Synthesis and cytotoxicity of novel (thiazol-2-yl)hydrazine derivatives as promising anti-Candida agents

Article abstract of DOI:10.1016/j.ejmech.2013.04.042

Thirty-eight new (4-(4-substituted-phenyl/2,4-disubstituted-phenyl)- thiazol-2-yl)hydrazine derivatives were synthesized in good yield and assayed for their in vitro anti-Candida activity, compared to topical and systemic antifungal drugs, against twenty-two clinical isolates of Candida spp. The concurrent presence of aliphatic chains or cycloaliphatic rings at N1-hydrazine and a 4-methyl/4-methoxyphenyl at C4 position of the thiazole nucleus exhibited an interesting anti-Candida inhibitory activity. Moreover, some of the most active compounds showed synergistic antifungal effects and lower cell toxicity when combined with clotrimazole.

Full text of DOI:10.1016/j.ejmech.2013.04.042

European Journal of Medicinal Chemistry 65 (2013) 102e111
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and cytotoxicity of novel (thiazol-2-yl)hydrazine derivatives
as promising anti-Candida agents
,
a
a
b
Simone Carradori a , Daniela Secci , Adriana Bolasco , Daniela Rivanera ,
*
Emanuela Mari c, Alessandra Zicari c, Lavinia Vittoria Lotti c, Bruna Bizzarri a
,
*
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:
Thirty-eight new (4-(4-substituted-phenyl/2,4-disubstituted-phenyl)-thiazol-2-yl)hydrazine derivatives
were synthesized in good yield and assayed for their in vitro anti-Candida activity, compared to topical
and systemic antifungal drugs, against twenty-two clinical isolates of Candida spp. The concurrent
presence of aliphatic chains or cycloaliphatic rings at N1-hydrazine and a 4-methyl/4-methoxyphenyl at
C4 position of the thiazole nucleus exhibited an interesting anti-Candida inhibitory activity. Moreover,
some of the most active compounds showed synergistic antifungal effects and lower cell toxicity when
combined with clotrimazole.
Received 12 January 2013
Received in revised form
24 April 2013
Accepted 25 April 2013
Available online 1 May 2013
Keywords:
Ó 2013 Elsevier Masson SAS. All rights reserved.
Antifungal drugs
Candida spp.
Cytotoxicity
FIC index
Hantzsch reaction
Thiazole
1. Introduction
correctly to topical/systemic treatment [2,3]. Candida albicans,
Candida glabrata, and Candida krusei are the most frequent species
Opportunistic Candida species represent the most common
fungal pathogens that affect humans and the mortality associated
with invasive mycosis increased in the last decade. The pathogen-
esis of Candida spp. produces a wide spectrum of diseases, which
depend primarily on the immune status of the host, ranging from
superficial mucocutaneous diseases to invasive illnesses [1]. In
healthy individuals, Candida infections are usually due to impaired
epithelial barrier functions and statistically occur in all age groups,
but are more frequent and harmful in the newborn and the elderly.
The growing problem of mucosal and systemic candidiasis reflects
the enormous increase in the number of risk factors (patients with
cell-mediated immune deficiency and those receiving aggressive
cancer therapies, excessive use of broad spectrum antibiotics,
immunosuppression, or transplantation therapy). As it is often not
possible to correct the underlying predisposing conditions, in-
fections are usually more severe and generally do not respond
isolated from clinical practice. Moreover, others species such as
Candida parapsilosis, Candida tropicalis, and Candida sakè have also
emerged as new opportunistic fungi threatening patients’ life.
Clinically, azoles are first-line agents in treating fungal infections,
but their extensive use has led to the occurrence of resistant strains
which greatly limited the therapeutic options. Antifungal resistance
is a serious concern due to the limited number of available agents,
therefore there is an emergent demand for the discovery of new
antifungal agents to open the possibility of a novel therapeutic
approach [4].
In the literature, it emerged that (4-aryl-thiazol-2-yl)hydrazines
were endowed with potent anti-Candida activity against a large
number of clinically relevant fungal strains [5,6]. In an attempt to
improve the antifungal spectrum activityand drug-like properties of
this scaffold, we have recently reported on new antifungal (thiazol-
2-yl)hydrazine skeleton, with different substitutions at C2 and C4
positions of the thiazole nucleus [7e10], and their ability to inhibit
growth of several fungal pathogens. As compared with the lead
structures, the synthesized molecules showed promising antifungal
activity, additive effects with clotrimazole, and low cell toxicity.
* Corresponding authors. Tel./fax: þ39 06 49913772.
E-mail addresses: simone.carradori@uniroma1.it (S. Carradori), bruna.bizzarri@
uniroma1.it (B. Bizzarri).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.04.042

S. Carradori et al. / European Journal of Medicinal Chemistry 65 (2013) 102e111
103
Starting from the structureeactivity relationships (SARs)
extrapolated from biological results, we decided to design and
evaluate a large number of such derivatives keeping constant the
significant (thiazol-2-yl)hydrazine pharmacophore and intro-
ducing different moieties at N1-hydrazine (aliphatic chains and
cycloaliphatic rings) and 4-substituted-phenyl at C4 position of
thiazole nucleus (Table 1). According to the established guidelines
of Clinical and Laboratory Standards Institute (CLSI) and the Euro-
pean Committee for Antimicrobial Susceptibility Testing (EUCAST,
version 4.1 valid from 2012-03-14), we assayed the susceptibility of
clinical Candida spp. and bacterial isolates to our novel compounds
by determining their minimal inhibitory concentration (MIC) [11]
thus providing a first indication of the potency and selectivity of
such compounds.
analytical, spectral, and mass data (Experimental protocols and
Supplementary material). In general in the IR spectrum, they showed
strong bands at about 3220 and 1570 cmꢀ1 due to the presence of NH
and C]N group, respectively.
3. Microbiology
Firstly, derivatives of AeD series, dissolved in dimethylsulfoxide
(DMSO), were evaluated for their antibacterial activity (Table S1,
Supplementary material). Organisms from routine clinical Gram-
positive (Staphylococcus aureus, Staphylococcus warneri, Strepto-
coccus faecalis, Streptococcus
a-hemolyticus) and Gram-negative
isolates (Escherichia coli, Proteus mirabilis, Enterobacter spp., Kleb-
siella oxytoca) 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
conventional 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) [12] with Mueller-Hinton II broth (BBL Micro-
biology Systems, Cockeysville, MD).
2. Chemistry
The chemical synthesis of AeD compounds is outlined in
Scheme 1. Different carbonyl compounds were reacted directly with
thiosemicarbazide with catalytic amounts of acetic acid in ethanol
and the obtained thiosemicarbazones subsequently were condensed
with newly synthesized
a-bromo-4-substituted- or a-bromo-2,4-
disubstituted-acetophenone (obtained by direct bromination of the
corresponding 4-substituted or 2,4-disubstituted-acetophenone
with Br2 in CHCl3) to 2,4-disubstituted-1,3-thiazoles (Hantzsch re-
action). All synthesized compounds were fully characterized by
Then, the antifungal activity was evaluated against twenty-two
clinical fungal isolates of Candida spp. (C. albicans, C. glabrata,
C. tropicalis, C. krusei, C. parapsilosis, and C. sakè) and compared with
Table 1
Minimal inhibitory concentration (MIC) of newly synthesized compounds (AeD series), clotrimazole (C), fluconazole (F), and griseofulvin (G) against 22 clinical strains of
Candida species.
Tested fungi (MIC values mg/mL)
Comp
Ar
C. albicans
C. glabrata
C. tropicalis
C. krusei
C. parapsilopis
C. sakè
(8 strains)
(4 strains)
(3 strains)
(3 strains)
(2 strains)
(2 strains)
A1
A2
A3
B1
B2
B3
B4
B5
B6
B7
4-CH3-Ph
0.50e2
0.50e2
0.50e2
0.25e2
0.25e2
0.25e2
16e64
4e16
0.50e2
0.25e2
2
2
2
2
2
4-CH3-Ph
2
2
2
2
4-CH3-Ph
4
8
8
8
4-OCH3-Ph
4-OCH3-Ph
4-OCH3-Ph
4-OCH3-Ph
4-OCH3-Ph
4-OCH3-Ph
4-OCH3-Ph
0.50e2
2
2
16
2
16
2
16
2
2
2
2
2
2
2
32e64
8e16
0.50e2
4e8
64
16
2
256
16
2
256
16
2
256
16
2
0.50e2
2e8
8
32
32
32
(continued on next page)

104
S. Carradori et al. / European Journal of Medicinal Chemistry 65 (2013) 102e111
Table 1 (continued )
Tested fungi (MIC values mg/mL)
Comp
Ar
C. albicans
C. glabrata
C. tropicalis
C. krusei
C. parapsilopis
C. sakè
(8 strains)
(4 strains)
(3 strains)
(3 strains)
(2 strains)
(2 strains)
B8
B9
4-OCH3-Ph
4-OCH3-Ph
0.25e2
8e32
0.50e2
8e32
2
2
2
2
32
16
16
16
B10
4-OCH3-Ph
2e8
4e8
16
16
16
16
B11
B12
4-OCH3-Ph
4-OCH3-Ph
2e8
4e8
16
16
16
64
16
8e64
32e64
256
256
256
C1
C2
C3
C4
C5
C6
C7
C8
C9
2,4-OCH3-Ph
2,4-OCH3-Ph
2,4-OCH3-Ph
2,4-OCH3-Ph
2,4-OCH3-Ph
2,4-OCH3-Ph
2,4-OCH3-Ph
2,4-OCH3-Ph
2,4-OCH3-Ph
128e256
64e256
128e256
128e256
128e256
64e256
64e256
128e256
64e256
128e256
128e256
128e256
128e256
128e256
128e256
128e256
128e256
64e256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
C10
C11
2,4-OCH3-Ph
2,4-OCH3-Ph
64e256
64e256
256
256
256
256
256
256
256
256
128e256
128e256
D1
D2
2,4-Cl-Ph
2,4-Cl-Ph
2e4
2e4
2e4
4
4
8
8
8
8
8
8
4

S. Carradori et al. / European Journal of Medicinal Chemistry 65 (2013) 102e111
105
Table 1 (continued )
Tested fungi (MIC values mg/mL)
Comp
Ar
C. albicans
C. glabrata
C. tropicalis
C. krusei
C. parapsilopis
C. sakè
(8 strains)
(4 strains)
(3 strains)
(3 strains)
(2 strains)
(2 strains)
256
256
256
256
16
D3
D4
D5
D6
D7
D8
D9
2,4-Cl-Ph
2,4-Cl-Ph
2,4-Cl-Ph
2,4-Cl-Ph
2,4-Cl-Ph
2,4-Cl-Ph
2,4-Cl-Ph
2e4
0.50e4
256
2e4
4
256
256
256
8
256
256
256
256
16
4
4
128e256
256
8
2e8
2e4
2e4
2e4
4e8
2e4
4
4
4
4
4
8
8
4
4
4
32
32
D10
2,4-Cl-Ph
0.25e2
2
2
2
2
2
D11
D12
2,4-Cl-Ph
2,4-Cl-Ph
0.50e2
2e4
2
2
4
2
4
2
8
2
8
2e4
C
F
G
0.50e2
4e64
4e8
2
2
4
4
4
4e16
4e8
4e16
8
4e16
8
4e16
8
4e16
8
topical and systemic reference drugs clotrimazole, fluconazole, 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 identified by con-
ventional methodologies. Prior to testing, each isolate was sub-
cultured on a qualified medium to ensure purity and optimal
Scheme 1. Synthesis of novel compounds (AeD series). Reagents: (i) thiosemicarbazide, EtOH, acetic acid (cat.), rt; (ii) Br2, CHCl3; (iii)
a-bromo-4-substituted- or a-bromo-
2,4-disubstituted-acetophenone, EtOH, rt.

106
S. Carradori et al. / European Journal of Medicinal Chemistry 65 (2013) 102e111
Table 3
growth. All derivatives were dissolved in DMSO. The in vitro anti-
fungal activities were determined by the broth microdilution
method with Sabouraud dextrose broth (BBL Microbiology Sys-
tems, Cockeysville, MD) as recommended by the NCCLS [13].
Furthermore, the most active and representative compounds A1,
Cytotoxic effect (CC50) of the eight most active compounds and clotrimazole tested
on Hep2 cells after 24 h of incubation at 37 ꢁC using Trypan blue exclusion test
expressed as cell survival fraction (%).a
Comp
Trypan Blue exclusion test
Trypan Blue
exclusion test
CC50 (mg/mL)
Concentration (
mg/mL)
A2, B2, B3, B6, B8, D10, and D11 (concentration range 0.5e4 mg/mL)
were tested, by broth microdilution susceptibility test method,
100 50 10
5
0.5
0.05 0.005
against clinical isolates of C. albicans and C. krusei in combination
with clotrimazole (concentration range 0.125e256 mg/mL), to study
A1
A2
B2
B3
B6
B8
D10
D11
62.0 72.8 83.2 93.0 91.0 94.0 97.0
77.0 67.0 72.4 84.0 86.0 92.2 94.6
ꢄ100
ꢄ100
25
36
35
35
42
41
38
the effects of these additions on the Candida strains’ growth. The
yeast inoculums were adjusted to a concentration of 0.5 ꢂ 103 to
2.5 ꢂ 103 CFU/mL in Sabouraud dextrose broth and an aliquot of
0.1 mL was added to each well of the microdilution plate. The plate
was incubated at 35 ꢁC. The MIC end points were read visually
following 24 h of incubation as described for in vitro susceptibility
testing (Table 2). The simultaneous use of two or more antimicrobial
agents has certain rationale and is recommended in specifically
defined situations. Combination of two or more drugs has been
recognized as an important approach to delay the emergence of
fungal resistance, to produce desirable synergistic effects expanding
the spectrum of activity, and to limit single-drug toxicity lowering
the dosage of each of them. However, the selection of an appropriate
combination requires an understanding of the potential for inter-
action between the antifungal agents. In vitro antifungal combina-
tions are usually assessed on the basis of the fractional inhibitory
concentration (FIC) index, which represents the sum of the FICs of
each drug tested, where the FIC is determined for each drug by
dividing the MIC of a drug when used in combination by the MIC of
each drug when used alone. For all of the wells of the microtitration
plates that corresponded to an MIC, the sum of the FICs (SFIC) was
calculated for each well with the equation SFIC ¼ FIC (A þ B) ¼ (MIC
of A in combination/MIC of A alone) þ (MIC of B in combination/MIC
of B alone) [14].
0
0
0
0
0
0
0
0
0
0
0
0
0
0
64.0 69.0 64.0 71.0 78.0
75.5 83.0 88.0 90.0 92.0
72.4 84.0 83.0 91.2 94.0
70.2 81.0 84.0 90.2 93.0
88.0 94.0 95.0 96.0 97.2
87.5 90.0 92.4 93.0 94.0
68.0 92.5 93.4 93.0 94.4
Clotrimazole
a
Cells incubated with culture medium alone represented the controls. Viability
L of DMSO ¼ 94%, viability with 20 L of DMSO ¼ 87%. Data represent cell
survival fraction (%) of at least three independent experiments. CC50: concentration
g/mL) required to reduce cell viability by 50%.
with 2
m
m
(
m
[15], followed by optical microscopy examination of the most
representative derivatives A1 and B2 (Fig. 1).
5. Results and discussion
A large amount of (thiazol-2-yl)hydrazine derivatives were
synthesized in high yields and evaluated in vitro for their broad
antimicrobial effect against several clinical isolates. All compounds
did not show antibacterial activity (MIC values > 256
Supplementary material) thus determining a prominent selectivity
against fungal pathogens.
mg/mL,
On the basis of the in vitro antifungal activity assay, preliminary
SARs of the synthesized compounds were obtained. In general, the
introduction of 4-substituted and 2,4-disubstituted (with the only
exception of 2,4-OCH3) aryl groups at the C4 of the thiazole nucleus
brought a comparable anti-Candida activity, especially against
C. albicans, C. glabrata, and C. tropicalis, with respect to clotrimazole.
The other species are less susceptible to these compounds, even
though some derivatives (A1, A2, B2, B3, B6, B8, D10, and D11) also
displayedadiscreteantifungalactivityagainstC.krusei, C. parapsilosis,
and C. sakè.
4. Cell toxicity
Compounds that have been demonstrated to achieve the
strongest anti-Candida activity (A1, A2, B2, B3, B6, B8, D10, and
D11) were also analyzed to estimate their ability to induce cytotoxic
effects, at seven concentrations ranging from 0.005 to 100 mg/mL
(Table 3), on a cultured cell line derived from a human laryngeal
epidermoid carcinoma of the larynx (Hep2). Cell viability was
examined using Trypan Blue exclusion test, as previously described
As stated above, derivatives C1eC11, with 2,4-OCH3-aryl ring at
C4 of the thiazole, were completely inactive (MIC ¼ 256
the change with chlorine atoms increased the biological activity up
to MIC values of 2 g/mL (compare C10/D10 and C11/D11). With this
kind of disubstitution, compounds with a cycloaliphatic ring on the
g/mL).
Monosubstitution with a 4-CH3-aryl (A1eA3) or 4-OCH3-aryl (B1e
B12) moieties showed significant effect on improving the anti-
fungal activity. The better activity could be attributed to the portion
of the molecule on the hydrazone linker. Linear aliphatic chains
were found to be more active than cycloaliphatic rings (B10, B11,
C10, C11) or hindered aliphatic pendants (B5, C5, D5).
To quantify the potential synergistic effect of the most active
compounds (A1, A2, B2, B3, B6, B8, D10, and D11) in association
with clotrimazole, we assayed combinations of them in a micro-
dilution assay. Our interpretation of the FIC results, according to
accepted criteria of The American Society of Microbiology for
determining drug interactions were as follows. For compounds A1,
A2, B2, B3, B8, and D10, the calculated SFIC was ꢃ0.5 against
C. albicans and only for compound B8 the calculated SFIC was ꢃ0.5
against C. krusei, thus indicating synergistic effect. On the opposite,
mg/mL) but
Table 2
m
Synergistic effect (FIC index) of the most active compounds in combination with
clotrimazole (C) on the growth of C. albicansa and C. krusei.b
hydrazone linker must be preferred (D10 and D11, MIC ¼ 2
m
Comp C. albicans
MIC ( g/mL) SFIC (effect)
C. krusei
MIC ( g/mL) SFIC (effect)
m
m
Alone Combo
Alone Combo
4
C
2
A1
A2
B2
B3
B6
B8
D10
D11
2
2
2
2
2
2
2
2
0.5
0.5
0.5 (Synergism)
0.5 (Synergism)
2
2
2
2
2
2
2
2
2
1
1
1
2
0.5
2
1.5 (No interaction)
0.75 (No interaction)
0.75 (No interaction)
0.75 (No interaction)
1.5 (No interaction)
0.375 (Synergism)
1.5 (No interaction)
1.5 (No interaction)
0.125 0.125 (Synergism)
0.5
2
0.125 0.125 (Synergism)
0.125 0.125 (Synergism)
2
0.5 (Synergism)
2 (No interaction)
2 (No interaction)
2
a
C. albicans strains isolated from bronchial washing of transplant patient and
from blood culture of surgery unit’s patient.
b
C. krusei strains isolated from blood culture of coronary unit’s patient and from
wound’s swab of surgery unit’s patient.

S. Carradori et al. / European Journal of Medicinal Chemistry 65 (2013) 102e111
107
Fig. 1. Ultrastructural alterations in Hep2 cells exposed to the most active compounds A1 and B2 versus clotrimazole (reference compound). Panels a, d, and g show control
unexposed Hep2 cells. Panels b and c show Hep2 cells exposed to compound A1 at concentration, respectively, of 5 and 100 g/mL. Panels e and f show Hep2 cells exposed to
g/mL of clotrimazole. N ¼ nucleus, M ¼ mitochondria, er ¼ endoplasmic
m
compound B2 at concentration, respectively, of 5 and 100 mg/mL. Panel h shows cells treated with 5 m
reticulum.
no interaction (0.5< SFIC <4) has been registered for the other
derivatives (Table 2).
observations, cells were treated with compounds A1, B2, and clo-
trimazole and analyzed by Transmission Electron microscopy. As
shown in Fig. 1 (Panels a, d, and g), the unexposed Hep2 cells had
the typical morphology, with a well developed endoplasmic retic-
ulum (ER) membrane system, numerous elongated mitochondria
Considering this, the same selected derivatives (A1, A2, B2, B3,
B6, B8, D10, and D11) and clotrimazole were investigated for their
cytotoxic effects (CC50) by Trypan blue exclusion test at seven
different concentrations (Table 3) and by electron microscopy
(Fig. 1).
and well preserved nuclei. After treatment with 5 mg/mL of com-
pound A1 (Panel b) some autophagic vacuoles appeared in the
cytoplasm, often located in the proximity of mitochondria (black
arrows). Autophagic vacuoles were more prominent in cells
It is evident (Table 3) that at the highest concentrations used
(50e100 mg/mL) compounds A1 and A2 showed the lowest cyto-
toxicity on Hep2 cells, whereas compound B2 was the most cyto-
toxic. No significative differences were noted among the other
compounds and clotrimazole (reference drug). On the basis of these
exposed to 100
resulted in similar alterations (Panel e), with more pronounced
mitochondrial damage at 5 g/mL (loss of cristae and swelling)
mg/mL of A1 (Panel c). Exposure to compound B2
m

108
S. Carradori et al. / European Journal of Medicinal Chemistry 65 (2013) 102e111
with respect to compound A1. At a 100
m
g/mL concentration of B2
acetophenone (50 mmol), both suspended in 50 mL of ethanol,
were reacted at room temperature under magnetic stirring for 8e
16 h. The precipitate was filtered and purified by chromatography
(SiO2, ethyl acetate/n-hexane 1/3) to give the final compounds in
high yield.
(Panel f), dilatation of the ER was also detectable. On the other
hand, cells treated with 5 g/mL of clotrimazole (Panel h) showed
m
finely granular cytoplasm, due to extensive ER degranulation, and
prominent vacuolization in the proximity of mitochondria. No live
cells were observed at 100 mg/mL of clotrimazole (data not shown).
Taken together, the ultrastructural analysis showed clear signs of
progressive cell damage in Hep2 cells exposed to the stress induced
by the chemical compounds tested. The presence of autophagic
vacuoles in cells exposed at low concentrations of A1 might point to
a protective effect, since autophagy may act as a survival pathway,
delaying or preventing cell death by removal of damaged mito-
chondria. Cells exposed to B1 (and to higher concentrations of A1)
displayed more profound mitochondrial damage associated to an
increased cytoplasmic vacuolization that are both consistent with
the more severe cellular damage observed. Thus the morphological
data support and strengthen the results of the cytotoxicity assay,
but only at concentrations higher than their corresponding MIC
values. Moreover, these compounds displayed lower cytotoxicity
than clotrimazole (reference drug).
In conclusion, we investigated the synthesis and in vitro anti-
microbial activity of thirty-eight new (thiazol-2-yl)hydrazine de-
rivatives by determining the minimal inhibitory concentrations
with the EUCAST broth microdilution method. The substitution of
the nucleus linked to the N1-hydrazine had a deep impact on the
selective antifungal activity on this scaffold with no effects on
Gram-positive and Gram-negative bacteria. Derivatives A1, A2, B2,
B3, B8, and D10 could be also used in association with established
antifungal azole derivatives to enhance their inhibitory activity
against clinical isolates of Candida spp. (FIC index ꢃ 0.5). Moreover,
displaying a better activity and a lower cytotoxicity compared to
the drug reference clotrimazole, they represent good lead com-
pounds for further optimization of novel broad spectrum anti-
Candida spp. agents.
6.2. Characterization data for new compounds
6.2.1. 1-(Pentan-2-ylidene)-2-(4-p-tolylthiazol-2-yl)hydrazine (A1)
Mp 209e210 ꢁC, 83% yield; 1H NMR (CDCl3, 400 MHz)
d (ppm):
0.95e0.99 (t, J ¼ 7.3 Hz, 3H, CH2CH3), 1.60e1.66 (m, 2H, CH2CH3),
2.20 (s, 3H, CH3C]), 2.33e2.39 (m, 2H, CH2C]), 2.40 (s, 3H, 4-CH3e
Ar), 6.62 (s, 1H, C5H-thiaz.), 7.28e7.30 (d, Jo ¼ 7.8 Hz, 2H, Ar), 7.60e
7.62 (d, Jo ¼ 8.0 Hz, 2H, Ar), 12.33 (bs, 1H, NH, D2O exch.); ESI-MS:
m/z 274.1 [M þ H]þ.
6.2.2. 1-(Pentan-3-ylidene)-2-(4-p-tolylthiazol-2-yl)hydrazine (A2)
Mp 100e103 ꢁC, 90% yield; 1H NMR (CDCl3, 400 MHz)
d (ppm):
1.13e1.17 (t, J ¼ 7.3 Hz, 3H, CH2CH3), 1.22e1.26 (t, J ¼ 7.8 Hz, 3H,
CH2CH3), 2.33e2.36 (m, 2H, CH2CH3), 2.40 (s, 3H, 4-CH3eAr), 2.54e
2.56 (m, 2H, CH2CH3), 6.63 (s, 1H, C5H-thiaz.), 7.28e7.30 (d,
Jo ¼ 8.2 Hz, 2H, Ar), 7.59e7.61 (d, Jo ¼ 8.2 Hz, 2H, Ar), 12.41 (bs, 1H,
NH, D2O exch.); ESI-MS: m/z 274.1 [M þ H]þ.
6.2.3. 1-(Heptan-2-ylidene)-2-(4-p-tolylthiazol-2-yl)hydrazine (A3)
Mp 97e100 ꢁC, 98% yield; 1H NMR (DMSO-d6, 400 MHz)
d
(ppm): 0.85e0.88 (m, 3H, CH2CH3), 1.28e1.30 (m, 2H, CH2), 1.53e
1.56 (m, 4H, 2 ꢂ CH2), 1.91e1.93 (m, 2H, CH2), 2.22 (s, 3H, CH3C]),
2.50 (s, 3H, 4-CH3-Ar), 7.14 (s, 1H, C5H-thiaz.), 7.18e7.20 (d,
Jo ¼ 7.8 Hz, 2H, Ar), 7.70e7.72 (d, Jo ¼ 7.9 Hz, 2H, Ar), 12.35 (bs, 1H,
NH, D2O exch.); ESI-MS: m/z 302.1 [M þ H]þ.
6.2.4. 1-(4-(4-Methoxyphenyl)thiazol-2-yl)-2-(propan-2-ylidene)
hydrazine (B1)
Mp 230e231 ꢁC, 92% yield; 1H NMR (CDCl3, 400 MHz)
d (ppm):
6. Experimental protocols
2.12 (s, 3H, CH3C]), 2.21 (s, 3H, CH3C]), 3.85 (s, 3H, 4-OCH3-Ar),
6.53 (s, 1H, C5H-thiaz.), 6.98e7.00 (d, Jo ¼ 8.0 Hz, 2H, Ar), 7.64e7.66
(d, Jo ¼ 8.0 Hz, 2H, Ar), 12.29 (bs, 1H, NH, D2O exch.); ESI-MS: m/z
262.1 [M þ H]þ.
The chemicals, solvents for synthesis, and spectral grade sol-
vents were purchased from Aldrich (Italy) and used without
further purification. Melting points (uncorrected) were deter-
mined 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
6.2.5. 1-(Butan-2-ylidene)-2-(4-(4-methoxyphenyl)thiazol-2-yl)
hydrazine (B2)
Mp 192e193 ꢁC, 96% yield; 1H NMR (CDCl3, 400 MHz)
d (ppm):
as
d
units (parts per millions) relative to the solvent peak. Coupling
1.13e1.17 (t, J ¼ 7.3 Hz, 3H, CH2CH3), 2.19 (s, 3H, CH3C]), 2.39e2.41
(m, 2H, CH2CH3), 3.85 (s, 3H, 4-OCH3-Ar), 6.53 (s, 1H, C5H-thiaz.),
6.98e7.00 (d, Jo ¼ 8.0 Hz, 2H, Ar), 7.65e7.67 (d, Jo ¼ 8.0 Hz, 2H, Ar),
12.20 (bs, 1H, NH, D2O exch.); ESI-MS: m/z 276.1 [M þ H]þ.
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). Mass spectra were recorded on Micro-
mass LCT Premier Spectrometer under conditions of electrospray
ionization (ESI) (Waters, UK). Elemental analyses for C, H, and N
were recorded on a PerkineElmer 240 B microanalyzer and the
analytical results were within ꢅ0.4% of the theoretical values for
all compounds (Supplementary material). All reactions were
monitored by TLC performed on 0.2 mm thick silica gel plates (60
F254 Merck).
6.2.6. 1-(4-(4-Methoxyphenyl)thiazol-2-yl)-2-(pentan-2-ylidene)
hydrazine (B3)
Mp 171e172 ꢁC, 95% yield; 1H NMR (CDCl3, 400 MHz)
d (ppm):
0.95e0.99 (t, J ¼ 7.2 Hz, 3H, CH2CH3), 1.61e1.63 (m, 2H, CH2), 2.17 (s,
3H, CH3C]), 2.33e2.37 (m, 2H, CH2), 3.84 (s, 3H, 4-OCH3-Ar), 6.53
(s, 1H, C5H-thiaz.), 6.98e7.00 (d, Jo ¼ 8.0 Hz, 2H, Ar), 7.65e7.67 (d,
Jo ¼ 8.0 Hz, 2H, Ar), 12.23 (bs, 1H, NH, D2O exch.); ESI-MS: m/z 290.1
[M þ H]þ.
6.1. General procedure for the synthesis of AeD series
The appropriate carbonyl compound (50 mmol) was dissolved
in 50 mL of ethanol and magnetically stirred with an equimolar
quantity of thiosemicarbazide for 8e18 h at room temperature with
catalytic amounts of acetic acid. The desired thiosemicarbazone
precipitated from reaction mixture, was filtered, crystallized from
suitable solvent (EtOH or i-PrOH), and dried under vacuum.
Equimolar amounts of the prepared thiosemicarbazone (50 mmol)
6.2.7. 1-(4-(4-Methoxyphenyl)thiazol-2-yl)-2-(pentan-3-ylidene)
hydrazine (B4)
Mp 260e261 ꢁC, 99% yield; 1H NMR (CDCl3, 400 MHz)
d (ppm):
1.09e1.11 (m, 3H, CH2CH3), 1.15e1.18 (m, 3H, CH2CH3), 2.30e2.34
(m, 2H, CH2CH3), 2.50e2.54 (m, 2H, CH2CH3), 3.73 (s, 3H, 4-OCH3-
Ar), 6.87 (s, 1H, C5H-thiaz.), 7.28e7.30 (d, Jo ¼ 8.0 Hz, 2H, Ar), 7.53e
7.57 (d, Jo ¼ 8.0 Hz, 2H, Ar), 12.01 (bs, 1H, NH, D2O exch.); ESI-MS:
m/z 290.1 [M þ H]þ.
and
a
-bromo-4-substituted- or
a-bromo-2,4-disubstituted-

S. Carradori et al. / European Journal of Medicinal Chemistry 65 (2013) 102e111
109
6.2.8. 1-(4-(4-Methoxyphenyl)thiazol-2-yl)-2-(4-methylpentan-2-
2 ꢂ CH]), 7.07e7.09 (m, 2H, Ar), 7.78e7.80 (m, 2H, Ar), 11.05
(bs, 1H, NH, D2O exch.); ESI-MS: m/z 328.1 [M þ H]þ.
ylidene)hydrazine (B5)
Mp 188e190 ꢁC, 86% yield; 1H NMR (CDCl3, 400 MHz)
d (ppm):
0.94e0.97 (m, 6H, 2 ꢂ CH3), 0.97e2.01 (m, 1H, CH), 2.17 (s, 3H,
CH3C]), 2.23e2.24 (m, 2H, CH2), 3.84 (s, 3H, 4-OCH3-Ar), 6.54 (s,
1H, C5H-thiaz.), 6.98e7.00 (d, Jo ¼ 6.9 Hz, 2H, Ar), 7.64e7.66 (d,
Jo ¼ 6.9 Hz, 2H, Ar), 12.21 (bs, 1H, NH, D2O exch.); ESI-MS: m/z 326.1
[M þ Na]þ.
6.2.16. 1-(4-(2,4-Dimethoxyphenyl)thiazol-2-yl)-2-(propan-2-
ylidene)hydrazine (C1)
Mp 110e112 ꢁC, 95% yield; 1H NMR (CDCl3, 400 MHz)
d (ppm):
0.93 (s, 3H, CH3C]), 0.96 (s, 3H, CH3C]), 3.73 (s, 3H, OCH3-Ar), 3.85
(s, 3H, OCH3-Ar), 6.02 (s, 1H, C5H-thiaz.), 7.35e7.41 (m, 1H, Ar), 7.53
(s, 1H, Ar), 7.62e7.68 (m, 1H, Ar), 11.06 (bs, 1H, NH, D2O exch.); ESI-
MS: m/z 292.1 [M þ H]þ.
6.2.9. 1-(Hex-5-en-2-ylidene)-2-(4-(4-methoxyphenyl)thiazol-2-
yl)hydrazine (B6)
Mp 178e179 ꢁC, 99% yield; 1H NMR (DMSO-d6, 400 MHz)
6.2.17. 1-(Butan-2-ylidene)-2-(4-(2,4-dimethoxyphenyl)thiazol-2-
d
(ppm): 1.94 (s, 3H, CH3C]), 2.28e2.33 (m, 2H, CH2), 2.35e2.39
yl)hydrazine (C2)
(m, 2H, CH2), 3.77 (s, 3H, 4-OCH3-Ar), 5.05e5.09 (m, 2H, CH2]),
5.81e5.85 (m, 1H, CH]), 6.96e6.98 (d, Jo ¼ 7.5 Hz, 2H, Ar), 7.09 (s,
1H, C5H-thiaz.), 7.73e7.75 (d, Jo ¼ 7.5 Hz, 2H, Ar), 12.19 (bs, 1H, NH,
D2O exch.); ESI-MS: m/z 302.1 [M þ H]þ.
Mp 190e192 ꢁC, 90% yield; 1H NMR (CDCl3, 400 MHz)
d (ppm):
1.13e1.16 (t, J ¼ 7.2 Hz, 3H, CH2CH3), 2.24 (s, 3H, CH3C]), 2.38e2.40
(m, 2H, CH2CH3), 3.96 (s, 3H, OCH3-Ar), 4.17 (s, 3H, OCH3-Ar), 5.99 (s
1H, C5H-thiaz.), 6.53e6.55 (m,1H, Ar), 6.59e6.61 (m,1H, Ar), 7.69 (s,
1H, Ar), 13.02 (bs, 1H, NH, D2O exch.); ESI-MS: m/z 328.1 [M þ Na]þ.
6.2.10. 1-(Heptan-2-ylidene)-2-(4-(4-methoxyphenyl)thiazol-2-yl)
hydrazine (B7)
6.2.18. 1-(4-(2,4-Dimethoxyphenyl)thiazol-2-yl)-2-(pentan-2-
Mp 139e140 ꢁC, 91% yield; 1H NMR (DMSO-d6, 400 MHz)
(ppm): 0.89e0.93 (m, 3H, CH2CH3), 1.27e1.33 (m, 2H, CH2), 1.58e
ylidene)hydrazine (C3)
d
Mp 85e87 ꢁC, 92% yield; 1H NMR (CDCl3, 400 MHz)
d (ppm):
1.62 (m, 2H, CH2), 2.16 (s, 3H, CH3C]), 2.41e2.44 (m, 2H, CH2),
2.56e2.59 (m, 2H, CH2), 3.89 (s, 3H, 4-OCH3-Ar), 6.98 (s, 1H, C5H-
thiaz.), 7.77e7.80 (m, 2H, Ar), 7.95e7.97 (m, 2H, Ar), 12.61 (bs, 1H,
NH, D2O exch.); ESI-MS: m/z 318.1 [M þ H]þ.
0.95e0.97 (m, 3H, CH2CH3), 1.30e1.49 (m, 4H, 2 ꢂ CH2), 2.11 (s, 3H,
CH3C]), 3.73 (s, 3H, OCH3-Ar), 3.81 (s, 3H, OCH3-Ar), 6.60 (s, 1H,
C5H-thiaz.), 6.74 (s, 1H, Ar), 6.78e6.81 (m, 1H, Ar), 7.25e7.29 (m, 1H,
Ar), 12.01 (bs, 1H, NH, D2O exch.); ESI-MS: m/z 342.1 [M þ Na]þ.
6.2.11. 1-(Heptan-3-ylidene)-2-(4-(4-methoxyphenyl)thiazol-2-yl)
6.2.19. 1-(4-(2,4-Dimethoxyphenyl)thiazol-2-yl)-2-(pentan-3-
hydrazine (B8)
ylidene)hydrazine (C4)
Mp 98e99 ꢁC, 99% yield; 1H NMR (CDCl3, 400 MHz)
d
(ppm):
Mp 102e103 ꢁC, 93% yield; 1H NMR (CDCl3, 400 MHz)
d (ppm):
0.95e0.98 (m, 3H, CH2CH3), 1.12e1.16 (m, 2H, CH2), 1.20e1.23 (m,
2H, CH2), 1.52e1.59 (m, 3H, CH2CH3), 2.38e2.40 (m, 2H, CH2), 2.49e
2.53 (m, 2H, CH2), 3.84 (s, 3H, 4-OCH3-Ar), 6.53 (s, 1H, C5H-thiaz.),
6.98e7.00 (d, Jo ¼ 8.3 Hz, 2H, Ar), 7.64e7.66 (d, Jo ¼ 8.3 Hz, 2H, Ar),
12.24 (bs, 1H, NH, D2O exch.); ESI-MS: m/z 340.1 [M þ Na]þ.
1.13e1.16 (t, J ¼ 7.2 Hz, 3H, CH2CH3), 1.21e1.25 (t, J ¼ 7.3 Hz, 3H,
CH2CH3), 2.37e2.41 (m, 2H, CH2CH3), 2.59e2.63 (m, 2H, CH2CH3),
3.96 (s, 3H, OCH3-Ar), 4.17 (s, 3H, OCH3-Ar), 5.96 (s, 1H, C5H-thiaz.),
6.53e6.55 (m, 1H, Ar), 6.60e6.62 (m, 1H, Ar), 7.70 (s, 1H, Ar), 12.49
(bs, 1H, NH, D2O exch.); ESI-MS: m/z 342.1 [M þ Na]þ.
6.2.12. 1-(4-(4-Methoxyphenyl)thiazol-2-yl)-2-(octan-2-ylidene)
6.2.20. 1-(4-(2,4-Dimethoxyphenyl)thiazol-2-yl)-2-(4-
hydrazine (B9)
methylpentan-2-ylidene)hydrazine (C5)
Mp 143e145 ꢁC, 99% yield; 1H NMR (CDCl3, 400 MHz)
d
(ppm):
Mp 70e72 ꢁC, 90% yield; 1H NMR (CDCl3, 400 MHz)
d (ppm): 0.91
0.91e0.96 (m, 3H, CH2CH3), 1.29e1.96 (m, 10H, 5 ꢂ CH2), 2.15 (s, 3H,
CH3C]), 3.73 (s, 3H, 4-OCH3-Ar), 6.51 (s, 1H, C5H-thiaz.), 6.80e6.83
(m, 2H, Ar), 7.25e7.29 (m, 2H, Ar), 13.00 (bs, 1H, NH, D2O exch.);
ESI-MS: m/z 354.1 [M þ Na]þ.
(s, 3H, CH3C]), 1.01 (s, 6H, 2 ꢂ CH3), 1.29e1.32 (m, 2H, CH2), 1.78e
1.82 (m, 1H, CH), 3.71 (s, 3H, OCH3-Ar), 3.85 (s, 3H, OCH3-Ar), 6.59
(s,1H, C5H-thiaz.), 6.72 (s,1H, Ar), 6.75e6.79 (m,1H, Ar), 7.13-7.15 (m,
1H, Ar), 11.89 (bs, 1H, NH, D2O exch.); ESI-MS: m/z 356.1 [M þ Na]þ.
6.2.13. 1-(1-Cyclohexylethylidene)-2-(4-(4-methoxyphenyl)thiazol-
6.2.21. 1-(4-(2,4-Dimethoxyphenyl)thiazol-2-yl)-2-(hex-5-en-2-
2-yl)hydrazine (B10)
ylidene)hydrazine (C6)
Mp 200e202 ꢁC, 85% yield; 1H NMR (CDCl3, 400 MHz)
d
(ppm):
Mp 185e187 ꢁC, 92% yield; 1H NMR (CDCl3, 400 MHz)
d (ppm):
1.29e1.82 (m, 10H, 5 ꢂ CH2), 2.14 (s, 3H, CH3C]), 2.16e2.19 (m, 1H,
CH), 3.84 (s, 3H, 4-OCH3-Ar), 6.54 (s, 1H, C5H-thiaz.), 6.98e7.00 (d,
Jo ¼ 8.6 Hz, 2H, Ar), 7.64e7.66 (d, Jo ¼ 8.6 Hz, 2H, Ar), 12.09 (bs, 1H,
NH, D2O exch.); ESI-MS: m/z 330.1 [M þ H]þ.
2.25 (s, 3H, CH3C]), 2.35e2.38 (m, 2H, CH2), 2.45e2.49 (m, 2H,
CH2), 3.98 (s, 3H, OCH3-Ar), 4.17 (s, 3H, OCH3-Ar), 5.01e5.06 (m, 2H,
CH2]), 5.78e5.81 (m, 1H, CH]), 5.98 (s, 1H, C5H-thiaz.), 6.53e6.55
(m, 1H, Ar), 6.59e6.61 (m, 1H, Ar), 7.69 (s, 1H, Ar), 13.08 (bs, 1H, NH,
D2O exch.); ESI-MS: m/z 332.1 [M þ H]þ.
6.2.14. 1-Cyclooctylidene-2-(4-(4-methoxyphenyl)thiazol-2-yl)
hydrazine (B11)
6.2.22. 1-(4-(2,4-Dimethoxyphenyl)thiazol-2-yl)-2-(heptan-2-
Mp 177e179 ꢁC, 89% yield; 1H NMR (CDCl3, 400 MHz)
d
(ppm):
1.55e2.67 (m, 14H, 7 ꢂ CH2), 3.87 (s, 3H, 4-OCH3-Ar), 6.54 (s, 1H,
C5H-thiaz.), 7.00e7.02 (d, Jo 8.8 Hz, 2H, Ar), 7.67e7.69
ylidene)hydrazine (C7)
Mp 211e212 ꢁC, 95% yield; 1H NMR (CDCl3, 400 MHz)
d (ppm):
¼
0.91e0.96 (m, 3H, CH2CH3), 1.29e1.33 (m, 8H, 4 ꢂ CH2), 2.12 (s, 3H,
CH3C]), 3.94 (s, 3H, OCH3-Ar), 4.12 (s, 3H, OCH3-Ar), 6.60 (s, 1H,
C5H-thiaz.), 7.21e7.23 (m, 1H, Ar), 7.34 (s, 1H, Ar), 7.42e7.44 (m, 1H,
Ar), 12.29 (bs, 1H, NH, D2O exch.); ESI-MS: m/z 348.1 [M þ H]þ.
(d, Jo ¼ 8.8 Hz, 2H, Ar), 12.31 (bs, 1H, NH, D2O exch.); ESI-MS: m/z
330.1 [M þ H]þ.
6.2.15. 1-(4,4-Dimethylcyclohex-2-enylidene)-2-(4-(4-
methoxyphenyl)thiazol-2-yl)hydrazine (B12)
6.2.23. 1-(4-(2,4-Dimethoxyphenyl)thiazol-2-yl)-2-(heptan-3-
Mp 211e213 ꢁC, 88% yield; 1H NMR (DMSO-d6, 400 MHz)
ylidene)hydrazine (C8)
d
(ppm): 1.02e1.04 (m, 6H, 2 ꢂ CH3), 1.75e2.48 (m, 4H, 2 ꢂ CH2),
Mp 85e87 ꢁC, 92% yield; 1H NMR (CDCl3, 400 MHz)
d
(ppm):
4.14 (s, 3H, 4-OCH3-Ar), 5.97 (s, 1H, C5H-thiaz.), 6.48e6.50 (m, 2H,
1.01e1.03 (m, 3H, CH2CH3),1.33e1.40 (m, 8H, 4 ꢂ CH2),1.57e1.59 (m,

110
S. Carradori et al. / European Journal of Medicinal Chemistry 65 (2013) 102e111
3H, CH2CH3), 3.91 (s, 3H, OCH3-Ar), 4.18 (s, 3H, OCH3-Ar), 6.87 (s, 1H,
C5H-thiaz.), 7.40e7.41 (m, 1H, Ar), 7.52 (s, 1H, Ar), 7.65e7.66 (m, 1H,
Ar), 12.79 (bs, 1H, NH, D2O exch.); ESI-MS: m/z 348.1 [M þ H]þ.
7.52 (m, 1H, Ar), 7.68 (s, 1H, Ar), 7.88e7.90 (m, 1H, Ar), 10.89 (bs, 1H,
NH, D2O exch.); ESI-MS: m/z 342.0 [M þ H]þ.
6.2.32. 1-(4-(2,4-Dichlorophenyl)thiazol-2-yl)-2-(hex-5-en-2-
6.2.24. 1-(4-(2,4-Dimethoxyphenyl)thiazol-2-yl)-2-(octan-2-
ylidene)hydrazine (D6)
ylidene)hydrazine (C9)
Mp 113e115 ꢁC, 98% yield; 1H NMR (CDCl3, 400 MHz)
d (ppm):
Mp 198e200 ꢁC, 92% yield; 1H NMR (CDCl3, 400 MHz)
d
(ppm):
2.16 (s, 3H, CH3C]), 2.36e2.39 (m, 2H, CH2), 2.47e2.50 (m, 2H,
CH2), 5.01e5.06 (m, 2H, CH2]), 5.85e5.87 (m, 1H, CH]), 7.00 (s,
1H, C5H-thiaz.), 7.41e7.43 (d, Jo ¼ 8.3 Hz, 1H, Ar), 7.53 (s, 1H, Ar),
7.62e7.64 (d, Jo ¼ 8.3 Hz, 1H, Ar), 12.71 (bs, 1H, NH, D2O exch.); ESI-
MS: m/z 362.0 [M þ Na]þ.
0.89e0.91 (m, 3H, CH2CH3),1.26e1.65 (m, 10H, 5 ꢂ CH2), 2.23 (s, 3H,
CH3C]), 3.96 (s, 3H, OCH3-Ar), 4.14 (s, 3H, OCH3-Ar), 5.97 (s, 1H,
C5H-thiaz.), 6.53e6.55 (m,1H, Ar), 6.59e6.61 (m,1H, Ar), 7.68 (s,1H,
Ar), 13.00 (bs, 1H, NH, D2O exch.); ESI-MS: m/z 362.1 [M þ H]þ.
6.2.25. 1-(1-Cyclohexylethylidene)-2-(4-(2,4-dimethoxyphenyl)
6.2.33. 1-(4-(2,4-Dichlorophenyl)thiazol-2-yl)-2-(heptan-2-
thiazol-2-yl)hydrazine (C10)
ylidene)hydrazine (D7)
Mp 102e103 ꢁC, 90% yield; 1H NMR (CDCl3, 400 MHz)
d
(ppm):
Mp 160e162 ꢁC, 97% yield; 1H NMR (CDCl3, 400 MHz)
d (ppm):
1.30e1.60 (m, 10H, 5 ꢂ CH2), 2.12 (s, 3H, CH3C]), 2.19e2.21 (m, 1H,
CH), 3.88 (s, 3H, OCH3-Ar), 4.11 (s, 3H, OCH3-Ar), 6.89 (s, 1H, C5H-
thiaz.), 7.44e7.45 (m, 1H, Ar), 7.56 (s, 1H, Ar), 7.67e7.70 (m, 1H, Ar),
12.73 (bs, 1H, NH, D2O exch.); ESI-MS: m/z 360.1 [M þ H]þ.
0.90e0.93 (m, 3H, CH2CH3), 1.32e1.36 (m, 4H, 2 ꢂ CH2), 1.57e1.59
(m, 2H, CH2), 2.16 (s, 3H, CH3C]), 2.37e2.38 (m, 2H, CH2), 6.99 (s,
1H, C5H-thiaz.), 7.41e7.43 (d, Jo ¼ 8.4 Hz, 1H, Ar), 7.54 (s, 1H, Ar),
7.62e7.64 (d, Jo ¼ 8.4 Hz, 1H, Ar), 12.72 (bs, 1H, NH, D2O exch.); ESI-
MS: m/z 378.0 [M þ Na]þ.
6.2.26. 1-Cyclooctylidene-2-(4-(2,4-dimethoxyphenyl)thiazol-2-yl)
hydrazine (C11)
6.2.34. 1-(4-(2,4-Dichlorophenyl)thiazol-2-yl)-2-(heptan-3-
Mp 204e206 ꢁC, 95% yield; 1H NMR (CDCl3, 400 MHz)
d
(ppm):
ylidene)hydrazine (D8)
1.41e1.42 (m, 2H, CH2), 1.55e1.57 (m, 2H, CH2), 1.81e1.83 (m, 2H,
CH2), 1.94e1.96 (m, 2H, CH2), 2.45e2.47 (m, 2H, CH2), 2.69e2.72 (m,
4H, 2 ꢂ CH2), 3.96 (s, 3H, OCH3-Ar), 4.16 (s, 3H, OCH3-Ar), 5.95 (s,
1H, C5H-thiaz.), 6.53e6.55 (m, 1H, Ar), 6.59e6.61 (m, 1H, Ar), 7.69
(s, 1H, Ar), 12.98 (bs, 1H, NH, D2O exch.); ESI-MS: m/z 360.1
[M þ H]þ.
Mp 155e156 ꢁC, 88% yield; 1H NMR (CDCl3, 400 MHz)
d (ppm):
1.02e1.05 (m, 3H, CH2CH3), 1.41e1.53 (m, 4H, 2 ꢂ CH2), 1.59e1.61
(m, 3H, CH2CH3), 2.36e2.52 (m, 4H, 2 ꢂ CH2), 7.00 (s, 1H, C5H-
thiaz.), 7.42e7.43 (m, 1H, Ar), 7.55 (s, 1H, Ar), 7.64e7.66 (m, 1H, Ar),
12.88 (bs, 1H, NH, D2O exch.); ESI-MS: m/z 378.0 [M þ Na]þ.
6.2.35. 1-(4-(2,4-Dichlorophenyl)thiazol-2-yl)-2-(octan-2-ylidene)
6.2.27. 1-(4-(2,4-Dichlorophenyl)thiazol-2-yl)-2-(propan-2-
ylidene)hydrazine (D1)
hydrazine (D9)
Mp 112e113 ꢁC, 91% yield; 1H NMR (CDCl3, 400 MHz)
d (ppm):
Mp 155e156 ꢁC, 95% yield; 1H NMR (DMSO-d6, 400 MHz)
0.85e0.91 (m, 3H, CH2CH3), 1.22e1.61 (m, 10H, 5 ꢂ CH2), 2.27 (s, 3H,
CH3C]), 5.94 (s, 1H, C5H-thiaz.), 6.51e6.53 (m, 1H, Ar), 6.62e6.65
(m,1H, Ar), 7.58 (s,1H, Ar),12.85 (bs,1H, NH, D2O exch.); ESI-MS: m/
z 370.0 [M þ H]þ.
d
(ppm): 1.93 (s, 3H, CH3C]), 1.96 (s, 3H, CH3C]), 7.30 (s, 1H, C5H-
thiaz.), 7.48e7.51 (m, 1H, Ar), 7.68 (s, 1H, Ar), 7.85e7.88 (m, 1H, Ar),
10.48 (bs, 1H, NH, D2O exch.); ESI-MS: m/z 321.9 [M þ Na]þ.
6.2.28. 1-(Butan-2-ylidene)-2-(4-(2,4-dichlorophenyl)thiazol-2-yl)
6.2.36. 1-(1-Cyclohexylethylidene)-2-(4-(2,4-dichlorophenyl)
hydrazine (D2)
thiazol-2-yl)hydrazine (D10)
Mp 158e160 ꢁC, 90% yield; 1H NMR (CDCl3, 400 MHz)
d
(ppm):
Mp 173e175 ꢁC, 95% yield; 1H NMR (CDCl3, 400 MHz)
d (ppm):
1.13e1.16 (m, 3H, CH2CH3), 2.24 (s, 3H, CH3C]), 2.38e2.40 (m, 2H,
CH2CH3), 5.99 (s, 1H, C5H-thiaz.), 6.53e6.55 (m, 1H, Ar), 6.59e6.61
(m,1H, Ar), 7.69 (s,1H, Ar),13.02 (bs,1H, NH, D2O exch.); ESI-MS: m/
z 314.0 [M þ H]þ.
1.33e1.84 (m, 10H, 5 ꢂ CH2), 2.14 (s, 3H, CH3C]), 2.20e2.22 (m, 1H,
CH), 6.99 (s, 1H, C5H-thiaz.), 7.41e7.43 (m, 1H, Ar), 7.54 (s, 1H, Ar),
7.62-7.64 (m, 1H, Ar), 12.68 (bs, 1H, NH, D2O exch.); ESI-MS: m/z
368.0 [M þ H]þ.
6.2.29. 1-(4-(2,4-Dichlorophenyl)thiazol-2-yl)-2-(pentan-2-
6.2.37. 1-Cyclooctylidene-2-(4-(2,4-dichlorophenyl)thiazol-2-yl)
ylidene)hydrazine (D3)
hydrazine (D11)
Mp 159e160 ꢁC, 95% yield; 1H NMR (CDCl3, 400 MHz)
d
(ppm):
Mp 161e162 ꢁC, 92% yield; 1H NMR (CDCl3, 400 MHz)
d (ppm):
0.99e1.02 (m, 3H, CH2CH3), 1.59e1.61 (m, 2H, CH2), 2.15 (s, 3H,
CH3C]), 2.31e2.35 (m, 2H, CH2), 6.09 (s, 1H, C5H-thiaz.), 6.96e7.00
(m, 2H, Ar), 7.05e7.10 (m, 1H, Ar), 7.67 (s, 1H, Ar), 12.21 (bs, 1H, NH,
D2O exch.); ESI-MS: m/z 328.0 [M þ H]þ.
1.44e1.57 (m, 6H, 3 ꢂ CH2), 1.84e1.98 (m, 4H, 2 ꢂ CH2), 2.47e2.49
(m, 2H, CH2), 2.61e2.63 (m, 2H, CH2), 6.99 (s, 1H, C5H-thiaz.), 7.53e
7.55 (m, 1H, Ar), 7.57 (s, 1H, Ar), 7.61e7.63 (m, 1H, Ar), 12.73 (bs, 1H,
NH, D2O exch.); ESI-MS: m/z 368.0 [M þ H]þ.
6.2.30. 1-(4-(2,4-Dichlorophenyl)thiazol-2-yl)-2-(pentan-3-
ylidene)hydrazine (D4)
6.2.38. 1-(4-(2,4-Dichlorophenyl)thiazol-2-yl)-2-(ferrocen-2-
ylidene)hydrazine (D12)
Mp 217e218 ꢁC, 92% yield; 1H NMR (DMSO-d6, 400 MHz)
Mp 170e171 ꢁC, 85% yield; 1H NMR (DMSO-d6, 400 MHz)
d
(ppm): 2.48e2.51 (m, 6H, 2 ꢂ CH2CH3), 4.15e4.18 (m, 4H,
d (ppm): 2.24 (s, 3H, CH3C]), 4.23 (s, 5H, ferrocene), 4.38 (s, 2H,
2 ꢂ CH2CH3), 6.18 (s, 1H, C5H-thiaz.), 7.67e7.68 (m, 2H, Ar), 7.85 (s,
ferrocene), 4.66 (s, 2H, ferrocene), 7.33 (s, 1H, C5H-thiaz.), 7.51e7.53
(m, 1H, Ar), 7.71 (s, 1H, Ar), 7.87e7.90 (m, 1H, Ar), 11.48 (bs, 1H, NH,
D2O exch.); ESI-MS: m/z 491.9 [M þ Na]þ.
1H, Ar), 10.38 (bs, 1H, NH, D2O exch.); ESI-MS: m/z 328.0 [M þ H]þ.
6.2.31. 1-(4-(2,4-Dichlorophenyl)thiazol-2-yl)-2-(4-methylpentan-
2-ylidene)hydrazine (D5)
6.3. Antibacterial activity
Mp 159e160 ꢁC, 90% yield; 1H NMR (DMSO-d6, 400 MHz)
d
(ppm): 0.49e0.51 (s, 6H, 2 ꢂ CH3), 1.91e1.95 (m, 2H, CH2), 2.11e
Microtiter plates containing serial dilutions of each compound
ranging from 256 to 0.5 mg/mL were inoculated with each organism
2.13 (m, 1H, CH), 2.51 (s, 3H, CH3C]), 7.31 (s, 1H, C5H-thiaz.), 7.50e

S. Carradori et al. / European Journal of Medicinal Chemistry 65 (2013) 102e111
111
to yield the appropriate density (105/mL) in a 100
mL final volume;
scraper, washed a couple of time with phosphate buffer saline (PBS)
and fixed overnight in a mixture of 2% paraformaldehyde-2%
glutaraldehyde in PBS (pH 7.4), post-fixed in 1% osmium
tetroxide in veronal acetate buffer (pH 7.4) for 1 h at 25 ꢁC. Samples
were then stained with 0.1% tannic acid in the same buffer for
30 min at 25 ꢁC and with uranyl acetate (5 mg/mL) for 1 h at 25 ꢁC,
then dehydrated with graded acetone and embedded in Epon-
812 (Electron Microscopy Science, Società Italiana Chimici,
Rome, Italy). Ultrathin sections were cut using a Reichert ultrami-
crotome, counterstained with uranyl acetate and lead citrate, and
examined under a Philips CM10 transmission electron microscope
(FEI, Eindhoven, The Netherlands) [17].
each plate included positive controls (bacteria without a com-
pound), and a negative control (medium only). The plates were
incubated for 18e22 h at 35 ꢁC. The Minimal Inhibitory Concen-
tration (MIC) for all isolates was defined as the lowest concentra-
tion of antibacterial agent that completely inhibited the growth of
the organism, as detected by the unaided eye.
6.4. 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
positive controls (fungi without a compound) and a negative con-
trol (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 agent that completely inhibited the growth of the or-
ganism, as detected by unaided eye.
Acknowledgments
This work was supported by grants from MIUR (Italy).
Appendix A. Supplementary material
6.5. Broth microdilution susceptibility test method
Supplementary material related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2013.04.042.
The yeast inoculum was adjusted to a concentration of 0.5e
2.5 ꢂ 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
24 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.
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Hep2 cells, at the end of the incubation time with the different
compounds, have been detached from the flasks using a manual