBr, cmꢀ1
)
d
3410, 3052, 2922, 2849, 1590, 1548, 1521, 1447, 1434, 1372, 750, 700,
565, 509, 438, 274; Anal. Calcd. for C40N6H44O2Cu (704.36): C,
68.21; H, 6.33; N, 11.93; Cu, 9.02; Found: C, 67.93; H, 6.12; N, 12.10;
Cu, 8.89; meff, 1.62 BM.
(2C), 127.0, 127.4, 128.2 (2C), 129.3 (2C), 130.2 (2C), 130.4, 131.8,
132.7, 133.4, 135.8, 136.6, 137.3 (AromaticeC), 156.1 (C, CN3), 178.4
(C, C]O); Anal. Calcd. for C20N3H15OCl2 (384.26): C, 62.51; H, 3.93;
N, 10.94; Found: C, 62.26; H, 3.91; N, 10.98.
4.3.5. Bis(N-phenyl-N0-(1-naphthyl)-N00-benzoylguanidinato)
4.2.8. N-phenyl-N0-(3,5-dichlorophenyl)-N00-benzoylguanidine (8)
Quantities used were 5.6 ml (40 mmol) triethylamine, 3.3 g
(20 mmol) 3,5-dichloroaniline, 5.12 g (20 mmol) N-benzoyl-N0-
phenylthiourea and 5.44 g (20 mmol) mercuric chloride; Yield
5.53 g, 72%; yellow solid; m.p. 112e114 ꢁC; FTeIR (KBr, cmꢀ1) 3382,
3194, 3056, 1609, 1565, 1508, 1369, 1258, 1114, 908, 803, 757, 687,
copper(II) (5a)
Quantities used were 1.46
g
(4 mmol) N-phenyl-N0-(1-
naphthyl)-N00-benzoylguanidine (5) and 0.4 g (2 mmol) Cu(CH3
-
COO)2.H2O; Yield 76%; dirty greenish solid; m.p. 204e205 ꢁC; FTeIR
(KBr, cmꢀ1) 3397, 3053, 1589, 1542, 1518, 1424, 1372, 781, 746, 704,
578, 484, 419, 276; Anal. Calcd. for C48N6H36O2Cu (792.38): C, 72.76;
H, 4.58; N, 10.61; Cu, 8.02; Found: C, 72.46; H, 4.48, N, 10.82; Cu,
7.58; meff, 1.60 BM.
582, 490; 1H NMR (300 MHz, CDCl3, 25 ꢁC)
ArH), 8.24 (d, 2H, J ¼ 7.5 Hz, ArHc¼c’), 10.51 (br, s, 1H, ReNH), 12.02
(br, s, 1H, PheNH); 13C NMR (75.47 MHz, CDCl3, 25 ꢁC)
124.3 (2C),
d 7.06e7.78 (m, 11H,
d
126.3 (2C), 126.7 (2C), 127.1, 129.0 (2C), 129.4, 130.2 (2C), 130.6,
133.9 (2C), 134.3, 137.4, 137.8 (AromaticeC), 157.3 (C, CN3), 176.7 (C,
C]O); Anal. Calcd. for C20N3H15OCl2 (384.26): C, 62.51; H, 3.93; N,
10.94; Found: C, 62.32; H, 4.06; N, 11.05.
4.3.6. Bis(N-phenyl-N0-(2,4-dichlorophenyl)-N00-benzoylguanidinato)
copper(II) (6a)
Quantities used were 1.54
g
(4 mmol) N-phenyl-N0-(2,4-
dichlorophenyl)-N00-benzoylguanidine (6) and 0.4 g (2 mmol)
Cu(CH3COO)2.H2O; Yield 80%; blue solid; m.p. 222e224 ꢁC; FTeIR
(KBr, cmꢀ1) 3381, 3056, 1591, 1546, 1519, 1434, 1369, 745, 698, 612,
584, 509, 485, 443, 273; Anal. Calcd. for C40N6H28O2Cl4Cu (830.05):
C, 57.88; H, 3.40; N, 10.12; Cu, 7.66; Found: C, 57.39; H, 3.31; N,
10.17; Cu, 7.35; meff, 1.67 BM.
4.3. Synthesis of complexes (1ae8a)
The solution of Cu(CH3COO)2.H2O (1 mmol) in dry methanol
(20 ml) was added into the solution of an appropriate guanidine
(2 mmol) in methanol (15 ml) at room temperature [25b]. The reac-
tion mixture was stirred for 4 h under an inert atmosphere; then the
precipitates were filtered and washed with methanol (Scheme 1).
These precipitates were dissolved in a CHCl3/n-hexane mixture (1:1)
and kept for slow evaporation to give the crystals of the desired
complexes which were isolated mechanically from the mother liquor.
4.3.7. Bis(N-phenyl-N0-(3,4-dichlorophenyl)-N00-benzoylguanidinato)
copper(II) (7a)
Quantities used were 1.54
g
(4 mmol) N-phenyl-N0-(3,4-
dichlorophenyl)-N00-benzoylguanidine (7) and 0.4 g (2 mmol)
Cu(CH3COO)2.H2O; Yield 76%; blue solid; m.p. 213e214 ꢁC; FT-IR
(KBr, cmꢀ1) 3399, 3056, 1586, 1550, 1516, 1438, 1370, 745, 699,
610, 584, 500, 479, 439, 275; Anal. Calcd. for C40N6H28O2Cl4Cu
(830.05): C, 57.88; H, 3.40; N, 10.12; Cu, 7.66; Found: C, 57.38; H,
3.39; N, 10.24; Cu, 7.56; meff, 1.66 BM.
4.3.1. Bis(N,N0-diphenyl-N00-benzoylguanidinato)copper(II) (1a)
Quantities used were 1.26 g (4 mmol) N,N0-diphenyl-N"-ben-
zoylguanidine (1) and 0.4 g (2 mmol) Cu(CH3COO)2.H2O; Yield
1.11 g, 80%; blue solid; m.p. 226e228 ꢁC (decompose); FTeIR (KBr,
cmꢀ1) 3404, 3059, 1591, 1557, 1516, 1420, 1369, 1217, 1069, 745, 695,
555, 480, 429, 274; Anal. Calcd. for C40N6H32O2Cu (692.27): C,
69.40; H, 4.66; N, 12.15; Cu, 9.18; Found: C, 69.75; H, 4.54; N, 12.48;
Cu, 8.86; meff, 1.64 BM.
4.3.8. Bis(N-phenyl-N0-(3,5-dichlorophenyl)-N00-benzoylguanidinato)
copper(II) (8a)
Quantities used were 1.54
g
(4 mmol) N-phenyl-N0-(3,5-
dichlorophenyl)-N00-benzoylguanidine (8) and 0.4 g (2 mmol)
Cu(CH3COO)2.H2O; Yield 83%; blue solid; m.p. 218e220 ꢁC; FT-IR
(KBr, cmꢀ1) 3391, 3061, 1586, 1554, 1518, 1460, 1437, 1364, 745,
696, 669, 566, 502, 465, 430, 274; Anal. Calcd. for C40N6H28O2Cl4Cu
(830.05): C, 57.88; H, 3.40; N, 10.12; Cu, 7.66; Found: C, 57.55; H,
3.07; N, 9.84; Cu, 7.27; meff, 1.65 BM.
4.3.2. Bis(N-phenyl-N’-(n-butyl)-N00-benzoylguanidinato)copper(II)
(2a)
Quantities used were 1.18 g (4 mmol) N-phenyl-N0-(n-butyl)-
N00-benzoylguanidine (2) and 0.4 g (2 mmol) Cu(CH3COO)2.H2O;
Yield 1.13 g, 87%; blue solid; m.p. 203e205 ꢁC; FTeIR (KBr, cmꢀ1
)
3426, 3055, 2953, 2904, 2867, 1592, 1560, 1518, 1440, 1373, 753,
709, 527, 480, 444, 273; Anal. Calcd. for C36N6H40O2Cu (652.29): C,
66.29; H, 6.18; N, 12.88; Cu, 9.74; Found: C, 65.97; H, 5.99; N, 13.05;
Cu, 9.20; meff, 1.61 BM.
4.4. Data collection and structural refinement of 1, 2a and 4a
Crystals of the guanidine 1 and complexes 2a and 4a were
grown by the slow evaporation method. The solvent systems for
crystal growth were ethanol for the guanidines and petroleum-
ether/chloroform (1:1) for the complexes. The yellowish guanidine
and blue complex crystals were mounted in a random orientation
on a glass fiber and the reflection data were collected on a Rigaku
4.3.3. Bis(N-phenyl-N0-(sec-butyl)-N00-benzoylguanidinato)
copper(II) (3a)
Quantities used were 1.18 g (4 mmol) N-phenyl-N0-(sec-butyl)-
N00-benzoylguanidine (3) and 0.4 g (2 mmol) Cu(CH3COO)2.H2O;
AFC7R Mercury CCD diffractometer with
chromated MoK radiation (
solved by using direct methods Sir 97 [42] and refined with Shelxl
97 [43]. All the non-hydrogen atoms were refined anisotropically
a graphite mono-
¼ 0.71073Å). The structures were
Yield 1.06 g, 81%; blue solid; m.p. 192e194 ꢁC; FTeIR (KBr, cmꢀ1
)
a
l
3420, 3053, 2963, 2930, 2874, 1592, 1561,1524, 1447, 1377, 750, 704,
583, 488, 450, 275; Anal. Calcd. for C36N6H40O2Cu (652.29): C,

34
G. Murtaza et al. / European Journal of Medicinal Chemistry 48 (2012) 26e35
for all the compounds. The H atoms attached to nitrogen in 1, 2a
and 4a were refined isotropically. Other hydrogen atoms were
located at the calculated positions and refined as riding atoms.
Table 1 summarizes the crystal data and refinement description.
(ATCC 15224), S. setubal (ATCC 19196) strains. Cefixime and Roxi-
thromycin were used as standard antibiotics.
An amount of 1 mg of the each compound was dissolved in 1 ml
of DMSO. Nutrient agar medium was prepared by suspending
nutrient agar (Merck) 2 g in 100 ml distilled water; pH 7.0, auto-
claved, cooled and then seeded with 1 ml of prepared inoculum to
have 106 CFU per ml. The Petri dishes were prepared by pouring
75 ml of seeded nutrient agar and allowed to solidify. Eleven wells
per dish were made with sterile cork borer (8 mm), and labeled.
4.5. Biological assays
4.5.1. Brine shrimps lethality assay
All the synthesized ligands and complexes were screened for
their cytotoxicity by the brine shrimps lethality assay method with
a little modification [44]. In short, stock solution of the test
compounds was prepared (10 mg/ml of DMSO), artificial sea water
with salt concentration 34 g/L in distilled H2O was also prepared in
which brine-shrimp (Artemia salina) eggs were hatched at room
temperature, which were then transferred to vials containing sea
water and final concentrations of test compounds (1000, 100 and
Using a micropipette, 100 ml of test solutions were poured into the
respective wells. Finally, the Petri dishes were incubated at 37 ꢁC for
24 h. Triplicate plates were prepared for each sample. The diameter
of the clear zone (showing no bacterial growth) around each well
was measured with the help of vernier caliper. Antibacterial
activities were calculated as a mean of 3 replicates.
10
m
g/ml) were obtained. After 24 h, the number of surviving
4.5.4. Antifungal activity
shrimps was determined and LD50 was calculated by analyzing the
data with the computer program Finny.
The synthesized ligands and complexes were investigated for
their antifungal activity against four fungal strains: M. species
(0300), A. niger (0198), Aspergillus flavus (0064) and F. solani (0291).
A susceptibility test was performed [49] with some modifications
[50]. The samples were prepared in DMSO with concentration of
12 mg/ml. Sabouraud dextrose agar (Merck) was prepared in
distilled water having the concentration of 65 g/L, pH was adjusted
to 5.6, then autoclaved and cooled to 50 ꢁC. A volume of 4 ml of this
4.5.2. In vitro cytotoxicity screening
The test and reference compounds were dissolved to a concen-
tration of 250,000 ng/ml in full medium, by a 20 fold dilution of
a stock solution which contained 1 mg/200
ml. The compounds
were taken into DMSO. In vitro cytotoxicity was estimated by the
microculture sulforhodamine B (SRB) test [45]. The seven human
cancer cell lines A498 (renal cancer), MCF-7 (estrogen receptor
(ER)þ/progesterone receptor (PgR)þ breast cancer), EVSA-T
(estrogen receptor (ER)ꢀ/progesterone receptor (PgR) ꢀ breast
cancer), H226 (lung cancer), IGROV (ovarian cancer), M19 MEL
(melanoma) and WIDR (colon cancer), included in the anticancer
screening panel of the National Cancer Institute, USA, were used in
the present study.
The experiment was started on day 0. On day 0, 10,000 cells per
well were seeded into 96-wells flat bottom microtiter plates (falcon
3072, DB). The plates were incubated overnight at 37 ꢁC, 5% CO2 to
allow the cells to adhere to the bottom. On day 1, a three-fold dilution
sequence of ten steps was made in full medium, starting with the
250,000 ng/mL stock solution. Every dilution was used in quadru-
medium was poured into labeled test tubes and 67 ml of the each
sample was added to the respective test tubes. The tubes were then
allowed to solidify in a slanting position at room temperature. The
tubes were prepared in triplicate for each fungus species. Other
media supplemented with DMSO and reference antifungal drug
(Terbinafine) were used as negative and positive controls, respec-
tively. Each tube was inoculated with a 4 mm diameter piece of
inoculum, removed from a seven days old culture of fungus. The
tubes were incubated at 28 ꢁC for 7 days. Cultures were examined
twice weekly during the incubation. The growth in the medium was
determined by measuring the linear growth (mm) and growth
inhibition was calculated with reference to the negative control,
using the following formula.
Percentage inhibition of fungal growth
plicate by adding 200 ml to a column of four wells. This procedure
resultsina highest concentration of625,000 ng/ml present incolumn
1e2. Column 2 was used for the blank. After incubation of 3 days, the
plates were washed with PBS (phosphate buffered saline) twice. The
Linear growth in testðmmÞ
¼ 100 ꢀ
ꢂ 100
Linear growth in controlðmmÞ
fluorescein diacetate (FDA) stock solutionwas diluted to 2
mg/ml with
PBS and 200 l of this solution was added to each of the control,
m
Supplementary data
experimental and blank wells. The plates were incubated for 30 min
at37 ꢁC and thefluorescence generated from each well was measured
at an excitationwavelength of 485 nm and an emissionwavelength of
535 nm using an automated microplate reader (Labsystems Multis-
kan MS). Data was used for construction of concentration-response
curves and determination of the ID50 value using the Deltasoft 3
software. The variability of the in vitro cytotoxicity test depends on
inter alia the cell lines used and the serum applied. With the same
batch ofcelllines and the same batch of serum theinter-experimental
CV (coefficient of variation) is 1e11% depending on the cell line and
the intra-experimental CV is 2e4%. These values may be higher with
other batches of cell lines and/or serum [46].
Crystallographic data (excluding structure factors) for the
structures reported in this paper have been deposited to the
Cambridge Crystallographic Data Centre as supplementary publi-
cation no. CCDC-815699 for 1, CCDC-815700 for 2a and CCDC-
815698 for 4a. Copies of available materials can be obtained, free of
charge, on application to the director, CCDC, 12 Union Road, Cam-
bridge CB21EZ, UK, (Fax: +44 (0) 1223 336033 or e-mail: deposit@
ccdc.cam.ac.uk).
Acknowledgments
In vitro cytotoxicity data for complexes and reference
compounds are given in Table 7.
The authors are thankful to the Higher Education Commission of
Pakistan for the grant of funds for the research project at Quaid-i-
Azam University, Islamabad. The in vitro cytotoxicity screenings
were carried out by Ms. P. F. Van Cuijk in the Laboratory of Trans-
national Pharmacology, Department of Medical Oncology, Erasmus
Medical Center, Rotterdam, The Netherlands, under the supervision
of Dr. E. A. C. Wiemer and Prof. G. Stoter.
4.5.3. In vitro antibacterial activity
Synthesized ligands and complexes were investigated for their
antibacterial activity by the agar well diffusion method [47,48]
against four strains of bacteria, two Gram positive, S. aureus
(ATCC 6538), M. luteus (ATCC 10240) and two Gram negative, E. coli

G. Murtaza et al. / European Journal of Medicinal Chemistry 48 (2012) 26e35
35
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Supplementary data associated with this article can be found in
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include MOL files and InChiKeys of the most important compounds
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