Synthesis, anti-Candida activity, and cytotoxicity of new (4-(4-iodophenyl)thiazol-2-yl)hydrazine derivatives

Article abstract of DOI:10.1016/j.ejmech.2012.04.006

Novel (4-(4-iodophenyl)-thiazol-2-yl)hydrazine derivatives were assayed for their in vitro anti-Candida activity, compared to topical and systemic antifungal drugs, against twenty-seven clinical isolates. The presence of aliphatic chains or specific heteroaromatic rings on hydrazone moiety at position C2 and a 4-iodophenyl at C4 of the thiazole ring gave a promising inhibitory activity especially against Candida albicans and Candida krusei. The most active compounds have been also evaluated for their cytotoxicity and in association with clotrimazole for anti-Candida activity.

Full text of DOI:10.1016/j.ejmech.2012.04.006

European Journal of Medicinal Chemistry 53 (2012) 246e253
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, anti-Candida activity, and cytotoxicity of new
(4-(4-iodophenyl)thiazol-2-yl)hydrazine derivatives
,
a
a
a
Daniela Secci a, Bruna Bizzarri a , Adriana Bolasco , Simone Carradori , Melissa D’Ascenzio ,
*
Daniela Rivanera b, Emanuela Mari c, Lucia Polletta c, Alessandra Zicari c
a Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy
b Dipartimento Sanità Pubblica e Malattie Infettive, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy
c Dipartimento di Medicina Sperimentale, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel (4-(4-iodophenyl)-thiazol-2-yl)hydrazine derivatives were assayed for their in vitro anti-Candida
activity, compared to topical and systemic antifungal drugs, against twenty-seven clinical isolates. The
presence of aliphatic chains or specific heteroaromatic rings on hydrazone moiety at position C2 and a 4-
iodophenyl at C4 of the thiazole ring gave a promising inhibitory activity especially against Candida
albicans and Candida krusei. The most active compounds have been also evaluated for their cytotoxicity
and in association with clotrimazole for anti-Candida activity.
Received 16 November 2011
Received in revised form
6 March 2012
Accepted 6 April 2012
Available online 14 April 2012
Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords:
Antifungal drugs
Candida spp.
Cytotoxicity
Hydrazone
Hantzsch reaction
Thiazole
1. Introduction
leukemia and in those who have undergone transplantation [7].
Epidemiologic studies suggest that the problem is not under
Candida species are the most common opportunistic fungal
pathogens in humans, with Candida albicans being the most prev-
alent microbial population in mucosal and systemic infections.
Candidiasis encompasses infections that range from superficial,
such as oral thrush and vaginitis, to systemic and potentially life-
threatening diseases [1,2]. Candida infections are usually confined
to severely immunocompromised people, cancer, transplants as
well as surgery patients [3]. C. albicans and C. glabrata account for
approximately 80% of Candida spp. recovered from patients with
candidemia or invasive candidiasis [4]. Candida glabrata has
recently become very important because of its increasing incidence
worldwide, its association with fluconazole resistance in up to 20%
of clinical specimens, and its overall decreased susceptibility to
other azoles and polyenes [5]. Candida parapsilosis is also an
important species to consider in hospitalized patients because it is
especially common in infections associated with vascular catheters
and prosthetic devices [6]. Finally, Candida tropicalis has frequently
been considered an important cause of candidemia in patients with
control and, in fact, the incidence of drug resistance to antifungal
agents shows there is an urgent need for new chemical entities
with alternative mode of action.
From an overview of the most recent literature, it emerged that
(4-aryl-thiazol-2-yl)hydrazines have been investigated for their
significant antimicrobial activity against a large number of clinically
relevant fungal [8e10] and (myco)bacterial [11] strains. These
structures are endowed with a different mechanism of action in
relation to azoles and amphotericin B (inhibition of the fungal
biofilm growth, low toxicity profile) [12], and an encouraging
inhibitory activity against Escherichia coli FabH enzyme. Thus, the
(thiazol-2-yl)hydrazine nucleus has attracted much interest in the
development of pharmacologically active compounds. Keeping this
in view and in continuation of our research on biologically potent
molecules in the field [13e16], in this paper we report on the
synthesis and the biological evaluation (in vitro antifungal and
antibacterial activity, association with clotrimazole, and cytotox-
icity) of a new series of 2,4-disubstituted-1,3-thiazoles bearing
different moieties on double bond C]N and a 4-iodophenyl in
position C2 and C4 of thiazole nucleus, respectively (Table 1). Our
choice of an iodine atom derived from the good results obtained
* Corresponding author. Tel./fax: þ39 06 49913772.
E-mail address: bruna.bizzarri@uniroma1.it (B. Bizzarri).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.04.006

D. Secci et al. / European Journal of Medicinal Chemistry 53 (2012) 246e253
247
Table 1
Structures and minimal inhibitory concentration (MIC) of compounds 1e25, clotrimazole (C), fluconazole (F), and griseofulvin (G) against twenty-seven clinical strains of
Candida spp.
Aliphatic and cycloaliphatic derivatives
Tested fungi (MIC values mg/mL)
I
C. albicans
C. krusei
C. sakè
C. glabrata
C. tropicalis
C. parapsilosis
(10 strains)
(3 strains)
(2 strains)
(4 strains)
(4 strains)
(4 strains)
N
HN
R
N
S
R
1
2
3
4
5
6
7
8
Pentan-2-yl
Pentan-3-yl
Heptan-2-yl
Cyclopentyl
2-CH3ecyclopentyl
3-CH3ecyclopentyl
Cyclohexyl
2-CH3ecyclohexyl
3-CH3ecyclohexyl
4-CH3ecyclohexyl
Cycloheptyl
0.25e2
0.25e2
0.25e2
2e4
0.25e2
0.25e2
0.50e2
0.25e2
2e4
4
256
2
2
2
2
2
128
128
64
64
32
256
32
32
32
4
16
128
128
64
2e8
2e8
2e8
2e4
2e8
2e8
2e4
2e8
4e8
2e4
2e8
0.50e2
0.50e2
0.50e2
0.50e2
0.50e2
0.50e2
2e4
0.50e2
2e4
2e8
2e8
2e8
2e8
2e4
2e8
2e8
2e4
2e8
2e4
2e4
2e8
9
10
11
0.50e2
0.25e2
64
0.50e2
I
N
S
(Hetero)aryl derivatives
Cy
R1
HN
N
Cy
Fur-2-yl
Fur-2-yl
R1
H
CH3
H
CH3
CH3
H
CH3
H
12
13
14
15
16
17
18
19
20
21
22
23
24
25
C
0.50e2
0.25e2
2e8
2e4
0.25e2
2e4
0.25e2
0.25e2
0.25e2
0.50e2
2e4
0.25e2
2e4
2e8
64
128
128
2
64
64
64
128
2
4
2
64
64
2
64
128
128
4
64
64
64
128
4
4
4
64
64
4
4e8
2e8
4e8
4e8
2e8
4e8
2e8
2e8
2e8
4e8
4e8
2e8
4e8
4e8
0.25e2
4e16
8
2e8
0.50e2
2e4
2e4
0.50e2
2e4
0.50e2
0.50e2
0.50e2
2e4
2e4
0.50e2
2e4
2e4
2e8
2e4
0.50e2
2e8
2e4
2e8
2e8
2e8
0.50e2
2e4
2e8
2e4
2e4
0.50e2
4e16
8
Thiophen-2-yl
Thiophen-2-yl
Pyridin-2-yl
Pyridin-3-yl
Pyridin-3-yl
Pyridin-4-yl
Pyridin-4-yl
1H-Indol-3-yl
CH3
H
Benzodioxol-5-yl
Naphthalen-1-yl
Naphthalen-2-yl
Coumarin-3-yl
H
H
CH3
CH3
2e4
1e4
4e16
8
2e4
4e64
8
2
2
F
G
4e16
8
4e16
8
with other halogens (F, Cl, Br) and the possibility to increase the
lipophilicity of this scaffold. According to the established guidelines
of Clinical and Laboratory Standards Institute (CLSI) and the Euro-
pean Committee for Antimicrobial Susceptibility Testing (EUCAST),
we assayed the susceptibility of twenty-seven clinical Candida spp.
and bacterial strains to our compounds by determining their
minimum inhibitory concentration (MIC) [17].
spectrum, they showed strong bands at about w3200 and
w1595 cmꢀ1 due to the presence of NH and C]N group, respec-
tively. In MS spectra, the fragment peaks which correspond to loss
of eI (M ꢀ 127) from the molecular ion are consistent with the
postulated structure. Furthermore, because of the hindered rota-
tion of the double bond around the C]N group, the synthesized
products could exist as a couple of conformers E/Z, each interacting
in different way with the biological target. Diastereoselective
dynamic HPLC and theoretical studies about the E/Z interconver-
sion and the configurational stability of structurally similar thio-
semicarbazone derivatives showed that E-form dominated on Z-
form [18].
2. Chemistry
The new derivatives were synthesized as reported in our
previous communication [16]. Different carbonyl compounds were
reacted directly with thiosemicarbazide with catalytic amounts of
acetic acid in ethanol. These thiosemicarbazones were subse-
3. Pharmacology
quently condensed with newly synthesized a-bromo-4-iodo-ace-
tophenone (obtained by bromination of 4-iodo-acetophenone with
Br2 in CHCl3) to 2,4-disubstituted-1,3-thiazoles (Hantzsch reaction)
as shown in Scheme 1. All synthesized compounds were fully
characterized by analytical and spectral data. In general in the IR
First, derivatives 1e25, dissolved in dimethylsulfoxide (DMSO),
were evaluated for their antibacterial activity (Table S1, Supporting
Information). Organisms from routine clinical Gram-positive
(Staphylococcus aureus, Staphylococcus warneri, Streptococcus

248
D. Secci et al. / European Journal of Medicinal Chemistry 53 (2012) 246e253
NH2
H
N
i
I
N
O
S
NH2
H
N
H2N
S
N
S
iii
HN
N
I
I
1-25
ii
O
O
H3C
Br
Scheme 1. Synthesis of compounds 1e25. Reagents: (i) thiosemicarbazide, EtOH, acetic acid (cat.), rt; (ii) Br2, CHCl3; (iii) a-bromo-4-iodo-acetophenone, EtOH, rt.
pyogenes, Enterococcus faecalis) and Gram-negative isolates (E. coli,
Klebsiella pneumoniae, Enterobacter aerogenes, Proteus vulgaris)
from the respiratory tract were collected from specimens of
patients at the Hospital ‘Azienda Policlinico Umberto Iꢁ’ (Sapienza
University of Rome). The isolates were subcultured on a qualified
medium to ensure purity. The isolates were identified by conven-
tional methodologies; all isolates were subcultured to ensure
optimal growth. The in vitro antibacterial activities of the
compounds were determined by the broth microdilution method,
as recommended by the National Committee for Clinical Laboratory
Standards (NCCLS) [19] with Mueller-Hinton II broth (BBL Micro-
biology Systems, Cockeysville, MD).
Then, the antifungal activity was evaluated against twenty-
seven clinical fungal isolates of Candida spp. (C. albicans, C. krusei,
C. parapsilosis, C. tropicalis, C. sakè, and C. glabrata) and compared
with topical and systemic reference drugs clotrimazole, flucona-
zole, and griseofulvin (Table 1). The used clinical isolates were
recently collected from specimens of patients at the ‘Azienda
Policlinico Umberto Iꢁ’ (Sapienza University of Rome) and were
obtained from haematology/oncology and surgery departments,
which also included an intensive care unit. In particular, the
samples were isolated from the upper and lower respiratory tract,
blood, and indwelling venous catheters; the isolates were identi-
fied by conventional methodologies. Prior to testing, each isolate
was subcultured on a qualified medium to ensure purity and
optimal growth. All derivatives were dissolved in DMSO. The
in vitro antifungal activities were determined by the broth micro-
dilution method with Sabouraud dextrose broth (BBL Microbiology
Systems, Cockeysville, MD) as recommended by the NCCLS [20].
Furthermore, the most active and representative compounds
0.125e256 mg/mL), to evaluate a possible association (additive/
synergistic effect) between the substances on the fungal strains’
growth [20] and to have in this way a preliminary insight into the
possible mechanism of action of this scaffold (Table 2).
4. Cell toxicity
Compounds that have been demonstrated to achieve the
strongest anti-Candida activity were also analyzed in two steps to
estimate their ability to induce cytotoxic effects, at concentrations
ranging from 0.05 to 100 mg/mL (Table S2, Supporting Information),
on a cultured cell line derived from a human laryngeal epidermoid
carcinoma of the larynx (Hep2). Cell viability (EC50) was first
examined using Trypan Blue exclusion test (Table 3), as previously
described [21], followed by optical microscopy examination (Fig. 1).
Afterwards, MTT Cell Proliferation Assay was performed in the
same conditions used for the cytotoxicity tests to analyze variation
in cell proliferation rate and conversely, when metabolic events
lead to apoptosis or necrosis, the reduction in cell viability (Table 3).
5. Results and discussion
Derivatives 1e25 displayed a good inhibitory activity against all
clinical fungal isolates, with a selectivity oriented towards C. albi-
cans and C. tropicalis (MIC values lower than ones obtained after
treatment with reference drugs) (Table 1). A discrete antifungal
activity was also reported against other Candida spp. (MIC values
comparable with those obtained with reference drugs). With
respect to the other series of (thiazol-2-yl)hydrazines previously
synthesized by us [13ꢀ16], the presence of a 4-iodophenyl at C4
improved the antimicrobial activity of these 2,4-disubstituted-1,3-
thiazoles. The introduction of this halogen increased the
Clog P values of the newly synthesized molecules (Supporting
1, 2, 3, 5, 6, 20, and 23 (concentration range 0.5e4 mg/mL) were
tested against a number of clinical isolates of C. albicans and
C. krusei in combination with clotrimazole (concentration range
Table 2
Effects of the association between the most active compounds (2
m
g/mL) and clotrimazole (C) (2
24 h, MIC ( g/mL)
m
g/mL) on C. albicansa and C. krusei.b
Comp.
18 h, MIC (
m
g/mL)
m
48 h, MIC (mg/mL)
C. albicans (4 strains)
C. krusei (2 strains)
C. albicans (4 strains)
C. krusei (2 strains)
C. albicans (4 strains)
C. krusei (2 strains)
C
2
0.5
1
0.5
0.5
1
4
2
4
2
2
2
2
4
2
1
2
1
1
2
1
0.5
4
2
4
2
2
4
2
4
2
2
2
2
2
2
2
2
4
2
4
2
2
4
2
4
C þ 1
C þ 2
C þ 3
C þ 5
C þ 6
C þ 20
C þ 23
1
0.5
a
C. albicans strains isolated from bronchial washing of transplant patient and from blood culture of surgery unit’s patient.
C. krusei strains isolated from blood culture of coronary unit’s patient and from wound’s swab of surgery unit’s patient.
b

D. Secci et al. / European Journal of Medicinal Chemistry 53 (2012) 246e253
249
Table 3
and the reference drug clotrimazole (a well-known drug widely
used in therapy) exert the same cytotoxicity trend. For a better
comprehension of this behavior, cells after treatment with
compound 23 (50 and 100 mg/mL) have been analyzed by optical
microscopy (40X) to assess the biological effects on cell structural
elements. As reported in Fig. 1, at concentrations higher (50 and
Anti-proliferative and cytotoxic effect (EC50) of the most active compounds tested on
Hep2 cells after 24 h of incubation at 37 ꢁC.a
Comp.
MTT Cell Proliferation
Trypan Blue exclusion
test EC50 (mg/mL)
assay EC50
(
mg/mL)
1
2
3
5
6
20
23
45
50
42
30
50
25
35
48
nd
nd
62
30
51
30
40
40
100
mg/mL) than its best MIC value (0.25
mg/mL), compound 23
exerts
a
cytotoxic effect and induces cellular modifications
involving the changing of cell shape and volume, presence of
apoptotic blebs and of intra-cytoplasmatic vacuoles, and induction
of nuclear fragmentation. The reported data suggested that the
combined treatment could lead to an encouraging synergic effect
on the fungal strains’ growth which might be useful to limit the
induction of resistance to clotrimazole and its toxicity.
Clotrimazole
nd ¼ not determined.
a
Data represent arithmetic means
ꢂ
SD of at least three independent
experiments.
In this study we investigated the synthesis and in vitro antimi-
crobial activity of new thiazolylhydrazine derivatives by deter-
mining the minimum inhibitory concentrations with the EUCAST
broth microdilution method. The substitution of the nucleus linked
to the hydrazone moiety had a deep impact on the antifungal
activity on this scaffold. Based on the reported results, derivatives
1e3, 5, 6, 20, and 23 could be also used in association with azole
derivatives to enhance their antifungal activity. Moreover, dis-
playing a better activity and a lower cytotoxicity compared to the
drug reference clotrimazole, they represent good templates for the
development of novel broad-spectrum anti-Candida spp. agents.
information) and confirmed an interesting correlation between
Clog P values >5.34 (i.e. 7.11 for compound 23) and a stronger
antifungal activity in this scaffold. In addition, some of the most
active and representative compounds (1e3, 5, 6, 20, and 23) were
tested in association with clotrimazole to evaluate a potential
synergistic/additive effect with the azoles class (Table 2). The best
concentrations of the selected compounds and clotrimazole resul-
ted in the lowest MIC values reported in Table 1. In particular,
compounds 5, 23 and 20 were the most potent in inhibiting
C. albicans with MIC values of 0.5
compounds 5 and 23) and 1 g/mL (1-fold reduction for compound
g/mL) at 18 h
and 24 h, while compounds 1e3 and 6 showed a similar behavior
only at 18 h. When the MIC end points were determined following
48 h of incubation, the observed anti-Candida effect was slightly
lower. As regards C. krusei strains, the combined treatment of clo-
trimazole plus selected derivatives, after 18e48 h, gave a slight
mg/mL (2-fold reduction for
m
6. Experimental protocols
20) in comparison with clotrimazole alone (MIC ¼ 2
m
The chemicals, solvents for synthesis, and spectral grade
solvents were purchased from Aldrich (Italy) and used without
further purification. Melting points (uncorrected) were determined
automatically on an FP62 apparatus (Mettler-Toledo). 1H NMR
spectra were recorded at 400 MHz on a Bruker spectrometer using
CDCl3 or DMSO-d6 as solvent. Chemical shifts are expressed as
reduction of the MIC values (2e4
mg/mL) with respect to those of
the control (MIC ¼ 4
m
g/mL). In general, MIC values obtained from
d units (parts per millions) relative to the solvent peak. Coupling
this drugs association may suggest the possibility of using them in
combination, in order to lower their single dosage and so to limit
their corresponding toxicity.
constants J are valued in Hertz (Hz). IR spectra were registered on
a Perkin Elmer FT-IR Spectrometer Spectrum 1000 in anhydrous
potassium bromide (KBr). Elemental analyses for C, H, and N were
recorded on a PerkineElmer 240 B microanalyzer and the analytical
results were within ꢂ0.4% of the theoretical values for all
compounds. All reactions were monitored by TLC performed on
0.2 mm thick silica gel plates (60 F254 Merck). The GC/MS unit
Considering this, the same selected derivatives (1e3, 5, 6, 20,
and 23) and clotrimazole were investigated for their cytotoxic
effects (EC50) by Trypan blue exclusion test and MTT assay (Table 3).
The results suggested that both the newly synthesized compounds
Fig. 1. Morphological changes of Hep2 cells after treatment with compound 23. Cells have been analyzed after 24 h of incubation at 37 ꢁC by optical microscopy (40X): A) Hep2 cells
incubated for 24 h with culture medium alone represents the control. Cells are all adherent to the flask, plasma membranes are intact, cell shape is regular, and one or more nuclei
are well evident in each cell. Cells are strictly near one to each other because of cell to cell contacts. The black arrows indicate cells in mitosis. B) Hep2 cells after incubation for 24 h
with 50
the flask surface. Cells show various dimensions and shapes, plasma membranes are not intact and blebs are present (black arrow). In several cells intra-cytoplasmatic vacuoles are
evident (red arrows). C) Hep2 cells after incubation with 100 g/mL of compound 23. Cells are all detached by the flask surface and are floating in the supernatant. Apoptotic blebs
mg/mL of compound 23. The number of cells is significantly reduced and cell shape is completely changed. Cells are losing their cell to cell contacts and getting detached by
m
are largely evident as well as the presence of intra-cytoplasmatic vacuoles (red arrows). Nuclei are reduced in small fragments (black arrow). (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)

250
D. Secci et al. / European Journal of Medicinal Chemistry 53 (2012) 246e253
consisted of a PerkineElmer Autosystem XL gas chromatograph.
The samples were introduced directly into the ion source. Electron
ionization (EI) mass spectra were obtained by a Faisons QMD 1000
6.1.6. 1-(4-(4-Iodophenyl)thiazol-2-yl)-2-(3-
methylcyclopentylidene)hydrazine (6)
Yield 88%, mp 177e179 ꢁC; 1H NMR (CDCl3, 400 MHz)
mass spectrometer (70 eV, 200
m
A, ion source temperature of
d (ppm): 1.09e1.14 (m, 3H, CH3ecyclopentyl), 1.48e1.53 (m, 1H,
200 ꢁC, carrier gas helium).
CHecyclopentyl), 2.10e2.21 (m, 2H, CH2ecyclopentyl), 2.54e2.66
(m, 2H, CH2ecyclopentyl), 2.82e2.90 (m, 2H, CH2ecyclopentyl),
6.72 (s, 1H, C5Hethiazole), 7.44e7.46 (d, J ¼ 8.7 Hz, 2H, Ar),
7.81e7.83 (d, J ¼ 8.7 Hz, 2H, Ar), 12.11 (bs, 1H, NH, D2O exch.).
6.1. General procedure for the synthesis of derivatives 1e25
The appropriate carbonyl compound (50 mmol) was dissolved
in 50 mL of ethanol and magnetically stirred with an equimolar
quantity of thiosemicarbazide for 24 h at room temperature with
catalytic amounts of acetic acid. The desired thiosemicarbazone
precipitated from reaction mixture, was filtered, crystallized from
suitable solvent, and dried. Equimolar quantities of 4-iodo-aceto-
phenone and bromine, both dissolved in chloroform, were stirred
for 4 h at room temperature until the presence of HBr disappeared.
The solution was evaporated under vacuum and the obtained pale
6.1.7. 1-Cyclohexylidene-2-(4-(4-iodophenyl)thiazol-2-yl)
hydrazine (7)
Yield 84%, mp 186e188 ꢁC; 1H NMR (CDCl3, 400 MHz)
d
(ppm):
1.62e1.69 (m, 4H,
2
ꢃ
CH2ecyclohexyl), 1.71e1.78 (m, 2H,
CH2ecyclohexyl), 2.48e2.54 (m, 2H, CH2ecyclohexyl), 2.63e6.70
(m, 2H, CH2ecyclohexyl), 6.72 (s, 1H, C5Hethiazole), 7.45e7.47 (d,
J ¼ 8.6 Hz, 2H, Ar), 7.81e7.83 (d, J ¼ 8.6 Hz, 2H, Ar), 12.03 (bs, 1H,
NH, D2O exch.).
yellow solid was washed with petroleum ether to give
iodo-acetophenone in good yield (94%). Equimolar amounts of the
prepared thiosemicarbazone (50 mmol) and -bromo-4-iodo-ace-
tophenone (50 mmol), both suspended in 50 mL of ethanol, were
reacted at room temperature under magnetic stirring for 10 h. The
precipitate was filtered and purified by chromatography to give
compounds 1e25 in high yield.
a-bromo-4-
6.1.8. 1-(4-(4-Iodophenyl)thiazol-2-yl)-2-(2-
methylcyclohexylidene)hydrazine (8)
a
Yield 96%, mp 157e159 ꢁC; 1H NMR (DMSO-d6, 400 MHz)
d
(ppm): 1.06e1.08 (m, 3H, CH3ecyclohexyl), 1.12e1.17 (m, 1H,
CHecyclohexyl), 1.43e1.52 (m, 2H, CH2ecyclohexyl), 1.69e1.76
(m, 2H, CH2ecyclohexyl), 1.78e1.85 (m, 2H, CH2ecyclohexyl),
2.37e2.39 (m, 1H, CHecyclohexyl), 2.97e2.99 (m, 1H,
CHecyclohexyl), 7.29 (s, 1H, C5Hethiazole), 7.62e7.64 (d, J ¼ 7.8 Hz,
2H, Ar), 7.73e7.75 (d, J ¼ 7.8 Hz, 2H, Ar), 10.98 (bs, 1H, NH, D2O
exch.).
6.1.1. 1-(4-(4-Iodophenyl)thiazol-2-yl)-2-(pentan-2-ylidene)
hydrazine (1)
Yield 89%, mp 210e211 ꢁC; 1H NMR (CDCl3, 400 MHz)
d (ppm):
0.95e0.99 (t, J ¼ 7.3 Hz, 3H, CH3),1.60e1.66 (m, 2H, CH2), 2.19 (s, 3H,
CH3C]N), 2.34e2.37 (m, 2H, CH2), 6.73 (s, 1H, C5Hethiazole),
7.45e7.47 (d, J ¼ 7.7 Hz, 2H, Ar), 7.82e7.84 (d, J ¼ 7.7 Hz, 2H, Ar),
12.23 (bs, 1H, NH, D2O exch.).
6.1.9. 1-(4-(4-Iodophenyl)thiazol-2-yl)-2-(3-
methylcyclohexylidene)hydrazine (9)
Yield 99%, mp 168e170 ꢁC; 1H NMR (DMSO-d6, 400 MHz)
d
(ppm): 1.01e1.03 (m, 3H, CH3ecyclohexyl), 1.23e1.26 (m, 1H,
6.1.2. 1-(4-(4-Iodophenyl)thiazol-2-yl)-2-(pentan-3-ylidene)
hydrazine (2)
CHecyclohexyl), 1.78e1.84 (m, 2H, CH2ecyclohexyl), 1.92e1.97 (m,
2H, CH2ecyclohexyl), 2.13e2.19 (m, 2H, CH2ecyclohexyl),
2.49e2.51 (m, 1H, CHecyclohexyl), 3.02e3.04 (m, 1H,
CHecyclohexyl), 6.73 (s, 1H, C5Hethiazole), 7.43e7.47 (m, 2H, Ar),
7.80e7.83 (m, 2H, Ar), 12.44 (bs, 1H, NH, D2O exch.).
Yield 77%, mp 184e185 ꢁC; 1H NMR (DMSO-d6, 400 MHz)
d
(ppm): 1.01e1.06 (m, 6H, 2 ꢃ CH3), 2.37e2.41 (m, 4H, 2 ꢃ CH2),
6.78 (s, 1H, C5Hethiazole), 7.62e7.64 (m, 2H, Ar), 7.78e7.80 (m, 2H,
Ar), 11.01 (bs, 1H, NH, D2O exch.).
6.1.10. 1-(4-(4-Iodophenyl)thiazol-2-yl)-2-(4-
methylcyclohexylidene)hydrazine (10)
6.1.3. 1-(Heptan-2-ylidene)-2-(4-(4-iodophenyl)thiazol-2-yl)
hydrazine (3)
Yield 95%, mp 151e153 ꢁC; 1H NMR (CDCl3, 400 MHz)
Yield 92%, mp 212e213 ꢁC; 1H NMR (CDCl3, 400 MHz)
d
(ppm):
d
(ppm): 0.97e0.99 (m, 3H, CH3ecyclohexyl), 1.19e1.22 (m,
2H, CHecyclohexyl), 1.73e1.75 (m, 1H, CHecyclohexyl),
1.97e2.00 (m, 2H, CHecyclohexyl), 2.18e2.21 (m, 2H,
0.90e0.93 (t, J ¼ 7.1 Hz, 3H, CH3), 1.33e1.38 (m, 4H, 2 ꢃ CH2),
1.57e1.61 (m, 2H, CH2), 2.18 (s, 3H, CH3), 2.35e2.38 (t, J ¼ 7.1 Hz, 2H,
CH2), 6.73 (s, 1H, C5Hethiazole), 7.45e7.47 (d, J ¼ 7.9 Hz, 2H, Ar),
7.82e7.84 (d, J ¼ 7.9 Hz, 2H, Ar), 12.21 (bs, 1H, NH, D2O exch.).
2
ꢃ
2
ꢃ
2 ꢃ CHecyclohexyl), 2.51e2.54 (m, 2H, 2 ꢃ CHecyclohexyl), 6.71 (s,
1H, C5Hethiazole), 7.45e7.47 (d, J ¼ 8.2 Hz, 2H, Ar), 7.82e74 (d,
J ¼ 8.2 Hz, 2H, Ar), 12.36 (bs, 1H, NH, D2O exch.).
6.1.4. 1-Cyclopentylidene-2-(4-(4-iodophenyl)thiazol-2-yl)
hydrazine (4)
6.1.11. 1-Cycloheptylidene-2-(4-(4-iodophenyl)thiazol-2-yl)
Yield 86%, mp 213e215 ꢁC; 1H NMR (CDCl3, 400 MHz)
d
(ppm):
hydrazine (11)
1.84e1.87 (m, 2H, CH2ecyclopentyl), 1.93e1.96 (m, 2H,
CH2ecyclopentyl), 2.51e2.55 (m, 2H, CH2ecyclopentyl), 2.62e2.66
(m, 2H, CH2ecyclopentyl), 6.72 (s, 1H, C5Hethiazole), 7.45e7.47 (d,
J ¼ 8.7 Hz, 2H, Ar), 7.82e7.84 (d, J ¼ 8.7 Hz, 2H, Ar), 12.07 (bs, 1H,
NH, D2O exch.).
Yield 80%, mp 200e201 ꢁC; 1H NMR (CDCl3, 400 MHz)
d
(ppm):
1.61e1.69 (m, 6H,
3
ꢃ
CH2ecycloheptyl), 1.82e1.86 (m, 2H,
CH2ecycloheptyl), 2.55e2.58 (m, 2H, CH2ecycloheptyl), 2.68e2.71
(m, 2H, CH2ecycloheptyl), 6.72 (s, 1H, C5Hethiazole), 7.46e7.48 (d,
J ¼ 8.6 Hz, 2H, Ar), 7.82e7.84 (d, J ¼ 8.6 Hz, 2H, Ar), 12.23 (bs, 1H,
NH, D2O exch.).
6.1.5. 1-(4-(4-Iodophenyl)thiazol-2-yl)-2-(2-
methylcyclopentylidene)hydrazine (5)
6.1.12. 1-(Furan-2-ylmethylene)-2-(4-(4-iodophenyl)thiazol-2-yl)
hydrazine (12)
Yield 87%, mp 148e150 ꢁC; 1H NMR (CDCl3, 400 MHz)
d
(ppm): 1.21e1.28 (m, 3H, CH3ecyclopentyl), 1.73e1.78 (m, 1H,
Yield 84%, mp 271e272 ꢁC; 1H NMR (DMSO-d6, 400 MHz)
CHecyclopentyl), 1.99e2.11 (m, 2H, CH2ecyclopentyl), 2.55e2.65
(m, 2H, CH2ecyclopentyl), 3.70e3.76 (m, 2H, CH2ecyclopentyl),
6.78 (s, 1H, C5Hethiazole), 7.48e7.50 (d, J ¼ 8.6 Hz, 2H, Ar),
7.76e7.78 (d, J ¼ 8.6 Hz, 2H, Ar), 12.14 (bs, 1H, NH, D2O exch.).
d (ppm): 6.53 (m, 1H, C4Hefuran), 6.87 (s, 1H, C5Hethiazole),
7.33 (m, 1H, C5Hefuran), 7.62e7.64 (m, 2H, Ar), 7.68e7.70 (m,
1H, C3Hefuran), 7.79e7.81 (m, 4H, Ar), 11.15 (bs, 1H, NH, D2O
exch.).

D. Secci et al. / European Journal of Medicinal Chemistry 53 (2012) 246e253
251
6.1.13. 1-(1-(Furan-2-yl)ethylidene)-2-(4-(4-iodophenyl)thiazol-2-
yl)hydrazine (13)
J ¼ 7.2 Hz, 2H, C3He and C5Hepyridine), 8.82e8.84 (d, J ¼ 7.2 Hz,
2H, C3He and C5Hepyridine), 12.09 (bs, 1H, NH, D2O exch.).
Yield 84%, mp 272e273 ꢁC; 1H NMR (DMSO-d6, 400 MHz)
d
(ppm): 2.24 (s, 3H, CH3), 6.57 (m, 1H, C4Hefuran), 6.83 (s, 1H,
6.1.21. 1-((1H-Indol-3-yl)methylene)-2-(4-(4-iodophenyl)thiazol-
2-yl)hydrazine (21)
C5Hethiazole), 7.37 (m, 1H, C5Hefuran), 7.65e7.67 (d, J ¼ 8.0 Hz,
2H, Ar), 7.71e7.73 (m, 1H, C3Hefuran), 7.75e7.77 (d, J ¼ 8.0 Hz, 2H,
Ar), 11.04 (bs, 1H, NH, D2O exch.).
Yield 90%, mp 230e231 ꢁC; 1H NMR (DMSO-d6, 400 MHz)
d
(ppm): 7.22e7.25 (m, 3H, indole), 7.34 (s, 1H, C5Hethiazole),
7.48e7.50 (m, 1H, indole), 7.66e7.68 (m, 2H, Ar), 7.75e7.77 (m,
2H, Ar), 8.23e8.25 (m, 1H, indole), 8.27 (s, 1H, HeC]N), 11.56 (bs,
1H, NH, D2O exch.), 11.88 (bs, 1H, NH-indole, D2O exch.).
6.1.14. 1-(4-(4-Iodophenyl)thiazol-2-yl)-2-(thiophen-2-
ylmethylene)hydrazine (14)
Yield 93%, mp 190e192 ꢁC; 1H NMR (DMSO-d6, 400 MHz)
d
(ppm): 7.08e7.10 (m, 1H, C4Hethiophene), 7.35e7.36 (m, 1H,
6.1.22. 1-(Benzo[d][1,3]dioxol-5-ylmethylene)-2-(4-(4-iodophenyl)
thiazol-2-yl)hydrazine (22)
C3Hethiophene), 7.37 (s, 1H, C5Hethiazole), 7.57e7.58 (m,
C5Hethiophene), 7.63e7.65 (d, J ¼ 8.5 Hz, 2H, Ar), 7.74e7.76 (d,
J ¼ 8.5 Hz, 2H, Ar), 8.20 (s, 1H, HeC]N), 12.23 (bs, 1H, NH, D2O
exch.).
Yield 80%, mp 218e220 ꢁC; 1H NMR (DMSO-d6, 400 MHz)
d
(ppm): 6.07 (s, 2H, OCH2O), 6.98e7.01 (m, 1H,
C7Hebenzodioxole), 7.11e7.12 (m, 1H, C4H-benzodioxole),
7.24e7.27 (m, 1H, C6Hebenzodioxole), 7.36 (s, 1H, C5Hethiazole),
7.65e7.67 (d, J ¼ 7.9 Hz, 2H, Ar), 7.74e7.76 (d, J ¼ 7.9 Hz, 2H, Ar),
7.96 (s, 1H, HeC]N), 12.05 (bs, 1H, NH, D2O exch.).
6.1.15. 1-(4-(4-Iodophenyl)thiazol-2-yl)-2-(1-(thiophen-2-yl)
ethylidene)hydrazine (15)
Yield 95%, mp 263e265 ꢁC; 1H NMR (DMSO-d6, 400 MHz)
d
(ppm): 2.32 (s, 3H, CH3C]N), 7.06e7.07 (m, 1H, C4Hethiophene),
6.1.23. 1-(4-(4-Iodophenyl)thiazol-2-yl)-2-(naphthalen-1-
ylmethylene)hydrazine (23)
7.34e7.35 (m, 1H, C3Hethiophene), 7.37 (s, 1H, C5Hethiazole),
7.50e7.52 (m, 1H, C5Hethiophene), 7.65e7.67 (d, J ¼ 8.1 Hz, 2H,
Ar), 7.75e7.77 (d, J ¼ 8.1 Hz, 2H, Ar), 12.25 (bs, 1H, NH, D2O exch.).
Yield 84%, mp 191e193 ꢁC; 1H NMR (DMSO-d6, 400 MHz)
d
(ppm): 7.43 (s, 1H, C5Hethiazole), 7.62e7.64 (m, 2H, Ar),
7.65e7.67 (m, 3H, Ar), 7.70e7.72 (m, 2H, Ar), 7.76e7.80 (m, 4H, Ar),
8.01 (s, 1H, HeC]N), 10.19 (bs, 1H, NH, D2O exch.).
6.1.16. 1-(4-(4-Iodophenyl)thiazol-2-yl)-2-(1-(pyridin-2-yl)
ethylidene)hydrazine (16)
Yield 99%, mp 168e170 ꢁC; 1H NMR (DMSO-d6, 400 MHz)
6.1.24. 1-(4-(4-Iodophenyl)thiazol-2-yl)-2-(1-(naphthalen-2-yl)
d
(ppm): 2.42 (s, 3H, CH3C]N), 7.50 (s, 1H, C5Hethiazole),
ethylidene)hydrazine (24)
7.54e7.57 (m, 1H, C5Hepyridine), 7.67e7.69 (d, J ¼ 8.5 Hz, 2H,
Ar), 7.77e7.69 (d, J ¼ 8.5 Hz, 2H, Ar), 8.13e8.15 (m, 2H, C3He and
C4Hepyridine), 8.67e8.68 (m, 1H, C6Hepyridine), 11.75 (bs, 1H, NH,
D2O exch.).
Yield 75%, mp 228e230 ꢁC; 1H NMR (CDCl3, 400 MHz)
d (ppm):
2.40 (s, 3H, CH3C]N), 6.95 (s, 1H, C5Hethiazole), 7.52e7.55 (m, 2H,
Ar), 7.56e7.58 (d, J ¼ 6.7 Hz, 2H, Ar), 7.72e7.74 (d, J ¼ 6.7 Hz, 2H, Ar),
7.94e7.97 (m, 3H, Ar), 8.04e8.05 (m, 1H, Ar), 8.12e8.14 (m, 2H, Ar),
11.57 (bs, 1H, NH, D2O exch.).
6.1.17. 1-(4-(4-Iodophenyl)thiazol-2-yl)-2-(pyridin-3-ylmethylene)
hydrazine (17)
6.1.25. 1-(2H-Chromen-2-oxo-3-ylethylidene)-2-(4-(4-iodophenyl)
Yield 88%, mp 221e223 ꢁC; 1H NMR (DMSO-d6, 400 MHz)
thiazol-2-yl)hydrazine (25)
d
(ppm): 7.47 (s,1H, C5Hethiazole), 7.64e7.67 (m, 2H, Ar), 7.76e7.78
Yield 77%, mp 153e155 ꢁC; 1H NMR (CDCl3, 400 MHz)
d (ppm):
(m, 2H, Ar) 7.79e7.81 (m, 1H, C5Hepyridine), 8.11e8.14 (m, 1H,
C4Hepyridine), 8.37e8.39 (m, 1H, C6Hepyridine), 8.69e8.71 (m,
1H, C2Hepyridine), 8.96 (s, 1H, HeC]N), 12.56 (bs, 1H, NH, D2O
exch.).
2.38 (s, 3H, CH3C]N), 6.91 (s, 1H, C5Hethiazole), 7.31e7.33 (m, 1H,
Ar), 7.55e7.57 (d, J ¼ 8.3 Hz, 2H, Ar), 7.60e7.63 (m, 2H, Ar),
7.74e7.76 (d, J ¼ 8.3 Hz, 2H, Ar), 8.06 (s, 1H, Ar), 12.52 (bs, 1H, NH,
D2O exch.).
6.1.18. 1-(4-(4-Iodophenyl)thiazol-2-yl)-2-(1-(pyridin-3-yl)
ethylidene)hydrazine (18)
6.2. Antibacterial activity
Yield 70%, mp 228e230 ꢁC; 1H NMR (DMSO-d6, 400 MHz)
Microtiter plates containing serial dilutions of each compound
d
(ppm): 2.39 (s, 3H, CH3C]N), 7.53 (s, 1H, C5Hethiazole),
ranging from 128 to 0.5
m
g/mL were inoculated with each organism
L final volume;
7.69e7.71 (m, 2H, Ar), 7.74e7.76 (m, 2H, Ar), 7.83e7.85 (m, 1H,
C5Hepyridine), 8.16e8.18 (m,1H, C4Hepyridine), 8.42e8.44 (m,1H,
C6Hepyridine), 8.77e8.79 (m, 1H, C2Hepyridine), 12.16 (bs, 1H, NH,
D2O exch.).
to yield the appropriate density (105/mL) in a 100
m
each plate included positive controls (bacteria without
a compound), and a negative control (medium only). The plates
were incubated for 18e22 h at 35 ꢁC. The Minimal Inhibitory
Concentration (MIC) for all isolates was defined as the lowest
concentration of antibacterial agent that completely inhibited the
growth of the organism, as detected by the unaided eye.
6.1.19. 1-(4-(4-Iodophenyl)thiazol-2-yl)-2-(pyridin-4-ylmethylene)
hydrazine (19)
Yield 99%, mp >290 ꢁC; 1H NMR (DMSO-d6, 400 MHz)
d (ppm):
7.58 (s, 1H, C5Hethiazole), 7.65e7.67 (d, J ¼ 7.9 Hz, 2H, Ar),
7.77e7.79 (d, J ¼ 7.9 Hz, 2H, Ar), 8.08e8.09 (m, 2H, C3He and
C5Hepyridine), 8.13 (s, 1H, HeC]N), 8.80e8.81 (m, 2H, C2He and
C6Hepyridine), 13.11 (bs, 1H, NH, D2O exch.).
6.3. Antifungal activity
Microtiter plates containing serial dilutions of each compound
were inoculated with each organism to yield the appropriate
density (103/mL) in a 100
mL final volume; each plate included
6.1.20. 1-(4-(4-Iodophenyl)thiazol-2-yl)-2-(1-(pyridin-4-yl)
positive controls (fungi without a compound) and a negative
control (medium only). The plates were incubated for 24 h at 37 ꢁC.
The MIC for all isolates was defined as the lowest concentration of
antifungal agents that completely inhibited the growth of the
organism, as detected by unaided eye.
ethylidene)hydrazine (20)
Yield 92%, mp >290 ꢁC; 1H NMR (DMSO-d6, 400 MHz)
d (ppm):
2.36 (s, 3H, CH3C]N), 7.56 (s, 1H, C5Hethiazole), 7.67e7.69 (d,
J ¼ 8.3 Hz, 2H, Ar), 7.78e7.80 (d, J ¼ 8.3 Hz, 2H, Ar), 8.14e8.16 (d,

252
D. Secci et al. / European Journal of Medicinal Chemistry 53 (2012) 246e253
6.4. Broth microdilution susceptibility test method
The yeast inoculum was adjusted to concentration of
650 nm (OD650) was subtracted from the OD550 to eliminate
nonspecific background.
a
0.5e2.5 ꢃ 103 CFU/mL in RPMI medium, and an aliquot of
0.1 mL was added to each well of the microdilution plate. The
plates were incubated at 35 ꢁC, the MIC end points were read
visually following 18, 24, and 48 h of incubation and were
defined as the lowest concentration that produced a prominent
decrease in turbidity compared with that of the drug-free growth
control.
6.7. Optical microscopy
Hep2 cells, at the end of the incubation time with the different
compounds, have been detached from the flasks using a manual
scraper, washed a couple of time with phosphate buffer saline
(PBS) and fixed overnight at þ4 ꢁC with a solution of 2.5% glutar-
aldehyde in 0.1 M PBS (pH 7.3). Cells were then fixed with 2%
osmium tetroxide in the same buffer for 2 h at room temperature.
After that, cells were dehydrated at þ4 ꢁC in graded steps of ethanol
(30% up to 100%) 100 at step, treated with toluene and embedded
6.5. Cytotoxicity assay
48 h at 60 ꢁC in epossidic resin (Epon 812). Thin sections (30
mm),
The Hep2 cell line, delivered from
a human laryngeal
obtained using an ultra microtome (Ultra Cut Leica), were pro-
cessed for Azure II staining plus Fuchsin and analyzed with an
optical microscopy equipped with a camera to acquire images
(NIKON E-1000) [23].
epidermoid carcinoma of the larynx and purchased from
a Korean Cell Line Bank (KCLB No. 10023), was a kind gift by Prof.
R. Misasi (Department of Experimental Medicine e Sapienza
University of Rome). Cells were cultured in Dulbecco’s modified
eagle media (DMEM), supplemented with 10% fetal bovine
serum (FBS; Gibco, USA) and 1% streptomycinepenicillin. Cell
lines were maintained as adherent type cultures under humid-
ified atmosphere in 5% CO2 at 37 ꢁC in Dulbecco’s modified
Eagle’s culture medium. Experiments were performed on cells
grown to 60e70% confluency. The stock solutions of the inves-
tigated compounds were prepared in sterile DMSO and succes-
sive dilutions were made in culture medium; the DMSO percent
present in culture medium never exceeded 0.5%. Hep2 cells in
the exponential phase of growth (1 ꢃ 105/mL) were seeded into
24-well microplate and incubated for 24 h with six different
Acknowledgments
This work was supported by grants from MURST (2008e2009).
Appendix A. Supplementary material
Supplementary material associated with this article can
be found, in the online version, at doi:10.1016/j.ejmech.2012.
04.006.
concentrations of the compounds (100e0.05
mg/mL). Some
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