Structural studies and investigation on the activity of imidazole-derived thiosemicarbazones and hydrazones against crop-related fungi

Article abstract of DOI:10.3390/molecules181012645

New imidazole derived thiosemicarbazones and hydrazones were prepared by condensation of 4(5)-imidazole carboxaldehyde, 4-(1H-imidazole-1-yl)benzaldehyde and 4-(1H-imidazole-1-yl)acetophenone with a thiosemicarbazide or hydrazide. All compounds were characterized by quantitative elemental analysis, IR and NMR techniques. Eight structures were determined by single crystal X-ray diffraction. The antifungal activities of the compounds were evaluated. None of the compounds exhibited significant activity against Aspergillus flavus and Candida albicans, while 4(5)-imidazolecarboxaldehyde thiosemicarbazone (ImT) and 4-(1H-imidazole-1-yl)benzaldehyde thiosemicabazone (4ImBzT) were highly and selectively active against Cladosporium cladosporioides. 4(5)- Imidazolecarboxaldehyde benzoyl hydrazone (4(5)ImPh), 4(5)- imidazolecarboxaldehydepara- chlorobenzoyl hydrazone (4(5)ImpClPh), 4(5)-imidazolecarboxaldehyde-paranitrobenzoyl hydrazone (4(5)ImpNO ₂Ph), 4-(imidazole-1-yl)acetophenone-para-chlorobenzoyl hydrazone (4ImAcpClPh) and 4-(imidazole-1-yl)acetophenone-para-nitrobenzoylhydrazone (4ImAcpNO₂Ph) were highly active against Candida glabrata. 4(5)ImpClPh and 4(5)ImpNO₂Ph were very effective against C. cladosporioides. In many cases, activity was superior to that of the reference compound nystatin.

Full text of DOI:10.3390/molecules181012645

Molecules 2013, 18, 12645-12662; doi:10.3390/molecules181012645
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Structural Studies and Investigation on the Activity of
Imidazole-Derived Thiosemicarbazones and Hydrazones
against Crop-Related Fungi
Débora C. Reis, Angel A. Recio Despaigne, Jeferson G. Da Silva, Nayane F. Silva, Camila F. Vilela,
Isolda C. Mendes, Jacqueline A. Takahashi and Heloisa Beraldo *
Departamento de Química, Universidade Federal de Minas Gerais, Belo Horizonte 31270-901,
MG, Brazil
* Author to whom correspondence should be addressed; E-Mail: hberaldo@ufmg.br;
Tel.: +55-31-3409-5740; Fax: +55-31-3409-5700.
Received: 14 August 2013; in revised form: 25 September 2013 / Accepted: 26 September 2013 /
Published: 14 October 2013
Abstract: New imidazole derived thiosemicarbazones and hydrazones were prepared by
condensation of 4(5)-imidazole carboxaldehyde, 4-(1H-imidazole-1-yl)benzaldehyde and
4-(1H-imidazole-1-yl)acetophenone with a thiosemicarbazide or hydrazide. All compounds
were characterized by quantitative elemental analysis, IR and NMR techniques. Eight
structures were determined by single crystal X-ray diffraction. The antifungal activities
of the compounds were evaluated. None of the compounds exhibited significant activity
against Aspergillus flavus and Candida albicans, while 4(5)-imidazolecarboxaldehyde
thiosemicarbazone (ImT) and 4-(1H-imidazole-1-yl)benzaldehyde thiosemicabazone
(4ImBzT) were highly and selectively active against Cladosporium cladosporioides.
4(5)-Imidazolecarboxaldehyde benzoyl hydrazone (4(5)ImPh), 4(5)-imidazolecarboxaldehyde-
para-chlorobenzoyl hydrazone (4(5)ImpClPh), 4(5)-imidazolecarboxaldehyde-para-
nitrobenzoyl hydrazone (4(5)ImpNO2Ph), 4-(imidazole-1-yl)acetophenone-para-chloro-
benzoyl hydrazone (4ImAcpClPh) and 4-(imidazole-1-yl)acetophenone-para-nitro-
benzoylhydrazone (4ImAcpNO2Ph) were highly active against Candida glabrata.
4(5)ImpClPh and 4(5)ImpNO2Ph were very effective against C. cladosporioides. In many
cases, activity was superior to that of the reference compound nystatin.
Keywords: imidazole; hydrazones; thiosemicarbazones; antifungal activity

Molecules 2013, 18
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1. Introduction
The imidazole nucleus is well known to play an important role in living organisms since it is
incorporated into the histidine molecule and many other important biological systems. Imidazole
derivatives are the most used class of antifungal drugs [1], being active against pathogenic and
nonpathogenic fungi [2]. Due to their antifungal properties imidazole-derived compounds have been
used in agriculture as effective ingredients for controlling plants pests. Imidazole derivatives are
employed in the control of spoilage microorganisms or organisms potentially harmful to man, in the
protection of wood against fungi and also in food storage [3]. In addition, imidazole compounds were
reported to be active against Fungi imperfecti, Basidiomycetes, Ascomycetes and Oomycetes.
Outstanding activity was observed against powdery mildews (e.g., Erysiphenecator) and leaf spots
(e.g., Mycosphaerella spp.). Furthermore, these compounds are effective against phytopathogenic
gram-negative (e.g., Xanthomona spp., Pseudomonas spp., Erwinia amylovora, Ralstonia spp.) and
gram-positive bacteria and viruses (e.g., tobacco mosaic virus) [4].
A. flavus is a worrying plant pathogen since it can produce aflatoxins on certain crops like oilseeds,
corn and nuts (e.g., peanuts). Fungi unknown to produce aflatoxins can also cause agricultural
damages, like C. cladosporioides that acts in the post-harvesting spoilage of fruits and vegetables. In
vineyards, C. cladosporioides can also cause grape rot [5]. Both species have been reported to be
among the major species normally found in several commodities like soybeans [6]. These species can
become still more harmful since they can be spread by air from industrial food processing
environments, such as grain mills or organic waste recycling facilities [7]. In contact with humans,
they act as allergenic factors, causing a series of respiratory related diseases [8]. It has been reported
that C. cladosporioides can play a role as opportunistic fungi causing ocular infections [9]. A. flavus
has also been reported to be an opportunistic fungus especially in immuno-compromised patients [10].
Thiosemicarbazones [11,12] and hydrazones [13] are reported as compounds which present
significant antifungal activity. Their metal complexes also exhibit antifungal properties [14–16].
Hydrazones have previously been reported as active against some species of Candida sp [16–18].
In the present work we synthesized a series of imidazole-derived thiosemicarbazones and
hydrazones and tested the potential of these compounds as antifungal agents to fight A. flavus and C.
cladosporioides. Since pathogens like Candida sp. have a huge importance as a clinical target of new
fungicides, we extended the screening in order to evaluate the potential of the studied compounds
against Candida albicans and Candida glabrata.
2. Results and Discussion
2.1. Characterization of the Thiosemicarbazone Derivatives
Eight imidazole-derived thiosemicarbazones (Figure 1) were obtained. Among them seven
compounds (2–8) were original. Microanalyses were compatible with the proposed formulations. The
infrared spectra of the thiosemicarbazones showed a very strong band at 3,446–3,145 cm−1 which was
attributed to the ν(N–H) vibration. The ν(C=N) and ν(C=S) vibrations appeared at 1,638–1,601 cm−1
and 848–830 cm−1, respectively [19,20].

Molecules 2013, 18
Figure 1. Generic representation for imidazole-derived thiosemicarbazones.
12647
1
The NMR spectra of all thiosemicarbazones were recorded in DMSO-d6. The H resonances were
assigned on the basis of chemical shifts and multiplicities. The carbon type (C, CH) was determined by
using distortionless enhancement by polarization transfer (DEPT 135) experiments. The assignments of the
protonated carbons were made by 2D hetero-nuclear multiple quantum coherence experiments (HMQC).
1
13
In the H- and C-NMR spectra of thiosemicarbazone 1 duplicated signals were observed for all
hydrogens and carbons, indicating the presence of the Z (60%) and E (40%) configurational isomers in
the DMSO-d6 solution, as frequently occurs with thiosemicarbazones [21]. In the spectra of 2 three
signals were observed for each hydrogen and each carbon. In the 13C-NMR the signals at δ 175.29 and
δ 175.47 were assigned to C=S of the E and Z isomers, respectively, and the signal at δ 172.05 to
C–SH. The proportion of E and Z isomers is 1:1, but it was not possible to determine the percentage of
the thiol form in solution due to overlapping signals in the S–H spectral region. The thione-thiol
tautomerism had been previously observed for other thiosemicarbazones [22].
1
In the H- and 13C-NMR spectra of 3–8 only one signal was observed for each hydrogen and
carbon; the chemical shifts indicate the presence of only the E isomer in solution. Generally when
N5–Hb is replaced by a methyl or a phenyl group in 4, 5, 7 and 8 the signal of N5–Ha shifts upfield.
For both formyl and acetyl compounds the signals of the imidazole and benzene rings were observed
in the same region.
The N4–H signals at δ 11.93–11.53 (formyl derivatives) and δ 10.66–10.27 (acetyl derivatives)
indicate hydrogen bonding with the solvent [21]. In the spectra of the acetyl derivatives the signals of
N4–H appear at lower frequencies due to the gamma-gauche effect of the CH3 group in C10, which
makes more difficult the interaction of DMSO-d6 with N4–H [23].
Crystals suitable for X-ray diffraction studies were obtained by slow evaporation from the mother
liquor in the synthesis of 1, 2, 3, 4 and 5. Interestingly, 1 crystallized with one water molecule as
ImT·H2O (1a). The crystal data and refinement results for imidazole-derived thiosemicarbazones are
given in Table S1 of the Supplementary Material. The ORTEP diagrams for 1a and 2–5 and the

Molecules 2013, 18
12648
numbering scheme are shown in Figure 2. Selected bonds distances and angles are listed in Table S2.
The geometric parameters for hydrogen bonds in 1a and 2–5 are listed in Table 1.
Compounds 1a, 2, 3 and 5 crystallized in the monoclinic system with one molecule of the
thiosemicarbazone per asymmetric unit, while 4 crystallized in the triclinic system with two molecules
per asymmetric unit (see Figure 2). In all structures the CNNC(S)N backbone is almost planar with
rms distance of atoms from the least-squares plane of 0.0277 Å (1a), 0.0137 Å (2), 0.0705 Å (3),
0.0438 and 0.0669 Å (for C10N3N4C11S1N5 and C30N23N24C31S21N25, respectively, in 4) and
0.0311 Å (5). The bond lengths in the thiosemicarbazone backbone are similar to those previously
described in the literature [19,20,24,25].
Figure 2. Molecular plots of ImT·H2O (1a), ImTPh (2), 4ImBzT (3), 4ImBzTM (4) and
4ImBzTPh (5), showing the labeling scheme of the non-H atoms and their displacement
ellipsoids at the 50% probability level.
The thiosemicarbazone backbone of 1a and 3–5 adopt the EE conformation in relation to the C4–N3 or
C10–N3 and N4–C5 or N4–C11 bonds. The N5–H⋯N3 hydrogen bond probably hinders rotation
around the N4–C5 or N4–C11 bonds and might contribute to the stability of the E conformation. In
fact, the EE conformation is commonly observed in 2-formylpyridine- and 2-acetylpyridine-derived
thiosemicarbazones in the solid state [26,27]. Furthermore, the EE conformation allows the formation
of dimmers via intermolecular N4–H⋯S1 hydrogen bonds (see Table 1). In addition, in the case of 1a
a hydration water molecule forms intermolecular hydrogen bonds (see Table 1).

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Table 1. Hydrogen bonds geometric parameters (Å, °) for ImT·H2O (1a), ImTPh (2),
4ImBzT (3), 4ImBzTM (4) and 4ImBzTPh (5).
D–H…A
d(D–H)
d(H⋯A)
d(D⋯A)
< (DHA)
1a
O1W–H1W⋯N2 i
O1W–H2W⋯N2 ii
N1–H1⋯S1 iii
0.8599(10)
0.8599(10)
0.86
2.158(10)
2.200(5)
2.58
2.950(2)
153(2)
169(2)
149.6
157.2
161.0
164.2
3.0487(19)
3.3540(15)
3.4168(14)
3.2804(19)
2.9429(19)
N4–H4A⋯S1 iv
N5–H5A⋯O1W ii
N5–H5B⋯O1W
0.86
2.61
0.86
2.46
0.86
2.11
2
3
4
N1–H1⋯S1 v
N4–H4⋯N2
N5–H5⋯N3
0.86
0.86
0.86
2.61
2.02
2.12
3.3632(14)
2.7158(18)
2.5866(17)
147.1
137.2
113.9
N4–H4 ⋯S1 vi
N5–H5A⋯S1 vii
N5–H5B⋯N1 viii
0.86
0.86
0.86
2.60
2.79
2.12
3.4424(13)
3.4162(14)
2.9405(18)
166.4
130.6
159.4
N24–H2⋯S1 ix
N25–H25A⋯N9
N4–H4⋯S21 x
N5–H5A⋯N21
0.86
0.86
0.86
0.86
2.56
2.22
2.55
2.21
3.3890(18)
2.999(2)
162.7
150.9
154.8
149.2
3.3501(19)
2.984(2)
5
N4–H4⋯S1 xi
N5–H5⋯N1 xii
0.86
0.86
2.56
2.61
3.3950(17)
3.309(3)
164.0
139.1
Symmetry transformations used to generate equivalent atoms: i = −x + 1, y+1/2, −z + 1/2; ii = −x+1, −y + 1,
−z + 1; iii = x, y − 1, z; iv = −x, −y + 1, −z; v = −x + 2, y−1/2, −z + 3/2; vi = −x + 2, −y, −z + 2;
vii = x, −y + 1/2, z + 1/2; viii = x−1, −y + 1/2, z−3/2; ix = x, y, z − 1; x = x, y, z + 1; xi = −x + 1, −y + 2, −z + 1
and xii = −x + 2, −y, −z + 1.
In contrast, 2 adopts the ZE conformation in relation to the C4–N3 and N4–C5 bonds (see Figure 2).
This probably occurs due to the presence of a weak N1-H⋯N3 hydrogen bond (see Table 1). This
conformation is often observed in 2-benzoylpyridine-derived thiosemicarbazones [28].
2.2. Characterization of the Hydrazone Derivatives
Twelve new imidazole-derived hydrazones (Figure 3) were obtained. Microanalyses were compatible
with the proposed formulations. In the infrared spectra of the hydrazones the absorption at
3,240–3,139 cm−1 was attributed to the ν(N-H) vibration mode. Absorptions at 3,100–3,020 cm−1 in the
spectra of compounds 9–12 are characteristic of N4-H⋯N2 hydrogen bonds. Very strong bands at
1,687–1,659 cm−1 were assigned to ν(C=O) and absorptions at 1,626–1,606 cm−1 were attributed
to ν(C=N).

Molecules 2013, 18
Figure 3. Generic representation for imidazole-derived hydrazones.
12650
1
5
6
1
H
R1
2
1
N
N
4
7
4
10
N
2
3
2
2
N
N
NH
N
NH
3
H
3
4
9
8
3
4
5
11
1
R
R2
O
O
(9)-R= CH3
(13)-R1=H; R2= CH3
(14)-R1= H; R2= C6H5
(10)-R= C6H5
(11)-R=pCl-C6H4
(15)-R1= H; R2= pCl-C6H4
(16)-R1= H; R2= pNO2-C6H4
(17)-R1=R2= CH3
(12)-R=pNO2-C6H4
(18)-R1=CH3; R2= C6H5
(19)-R1=CH3; R2= pCl-C6H4
(20)-R1=CH3; R2= pNO2-C6H4
As for the thiosemicarbazones the NMR spectra of all hydrazones were recorded in DMSO-d6.
Three signals for all hydrogens and carbons were found in the 1H- and 13C-NMR spectra of compound 9,
suggesting the existence of three isomers in solution. The signals of C5 at δ 171.89 and δ 171.57 were
attributed to C=O of the E and Z isomers, respectively, and the signal at δ 168.35 was assigned to C–OH
in the enol tautomer [29].
The signals at δ 11.22, δ 11.03 and δ 9.76 were attributed to N1–H of the imidazole ring in the enol,
E and Z isomers, respectively. Signals at δ 13.30 and δ 12.93 were assigned to N4–H in the Z and E
isomers. The O–H signal of the enol tautomer is overlapped with the N4–H signal of the E isomeric form.
In the 1H-NMR spectra of compounds 10–12 only one signal was observed for each hydrogen. The
signals attributed to N1–H were found at δ 8.38 (10), δ 8.39 (11) and δ 8.37 (12) and those of N4–H
were found at δ 14.29 (10), δ 14.34 (11) and δ 14.54 (12), indicating that they are in the Z
configuration in solution. In the 13C-NMR spectra of 10 and 11 one signal was verified for each
carbon, confirming the existence of only one isomer in solution. The signals of C5=O were observed at
δ 162.81 and δ 162.26 in the spectra of 10 and 11, respectively. Due to the low solubility of 12 it was
impossible to record its 13C-NMR spectrum.
1
The H and 13C-NMR spectra of compounds 13 and 17 showed duplicated signals for each
hydrogen and carbon indicating the presence of two isomeric forms in solution. The signals at δ 172.00
and δ 165.67 for 13 and δ 171.82 and δ 166.13 for 17 corresponded to C11=O and C11–OH of the keto
and enol tautomers, respectively [29]. The signals at δ 11.49 and δ 10.51 were attributed to N4–H in
the keto tautomer for 13 and 17, and those at δ 11.35 and δ 10.41 to O–H in the enol tautomer,
respectively. The keto tautomers adopt the E configuration in solution.
In the spectra of compounds 14–16, 18 and 19, only one signal was observed for each hydrogen and
carbon. The signals of N4–H appear at δ 12.22–10.84, indicating the presence of E configuration for
these compounds in solution. The signal at δ 163.82–161.13 for 14–16 and the signal at δ 166.13 and δ

Molecules 2013, 18
12651
171.82 for 18 and 19 were assigned to C11=O. Due to the low solubility of compound 20 it was
impossible to record its NMR spectrum.
Crystals were obtained by slow evaporation from the mother liquor in the synthesis of 11, 14 and 15.
Interestingly, 11 crystallized with one hydration water molecule as 4(5)ImpClPh·H2O (11a). The
crystal data and refinement results for the imidazole-derived hydrazones are given in Table S3 of the
Supplementary Material. The ORTEP diagrams for 11a, 14 and 15 and the numbering scheme are
shown in Figure 4. The selected bonds and angles are listed in Table S4. The geometric parameters for
hydrogen bonds in 11a, 14 and 15 are listed in Table 2.
The three compounds crystallized in the monoclinic system with one molecule of the hydrazone per
asymmetric unit (see Figure 4). In all structures the CNNC(O)C backbone is almost planar with rms
distance of atoms from the least-squares plane of 0.0402 Å (11a), 0,0476Å (14) and 0.0287 Å (15).
Figure 4. Molecular plots of 4(5)ImpClPh·H2O (11a), 4ImBzPh (14) and 4ImBzpClPh
(15), showing the labeling scheme of the non-H atoms and their displacement ellipsoids at
the 50% probability level.
The hydrazone backbone of 11a adopts the ZZ conformation in relation to the C4–N3 and N4–C5
bonds. The intramolecular N4–H⋯N2 hydrogen bond probably hinders rotation around C4–N3
contributing to the stability of the Z configuration. This conformation was also observed by us in
2-benzoylpyridine-derived hydrazones [30]. The crystal packing of 11a is dominated by N–H⋯N and
N–H⋯O hydrogen bonds (see Table 2).
In contrast, 14 and 15 adopt the EZ conformation in relation to the C10–N3 and N4–C11 bonds.
The crystal packings of 14 and 15 are very similar and are dominated by intermolecular N4–H⋯N1
hydrogen bond (see Table 2).

Molecules 2013, 18
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Table 2. Hydrogen bonds geometric parameters (Å, °) of for 4(5)ImpClPh·H2O (11a),
4ImBzPh (14) and 4ImBzpClPh (15).
D–H…A
d(D–H)
d(H⋯A)
d(D⋯A)
<(DHA)
1a
N1–H1A⋯O1 i
N1–H1A⋯N3 i
N4–H4A⋯N2
0.86
0.86
0.86
1.96
2.67
2.00
2.799(5)
3.242(5)
2.698(5)
164.4
125.4
137.0
14
15
N4–H4⋯N1 ii
N4–H4⋯N1 ii
0.86
0.86
2.26
2.30
3.079(3)
158.8
166.9
3.1411(16)
Symmetry transformations used to generate equivalent atoms: i = −x + 1\2, y − 1/2, −z + 3/2 and ii = x − 1,
−y + 1/2, z + 1/2.
2.3. Antifungal Activity
The results of the biological assays are reported in Table 3. In general, the thiosemicarbazones
presented lower activity than the hydrazones. However, high activity was found for
thiosemicarbazones 1 and 3 against C. cladosporioides, with MIC50 = 5.79 and 2.00 µM, respectively.
These values were significantly lower than those determined for nystatin (MIC50 = 269 µM) which was
used as positive control. Compounds 1 and 3 were selectively active against C. cladosporioides, since
their MIC50 values against A. flavus, C. albicans and C. glabrata were much higher than those
determined against C. cladosporioides. Derivatives 1–8 presented the same pattern of activity against
the two Candida species.
From twelve hydrazones tested, only five showed some antifungal activity. Compounds 10, 19 and
20 proved to be active against C. glabrata, suggesting some selectivity. Compound 10 was very active,
with MIC50 < 1.20 µM. 11 and 12 were active against C. glabrata and C. cladosporioides. Both were
more active than nystatin against C. cladosporioides. Compounds 12 and 20 proved to be more active
than nystatin against C. glabrata. 12, which bears a pNO2Ph substituent, showed higher activity
against C. glabrata (MIC50 < 0.9 µM) and C. cladosporioides (MIC50 = 1.6 µM) than nystatin
(MIC50 = 1.05 µM and > 269 µM, respectively).
Since compounds 1 and 3 have in common the presence of a primary amine group at N5 we can
presume that steric hindrance at this position in the other thiosemicarbazones could lead to weaker
interaction with the biological target, with decreasing of activity.
None of the tested thiosemicarbazones presented a significant activity against A. flavus. This is an
important finding, since selectivity is a very welcome property of a given antifungal, allowing an optimum
exploitation of its capacity without affecting beneficiating microorganisms present in a given crop.
Evaluation of activity for the imidazole-carboxaldehyde-derived hydrazones 9–12 reveals that the
presence of the phenyl group in 10–12 significantly improves antifungal activity in relation to the
presence of the methyl group in 9. Among the imidazole-acetophenone-derived hydrazones the
presence of the p-ClPh or p-NO2Ph groups seems to favor antifungal activity against C. glabrata in
compounds 19 and 20.The presence of the p-NO2Ph group in 12 and 20 and of the p-ClPh group in 11
and 19 seems to be very effective to increase the antifungal effect of the compounds.

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Table 3. MIC50 of imidazole-derived thiosemicarbazones and hydrazones against
phytopathogenic and human pathogenic fungi.
MIC50 (µM)
C. albicans A. flavus
Compounds
C. glabrata
>1477.45
>1019.16
509.58
C. cladosporioides
5.79
ImT (1)
ImTPh (2)
>1477.45
>1019.16
>509.58
>964.02
>777.99
>964.02
>914.54
>745.33
>1643
>1477.45
>1019.16
>1019.16
>964.02
>777.99
>964.02
>914.54
>745.33
1643
1019.16
2.00
4ImBzT (3)
4ImBzTM (4)
4ImBzTPh (5)
4ImAcT (6)
>964.02
>777.99
>964.02
>914.54
>745.33
>1643
>964.02
>777.99
>964.02
>914.54
>745.33
>1643
4ImAcTM (7)
4ImAcTPh(8)
4(5)ImMe (9)
4(5)ImPh (10)
4(5)ImpClPh (11)
4(5)ImpNO2Ph (12)
4ImBzMe (13)
4ImBzPh (14)
4ImBzpClPh (15)
4ImBzpNO2Ph (16)
4ImAcMe (17)
4ImAcPh (18)
4ImAcpClPh (19)
4ImAcpNO2Ph (20)
Nystatin
<1.20
>1167
>1167
1167
15.70
1005.20
>964.40
>1095.20
>861.10
>769.80
>745.60
>1031.90
>821.40
>737.90
>715.60
>269
>1005.20
482.20
7.80
<0.90
1.90
>1095.20
>861.10
769.80
>1095.20
>861.10
>769.80
>745.60
>1031.90
>821.40
>737.90
>715.60
>269
>1095.20
>861.10
>769.80
>745.60
>1031.90
>821.40
>737.90
>715.60
>269
>745.60
>1031.90
>821.40
1.40
<0.68
1.05
3. Experimental
3.1. General
All common chemicals were purchased from Aldrich and used without further purification.
Thiosemicarbazones and hydrazones were prepared according to procedures previously employed by
our group [15,19,31–33]. Melting points were determined using the digital Mettler Toledo FP90
equipment. Partial elemental analyses were performed on a Perkin Elmer CHN 2400 analyzer. Infrared
spectra were recorded on a Perkin Elmer FT-IR Spectrum GX spectrometer using KBr plates
(4000–400 cm−1). NMR spectra were obtained with a Bruker DPX-200 Avance (200 MHz)
spectrometer using DMSO-d6 as the solvent and TMS as internal reference. X-ray diffraction data
collection was performed on an Oxford-Diffraction GEMINI diffractometer (LabCri-UFMG) using
graphite-Enhance Source Mo Kα radiation (λ = 0.71069 Å or Cu Kα radiation (λ = 1.54180 Å Data
integration and scaling of the reflections were performed with the Crysalis suite [34]. Final unit cell
parameters were based on the fitting of all reflections positions.

Molecules 2013, 18
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3.2. Synthesis of Thiosemicarbazone Derivatives (1–8)
The imidazole-derived thiosemicarbazones were prepared by reacting equimolar amounts (2 mmol)
of 4(5)-imidazole-carboxaldehyde, 4-(1H-imidazole-1-yl)-benzaldehyde or 4-(1H-imidazole-1-yl)-
acetophenone with the suitable thiosemicarbazide using methanol as solvent. The reaction mixture was
kept under reflux for 6 h. After cooling to room temperature the resulting solids were filtered off,
washed with ethanol and ether and dried in vacuum.
4(5)-Imidazole-carboxaldehyde-thiosemicarbazone (ImT) (1): Yield: 89%. White solid. M.p.: 201–202 °C.
Anal. Calc. for C5H7N5S: C, 35.49; H, 4.17; N, 41.39. Found: C, 35.20; H, 4.04; N, 40.83%. FW:
1
169.21 g mol−1. IR (KBr, cm−1): ν(NH) 3446s, ν(C=N) 1616s, ν(C=S) 848m. H-NMR (DMSO-d6, δ,
E, Z configuration): 13.21 (s, 1H, N4H, Z), 12.89 (s, 1H, N1H, E,Z), 11.43 (s, 1H, N4H, E), 8.28
(s, 1H, N5Ha, Z), 8.21 (s, 1H, N5Hb, Z), 8.08 (s, 1H, H4, E), 8.01 (s, 1H, H2, Z), 7.91 (s, 1H, N5Ha, E),
7.91 (s, 1H, N5Hb, E), 7.73 (s, 1H, H1, E), 7.47 (s, 1H, H1, Z), 7.28 (s, 1H, H2, E). 13C-NMR (DMSO-d6,
δ, E, Z configuration): 177.80 (C5, E), 177.58 (C5, Z), 138.18 (C4, Z), 135.54 (C3, E, Z), 135.47 (C4, E),
134.76 (C2, Z), 131.14 (C2, E), 121.56 (C1, E), 121.57 (C1, Z).
4(5)-Imidazole-carbaldehyde-N(5)-phenylthiosemicarbazone (ImTPh) (2): Yield: 80%. White solid.
Mp.: 219–220 °C. Anal. Calc. for C11H11N5S: C, 53.86; H, 4.52; N, 28.55. Found: C, 53.93; H, 4.52;
N, 28.52%. FW: 245.30 g mol−1. IR (KBr, cm−1): ν(NH) 3358s, ν(C=N) 1635s, ν(C=S) 841m. 1H-NMR
[DMSO-d6, δ, J(Hz), E, Z configuration, thiol]: 13.41(s, 1H, N4H, Z); 12.89 (s, 1H, N1H, E, Z, thiol);
11.78 (s, 1H, N4H, E); 10.18 (s, 1H, N5Hb, E); 10.06 (s, 1H, N5Hb, Z); 8.79 (s, 1H, H4, thiol); 8.07
(s, 1H, H4, Z); 7.89 (s, 1H, H2, Z); 7.87 (s, 1H, H1, thiol); 7.87 (s, 1H, H2, thiol); 7.87 (s, 1H, H4, E); 7.86
(t, 2H, H7, H11, 8.56, E,Z); 7.75 (s, 1H, H1, E, Z); 7.35 (s, 1H, H2, E); 7.61 (t, 2H, H8, H10, 8.55, E,
Z, thiol); 7.17 (t, 1H, H9, 7.89, E, Z, thiol). 13C-NMR (DMSO-d6, δ, E, Z configuration, thiol): 175.47
(C5, Z); 175.29 (C5, E); 172.05 (C5, thiol); 165.20 (C6, thiol); 138.99 (C6, E, Z); 138.84 (C1, thiol);
137.57 (C4, E); 136.68 (C4, Z); 135.74 (C4, thiol); 134.70 (C2, Z); 135.36 (C3, E, Z, thiol); 131.28
(C2, E); 128.70 (C7, C11, thiol); 128.61 (C2, thiol ); 127.99 (C7, C11, E, Z); 125.15 (C8, C10, thiol);
124.96 (C8, C10, E, Z); 124.73 (C9, E, Z); 121.96 (C1, E, Z); 118.80 (C9, thiol).
4-(1H-Imidazole-1-yl)-benzaldehyde-thiosemicarbazone (4ImBzT) (3): Yield: 96%. White solid. M.p.:
244–245 °C. Anal. Calc. for C11H11N5S: C, 53.86; H, 4.52; N, 28.55. Found: C, 53.68; H, 4.19; N,
1
28.75%. FW: 245.30 g mol−1. IR (KBr, cm−1): ν(NH) 3444s, ν(C=N) 1616s, ν(C=S) 848m. H-NMR
[DMSO-d6, δ, J (Hz)]: 11.53 (s, 1H, N4H); 8.38 (s, 1H, H10); 8.28 (s, 1H, N5Ha); 8.14 (s, 1H, N5Hb);
8.02 (s, 1H, H1); 7.98 (d, 2H, H5, H9, 8.30); 7.85 (s, 1H, H3); 7.72 (d, 2H, H6, H8, 8.31); 7.16 (s, 1H, H2).
13C-NMR (DMSO-d6, δ): 177.96 (C11); 141.25 (C1); 137.56 (C4); 135.49 (C10); 132.62 (C7); 129.99
(C2); 128.74 (C6); 128.74 (C8); 120.11 (C5); 120.11 (C9); 117.79 (C3).
4-(1H-Imidazole-1-yl)-benzaldehyde-N(5)-methylthiosemicarbazone (4ImBzTM) (4): Yield: 76%.
White solid. M.p.: 204–206 °C. Anal. Calc. for C12H13N5S: C, 55.58; H, 5.05; N, 27.01. Found: C,
55.88; H, 4.43; N, 26.95%. FW: 259.33 g mol−1. IR (KBr, cm−1): ν(NH) 3145s, ν(C=N) 1610s, ν(C=S)
835m. 1H-NMR [DMSO-d6, δ, J (Hz)]: 11.57 (s, 1H, N4H); 8.60 (s, 1H, N5Ha); 8.36 (s, 1H, H10); 8.07 (s,
1H, H1); 7.93 (d, 2H, H5, H9, 7.52); 7.82 (s, 1H, H3); 7.72 (d, 2H, H6, H8, 8.53); 7.13 (s, 1H, H2);

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13
3.03 (d, 3H, H12, 4.37). C-NMR (DMSO-d6, δ): 177.67 (C11); 140.49 (C1); 137.49 (C4); 135.49
(C10); 132.69 (C7); 129.99 (C2); 128.58 (C6); 128.58 (C8); 120.10 (C5); 120.10 (C9); 117.78 (C3);
30.81 (C12).
4-(1H-Imidazole-1-yl)-benzaldehyde-N(5)-phenylthiosemicarbazone (4ImBzTPh) (5): Yield: 83%.
White solid. M.p.: 194–196 °C. Anal. Calc. for C17H15N5S: C, 63.53; H, 4.70; N, 21.79. Found: C,
63.03; H, 4.70; N, 21.82%. FW: 321.34 g mol−1. IR (KBr, cm−1): ν(NH) 3358 s, ν(C=N) 1635s,
1
ν(C=S) 837m. H-NMR [DMSO-d6, δ, J (Hz)]: 8.38 (s, 1H, H10); 8.20 (s, 1H, H1); 8.06 (d, 2H, H6,
H8, 8.41); 7.84 (s, 1H, H3); 7.70 (d, 2H, H5, H9, 8.42); 7.58 (d, 2H, H13, H17, 7.67); 7.57 (t, 1H,
13
H16); 7.38 (t, 2H, H14, H16, 7.57); 7.14 (s, 1H, H2). C NMR (DMSO-d6, δ): 176.01 (C11); 141.68
(C1), 139.00 (C12); 137.69 (C4); 135.49 (C10); 132.40 (C7); 130.01 (C2), 129.05 (C8); 129.05 (C6);
127.99 (C14, C16); 125.97 (C13, C17); 125.34 (C15); 120.04 (C5); 120.03 (C9); 117.75 (C3).
4-(1H-Imidazole-1-yl)-acetophenone-N(5)-phenylthiosemicarbazone (4ImAcT) (6): Yield: 83%. White
solid. M.p.: 249–251 °C. Anal. Calc. for C12H13N5S: C, 55.58; H, 5.05; N, 27.01. Found: C, 55.31; H,
4.98; N, 27.43%. FW: 295.33 g mol−1. IR (KBr, cm−1): ν(NH) 3444s, ν(C=N) 1616s, ν(C=S) 848m.
1H-NMR [DMSO-d6, δ, J (Hz)]: 10.27 (s, 1H, N4H); 8.35 (s, 1H, H1); 8.33 (s, 2H, N5H); 8.08 (d, 2H,
H6, H8, 8.67); 7.82 (s, 1H, H3); 7.66 (d, 2H, H5, H9, 8.66); 7.12 (s, 1H, H2); 2.32(s, 3H, H12).
13CNMR (DMSO-d6, δ): 178.83 (C11); 145.85 (C4); 137.25 (C7); 135.93 (C10); 135.52 (C1); 130.00
(C2); 128.15 (C6); 128.15 (C8); 119.63 (C5); 119.63 (C9); 117.85 (C3); 13.91 (C12).
4-(1H-Imidazole-1-yl)-acetophenone-N(5)-methylthiosemicarbazone (4ImAcTM) (7): Yield: 76%.
White solid. M.p.: 215–216 °C. Anal. Calc. for C13H15N5S: C, 57.12; H, 5.53; N, 25.62. Found: C,
57.17; H, 5.57; N, 25.55%. FW: 273.36 g mol−1. IR (KBr, cm−1): ν(NH) 3358s, ν(C=N) 1615s, ν(C=S)
830m. 1H-NMR [DMSO-d6, δ, J (Hz)]: 10.28 (s, 1H, N4H); 8.54 (s, 1H, N5Ha); 8.37 (s, 1H, H1); 8.08
(d, 2H, H6, H8, 8.70); 7.83 (s, 1H, H3); 7.67 (d, 2H, H5, H9, 8.72); 7.13 (s, 1H, H2); 3.05 (d, 3H,
13
H13); 2.32 (s, 3H, H12). C-NMR (DMSO-d6, δ): 175.56 (C11); 146.50 (C4); 137.18 (C7); 135.96
(C10); 135.50 (C1); 129.97 (C2); 128.06 (C8); 128.06 (C6); 119.62 (C9); 119.62 (C5); 117.82 (C3);
13.91 (C12); 31.10 (C13).
4-(1H-Imidazole-1-yl)-acetophenone-N(5)-phenylthiosemicarbazone (4ImAcTPh) (8): Yield: 70%.
White solid. M.p.: 191–192 °C. Anal. Calc. for C18H17N5S: C, 64.45; H, 5.11; N, 20.88. Found: C,
64.39, H, 5.98; N, 20.60%. FW: 335.42 g mol−1. IR (KBr, cm−1): ν(NH) 3286s, ν(C=N) 1612s, ν(C=S)
1
832m. H-NMR [DMSO-d6, δ, J (Hz)]: 8.39 (s, 1H, H1); 8.18 (d, 2H, H6, H8, 8.57); 7.85
(s, 1H, H3); 7.70 (d, 2H, H5, H9, 8.60); 7.58 (d, 2H, H14, H18, 7.80); 7.39 (t, 2H, H15, H17, 7.66);
13
7.23 (t, 1H, H16, 7.38); 7.16 (s, 1H, H2); 2.42 (s, 3H, H12). C-NMR (DMSO-d6, δ): 176.99 (C11);
147.89 (C4); 139.13 (C13); 137.35 (C7); 135.78 (C10); 135.52 (C1), 129.89 (C2), 128.40 (C6, C8);
128.06 (C15, C17); 125.97 (C14, C18), 125.39 (C16); 119.64 (C5, C9); 117.84 (C3); 14.33 (C12).
3.3. Synthesis of Hydrazone Derivatives (9–20)
The imidazole-derived hydrazones were prepared by mixing equimolar amounts (2 mmol) of
4(5)-imidazole-carboxaldehyde, 4-(1H-imidazole-1-yl)-benzaldehyde and 4-(1H-imidazole-1-yl)-

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acetophenone with the desired hydrazide in methanol with addition of three drops of acetic acid as
catalyst. The reaction mixture was kept under reflux for 6 h. After cooling to room temperature the
resulting solids were filtered off, washed with ethanol and ether and dried in vacuum. In the case of
compound 9, the synthesis was carried out using acetonitrile as solvent.
4(5)-Imidazole-carboxaldehyde-acetylhydrazone [4(5)ImM] (9): Yield: 76%. White solid. M.p.:
163–164 °C. Anal. Calc. for C6H8N4O: C, 47.63; H, 5.30; N, 36.82. Found C, 47.45; H, 5.52; N,
36.86%. FW: 152.15 g mol−1. IR (KBr, cm−1): ν(N-H) 3210 s; ν(N-H)imidazole 3097–3004 m; ν(CH3ass
)
1
2930 m; ν(CH3s) 2864 m; ν(C=O) 1675 s; ν(C=N) 1615 m. H-NMR (DMSO-d6, δ, E, Z
configuration, enol): 13.30 (s, 1H, N4H, Z); 12.93 (s, 1H, N4H, E); 12.93 (s, 1H, OH, enol); 11.22
(s, 1H, N1H, enol); 11.03 (s, 1H, N1H, E); 9.76 (s, 1H, N1H, Z); 8.10 (s, 1H, H4, enol); 8.03 (s, 1H,
H4, Z), 7.94 (s, 1H, H4, E); 7.76 (s, 1H, H1, Z); 7.68 (s, 1H, H1, E); 7.43 (s, 1H, H1, enol); 7.37
(s, 1H, H2, Z); 7.24 (s, 1H, H2, E, enol); 2.02 (s, 3H, H6, Z), 2.16 (s, 3H, H6, E), 1.94 (s, 3H, H6,
enol). 13C-NMR (DMSO-d6, δ, E, Z configuration, enol): 171.89 (C5, E); 171.57 (C5, Z); 168.35
(C5, enol); 139.55 (C4, Enol); 137,0 (C4, E); 136.33 (C4, Z); 137.80 (C2, enol); 134.13 (C2, Z);
131.80 (C2, E); 137.36 (C3, Z); 121.25 (C3, enol); 120.19 (C3, E); 135.74 (C1, E); 135.53 (C1, enol);
22.15 (C6, Z); 21.54 (C6, enol); 20.08 (C6, E).
4(5)-Imidazole-carboxaldehyde-benzoylhydrazone [4(5)ImPh] (10): Yield: 81%. White solid. M.p.:
276–278 °C. Anal. Calc. for C11H10N4O: C, 61.67; H, 4.71; N, 26.15. Found: C, 61.80; H, 4.68; N,
26.06%. FW: 214.22 g mol−1. IR (KBr, cm−1): ν(N-H) 3213 s; ν(N-H)imidazole 3100–3027 m; ν(C=O)
1644 s; ν(C=N) 1626 m. 1H-NMR [DMSO-d6, δ, J(Hz)]: 14.29 (s, 1H, N4H); 8.37 (s, 1H, N1H); 8.11
(s, 1H, H4); 7.89 (d, 2H, H7, H11, 6.86); 7.75 (s, 1H, H1); 7.58 (m, 1H, H9); 7.53 (m, 2H, H8, H10,
6.91); 7.51(s, 1H, H2). 13C-NMR (DMSO-d6, δ): 162.81 (C5); 136.61 (C4); 135.67 (C2); 135.03 (C1);
133.55 (C6); 131.54 (C9); 128.14 (C8, C10); 127.44 (C7, C11); 121.26 (C3).
4(5)-Imidazole-carboxaldehyde-para-chloro-benzoylhydrazone [4(5)ImpClPh] (11): Yield: 56%.
White solid. M.p.: 277–280 °C. Anal. Calc. for C11H9ClN4O: C, 53.13; H, 3.65; N, 22.53. Found C, 53.08;
H, 3.59; N, 22.32%. FW: 248.67 g mol−1. IR (KBr, cm−1): ν(N-H) 3200 s; ν(N-H)imidazole 3100–3058 m;
1
ν(C=O) 1660 s; ν(C=N) 1615 m. H-NMR [DMSO-d6, δ, J(Hz)]: 14.34 (s, 1H, N4H); 8.39 (s, 1H,
N1H); 8.12 (s, 1H, H4); 7.95 (d, 2H, H7, H11, 8.55); 7.79 (s, 1H, H1); 7.65(d, 2H, H8, H10, 8.64);
7.55(s, 1H, H2). 13C-NMR (DMSO-d6, δ): 161.26 (C5); 136.50 (C4); 136.61 (C9); 135.99 (C2);
135.59 (C1); 132.23 (C6); 128.99 (C8, C10); 128.81 (C7, C11); 121.61 (C3).
4(5)-Imidazole-carboxaldehyde-para-nitro-benzoylhydrazone [4(5)ImpNO2Ph] (12): Yield: 76%.
Yellow solid. M.p.: >300 °C. Anal. Calc. for C11H9N5O3: C, 50.97; H, 3.50; N, 27.02. Found: C,
50.58; H, 3.43; N, 26.96%; mol wt, 259.22 g mol−1. IR (KBr, cm−1): ν(N-H) 3159 s; ν(N-H)imidazole
1
3127–3063 m; ν(C=O) 1687 s; ν(C=N) 1619 m. H-NMR [DMSO-d6, δ, J(Hz)]: 14.54 (s, 1H, N4H);
8.41 (d, 2H, H8, H10, 6.89); 8.37 (s, 1H, N1H); 8.18 (m, 2H, H7, H11); 8.13 (m, 1H, H4); 7.82
(s, 1H, H1); 7.61(s, 1H, H2). Due the low solubility of compound 12 was impossible to record the
13C-NMR spectrum.

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4-(1H-Imidazole-1-yl)-benzaldehyde-acetylhydrazone (4ImBzM) (13): Yield: 65%. White solid. M.p.:
148–150 °C. Anal. Calc. for C12H12N4O: C, 63.14; H, 5.30; N, 24.55. Found C, 63.07; H, 5.23; N,
24.66%. FW: 228.25 g mol−1. IR (KBr, cm−1): ν(N-H) 3171 s; ν(CH3ass) 2978m; ν(CH3s) 2863 m;
1
ν(C=O) 1659 s; ν(C=N) 1618 m. H NMR [DMSO-d6, δ, J (Hz), keto, enol tautomers]: 11.49 (s, 1H,
N4H, keto); 11.35 (s, 1H, OH, enol); 8.36 (s, 1H, H1, keto); 8.20 (s, 1H, H10, keto); 8.03 (s, 1H, H10,
enol); 7.83 (s, 1H, H3, keto); 7.81 (m, 2H, H6, H8, keto); 7.76 (m, 2H, H5, H9, keto); 7.15 (s, 1H, H2,
keto); 2.23 (s, 3H, H12, enol); 1.99 (s, 3H, H12, keto). 13C-NMR (DMSO-d6, δ, keto, enol tautomers):
172.00 (C11, keto); 165.67 (C11, enol); 144.49 (C10, keto); 141.38 (C10, enol); 137.63 (C4, keto);
135.45 (C1, keto); 132.70 (C7, keto); 130.02 (C2, keto); 127.97 (C6, C8, keto); 120.30 (C5, C9, keto);
117.75 (C3, keto); 21.58 (C12, keto), 20.12 (C12, enol).
4-(1H-Imidazole-1-yl)-benzaldehyde-benzoylhydrazone (4ImBzPh) (14): Yield: 84%. White solid.
M.p.: 250-252 °C. Anal. Calc. for C17H14N4O: C, 70.33; H, 4.86; N, 19.30. Found: C, 70.10; H, 4.70;
N, 19.47%. FW: 290.32 g mol−1. IR (KBr, cm−1): ν(N-H) 3195 s; ν(C=O) 1678 s; ν(C=N) 1610 m.
1H-NMR [DMSO-d6, δ, J(Hz)]: 11.95 (s, 1H, N4H); 8.52 (s, 1H, H10); 8.38 (s,1H, H1); 7.96 (d, 2H,
H13, H17, 7.55); 7.90 (m, 2H, H6, H8); 7.86 (s, 1H, H3); 7.80 (m, 2H, H5, H9, 8.46); 7.60 (t, 3H,
H14, H15, H16, 7.58); 7.16 (s, 1H, H2). 13C-NMR (DMSO-d6, δ): 163.12 (C11); 146.62 (C10); 137.77
(C4); 135.45 (C1); 133.30 (C12); 132.66 (C7); 131.70 (C15); 130.05 (C2); 128.41 (C6, C8 and C14,
C16); 127.58 (C13, C17); 120.27 (C5, C9); 117.72 (C3).
4-(1H-Imidazole-1-yl)-benzaldehyde-para-chloro-benzoylhydrazone (4ImBzpClPh) (15): Yield: 92%.
White solid. M.p.: 244–245 °C. Anal. Calc. for C17H13ClN4O: C, 62.87; H, 4.03; N, 17.25. Found: C,
62.94; H, 4,01; N, 17.05%. FW: 324.76 g mol−1. IR (KBr, cm−1): ν(N-H) 3189 s; ν(C=O) 1678 s;
ν(C=N) 1608 m. 1H-NMR [DMSO-d6, δ, J (Hz)]: 12.01 (s, 1H, N4H); 8.51 (s, 1H, H10); 8.38 (s,1H, H1);
7.97 (d, 2H, H13, H17, 8.27); 7.89 (d, 2H, H6, H8, 8.50); 7.85 (m, 1H, H3); 7.78 (m, 2H, H14, H16, 8.49);
13
7.64 (d, 2H, H5, H9, 8.29); 7.16 (s, 1H, H2). C-NMR (DMSO-d6, δ): 162.03 (C11); 146.96 (C10);
137.84 (C15); 136.55 (C4); 135.45 (C1); 132.54 (C7); 131.97 (C12); 130.05 (C2); 129.49 (C6, C8);
128.49 (C13, C17 and C14, C16); 120.29 (C5, C9); 117.71 (C3).
4-(1H-Imidazole-1-yl)-benzaldehyde-para-nitro-benzoylhydrazine (4ImBzpNO2Ph) (16): Yield: 96%.
Yellow solid. M.p.: 261–263 °C. Anal. Calc. for C17H13N5O3: C, 60.89; H, 3.91; N, 20.89. Found: C,
60.51; H, 3.92; N, 20.78%. FW: 335.31 g mol−1. IR (KBr, cm−1): ν(N-H) 3194 s; ν(C=O) 1687 s;
ν(C=N) 1606 m. 1H NMR [DMSO-d6, δ, J (Hz)]: 12.22 (s, 1H, N4H); 8.52 (s, 1H, H10); 8.41 (d, 2H,
H14, H16, 7.12); 8.38 (s,1H, H1); 8.18 (d, 2H, H13, H17, 8.49); 7.92 (d, 2H, H6, H8, 8.58); 7.85 (s,
1H, H3); 7.81 (d, 2H, H5, H9, 8.22); 7.16 (s, 1H, H2). 13C NMR (DMSO-d6, δ): 161.46 (C11); 147.72
(C10); 149.19 (C15); 138.93 (C4); 137.99 (C12); 135.42 (C1); 132.34 (C7); 130.11 (C2); 129.10 (C13,
C17); 128.65 (C6, C8); 123.27 (C14, C16); 120.30 (C5, C9); 117.70 (C3).
4-(Imidazole-1-yl)-acetophenone-acetylhydrazone (4ImAcM) (17): Yield: 68%. White solid. M.p.:
210–211 °C. Anal. Calc. for C13H14N4O: C 64.45; H, 5.82; N, 23.13. Found: C, 64.30; H, 5.75; N,
23.24%. FW: 242.27 g mol−1. IR (KBr, cm−1): ν(N-H) 3183 s; ν(CH3ass) 2933m; ν(CH3s) 2873 m;
1
ν(C=O) 1674 s; ν(C=N) 1615 m. H-NMR [DMSO-d6, δ, J (Hz), keto, enol tautomers]: 10.51 (s, 1H,
N4H, keto); 10.41 (s, 1H, OH, enol); 8.33 (s, 1H, H1, keto); 7.90 (d, 2H, H6, H8, 8.64, keto); 7.80

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(s, 1H, H3, keto); 7.70 (d, 2H, H5, H9, 8.64, keto); 7.14 (s, 1H, H2, keto); 2.29 (s, 3H, H12, enol); 2.27
(s, 3H, H12, keto); 2.26 (s, 3H, H13, enol); 2.06 (s, 3H, H13, keto). 13C-NMR (DMSO-d6, δ, keto, enol
tautomers): 171.82 (C11, keto); 166.13 (C11, keto); 149.31 (C10, enol); 145.78 (C10, keto); 137.14
(C7, enol); 137.00 (C7, keto); 136.53 (C4, keto); 135.42 (C1, keto); 129.96 (C2, keto); 127.60 (C6, C8,
enol); 127.28 (C6, C8, keto), 119.84 (C5, C9, keto); 119.70 (C5, C9, enol); 117.76 (C3, keto); 20.84
(C13, keto); 21.65 (C13, enol); 13.38 (C12, keto); 13.83 (C12, enol).
4-(Imidazole-1-yl)-acetophenone-benzoylhydrazone (4ImAcPh) (18): Yield: 92%. White solid. M.p.:
242–243 °C. Anal. Calc. for C18H15N4O: C, 71.04; H, 5.30; N, 18.41. Found: C, 71.10; H, 5.47; N,
18.32%. FW: 304.34 g mol−1. IR (KBr, cm−1): ν(N-H) 3139 s; ν(C=O) 1663 s; ν(C=N) 1614 m.
1H-NMR [DMSO-d6, δ, J (Hz)]:10.84 (s, 1H, N4H); 8.36 (s, 1H, H1); 7.95 (d, 2H, H6, H8, 6.36); 7.89
(d, 2H, H14, H18, 6.62); 7.83 (s, 1H, H3); 7.74 (d, 2H, H5, H9, 8.07); 7.56 (t, 3H, H15, H16, H17, 7.98);
7.14 (s, 1H, H2); 2.40 (s, 3H, H12).13C-NMR (DMSO-d6, δ): 168.03 (C11); 153.79 (C10); 137.37
(C7); 136.29 (C4); 135.43 (C1); 134.02 (C13); 131.23 (C16); 129.88 (C2); 128.05 (C15, C17); 127.66
(C6, C8); 127.62 (C14, C18); 117.72 (C3); 119.75 (C5, C9); 14.49 (C12).
4-(Imidazole-1-yl)acetophenone-para-chloro-benzoylhydrazone (4ImAcpClPh) (19): Yield: 96%.
White solid. M.p.: 240–243 °C. Anal. Calc. for (C18H15ClN4O): C, 63.81; H, 4.46; N, 16.54. Found: C,
63.79; H, 4.38; N, 16.99%. FW: 338.79 g mol−1. IR (KBr, cm−1): ν(N-H) 3240 s; ν(C=O) 1661 s;
1
ν(C=N) 1611 m. H-NMR [DMSO-d6, δ, J (Hz)]:10.91 (s, 1H, N4H); 8.36 (s, 1H, H1); 7.92 (m, 4H,
H6, H8, H14, H18); 7.83 (s, 1H, H3); 7.75 (d, 2H, H5, H9, 7.60); 7.60 (d, 2H, H15, H17, 8.17); 7.14
(s, 1H, H2); 2.40 (s, 3H, H12).13C-NMR (DMSO-d6, δ): 167.27 (C11); 155.34 (C10); 141.40 (C16);
137.49 (C7); 136.19 (C4); 135.46 (C1); 132.66 (C13); 130.10 (C2); 130.01 (C6, C8); 128.27 (C15,
C17); 127.86 (C14, C18); 119.76 (C5, C9); 117.76 (C3); 14.50 (C12).
4-(Imidazole-1-yl)-acetophenone-para-nitro-benzoylhydrazone (4ImAcpNO2Ph) (20): Yield: 76%.
Yellow solid. M.p.: 279–281 °C. Anal. Calc. for C18H15N5O3: C, 61.89; H, 4.33; N, 20.05. Found: C,
61.72; H, 4.28; N, 20.10%. FW: 349.34 g mol−1. IR (KBr, cm−1): ν(N-H) 3136 s; ν(C=O) 1665 s;
1
ν(C=N) 1616 m. Due to the low solubility of the compound it was impossible to record its H and
13C-NMR spectrum.
3.4. X-ray Crystallography
Crystals of ImT·H2O (1a), ImTPh (2), 4ImBzT (3), 4ImBzTM (4), 4ImBzTPh (5),
4(5)ImpClPh·H2O (11a), 4ImBzPh (14) and 4ImBzpClPh (15) were mounted on Mitgen loops in
random orientations and used for data collection. The structures were solved by direct methods using
SHELXS-97 [35] and refined by full-matrix least-squares techniques against F2 using
SHELXL-97 [36]. Positional and anisotropic atomic displacement parameters were refined for
non-hydrogen atoms. Although some hydrogen atoms could be identified in a Fourier difference map,
in the final model they were geometrically positioned and refined using a riding model. Molecular
graphics were obtained from ORTEP [37,38]. Crystals of 11a have shown to be twinned. Moreover,
the crystal structure presented disordered water molecule in four different positions with equal site
occupation factor of 0.25; the corresponding oxygen atoms were refined isotropically.

Molecules 2013, 18
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CCDC 903502, CCDC 952430, CCDC 903501, CCDC 952431, CCDC 952432, CCDC 903499,
CCDC 952433 and CCDC 903500 contain the supplementary crystallographic data for 1a, 2–5, 11a,
14 and 15, respectively. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/
data_request/cif, or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge
CB2 1EZ, UK; fax: +44-1223-336-033; or e-mail:deposit@ccdc.cam.ac.uk.
3.5. Biological Assays
Filamentous fungi, Cladosporium cladosporioides (LABB 6) and Aspergillus flavus (LABB44)
were from Biotechnology and Bioassays Laboratory (LABB, Chemistry Department, Universidade
Federal de Minas Gerais, Belo Horizonte, Brazil) and were maintained in potato dextrose agar (PDA)
under refrigeration at 7 °C. Candida glabrata (ATCC 2001) and Candida albicans (ATCC 18804)
were maintained in broth heart infusion (BHI). For the experiments, fungi were grown at room
temperature in PDA or BHI until abundant sporulation. Spores were harvested and suspended in sterile
water, counted on a Neubauer chamber and diluted to give a suspension containing a final
concentration of 5 × 103 spores/mL. An initial screening was carried out in microtiter plates, for all
compounds, in duplicate, using a concentration of 100 µg/well (in DMSO). The final concentrations of
DMSO in the assays did not exceed 2%. Positive (inoculum plus medium) and negative (inoculum plus
reference compound nystatin) controls were run simultaneously. Extracts showing activity in the
screening step were also tested in the microdilution assay in 12 serial concentrations (250, 125, 62.5,
31.3, 15.6, 7.81, 3.91, 1.95, 0.98, 0.49, 0.24 and 0.12 µg/mL), in duplicate. Fungal inhibition was
assessed by using a microplate TP-reader (Thermoplate, Palm City, FL, USA). The minimum
inhibitory concentration (MIC) value was defined as the lowest concentration of the compound
showing 100% of fungal growth inhibition after incubation time (48 h). MIC50 values were assessed
and express the lowest concentration of the compounds able to inhibit 50% of fungal growth.
Experiments were carried out according to Zacchino and Gupta [39].
4. Conclusions
The imidazole-derived thiosemicarbazones were inactive against all fungi strains except for
compounds 1 and 3 which proved to be selectively active against C. cladosporioides. In general the
imidazole-derived hydrazones showed antifungal activity against C. glabrata and C. cladosporioides.
Compounds 19 and 20 were selectively active against C. glabrata. All active compounds were more
effective than the control drug nystatin. As already mentioned, selectivity is a very important property
of an antifungal, which allows an optimum exploitation of its capacity without affecting beneficiating
microorganisms. Hence, some of the studied compounds could constitute novel antifungal drug
candidate prototypes.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/18/10/12645/s1.

Molecules 2013, 18
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Acknowledgments
This work was supported by PNPD-CAPES, CNPq and INCT-INOFAR (Proc. CNPq
988573.364/2008-6) from Brazil.
Conflicts of Interest
The authors declare no conflict of interest.
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© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).