Synthesis and in vitro activity of 2-thiazolylhydrazone derivatives compared with the activity of clotrimazole against clinical isolates of Candida spp.

Article abstract of DOI:10.1016/j.bmcl.2007.05.078

In this paper, we report on the synthesis of a novel series of 2-thiazolylhydrazone derivatives and the influence of the substituents on the thiazole ring on antifungal activity. All synthesized compounds were screened for their in vitro activities agains

Full text of DOI:10.1016/j.bmcl.2007.05.078

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4635–4640
Synthesis and in vitro activity of 2-thiazolylhydrazone
derivatives compared with the activity of clotrimazole
against clinical isolates of Candida spp.
Franco Chimenti,a Bruna Bizzarri,a,* Elias Maccioni,b Daniela Secci,a Adriana Bolasco,a
Rossella Fioravanti,a Paola Chimenti,a Arianna Granese,a Simone Carradori,a
Daniela Rivanera,c Daniela Lilli,c Alessandra Zicarid and Simona Distintob
a
`
Dipartimento di Studi di Chimica e Tecnologia delle Sostanze Biologicamente Attive Universita degli Studi di Roma ‘‘La Sapienza’’,
P.le A. Moro 5, 00185 Rome, Italy
b
`
Dipartimento Farmaco Chimico Tecnologico, Universita degli Studi di Cagliari, Via Ospedale 72, 09124 Cagliari, Italy
c
`
Dipartimento di Scienze di Sanita Pubblica, Universita ‘‘La Sapienza’’, P.le A. Moro 5, 00185 Rome, Italy
Dipartimento di Medicina Sperimentale, Universita ‘‘La Sapienza’’, V.le Regina Elena 324, 00161 Rome, Italy
`
d
`
Received 17 April 2007; revised 23 May 2007; accepted 24 May 2007
Available online 27 May 2007
Abstract—In this paper, we report on the synthesis of a novel series of 2-thiazolylhydrazone derivatives and the influence of the
substituents on the thiazole ring on antifungal activity. All synthesized compounds were screened for their in vitro activities against
22 clinical isolates of Candida spp., representing six different species, compared to clotrimazole as a reference compound. Some of
the tested compounds were found to possess significant antifungal activity when compared to clotrimazole, in particular compound
14 which exhibited higher potency against most of the Candida spp. considered. The compounds that were most active as anti-Can-
dida agents were also submitted to cytotoxic screening by the Trypan Blue dye exclusion assay and in general they were shown to
induce low cytotoxic effects.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Although Candida spp. are present as commensal flora
in 30–60% of healthy individuals,1 they can become
infective depending on predisposing conditions related
to the host, such as systemic disease leading to immun-
osupression, and local conditions.2 As a matter of fact,
the pathogenicity of Candida spp. is affected by several
virulence factors, such as the ability to adhere to epithe-
lial and endothelial cells,3 germination, extracellular
proteinases and phospholipases, and phenotypic
switching.4
in supportive therapy and an increasing elderly
population.
In addition, the widespread use of antimicrobial agents
for prophylaxis and treatment has led to less effective
antibiotics, not only because many induced side effects
but also due to the emergence of drug resistant microor-
ganisms.5–7
Large-scale surveillance for fungal bloodstream infec-
tions has been performed worldwide by a number of
organizations, including the European Confederation
of Medical Mycology (ECMM),8 the Centres for Dis-
ease Control and Prevention (CDC),9 and the National
Epidemiology of Mycoses Survey (NEMIS).10 These
studies have demonstrated an increasing incidence of
drug-resistant fungal pathogens. As a matter of fact, a
significant number of yeast species (Candida glabrata,
Candida krusei, Candida guilliermondii, Candida lusita-
niae) have been shown to exhibit primary resistance to
amphotericin B, while C. glabrata and C. krusei have
Over the past few decades, the frequency of systematic
fungal infections has progressively increased, due to the
larger population of immunologically compromised
hosts (AIDS, cancer, and transplant), thanks to advances
Keywords: 2-Thiazolylhydrazone derivatives; Antifungal agents; Can-
dida spp.
*
Corresponding author. Tel.: +39 06 49913975; fax: +39 06
49913772; e-mail: bruna.bizzarri@uniroma1.it
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.05.078

4636
F. Chimenti et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4635–4640
been found to be intrinsically less susceptible to triazoles
than the same Candida albicans. It has also been found
that Candida dubliniensis can rapidly develop in vitro
stable resistance to fluconazole.11,12 Furthermore,
C. albicans intrinsically resistant strains have been
observed as part of a commensal microflora or due to
acquisition from the environment or from other
individuals.13
infections today can reach 90% in immunocompromised
patients especially.15
Apart from this, antifungal chemotherapy could be
associated with a low toxicological profile, determining
termination of the treatment.16
Another important issue is the clinical outcome due to
several factors such as host defenses, the virulence of
the fungal pathogen, drug efficacy/toxicity, and drug
interactions.17,18 Besides, pharmacokinetic studies have
shown a variability of at least 50% between individu-
als;19,20 in particular, plasma levels for voriconazole
can vary from 74% to 100%.21 These variations could
account for the lack of efficacy and the toxicity.
In addition, despite advances in therapy thanks to the
introduction of fluconazole and itraconazole, the devel-
opment of new, more potent azoles, such as voriconazole
and posaconazole, and the discovery of a new class of
echinocandins,14 mortality rates for invasive fungal
As a consequence of the toxicity of the currently used
polyene antifungal drugs and the emergence of candidal
species resistant to azole-based agents, there is an urgent
need for investigating alternative drug therapies.
n(H2C)
R
S
S
n(H2C)
R
i
N-NH
O
NH2-NH
+
NH2
NH2
O
Br
It has recently been reported in the literature that phe-
nyl-thiazole analogues can be used as efficient antifungal
agents.22 Moreover, a large number of substituted ben-
zothiazole,23 thiazole derivatives of triazoles,24 thia-
zolylhydrazones25 have been shown to exhibit
significant antimicrobial activity against a variety of
fungal strains.
ii
R1
S
n(H2C)
R
N-NH
N
R1
1-20
n = 1,2,3
R = H, 2-CH3, 3-CH3, 4-CH3
Moving from these literature indications and pursuing
our research in the field,26,27 in this report we describe
the synthesis and antimicrobial evaluation of a new ser-
ies of 2-thiazolylhydrazone derivatives.
Scheme 1. Synthesis of 2-thiazolylhydrazone derivatives 1–20.
Reagents: (i) isopropyl alcohol, AcOH; (ii) isopropyl alcohol.
Table 1. Chemical and physical data of derivatives 1–20
H
R
N
R1
N
N
S
Compound
R
R1
4-CH3
M.W.
Mp (ꢁC)
% Yield
1
2
Cyclopentyl
Cyclopentyl
352.27a
368.27a
383.27a
383.27a
363.28a
372.70a
417.16a
414.35a
380.34a
396.34a
411.31a
445.21a
442.41a
380.34a
396.34a
391.32a
400.76a
339.85b
400.76a
285.41
218–220
214–217
187–190
219–220
207–208
223–225
213–215
235–237
160–162
135–137
165–166
130–133
150–152
171–174
162–165
189–190
195–199
192–195
177–179
184–185
71
88
69
80
67
58
66
58
64
94
60
71
87
95
84
98
84
77
92
97
4-OCH3
3-NO2
4-NO2
4-CN
4-Cl
3
Cyclopentyl
Cyclopentyl
4
5
Cyclopentyl
Cyclopentyl
6
7
Cyclopentyl
Cyclopentyl
4-Br
8
4-C6H5
4-CH3
4-OCH3
3-NO2
4-Br
9
Cyclohexyl-2-CH3
Cyclohexyl-2-CH3
Cyclohexyl-2-CH3
Cyclohexyl-2-CH3
Cyclohexyl-2-CH3
Cyclohexyl-3-CH3
Cyclohexyl-3-CH3
Cyclohexyl-3-CH3
Cyclohexyl-3-CH3
Cyclohexyl-3-CH3
Cyclohexyl-4-CH3
Cycloheptyl
10
11
12
13
14
15
16
17
18
19
2028
4-C6H5
4-CH3
4-OCH3
4-CN
4-Cl
4-F
4-Cl
H
a Chloridrate.
b Bromidrate.

F. Chimenti et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4635–4640
4637
The cytotoxic activity of selected compounds that
showed good activity against Candida species was eval-
uated: we incubated them in the presence of an immor-
talized hybrid cell line displaying an endothelial
phenotype, EAhy 926, derived from the fusion of human
umbilical vein endothelial cells (HUVEC) with a lung
carcinoma cells and determined their cell viability by
the Trypan Blue dye exclusion assay.
Table 2. 1H NMR data of derivatives 1–20
Compound
1H NMR d (ppm)
1a
1.80 (q, 2H, cyclopentyl), 1.92 (q, 2H, cyclopentyl), 2.35 (s, 3H, 40-CH3-phenyl), 2.50 (t, 2H, cyclopentyl), 2.62 (t, 2H,
cyclopentyl), 6.67 (s, 1H, C5H-thiaz.), 7.22 (d, J = 7.9 Hz, 2H, Ar), 7.55 (d, J = 7.9 Hz, 2H, Ar), 12.13 (br s, 1H, NH, D2O
exch.)
2a
3a
4a
5b
6b
7b
8a
1.85 (q, 2H, cyclopentyl), 1.90 (q, 2H, cyclopentyl), 2.50 (t, 2H, cyclopentyl), 2.63 (t, 2H, cyclopentyl), 3.83 (s, 3H, 40-OCH3-
phenyl), 6.51 (s, 1H, C5H-thiaz.), 6.96 (d, J = 7.9 Hz, 2H, Ar), 7.63 (d, J = 7.9 Hz, 2H, Ar), 12.13 (br s, 1H, NH, D2O exch.)
1.90 (q, 2H, cyclopentyl), 1.97 (q, 2H, cyclopentyl), 2.58 (t, 2H, cyclopentyl), 2.68 (t, 2H, cyclopentyl), 6.96 (s, 1H, C5H-thiaz.),
7.77 (t, 1H, Ar), 8.20 (t, 1H, Ar), 8.31 (d, J = 7.9 Hz, 1H, Ar), 8.53 (d, J = 1.68 Hz, 1H, Ar), 12.13 (br s, 1H, NH, D2O exch.)
1.91 (q, 2H, cyclopentyl), 2.01 (q, 2H, cyclopentyl), 2.57 (t, 2H, cyclopentyl), 2.66 (t, 2H, cyclopentyl), 6.97 (s, 1H, C5H-thiaz.),
7.94 (d, J = 8.8 Hz, 2H, Ar), 8.35 (d, J = 8.8 Hz, 2H), 12.40 (br s, 1H, NH, D2O exch.)
1.83 (q, 2H, cyclopentyl), 1.87 (q, 2H, cyclopentyl), 2.37 (t, 4H, cyclopentyl), 7.66 (s, 1H, C5H-thiaz.), 7.96 (d, J = 8.4 Hz, 2H,
Ar), 8.12 (d, J = 8.4 Hz, 2H, Ar), 10.83 (br s, 1H, NH, D2O exch.)
1.72 (q, 2H, cyclopentyl), 1.78 (q, 2H, cyclopentyl), 2.37 (t, 4H, cyclopentyl), 7.30 (s, 1H, C5H-thiaz.), 7.44 (d, J = 8.4 Hz, 2H,
Ar), 7.84 (d, J = 8.4 Hz, 2H, Ar), 10.70 (br s, 1H, NH, D2O exch.)
1.72 (q, 2H, cyclopentyl), 1.80 (q, 2H, cyclopentyl), 2.37 (t, 4H, cyclopentyl), 7.31 (s, 1H, C5H-thiaz.), 7.57 (d, J = 8.4 Hz, 2H,
Ar), 7.77 (d, J = 8.4 Hz, 2H, Ar), 10.65 (br s, 1H, NH, D2O exch.)
1.87 (q, 2H, cyclopentyl), 1.94 (q, 2H, cyclopentyl), 2.52 (t, 2H, cyclopentyl), 2.66 (t, 2H, cyclopentyl), 6.72 (s, 1H, C5H-thiaz.),
7.38 (d, J = 7.1 Hz, 1H, Ar), 7.47 (t, 2H, Ar), 7.59 (d, J = 7.1 Hz, 2H, Ar), 7.69 (d, J = 7.9 Hz, 2H, Ar), 7.77 (d, J = 7.9 Hz, 2H,
Ar), 12.24 (br s, 1H, NH, D2O exch.)
9a
1.16 (d, J = 6.3 Hz, 3H, CH3), 1.24–1.42 (m, 1H, cyclohexyl), 1.57–1.66 (m, 2H, cyclohexyl), 1.79–1.86 (m, 1H, cyclohexyl),
1.98–2.03 (m, 2H, cyclohexyl), 2.17–2.27 (m, 1H, cyclohexyl), 2.41 (s, 3H, 40-CH3-phenyl), 2.43–2.49 (m, 1H, cyclohexyl),
3.04–3.10 (m, 1H, cyclohexyl), 6.66 (s, 1H, C5H-thiaz.), 7.28 (d, J = 8.4 Hz, 2H, Ar), 7.60 (d, J = .4 Hz, 2H, Ar), 12.50 (s, 1H,
NH, D2O exch.)
10a
1.11 (d, J = 6.3 Hz, 3H, CH3,), 1.17–1.37 (m, 1H, cyclohexyl), 1.48–1.61 (m, 2H, cyclohexyl), 1.75–1.81 (m, 1H, cyclohexyl),
1.95–1.99 (m, 2H, cyclohexyl), 2.11–2.21 (m, 1H, cyclohexyl), 2.35–2.44 (m, 1H, cyclohexyl), 2.97–3.03 (m, 1H, cyclohexyl),
3.81 (s, 3H, 40-OCH3-phenyl), 7.53 (s, 1H, C5H-thiaz.), 6.94 (d, J = 8.8 Hz, 2H, Ar), 7.60 (d, J = 8.8 Hz, 2H, Ar), 12.37 (s, 1H,
NH, D2O exch.)
11b
12a
13a
1.08 (d, J = 6.5 Hz, 3H, CH3), 1.16–1.26 (m, 1H, cyclohexyl), 1.37–1.59 (m, 2H, cyclohexyl), 1.69–2.02 (m, 4H, cyclohexyl),
2.31–2.42 (m, 1H, cyclohexyl), 2.91–2.96 (m, 1H, cyclohexyl), 7.56 (s, 1H, C5H-thiaz.), 7.70 (t, 1H, Ar), 8.12 (d, J = 8.1 Hz,
1H, Ar), 8.27 (d, J = 8.1 Hz, 1H, Ar), 8.68 (s, 1H, Ar), 10.95 (s, 1H, NH, D2O exch.)
1.15 (d, J = 6.3 Hz, 3H, CH3), 1.26–1.38 (m, 1H, cyclohexyl), 1.54–1.65 (m, 2H, cyclohexyl), 1.75–1.81 (m, 1H, cyclohexyl),
1.92–1.98 (m, 2H, cyclohexyl), 2.13–2.23 (m, 1H, cyclohexyl), 2.39–2.47 (m, 1H, cyclohexyl), 2.97–3.02 (m, 1H, cyclohexyl),
6.76 (s, 1H, C5H-thiaz.), 7.59 (s, 4H, Ar), 12.41 (s, 1H, NH, D2O exch.)
1.13 (d, J = 6.3 Hz, 3H, CH3), 1.26–1.39 (m, 1H, cyclohexyl), 1.54–1.61 (m, 2H, cyclohexyl), 1.79–1.83 (m, 1H, cyclohexyl),
1.89–1.97 (m, 2H, cyclohexyl), 2.17–2.22 (m, 1H, cyclohexyl), 2.38–2.41 (m, 1H, cyclohexyl), 3.01–3.05 (m, 1H, cyclohexyl),
6.75 (s, 1H, C5H-thiaz.), 7.36 (d, J = 7.1 Hz, 1H, Ar), 7.46 (t, 2H, Ar), 7.59 (d, J = 7.1 Hz, 2H, Ar), 7.69 (d, J = 8.4 Hz, 2H,
Ar), 7.78 (d, J = 8.4 Hz, 2H, Ar), 12.48 (s, 1H, NH, D2O exch.)
14a
15a
16a
17a
18a
19a
20a
1.16 (d, J = 6.4 Hz, 3H, CH3), 1.35 (s, 1H, cyclohexyl), 1.60–1.62 (m, 2H, cyclohexyl), 1.81 (s, 2H, cyclohexyl), 1.98–1.99 (m,
2H, cyclohexyl), 2.20–2.22 (m, 1H, cyclohexyl), 2.42 (d, J = 6.1 Hz, 3H, 40-CH3-phenyl), 3.03–3.04 (m, 1H, cyclohexyl), 6.63
(s, 1H, C5H-thiaz.), 7.28 (s, 2H, Ar), 7.60 (s, 2H, Ar), 12.52 (s, 1H, NH, D2O exch.), 13.56 (s, 1H, NH, D2O exch.)
1.06 (d, J = 6.2 Hz, 3H, CH3), 1.20–1.21 (m, 1H, cyclohexyl), 1.81–1.95 (m, 6H, cyclohexyl), 2.52 (t, 1H, cyclohexyl), 3.05–3.06
(m, 1H, cyclohexyl), 3.85 (s, 3H, 40-OCH3-phenyl), 6.52 (s, 1H, C5H-thiaz.), 6.99 (d, J = 8.0 Hz, 2H, Ar), 7.65 (d, J = 8.6 Hz,
2H, Ar), 12.44 (s, 1H, NH, D2O exch.), 13.58 (s, 1H, NH, D2O exch.)
1.19 (d, J = 6.4 Hz, 3H, CH3), 1.57–1.61 (m, 3H, cyclohexyl), 1.83–1.85 (m, 1H, cyclohexyl), 2.00–2.03 (m, 2H, cyclohexyl),
2.19–2.21 (m, 1H, cyclohexyl), 2.50–2.52 (m, 1H, cyclohexyl), 3.00–3.01 (m, 1H, cyclohexyl), 6.87 (s, 1H, C5H-thiaz.), 7.79 (d,
J = 7.8 Hz, 2H, Ar), 7.86 (s, 2H, Ar), 12.48 (s, 1H, NH, D2O exch.), 14.01 (br s, 1H, NH, D2O exch.)
1.64 (d, J = 6.5 Hz, 3H, CH3), 1.25–1.26 (m, 1H, cyclohexyl), 1.69–1.71 (m, 1H, cyclohexyl), 1.98–1.99 (m, 2H, cyclohexyl),
2.20–2.21 (m, 2H, cyclohexyl), 2.50–2.52 (m, 2H, cyclohexyl), 2.95–2.96 (m, 1H, cyclohexyl), 6.68 (s, 1H, C5H-thiaz.), 7.47 (d,
J = 8.6 Hz, 2H, Ar), 7.67 (d, J = 8.5 Hz, 2H, Ar), 12.50 (s, 1H, NH, D2O exch.), 13.80 (s, 1H, NH, D2O exch.)
1.16 (d, J = 6.5 Hz, 3H, CH3), 1.58–1.59 (m, 3H, cyclohexyl), 1.98–1.99 (m, 1H, cyclohexyl), 2.05–20.7 (m, 2H, cyclohexyl),
2.25–2.27 (m, 1H, cyclohexyl), 2.40–2.41 (m, 1H, cyclohexyl), 3.00–3.01 (m, 1H, cyclohexyl), 6.62 (s, 1H, C5H-thiaz.), 7.18 (d,
J = 6.1 Hz, 2H, Ar), 7.70 (d, J = 5.3 Hz, 2H, Ar), 12.71 (s, 1H, NH, D2O exch.), 14.30 (s, 1H, NH, D2O exch.)
0.99 (d, J = 6.1 Hz, 3H, CH3), 1.20–1.21 (m, 2H, cyclohexyl), 1.60–1.61 (m, 1H, cyclohexyl), 2.00–2.01 (t, 2H, cyclohexyl),
2.20–2.30 (m, 2H, cyclohexyl), 2.60–2.61 (m, 1H, cyclohexyl), 3.09–3.10 (m, 1H, cyclohexyl), 6.68 (s, 1H, C5H-thiaz.), 7.47–
7.51 (m, 2H, Ar), 7.71 (d, J = 6.6 Hz, 2H, Ar), 12.55 (s, 1H, NH, D2O exch.), 13.75 (s, 1H, NH, D2O exch.)
1.58–1.63 (m, 6H, cycloheptyl), 1.70–1.71 (m, 2H, cycloheptyl), 2.55–2.56 (m, 2H, cycloheptyl), 2.70–2.72 (m, 2H,
cycloheptyl), 6.80 (s, 1H, C5H-thiaz.), 7.48–7.50 (m, 3H, Ar), 7.73 (d, J = 7.9 Hz, 2H, Ar), 12.40 (s, 1H, NH, D2O exch.), 13.80
(br s, 1H, NH, D2O exch.)
a CDCl3.
b DMSO-d6.

4638
F. Chimenti et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4635–4640
2-Thiazolylhydrazone derivatives 1–20 were synthesized
as reported in our previous communications.28,29
unit accounted for 65% of the cases (15/22 isolates).
The isolates were identified by conventional methodol-
ogies. Prior to testing, each isolate was subcultured on
a qualified medium to ensure purity and optimal
growth.
Cyclic ketones or aryl aldehydes reacted directly with
thiosemicarbazide and the obtained thiosemicarbaz-
ones subsequently reacted with a-halogenoketones to
yield the 4-substituted thiazole ring derivatives as
shown in Scheme 1. In the synthesis of all compounds
isopropyl alcohol proved to be the best solvent for
our purpose. As a matter of fact, the reaction prod-
ucts precipitate on cooling down and can be filtered
and purified by crystallization from ethanol or etha-
nol/isopropanol. All synthesized compounds were fully
characterized by analytical and spectral data as listed
in Tables 1 and 2.
The data reported in Table 3 show that cyclopentyl deriv-
atives 1–6 had good anti-C. albicans and anti-C. glabrata
activity. In particular compounds 1, 2, and 6 were very
active against both strains, while compounds 3, 4, and 5
showed good activity only against C. glabrata. Further-
more, cyclohexyl substituted derivatives 9–19 showed
good anti-C. albicans activity, in particular with com-
pounds 9–11 and 14–19. Some derivatives, especially 9
and 10, also showed good anti-C. glabrata activity. Cyclo-
heptyl derivative 20 showed good anti-C. albicans activity.
Among all synthesized compounds, we found the anti-
fungal activity showed by compound 14, 2-(3-methyl-
cyclohexyl)-1-[4-(4-methylphenyl)-2-thiazolyl]hydrazone,
particularly interesting. Compound 14wasmoreactivethan
the clotrimazole reference compound against C. albicans,
All synthesized compounds were evaluated for anti-
fungal activity and compared with the reference com-
pound clotrimazole (Table 3).30,31
The newly prepared compounds were dissolved in
dimethylsulfoxide (DMSO) and their in vitro activity
was evaluated against a total of 22 strains of Candida
species. The included isolates were Candida albicans (8
strains), Candida glabrata (4), Candida krusei (3), Can-
`
C. tropicalis, C. krusei, C. parapsilosis, and C. sake.
The cytotoxic profile of the compounds showed that the
derivatives were less toxic at concentrations below
0.5 lg/mL with a percentage of viable cells of 73.0 and
94.7 (Table 4).32–34
`
dida tropicalis (3), Candida sake (2), and Candida
parapsilosis (2). The isolates were collected from
specimens of patients at the ‘Azienda Policlinico
Umberto Iꢁ’ of Rome ‘La Sapienza’ University and
were obtained from haematology/oncology and sur-
gery 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 intensive care
In conclusion, a series of novel 2-thiazolylhydrazone
derivatives was assessed for antifungal activity on 22
Candida strains. Compound 14 was found to have good
antifungal activity and may be used as a good reference
for identifying features of the structure that could be
important for antifungal activity.
Table 3. Minimal inhibitory concentration (MIC)a of compounds 1–20 and clotrimazole against 22 strains of Candida species
Compound
Tested fungi (MICa lg/mL)
`
C. sake
(2 strains)
C. albicans
(8 strains)
C. glabrata
(4 strains)
C. tropicalis
(3 strains)
C. krusei
(3 strains)
C. parapsilosis
(2 strains)
1
2
0.25–2
0.50–2
1–8
0.50–2
2–4
4–16
64–128
64–128
64–128
32–64
32–64
128–>128
64–128
2–4
64–128
32–64
>128
32–64
32–64
64–128
>128
>128
64
128
8–16
>128
>128
128
8–16
128
>128
8
16–32
32
3
0.50–2
0.50–2
0.50–2
0.50–2
2–8
16–32
16
32
4
8–32
2–8
5
6
0.25–2
2–8
16
32
7
8
16–64
0.25–2
0.50–2
0.50–2
4–8
>128
0.50–2
0.25–2
16–64
2–4
32
32
9
10
11
12
13
14
15
16
17
18
19
20
Clob
8
64
16
8
32
16–32
16
>128
64–128
16
8–16
8–16
16
32–64
64
32–64
0.50–2
0.50–2
0.50–2
0.50–2
0.50–4
0.50–2
0.50–2
0.50–4
64–128
32–64
>128
>128
>128
>128
32–64
>128
4–8
16–32
2–4
8–16
4–8
2
0.50–2
2–4
2–4
0.50–2
2–4
4–8
0.50–1
1–2
0.50–1
1
2–4
4–8
4–16
2–4
4–16
05–1
8–16
4–8
1
8
8
2
8–16
4–8
8–16
4–8
16–32
8–16
a Range value.
b Clotrimazole.

F. Chimenti et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4635–4640
4639
Table 4. Cytotoxic effect of selected compounds tested on EAhy 926
cells after 24 h of incubation at 37 ꢁC, using Trypan Blue exclusion
test, expressed as cell survival fractiona (%)
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Compound
Concentrationb (lg/mL)
50
5
0.5
0.05
1
2
75.0 3.2
66.0 3.2
76.0 4.6
64.9 4.2
64.0 3.2
75.0 4.0
83.3 3.6
38.5 4.3
54.5 3.2
25.0 1.9
28.6 2.8
52.9 2.7
54.5 5.0
80.0 4.1
76.6 4.0
81.0 3.2
81.0 3.0
63.6 3.6
83.3 3.6
85.7 4.2
72.7 3.1
72.7 3.8
50.0 2.5
50.0 1.9
60.0 1.5
80.0 3.9
82.0 4.6
82.4 2.8
84.7 2.8
89.2 2.8
78.5 4.2
84.0 3.2
86.0 3.8
83.3 3.6
80.0 1.9
68.7 2.9
70.0 4.2
76.9 1.7
87.5 1.8
85.4 5.0
85.0 2.2
88.9 6.0
86.0 2.6
85.7 3.6
86.0 4.8
88.8 2.2
84.0 2.0
83.3 2.2
68.8 1.4
73.0 3.1
80.0 2.0
94.7 2.5
5
6
9
10
11
14
15
16
17
18
20
19. Courtney, R.; Radwanski, E.; Lim, J.; Laughlin, M.
Antimicrob. Agents Chemother. 2004, 48, 804.
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a Cells incubated with culture medium alone represented the controls
and the cell viability was always greater than 97%.
b Data represent the arithmetic means SD of at least three indepen-
dent experiments.
21. Hoffman, H. L.; Rathbun, R. C. Exp. Opin. Investig..
Drugs 2002, 11, 409.
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Moreover other tested compounds may be good candi-
dates for further investigation.
Acknowledgments
24. Turan-Zitouni, G.; Kaplancikli, Z. A.; Yildiz, M. T.;
Chevallet, P.; Kaya, D. Eur. J. Med. Chem. 2005, 40, 607.
25. Cukurovali, A.; Yilmaz, I.; Gur, S.; Kazaz, C. Eur. J.
Med. Chem. 2006, 41, 201.
26. Chimenti, F.; Bizzarri, B.; Manna, F.; Bolasco, A.; Secci,
D.; Chimenti, P.; Granese, A.; Rivanera, D.; Lilli, D.;
Scaltrito, M. M.; Brenciaglia, M. I. Bioorg. Med. Chem.
Lett. 2005, 15, 603.
27. Chimenti, F.; Bizzarri, B.; Bolasco, A.; Secci, D.; Chim-
enti, P.; Carradori, S.; Granese, A.; Rivanera, D.; Lilli, D.;
Scaltrito, M. M.; Brenciaglia, M. I. Eur. J. Med. Chem.
2006, 41, 208.
This work was supported by grants from MURST. We
acknowledge Anton Gerada, a professional translator
Fellow of the Institute of Translation and Interpreting
of London and Member of AIIC (Association Interna-
ˆ
tionale des Interpretes de Conferences—Geneva) for
revision of the manuscript.
´
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4640
F. Chimenti et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4635–4640
32. In vitro cytotoxicity: The cytotoxicity of the newly
synthesized compounds under investigation was tested
against EAhy, human cell line obtained from a hybridoma
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was estimated by the Trypan Blue dye exclusion assay.
Cell lines were maintained as adherent type cultures under
humidified atmosphere in 5% CO2 at 37 ꢁC, Dulbecco’s
modified Eagle’s culture medium (high glucose) supple-
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containing antibiotic mixture. Experiments were per-
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solutions of the investigated compounds were prepared in
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dilutions were made in culture medium; the DMSO
percent present in culture medium never exceeded 0.5%.
EAhy cells in the exponential phase of growth (1 · 105/
mL) were seeded into 24-well microplate. and incubated
for 24 h with four different concentrations of the com-
pounds (50–0.05 lg/mL). Some plates containing cells
alone or cells and DMSO represented the controls. After
the incubation period, cells were mechanically scraped off
from the plates and an aliquot was diluted (1:1) with a
solution 0.4% Trypan Blue Stain. After few minutes at rt
cells were counted under an optical microscope in a
Thoma hemocytometer chamber by two different opera-
tors. On the basis that Trypan Blue is a vital dye34 and can
enter and interact with the cells unless the plasmatic
membrane is damaged, blue stained cells were considered
as dead. Values are expressed as % of viable cells. Cell
viability in control samples was always 97–98%.
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