A versatile salicyl hydrazonic ligand and its metal complexes as antiviral agents

Article abstract of DOI:10.1016/j.jinorgbio.2015.05.013

Acylhydrazones are very versatile ligands and their coordination properties can be easily tuned, giving rise to metal complexes with different nuclearities. In the last few years, we have been looking for new pharmacophores able to coordinate simultaneous

Full text of DOI:10.1016/j.jinorgbio.2015.05.013

Journal of Inorganic Biochemistry 150 (2015) 9–17
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
A versatile salicyl hydrazonic ligand and its metal complexes as
antiviral agents
a,
a
a
b
b
b
a
c
D. Rogolino ⁎, M. Carcelli , A. Bacchi , C. Compari , Laura Contardi , E. Fisicaro , A. Gatti , M. Sechi ,
d
d
A. Stevaert , L. Naesens
a
Dipartimento di Chimica, Parco Area delle Scienze 17/A, 43124 Parma, Italy
b
c
Dipartimento di Farmacia Università di Parma, Parco Area delle Scienze 17/A, 43124 Parma, Italy
Dipartimento di Chimica e Farmacia, Università di Sassari, Via Vienna 2, I-07100 Sassari, Italy
Rega Institute for Medical Research, KU Leuven — University of Leuven, B-3000 Leuven, Belgium
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Acylhydrazones are very versatile ligands and their coordination properties can be easily tuned, giving rise to
metal complexes with different nuclearities. In the last few years, we have been looking for new pharmacophores
able to coordinate simultaneously two metal ions, because many enzymes have two metal ions in the active site
and their coordination can be a successful strategy to inhibit the activity of the metalloenzyme. As a part of this
Received 9 March 2015
Received in revised form 21 May 2015
Accepted 24 May 2015
Available online 27 May 2015
2
ongoing research, we synthesized the acylhydrazone H L and its complexes with Mg(II), Mn(II), Co(II), Ni(II),
Cu(II) and Zn(II). Their characterization, both in solution – also by means of potentiometric studies – and in
the solid state, evidenced the ability of the o-vanillin hydrazone scaffold to give rise to different types of metal
complexes, depending on the metal and the reaction conditions. Furthermore, we evaluated both the free ligand
and its metal complexes in in vitro studies against a panel of diverse DNA- and RNA-viruses. In particular, the
Mg(II), Mn(II), Ni(II) and Zn(II) complexes had EC50 values in the low micromolar range, with a pronounced ac-
tivity against vaccinia virus.
Keywords:
Acylhydrazones
Chelating ligand
Metal complexes
Antiviral activity
©
2015 Elsevier Inc. All rights reserved.
1
. Introduction
atom. Their degree of deprotonation depends on the reaction conditions
and the metal employed and they can give rise to monometallic, bis-
chelated complexes or to multimetallic assemblies held together by in-
termolecular forces [2,3,12].
Acylhydrazone-based ligands and their transition metal complexes
are widely studied in catalysis [1], as building blocks in supramolecular
chemistry [2,3], as anticancer agents [4,5], and, particularly interesting
for our research, antiviral molecules [6,7]. They are often used to build
multinuclear metal complexes since they are versatile ligands and
their coordination properties can be easily tuned from an electronic
and a steric point of view [8–10]. Acylhydrazone ligands, in fact,
can act as bidentate or tridentate chelating ligands and even as
tetradentates, and can coordinate one or more metal centers [1,3,10,
In this context, we synthesized an acylhydrazone ligand (H
2
L)
(Fig. 1) by condensation of o-vanillin with salicyl hydrazide. H L is
2
diprotic and a potentially ONOO tetradentate ligand; it can coordinate
one but also two metal ions (Fig. 1).
Its coordination compounds with Mg(II), Mn(II), Co(II), Ni(II),
Cu(II) and Zn(II) were isolated and characterized by IR, elemental
analysis and mass spectrometry (complexes 1–6, Fig. 2); the X-ray
1
1]. Complexes of salicylaldehyde hydrazones with Mn(II), Cu(II),
lanthanide(III), Pd(II), and Ni(II) have been previously studied [1–3,
–11] and they represent a challenging topic from the coordination
crystal structures of the tetranuclear copper complex Cu
and of the bis-chelated cobalt(III) complex [CoL ]NHEt
discussed. Moreover, the solution behavior of H L towards Mg(II),
4
L
4
·4CH
3
CN
2
3
(3) are also
8
2
point of view. These ligands typically coordinate to the metal ion
through the phenolic and carbonyl oxygens and the imine nitrogen
Mn(II), Ni(II), Cu(II) and Zn(II) was investigated by means of poten-
tiometric experiments.
It is well known that the metal complexes of hydrazones have di-
verse biological and pharmaceutical properties: in fact, they were stud-
ied as antimicrobial, anti-inflammatory, antifungal, anti-tubercular,
anticancer, antimalarial and antiviral agents [13–16]. In the last few
years, the research of new metal-chelating pharmacophores with anti-
viral activity is at the center of our interest [17–20]. Many enzymes
⁎
Corresponding author at: Dipartimento Chimica, Università di Parma Campus
Universitario — Parco Area delle Scienze 17/A, 43124 Parma, Italy.
E-mail address: dominga.rogolino@unipr.it (D. Rogolino).
http://dx.doi.org/10.1016/j.jinorgbio.2015.05.013
0162-0134/© 2015 Elsevier Inc. All rights reserved.

10
D. Rogolino et al. / Journal of Inorganic Biochemistry 150 (2015) 9–17
2
(
5 °C), δ: 3.91 (s, 3H, OCH
m, 2H, ArH), 7.06 (d, J = 8.2 Hz, 1H, ArH), 7.21 (d, J = 8.2 Hz, 1H;
ArH), 7.47 (t, 2H; J = 7.4, ArH), 7.92 (d, 2H; J = 7.7, ArH), 8.60 (s, 1H;
HC = N). 13C-NMR (MeOD-d
, 25 °C), δ: 55.27; 113.79; 114.78;
17.10; 118.63; 119.01; 119.06; 121.25; 128.08; 134.02; 148.11;
3
), 6.90 (t, J = 7.6 Hz, 1H, ArH); 6.96–7.00
4
1
1
+
49.48; 159.73. MS (EI, 70 eV) m/z (%) = 286.0 ([M] , 100);. IR
−
1
(
cm ): νNH+OH = 3202 (br); νC_O = 1606; νC_N = 1560; νOCH3 =
1
9
256, 1079. Anal. Calcd. for C15
.49. Found: C 61.20, H 4.89, N 9.58.
Synthesis of the complexes 1–6, general procedure. The ligand H
0.5 mmol) was dissolved in 30 ml of methanol and 1 eq. of NEt was
14 2 4 2
H N O ·1/2H O: C 61.01; H 5.12; N
2
L
(
3
Fig. 1. Chemical structure of the acylhydrazonic ligand H
L can coordinate one or two metal ions (right).
2
L (left); the polydentate ligand
added. The yellow solution was stirred at 65 °C for 30 min. 0.5 eq. of ac-
etate of the metal were added and then the reaction mixture was stirred
at reflux for 4 h, concentrated in vacuum and cooled overnight. The pre-
cipitate was filtered off, washed with water and dried under vacuum.
H
2
have two metal ions in their active site and coordination of these cofac-
tors can be a successful strategy to inhibit the activity of a given
metalloenzyme [21,22]. As a part of this ongoing research, we evaluated
H L and its metal complexes 1–6 in in vitro studies against a panel of di-
2
verse DNA- and RNA-viruses, discovering promising activity of this scaf-
fold against herpes simplex virus (HSV) and vaccinia virus (VV).
2
.2.2. Mg(HL)
Light yellow powder. Yield: 70%. H-NMR (MeOD, 25 °C), δ: 3.91 (s,
H, OCH ); 6.88–7.07 (m, 4H, ArH); 7.20 (d, J = 7.4 Hz, 1H, ArH); 7.46 (t,
2 2
·2H O (1)
1
3
3
J = 7.9 Hz, 1H); 7.92 (d, J = 7.9 Hz, 1H); 8.60 (s, 1H; HC = N). MS-ESI,
+
−1
m/z (%) = 595 ([Mg(HL)
νC_O 1627, 1609; νOCH3
Mg · 2H O: C 57.11, H 4.79, N 8.88. Found: C 57.33, H 4.33,
2
+ H] , 40). IR (cm ): νNH = 3267 (br);
2
. Experimental
=
=
1211, 1082. Anal. Calcd. for
C H
30 26
4
N O
8
2
2
.1. Material and methods
N 9.00.
All reagents of commercial quality were used without further purifi-
2
.2.3. Mg
.5 mmol of H
NaOH 4 M were added. The yellow solution was stirred at 65 °C for
0 min. 1 eq. of magnesium acetate was added and the reaction mixture
2
L
2
·4H
2
O (1a)
cation. Purity of compounds was determined by elemental analysis and
verified to be ≥96% for all synthesized molecules. NMR spectra were re-
corded at 25 °C on a Bruker Avance 400 FT spectrophotometer. The ATR-
IR spectra were recorded by means of a Nicolet-Nexus (Thermo Fisher)
spectrophotometer by using a diamond crystal plate in the range of
0
2
L was dissolved in 30 ml of methanol and 2.5 eq. of
3
was stirred at reflux for 4 h, concentrated in vacuum and cooled over-
night. The precipitate was filtered off and washed with water. Intense
yellow powder. Yield: 64%. H-NMR (MeOD, 25 °C), δ: 3.84 (s, br, 3H,
OCH
1
3
−
1
4
000–400 cm . Elemental analyses were performed by using a
1
FlashEA 1112 series CHNS/O analyzer (Thermo Fisher) with gas-
chromatographic separation. Electrospray Ionization mass spectral
analyses (ESI-MS) were performed with an ESI-TOF (electrospray ioni-
zation time-of-flight) Micromass 4LCZ spectrometer. MS spectra were
acquired in positive EI mode by means of a DEP-probe (Direct Exposure
Probe) mounting on the tip a Re-filament with a DSQII Thermo Fisher
apparatus, equipped with a single quadrupole analyzer.
3
); 6.45 (s, br, 1H, ArH), 6.68–6.82 (m, br, 5H, ArH); 7.20 (s, br,
H, ArH); 7.90 (s, br, 1H); 8.24 (s, br, 1H; HC_N). MS-ESI, m/z (%) =
+
+
+
2 2 2 2
09 ([MgL] , 100); 617 ([Mg L ] , 50); 640 ([Mg L + Na] , 40). IR
−
1
(
cm ): νOH = 3420–3500 (br); νC_O = 1609; νOCH3 = 1211, 1082.
Anal. Calcd. for C30
C 52.67, H 4.63, N 8.03.
H N O Mg
24 4 8 2
2
· 4H O: C 52.28, H 4.68, N 8.13. Found:
2
.2.4. Mn
2 2 2
L ·2H O (2)
2
2
.2. Synthesis
−
Brown powder. Yield: 70%. MS-ESI, m/z (–, %) = 624 ([Mn(HL)
5); 677 ([Mn
OCH3 = 1208. Anal. Calcd. for C30
2
] ,
−
−1
9
ν
L
2 2
] , 100). IR (cm ): νNH = 3267 (br); νC_O = 1607;
Mn · 2H O: C 50.43, H 3.95,
.2.1. N'-(2-hydroxy-3-methoxybenzylidene)-2-hydroxybenzoylhydrazone
H
24
N O
4 8
2
2
2
(H L)
N 7.84. Found: C 50.68, H 4.27, N 7.87.
2
H L was obtained by slight modifications of reported proce-
dures [23]. Briefly, an equimolar amount of salicyl hydrazide and alde-
hyde are dissolved in absolute ethanol. The mixture was refluxed for
2
.2.5. [CoL
Brown powder. Yield: 64%. H-NMR (DMSO-d
H, CH3, NEt ), 2.50 (overlapping with solvent signal, CH2, NEt
.36 (s, 3H, OCH ), 6.41 (t, 1H; J = 7.6, ArH), 6.51 (d, 1H; J = 7.7,
2 3
](NHEt ) (3)
6
h, cooled at room temperature and concentrated in vacuum. The
1
6
, 25 °C), δ: 0.99 (t,
),
resulting precipitate was filtered off, washed with cold ethanol and
9
3
3
3
1
dried in vacuum. Yield = 89%. H-NMR (DMSO-d
H, OCH ), 6.85–7.06 (m, 4H, ArH), 7.17 (d, J = 7.5 Hz, 1H, ArH), 7.45
t, J = 7.6 Hz, 1H; ArH), 7.89 (d, 2H; J = 7.5, ArH), 8.69 (s, 1H; HC =
6
, 25 °C), δ: 3.82 (s,
3
3
(
3
ArH), 6.67 (t, 1H, J = 8, ArH), 7.22 (m, 2H, ArH), 7.47 (d, 1H, J =
7
1
ν
C
9
.9, ArH),7.70 (d, 1H, J = 8, ArH), 8.93 (s, 1H; HC = N), 12.64 (s,
1
N), 10.87 (s, br, 1H; NH), 11.99 (s, br, 2H; OH). H-NMR (MeOD-d
4
,
−
−1
H, NHEt
NH = 3206 (br); νC_O = 1624; νC_N = 1598. Anal. Calcd. for
Co: C 59.26, H 5.53, N 9.60. Found: C 59.55, H 5.48, N
.41. Crystals suitable for X-ray diffraction analysis were obtained
3 2
). MS-ESI, m/z (−, %) = 627 ([CoL ] , 100). IR (cm ):
36
40 5 8
H N O
by slow evaporation of a methanol solution of the complex.
2
.2.6. Ni(HL)
2 2
·2H O (4)
+
Green powder. Yield: 87%. MS-ESI, m/z (+,%) = 629 ([Ni(HL)
2
] ,
+ Na] , 50). IR (cm ): νNH+OH = 3150–3200
1629; 1201. Anal. Calcd. for
30 26 4 8 2
H N O Ni · 2H O: C 54.16, H 4.55, N 8.42. Found: C 54.51, H 4.84,
+
−1
7
0); 652 ([Ni(HL)
2
(
br);
ν
C_O
=
ν
OCH3
=
C
Fig. 2. Synthesis of complexes 1–6.
N 8.34.

D. Rogolino et al. / Journal of Inorganic Biochemistry 150 (2015) 9–17
11
2
.2.7. Cu
4
L
4
·3H
2
O (5)
Table 1
+
4 4 3
Crystal data and structure refinement for (3) and Cu L ·4CH CN.
Green powder. Yield: 46%. MS-ESI, m/z (+,%) = 348 ([CuL] , 100);
+
+
−1
7
17 ([Cu
NH+OH = 3150–3200 (br); νC_O = 1629; νOCH3 = 1201. Anal. Calcd.
16Cu 3H O: C 49.86, H 3.77, N 7.75. Found: C 50.00, H
.42, N 7.60. Crystals of Cu ·4CH CN suitable for X-ray diffraction
analysis were obtained by recrystallization of (5) from acetonitrile.
2
L
2
+ Na] , 40); 1392 ([Cu
4
L
4
+ H] , 10). IR (cm ):
Cu L ·4CH CN
3
C
4
4
3
ν
Empirical formula
Formula weight
Temperature/K
Crystal system
(C15
H12CuN
2
O
4
4
) ·4CH
3
CN
36 5 8
H40CoN O
for C60
3
H
48
N
8
O
4
2
1555.44
293(2)
Tetragonal
729.66
293(2)
Monoclinic
P2 /n
4
L
4
3
Space group
I4
1
/a
1
a/Å
b/Å
c/Å
α/°
β/°
γ/°
13.470(1)
13.470
38.680(4)
90
90
90
7018.6(16)
16
1.472
1.271
3184.0
MoKα (λ = 0.71073)
3.202 to 48.052
34,451
10.375(2)
17.306(3)
19.457(3)
90
106.201(3)
90
3355(1)
4
1.445
0.573
1528.0
MoKα (λ = 0.71073)
3.208 to 49.65
13,784
2
.2.8. Zn(HL)
2 2
·H O (6)
−
Yellow powder. Yield: 86%. MS-ESI, m/z (−,%) = 635 ([Zn(HL)
2
] ,
5). IR (cm ): νNH = 3170 (br); νC_O = 1611; νC_N = 1534. Anal.
Zn · H O: C 55.10, H 4.32, N 8.57. Found: C 55.10,
−
1
6
Calcd. for C30
H 4.00, N 8.52.
H N O
26 4 8
2
3
Volume/Å
Z
3
2
.3. Potentiometric studies
ρ
calc g/cm
−
1
μ/mm
F(000)
Radiation
Θ range/°
The metal stock solutions were prepared from MgCl
drich), MnCl ·4H O (Janssen), CuCl ·2H O (Merck), ZnCl
Carlo Erba) and NiCl ·6H O (Merck). Their concentrations were deter-
2
·6H
2
O (Al-
(Analyticals
2
2
2
2
2
2
2
2
Reflections collected
mined by using EDTA as a titrant. Mn(II), Mg(II) and Zn(II), were titrat-
ed at pH 10 (ammonia/ammonium chloride as buffer; indicator: sodium
salt of Eriochrome Black T). Mn(II) was titrated in the presence of
triethanolamine and hydroxylamine chloride. For Ni(II), murexide in
potassium nitrate was used as an indicator. For Cu(II), the titrations
were performed in the presence of concentrated ammonia using Fast
Sulfon Black as indicator. Equilibrium constants at 25 ± 0.1 °C for pro-
tonation and complexation reactions were determined by means of po-
tentiometric measurements in methanol/water = 9:1 v/v solution at
ionic strength 0.1 M KCl, carried out under nitrogen in the pH range
Independent reflections
Data/restraints/parameters
Goodness-of-fit on F
2760
2760/0/231
1.024
13,784
13,784/0/458
1.120
2
Final R indexes [I ≥ 2σ (I)]
Final R indexes [all data]
Largest ΔF max/min/e Å−
R
1
= 0.0379
wR = 0.0844
= 0.0678,
wR = 0.0965
0.34/−0.21
R
1
= 0.1484
wR = 0.3472
= 0.1881,
wR = 0.3853
3.75/−1.09
2
2
R
1
R
1
2
2
3
structure was solved with the SIR2004 [28] structure solution program
using Direct Methods and refined with the ShelXL refinement package
[29] using Least Squares minimization. Anisotropic displacement pa-
rameters were refined for all non-hydrogen atoms. Hydrogen atoms
were partly introduced in calculated positions riding on their carrier
atoms. Compound (3) was seriously twinned, as shown by the high
final R factors and by the high residuals in the final difference Fourier
map, located close to the metal atom. Nevertheless, the structure gave
useful information about the connectivity and packing organization. Hy-
drogen bonds were analyzed with PARST97 [30] and the Cambridge
Structural Database software [31,32] was used for the analysis of the
2
.5–11. Potentiometric titrations were carried out by a fully automated
apparatus equipped with a CRISON GLP 21–22 digital voltmeter (resolu-
tion, 0.1 mV) and a 5 mL Metrohm Dosimat 655 autoburet, both con-
trolled by a homemade software in BASIC, working on an IBM
computer. Temperature was controlled to ± 0.1 °C by using a thermo-
static circulating water bath (ISCO GTR 2000 II×). Appropriate aliquots
2
of ligand H L solution, prepared by weight, were titrated with standard
KOH (methanol/water = 9:1 v/v, I = 0.1 M KCl) with and without metal
ions, applying constant speed magnetic stirring. Freshly boiled metha-
nol and double-distilled water, kept under nitrogen, were used
throughout. The experimental procedure to reach high accuracy in the
determination of the equilibrium constants in this mixed solvent has
been described in detail elsewhere [24]. The protonation constants of
4 4 3
crystal packing. Crystallographic data for (3) and Cu L ·4CH CN have
been deposited with the Cambridge Crystallographic Data Centre as
supplementary publication no. CCDC 1050872 and CCDC 1050873. Cop-
ies of the data can be obtained free of charge on application to CCDC, 12
Union Road, Cambridge CB2 1EZ, UK (fax: (+44) 1223-336-033; e-
mail: deposit@ccdc.cam.ac.uk).
2
H L were obtained by titrating 20 mL of samples of the ligand
−
3
(
5 × 10 M). The speciation was defined by performing the titrations
at different ligand/metal ratios (1 up to 4) and using a halved concentra-
tion of the ligand, to avoid solubility problems. At least two measure-
ments (about 60 experimental points each) were performed for each
system. The electrodic chain (Crison 5250 glass electrode and 0.1 M
KCl in methanol/water = 9/1 v/v calomel electrode, Radiometer 401)
2.5. Biological studies
The compounds' antiviral activity in cell culture was determined by
cytopathic effect (CPE) reduction assays with a broad and diverse panel
of viruses. The following viruses were investigated on human embryon-
ic lung fibroblast cells: herpes simplex virus type 1 (HSV-1) strain KOS;
+
was calibrated in terms of [H ] by means of a strong acid–strong base
titration by Gran's method [25] allowing the determination of the stan-
dard potential, E° (371.5 ± 0.4 mV), and of the ionic product of water,
−
K
w
(pK
ware HYPERQUAD [26] was used to obtain the speciation and the loga-
rithm of the stability constants (log βpqr) from titration data. βpqr
w
= 14.40 ± 0.05) in the experimental conditions used. The soft-
a thymidine kinase-deficient (TK ) HSV-1 KOS strain resistant to acy-
clovir; herpes simplex virus type 2 (HSV-2) strain G; vaccinia virus
(Lederle strain); a clinical isolate of human adenovirus type 2 (Ad2);
and vesicular stomatitis virus (VSV). The viruses assessed on human
cervix carcinoma HeLa cells were: VSV; Coxsackie B4 virus; and respira-
tory syncytial virus (RSV). African Green Monkey Vero cells were used
to study the antiviral effect on para-influenza-3 virus; reovirus-1;
Sindbis virus; Coxsackie B4 virus and Punta Toro virus. Human influenza
A/H1N1, A/H3N2 and B viruses were examined on Madin–Darby canine
kidney (MDCK) cells. Finally, the activity against human immunodefi-
ciency virus type 1 and type 2 (HIV-1 and HIV-2) was assessed in
human MT-4 lymphoblast cells.
=
p
q
r
[
M
p
L
q
H
r
]/[M] [L] [H] is the equilibrium constant for the reaction
pM + qL + rH = M , in which M indicates the metal, L the
p q r
L H
completely deprotonated ligand and H the proton. Charges are omitted
for simplicity.
2
.4. X-ray diffractometry
4 4 3
Single crystals of (3) and of Cu L ·4CH CN were selected and
mounted on glass fibers to collect data on a SMART Breeze diffractome-
ter. The crystals were kept at 293 K during data collection. Table 1 re-
ports crystal data and structure analysis. Using Olex2 [27], the
Semiconfluent cell cultures in 96-well plates were inoculated with
the virus at a multiplicity of infection of 100 CCID50 (50% cell culture

12
D. Rogolino et al. / Journal of Inorganic Biochemistry 150 (2015) 9–17
infective dose) or 20 PFU (plaque forming units) per well. Simulta-
neously with the virus, serial dilutions of the test or reference com-
pounds were added. The plates were incubated at 37 °C (or 35 °C in
the case of influenza virus) until clear CPE was reached, i.e., during 3
to 6 days, except for Ad2 which required 10 days incubation. Microscop-
ic scoring was then performed to determine the antiviral activity
[
[
expressed as 50% effective concentration (EC50)] and cytotoxicity
expressed as minimum cytotoxic concentration (MCC)]. In the case of
HIV-1 and HIV-2, virus-induced CPE was determined by a colorimetric
formazan-based cell viability assay.
3
. Results and discussion
3
.1. Chemistry
Fig. 3. Proposed coordinating mode of H L in the complexes (1a) and (2).
2
2
The ligand H L (Fig. 1) was easily prepared in high yields by conden-
sation of salicyl hydrazide and o-vanillin [23] and it was satisfactorily
characterized. It is present in the E form in solution, as evidenced by
the chemical shift values of the HC_N and NH proton in the H-NMR
spectrum and some examples in the literature [3], the formation of a di-
nuclear Mg
L
2 2
structure is proposed (Fig. 3).
1
1
In the H-NMR spectrum in d -DMSO of 1 and 1a there are two sets
6
spectrum registered in DMSO-d
tri- or tetradentate; in fact, there are diverse O and N donor atoms in
suitable positions to coordinate one or several metal centers. To explore
6
[33]. The ligand can behave as bi-,
of signals: one attributable to the free ligand, and one to the
magnesium(II) complex. The use of a coordinating solvent, therefore,
causes partial decoordination of the ligand. On the contrary, in the spec-
1
its coordination possibilities, H
divalent metal ions (Mg, Mn, Co, Ni, Cu and Zn) in the presence of a base
NEt ), yielding the corresponding metal complexes 1–6 (Fig. 2). Both
2
L was reacted with the acetates of some
tra of (1) and (1a) in MeOD there is a unique set of signals. In the H-
NMR spectrum of (1), the signals are sharp, with only a 0.01–
0.02 ppm shift vs the free ligand; in the spectrum of (1a), instead, the
signals are very broad and the chemical shifts are slightly different. In
particular, the aromatic proton in ortho to the methoxy group and the
methoxy protons themselves are shifted downfield of 0.4 and
0.07 ppm respectively, thus the involvement of the methoxy moiety in
coordination is probable (Fig. 3).
(
3
the 1:1 and the 1:2 metal to ligand ratio were used, but in all cases, in
the presence of one equivalent of base the same chemical species (i.e.,
complexes 1–6, Fig. 2) were recovered. The IR spectra of the complexes
were analyzed in comparison with that of H
magnesium, nickel and zinc complexes (1, 4 and 6) the absorptions as-
2
L. In the IR spectra of the
−
1
sociated with the NH group (2993–3211 cm in the ligand) are still
present, which indicates that NH is not deprotonated upon complexa-
tion. In the manganese, cobalt and copper complexes (2), (3) and (5),
instead, the NH absorption band is absent, and the ligand results
bideprotonated (OH and NH). It is more difficult to rationalize the be-
havior of the C_O absorption. This is a strong band around
ESI-MS data and solution studies (see below) suggest that also the
manganese complex (2) is a dinuclear species Mn L , with the ligand
2 2
bideprotonated (Fig. 3). In the literature there are some examples of dinu-
clear and multinuclear clusters of manganese with 2-hydroxy-3-
methoxy-benzoylhydrazone [12] or 2-hydroxy-3-methoxy-benzylidene
acetohydrazide [3] and, even when a mononuclear stoichiometry is isolat-
ed at the solid state, multinuclear systems are proposed in solution [11].
Some examples are present in the literature of cobalt complexes with
acylhydrazonic ligands, with formation of mononuclear and polynuclear
−
1
1
605 cm
2
in H L; it is not significantly shifted in (2) and (6), while
−
1
there is an upper shift of about 20 cm in the other metal complexes.
On the contrary, the C_O band in acylhydrazones usually undergoes a
downshift upon complexation [34–36]. It has to be taken into account
metal complexes [39–41]. During the synthesis in the presence of NEt
Co(II) is oxidized to Co(III) and the ionic complex [CoL ](NHEt ) (3)
was isolated. The presence of a diamagnetic compound was confirmed
3
,
2
that in H L the C_O group is probably involved in an intramolecular hy-
2
3
drogen bond, as observed in the crystal structure of analogous mole-
cules [37,38], and this results in an abnormally lower IR value
1
by the registration of the H-NMR spectrum in d
sible to observe the signals of the triethylammonium counterion and the
bideprotonated ligand. Compared to H L, the aromatic protons have a
6
-DMSO, where it is pos-
1
2
(ν L and about 1650 cm , generally, in
C_O = 1605 cm− in H
−1
acylhydrazones) [34–36]. The shift of the band due to coordination to
2
the metal ion could be comparable to the lowering induced by intramo-
shift to higher fields of about 0.3–0.4 ppm and to lower fields of about
−
1
lecular hydrogen interaction (about 30–50 cm ). Therefore, it is rea-
sonable to consider that in (2) and (6) the carbonyl oxygen is
coordinated to the metal. X-ray diffraction analysis has confirmed this
hypothesis also for (3) and (5), where the ligand effectively coordinates
the metal in the keto form.
0.35 ppm for the iminic proton. In the MS-ESI(−) spectrum there is the
−
signal relative to the [CoL
2
]
species. Diffraction analysis on a single crys-
tal of 3 confirmed definitively the formation of a bis-chelated ionic com-
pound (see below).
As also recently reported in the literature, acylhydrazones could be
suitable ligands for the synthesis of multinuclear copper complexes [8,
For (1), (4) and (6), it is possible to suggest the formation of a bis-
chelated compound M(HL)
and coordinates to the metal by means of the phenolic oxygen, the
2
, where the ligand is monodeprotonated
42]. The reaction of copper(II) acetate with H
2
L afforded the green
·4CH CN were
tetranuclear complex Cu ·3H O (5); crystals of Cu
L
4 4
2
4
L
4
3
iminic nitrogen and the carbonylic oxygen. For complex (2), instead, a
obtained by recrystallization of (5) from acetonitrile and characterized
dinuclear metal complex of type Mn
L
2 2
was isolated. ESI-MS and ele-
by X-ray diffraction analysis. It is interesting to note that the presence
mental analysis support such hypothesis. The potentiometric measure-
ments discussed in the following section indicate for magnesium the
presence of another species in solution with a Mg:ligand 1:1 ratio. To
4 4
of the Cu L species was confirmed in solution by means of ESI-MS
(see Supplementary material). In (5) the ligand is bideprotonated, as in-
ferred also by IR data, and coordinates to the metal in an ONO fashion,
through the deprotonated phenolic oxygen, the iminic nitrogen and
the carbonylic oxygen. It is well known [43–47] that acylhydrazones ex-
hibit keto–enol tautomerism in solution and that, if complexation is
followed by deprotonation of the ligand, the enol form is stabilized. In
the case of (5), instead, the ligand coordinates to the metal center in
2
isolate this species, magnesium acetate was allowed to react with H L
in the presence of 2.5 equivalents of NaOH (pH 8–9) in a 1:1 metal to li-
gand ratio: in these conditions, the new complex (1a) was effectively
isolated. In (1a) the ligand is bideprotonated (in the IR spectrum, the
−
1
band associated to the NH absorption at 3267 cm , which is clearly vis-
−
1
ible in the spectrum of (1), is absent). On the basis of the ESI-MS
the keto form (see crystal structure analysis and νC_O = 1629 cm ).

D. Rogolino et al. / Journal of Inorganic Biochemistry 150 (2015) 9–17
13
3
.2. X-ray analysis
4 4 3
Compound Cu L ·4CH CN is obtained by crystallization from aceto-
nitrile, that is found in the packing of the crystal structure together with
the tetranuclear complex. Cu ·4CH CN crystallizes in the tetragonal
space group I4 /a, and the molecular structure of the complex is
shown in Figs. 4 and 5.
The Cu neutral molecule is based on the monomeric unit shown
L
4 4
3
1
4 4
L
in Fig. 4, built by the coordination of the bideprotonated ligand to one
copper(II) ion, in a tridentate O\\N\\O chelation that generates one
five-membered and one six-membered chelation ring with bite angles
of 82(1)° and 93(1)°, respectively. The unit is practically planar (rms de-
viation from planarity = 0.18 Å), since the rotatable terminal 2-
hydroxy-phenyl ring is locked in the plane by an intramolecular hydro-
gen bond to the amidic nitrogen [O4\\H…N1 = 2.593(6) Å, 128(6)°].
This intramolecular hydrogen bond in acylhydrazonic ligands similar
Fig. 5. Left: Cu
4
O
4
molecular core of the complex Cu
4
L
4
·4CH
3
CN. The elongation of the
dotted bonds is due to the repulsion between ligand evidenced in the image on the right.
2
to H L has been observed in analogous coordination compounds, and,
interestingly, it points to the stabilization of the keto form in the keto-
enol tautomerism of the ligand [48]. The tetranuclear neutral complex
much longer contact (2.612(3)Å). The methoxy oxygen O
ed to the second copper ion generating a five-membered chelation ring
involving O and O , slightly distorted in an envelope conformation with
2
is also bond-
(
Fig. 5) is generated by a fourfold rotoinversion axis whereby the
hydrazonic carbonyl O bridges three copper ions, the first through a
3
2
3
short bond [1.957(3) Å, belonging to the O\\N\\O chelation], the sec-
ond to the metal in the position (1/4 − y, x − 3/4,1/4 − z) by a slightly
longer bond (1.991 Å), and the third at (1 − x, −1/2 − y, z) through a
the metal at the flap deviating 0.45 Å form the plane defined by the che-
lated system. The copper ion shows a distorted square pyramidal coor-
dination with a longer contact completing a potential octahedron. The
4 4 3
Fig. 4. Molecular structure and labeling, with thermal ellipsoids at the 50% probability level, of the monomeric unit in the complex Cu L ·4CH CN.

14
D. Rogolino et al. / Journal of Inorganic Biochemistry 150 (2015) 9–17
resulting complex shows a Cu
4
L
4
core with eight short (1.957 and
divalent metal ions (Mg2+, Mn2+, Zn2+, Ni2+, Cu2+) were carried out
1
.991 Å) and four long (2.612 Å) Cu\\O distances (Fig. 5). The elonga-
in mixed solvent methanol/water = 9/1 v/v, in which the ligand and al-
most all the complexes are soluble. The ionic strength was adjusted to
0.1 M KCl. H L is diprotic with pKa1 = 8.43 ± 0.01 and pKa2 =
2
tion of these contacts, that hinders the completion of the octahedral co-
ordination, is due to the repulsion between the ligand skeletons that
face each other and whose average plane diverge towards the outer
part of the complex (Fig. 5). The same complex has been previously de-
scribed as the DMF solvate [49], showing the same arrangement, the
same space group, but a different cell and a different crystal packing;
12.11 ± 0.01; the more acidic group is probably the OH of the o-
vanillin moiety, and pKa2 should be attributed to the dissociation of
the salicylic OH. The best fit of the experimental titration curves, carried
out in the presence of the metal ions, has been obtained by the
Hyperquad software with the sets of species shown in Table 2. The pro-
posed models are corroborated by very good statistical parameters and
by nice overlapping between experimental and computed titration
curves. The sets reported in Table 2 have been chosen following both
the criteria of the minimum standard deviation and of chemical
soundness.
4 4 3
the crystal packing of Cu L ·4CH CN is shown in Fig. 6.
Compound (3) has been obtained as extremely twinned and weakly
diffracting crystals, which nevertheless allowed unambiguous determi-
nation of the structure of the salt [CoL ](NHEt ), shown in Fig. 7.
2 3
In the octahedral anionic bischelate complex, the two deprotonated
ligands are coordinated by the same tridentate O\\N\\O chelation
observed in the copper tetranuclear complex, forming one six-
membered and one five-membered chelate ring with bite angles of
For Mg2+, Mn2+, Zn2+, and Ni2+, the species found in solution are
MLH, ML
state the complexes ML
by changing the reaction conditions, it is also possible to obtain the spe-
cies with a stoichiometric ratio 1:1 Mg (1a). ZnL can be found, but
2
H, ML, and ML
2
(charges are omitted for simplicity). In solid
9
5° and 83°, respectively, for both ligands. As above, the structure of
2 2
H are isolated for Mg, Ni and Zn, but, simply
the complex unit is stabilized by an intramolecular OH…N hydrogen
bond, with the nitrogen atom of the amide group acting as the hydrogen
L
2 2
2 2
H
bond acceptor [O5\\H…N3 = 2.56(1) Å, 148(1)°; O1\\H…N1 =
in low quantities and with a high standard deviation, so it was excluded
for statistical reasons.
+
2
.58(1) Å, 145(1)°]. The NHEt
3
cation forms a hydrogen bond to the
methoxy group of one of the two coordinated ligands [N5\\H…O4 =
We have tried sets containing other species, in particular bimetallic
3
.19(2) Å, 170(1)°]. Table 1 reports crystal data and structure analysis.
2 2
species such as M LH and M L and we can definitely exclude them for
Zn . To obtain a good fit between experimental and computed titration
2
+
3
.3. Potentiometric measurements
curves, it is necessary to add to the model the oxydrilated species, formed
in small quantities in basic environment. For Mg2 and Zn , at high pH
+
2+
Acylhydrazones represent an interesting class of polydentate, che-
2 2
the hydroxide Mg(OH) and Zn(OH) (in our notation MH−2) are found,
whereas for Mn2 and Ni , the dissociation of a water molecule in the
+
2+
lating agents with a great variety of complexation modes, due to the
possible keto-enol tautomerization and the presence of several poten-
tial donor sites. These peculiarities make the study of their speciation
in solution very attractive, but at the same time quite complicated to un-
ravel; however this information is essential to try to understand their
biological activity and mechanism of action [50]. Potentiometric studies
coordination sphere of the metal is suggested, giving rise to MLH−1 and
ML
2
H−1. Oxydrilated species can be disregarded at L/M ratio greater
2
+
than 1 and at pH lower than 10. The behavior of the ligand with Cu
in solution is different, in fact the only species that we are able to find is
CuL; evidently, as also confirmed by the X-ray structure, the copper(II)
ion greatly increases the acidity of the amidic hydrogen and induces
2
to examine the coordinating behavior of H L in solution with a variety of
4 4 3
Fig. 6. Crystal packing of Cu L ·4CH CN with acetonitrile highlighted in spacefilling representation.

D. Rogolino et al. / Journal of Inorganic Biochemistry 150 (2015) 9–17
15
Fig. 7. Crystal structure and labeling of compound (3), with thermal ellipsoids at the 50% probability level. The cation has not been shaded for clarity.
bideprotonation of the ligand. It seems therefore that the formation of a
tetranuclear cluster is particularly favored by thermodynamic
parameters.
Note that we cannot exclude the presence of species with the gener-
n n
al formula M L : the mathematical processing cannot distinguish be-
the different divalent metal ion and by the ligand under investigation
at M/L = 1/2 are compared. It is noteworthy that Mg2 at physiological
pH is almost completely present as aqua ion and that the complexation
takes place only in basic environment, unlike the other metal ions, for
which the complexation already starts at acidic pH.
+
tween the different n values, because the correlation between the
species does not allow the convergence of the iterative calculation. Ef-
fectively, as already discussed, mass spectrometric data confirm the for-
3.4. Biological activity
mation of Cu
L
4 4
also in solution, in agreement with the structure of the
As a part of an antiviral testing program conducted in our laboratory
complex (5) isolated in the solid state. The same argument is valid also
[17–20], we evaluated the inhibitory activity of H L and its metal com-
2
with the other cations under study: we cannot exclude the formation in
plexes 1–6 against a broad panel of DNA- [i.e., herpes simplex virus
solution of dimeric M
ed at the solid state in (1a) (Mg
2
L
2
and/or M
n
L
n
species, as then effectively isolat-
type 1 (HSV-1) and type 2 (HSV-2), VV and adenovirus (Ad-2), evaluat-
ed in human embryonic lung fibroblast HEL cells] and RNA-viruses (i.e.,
Coxsackie B4 virus and respiratory syncytium virus, tested in HeLa cells;
parainfluenza-3 virus and Punta Toro virus, tested in Vero cells; influen-
za virus, tested in MDCK cells; human immunodeficiency virus type 1
and type 2, tested in human MT-4 lymphoblast cells; vesicular
2
L
2
·4H O) and in (2) (Mn ·2H O).
2
2
L
2
2
The values of the formation constants of the various species differ great-
ly (Table 2). In particular, as expected, the stability constant for the com-
2
+
2+
plexes of Mg
are the lowest, whereas Ni
shows the highest
affinities. In Fig. 8 the distribution diagrams of the systems formed by
Table 2
p q r
L H .
]/[M] [L] [H] ) in methanol/water = 9:1 v/v, I = 0.1 M KCl at 25 °C for the ligand under study with Mg2+, Mn2+, Zn2+, Ni2+, and Cu2+
p
q
r
Logarithms of formation constants (βpqr = [M
SDs are given in parentheses.
p
q
r
Mg(II)
Log ßpqr
Mn(II)
Zn(II)
Ni(II)
Cu(II)
Log ßpqr
Log ßpqr
Log ßpqr
Log ßpqr
1
1
1
1
1
2
2
1
1
1
0
0
14.87 (0.17)
22.04 (0.13)
11.76 (0.07)
7.64 (0.02)
17.45 (0.05)
26.23 (0.17)
17.32 (0.19)
10.41 (0.04)
17.04 (0.13)
20.58 (0.02)
32.72 (0.36)
24.8 (0.59)
14.51 (0.05)
27.64 (0.14)
16.28 (0.19)
10.74 (0.06)
14.12
(
0.01)
1
0
−2
−21.42
−15.52 (0.18)
(0.09)
1
1
1
2
−1
−1
−1.61 (0.05)
5.67 (0.2)
3.78 (0.06)
12.62 (0.59)
0
0
1
1
1
2
L + H = LH
L + 2H = LH
Log ß011 = 8.43 (0.01)
Log ß012 = 12.11
2
(0.01)

16
D. Rogolino et al. / Journal of Inorganic Biochemistry 150 (2015) 9–17
Fig. 8. Distribution diagrams for the systems under investigation at M/L = 1/2 (methanol/water = 9:1 v/v, I = 0.1 M KCl).
stomatitis virus, tested in human embryonic lung fibroblast cells). None
of the compounds showed activity against any of the RNA viruses tested,
including human immunodeficiency virus type 1 and type 2, and influ-
enza virus (data not shown). For the latter virus, the activities of the
compounds were evaluated in a virus yield assay in MDCK cells, as
well as in an enzymatic assay with the influenza virus PA endonuclease,
which is a Mg (or Mn ) -dependent metalloenzyme [17,20,53].
On the other hand, the tested compounds showed very interesting
activity against the DNA-viruses herpes simplex virus (i.e., HSV-1,
HSV-2, and an acyclovir-resistant thymidine-kinase deficient HSV-1
strain) and VV. The ligand and metal complexes were not active against
Ad-2, which is a naked DNA-virus. To the best of our knowledge, no data
are available in the literature about activity of preformed metal com-
plexes with hydrazonic ligands against these viruses.
The antiviral activity and cytotoxicity of H
are summarized in Table 3.
2
L and its complexes 1–6
It is worth noting that, while (3) and (5) were inactive, H
2
L and its
complexes (1), (2), (4) and (6) had EC50 values in the low micromolar
range. Complexes (1), (2), (6) had particularly pronounced activity
against VV, with EC50 values of 0.6, 0.5 and 0.4 μM, respectively. They
had a favorable selectivity index (i.e., ratio of cytotoxic concentration
to antivirally effective concentration for VV) in the range of 33–50.
One compound in the series, i.e., the copper complex (5) displayed
prominent cytotoxicity yet was devoid of antiviral activity.
The biological properties of acylhydrazones and thiosemicarbazones
are often related to their coordination abilities [52], or, for example, to
the possibility that they may act as carriers for metal ions, allowing pen-
etration of the viral envelope [53].
2
+
2+
Clearly, the mechanism of action and precise viral target of the com-
pounds presented here remain to be identified. One possibility worth
further investigation is the D10 decapping enzyme of vaccinia virus,
which was recently reported to act through a two-metal-ion mecha-
nism [54] and, hence, may be sensitive to metal-chelating inhibitors.
An explanation for the inactivity of the copper and cobalt complexes
(5 and 3) can be found on structural basis. In fact, the Mg, Mn, Ni and Zn
Table 3
Antiviral activity and cytotoxicity of compounds H
onic lung fibroblast cells.
2
L and 1–6 in cultures of human embry-
a
Compound
Antiviral activity (EC50 in μM)
Cytotoxicity
MCC in μM
b
HSV-1 HSV-1/TK− HSV-2
VV
VSV
Ad-2
2
or
complexes (1), (2), (4) and (6) are neutral, with formulas M(HL)
Mn . On the other hand, (5) is a tetranuclear cluster Cu and this
cluster is stable also in solution, and (3) is the ionic Co(III) complex
[CoL ](NHEt ). Therefore, their antiviral inactivity could be related, for
2
L
2
4 4
L
H
1
2
3
4
5
6
2
L
1.6
1.3
2.6
0.8
2.2
1.1
1.3
0.6
0.5
N100 N100
N100 N100
N100 N100
60
≥20
≥20
1.1
1.6
0.6
2
3
N100
1.8
N100
1.8
N100
4.3
N100 N100 N100
2.9 N100 N100
N100 N100 N100
20
100
0.8
example, to insufficient cellular uptake.
Paradoxically, this marked difference in biological activity in relation
to a striking structural difference, can be considered as proof that the
complexes are stable in the assay conditions.
N100
1.1
N100
0.8
N100
0.8
0.4
17
N100 N100
≥20
Brivudin
Acyclovir
Ganciclovir 0.025
Cidofovir
Zalcitabine
Alovudine
0.05
0.25
30
10
173
0.09
N250
ND
ND
ND
7.9
7.5
16
N250
N250
N100
N250
N250
N250
N250 N250
0.025 N100 N100
0.5
4. Conclusions
0.95
ND
ND
1.1
ND
ND
0.65
ND
ND
16
ND
ND
N250
ND
We have started a project focused on the synthesis and biological
ND
evaluation of chemical scaffolds that could be able to bind the metal co-
factors in the active site of some viral enzymes and, in this way, can in-
hibit virus replication [17–20,51]. From this perspective, hydrazones
seem promising, because of their well-known coordinating abilities
and biological properties [4–11,13–16]. We have focused our attention
on the o-vanillin acylhydrazone scaffold: the potentially tetradentate li-
Values shown are the average of two independent tests.
ND: not determined.
HSV-1: herpes simplex virus type 1; HSV-1/TK−: acyclovir-resistant thymidine-kinase de-
ficient HSV-1; HSV-2: herpes simplex virus type 2;VV: vaccinia virus; VSV: vesicular sto-
matitis virus; Ad-2: human adenovirus type 2.
a
EC50: 50% effective concentration, i.e., compound concentration producing 50% inhi-
bition of virus-induced CPE, determined by microscopy.
b
gand H
L shows a great versatility. H
L promotes the formation of bis-
with Mg(II), Ni(II), and
MCC: minimum cytotoxic concentration, i.e., compound concentration producing mini-
2
2
mal alterations in cell morphology, determined by microscopy.
chelated, tridentate complexes of type M(HL)
2

D. Rogolino et al. / Journal of Inorganic Biochemistry 150 (2015) 9–17
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