DNA and BSA binding, anticancer and antimicrobial properties of Co(II), Co(II/III), Cu(II) and Ag(i) complexes of arylhydrazones of barbituric acid

Article abstract of DOI:10.1039/c5ra20157h

Two new cocrystalline compounds, (Hen)(H₂L²)·2/3H₂O (2) and (Him)(H₃L³)·2H₂O (8), were prepared by the reaction of 5-(2-(4-chlorophenyl)hydrazono)pyrimidine-2,4,6(1H,3H,5H)-trione (H_3<

Full text of DOI:10.1039/c5ra20157h

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Mahmudov, M. F. C. Guedes da Silva, F. Marchetti, C. Pettinari, D. Petrelli, L. A. Vitali, L. Quassinti, M.
Bramucci, G. Lupidi and A. J. L. Pombeiro, RSC Adv., 2015, DOI: 10.1039/C5RA20157H.
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Page 1 of 26
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DNA and BSA Binding, Anticancer and Antimicrobial Properties
of Co(II), Co(II/III), Cu(II) and Ag(I) Complexes of Arylhydrazones
of Barbituric Acid
a,b
a,c,
a,
Jessica Palmucci, Kamran T. Mahmudov, * M. Fátima C. Guedes da Silva, *
b
d
e
f
f
Fabio Marchetti, Claudio Pettinari, Dezemona Petrelli, Luca A. Vitali, Luana Quassinti,
d
f,*
a,
Massimo Bramucci, Giulio Lupidi, Armando J. L. Pombeiro *
a
Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco
Pais, 1049–001 Lisbon, Portugal
b
School of Science and Technology, University of Camerino, Chemistry Section, via S. Agostino 1,
6
2032 Camerino, Italy
c
Department of Chemistry, Baku State University, Z. Xalilov Str. 23, Az 1148 Baku, Azerbaijan
School of Pharmacy, University of Camerino, Chemistry Section, via S. Agostino 1, 62032
d
Camerino, Italy
e
School of Biosciences and Veterinary Medicine, University of Camerino, Piazza dei Costanti 4,
6
2032 Camerino, Italy
f
School of Pharmacy, University of Camerino, Biological Section, via Gentile III da Varano, 62032
Camerino, Italy
∗Corresponding authors
Eꢀmail addresses:
kamran_chem@mail.ru, kamran_chem@yahoo.com (Kamran T. Mahmudov)
fatima.guedes@tecnico.ulisboa.pt (M. Fátima C. Guedes da Silva)
giulio.lupidi@unicam.it (Giulio Lupidi)
pombeiro@tecnico.ulisboa.pt (Armando J. L. Pombeiro)
1

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Graphical abstract
Abstract
Two new cocrystaline compounds, (Hen)(H
2
3
2
L )ꢀ2/3H
2
O (2) and (Him)(H
3
L )ꢀ2H
2
O (8), were
2
prepared by reaction of 5ꢁ(2ꢁ(4ꢁchlorophenyl)hydrazono)pyrimidineꢁ2,4,6(1H,3H,5H)ꢁtrione (H
3
L )
and sodium salt of 2ꢁ(2ꢁ(2,4,6ꢁtrioxotetrahydropyrimidinꢁ5(2H)ꢁylidene)hydrazinyl)benzenesulfonic
3
3
acid (H
4 3 2 2 2 2
L ), [Na(H L )(ꢁꢁH O)(H O) ] (1) with protonated ethylenediamine (Hen) and imidazole
(Him), respectively. By using 5ꢁ(2ꢁ(2ꢁhydroxyphenyl)hydrazono)pyrimidineꢁ2,4,6(1H,3H,5H)ꢁ
1
II
II
II/III
I
trione (H L ) and 1, several known Cu , Co , Co
and a new Ag complexes,
4
1
1
3
[
[
Cu(H
2
2
L )(H
2
O)(im)]ꢀ3H
2
O
(3), [Co(H
2
O)
3
6
][Co(H
2
L )
2
]
2
ꢀ8H
2
O
(4), [Co(H
2
L )(im)
3
]
(5),
3
3
Cu(H
L )(im)
2
]ꢀH O (6), [Ag(H
2
2
O)(µꢁH
3
L )]
n
(7) and [Co(H
2
O)
6
][H
3
L ]
2
ꢀ8H
2
O (9), were also
prepared in order to study their DNA and BSA binding, anticancer and antimicrobial properties.
The complexes are able to interact with DNA and BSA with high binding constant values. In
particular, complex 4 strongly intercalates DNA and binds BSA. The antimicrobial activity of all
compounds, determined against a panel of reference bacterial and fungal strains, indicates that only
2
and 7 possess antimicrobial activity. The same compounds 2 and 7 show a pronounced
antiproliferative activity against the human tumor cell lines A375, MDAꢁMB 231 and HCT116.
+
2+
2+
Keywords: Arylhydrazones of barbituric acid; drug cocrystal design; Ag , Cu or Co complexes;
DNA binding; bovine serum albumin binding; antimicrobial activity; antitumor activity.
Introduction
Although barbituric acid is pharmacologically inactive, its derivatives possess very interesting
pharmaceutical properties and can lead to drugs that act as central nervous system depressants, as
sedative (in small doses)/hypnotic (in larger doses) pharmaceuticals, anticonvulsants and
1
−5
anesthetics. Currently, therapeutic applications of barbiturates are expanding in connection with
2
−5
their antitumor, antiꢁinvasive and antiꢁangiogenic effects. The pharmaceutical properties of
barbiturates mainly depend on the side groups attached to the C5 atom of the pyrimidine ring
1
−5
(
Scheme 1a). Several methods for the modification/functionalization of barbituric acid have been
6−11
reported,
and one of the most common and widely used concerns the JappꢁKlingemann reaction,
2

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in which barbituric acid reacts with diazonium salts leading to arylhydrazones of barbituric acid
6
,8
(
AHBAs) (Scheme 1b) in basic medium. However, to our knowledge, pharmaceutical properties
of AHBAs have not yet been studied, what is explored in this work, including their cocrystals and
metal complexes.
Scheme 1. Molecular structure of barbituric acid (a) and AHBAs (b).
Poor aqueous solubility is one of the most serious formulation challenges facing the
pharmaceutical industry today. Approximately 40% of the drugs discovered through combinatorial
12−14
chemistry and high throughput screening have an aqueous solubility of less than 10 ꢂM.
Limited aqueous solubility of drug molecules can lead to poor bioavailability and constitutes a
15
significant risk factor in low oral absorption. A number of strategies have been employed for
increasing the dissolution rate of poorly aqueous soluble pharmaceuticals, specifically in solid
dosage formulations, the most applied technique for dissolution enhancement being cocrystal/salt
16,17
formation.
As far as we know, before the current work, cocrystals of AHBAs had not yet been
isolated and structurally characterized, and their antimicrobial and antitumor activity and
DNA/protein binding studies had not been performed.
Thus, in this work we focused on the following aims: i) synthesis of new AHBA ligands and
I
their cocrystals with ethylenediamine (en) and imidazole (im); ii) preparation of new Ag and
II
II/III
II
18
known water soluble Co , Co
and Cu ꢁAHBA complexes (Scheme 2); iii) evaluation of the
II
II/III
antimicrobial and antiproliferative activities all AHBA ligands and their cocrystals and Co , Co
,
II
I
Cu and Ag complexes; iv) investigation of the binding mode of AHBAs and their cocrystals and
complexes to DNA and BSA albumin, that should play a role in the pharmacokinetics and
pharmacodynamics of such potential drugs.
Results and Discussion
II/III
II
I
Synthesis and characterization of AHBA ligands their cocrystals and Co , Cu and Ag complexes
1
Water soluble 5ꢁ(2ꢁ(2ꢁhydroxyphenyl)hydrazono)pyrimidineꢁ2,4,6(1H,3H,5H)ꢁtrione (H
4
L ),
sodium salt of 2ꢁ(2ꢁ(2,4,6ꢁtrioxotetrahydropyrimidinꢁ5(2H)ꢁylidene)hydrazinyl)benzeneꢁsulfonic
3
3
1
acid
Co(H
(H
4
L ),
[Na(H
3
L )(ꢁꢁH
2
O)(H
2
O)
2
]
2
(1),
[Cu(H
2
L )(H
2
O)(im)]ꢀ3H
2
O
(3),
1
3
3
[
2
O) ][Co(H
6
2
L )
2
]
2
ꢀ8H
2
O
(4), [Co(H
2
L )(im)
3
]
(5), [Cu(H
2
L )(im)
2
]ꢀH
2
O
(6) and
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3
18
[
Co(H
2
6
O) ][H
3
L ]
2
ꢀ8H
2
O (9) (Scheme 2) were reported earlier by us, and hence will not be
discussed.
HO
.
..
O
H
N
N
.
..
O
H
N
HN
O
Cl
HN
HN
N
O
HN
O
O
H3L2
H4L1
H
O
N
O
NH
O
O
N
N
HN
OH2
Co
2+
OH2
Co
N
NH2
H
N
O
H O
OH2
OH2
N
2
O
N
O
H
O
2
8H O
.
..
Cu
O
C
O
H
H2O
3H2O
O
C
C
N
Cl
2/3H2O
HN
HN
N
OH2
HN
HN
O
N
NH
O
C
N
HN
O
O
2
(4)
(
2)
(3)
[Cu(H2L )(imH)(H2O)] 3H2O
[Co(H O) ][Co(H L ) ] 8H O
1
.
2
6
2
2 2
2
2
.
1
.
[
NH NH ][H L ] 2/3H O
2
3
2
2
O
S
H
O
N
O
O
C
C
2
+
O
NH
OH2
O
C
N
H
C
N
O
H2O
OH2
OH2
O
N
2
8H O
S
O
Co
O
O
OH2 H2
O
OH2
O
N
S
H
2
H O
N
N
N
O
O
O
Na
OH2
HN
HN
O
Na
H2O
O
NH
O
Co
N
O
H
N
NH
S
H2O
H2
N
N
2
H
O
O
O
N
O
C
(
9)
C
N
HN
N
H
3
.
[
Co(H O) ][H L ] 8H O
H
C
2
6
3
2
2
C
O
N
(
5)
O
H
3
(1)
[
Co(H L )(imH) ]
2
3
3
[
Na(H L )(H O) ]
3
2
3 2
O
S
O
O
O
+
C
C
NH
O
HN
S
O
N
N
C
O
C
O
2
H
NH
O
N
N
N
O
N
NH
O
2H2O
HN
O
H2O
H
O
Cu
N
O
N
NH
O
S
OH2
HN
O
H
N
Ag
O
NH
O
N
H O
1
O
2
H
Ag
O
(
8)
O
N
(
6)
2
S
O
3 .
[
imH][H L ] 2H O
3 2
3
H
N
[
Cu(H L )(imH) ] H O
2
2
2
HN
C
C
C
1
O
C
n
HN
O
(
7)
3
[
Ag(H L )]
3
n
1
2
Scheme 2. Schematic representations of H
4
L , H
3
L and 1−9.
2
5
ꢁ(2ꢁ(4ꢁChlorophenyl)hydrazono)pyrimidineꢁ2,4,6(1H,3H,5H)ꢁtrione (H
3
L ) (Scheme 2) was
18−22
prepared by a modified known
aqueous diazotization of 4ꢁchloroaniline and subsequent
2
coupling with barbituric acid in basic medium. H
3
L is highly soluble in DMSO, DMF, water,
1
13
2
methanol, acetone and acetonitrile. The H and CꢁNMR spectra of H
3
L support the hydrazone
1
13
form and =N−NH− signal is observed at δ 14.03 in the HꢁNMR spectrum, while the CꢁNMR
4

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spectrum shows two separate singlets for the C=O and C=OꢀꢀꢀH of pyrimidine moiety at δ 159.76
2
and 161.92, respectively (Figures 1S and 2S). The IR spectrum of H
3
L shows ν(NH) vibrations at
–
1
3
1
260, 3085 and 2813 cm , while ν(C=O) at 1753 and 1653, ν(C=OꢀꢀꢀH) at 1588 and ν(C=N) at
–1
513 cm (Figure 3S), supporting the existence of the Hꢁbonded hydrazone structure in the solid
state.
Cocrystals of (Hen)(H L )ꢀ2/3H O (2) were obtained from reaction of H L with ethylenediamine
2
2
2
2
3
−1
(
en) in methanol (Schemes 2 and 1S). The IR spectrum of 2 displays ν(C=O) at 1693 cm , value
−1
1
that is significantly shifted in relation to that (1753 cm ) of the free ligand (Figure 4S). The Hꢁ
6
NMR spectrum of 2 in DMSOꢁd solution at room temperature confirms the disappearance of the
hydrazone proton (Figures 5S and 6S). The ESI mass spectrum of 2 dissolved in methanol shows
+
the parent peak at m/z = 327.2 [C12
H
15ClN
6
O
3
+H] . Elemental analyses are consistent with the
proposed formulation, which is also supported by Xꢁray crystallography (see below).
3
Another cocrystalinde compound, [(Him)(H
3
L )]ꢀ2H
2
O (8), was isolated by treatment of 1
I
with imidazole (im) in the presence of HNO
3
in methanol (Scheme 2). Whereas, the first Ag
3
complex with an AHBA ligand, [Ag(H O)(µꢁH L )] (7), is obtained in acidic medium (with
2
3
n
addition of HNO
3
) from reaction of 1 with AgNO
3
in acetoneꢁwater mixture (4:1, v/v) at room
1
temperature (Scheme 2). Both compounds, 7 and 8, were characterized by elemental analysis, H
13
and C NMR spectroscopies (Figures 7Sꢁ12S), ESIꢁMS, IR spectroscopy and single crystal Xꢁray
–
1
diffraction. In the IR spectra of 7 and 8, the ν(C=O) signal appears at 1689 and 1698 cm (Figures
1
3S and 14S), respectively, values that are significantly shifted in relation to the corresponding
–1
signal of 1 (1677 cm , Figure 15S). The ESI mass spectra of 7 and 8 dissolved in CH
3
OH show the
3
+
3
+
1
peaks at m/z = 839.1 [2{[Ag(H
2
O)(µꢁH
3
L )]
n
}−2H
2
O+H] and 312.1 [H
3
L +H] . In the H NMR
spectra of 7 and 8, the hydrazone protons are observed at 14.78 and 14.79 ppm, respectively
Figures 7S and 9S). Elemental analyses are consistent with the proposed formulations which are
(
also proved by Xꢁray analysis (see below). Both compounds are soluble in polar solvents such as
water, methanol, DMF and DMSO.
Xꢀray diffraction analyses
Crystals of 2 and 8 suitable for Xꢁray diffraction analysis were obtained upon crystallization from
methanol or waterꢁacetone mixtures (1:4, v/v) (7). Representative plots of 2, 7 and 8 are depicted in
Figure 1. Crystallographic data and refinement parameters are given in Table 1S, selected bond
distances and angles in Table 2S. Hꢁcontacts for 2, 7 and 8 are outlined in Table 3S and depicted in
Figures 16Sꢁ19S).
2 3
In 2 and 8 the (H L ) or (H
L3)− species cocrystalized with monoprotonated ethylene
2
−
diamine (2) or imidazolium (8) cations, respectively, apart from two water molecules, and can be
5

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roughly planar (in 8) or present a considerable twisting (in 2) as measured by the angles between
the least square plane of the heteronuclear ring (plane H) and the least square plane of the phenyl
ring (plane P; Table 3S).
Compound 2 crystallizes in the monoclinic space group P21/c with three monoprotonated
2
–
ethylene diamines, three (H
2
L ) anions and two water molecules. Due to deprotonation of
2
7
hydrazone unit of H L by en, its "resonance assisted hydrogen bond" (RAHB) system is converted
3
7
into "charge assisted hydrogen bond" (CAHB) (Scheme 1S), with mediumꢁtoꢁweak intermolecular
2
–
hydrogenꢁbond contacts involving the (H
2
L ) monoanion varying in the (donorꢀꢀꢀacceptor) range
distances of 2.785(8)−3.296(8) Å (Table 3S). Together with ketoꢁO atoms, every hydrazo/azo group
2
−
+
of (H
2
L ) in 2 acts as acceptor from two Hen or from one of these cations and a molecule of
water, to form two (or even three) bifurcated hydrogen bonds (Figure 16S and Table 3S). The
2
−
nitrogen and oxygen atoms of pyrimidine trione moieties of (H L ) are further linked via
2
intermolecular Hꢁcontacts giving rise to an intrincate 3D network (Figure 17S).
I
3 –
The assymetric unit of 7 contains one Ag cation, a (H
3
L ) anion and a coordinated water
molecule. The structure is a 1D polymer that runs along the crystallographic b direction with the
23
5
metal cation adopting a distorted square pyramidal geometry (τ = 0.35) filled by the oxygen atom
from water, two Osulfonylꢁatoms and two Oketoneꢁatoms from three adjacent symmetry generated
hydrazone moieties (Figure 18S). An interesting feacture of polymer 7 arises from the ꢀꢁOOSO
bridging mode of the sulfonyl groups of the organic ligands which results in the generation of
{
2 2 4
Ag S O } dimetallic cores (Figure 19S). These cores are then doubly bound by two Oketoneꢁatoms
from the pyrimidine trione moieties thus giving rise to an infinite 1D double chain. Therefore, this
double chain comprises additional cavities formed by repeating 12ꢁmembered dimetallic
~{AgOketoneCNCOketone
2
} ~, and 10ꢁmembered monometallic ~{AgOsulfonylSC
2 2 2
N C Oketone}~ rings.
3
−
In 7 the (H
3
L ) anion preserves the strong RAHB linking the NHꢁmoiety of the hydrazone unit
(N1ꢁH1N) in a threeꢁcentered interaction with coordinated Oketoneꢁ and Osulnonylꢁatoms. The nonꢁ
coordinated Osulfonylꢁatom (O12) is involved in an intraꢁchain Hꢁbond contact acting as acceptor
towards HN(3) (donor site) of pyrimidine moiety.
7
The structure of 8 contains threeꢁcentred RAHB/CAHB interactions in the anionic
hydrazone units involving the Oketoneꢁ and Osulnonylꢁatoms, which relates to the synergistic mutual
reinforcement of intramolecular hydrogen bonding due to πꢁelectron delocalization. The typical
double bond lengths of Cpyrimidine−Nhydrazone in the structure of 8 [1.326(4) Å] and the formation of
intramolecular N−HꢀꢀꢀO RAHB contacts indicate that hydrazone is the preferred tautomeric form in
these cases. The structure of 8 contains trapped water molecules, which interact among themselves
and with the imidazolium and pyrimidine trione moiety via intermolecular hydrogen bonds. Such
6

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interactions, together with others involving the Oꢁ and Nꢁatoms of pyrimidine trione rings (Table 3S
and Figure 20S), give rise to intrincate 3D supramolecular frameworks.
2
7
8
Figure 1. Xꢁray molecular structures of 2, 7 and 8 with atom numbering schemes. In 2, only one
hydrazine anion and one ethylenediamonium cation is shown.
Electronic absorption spectra
1
The electronic absorption spectra of the complexes 1−9 and the free proligands H
4
L and
2
1
2
3 4 3
H L have been recorded in DMSO (2−9, H L and H L ) and water solution (1) and all the
spectra are reported in Figure 21S (Supplementary material). The wavelength (λmax) and molar
absorption values (ɛ) of all free ligands, their metal complexes and cocrystals in the UVꢁVis region
1
2
2
20−600 nm are reported in Table 4S (Supplementary material). In the spectra of H L and H L
4 3
the bands at 262−266 nm are due to π–π* electronic transition in C=O, while the bands at 388−414
2
4−26
nm are mainly of n−π* type in N=N.
Similarly the sodium complex 1 shows analogous features,
even if the band at the lower wavelength seems splitted into two very close absorptions.
1
With respect to H
4
L , in the spectra of its complexes 3 and 4, containing Cu(II) and
Co(II/III) respectively, a new broad and very intense absorption band appears in the 420−480 nm
range, which suggests n−π* and π−π* electronic transitions in the obtained compounds. Thus, new
band can be attributed to a deprotonated form of the ligand moiety arising upon coordination to a
7

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metal centre and can be assigned to electronic transitions to bring about the conjugation between the
metallocycles and the aromatic ring system.
The spectra of metal the complexes 5−9 are essentially similar to that of the ligand in the sodium
salt 1, apart for the Co(II) and Cu(II) complexes 5 and 6, respectively, where a broadening of the
band at lower wavelength is observed, due to an additional band partially obscured by the formed
II
II
one. No dꢁd or CT transitions were observed in any of the Co and Cu complexes and this may be
ꢁ4
due to the weakness of the bands at such low concentrations of the solutions (ca. 10 M) or because
they are obscured by the intense bands.
DNA-Binding Mode and Affinity
The mechanisms of DNAꢁbinding and behaviour of metal complexes, as novel potential
therapeutic agents, are strongly attributed to the size, shape, and planarity of the intercalative
ligands. Metal complexes can bind to DNA via both covalent and/or nonꢁcovalent interactions. In
covalent binding the labile ligand of the complexes is replaced by a nitrogen atom from a DNA base
such as guanine N7. Nonꢁcovalent DNA interactions include intercalative, electrostatic and groove
(surface) binding. Intercalation can involve the partial insertion of aromatic heterocyclic rings of the
27
ligands between the DNA base pairs. When metal complexes intercalate to the base pair of DNA,
the π orbital of the intercalated ligands in the complexes can couple with π orbitals of the base
pairs, thus decreasing the π−π transition energies. On the other hand, the coupling π orbital can be
∗
∗
∗
28
partially filled by electrons, thus decreasing the transition probabilities. Thus, some basic
interaction studies of metal(or ligands)ꢁcomplex−DNA are of relevance and we have used different
1
methods to evaluate the DNA binding affinity of the complexes 1−9 and the proligands H
4
L and
2
H
3
L explored in this work.
Electronic absorption titration
Electronic absorption spectroscopy is universally employed to examine the binding mode of
DNA with small molecules. The interaction of such molecules with DNA is often characterized
through absorption spectral titration, followed by the changes in the absorbance and shift in the
wavelength. The absorption spectra of complex 4 in the absence and presence of calf thymus DNA
(ctꢁDNA), is given in Figure 2 while the results obtained for the titration of all complexes and
proligands are reported in Figure 22S (Supplementary material).
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Figure 2. Absorption spectral traces of complex 4 buffer (10mM, pH 7.2) after gradual addition of
calf thymus DNA (from a = 0 to h = 24 ꢀM). Inset shows the plot of [DNA]/|εa ꢀ εf| vs. [DNA]
(equation 1 in supplementary material).
As reported in Figure 2, fixed amounts of complex 4 were titrated with increasing amount of ctꢁ
DNA (0−26 ꢀM), resulting in an overall hyperchromism at the intraligand absorption bands in the
2
40−280 nm range of the complex. In general, the "hyperchromic effect" is related to corresponding
29
changes of DNA on its conformation and structure, after coordination with metal complexes.
Hypochromism results from contraction of DNA in the helix axis, while hyperchromism arises from
3
0,31
the damage of DNA double helix structure.
the tested compounds, while a moderate red shift of 1−4 nm was observed only for complexes 5 and
. The hyperchromic effect with red shift seems to suggest that complexes 5 and 7 bind to DNA by
external contact possibly via electrostatic binding, as previously observed for other metal
An "hyperchromic effect" has been observed for all
7
32
complexes.
For complexes 1, 3, 4, 6, 8 and 9, or proligands H
1
2
4
3
L and H L , no shift of the absorption
maximum was observed and this behaviour can be probably related to a different DNA binding
propensity. To further evaluate the binding affinity of complexes and cocrystals with ctꢁDNA, the
intrinsic binding constant has also been determined by using equation (1) (supplementary material),
detecting the changes of absorbance for of different concentrations of ctꢁDNA. The values of the
b
binding constant K , obtained from for compounds 1−9 are reported in Table 1 and can be resumed
1
1
according to the following trend: 4 >> 2 > 7 > 3 ≈ H
4
L ≈ 8 ≈ 5 ≈ 9 > 6 ≈ H
3
L ≈ 1.
Competitive studies with EB
The measurement of the ability of a complex to affect the ethidium bromide (EB) fluorescence
intensity in the EBꢁctꢁDNA adducts can also be used to determine of the affinity of the complex for
9

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33
DNA. EB is a sensitive fluorescence probe, emitting intense fluorescence at about 600 nm in the
presence of DNA, due to its strong intercalation between the adjacent DNA base pairs, and the
enhanced fluorescence could be quenched by the addition of another competitive binding molecule.
If a molecule can replace EB from the EBꢁDNA complex, a decrease of fluorescence of the solution
is observed due to the fact that free EB molecules are readily quenched by the surrounding water
molecules. Two mechanisms have been proposed to account for the reduction in the emission
34,35
intensity: the replacement of molecular fluorophores (EB in this case) and/or electron transfer.
Figure 3. Emission spectra of EB bound to ctꢁDNA in the presence of complex 4 with increasing
concentration (from a = 0 to I = 39ꢀM; ([EB] = 6 ꢀM, [ctꢁDNA] = 50 ꢀM). Inset: Stern Volmer plot
of F /F vs [complex 4] for the titration of EBꢁctꢁDNA system with complex 4.
0
Figure 3 reports the emission spectra of the EBꢁctꢁDNA in the presence of increasing amounts of
complex 4. The addition of 4 (different concentrations) to the EBꢁbound ctꢁDNA solution caused a
reduction in emission intensitiy, indicating that complex 4 substitutes ctꢁDNAꢁbound EB and binds
to ctꢁDNA with different affinity. In order to understand quantitatively the magnitude of the binding
1
2
strength of complexes 1−9 and proligands H
been then analyzed by the Stern−Volmer equation and KSV values are shown in Table 1. From data
on Table 1 a low correlation between K and KSV values was observed, probably due to the
4 3
L and H L with ctꢁDNA, the quenching ability has
b
different sensitivity of the two techniques used. Additionally we have evaluated the binding
1
constant Kapp (Table 1), and the values obtained for compounds 3, 4, 5, 9 and the proligand H L
4
suggest that they intercalated strongly to DNA. A similar mechanism is hypothesized for the other
compounds and the free proligand that bind to DNA with lower affinity.
1
0

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Table 1. Values of binding constants with DNA and BSA for the interaction of complexes 1−9 and
1
2
proligands H
4
L and H
3
L .
EBꢀctꢀ
DNA
Binding
ctꢀDNA
Binding
EBꢀctꢀ
DNA
Binding
DAPIꢀctꢀ
DNA
binding
Compound
BSA
Binding
BSA
Binding
K
sv
K
q
K
b
K
sv
K
app
K
sv
4
ꢀ1
13
ꢁ1 ꢁ1
3
ꢀ1
3
ꢀ1
5
ꢀ1
3
ꢀ1
1
0 M
10 M s
10 M
10 M
10 M
10 M
8
.80(±0.36)
0.88(±0.03)
0.75(±0.24)
13.6(±0.27)
1.76(±0.34)
54.9(±0.10)
1.14(±0.36)
0.85(±0.21)
3.33(±0.60)
1.17(±0.44)
1.12(±0.35)
0.78(±0.21)
1.36(±0.40)
ND
ND
18.65(±0.20)
13.61(±0.78)
9.45(±0.36)
70.05(±2.51)
13.47(±0.10)
13.38(±0.21)
8.55(±1.20)
28.60 (±0.06)
21.90 (±0.60)
1.39(±0.21)
27.33(±0.52)
1
2
3
4
5
6
7
8
1
1
9
2
2
1
2
1
6.42(±0,40)
0.48(±0.22)
6.82(±0.40)
6.31(±0.12)
9.64(±0.85)
7.28(±0.80)
5.55(±0.30)
6.50(±0.90)
1.64(±0.04)
1.04(±0.02)
9.68(±0.04)
2.63(±0.02)
2.96(±0.08)
1.72(±0.08)
2.55(±0.03)
1.65(±0.09)
1.82(±0.17)
1.64(±0.11)
4.95(±0.24)
11.73(±0,12)
28.54(±0.05)
21.66(±0,10)
5.42(±0.30)
0.53(±0,02)
1.56(±0.25)
21.84(±0.07)
0.88(±0.06)
8.93(±0.61)
2.47
5.86
14.27
10.83
2.71
0.26
0.78
1.09
0.44
4.46
9
2
18.24 (±1.76)
H
H
3
4
L
L
1
16.46(±1.16)
Figure 23S (Supplementary material) reports the emission quenching spectra of EBꢁctꢁDNA
solutions in the presence of all complexes and proligands. Data from Table 1 suggest a stronger
1
interaction of complexes 3, 4, 5 and 9 with EBꢁctꢁDNA (4 > 5 ≈ 9 > 3 > H
4
L ), than the remaining
compounds, showing a lower affinity for the EBꢁctꢁDNA complex in the following order: 6 ≈ 2 > 8
2
5
ꢁ1
>
3
H L > 7. Moreover, the high Kapp values obtained (about 10 M ) suggest that our compounds
intercalate strongly to DNA with similar mechanism, i.e. through the replacement of EB from DNA,
but with different affinity.
Competitive studies with DAPI
36
DAPI is a very well known fluorogenic probe that binds to the minor groove of DNA.
However, in the presence of another molecular species able to bind to the DNA minor groove, there
would be competition between this species and DAPI, and displacement of DAPI from ctꢁDNA
produces a decrease in the fluorescence intensity of the DAPIꢁctꢁDNA complex.
1
1

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Figure 4. Emission fluorescence spectra of DAPIꢁctꢁDNA complex upon excitation at 338 nm in
the presence of 4 with increasing concentration (from a = 0 to I = 36 ꢂM). Experiments were
0
performed in 10 mM phosphate buffer, pH 7.2. Inset: Stern Volmer plot of F /F vs [complex 4] for
the titration of DAPIꢁctꢁDNA complex with complex 4.
As shown in Figure 4, the addition of 4, with increasing concentration, to the DAPIꢁctꢁDNA
complex reduces its fluorescence intensity, thereby clearly suggesting that complex 4 can display
DAPI to a considerable extent and can be able to bind to the minorgroove of DNA. Results for all
complexes and proligands are reported in Figure 24S (Supplementary material). Of particular
interest is the behaviour of complex 7 and the cocrystaline 2: in fact they do not decrease the
fluorescence of DAPIꢁctꢁDNA but produce an hyperchromic effect with an increase of fluorescence
and a blue shift of the emission maximum. Compounds 2 and 7 interact with the system DAPIꢁctꢁ
DNA with different affinity, but the presence of isosbestic points indicates an equilibrium state
between the complexes and DAPIꢁctꢁDNA (Figure 24S). The significant hyperchromic effects, as
well as the moderate blue shift of the emission maximum, support the existence of a strong
interaction between compounds 2 and 7 and the whole system DAPIꢁctꢁDNA, without expulsion of
DAPI molecules from the minor grove of DNA. A possible interaction between the metal
compounds and the DAPIꢁctꢁDNA system can be through external electrostatic interaction between
the metal compounds and the DAPIꢁctꢁDNA system. A similar situation has been recently found in
the case of waterꢁsoluble tris(pyrazolyl) methanesulfonate silver(I) derivatives of N‑ methylꢁ1,3,5ꢁ
37
triazaꢁ7ꢁhosphaadamantane salt.
The SternꢁVolmer quenching constants for the interaction of complexes and proligands with
DAPIꢁctꢁDNA were evaluated and reported in Table 1. The values of K show also in this case that
sv
complex 4 has the highest affinity towards DNA. The affinity of the tested compounds follow the
1
order 4 >> 8 ≈ H L > 9 > 1 while a lower activity was observed for the remaining species (2 ≈ 5 ≈
4
2
6
> 3 ≈ 7 ≈ 9 > H
3
L ). It’s worth to note that, after 4, the coꢁcrystal 8 seems very able to display
1
2

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efficiently DAPI from the minor grove of DNA helix, in accordance with previous findings on
3
8
several imidazolium salts.
From all data of Table 1 we can conclude that complexes 3, 4, 5, 6, 7 and 8 display a very high
affinity for DNA, but probably with different types of association, based on the value of their
association constants. Interestingly, the Co(II) complex 4 was that with the highest activity which
preferentially seems to bind to minor groove of DNA.
The highest affinity of the ionic complex 4 for both EBꢁctꢁDNA and DAPIꢁctꢁDNA, with respect
2+
2 6
to all other compounds, is likely related to its bulkiness, being composed by a cationic [Co(OH ) ]
1
ꢁ
unit and two anionic [Co(H L ) ] counterparts. Such very big system displays the highest ability,
2
2
among the others, to replace EB from the major grove of DNA and also DAPI from the minor grove
of DNA. Moreover, also the binding constant K of 4 is the highest among the others, and ca. 50
times higher than that exhibited by compound 9, containing the same cationic unit [Co(OH ) ] ,
b
2+
2
6
3
ꢁ
but only an anionic (H
3
L ) ligand. This means that there is a contribution of the anionic
] unit of compound 4 in its ability to bind DNA and to replace EB and DAPI from
major and minor groves, respectively. Such contribution can be tentatively explained if we consider
1
ꢁ
[
2 2
Co(H L )
1
+
the possibility of ionic pairs formation such as {[Co(OH
2
)
6
][Co(H
L )} for compound 9, which may behave as single positive systems able to interact
with the negative backbone of phosphate groups in DNA through electrostatic interactions and then
2 2
L ) ]} for compound 4 and
3
+
{
2 6 3
[Co(OH ) ](H
3
ꢁ
1
ꢁ
3 2 2
intercalation. Differently to the simple anion (H L ) in compound 9, the anionic unit [Co(H L ) ]
6
in compound 4 contains a d low spin Co(III) center, which is able to strongly attract and
concentrate the whole negative change on it, by depleting the electronic cloud from the periphery of
the complex. Additionally, DNA double helix possesses many hydrogenꢁbonding sites positioned
1
ꢁ
on the edges of the DNA bases, so that it’s quite possible that the (H
2
L ) functionalities could
39
participate in Hꢁbonding with the DNA base pairs and intercalate the DNA helix, even if the
1
ꢁ
2
(H L ) ligands are part of an anionic complex.
Protein Binding Studies
The interactions between the most abundant blood proteins, as serum albumins, and metal
40
complexes have attracted immense interest because of their possible role in plasma delivery. Their
binding with a drug may actually result in an increase or decrease of the drug's efficacy, making it
important to investigate the interactions of prospective drugs with serum albumins. Simple and
effective methods for investigating the strength and mode of binding of small molecules to serum
4
1
albumin are available. BSA (bovine serum albumin) has a wellꢁknown structure consisting of a
single polypeptide chain containing two tryptophan residues, that are responsible for the majority of
the intrinsic fluorescence of the protein and shows a strong fluorescence emission with a peak near
42
3
40 nm upon excitation at 285 nm. This emission is sensitive to the changes in the local
1
3

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43
environment of the tryptophan, and can be attenuated by the binding of a small molecule at or near
4
4
this residue. The binding of complex 4 to BSA is reported in Figure 5: the addition of increasing
amounts of 4 decreases dramatically the intrinsic fluorescence of the protein. A similar behaviour is
observed for the other complexes and proligands with BSA (Figure 25S in Supplementary material).
The addition of the complexes to BSA results in a large reduction in the fluorescence intensity
without any shift of the emission maximum.
−6
Figure 5. Emission spectra of BSA (15 × 10 M) in 20 mM phosphate buffer (pH = 7.0) and 25 °C.
Excitation carried out at 285 nm. The arrow (with its direction) shows that the increasing
concentration of complex 4 (from a = 0 to m = 30 ꢀM) is accompanied by a quenching of BSA
fluorescence intensity. Inset, SternꢁVolmer plots of F /F vs [complex 4] for the titration of BSA
0
with complex 4.
The SternꢁVolmer constants of binding to BSA for all complexes and proligands are reported in
Table 1. Complex 4 is also in this case the most active complex towards the binding to BSA, with a
SternꢁVolmer constant about 3 times higher than those of the other complexes.
Also in this case there is a different ability in binding BSA by the ionic compounds 4 and 9
2+
1
ꢁ
containing the same cationic unit [Co(OH
2
)
6
] but two different anionic counterparts, [Co(H
2 2
L ) ]
3
ꢁ
and [(H L )] . So that, even if we can imagine that the primary interation with BSA protein chain
2
2
+
2 6
could be with the cationic unit [Co(OH ) ] , possibly through replacement of some water molecule
7
from a labile d Co(II) centre, we cannot esclude some steric contribution from the anionic
1
ꢁ
[
Co(H L ) ] unit. Beside this, compounds 5, 6 and 8 show essentially the same activity towards the
2 2
BSA (Table 1), likely due to the hydrophobicity of substituents in the ligand and not to the type of
metal in the complex. Whereas, compounds 2, 7 and 9 bind to BSA with moderate affinity, similar
1
2
4 3
to that of proligands H L and H L .
It is well known that there are two quenching mechanisms involved in quenching process, which
are usually classified as dynamic quenching and static quenching. Dynamic quenching refers to a
1
4

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process where the fluorophore and the quencher come into contact during the lifetime of the excited
4
5
state, whereas static quenching refers to fluorophore–quencher complex formation. According to
the literature, for dynamic quenching the maximum scatter collision quenching constant of various
10
ꢁ1 ꢁ1 46
quenchers with the biopolymer is 2.0 x10 M s
and the fluorescence lifetime of the biopolymer
ꢁ8
47
is 10 s.
Table 1 shows the values of KSV and k
q
0 q
= KSV/τ . The obtained values of k were larger than the
10
ꢁ1 ꢁ1
limiting diffusion rate constant of the biomolecule (2.0 x 10 M s ), which indicates that the
fluorescence quenching is caused by a specific interaction between BSA and the different
compounds tested. Therefore, the quenching mechanism mainly arises from the formation of BSA–
complex rather than dynamic quenching. In conclusion, the static quenching is dominant in these
48
system.
1
2
Biological Activity of proligands H L , H L , cocrystals 2, 8 and metal complexes
4
3
1
, 3−7, 9
Antiproliferative activity
The cytotoxic activity of compounds 1
−
9, silver nitrate, and cisplatin was evaluated on a
selection of human tumor cell lines. A375 human malignant melanoma, MDAꢁMB 231 human
breast adenocarcinoma, and HCT116 human colon carcinoma cell lines were tested by the MTT
assay. All cell lines were submitted to increasing concentrations of compounds, in the 0.39
−100 ꢂM
range, for 72 h. The IC50 values are shown in Table 2 and compared with AgNO . Compound 7
3
presents pronounced antiproliferative effects with IC50 values of 0.97, 1.14 and 1.78 µM, for the
A375, HCT116, and MDAꢁMB 231 cell lines, respectively. IC50 values of the silver complex 7
3
resulted statistically significant in comparison with IC50 values of AgNO and cisplatin. The
cocrystalline 2 also shows antiproliferative activity on the tested cell lines, with IC50 values ranging
from 17.3 µM to 24.8 µM for HCT116 and MDAꢁMB 231cell lines, respectively. All the other
1
compounds are inactive. H
4
L shows low activity on A375 cell line.
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1
2
Table 2. In vitro cytotoxic activity of proligands H
4
L , H
3
L , cocrystals 2, 8 and complexes 1, 3−7,
9
.
Cell line (IC µM)
50
a
b
c
A375
MDAꢀMB 231
HCT116
1
H
9
4
L
79.3
68.6 ꢁ 82.4
> 100
> 100
> 100
> 100
> 100
5% C.I.
2
H
3
L
> 100
9
1
9
2
9
3
9
4
9
5
9
6
9
7
9
8
9
9
9
5% C.I.
5% C.I.
5% C.I.
5% C.I.
5% C.I.
5% C.I.
5% C.I.
5% C.I.
5% C.I.
5% C.I.
> 100
> 100
17.43
16.60 ꢁ18.31
> 100
24.81
23.10 ꢁ26.66
> 100
17.28
16.74 ꢁ17.83
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
0.97*°
0.93 ꢁ1.00
> 100
1.78*§
1.65 ꢁ1.91
> 100
1.14*§
1.04 ꢁ1.17
> 100
> 100
> 100
> 100
AgNO
5% C.I.
Cisplatin
5% C.I.
3
2.73
2.37 ꢁ2.92
0.59
11.20
9.90 ꢁ 12.67
7.12
6.01
5.66 ꢁ 6.38
4.92
9
9
0.51 ꢁ 0.71
5.99 ꢁ 7.62
4.51 ꢁ 5.50
The IC50 value is the concentration of compound that affords a 50% reduction in cell growth (after 72 h of
incubation).
a
b
C.I. = confidence interval. Human malignant melanoma cell line. Human breast adenocarcinoma cell line.
c
Human colon carcinoma cell line.
*
§
3
The twoꢁtailed P value is < 0.0001, considered extremely significant (compound 7 vs AgNO ).
The twoꢁtailed P value is < 0.0001, considered extremely significant (compound 7 vs cisplatin).
°
The twoꢁtailed P value is 0.0002, considered extremely significant (compound 7 vs cisplatin).
Antimicrobial activity
1
2
4 3
The antimicrobial activity of proligands H L and H L and complexes 1−9 was determined by
both disk diffusion and microdilution method. Four reference bacterial species (S.aureus, E.faecalis,
E.coli and P.aeruginosa) and a fungal one (C.albicans) were included in this study. As shown in
Tables 3 and 4, the two complexes 2 and 7 showed good antimicrobial activity against all the tested
bacterial and fungal species.
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6

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1
2
4 3
Table 3. Disc Diffusion Test of proligands H L , H L , cocrystals 2, 8 and complexes 1, 3−7, 9
against a panel of reference bacterial and fungal strains. Diameters of zone inhibition are expressed
in millimeters.
S. aureus
ATCC 29213
6.0±0
E. faecalis
ATCC 29212
6.0±0
E. coli
ATCC 25922
6.0±0
P. aeruginosa
ATCC 27853
6.0±0
C. albicans
ATCC 24433
6.0±0
Compound
1
H
H
4
L
L
2
6.0±0
6.0±0
6.0±0
6.0±0
6.0±0
3
1
2
3
4
5
6
7
8
9
6.0±0
8.7±0.2
6.0±0
6.0±0
6.0±0
6.0±0
10.5±0.5
6.0±0
6.0±0
6.5±0.5
6.0±0
6.0±0
6.0±0
6.0±0
9.5±0.5
6.0±0
6.0±0
10.2±0.2
6.0±0
6.0±0
6.0±0
6.0±0
11.0±1.0
6.0±0
6.0±0
11.0±0.5
6.0±0
6.0±0
6.0±0
6.0±0
12.5±0.5
6.0±0
6.0±0
11.0±1.0
6.0±0
6.0±0
6.0±0
6.0±0
15.5±0.5
6.0±0
6.0±0
6.0±0
6.0±0
6.0±0
6.0±0
Table 4. MIC values of complexes 2 and 7 against a representative panel of reference strains. All
the concentrations are expressed in ꢀM.
Compound
S. aureus
E. faecalis
E. coli
P. aeruginosa
C. albicans
ATCC 29213
ATCC 29212
ATCC 25922
ATCC 27853
ATCC 24433
MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC
6
1
4ꢁ
28
32
2
7
256
64
128
64
256
64
128
16
128
16
64
8
64
16
32
4
32
4
2
One of them (2) is the cocrystal of H L with ethylenedamine (en), the second one (7) is the
3
complex of 1 with silver. MIC values range from 32 to 128 for the complex 2 and from 4 to 64 µM
1
4
in the case of complex 7. Both active complexes possess a bactericidial activity. Proligands H L
2
and H L and complexes 2−6, 8 and 9 do not possess antimicrobial activity against the tested
3
strains.
Conclusions
II
II,III
II
I
In the present work, for the new series of Co , Co , Cu and Ag complexes of AHBA and
cocrystalline species with ethylenedamine and imidazole, we evaluated different biological
properties such as anticancer and antimicrobial activity and DNA and BSA binding abilities.
I
Among all the compounds screened, only the Ag complex (7) showed remarkable cytotoxic and
3
antibacterial activity (higher of that of AgNO ) while a lower activity was found for the
cocrystalline (2), and no activity was revealed by the other tested derivatives. They are both
different to all other proligands and metal complexes, in fact they seem to afford external
electrostatic interactions with the DAPIꢁctꢁDNA system without a displacement of the day.
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II/III
Although the Co
complex 4 was able to bind strongly with DNA, it seems however to be not
active towards the growth of tumor cells and bacteria.
Experimental
Materials and instrumentation
1
13
The H and C NMR spectra were recorded at room temperature on a Bruker Avance II + 300
TM
(
UltraShield Magnet) spectrometer operating at 300.130 and 75.468 MHz for proton and carbonꢁ
1
3, respectively. The chemical shifts are reported in ppm using tetramethylsilane as the internal
−1
reference. The infrared spectra (4000–400 cm ) were recorded on a BIOꢁRAD FTS 3000MX
instrument in KBr pellets. Carbon, hydrogen, and nitrogen elemental analyses were carried out by
the Microanalytical Service of the Instituto Superior Técnico. Electrospray mass spectra (ESIꢁMS)
were run with an ionꢁtrap instrument (Varian 500ꢁMS LC Ion Trap Mass Spectrometer) equipped
with an electrospray ion source. For electrospray ionization, the drying gas and flow rate were
optimized according to the particular sample with 35 p.s.i. nebulizer pressure. Scanning was
performed from m/z 100 to 1200 in methanol solution. The compounds were observed in the
negative or positive mode (capillary voltage = 80–105 V).
2
3
Synthesis of H L
Diazotization: 2.55 g (20 mmol) of 4ꢁchloroaniline was dissolved in 50 mL water, and 0.40 g (10
mmol) of NaOH was added. The solution was cooled in an ice bath to 273 K and 1.38 g (20 mmol)
2
of NaNO were added; 4.00 mL HCl were then added in 0.5 mL portions for 1 h. The temperature
of the mixture should not exceed 278 K.
Azocoupling: NaOH (0.80 g, 20 mmol) was added to a mixture of 20 mmol (2.56 g) of barbituric
acid with 50.00 mL of water. The solution was cooled in an ice bath, and a suspension of 4ꢁ
chlorobenzenediazonium chloride (prepared according to the procedure described above) was added
2
in two equal portions under vigorous stirring for 1 h. The formed precipitate of the H
3
L was filtered
2
off, recrystallized from methanol and dried in air. The characterizations of the H
3
L was carried out
1
13
by IR, H and CꢁNMR spectroscopies and are given below
2
H
3
L : Yield, 3.41 g, 64 % (based on barbituric acid), yellow powder soluble in DMSO, methanol,
acetone and acetonitrile. Anal. Calcd for C10
H
7
ClN
4
O
3
(Mr = 266.64): C, 45.04; H, 2.65; N, 21.01;
+
found: C, 44.87; H, 2.55; N, 20.83 %. ESIꢁMS: m/z: 267.3 [Mr+H] . IR (KBr): 3260, 3085 and
−
1
1
2
813 ν(NH), 1753 and 1653 ν(C=O), 1588 ν(C=OꢀꢀꢀH), 1513 ν(C=N) cm . H NMR (300.130
MHz) in DMSOꢁd
6
, internal TMS, δ (ppm): 7.48−7.63 (4H, Ar−H), 11.31 (s, 1H, N−H), 11.52 (s,
1
3
1
1
1
H, N−H), 14.03 (s, 1H, N−H). C{ H} NMR (75.468 MHz, DMSOꢁd ). δ: 118.11 (Ar−Cl),
6
18.30 and 129.54 (4Ar−H), 129.70 (C=N), 140.46 (Ar−NHN=), 149.80 and 159.76 (C=O), 161.92
(C=OꢀꢀꢀH).
1
8

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2
Synthesis of 2. A methanol solution (30 mL) of H
3
L (0.266 g, 1.0 mmol) was added at room
temperature ethylenediamine (en) (0.06 mL, 1.0 mmol). The mixture was left for slow evaporation;
the yellowꢁorange crystals of 2 suitable for Xꢁrays started to form after ca. 1 d at room temperature;
they were then filtered off and dried in air.
2
2
: Yield, 284 mg, 84 % (based on H
3
L ). It is soluble in water, methanol, DMF, DMSO and only
slightly in acetonitrile, acetone and chloroform. Anal. Calcd for C H Cl N O (Mr = 1016.25):
36
49
3
18 11
C, 42.55; H, 4.86; N, 24.81; found: C, 42.27; H, 4.94; N, 24.88 %. ESIꢁMS: m/z: 327.2
+
[
C
12
H15ClN
6
O
3
+H] . IR (KBr): 3446 and 3355 ν(OH), 3157 and 3062 ν(NH), 1693 and 1655
−
1 1
ν(C=O), 1579 ν(C=N) cm . H NMR (300.130 MHz) in DMSOꢁd , internal TMS, δ (ppm): 2.66
6
+
13
1
(
4H, CH
2
), 4.68 (6H, NH
1.50 (2CH
), 116.75 (Ar−Cl) and (Ar−NHN=), 120.13 (2Ar−H), 128.80 (2Ar−H), 129.10 (C=N),
49.06, 151.85 and 161.13 (C=O).
3 6
), 7.38−7.49 (4H, Ar−H). C{ H} NMR (75.468 MHz, DMSOꢁd ). δ:
4
1
2
Synthesis of 7. 776 mg (1 mmol) of 1 were dissolved in waterꢁacetone mixture (1:4, v/v), then 200
µL of HNO (65 % w/w) were added at room temperature. The addition of 338 mg (2 mmol) of
3
AgNO together with 10 mL of distilled water resulted in a clear solution. The resulting solution
3
was allowed to stand in darkness room at room temperature and yellow crystals of 7 were obtained
after 1 day.
7
: Yield, 236 mg, 54 % (based on Ag). It is soluble in water, DMSO, methanol and DMF. Anal.
Calcd for C10 AgN S (Mr = 437.13): C, 27.48; H, 2.08; N, 12.82 %. Found: C, 27.51; H, 2.09;
N, 13.19%. IR (KBr): 3480 ν(OH), 3211 and 3067 ν(NH), 1736, 1689 and 1663 ν(C=O), 1579
H
9
4 7
O
−1
+ 1
ν(C=N) cm . ESIꢁMS: m/z: 839.1 [2Mr−2H
2 6
O+H] . H NMR (300.130 MHz) in DMSOꢁd ,
internal TMS, δ (ppm): 7.20−7.77 (4H, Ar−H), 11.21 (s, 1H, N−H), 11.33 (s, 1H, N−H), 14.78 (s,
1
3
1
1
H, N−H). C{ H} NMR (75.468 MHz, DMSOꢁd
6
). δ: 116.14, 118.16, 124.87 and 127.50 (Ar−H),
Ag), 150.00 and 160.22 (C=O), 160.62
1
30.31 (Ar−NHN=), 135.96 (C=N), 138.16 (Ar−SO
3
(C=OꢀꢀꢀH).
Synthesis of 8. 776 mg (1 mmol) of 1 was dissolved in 30 mL methanol, then 200 µL of HNO (65
3
%
w/w) and 136 mg (2 mmol) of imidazole (im) were added at room temperature. After ca. 1 d at
room temperature, yellow crystals precipitated which were then filtered off and dried in air.
: Yield, 254 mg, 61 % (based on im). It is soluble in water, methanol, DMF, DMSO and only
slightly in acetonitrile, acetone and chloroform. Anal. Calcd for C13 S (Mr = 416.37): C,
7.50; H, 3.87; N, 20.18 %. Found: C, 37.26; H, 3.62; N, 20.20 %. IR (KBr): 3446 ν(OH), 3202,
8
16 6 8
H N O
3
3
−
1
063 and 2862 ν(NH), 1739, 1698 and 1675 ν(C=O), 1581 ν(C=N) cm . ESIꢁMS: m/z: 312.1
3
+ 1
[
H
3
L +H] . H NMR (300.130 MHz) in DMSOꢁd
6
, internal TMS, δ (ppm): 7.19−7.77 (4H, Ar−H
and 2H, C−H im), 9.08 (s, 1H, C−H im), 11.20 (s, 1H, N−H), 11.33 (s, 1H, N−H), 14.21 (s, 1H,
13
1
N−H im), 14.79 (s, 1H, N−H). C{ H} NMR (75.468 MHz, DMSOꢁd ). δ: 116.14, 118.16, 124.87
6
1
9

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−
and 127.50 (Ar−H), 130.31 (Ar−NHN=), 135.96 (C=N), 138.16 (Ar−SO
3
), 150.00 and 160.22
(C=O), 160.62 (C=OꢀꢀꢀH).
Xꢀray structure determinations
Xꢁray quality single crystals of the compounds were mounted in a nylon loop and measured at 150
K (7 and 8) or at room temperature (2). Intensity data were collected using a Bruker AXSꢁKAPPA
APEX II diffractometer or a Bruker APEXꢁII PHOTON 100 diffractometer with graphite
monochromated MoꢁKα (λ 0.71069) radiation. Data were collected using phi and omega scans of
0
.5° per frame and a full sphere of data was obtained. Cell parameters were retrieved using Bruker
4
1
49
SMART software and refined using Bruker SAINT on all the observed reflections. Absorption
4
9
corrections were applied using SADABS. Structures were solved by direct methods by using the
50
50
SHELXS package and refined with SHELXLꢁ2014. Calculations were performed using the
5
1
WinGX System–Version 1.80.03. The hydrogen atoms of water, hydrazine or ammonium groups
were found in the difference Fourier map and the isotropic thermal parameters were set at 1.5 times
the average thermal parameters of the belonging oxygen or nitrogen atoms, frequently with their
distances restrained by using the DFIX and DANG commands. Coordinates of hydrogen atoms
bonded to carbon atoms were included in the refinement using the ridingꢁmodel approximation with
the Uiso(H) defined as 1.2Ueq of the parent aromatic or methylene atoms. Least square refinements
with anisotropic thermal motion parameters for all the nonꢁhydrogen atoms and isotropic for the
remaining atoms were employed. CCDC 1055318, 1055323 and 1055324 contain the
supplementary crystallographic data for this paper. These data can be obtained free of charge from
the Cambridge Crystallographic Data Centre via w.ccdc.cam.ac.uk/data_request/cif.
DNA interaction studies
A stock solution of calf thymus DNA (ctꢁDNA) (Sigma–Aldrich) was prepared by dissolving the
solid material overnight in 10 mM phosphate buffer pH 7.2 containing 5 mM NaCl at 4°C. The
5
2,53
concentration of DNA was determined by UV absorbance at 260 nm as described.
Electronic absorption titration
Calf thymus (ct) DNA binding experiments were performed in 10 mM and phosphate buffer pH 7.2
using DMSO solution of the complexes at room temperature. In UV–Vis absorption titration
phosphate buffer was used and the concentration of each complex was 13 ꢀM while the ctꢁDNA
concentration added was from 0 to 26 ꢀM. The complex solution was titrated with the DNA, and
changes on the intensity of the bands of the complex were monitored. The intrinsic binding constant
(K
b
a
) for the interaction of the complexes with ctꢁDNA was determined from a plot of [DNA]/(ε ꢀ
5
4
ε
f
) versus [DNA] using absorption spectral titration data and the equation (1):
2
0

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[
DNA]/|ε ꢁ ε | = [DNA]/ |ε ꢁ ε | + 1/K |ε ꢁ ε |
(1)
a
f
b
f
b
b
f
where [DNA] is the concentration of DNA, the apparent absorption coefficients ε
a
, ε
f
and ε
b
correspond to Aobsd/[complex], the extinction coefficient for the free metal complex and the
b
extinction coefficient for the metal complex in the fully bound form, respectively. The K value is
given by the ratio of the slope to the intercept.
Fluorescence spectral study
The competitive binding experiment was carried out by maintaining the ethidium bromide (EB)
Sigma–Aldrich) and ctꢁDNA concentration at 5 ꢂM and 55.7 ꢂM, respectively, while increasing
(
the concentration of different complexes (from a DMSO stock solution). Fluorescence quenching
spectra were recorded using a Hitachi 4500 spectrofluorimeter with an excitation wavelength of 490
nm and 500ꢁ700 nm emission spectrum. The fluorescence spectra of a series of solutions with
various concentrations of complex and a constant EBꢁctꢁDNA complex were measured and the
fluorescence value of decrease in emission spectra were corrected according to the relationship:
(
A1+A2)/2
F
c
= F
m
× e
(2)
where F and F are the corrected and measured fluorescence, respectively. A1 and A2 are the
c m
absorbance of tested compounds at the exciting and emission wavelengths. For fluorescence
quenching experiments, the SternꢁVolmer's equation was used (Eqn. 3):
F
0
/F
represent the fluorescence intensity in the absence and in the presence of drug. [C]
is the concentration of complex and Ksv is the SternꢁVolmer constant which is equal to k x τ
where k is the bimolecular quenching rate constant and τ is the average fluorescence lifetime of
c
= 1+ k
Q
τ
0
[C] = 1 + Ksv [C]
(3)
0 c
where F and F
Q
0
,
Q
0
the fluorophore in the absence of drug. All experiments involving ctꢁDNA were performed in buffer
solution (10 mM phosphate buffer pH 7.2) at room temperature. The SternꢁVolmer constant Ksv can
55
be obtained by the equation (4):
I / I = 1 + K [compound]
(4)
0
SV
0
where I and I are the emission intensities in the absence and presence of the compounds,
respectively. KSV dependes on the ratio of the concentration of bound EB to the concentration of
DNA and is called simply as the quenching constant.
The binding constant Kapp was obtained by the equation (5):
KEB[EB] = Kapp[compound]
(5)
2
1

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in which the compound concentration is the value at a 50% reduction of the fluorescence intensity
7
ꢁ1
56
of EB and KEB = 1.0 × 10 M ([EB] = 5.0 ꢀM).
Minor Groove Displacement Assay
A wellꢁknown minor groove binder, 4',6ꢁdiamidinoꢁ2ꢁphenylindole (DAPI), was used to perform
the minor groove displacement assays as reported earlier. In a typical experiment, the changes in the
emission spectra of DAPI complex with the DNA in 10 mM phosphate buffer pH 7.2, were
monitored upon addition of increasing concentrations of compounds at room temperature.
Fluorescence quenching data were evaluated after excitation of DAPIꢁctꢁDNA complex at 338nm
and recording the spectra from 400ꢁ600 nm. Values of K constants were evaluated using equation
sv
3
after that all quenching fluorescence values obtained were corrected according to the relationship
2
. In the presence of enhancment of fluorescence the values of Ksv was evaluate as previously
57,58
described.
BSA binding studies
The proteinꢁbinding study was performed by tryptophan fluorescence quenching experiments using
bovine serum albumin (BSA). Bovine serum albumin (Sigma–Aldrich) stock solutions were
prepared in 20 mM potassium phosphate buffer pH 7.4. Fluorescence measurements were carried
out with a Hitachi 4500 spectrofluorometer by keeping the concentration of BSA constant (15 ×
−6
−6
1
0
M) while varying the complex concentration (from 0 to 40 × 10 M), at room temperature.
Fluorescence intensities were recorded after each successive addition of complex solution and
equilibration (ca. 5 min). Fluorescence spectra were recorded from 300 to 450 nm at an excitation
wavelength of 285 nm. The value of Stern Volmer constants and association binding constant of the
different metal complexes to HSA were evaluated following the equation 3 and fluorescent values
were corrected by equation 2.
Antiproliferative activity
A375 (human malignant melanoma cell line) and MDAꢁMB 231 cells (human breast
adenocarcinoma cell line) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) with 2
mM Lꢁglutamine, 100 IU/mL penicillin, 100 µg/mL streptomycin, and supplemented with 10%
heatꢁinactivated foetal bovine serum (HIꢁFBS). HCT116 cells (human colon carcinoma cell line)
were cultured in RPMI1640 medium with 2 mM Lꢁglutamine, 100 IU/mL penicillin, 100 ꢂg/mL
streptomycin, and supplemented with 10% HIꢁFBS. Cells were cultured in a humidified atmosphere
2
at 37° C in the presence of 5% CO . The MTT assay was used as a relative measure of cell
viability. Cellꢁviability assays were carried out as described. Briefly, cells were seeded at the
4
density of 2 × 10 cells/ml. After 24 h, samples were exposed to different concentrations of
2
2

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compounds 1−9 (0.39 ꢁ 100 ꢂM). Cells were incubated for 72 h in a humidified atmosphere of 5%
32
2
CO at 37 °C. At the end of incubation, the cytotoxic activity was evaluated by the MTT assay.
Experiments were conducted in triplicate. Cytotoxicity is expressed as the concentration of
compound inhibiting cell growth by 50% (IC50). The IC50 values were determined with GraphPad
Prism 4 computer program (GraphPad Software, S. Diego, CA, USA). Analysis of variance was
performed in InStat, GraphPad software, San Diego CA, USA. Unpaired t test was used for these
analyses, and p < 0.001 was considered to be statistically significant.
Antimicrobial activity
The antimicrobial activity of ligands, cocrystals and complexes was determined by both disc
diffusion and by microdilution methods following the general guidelines of the Clinical Laboratory
59
Standard Institute (CLSI). Microorganisms included in this study were: Staphylococcus aureus
ATCC 29213, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853,
Enterococcus faecalis ATCC 29212, Candida albicans ATCC 24433. Bacterial strains were
cultured overnight at 37 °C on blood agar plates and C. albicans was grown in RPMI1640. For the
8
6
disk diffusion method a suspension of 10 cells per mL prepared in saline (10 per mL for C.
albicans) was spread on the solid media plates (MHA for bacteria and MH supplemented with 2%
glucose and 0.5 mg/ml of methylene blue for Candida) using a sterile cotton swab. Sterile filter
paper discs (6 mm in diameter) were placed on the surface of inoculated plates and spotted with 10
ꢂL of each compound (20 mM in DMSO). The plates were incubated for 24 h at 35 ± 2°C (48 h in
the case of Candida). The known antimicrobials ciprofloxacin (5 ꢂg per disc) and nystatin (100 U
per disc) (CLSI) were used as positive control against bacteria and fungus, respectively. Activity
was determined by measuring the diameter of the growth inhibition zone (inhibition zone diameter,
IZD) visible around the paper disc (expressed in mm) using a caliper. Reported IZDs are inclusive
of the paper disc diameter (6 mm). Therefore, a 6ꢁmm IZD means no activity. No zone of inhibition
was observed using DMSO alone. Each test was repeated three times. For the microdilution method
twoꢁfold serial dilutions of each compound were prepared in 96ꢁwell plates, starting from 256 ꢂM
in Cation Adjusted Mueller Hinton Broth or RPMI 1640 medium, depending on the microorganism
6
−1
3
−1
tested. An equal volume of bacterial (10 cfu mL ) or fungal (10 cfu mL ) inoculum was added to
each well of the microtiter plate containing 0.1 mL of the serially diluted test molecule. Incubation
followed for 18−24 h at 35 °C (24−48 h in the case of C. albicans) in a normal atmosphere. The
Minimum Inhibitory Concentrations (MICs) were defined as the lowest concentration of the
compound able to inhibit the growth of the microorganisms. All tests were done in triplicate.
Minimum Bactericidal Concentration (MBC) was subsequently determined as recommended by the
CLSI guidelines.
2
3

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Acknowledgements
This work has been partially supported by the Foundation for Science and Technology (FCT),
Portugal (UID/QUI/00100/2013 project). M.F.C.G.S. and A.J.L.P. acknowledge the Russian
Science Foundation for support in the structural analysis and help in organizing the International
Laboratory (grant 14ꢁ43ꢁ00017). The authors acknowledge the Portuguese NMR Network (ISTꢁ
UTL Centre) for access to the NMR facility, and the IST Node of the Portuguese Network of massꢁ
spectrometry (Dr. Conceição Oliveira) for the ESIꢁMS measurements. The authors also
acknowledge the financial support by the University of Camerino (Fondo di Ateneo per la Ricerca
2
011–2012) and Nuova Simonelli Company.
Appendix A. Supplementary data
Crystal data, experimental parameters and selected details of the refinement calculations (for 2 and
8
), electronic absorption and emission fluorescence spectra of compounds 1−9. This material is
available free of charge via the Internet at http://pubs.rsc.org/ or from the authors. CCDC 1055318,
055323 and 1055324 contain the supplementary crystallographic data for compounds 2, 7 and 8,
respectively. These data can be obtained free of charge via
1
http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic Data
Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223ꢁ336ꢁ033; or eꢁmail:
deposit@ccdc.cam.ac.uk.
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