Versatile coordinating behaviour of bis(acylhydrazone) ligands derived from imino- and methyl-iminodiacetic acid diethyl ester. Antimicrobial properties of their trinuclear copper(II) complexes

Article abstract of DOI:10.1007/s11243-009-9321-x

The coordinating properties of a new bis(pyr- idylhydrazone) ligand derived from iminodiacetic acid diethyl ester and 2-pyridinecarboxaldehyde (picolinalde- hyde) H₃Imdp and of the bis(salicylhydrazone) H ₅Imds and H₄MeImds ligands derived, respectively, from iminodi- acetic acid diethyl ester and from methyl-iminodiacetic acid diethyl ester and salicylaldehyde were considered, by means of analytical and spectroscopic methods, towards first row transition metal ions. These ligands showed various coordination modes in complexation with Cu(II), Co(II), Mn(II) and Zn(II) ions. In particular, we have synthesized and characterized, by analytical, ¹H NMR and IR techniques, tri-, di- and mononuclear metal complexes of formula Co₃(HImdp)(NO₃)₄2H ₂O, Cu₃(HImdp)(NO₃)₄ C ₂H₅OHH₂O, Cu₃(HImdp)Cl₄, Zn₂(H₃Imdp)(ClO₄)₄2H₂O, Co₃(HImds)Cl₂CH₃OHH₂O, Zn ₂(H₃Imds)Cl₂2H₂O, Co-(H ₄Imds)NO₃ 2H₂O, Mn(H₄Imds)ClCH ₃OHH₂O, Cu(H_3- Imds)CH₃OHH ₂O and Cu(H₂MeImds)CH₃OH3H₂O. Antibacterial, antifungal and antiprotozoal properties of H₅Imds and H₃Imdp together with three copper(II) trinuclear species of H ₅Imds of formula Cu₃(HImds)(NO₃) ₂2CH₃OH 2H₂O, Cu₃(HImds)(ClO ₄)₂EtOH2H₂Oand Cu3(HImds)SO₄ 4H ₂O are also discussed. The H₅Imds ligand and their trinuclear copper(II) complexes showed good activities versus Trichomonas vaginalis, Staphylococcus epidermidis and Acanthamoeba castellanii. Springer Science+Business Media B.V. 2009.

Full text of DOI:10.1007/s11243-009-9321-x

Transition Met Chem (2010) 35:253–261
DOI 10.1007/s11243-009-9321-x
Versatile coordinating behaviour of bis(acylhydrazone) ligands
derived from imino- and methyl-iminodiacetic acid diethyl ester.
Antimicrobial properties of their trinuclear copper(II) complexes
•
Claudia Juliano Antonella Mattana
•
Costantino Solinas
Received: 3 November 2009 / Accepted: 1 December 2009 / Published online: 24 December 2009
ꢀ Springer Science+Business Media B.V. 2009
Abstract The coordinating properties of a new bis(pyr-
idylhydrazone) ligand derived from iminodiacetic acid
diethyl ester and 2-pyridinecarboxaldehyde (picolinalde-
hyde) H3Imdp and of the bis(salicylhydrazone) H5Imds and
H4MeImds ligands derived, respectively, from iminodi-
acetic acid diethyl ester and from methyl-iminodiacetic
acid diethyl ester and salicylaldehyde were considered, by
means of analytical and spectroscopic methods, towards
first row transition metal ions. These ligands showed var-
ious coordination modes in complexation with Cu(II),
Co(II), Mn(II) and Zn(II) ions. In particular, we have
Introduction
Di- and poly-nuclear metal complexes of hydrazonic
ligands play a significant role in the study of multi-electron
redox reactions, magnetic exchange interactions and
intramolecular binding [21, 24]. Acylhydrazone and
semicarbazone derivative compounds occupy an important
position as ligands in metal coordination chemistry,
because this general class of chelating agents shows dif-
ferent stereochemistries and nuclearity in complexation
with transition metal ions [4, 16]. In this context, our
attention was mainly attracted by bis(acylhydrazone)
ligands derived, in particular, from 2,6-diacetylpyridine,
whose interest as chelating agents is due to their versatility
in complexation with different metal ions giving a wide
variety of metal complexes that could range from mono- to
di-nuclear [3, 8] and polynuclear species [9, 15]. We are
currently interested in the coordinating behaviour of some
compartmental bis(acylhydrazones) derived from imino-
and methyl-iminodiacetic acid diethyl ester towards first
row transition metal ions. To date, we considered the
H5Imds and H4MeImds bis(salicylhydrazone) ligands
(Fig. 1), prepared by the reaction of the bis(hydrazide) of
imino- and methyl-iminodiacetic acid diethyl ester with
salicylaldehyde as previously described [2, 6], and the new
bis(pyridylhydrazone) H3Imdp (Fig. 2), derived from the
bis(hydrazide) of iminodiacetic acid diethyl ester with
2-pyridinecarboxaldehyde (picolinaldehyde). Previously, we
reported the synthesis of some linear trinuclear copper(II)
complexes of the bis(salicylhydrazone) H5Imds ligand,
together with the X-ray crystal structure of a coordination
polymer, of formula [Cu6(HImds)2(dmso)5(EtOH)(H2O)2]-
(ClO4)4Á2H2O [2] and the crystal structure of a sulphate-bridged
hexanuclear dimer complex [Cu3(MeImds)(SO4)(H2O)3]Á
5H2O from H4MeImds bis(salicylhydrazone) [6]. Regarding
1
synthesized and characterized, by analytical, H NMR and
IR techniques, tri-, di- and mononuclear metal complexes
of formula Co3(HImdp)(NO3)4Á2H2O, Cu3(HImdp)(NO3)4Á
C2H5OHÁH2O, Cu3(HImdp)Cl4, Zn2(H3Imdp)(ClO4)4Á2H2O,
Co3(HImds)Cl2ÁCH3OHÁH2O, Zn2(H3Imds)Cl2Á2H2O, Co-
(H4Imds)NO3Á2H2O, Mn(H4Imds)ClÁCH3OHÁH2O, Cu(H3-
Imds)ÁCH3OHÁH2O and Cu(H2MeImds).CH3OHÁ3H2O.
Antibacterial, antifungal and antiprotozoal properties of
H5Imds and H3Imdp together with three copper(II) trinuclear
species of H5Imds of formula Cu3(HImds)(NO3).22CH3OHÁ
2H2O, Cu3(HImds)(ClO4).2EtOHÁ2H2O and Cu3(HImds)SO4Á
4H2O are also discussed. The H5Imds ligand and their
trinuclear copper(II) complexes showed good activities
versus Trichomonas vaginalis, Staphylococcus epidermidis
and Acanthamoeba castellanii.
C. Juliano Á A. Mattana Á C. Solinas (&)
`
Dipartimento di Scienze del Farmaco, Universita di Sassari,
via Muroni 23/a, 07100 Sassari, Italy
e-mail: dsfcosta@uniss.it
123

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Transition Met Chem (2010) 35:253–261
and antiprotozoal properties of H5Imds, H3Imdp ligands
together with the trinuclear copper(II) complexes Cu3-
(HImds)(ClO4)2. EtOHÁ2H2O, Cu3(HImds)(NO3)2. 2CH3OHÁ
2H2O and Cu3(HImds)SO4Á4H2O, previously reported, are
here discussed.
O
O
H
N
N
N
C
N
C
N
N
H
N
H
Studies concerning the coordinating behaviour of
H3Imdp, H5Imds and H4MeImds or derivatives towards
other transition metal ions, in order to obtain mono- or
poly-nuclear species and hetero-nuclear metal complexes,
together with the antimicrobial properties of metal com-
plexes, also reported in this paper, are now in progress.
Fig. 1 The compartmental acyclic H3Imdp ligand
HO
OH
R
N
O
O
C
N
C
N
N
H
N
H
Fig. 2 The compartmental acyclic ligands H5Imds (R = H) and
H4MeImds (R = methyl)
Experimental
All reactants and solvents were reagent grade and used
without further purification. Iminodiacetic acid, methyli-
minodiacetic acid, salicylaldehyde, 2-pyridinecarboxalde-
hyde (picolinaldehyde), hydrazine hydrate (98%) and metal
salts were purchased from Aldrich. Elemental analyses (C, H
and N) were performed on Carlo Erba instruments CHNS-
O EA 1108 automatic equipment. All infrared spectra were
recorded using KBr discs on a Perkin–Elmer mod. 781 IR
spectrophotometer. UV/Vis spectra were recorded on a
this, we observed that H5Imds and H4MeImds act as
aggressive coordinator ligands towards copper(II) ions,
giving only linear trinuclear species of general formula
Cu3(HImds)X2ÁnH2O (X = nitrate, chloride and perchlorate),
Cu3(HImds)XÁnH2O (X = sulphate), Cu3(MeImds)X2ÁnH2O
(X = nitrate, chloride and perchlorate) Cu3(MeImds)XÁnH2O
(X = sulphate), this independently of the ligand/metal
molar ratio. In contrast, we found, as here reported, that the
reaction of copper(II) acetate with these ligands in 1:1
molar ratio, gave only mononuclear complexes of formula,
respectively, Cu(H3Imds).CH3OHÁH2O and Cu(H2MeI-
mds).CH3OHÁ3H2O. As a continuation of this work, here,
we report preliminary investigations showing the versatile
coordinating behaviour, the synthesis and the characteriza-
tion of H3Imdp and their trinuclear Cu(II) and Co(II) com-
plexes of formula Co3(HImdp)(NO3)4Á2H2O, Cu3(HImdp)
(NO3)4.C2H5OHÁH2O and Cu3(HImdp)Cl4, the dinuclear
zinc(II) complex Zn2(H3Imdp)(ClO4)4Á2H2O together with
the synthesis and the characterization of dinuclear Zn(II),
trinuclear and mononuclear Co(II) and Mn(II) complexes
of H5Imds, respectively, of formula Zn2(H3Imds)Cl2Á3H2O,
Co3(HImds)Cl2. CH3OHÁH2O, Co(H4Imds)NO3Á2H2O, Mn-
(H4Imds)Cl.CH3OHÁH2O, under 3:1 metal/ligand molar
ratio conditions, and the above-mentioned Cu(II) mononu-
clear complexes of H5Imds and H4MeImds ligands. All the
compounds have been characterized by analytical and IR
spectroscopic techniques. It is interesting to note that the
Cu3 core complexes are of interest as potential copper
biosite modelling, whereas trinuclear copper clusters are
studied as models for the metallo-biosite of the ‘‘blue pro-
teins’’ such as ascorbate oxidase and laccase [1, 11]. In
addition, several studies emphasized that some bis-
acylhydrazone or semi- and thio-semicarbazone ligands and
their metal complexes with Cu(II), Zn(II) or Sn(IV) showed
significant pharmacological properties and, in particular, as
regards the antimicrobial activity [5, 10, 19]. In this context,
preliminary studies concerning the antibacterial, antifungal
Beckman DU-65 spectrophotometer. The 1H NMR and 13
C
NMR were performed on a Varian 200 XL instrument. The
spectra were recorded at 300 K in solution of DMSO-d6; all
chemical shifts are relative to internal tetramethylsilane.
Synthesis of the ligands
H3Imdp
The bis(hydrazide) was prepared from iminodiacetic acid
diethyl ester according to the literature [12]. H3Imdp was
obtained by dropwise addition of an absolute ethanol
(50 mL) solution of 2-pyridinecarboxaldehyde (1.52 mL,
16 mmol) to an hot absolute ethanol suspended solution
(90 mL) of bis(hydrazide) (1.29 g, 8 mmol). After addition
of the 2-pyridinecarboxaldehyde to the suspension of the
bis(hydrazide), the resulting solution was completely clear;
it was then refluxed for 5 h. After evaporation, in vacuo, of
the 50% of the solvent, a pale yellow precipitate was
obtained by addition of n-hexane (20 mL). After filtration,
the title compound was washed with CHCl3 and dried in
vacuo. (yield 90%). m.p. = 181–183 ꢁC. Anal. found: C,
56.7; H, 5.2; N, 28.8%. Calc. for C16H17N7O2 C, 56.6; H,
5.1; N, 29.0%. 1H NMR [(CD3)2SO, 25 ꢁC]: d 3.37 (s, 4H,
CH2), 7.39–8.29 (m, 8H, C6H4), 8.57 (s, 2H, CH=N), 11.61
(s, 2H, N–H hydrazonic, exchangeable). 13C NMR
[(CD3)2SO, 25 ꢁC]: d 169.53 (–NH–C=O), 152.617 (–C=N),
149.59, 144.95, 136.88, 124.53, 119.88 (pyridinics C),
47.80 (–CH2–N).
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Transition Met Chem (2010) 35:253–261
255
IR (KBr, cm-1): 3450, 3100 m(NH)ms, 3050m (aryl),
1680vs (amide I), 1618m m(C=N), 1530m (amide II), 1270s
(amide III). UV/Vis (dmso): kmax = 330 nm.
H5Imds complexes
The preparation of H5Imds complexes was carried out
following the procedure used in a previous work [2]: a
methanolic solution of the Co(II) (chloride and nitrate),
Zn(II) (chloride) and Mn(II) (chloride) (3:1 metal/ligand
molar ratio) was added to a methanol/chloroform (5:1, v/v)
solution of the ligand and refluxed for 1.5 h. In all cases,
the precipitate formed was isolated, dried and washed with
MeOH.
Co3(HImds)Cl.2CH3OHÁ3H2O, Anal. found: C, 34.4; H,
3.6; N, 10.9%. Calc. for C19H25N5O6Cl2Co3, C, 34.2; H,
3.8; N, 10.5%. IR (KBr, cm-1): 1610m m(C=N–N=C).
Co(H4Imds)NO3Á2H2O, Anal. Found: C, 42.1; H, 4.0;
N, 16.7%. Calc. For C18H22N6O8Co, C, 42.5; H, 4.4;
N, 16.5%. IR (KBr, cm-1): 3220–2960mb m(OH, hydrogen
bonded), 1688m and 1660ms (amide I), 1610–1605m
m(C=N), 1385vs m(NO3).
H5Imds and H4MeImds ligands
H5Imds and H4MeImds were prepared as described [2, 6].
IR spectra and 13C NMR
H5Imds; (KBr, cm-1): 3475, 3380, 3190m m(OH, NH),
1683vs (amide I), 1615s m(C=N), 1520ms (amide II), 1260s
(amide III). 13C NMR [(CD3)2SO, 25 ꢁC]: d 167.60 (–NH–
C=O), 157.46 (–C=N), 147.73, 131.43, 129.67, 119.43,
118.67, 116.47 (aromatics C), 51.16 (–CH2–N).
H4MeImds; (KBr, cm-1): 3420, 3200ms m(OH, NH),
3050, 1670vs (amide I), 1620s m(C=N), 1520m (amide II),
1270s (amide III). 13C NMR [(CD3)2SO, 25 ꢁC]: d 167.00
(–NH–C=O), 157.46 (–C=N), 148.33, 132.12, 129.87,
120.07, 118.92, 116.77 (aromatics C), 59.89 (–CH2–N),
43.27 (–N–CH3).
Mn(H4Imds)Cl.CH3OHÁH2O, Anal. found: C, 44.5; H,
4.6; N, 13.5%. Calc. for C19H24N5O6ClMn, C, 44.9; H, 4.8;
N, 13.8%. IR (KBr, cm-1): 3210–2950mb m(OH, hydrogen
bonded), 1665–1689vs (amide I), 1610s m(C=N).
Zn2(H3Imds)Cl2Á2H2O, Anal. found: C, 35.2; H, 3.7;
N, 11.7%. Calc. for C18H21N5O6Cl2Zn2, C, 35.7; H, 3.5;
N, 11.6%. IR (KBr, cm-1): 1640vs (amide I), 1610ms
m(C=N).
Synthesis of the complexes
H3Imdp complexes
An absolute ethanol solution of copper(II) (nitrate and
chloride), or cobalt(II) nitrate hydrate salts was added (3:1,
metal/ligand molar ratio) dropwise, under magnetic stir-
ring, to an hot absolute ethanol solution of the ligand. The
solution was heated at 70 ꢁC for about 2 h. The Zn(II)
perchlorate complex was prepared in the same conditions,
but using 2:1 metal/ligand molar ratio. In all cases, a pre-
cipitate was formed. The solution was filtered; the pre-
cipitate washed with MeOH and dried in vacuo.
Cu3(HImdp)(NO3)4.C2H5OHÁH2O, Anal. found: C, 25.3;
H, 2.6; N, 18.2%. Calc. for Cu3C18N11H23O16, C, 25.7; H,
2.8, N, 18.3%. IR (KBr, cm-1): 1615m m(C=N–N=C),
1380vs m(NO3).
Cu3(HImdp)Cl4, Anal. found: C, 28.9; H, 2.6; N, 14.0%.
Calc. for Cu3C16N7H15O2 Cl4, C, 28.7; H, 2.3; N, 14.6%.
IR (KBr, cm-1): 1615m m(C=N–N=C).
Co3(HImdp)(NO3)4Á2H2O, Anal. found: C, 23.1; H, 2.2;
N, 18.2%, Calc. for Co3C16H19N11O18, C, 23.2; H, 2.3; N,
18.6%. IR (KBr, cm-1): 1615m m(C=N–N=C), 1385vs
m(NO3).
Synthesis of Cu(H3Imds).CH3OHÁH2O and
Cu(H2MeImds).CH3OHÁ3H2O complexes
The
mononuclear
Cu(H3Imds).CH3OHÁH2O
and
Cu(H2MeImds).CH3OHÁ3H2O, derived, respectively, from
H5Imds and H4MeImds ligands, were obtained using cop-
per(II) acetate in 1:1 metal/ligand molar ratio, in MeOH/
CHCl3 solution for 1 h at 60 ꢁC. In all cases, a green
precipitate was formed and characterized as:
Cu(H3Imds).CH3OHÁH2O, Anal. found C, 47.5; H, 4.4;
N, 14.8%. Calc. for C19H22N5O6Cu, C, 47.6; H, 4.6; N,
14.6%. IR (KBr, cm-1): 3411m m(NH, hydrazonic), 1680ms
(amide I), 1610vs m(C=N).
Cu(H2MeImds).CH3OHÁ3H2O, Anal. found C, 46.0; H,
5.7; N, 13.8%. Calc. for C, C20H29N5O8Cu, C, 45.2; H, 5.5;
N, 13.2%. IR (KBr, cm-1): 1665ms (amide I), 1610vs
m(C=N).
Preparation of H5Imds copper(II) trinuclear complexes
for antimicrobial tests
Zn2(H3Imdp)(ClO4)4Á2H2O, Anal. found: C, 21.4; H,
2.3; N, 18.9%. Calc. for Zn2C16H21N7O20Cl4, C, 21.3; H,
2.3; N, 18.9%. IR (KBr, cm-1): 1660ms, m(amide I),
1610m m (C=N), 1100vs m(ClO4). 1H NMR [(CD3)2SO,
25 ꢁC] : d 3.28 (s, 4H, CH2), 7.47–8.72 (m, 8H, C6H4),
8.72 (s, 2H, C=N),12.20 (s, 2H, N–H hydrazonic).
Cu3(HImds)(NO3)2. 2CH3OHÁ2H2O, and Cu3(HImds)SO4Á
4H2O complexes, utilized for antimicrobial screenings,
were prepared as reported [2, 6]. The perchlorate complex
of formula Cu3(HImds)(ClO4).2EtOHÁ2H2O was prepared
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Transition Met Chem (2010) 35:253–261
as described [2] and used without recrystallization in
powder form.
mL and 100 lL of the calibrate suspension was added to
each well. Final concentrations obtained ranged from 250–
0.976 lg mL-1. Microtiter plates were incubated aerobi-
cally at 37 ꢁC, in a humid atmosphere containing 5% CO2.
At 24, 48, 72 and 96 h of incubation, they were examined
with an inverted microscope (OLIMPUS CK) and checked
for protozoan motility. The lowest concentration in which
Antimicrobial activity
Bis(acylhydrazones) H5Imds, H3Imdp and related cop-
per(II) complexes were dissolved in dimethyl sulfoxide
(DMSO), the resulting stock solutions (20 mg mL-1) were
stored at ?4 ꢁC. In all cases, preliminary tests with DMSO
were performed to assure that no microorganism inhibition
occurred at used concentrations. Tests were performed in
duplicate and were repeated at least three times.
no motile flagellates were seen was defined as MIC100
according to Meingassner and Thurner [17].
,
To study the antiamoebic activity of compounds, we
used trophozoites of Acanthamoeba castellanii, isolated
from a case of amoebic keratitis (in Ancona, Italy), axe-
nically grown at 25 ꢁC in PYG medium [14]. The species
identification of this isolate was based on cyst morphology
and PCR analysis including primer pair JDP1/JDP2 (tar-
geted to 18S rDNA stretch ASA.S1) [20]. Experiments
were performed in sterile 96-well plates. Serial twofold
Their antibacterial and antifungal activity was deter-
mined as minimum inhibitory concentration (MIC), by
using an agar dilution technique [7, 23]. Bacteria included
both Gram-positive (Bacillus spp., Staphylococcus aureus
ATCC 25923, Staphylococcus aureus ATCC 6538,
Staphylococcus epidermidis of clinical isolation) and
Gram-negative strains (Escherichia coli ATCC 25922,
Pseudomonas aeruginosa ATCC 27853, Citrobacter
freundii, Enterobacter cloacae, Klebsiella pneumoniae of
clinical isolation). Antifungal assays were performed
against Candida albicans ATCC 10231, Aspergillus niger
ATCC 16404, Pichia guillermondii and Rhodotorula glu-
tinis. Twofold serial dilutions of compound solutions (250–
3.9 lg mL-1) were prepared in molten (45 ꢁC) Mueller–
Hinton Agar (MHA, Oxoid) for bacteria or potato dextrose
agar (PDA, Oxoid) for fungi, and poured in 50-mm-
diameter Petri plates. Each plate was inoculated with drops
containing 1 9 104 bacteria or 103–104 conidia or yeast
cells. For bacteria, MICs were recorded after 18–24 h of
incubation at 35 ꢁC as the lowest concentration which
completely inhibit bacterial growth. For fungi, plates were
incubated at 25 ꢁC and daily checked for at least 4 days to
assess fungal growth.
dilution of the compounds, ranging from 100 to 25 lg mL-1
,
were prepared in PYG medium. Control wells received
100 ll of PYG medium in place of drug dilutions.
Amoebae, washed twice in PBS, were suspended in PYG
medium at a density of 8 9 103 cells mL-1. One hundred
microlitres of the calibrated trophozoite suspension was
added to each well, and then the plates were sealed and
incubated at 37 ꢁC in a 5% CO2 atmosphere. At 2, 3, and
6 days of incubation, amoebae were counted with a
hemacytometer (Nageotte chamber). Cell viability was
determined by the nigrosin dye exclusion method. The
lowest drug concentrations that caused complete (100%)
inhibition of trophozoite growth (MIC100) at days 2, 3, and
6 was evaluated in comparison with that of rokitamycin
(Formenti), used as reference drug (stock solution at
20 mg mL-1, in 95% ethanol).
Antiprotozoal activities were evaluated against Tricho-
monas vaginalis and Acanthamoeba castellanii. Our study
was performed by using a T. vaginalis strain, isolated in
Sassari (Italy) from a case of acute vaginal trichomoniasis,
axenically grown at 37 ꢁC in Diamond medium supple-
mented with 10% heat-inactivated fetal calf serum (FCS,
GIBCO). Stock solution (2 mg mL-1) of metronidazole
(SIGMA), used as reference drug, was prepared in DMSO,
and stored in small aliquots at 4 ꢁC. The susceptibility of
T. vaginalis was determined by the assessment of growth
and motility of flagellates exposed to different concentra-
tions of compounds. Tests were performed in sterile tissue-
culture microtiter 96-well plates (Corning). Serial twofold
dilution of the compounds were made in Diamond medium
containing 10% FCS. Control wells received 100 lL of
complete Diamond medium. Protozoa, washed twice in
phosphate-buffered saline (PBS, pH 7.2), were suspended
in the same medium at the density of 2.5 9 105 cells per
Results and discussion
The nonadentate, symmetric ‘‘tritopic’’ H3Imdp, H5Imds
and ‘‘ditopic’’ H4MeImds ligands belong to the class of
compartmental trinucleating ligands and in particular they
could be assimilated to one of that ‘‘end-off’’ acyclic
ligands that provided speculative models for the type-III
site in cuproproteins [13]. The ligands were synthesized
by condensation of bis(hydrazide) of the iminodiacetic
acid, with 2-pyridinecarboxaldehyde and salicylaldehyde,
respectively, in absolute EtOH/CHCl3 solution for H5Imds
[2] and in absolute EtOH as regarding H3Imdp, and by
the reaction of bis(hydrazide) of the methyl-iminodiacetic
acid with salicylaldehyde, in MeOH/CHCl3 solution for
H4MeImds ligand [6]. The H3Imdp compound, is air-
stable, obtained in good yield and characterized by IR,
1H NMR 13C NMR, UV/Vis spectroscopy and elemental
analysis. The H3Imdp, H5Imds and H4MeImds metal
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Transition Met Chem (2010) 35:253–261
257
bands centred at about 1530 and 1270 cm-1 for H3Imdp,
1520 and 1260 cm-1 for H5Imds, 1520 and 1270 cm-1 as
regards the H4MeImds, assigned to Amide II and Amide III
system, respectively.
dmso
dmso
Cu
N
N
N
N
N
O
H3Imdp complexes
O
O
O
N
Cu
Cu
dmso
O
H2O
O
H2O
dmso
In spite of the metal ligand/metal molar ratio used in
the reactions, only trinuclear metal complexes of formula
Cu3 (HImdp)(NO3)4.C2H5OHÁH2O, Cu3(HImdp)Cl4 and
Co3(HImdp)(NO3)4Á2H2O were obtained (Fig. 5).
Cu
Cu
O
O
N
As mentioned earlier, all the complexes have been, also,
characterized in analogy and on the basis of the informa-
tion obtained by IR and X-ray structural analysis of H5Imds
and H4MeImds trinuclear copper(II) complexes (Figs. 3,
4). However, with the exception of the absorptions due to
anionic groups, the IR spectra of the Co(II) and Cu(II)
trinuclear complexes show similar spectroscopic patterns
observed in the 4000–600 cm-1 region. These similarities
agree well with the IR spectra of the trinuclear copper(II)
complexes of H5Imds and H4MeImds. In particular, in
the IR spectra of Cu3(HImdp)(NO3)4.C2H5OHÁH2O,
Cu3(HImdp)Cl4 and Co3(HImdp)(NO3)4Á2H2O trinuclear
complexes, we found the absence of the amide I band
m(C=O), centred at 1670 cm-1 in the free ligand, and the
presence of a new band, at about 1615 cm-1, due to the
–C=N–N=C– diazinic system, in good agreement with
the bi-deprotonated form of H3Imdp upon coordination.
Then, a trimetallic coordination occurs by the central
amino atom, pyridinic nitrogen and by the hydrazonic
system in its enolic form (Fig. 5).
Fig. 3 The repeatunit in[Cu6(HImds)2(dmso)5(EtOH)(H2O)2] (ClO4)4Á
2H2O [2]
O
O
O
S
O
OH
2
N
N
Cu
N
N
O
O
O
N
Cu
Cu
O
CH
3
OH
OH
2
2
Fig. 4 Trimetallic monomeric unit [Cu3(MeImds)(SO4)(H2O)3]
structure [6]
complexes, showed decomposition above 300 ꢁC without
melting, are air-stable, obtained in satisfactory yields and
are generally soluble in MeOH, H2O or in MeOH/H2O
mixture. Unfortunately, the lack of information about the
molecular structure of the metal complexes, due to micro-
crystalline forms not suitable for X-ray analysis, constrains
us to suggest plausible structures by means of elemental
analysis and spectroscopic methods and in analogy with
the coordinating behaviour, unequivocally, established by
X-ray structural analysis of the polymeric [Cu6(HImds)2-
(dmso)5(EtOH)(H2O)2](ClO4)4Á2H2O (Fig. 3) and the dimeric
[Cu3(MeImds)(SO4)(H2O)3]Á5H2O trimetallic complexes
previously described (Fig. 4) [2, 6].
X
anionic groups
or
M
O
solvating molecules
N
N
anionic groups
HN
M
N
N
IR spectra
O
M
anionic groups
or
IR spectra of H3Imdp, H5Imds and H4MeImds are mainly
characterized by the absorptions due to O–H and N–H
stretching in the 3500–3100 cm-1 region. Amide I charac-
teristic band of m(C=O) at 1680 cm-1 (H3Imdp), 1683 cm-1
(H5Imds) and 1670 cm-1 (H4MeImds) indicating a keto-form
in the solid state. All ligand spectra show the (C=N)
absorption band in the appropriate region (1620–1615 cm-1).
Hydrazonic chain is represented by two medium-intense
solvating molecules
X
Fig. 5 Proposed coordination mode in Cu3(HImdp)(NO3).4C2H5OHÁ
H2O, Cu3(HImdp)Cl4, Co3(HImdp)(NO3)4Á2H2O and Co3(HImds)Cl2.
CH3OHÁ3H2O trinuclear complexes [X = N (H3Imdp), OH (H5Imds);
M = Cu(II), Co(II)]
123

258
Transition Met Chem (2010) 35:253–261
X
No spectroscopic evidence is attributable to a mono- or
H2O
O
bi-dentate ligand behaviour of the nitrate group in the
Cu3(HImdp)(NO3)4.C2H5OHÁH2O complex.
O
H
M
O
O
H
N
Zn2(H3Imdp)(ClO4)4Á2H2O dinuclear complex was
obtained with 1:2 ligand/metal molar ratio. The IR spec-
trum is mainly characterized by two broad bands in the
range 3400–3150 cm-1 that could be attributable to sol-
vating water molecules and NH absorptions and by a
medium strong broad absorption of amide I m(C=O) band
centred at 1660 cm-1 (1680 cm-1 in the free ligand). The
presence of a weak band at about 1610 cm-1 (1618 cm-1
in the free ligand), is associated with azomethinic –HC=N–
system upon coordination. The 1H NMR spectrum exhibits
an hydrazonic NH (D2O exchangeable in the free ligand)
singlet signal at 12.20 ppm, low-field shifted with respect
to the free ligand (11.61 ppm) and a broad multiplet signal
in the range 8.85–7.20 ppm associated to a low-field shif-
ted pyridine ring (7.39–8.29 ppm in the free ligand). The
inside of multiplet contains a shifted azomethinic –HC=N–
singlet signal at 8.72 ppm (7.59 ppm in the free ligand).
Therefore, the pyridinic nitrogen, azomethinic nitrogen and
carbonylic group are affected by coordination to zinc metal
N
C
C
N
N
H
N
H
Fig. 6 Mononuclear coordinating fashion proposed for Mn(H4Imds)-
Cl.CH3OHÁH2O and Co(H4Imds)NO3Á2H2O complexes (M = Mn(II),
Co(II); X = Cl, NO3)
overlapping the N–H, aliphatic and aromatic mCH bands,
could be associated with an intramolecular OH…N=C
hydrogen bond between phenolic OH and azomethinic
nitrogen [18]. As known, this six-membered hydrogen-
bonded species was found in many salicylaldimine sys-
tems. While the X-ray structure of H5Imds ligand has not
been determined, the OH…N=C hydrogen bond was evi-
denced by X-ray molecular structure of H4MeImds parent
ligand [6]. The weak absorptions at 1535 and 1270 cm-1
can be attributable, respectively, to the amide II and III
bands (1520 and 1260 cm-1 in the free ligand). Therefore,
‘‘NO2’’ coordination occurs by the azomethinic nitrogen,
carbonylic group and by the deprotonated phenolic OH, in
one of external ligand compartment (Fig. 6). No spectro-
scopic evidence is attributable to a mono- or bi-dentate
ligand behaviour of the nitrate group in the Co(H4Imds)-
NO3Á2H2O complex. Therefore, with exception due to
MO3 absorption group (1385 cm-1), marked similarities
with the IR spectrum of Co(H4Imds)NO3Á2H2O complex
are shown by the spectrum of the mononuclear
Mn(H4Imds)Cl.CH3OHÁH2O complex, also obtained by 3:1
metal/ligand molar ratio (see experimental section). The
absorptions of one co-ordinated and one un-coordinated
carbonylic groups in Mn(H4Imds)Cl.CH3OHÁH2O IR
spectrum are positioned, respectively, at 1670 and
1689 cm-1, together with a new broad band at about 3210–
2950 cm-1, overlapped by N–H, aromatics and aliphatic
bands, that could be associated with an intramolecular
hydrogen bond between the phenolic OH and the azome-
thinic nitrogen (Fig. 6).
1
ions. Moreover, the confirmed presence, in the H NMR
spectrum, of undeprotonated NH upon coordination, toge-
ther with the presence of one amide I band m(C=O) in the
IR spectrum, suggests that the ligand is present in keto-
form: both carbonylic groups are implicated in the coor-
dination and that ‘‘N2O’’ donor atom sets are involved in
the coordination in the external compartments of the
ligand, to form a symmetric Zn2-complex (Fig. 7).
The IR spectrum of Zn2(H3Imdp)(ClO4)4Á2H2O com-
plex shows no evidence of mono- or bi-dentate ligand
behaviour of the perchlorate group.
H5Imds complexes
The reactions of Co(II) chloride and Co(II) nitrate showed
a curious and unexpected behaviour in the complexation
with H5Imds ligand. We obtained, in spite of metal/ligand
3:1 molar ratio used, the trinuclear-chloride and the
mononuclear-nitrate complex, respectively. Therefore, a
dramatic difference is present in their IR spectra: while the
spectrum of Co3(HImds)Cl.2CH3OHÁH2O (the proposed
coordination mode is reported in Fig. 5), shows the char-
acteristic print of the trinuclear complexes described ear-
lier, the IR spectrum of Co(H4Imds)NO3Á2H2O is mainly
characterized by the presence of two strong amide I bands
m(C=O) at 1686 and 1660 cm-1 (1683 in the free ligand)
that can be attributable, respectively, to one un-coordinated
[C=O and to one metal-coordinated [C=O. Two weak
shouldered bands present at 1610–1600 cm-1 are attribut-
able to m(C=N) absorptions (1615 cm-1 in the free ligand).
In the case of the dinuclear Zn2(H3Imds)Cl2Á2H2O
complex, also, obtained with 1:3 ligand/metal molar ratio,
the first significant evidence is a lower shift of the amide
I band to 1640 cm-1 compared to the free ligand
(1683 cm-1), this could suggest that both[C=O groups are
involved in the coordination of metal ions. Thus, the
absence of –C=N–N=C– diazinic system band together
with the presence of amide II and amide III bands,
respectively, at 1530 and 1270 cm-1, are in agreement
with undeprotonated nature of the hydrazonic nitrogens
upon coordination. Moreover, the presence of a medium
strong band centred at 1610 cm-1 (1615 cm-1 in the free
ligand), attributable to azomethinic nitrogen absorption,
A new broad band in the range 3220–2960 cm-1
,
123

Transition Met Chem (2010) 35:253–261
259
anionic groups or solvating molecules
O
O
R
N
C
C
H
H
N
N
Zn
Zn
X
X
O
O
H
N
Cu
N
N
C
N
C
N
N
H
N
H
O
O
Fig. 7 Proposed coordination mode in Zn2(H3Imds)Cl2Á2H2O and
Zn2(H3Imdp)(ClO4)4Á2H2O dinuclear complexes. [X = N (H3Imdp);
OH (H5Imds)]
Fig. 8 Plausible mononuclear coordinating mode in Cu(H3Imds).-
CH3OHÁH2O, (R = H) and in Cu(H2MeImds).CH3OHÁ3H2O, (R =
CH3)
suggest their involvement in the metal coordination. So,
this leads us to conclude that the ligand is present in
bi-deprotonated form only through the phenolic OH groups,
upon coordination. Therefore, the two zinc ions are coor-
dinated in a dinuclear symmetric fashion in the external
compartments of the ligand (Fig. 7). Unfortunately, the
assignment of the m(NH) and the m(OH) bands, in the 3700–
3000 cm-1 region, are compromised by the presence of a
large band due to solvating water molecule absorptions. As
mentioned, the reaction between H5Imds and copper(II)
acetate (1:1 molar ratio) does not give the expected tri-
metallic species, but only the Cu(H3Imds).CH3OHÁH2O
mononuclear complex. This fact could be, probably, cor-
related to the different nature of acetate ion with respect to
the anionic species of copper salts (nitrate, sulphate,
chloride or perchlorate), used in the reactions described
earlier, whereas only trinuclear species were obtained
regardless of metal/ligand molar ratio. Therefore, related
IR spectrum, shows mainly significant evidences as con-
cerning the absence of the symmetric and antisymmetric
stretching bands, characteristic of acetate species, and a
medium strong band at 1680 cm-1 that could be associated
to uncoordinated carbonyl groups, suggesting that the
ligand is present in keto-form upon coordination. A med-
ium strong band at about 1610 cm-1 suggests that the
azomethinic nitrogens undergo a negative shift upon
coordination. Moreover, the IR spectrum shows a very
strong band at about 1530 cm-1 associated to amide II
absorption. The spectrum is, also, characterized by a
medium band at 3411 cm-1 that could be associated to
hydrazonic m(NH) absorption. Thus, as proposed in Fig. 8,
the H5Imds ligand coordinates, in a mononuclear fashion
‘‘closed’’ form, by the azomethinic nitrogens and both
deprotonated phenolic groups. As regards the parent
H4MeImds copper(II) complex, the marked similarities in
the spectra of the mononuclear Cu(H2MeImds).CH3OHÁ
3H2O and Cu(H3Imds).CH3OHÁH2O complexes, taken
together with the stoichiometric and preparative similari-
ties, lead us to conclude that the Cu(H2MeImds).CH3OHÁ
3H2O complex has the same structure as that proposed
for Cu(H3Imds).CH3OHÁH2O (Fig. 8).
Antimicrobial activity
About the biological screening, bis(acylhydrazones)
H5Imds, H3Imdp and related copper(II) complexes were
evaluated in vitro for antibacterial (Bacillus spp., S. aureus,
S. epidermidis, C. freundii, E. cloacae, E. coli, K. pneumoniae
and P. aeruginosa), antifungal (C. albicans, P. guillermon-
dii, R. glutinis and A. niger) and antiprotozoal (T. vaginalis
and A. castellanii) activities. The results obtained showed
that some of the compounds possess an interesting activity
against several tested strains.
With regard to antibacterial activity (Table 1), most of
the tested compounds exhibited low activity against Gram-
positive and Gram-negative bacteria showing MIC values
ranging from 62.5 to 250 lg mL-1 with a few exceptions.
Compound H5Imds was more active than H3Imdp against
Gram-positive bacteria (MIC = 7.8 lg mL-1 vs. S. epi-
dermidis). Similar activity was shown by their copper(II)
Cu3(HImds)(ClO4)2. EtOHÁ2H2O and Cu3(HImds)SO4Á4H2O
complexes. Among all compounds tested, only H5Imds
showed a moderate activity against Gram-negative bacteria
(MIC = 31.25 lg mL-1 vs. C. freundii).
Results of antifungal activity evaluation indicated that
the tested compounds, in our experimental conditions, were
unable to inhibit the growth of C. albicans, P. guiller-
mondii, R. glutinis and A. niger. Only H5Imds exerted a
very low inhibitory activity against C. albicans (Table 2).
About anti-trichomonas assay, the results obtained are
reported in Table 3. In all experimental conditions, control
cells were viable and motile. The susceptibility of the
clinical isolate to metronidazole, used as reference drug,
was scanty (MIC100 of 50, 12.5 lg mL-1, respectively, at
24 h of incubation and next times). The bis(salicylhyd-
razone) H5Imds and the copper(II) complexes tested
exhibited a general good activity against T. vaginalis. In
particular, compound H5Imds exhibited the best activity
(MIC = 1.9 lg mL-1). Therefore, the presence of the
phenolic ring instead of pyridinic ring, in the hydrazonic
123

260
Transition Met Chem (2010) 35:253–261
Table 1 MIC100 values (lg mL-1) of bis(acylhydrazones) and copper(II) complexes towards some bacteria evaluated after 18–24 h of incu-
bation at 35 ꢁC
Bacteria
H5Imds
H3Imdp
Cu3(HImds) X2
X = NO3
Cu3(HImds) X2
X = ClO4
Cu3(HImds) X
X = SO4
Bacillus spp.
125
[250
[250
[250
ND
62.5
62.5
62.5
62.5
62.5
62.5
S. aureus ATCC 25923
S. aureus ATCC 6538
S. epidermidis
62.5
[250
7.8
62.5
62.5
62.5
15.6
7.8
7.8
C. freundii
31.25
[250
[250
[250
[250
ND
[250
[250
[250
[250
[250
[250
[250
[250
[250
[250
[250
[250
[250
[250
[250
E. cloacae
ND
E. coli ATCC 25922
K. pneumoniae
[250
ND
P. aeruginosa ATCC 27853
[250
Table 2 MIC100 values of bis(acylhydrazones) and copper(II) complexes towards some fungi obtained after 4 days of incubation at 25 ꢁC
Fungi
H5Imds
H3Imdp
Cu3(HImds) X2
X = NO3
Cu3(HImds) X2
X = ClO4
Cu3(HImds) X
X = SO4
C. albicans ATCC 10231
P. guillermondii
125
[250
[250
[250
ND
[250
[250
[250
ND
[250
[250
[250
ND
[250
[250
[250
ND
[250
[250
ND
R. glutinis
A. niger ATCC 16404
Table 3 MIC100 values (lg mL-1) of bis(acylhydrazones) and cop-
per(II) complexes towards T. vaginalis and A. castellanii obtained,
respectively, after 96 and 144 h of incubation at 37 ꢁC and in humid
atmosphere containing 5% CO2 compared with MIC100 values
b
(lg mL-1) of control drugs (ametronidazole, rokitamycin) obtained
in the same experimental conditions
Protozoa
H5Imds
H3Imdp
Cu3(HImds) X2
X = NO3
Cu3(HImds) X2
X = ClO4
Cu3(HImds) X
X = SO4
Control drugs
T. vaginalis
1.9
50
125
15.6
25
ND
50
15.6
12.5a
18.75b
A. castellanii
[100
[25
skeleton, could explain a dramatic increase of the H5Imds
ligand antitrichomonal activity compared to the H3Imdp
ligand.
2H2O (MIC100 25 lg mL-1) (Fig. 9). Its effect when it was
used at the MIC100 was amoebistatic, with recovery
eventually occurring (data not shown).
The investigation on A. castellanii indicated that after
6 days of incubation at 37 ꢁC, control cultures produced
about 2.5 9 103 amoebae/well, with doubling times of
about 42 h. Under the same experimental conditions, cul-
tures of trophozoites treated with the macrolide rokitamy-
cin (75–2.34 lg mL-1), used as reference drug [22],
showed significant difference in growth rate and MICs100
of 4.68, 18.75 lg mL-1, respectively, at 24 and 144 h of
incubation. The lowest concentration of tested compounds
that caused complete inhibition of trophozoite growth
(MIC100) at 144 h are shown in Table 3. The susceptibility
of A. castellanii to bis(acylhydrazones) was slight (MIC100
[50 lg mL-1), but all the H5Imds copper(II) complexes
showed a moderate activity against trophozoites. The best
activity was exhibited by Cu3(HImds)(NO3).22CH3OHÁ
Conclusions
The H5Imds, H4MeImds and H3Imdp compartmental
ligands exhibit versatile behaviour in complexation with
first row transition metal ions, ranging from mono- to
di- and trinuclear metal complexes. Further studies aimed
to obtain ethero-nuclear, mono- or poly-nuclear metal
complexes of H5Imds, H4MeImds and H3Imdp ligands,
towards transition metal ions, are now in progress.
In the light of the results obtained from the screening of the
antimicrobial activities of bis(acylhydrazones) and copper(II)
complexes, a very interesting activity emerged against T.
vaginalis for compound H5Imds (MIC100 = 1.9 lg mL-1),
123

Transition Met Chem (2010) 35:253–261
261
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100 micrograms/ml
50 micrograms/ml
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0
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24 48 72 96 120 144 168
Incubation Time (hours)
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