Anti-Candida activity and cytotoxicity of a large library of new N-substituted-1,3-thiazolidin-4-one derivatives

Article abstract of DOI:10.1016/j.ejmech.2015.10.048

On the basis of the recent findings about the biological properties of thiazolidinones and taking into account the encouraging results about the antifungal activity of some (thiazol-2-yl)hydrazines, new N-substituted heterocyclic derivatives were designed combining the thiazolidinone nucleus with the hydrazonic portion. In details, 1,3-thiazolidin-4-ones bearing (cyclo)aliphatic or (hetero)aromatic moieties linked to the N1-hydrazine at C2 were synthesized and classified into three series according to the aromatic or bicyclic rings connected to the lactam nitrogen of the thiazolidinone. These molecules were assayed for their anti-Candida effects in reference to the biological activity of the conventional topic (clotrimazole, miconazole, tioconazole) and systemic drugs (fluconazole, ketoconazole, amphotericin B). Finally, we investigated the selectivity against fungal cells by testing the compounds endowed with the best MICs on Hep2 cells in order to assess their cell toxicity (CC₅₀) and we noticed that two derivatives were less cytotoxic than the reference drug clotrimazole. Moreover, a preliminary molecular modelling approach has been performed against lanosterol 14-α demethylase (CYP51A1) to rationalize the activity of the tested compounds and to specify the target protein or enzyme.

Full text of DOI:10.1016/j.ejmech.2015.10.048

European Journal of Medicinal Chemistry 107 (2016) 82e96
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Anti-Candida activity and cytotoxicity of a large library of new
N-substituted-1,3-thiazolidin-4-one derivatives
,
Celeste De Monte a, Simone Carradori b *, Bruna Bizzarri a, Adriana Bolasco a,
Federica Caprara a, Adriano Mollica b, Daniela Rivanera c, Emanuela Mari d,
Alessandra Zicari d, Atilla Akdemir e, Daniela Secci a
a Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy
b Department of Pharmacy, “G. D'Annunzio” University of Chieti-Pescara, Via dei Vestini 31, 66100 Chieti, Italy
c
ꢀ
Dipartimento di Sanita Pubblica e Malattie Infettive, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy
d Dipartimento di Medicina Sperimentale, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy
e Bezmialem Vakif University, Faculty of Pharmacy, Department of Pharmacology, Vatan Caddesi, 34093 Fatih, Istanbul, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
On the basis of the recent findings about the biological properties of thiazolidinones and taking into
account the encouraging results about the antifungal activity of some (thiazol-2-yl)hydrazines, new N-
substituted heterocyclic derivatives were designed combining the thiazolidinone nucleus with the
hydrazonic portion. In details, 1,3-thiazolidin-4-ones bearing (cyclo)aliphatic or (hetero)aromatic moi-
eties linked to the N1-hydrazine at C2 were synthesized and classified into three series according to the
aromatic or bicyclic rings connected to the lactam nitrogen of the thiazolidinone. These molecules were
assayed for their anti-Candida effects in reference to the biological activity of the conventional topic
(clotrimazole, miconazole, tioconazole) and systemic drugs (fluconazole, ketoconazole, amphotericin B).
Finally, we investigated the selectivity against fungal cells by testing the compounds endowed with the
best MICs on Hep2 cells in order to assess their cell toxicity (CC50) and we noticed that two derivatives
were less cytotoxic than the reference drug clotrimazole. Moreover, a preliminary molecular modelling
Received 27 May 2015
Received in revised form
22 October 2015
Accepted 28 October 2015
Available online 2 November 2015
Keywords:
Antimycotic effect
Candida spp.
Cytotoxicity
N-substituted thiazolidinones
approach has been performed against lanosterol 14-
of the tested compounds and to specify the target protein or enzyme.
© 2015 Elsevier Masson SAS. All rights reserved.
a demethylase (CYP51A1) to rationalize the activity
ꢀ
1. Introduction
sake. Particularly, candidemia caused by C. glabrata is rising [6], but
some epidemiologic studies revealed that systemic candidiasis also
provoked by Candida albicans, Candida tropicalis and C. parapsilosis
is emerging, especially in South America [7e10].
Opportunistic infections caused by common fungi such as
Candida species are increasing in the last decades, affecting the
lifestyle of the patients in a manner that is proportional to the
degree of the infection, which in turn depends on the state of health
of the patient [1]. In this regard, apart from the mucocutaneous
mycosis with a functional epithelial damage, in patients suffering
from serious diseases such as cancer, AIDS, or in those who received
transplantation therapy or a broad spectrum antibiotic therapy, the
immune system is compromised and this could lead to systemic
mycosis [2]. Among fungal infections, recent data showed that
there are many cases caused by non-albicans species [3e5] such as
Candida glabrata, Candida krusei, Candida parapsilosis and Candida
With regard to the therapeutic strategy, the most used drugs
belong to the class of azoles because of their good profile in terms of
safety and bioavailability, but an increasing incidence of drug
resistance occurs, due to the massive use of these azole-based
agents [11]. Therefore, it is of primary importance to discover
new candidates as antimycotic agents to cope with these alarming
data and to manage systemic candidiasis especially in immuno-
compromised patients. Among novel compounds, thiazolidinones
have proven to possess several biological properties, including the
antifungal activity [12e16]. Furthermore, in our previous works we
reported different scaffolds of [4-(4’-substituted-phenyl)thiazol-2-
yl]hydrazine derivatives that have shown to be active as anti-
Candida agents at very low micromolar concentrations, with low
* Corresponding author.
E-mail address: simone.carradori@unich.it (S. Carradori).
http://dx.doi.org/10.1016/j.ejmech.2015.10.048
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

C. De Monte et al. / European Journal of Medicinal Chemistry 107 (2016) 82e96
83
cell toxicity and a synergistic action with clotrimazole (Scheme 1).
In detail, compounds containing aliphatic and cycloaliphatic groups
at the hydrazonic portion and substituted phenyl rings at C4 of the
thiazole showed very low MIC values especially towards C. albicans
and C. glabrata. Similarly, other derivatives with heterocyclic and
aromatic substituents at C2 such as furan, thiophene, pyridine,
benzodioxole and naphthalene were very active towards C. albicans
[17e21].
On the basis of these findings about the (thiazol-2-yl)hydrazine
scaffold and taking into account the therapeutic importance of
thiazolidinone nucleus, especially with regard to its antimycotic
activity [22], we developed a large library of new compounds
belonging to three different series (1Ae26A, 1Be26B, 1Ce26C) as
promising anti-Candida agents combining three chemically
different, but pharmacologically compatible moieties such as a
thiazolidinone, a hydrazonic portion and an aromatic or a bicyclic
substituent linked at the lactam nitrogen (Scheme 1).
According to the established guidelines of Clinical and Labora-
tory Standards Institute (CLSI) and the European Committee for
Antimicrobial Susceptibility Testing (EUCAST), we evaluated the
susceptibility of twenty-two clinical Candida spp. strains to our
compounds by determining their minimum inhibitory concentra-
tion (MIC) [23]. Finally, some of the compounds endowed with the
best MICs were tested to assess their cell toxicity (CC50) on Hep2
cells in order to evaluate their selectivity against fungal cells and we
found that two derivatives were less cytotoxic than the conven-
tional drug clotrimazole.
Scheme 2. Synthesis of the derivatives 1Ae26A, 1Be26B and 1Ce26C. Reagents and
conditions: (i) EtOH, acetic acid (cat.), rt; (ii) MeOH, CH3COONa, rt; (iii) anhydrous
acetone, anhydrous K2CO3, reflux.
2. Chemistry
thiazolidinone derivatives (1Ae26A, 1Be26B and 1Ce26C). All
the synthesized compounds were purified by column chromatog-
raphy before characterization by spectroscopic methods
(Experimental section) and elemental analysis (Supporting
information).
We designed novel 1,3-thiazolidin-4-one derivatives in which
the portion supported by N1-hydrazine moiety consisted of
aliphatic chains (C3eC8, linear, branched, unsaturated), cyclic
structures, aromatic, bicyclic and (hetero)aromatic rings (furan,
thiophene, benzene, pyridine, benzodioxole, and naphthalene),
whereas the lactam NH at the 4-thiazolidinone nucleus was func-
tionalized with electrophiles endowed with different steric features
obtaining three scaffolds, each of twenty-six compounds, by reac-
tion with 4-nitrobenzyl bromide (A series), 1-(chloromethyl)
naphthalene (B series) and N-(chloromethyl)phthalimide (C
series).
The synthetic strategy is outlined in Scheme 2. Several carbonyl
compounds reacted directly with thiosemicarbazide in ethanol
using catalytic amounts of acetic acid. The resulting thio-
semicarbazones were cyclized with ethyl bromoacetate in meth-
anol and sodium acetate to give the 1,3-thiazolidin-4-one
derivatives [24,25]. Finally, by the reaction between the thiazoli-
dinones and 4-nitrobenzyl bromide, 1-(chloromethyl)naphthalene
and N-(chloromethyl)phthalimide in anhydrous acetone and po-
tassium carbonate we obtained the N-functionalized 4-
3. Antibacterial and anti-Candida activity
Derivatives 1Ae26A, 1Be26B and 1Ce26C, dissolved in dime-
thylsulfoxide (DMSO), were evaluated for their antibacterial activ-
ity. Organisms from routine clinical Gram-positive (Staphylococcus
aureus, Staphylococcus warneri, Streptococcus faecalis, Streptococcus
a-hemolyticus) and Gram-negative isolates (Escherichia coli, Proteus
mirabilis, Enterobacter spp., Klebsiella oxytoca) from the respiratory
tract were collected from specimens of patients at the Hospital
‘Azienda Policlinico Umberto Iꢀ’ (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
Scheme 1. Evolution from (thiazol-2-yl)hydrazine to N-substituted-1,3-thiazolidin-4-one scaffold.

84
C. De Monte et al. / European Journal of Medicinal Chemistry 107 (2016) 82e96
broth microdilution method, as recommended by the National
Committee for Clinical Laboratory Standards (NCCLS) [26] with
Mueller-Hinton II broth (BBL Microbiology Systems, Cockeysville,
MD).
Once verified the absence of antibacterial effects, the antifungal
activity was evaluated against twenty-two clinical fungal isolates of
Candida spp. (C. albicans, C. glabrata, C. tropicalis, C. krusei,
C. glabrata. Similarly to A series, some (hetero)aromatic compounds
were effective: the furan derivative 19B was active against C. non-
albicans spp. while the thiophene derivative 21B and the benzene
derivative 22B were effective also towards C. albicans. Unlike A
series, in this series the pyridine derivative 24B was active with the
MIC value of 2
observed the same trend of the previous A and B series: compounds
1C and 6C had MIC values of 1 g/mL against C. non-albicans spp.
and were effective also against C. albicans (MIC ¼ 4 g/mL and
MIC ¼ 2 g/mL, respectively); most of the (cyclo)aliphatic de-
mg/mL against all the species. Finally, in C series we
ꢀ
C. parapsilosis and C. sake) and compared with topical and systemic
m
reference drugs clotrimazole, fluconazole, ketoconazole, micona-
zole, tioconazole and amphotericin B (see Tables 1e3). The used
clinical isolates were recently collected from specimens of patients
at the ‘Azienda Policlinico Umberto Iꢀ’ (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 iso-
lates were identified 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 microdilution method with Sabouraud dextrose broth (BBL
Microbiology Systems, Cockeysville, MD) as recommended by the
NCCLS [27].
m
m
rivatives and compounds 19C, 21C, 22C and 24C were good anti-
fungal agents even if not against all Candida species. In
consideration of these results, some of the most active derivatives
against C. albicans and non-albicans (1C, 6A, 6B, 6C, 7A and 10A)
were tested for their cytotoxic effects (CC50) by Trypan Blue
exclusion test at five different concentrations, and compared to
clotrimazole (Table 4). While compound 1C was cytotoxic at 22
mL and compounds 6A and 7A showed CC50 values similar to the
one of clotrimazole (32 g/mL for 6A and 35 g/mL for 7A), the less
cytotoxic derivatives were compounds 6B and 6C with CC50 values
of 95 g/mL and 99 g/mL, respectively. These two molecules
mg/
m
m
m
m
demonstrated to be very selective against fungal species, especially
against C. glabrata, since they displayed cytotoxicity towards hu-
man cells at much higher concentrations than those required for
the pharmacological effect towards fungal cells (the selectivity in-
dex, defined as the ratio between CC50 and MIC value for C. glabrata,
was 95 for 6B and 99 for 6C).
4. Cell toxicity
Some of the compounds endowed with the strongest antifungal
effect against Candida spp., namely 1C, 6A, 6B, 6C, 7A and 10A, were
also assayed at five concentrations ranging from 0.05 to 100 mg/mL
To conclude, we designed and synthesized three series of new
N-substituted thiazolidinone derivatives and we found that many
compounds have proven to be good and selective antifungal agents.
Regarding two active compounds (6B and 6C), we observed that
they possessed a very low toxicity against human cells with respect
to clotrimazole. Therefore, these derivatives can be taken into ac-
count as new lead compounds for the development of promising
antifungal drugs.
(Supporting information) to evaluate their cytotoxic activity against
a cultured cell line derived from a human laryngeal epidermoid
carcinoma (Hep2) with respect to the reference drug (clotrima-
zole), as reported in Table 4. Cell viability was analysed using Try-
pan Blue exclusion test [28].
5. Results and discussion
6. Molecular modelling studies
Three new large series of N-substituted-1,3-thiazolidin-4-one
derivatives were synthesized in high yields and tested in vitro for
their antimicrobial effect against several bacterial and fungal clin-
ical isolates. None of the products had shown antibacterial activity,
therefore the products demonstrated to be selectively active to-
wards mycotic pathogens. According to the in vitro biological data,
we observed that most of the compounds bearing aliphatic and
cycloaliphatic moieties showed the best antifungal activity (MICs
Determining the molecular targets of the tested compounds
either by biological assays or by molecular modelling studies is an
extremely difficult task since organisms consist of thousands of
proteins and other macromolecules (e.g., DNA and lipids) that can
interact with the compounds under investigation. We focused on
three enzymes that are important targets for well established
antifungal drugs, i.e., lanosterol 14-a demethylase (CYP51A1),1,3-b-
values of 1e2
mg/mL), similar or better than the reference drugs;
D-glucan synthase and squalene-epoxidase. The sequences of the
only few derivatives with (hetero)aromatic moieties were effective,
while compounds with benzodioxole and naphthalene linked to
the hydrazine moiety were not active. In details, with regard to A
series provided with a 4-nitrobenzyl substituent at the lactam NH,
three enzymes were obtained for C. albicans and a default BLAST
search was performed to identify crystal structures of related en-
zymes in the Brookhaven Protein Databank (PDB server). No
structures were obtained for the latter two enzymes, but a high
compound 1A was active against C. albicans and glabrata at 4
mL; among the (cyclo)aliphatic compounds, also derivatives
2Ae4A, 8Ae12A, 14A and 18A possessed MIC values of 2 g/mL,
while only the heterocyclic derivatives 19A and 21A and the aro-
matic derivative 22A were active at low concentrations against C.
m
g/
resolution crystal structure of lanosterol 14-a demethylase was
obtained for Saccharomyces cerevisiae in complex with lanosterol
(pdb code: 4lxj; 1.90 Å; 65% sequence identity to the C. albicans
enzyme).
m
Afterwards, the sequences of lanosterol 14-a demethylase from
non-albicans spp. Three compounds of this series had MICs of 1
m
g/
C. glabrata, C. parapsilosis and C. tropicalis were obtained and the
sequences alignments were created for all four Candida enzymes
and S. cerevisiae enzyme. All sequence identities are higher than
60% and the residue conservation in the binding pocket (defined as
all residues within 4.5 Å of lanosterol) of the enzymes are high
mL, displaying their antifungal effect at lower concentrations than
those of the reference drugs: compound 6A against C. albicans and
glabrata, compound 7A towards C. glabrata, and compound 10A
against C. tropicalis. In B series, bearing a naphthalene nucleus
linked to the lactam nitrogen, compound 1B was biologically active
(Table 5). Therefore, S. cerevisiae lanosterol 14-a demethylase was
towards all Candida spp. (MICs of 2e4
2Be15B and 18B had anti-Candida effect at 2
all the studied species. The branched aliphatic compound 6B
possessed a minimum inhibitory concentration of 1 g/mL against
m
g/mL), also derivatives
used as a model protein for docking studies of a representative set
of compounds (i.e., compounds 1A, 1B, 1C and 20C). The endoge-
m
g/mL but not against
nous substrate of lanosterol 14-
a demethylase is the hydrophobic
m
lanosterol and the ligandeprotein binding interactions are mostly

C. De Monte et al. / European Journal of Medicinal Chemistry 107 (2016) 82e96
85
Table 1
Minimum inhibitory concentration (MIC) expressed as geometric mean of derivatives 1Ae26A and reference compounds against twenty-two clinical strains of Candida species.
Tested fungi (MIC mg/mL)
ꢀ
Compound
N1-hydrazine moiety
C. albicans (8)
C. glabrata (4)
C. tropicalis (3)
C. krusei (3)
C. parapsilosis (2)
C. sake (2)
1A
4
4
2
2
2
2
2
8
2A
3A
4A
5A
6A
7A
8A
9A
10A
11A
2
2
2
4
1
4
2
4
2
4
128
64
8
2
16
32
32
2
2
16
128
32
2
2
8
8
2
8
8
8
1
32
64
2
4
1
2
2
2
8
16
128
8
16
256
16
8
2
2
128
1
256
16
2
16
16
2
2
12A
13A
14A
2
8
16
32
32
128
8
256
16
2
64
8
256
16
4
16
4
16
15A
16A
32
16
32
32
16
8
4
8
8
4
4
128
17A
18A
4
4
16
16
8
16
2
16
32
16
32
32
19A
4
8
2
16
16
2
20A
21A
22A
23A
32
8
32
2
16
4
4
2
8
2
8
8
4
32
32
2
8
4
2
256
256
128
32
32
24A
4
32
4
32
4
4
(continued on next page)

86
C. De Monte et al. / European Journal of Medicinal Chemistry 107 (2016) 82e96
Table 1 (continued )
Tested fungi (MIC mg/mL)
ꢀ
Compound
N1-hydrazine moiety
C. albicans (8)
C. glabrata (4)
C. tropicalis (3)
C. krusei (3)
C. parapsilosis (2)
C. sake (2)
25A
256
32
16
8
16
16
26A
256
64
16
8
8
8
Fluconazole
Ketoconazole
Clotrimazole
Miconazole
Tioconazole
Amphotericin B
2
2
2
2
8
4
2
2
2
4
4
4
2
2
2
2
8
2
2
4
2
4
8
4
2
2
2
4
8
2
2
4
2
2
4
2
hydrophobic in nature. Van-der-Waals interactions are formed with
the side chains of Tyr126, Leu129, Phe134, Phe236, Pro238, Phe241,
Leu380, Phe384 and Met509 (Fig. 1A). Collectively, docking studies
suggest that the synthesized compounds could also form mainly
Van-der-Waals interactions with the hydrophobic binding pocket
(Fig. 1B) similar to lanosterol.
at room temperature under magnetic stirring for 24 h. The resulting
4-thiazolidinone was poured on ice, filtered or extracted with
chloroform (3 ꢂ 100 mL) and purified by column chromatography
(SiO2, ethyl acetate/n-hexane). Then, the obtained thiazolidinone
(50 mmol) was dissolved/suspended in 50 mL of anhydrous acetone
in the presence of anhydrous potassium carbonate (50 mmol), and
reacted with equimolar amounts of 4-nitrobenzyl bromide, 1-
(chloromethyl)naphthalene and N-(chloromethyl)phthalimide for
24e48 h. The product was poured on ice, filtered or extracted with
chloroform (3 ꢂ 50 mL) and purified by column chromatography
(SiO2, ethyl acetate/n-hexane) in order to obtain the title com-
pounds in high yields.
7. Experimental section
The chemicals, solvents for synthesis, and spectral grade sol-
vents were purchased from Aldrich (Italy) and used without further
purification. Melting points (uncorrected) were determined auto-
matically on an FP62 apparatus (Mettler-Toledo). 1H and 13C NMR
spectra were recorded at 400 MHz on a Bruker spectrometer using
CDCl3 or DMSO-d6 as solvent. Chemical shifts are expressed as
7.1.1. 3-(4-Nitrobenzyl)-2-(2-(propan-2-ylidene)hydrazono)
thiazolidin-4-one (1A)
White powder, mp 157e163 ꢀC, 79% yield; 1H NMR (400 MHz,
d
units (parts per millions) relative to the solvent peak. Coupling
CDCl3):
d 2.00 (s, 3H, CH3), 2.08 (s, 3H, CH3), 3.82 (s, 2H, CH2,
constants J are valued in Hertz (Hz). IR spectra (neat) were regis-
tered on a Perkin Elmer FT-IR Spectrometer Spectrum 1000.
Elemental analyses for C, H, and N were recorded on a Per-
kineElmer 240 B microanalyzer and the analytical results were
within 0.4% of the theoretical values for all compounds. All re-
actions were monitored by TLC performed on 0.2 mm thick silica
gel plates (60 F254 Merck).
thiazolidinone), 5.05 (s, 2H, ArCH2), 7.60e7.62 (d, J ¼ 8.4 Hz, 2H, Ar),
8.18e8.20 (d, J ¼ 8.4 Hz, 2H, Ar).
7.1.2. 3-(4-Nitrobenzyl)-2-(2-(butan-2-ylidene)hydrazono)
thiazolidin-4-one (2A)
Yellow powder, mp 108e112 ꢀC, 75% yield; 1H NMR (400 MHz,
In general, the IR spectrum (neat) for derivatives 1Ae26A,
1Be26B, and 1Ce26C showed a band at about 3027 cmꢁ1 due to the
stretching of Csp2-H, at about 1693 due to the stretching of C]O
bond, at about 1622 for the stretching of C]N, and at about 1582
and 1447 for C]C stretching.
DMSO-d6): d 1.01e1.05 (t, 3H, CH3), 1.85 (s, 3H, CH3), 2.23e2.29 (q,
2H, CH2), 4.01 (s, 2H, CH2, thiazolidinone), 4.98 (s, 2H, ArCH2),
7.59e7.61 (d, J ¼ 8.4 Hz, 2H, Ar), 8.20e8.22 (d, J ¼ 8.4 Hz, 2H, Ar).
7.1.3. 3-(4-Nitrobenzyl)-2-(2-(pentan-2-ylidene)hydrazono)
thiazolidin-4-one (3A)
Yellow powder, mp 103e105 ꢀC, 81% yield; 1H NMR (400 MHz,
7.1. General procedure for the synthesis of compounds 1Ae26A,
1Be26B and 1Ce26C
CDCl3):
d 0.96e1.00 (t, 3H, CH3), 1.59e1.68 (q, 2H, CH2), 1.97 (s, 3H,
CH3), 2.30e2.34 (t, 2H, CH2), 3.81 (s, 2H, CH2, thiazolidinone), 5.05
(s, 2H, ArCH2), 7.61e7.63 (d, J ¼ 8.8 Hz, 2H, Ar), 8.19e8.21 (d,
J ¼ 8.8 Hz, 2H, Ar).
The initial carbonyl compound (50 mmol) was dissolved/sus-
pended in ethanol (50 mL) and magnetically stirred with thio-
semicarbazide (50 mmol) and catalytic amounts of acetic acid for
8e24 h at room temperature. The obtained thiosemicarbazone was
filtered, washed with appropriate solvent (n-hexane, petroleum
ether or diethyl ether) and dried under vacuum. The intermediate
thiosemicarbazone (50 mmol) reacted with ethyl bromoacetate
(50 mmol), in methanol (50 mL) and sodium acetate (50 mmol)
7.1.4. 3-(4-Nitrobenzyl)-2-(2-(pentan-3-ylidene)hydrazono)
thiazolidin-4-one (4A)
Yellow powder, mp 95e96 ꢀC, 81% yield; 1H NMR (400 MHz,
CDCl3):
d 0.98e1.02 (t, 3H, CH3), 1.14e1.17 (t, 3H, CH3), 2.35e2.45 (q,
4H, 2 ꢂ CH2), 3.81 (s, 2H, CH2, thiazolidinone), 5.05 (s, 2H, ArCH2),

C. De Monte et al. / European Journal of Medicinal Chemistry 107 (2016) 82e96
87
Table 2
Minimum inhibitory concentration (MIC) expressed as geometric mean of derivatives 1Be26B against twenty-two clinical strains of Candida species.
Tested fungi (MIC mg/mL)
ꢀ
Compound
N1-hydrazine moiety
C. albicans (8)
C. glabrata (4)
C. tropicalis (3)
C. krusei (3)
C. parapsilosis (2)
C. sake (2)
1B
4
4
2
2
2
2
2
2B
3B
4B
5B
6B
7B
8B
9B
10B
11B
2
2
2
2
4
4
2
2
2
4
128
64
2
2
2
2
2
2
16
2
2
16
2
16
2
16
2
8
2
1
64
64
2
2
2
4
8
2
2
2
8
16
8
16
16
128
8
2
2
8
16
256
2
16
32
8
16
2
2
12B
13B
14B
2
2
4
16
32
8
128
8
256
16
64
8
256
32
2
4
16
16
15B
16B
2
8
16
16
128
8
8
4
8
4
4
4
17B
18B
4
8
32
8
8
4
2
4
4
256
16
16
19B
16
8
8
16
16
2
20B
21B
22B
23B
32
2
32
8
128
2
4
16
32
32
4
16
32
32
4
2
2
8
2
8
4
8
8
32
24B
2
2
2
2
2
2
(continued on next page)

88
C. De Monte et al. / European Journal of Medicinal Chemistry 107 (2016) 82e96
Table 2 (continued )
Tested fungi (MIC mg/mL)
ꢀ
Compound
N1-hydrazine moiety
C. albicans (8)
C. glabrata (4)
C. tropicalis (3)
C. krusei (3)
C. parapsilosis (2)
C. sake (2)
25B
8
4
16
8
16
8
26B
256
64
32
8
8
8
7.60e7.62 (d, J ¼ 8.4 Hz, 2H, Ar), 8.18e8.20 (d, J ¼ 8.4 Hz, 2H, Ar).
7.1.10. 3-(4-Nitrobenzyl)-2-(2-cyclopentylidenehydrazono)
thiazolidin-4-one (10A)
Yellow powder, mp 173e175 ꢀC, 87% yield; 1H NMR (400 MHz,
7.1.5. 3-(4-Nitrobenzyl)-2-(2-(hex-5-en-2-ylidene)hydrazono)
thiazolidin-4-one (5A)
DMSO-d6):
d 1.69e1.71 (m, 4H, cyclopentane), 2.32e2.36 (m, 4H,
cyclopentane), 4.02 (s, 2H, CH2, thiazolidinone), 4.96 (s, 2H, ArCH2),
7.59e7.61 (d, J ¼ 8.8 Hz, 2H, Ar), 8.20e8.22 (d, J ¼ 8.8 Hz, 2H, Ar).
Grey powder, mp 110e111 ꢀC, 83% yield; 1H NMR (400 MHz,
DMSO-d6):
d 1.85 (s, 3H, CH3), 2.25e2.29 (m, 2H, CH2), 2.33e2.36
(m, 2H, CH2) 4.01 (s, 2H, CH2, thiazolidinone), 4.94e4.98 (m, 1H, ]
CH2), 4.99 (s, 2H, ArCH2), 5.03e5.07 (d, J ¼ 16.8 Hz, 1H, ]CH2)
5.79e5.89 (m,1H, CH]), 7.59e7.61 (d, J ¼ 8.4 Hz, 2H, Ar), 8.20e8.22
(d, J ¼ 8.4 Hz, 2H, Ar).
7.1.11. 3-(4-Nitrobenzyl)-2-(2-(2-methylcyclopentylidene)
hydrazono)thiazolidin-4-one (11A)
Yellow powder, mp 130e133 ꢀC, 78% yield; 1H NMR (400 MHz,
CDCl3):
d
1.37e1.39 (d, J ¼ 7.6 Hz, 3H, CH3), 1.35e1.39 (m, 1H,
cyclopentane), 1.65e1.71 (m, 1H, cyclopentane), 1.88e1.92 (m, 1H,
cyclopentane), 2.05e2.09 (m, 1H, cyclopentane), 2.38e2.43 (m, 1H,
cyclopentane), 2.55e2.62 (m, 2H, cyclopentane), 3.81 (s, 2H, CH2,
thiazolidinone), 5.03 (s, 2H, ArCH2), 7.62e7.64 (d, J ¼ 8.4 Hz, 2H, Ar),
8.19e8.21 (d, J ¼ 8.4 Hz, 2H, Ar).
7.1.6. 3-(4-Nitrobenzyl)-2-(2-(4-methylpentan-2-ylidene)
hydrazono)thiazolidin-4-one (6A)
Yellow powder, mp 70e75 ꢀC, 69% yield; 1H NMR (400 MHz,
CDCl3):
d
0.95e0.97 (d, 6H, 2 ꢂ CH3),1.95 (s, 3H, CH3), 2.01e2.04 (m,
1H, CH), 2.21e2.23 (d, 2H, CH2), 3.81 (s, 2H, CH2, thiazolidinone),
5.05 (s, 2H, ArCH2), 7.61e7.63 (d, J ¼ 8.0 Hz, 2H, Ar), 8.19e8.21 (d,
J ¼ 8.0 Hz, 2H, Ar).
7.1.12. 3-(4-Nitrobenzyl)-2-(2-(3-methylcyclopentylidene)
hydrazono)thiazolidin-4-one (12A)
Yellow powder, mp 206e210 ꢀC, 84% yield; 1H NMR (400 MHz,
7.1.7. 3-(4-Nitrobenzyl)-2-(2-(heptan-2-ylidene)hydrazono)
thiazolidin-4-one (7A)
CDCl3):
d 1.09e1.11 (d, 3H, CH3), 1.36e1.41 (m, 1H, cyclopentane),
Yellow powder, mp 64e69 ꢀC, 72% yield; 1H NMR (400 MHz,
1.93e2.01 (m, 1H, cyclopentane), 2.07e2.20 (m, 2H, cyclopentane),
2.33e2.43 (m, 1H, cyclopentane), 2.61e2.72 (m, 2H, cyclopentane),
3.83 (s, 2H, CH2, thiazolidinone), 5.03 (s, 2H, ArCH2), 7.61e7.63 (d,
J ¼ 8 Hz, 2H, Ar), 8.19e8.21 (d, J ¼ 8 Hz, 2H, Ar).
CDCl3): d 0.87e0.98 (m, 3H, CH3), 1.27e1.42 (m, 4H, CH2), 1.52e1.61
(m, 2H, CH2), 1.97 (s, 3H, CH3), 2.32e2.36 (t, 2H, CH2), 3.82 (s, 2H,
CH2, thiazolidinone), 5.05 (s, 2H, ArCH2), 7.61e7.63 (d, J ¼ 8.0 Hz,
2H, Ar), 8.19e8.21 (d, J ¼ 8.0 Hz, 2H, Ar).
7.1.13. 3-(4-Nitrobenzyl)-2-(2-cyclohexylidenehydrazono)
thiazolidin-4-one (13A)
7.1.8. 3-(4-Nitrobenzyl)-2-(2-(heptan-3-ylidene)hydrazono)
thiazolidin-4-one (8A)
Grey powder, mp 147e150 ꢀC, 81% yield; 1H NMR (400 MHz,
Yellow oil, 63% yield; 1H NMR (400 MHz, CDCl3):
d 0.86e0.89 (t,
CDCl3):
d 1.66 (bs, 4H, cyclohexane), 1.76 (bs, 2H, cyclohexane),
3H, CH3), 0.93e1.01 (m, 2H, CH2), 1.13e1.18 (m, 1H, CH2), 1.22e1.31
(m, 2H, CH2), 1.35e1.42 (m, 1H, CH2), 1.54e1.62 (m, 1H, CH2),
2.31e2.43 (m, 1H, CH2, 3H, CH3), 3.81 (s, 2H, CH2, thiazolidinone),
5.07 (s, 2H, ArCH2), 7.56e7.61 (m, 2H, Ar), 8.18e8.20 (d, J ¼ 8.8 Hz,
2H, Ar).
2.37e2.40 (m, 2H, cyclohexane), 2.59 (bs, 2H, cyclohexane), 3.82 (s,
2H, CH2, thiazolidinone), 5.05 (s, 2H, ArCH2), 7.59e7.61 (d,
J ¼ 8.4 Hz, 2H, Ar), 8.18e8.20 (d, J ¼ 8.4 Hz, 2H, Ar).
7.1.14. 3-(4-Nitrobenzyl)-2-(2-(2-methylcyclohexylidene)
hydrazono)thiazolidin-4-one (14A)
7.1.9. 3-(4-Nitrobenzyl)-2-(2-(octan-2-ylidene)hydrazono)
thiazolidin-4-one (9A)
Yellow powder, mp 136e138 ꢀC, 80% yield; 1H NMR (400 MHz,
Yellow powder, mp 64e70 ꢀC, 69% yield; 1H NMR (400 MHz,
CDCl3):
d
1.16e1.18 (d, J ¼ 6.4 Hz, 3H, CH3), 1.35e1.47 (m, 3H,
CDCl3):
d
0.91e0.92 (t, 3H, CH3), 1.30e1.36 (m, 6H, CH2), 1.55e1.60
cyclohexane), 1.78e1.82 (m, 2H, cyclohexane), 1.95e2.00 (m, 2H,
cyclohexane), 2.41e2.44 (m, 1H, cyclohexane), 3.13e3.17 (m, 1H,
cyclohexane), 3.80 (s, 2H, CH2, thiazolidinone), 5.05 (s, 2H, ArCH2),
7.60e7.62 (d, J ¼ 8.4 Hz, 2H, Ar), 8.18e8.20 (d, J ¼ 8.4 Hz, 2H, Ar).
(m, 2H, CH2), 1.97 (s, 3H, CH3), 2.31e2.35 (t, 2H, CH2), 3.81 (s, 2H,
CH2, thiazolidinone), 5.05 (s, 2H, ArCH2), 7.61e7.63 (d, J ¼ 8.4 Hz,
2H, Ar), 8.19e8.21 (d, J ¼ 8.4 Hz, 2H, Ar).

C. De Monte et al. / European Journal of Medicinal Chemistry 107 (2016) 82e96
89
Table 3
Minimum inhibitory concentration (MIC) expressed as geometric mean of derivatives 1Ce26C against twenty-two clinical strains of Candida species.
Tested fungi (MIC mg/mL)
ꢀ
Compound
N1-hydrazine moiety
C. albicans (8)
C. glabrata (4)
C. tropicalis (3)
C. krusei (3)
C. parapsilosis (2)
C. sake (2)
1C
4
1
2
1
2
2
2
2C
3C
4C
5C
6C
7C
8C
9C
10C
11C
2
2
64
4
8
2
8
2
2
4
2
2
8
8
8
8
8
4
32
1
4
32
2
128
2
8
2
64
128
2
2
4
8
2
2
2
4
16
16
16
32
32
8
8
2
2
16
8
16
64
8
16
256
8
2
16
2
16
4
12C
13C
14C
2
8
4
32
16
8
8
8
2
16
16
16
8
8
16
32
2
16
15C
16C
2
4
32
16
8
8
8
4
4
32
16
16
17C
18C
4
32
16
8
4
4
4
256
256
16
16
16
19C
2
8
8
4
2
2
20C
21C
22C
23C
256
4
16
32
8
128
2
256
4
256
4
256
8
2
8
8
8
8
256
8
16
8
16
32
24C
2
32
2
32
4
4
(continued on next page)

90
C. De Monte et al. / European Journal of Medicinal Chemistry 107 (2016) 82e96
Table 3 (continued )
Tested fungi (MIC mg/mL)
ꢀ
Compound
N1-hydrazine moiety
C. albicans (8)
C. glabrata (4)
C. tropicalis (3)
C. krusei (3)
C. parapsilosis (2)
C. sake (2)
25C
8
8
8
32
128
128
26C
256
16
128
128
128
128
7.1.15. 3-(4-Nitrobenzyl)-2-(2-(4-methylcyclohexylidene)
cyclohexane), 1.85e1.98 (m, 3H, cyclohexane), 2.24e2.32 (m, 1H,
cyclohexane), 2.53e2.56 (m, 1H, cyclohexane), 3.24e3.28 (m, 1H,
cyclohexane), 3.81 (s, 2H, CH2, thiazolidinone), 5.05 (s, 2H,
ArCH2), 7.59e7.61 (d, J ¼ 8.8 Hz, 2H, Ar), 8.18e8.20 (d, J ¼ 8.8 Hz,
2H, Ar).
hydrazono)thiazolidin-4-one (15A)
White powder, mp 123e128 ꢀC, 78% yield; 1H NMR (400 MHz,
CDCl3):
d
0.97e0.99 (d, J ¼ 6.4 Hz, 3H, CH3), 1.08e1.18 (m, 1H,
cyclohexane), 1.21e1.31 (m, 1H, cyclohexane), 1.69e1.76 (m, 1H,
Table 4
Cytotoxic effect (CC50) of the 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 (%).
Compound
Trypan Blue exclusion testa CC50
22
(mg/mL)
1C
6A
6B
32
95
6C
7A
99
35
10A
28
38
Clotrimazole
a
Cells incubated with culture medium alone represented the controls. Viability with 2
m
L of DMSO ¼ 94%, viability with 20 L of DMSO ¼ 87%. Data represent arithmetic
m
means of at least three independent experiments.

C. De Monte et al. / European Journal of Medicinal Chemistry 107 (2016) 82e96
91
Table 5
The sequence identity of the binding pocket residues of selected CYP51A1 (lano-
sterol 14-
a
demethylase) enzymes.
Different identity sequence (single amino acid substitution)
is reported in bold
4lxj
C. albicans
C. glabrata
C. parapsilosis
C. tropicalis
Tyr72
Gly73
Leu95
Tyr126
His128
Leu129
Thr130
Phe134
Ile139
Tyr
Gly
Leu
Tyr
His
Leu
Thr
Phe
Ile
Tyr
Gly
Leu
Tyr
His
Leu
Thr
Phe
Ile
Tyr
Gly
Met
Tyr
His
Leu
Thr
Phe
Ile
Tyr
Gly
Leu
Tyr
His
Leu
Thr
Phe
Ile
Tyr140
Leu147
Lys151
Val154
Phe236
Pro238
Phe241
Val242
Leu306
Gly310
Val311
Met313
Gly314
Gly315
Thr318
Leu380
His381
Ser382
Leu383
Phe384
Cys470
Phe506
Thr507
Ser508
Met509
Val510
Thr511
Tyr
Leu
Lys
Ala
Phe
Pro
Phe
Val
Leu
Gly
Ile
Met
Gly
Gly
Thr
Leu
His
Ser
Ile
Phe
Cys
Tyr
Ser
Ser
Met
Val
Val
Tyr
Leu
Lys
Val
Phe
Pro
Phe
Val
Leu
Gly
Val
Met
Gly
Gly
Thr
Leu
His
Ser
Leu
Phe
Cys
Phe
Thr
Ser
Met
Val
Thr
Phe
Leu
Lys
Ala
Phe
Pro
Phe
Val
Leu
Gly
Val
Met
Gly
Gly
Thr
Leu
His
Ser
Ile
Phe
Cys
Tyr
Thr
Ser
Met
Val
Thr
Tyr
Leu
Lys
Ala
Phe
Pro
Phe
Val
Leu
Gly
Val
Met
Gly
Gly
Thr
Leu
His
Ser
Ile
Phe
Cys
Tyr
Gln
Ser
Met
Val
Thr
Fig. 1. A) The binding interactions of lanosterol with its endogenous hydrophobic
binding pocket in lanosterol 14-
pose of compound 1A in the binding pocket of lanosterol 14-
a
demethylase (pdb: 4lxj). B) A representative docked
demethylase. The
a
binding pocket cavity is indicated in green (apolar residues) and purple (polar resi-
dues). (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
The binding pocket is defined as all amino acids within 4.5 Å of lanosterol.
7.1.16. 3-(4-Nitrobenzyl)-2-(2-cycloheptylidenehydrazono)
thiazolidin-4-one (16A)
DMSO-d6):
d 4.09 (s, 2H, CH2, thiazolidinone), 5.03 (s, 2H, ArCH2),
Yellow powder, mp 106e110 ꢀC, 81% yield; 1H NMR (400 MHz,
6.65e6.67 (m, 1H, furan), 6.98e6.99 (d, J ¼ 3.2 Hz, 1H, furan),
7.61e7.63 (d, J ¼ 8.8 Hz, 2H, Ar), 7.90 (s, 1H, furan), 8.21e8.23 (d,
J ¼ 8.8 Hz, 2H, Ar), 8.26 (s, 1H, CH]).
CDCl3):
d 1.58e1.71 (m, 8H, cycloheptane), 2.54e2.62 (m, 4H,
cycloheptane), 3.81 (s, 2H, CH2, thiazolidinone), 5.04 (s, 2H, ArCH2),
7.61e7.63 (d, J ¼ 8.8 Hz, 2H, Ar), 8.18e8.20 (d, J ¼ 8.8 Hz, 2H, Ar).
7.1.20. 3-(4-Nitrobenzyl)-2-(2-(1-(furan-2-yl)ethylidene)
hydrazono)thiazolidin-4-one (20A)
7.1.17. 3-(4-Nitrobenzyl)-2-(2-cyclooctylidenehydrazono)
thiazolidin-4-one (17A)
Yellow powder, mp 182e187 ꢀC, 82% yield; 1H NMR (400 MHz,
Yellow powder, mp 124e126 ꢀC, 87% yield; 1H NMR (400 MHz,
CDCl3): d 2.33 (s, 3H, CH3), 3.87 (s, 2H, CH2, thiazolidinone), 5.12 (s,
CDCl3):
d 1.37e1.44 (m, 4H, cyclooctane), 1.55e1.64 (m, 4H, cyclo-
2H, ArCH2), 6.51 (bs, 1H, furan), 6.90e6.91 (m, 1H, furan), 7.56e7.61
(m, 1H, furan), 7.63e7.65 (d, J ¼ 8 Hz, 2H, Ar), 8.20e8.22 (d, J ¼ 8 Hz,
2H, Ar).
octane), 1.83e1.84 (m, 2H, cyclooctane), 2.44e2.50 (m, 4H, cyclo-
octane), 3.83 (s, 2H, CH2, thiazolidinone), 5.05 (s, 2H, ArCH2),
7.60e7.62 (d, J ¼ 8 Hz, 2H, Ar), 8.18e8.20 (d, J ¼ 8 Hz, 2H, Ar).
7.1.21. 3-(4-Nitrobenzyl)-2-(2-(1-(thiophen-2-yl)ethylidene)
hydrazono)thiazolidin-4-one (21A)
7.1.18. 3-(4-Nitrobenzyl)-2-(2-(1-cyclohexylethylidene)hydrazono)
thiazolidin-4-one (18A)
Yellow powder, mp 160e164 ꢀC, 81% yield; 1H NMR (400 MHz,
Yellow powder, mp 130e133 ꢀC, 85% yield; 1H NMR (400 MHz,
CDCl3): d 2.42 (s, 3H, CH3), 3.86 (s, 2H, CH2, thiazolidinone), 5.11 (s,
DMSO-d6):
d 1.25e1.35 (m, 5H, cyclohexane), 1.57e1.73 (m, 8H,
2H, ArCH2), 7.08 (bs, 1H, thiophene), 7.40 (m, 2H, thiophene),
cyclohexane þ CH3), 1.82e1.89 (m, 1H, cyclohexane), 4.02 (s, 2H,
CH2, thiazolidinone), 4.98 (s, 2H, ArCH2), 7.58e7.60 (d, J ¼ 8 Hz, 2H,
Ar), 8.20e8.22 (d, J ¼ 8 Hz, 2H, Ar).
7.63e7.66 (m, 2H, Ar), 8.20e8.22 (m, 2H, Ar).
7.1.22. 3-(4-Nitrobenzyl)-2-(2-(1-phenylethylidene)hydrazono)
thiazolidin-4-one (22A)
7.1.19. 3-(4-Nitrobenzyl)-2-(2-(furan-2-ylmethylene)hydrazono)
thiazolidin-4-one (19A)
Yellow powder, mp 152e155 ꢀC, 85% yield; 1H NMR (400 MHz,
CDCl3):
d 2.43 (s, 3H, CH3), 3.86 (s, 2H, CH2, thiazolidinone), 5.14 (s,
Yellow powder, mp 150e155 ꢀC, 76% yield; 1H NMR (400 MHz,
2H, ArCH2), 7.42e7.44 (m, 3H, Ar), 7.66e7.68 (d, J ¼ 8.8 Hz, 2H, Ar),

92
C. De Monte et al. / European Journal of Medicinal Chemistry 107 (2016) 82e96
7.87e7.89 (m, 2H, Ar), 8.21e8.23 (d, J ¼ 8.8 Hz, 2H, Ar).
7.1.31. 2-(2-(Hex-5-en-2-ylidene)hydrazono)-3-(naphthalen-1-
ylmethyl)thiazolidin-4-one (5B)
Orange oil, 63% yield; 1H NMR (400 MHz, CDCl3):
d 1.89 (s, 3H,
7.1.23. 3-(4-Nitrobenzyl)-2-(2-(pyridin-3-ylmethylene)hydrazono)
thiazolidin-4-one (23A)
CH3), 2.35e2.43 (m, 4H, CH2), 3.83 (s, 2H, CH2, thiazolidinone),
5.01e5.10 (m, 2H, ]CH2), 5.47 (s, 2H, ArCH2), 5.83e5.93 (m, 1H,
CH]), 7.41e7.45 (t, 1H, Ar), 7.50e7.58 (m, 3H, Ar), 7.80e7.82 (d,
J ¼ 8.4 Hz, 1H, Ar), 7.88e7.90 (d, J ¼ 8.4 Hz, 1H, Ar), 8.30e8.32 (d,
J ¼ 8.4 Hz, 1H, Ar).
Red powder, mp 231e233 ꢀC, 83% yield; 1H NMR (400 MHz,
DMSO-d6):
d 4.15 (s, 2H, CH2, thiazolidinone), 5.06 (s, 2H, ArCH2),
7.47e7.54 (m,1H, Ar), 7.62e7.64 (d, J ¼ 8.0 Hz, 2H, Ar), 8.14e8.18 (m,
1H, Ar), 8.22e8.24 (d, J ¼ 8.0 Hz, 2H, Ar), 8.51 (s, 1H, CH]),
8.62e8.70 (m, 1H, Ar), 8.91 (s, 1H, Ar).
7.1.32. 2-(2-(4-Methylpentan-2-ylidene)hydrazono)-3-
(naphthalen-1-ylmethyl)thiazolidin-4-one (6B)
7.1.24. 3-(4-Nitrobenzyl)-2-(2-(1-(pyridin-2-yl)ethylidene)
hydrazono)thiazolidin-4-one (24A)
Orange oil, 61% yield; 1H NMR (400 MHz, CDCl3):
d 0.64e0.66 (d,
6H, 2 ꢂ CH3), 1.74e1.81 (m, 1H, CH), 2.01 (s, 3H, CH3), 2.19e2.21 (d,
2H, CH2), 3.83 (s, 2H, CH2, thiazolidinone), 5.46 (s, 2H, ArCH2),
7.41e7.49 (m, 1H, Ar), 7.50e7.60 (m, 3H, Ar), 7.80e7.92 (m, 1H, Ar),
8.15e8.17 (d, J ¼ 8.0 Hz, 1H, Ar), 8.25e8.27 (d, J ¼ 8.0 Hz, 1H, Ar).
Yellow powder, mp 227e230 ꢀC, 86% yield; 1H NMR (400 MHz,
DMSO-d6): d 2.40 (s, 3H, CH3), 4.12 (s, 2H, CH2, thiazolidinone), 5.09
(s, 2H, ArCH2), 7.44e7.47 (m, 1H, Ar), 7.65e7.67 (d, J ¼ 8.4 Hz, 2H,
Ar), 7.86e7.90 (m, 1H, Ar), 8.05e8.07 (d, J ¼ 8.0 Hz, 1H, Ar),
8.23e8.25 (d, J ¼ 8.4 Hz, 2H, Ar), 8.63e8.64 (m, 1H, Ar).
7.1.33. 2-(2-(Heptan-2-ylidene)hydrazono)-3-(naphthalen-1-
ylmethyl)thiazolidin-4-one (7B)
White powder, mp 148e153, 77% yield; 1H NMR (400 MHz,
7.1.25. 3-(4-Nitrobenzyl)-2-(2-(benzo[d][1,3]dioxol-5-ylmethylene)
hydrazono)thiazolidin-4-one (25A)
CDCl3):
d 1.58e1.59 (m, 2H CH2, 6H CH3), 1.72e1.74 (m, 2H, CH2),
Grey powder, mp 185e187 ꢀC, 79% yield; 1H NMR (400 MHz,
2.35e2.38 (t, 2H, CH2), 2.48e2.51 (t, 2H, CH2), 3.84 (s, 2H, CH2,
thiazolidinone), 5.47 (s, 2H, ArCH2), 7.40e7.45 (t, 1H, Ar), 7.50e7.58
(m, 3H, Ar), 7.80e7.82 (d, J ¼ 8.0 Hz,1H, Ar), 7.88e7.90 (d, J ¼ 8.0 Hz,
1H, Ar), 8.30e8.32 (d, J ¼ 8.0 Hz, 1H, Ar).
DMSO-d6):
d 4.09 (s, 2H, CH2, thiazolidinone), 5.03 (s, 2H, ArCH2),
6.10 (s, 2H, CH2 benzodioxole), 7.00e7.02 (d, J ¼ 7.6 Hz, 2H, Ar),
7.25e7.30 (m, 2H, Ar), 7.60e7.62 (d, J ¼ 8.8 Hz, 2H, Ar), 8.21e8.23 (d,
J ¼ 8.0 Hz, 1H, Ar), 8.34 (s, 1H, CH]).
7.1.34. 2-(2-(Heptan-3-ylidene)hydrazono)-3-(naphthalen-1-
7.1.26. 3-(4-Nitrobenzyl)-2-(2-(naphthalen-1-ylmethylene)
hydrazono)thiazolidin-4-one (26A)
ylmethyl)thiazolidin-4-one (8B)
Orange oil, 65% yield; 1H NMR (400 MHz, CDCl3):
d 0.83e0.87 (t,
Yellow powder, mp 185e191 ꢀC, 84% yield; 1H NMR (400 MHz,
3H, CH3), 0.92e0.96 (t, 3H, CH3), 1.23e1.37 (m, 5H, CH2), 2.26e2.34
(m, 3H, CH2), 3.84 (s, 2H, CH2, thiazolidinone), 5.46 (s, 2H, ArCH2),
7.40e7.58 (m, 4H, Ar), 7.80e7.92 (m, 2H, Ar), 8.21e8.27 (m, 1H, Ar).
CDCl3):
d 3.92 (s, 2H, CH2, thiazolidinone), 5.17 (s, 2H, ArCH2),
7.52e7.58 (m, 2H, Ar), 7.60e7.66 (m, 1H, Ar), 7.68e7.70 (d,
J ¼ 8.0 Hz, 2H, Ar), 7.92e7.98 (m, 3H, Ar), 8.20e8.22 (d, J ¼ 8.0 Hz,
2H, Ar), 8.93e8.95 (d, J ¼ 8.0 Hz, 1H, Ar), 9.05 (s, 1H, CH]).
7.1.35. 3-(Naphthalen-1-ylmethyl)-2-(2-(octan-2-ylidene)
hydrazono)thiazolidin-4-one (9B)
Pink powder, mp 64e68 ꢀC, 82% yield; 1H NMR (400 MHz,
7.1.27. 3-(Naphthalen-1-ylmethyl)-2-(2-(propan-2-ylidene)
CDCl3):
d 0.90e0.94 (t, 3H, CH3), 1.03e1.08 (m, 1H, CH2), 1.24e1.32
hydrazono)thiazolidin-4-one (1B)
Orange oil, 67% yield; 1H NMR (400 MHz, CDCl3):
d 1.67 (s, 3H,
(m, 5H, CH2), 1.53e1.58 (m, 1H, CH2), 1.88 (s, 3H, CH3), 2.02 (bs, 1H,
CH2), 2.28e2.33 (m, 2H, CH2), 3.83 (s, 2H, CH2, thiazolidinone), 5.47
(s, 2H, ArCH2), 7.41e7.58 (m, 4H, Ar), 7.80e7.82 (d, J ¼ 8.0 Hz, 1H,
Ar), 7.88e7.90 (d, J ¼ 8.0 Hz, 1H, Ar), 8.30e8.33 (d, J ¼ 8.0 Hz, 1H,
Ar).
CH3), 1.81 (s, 3H, CH3), 3.60 (s, 2H, CH2, thiazolidinone), 5.23 (s, 2H,
ArCH2), 7.17e7.21 (t, 1H, Ar), 7.23e7.36 (m, 3H, Ar), 7.56e7.58 (d,
J ¼ 8.0 Hz, 2H, Ar), 8.05e8.07 (d, J ¼ 8.0 Hz, 1H, Ar).
7.1.28. 2-(2-(Butan-2-ylidene)hydrazono)-3-(naphthalen-1-
7.1.36. 2-(2-Cyclopentylidenehydrazono)-3-(naphthalen-1-
ylmethyl)thiazolidin-4-one (10B)
ylmethyl)thiazolidin-4-one (2B)
Orange oil, 63% yield; 1H NMR (400 MHz, CDCl3):
d 1.12e1.20 (m,
Orange powder, mp 60e65 ꢀC, 81% yield; 1H NMR (400 MHz,
3H, CH3), 1.89 (s, 3H, CH3), 2.30e2.38 (m, 2H, CH2), 3.83 (s, 2H, CH2,
thiazolidinone), 5.48 (s, 2H, ArCH2), 7.41e7.45 (m, 1H, Ar),
7.50e7.59 (m, 3H, Ar), 7.80e7.82 (d, J ¼ 8.0 Hz,1H, Ar), 7.88e7.90 (d,
J ¼ 8.0 Hz, 1H, Ar), 8.29e8.32 (m, 1H, Ar).
CDCl3):
d 1.27e1.35 (m, 1H, cyclopentane), 1.73e1.79 (m, 3H,
cyclopentane), 2.36e2.40 (m, 2H, cyclopentane), 2.47e2.50 (m, 2H,
cyclopentane), 3.84 (s, 2H, CH2, thiazolidinone), 5.45 (s, 2H, ArCH2),
7.41e7.45 (t, 1H, Ar), 7.48e7.59 (m, 3H, Ar), 7.80e7.82 (d, J ¼ 8.0 Hz,
1H, Ar), 7.86e7.92 (m, 1H, Ar), 8.32e8.34 (d, J ¼ 8.0 Hz, 1H, Ar).
7.1.29. 3-(Naphthalen-1-ylmethyl)-2-(2-(pentan-2-ylidene)
hydrazono)thiazolidin-4-one (3B)
7.1.37. 2-(2-(2-Methylcyclopentylidene)hydrazono)-3-
(naphthalen-1-ylmethyl)thiazolidin-4-one (11B)
Orange oil, 65% yield; 1H NMR (400 MHz, CDCl3):
d 0.95e0.98 (t,
Yellow powder, mp 95e96 ꢀC, 94% yield; 1H NMR (400 MHz,
3H, CH3), 1.28e1.36 (m, 2H, CH2), 1.87 (s, 3H, CH3), 2.27e2.31 (t, 2H,
CH2), 3.83 (s, 2H, CH2, thiazolidinone), 5.47 (s, 2H, ArCH2), 7.41e7.63
(m, 4H, Ar), 7.80e7.93 (m, 2H, Ar), 8.27e8.32 (m, 1H, Ar).
CDCl3):
d
1.20e1.21 (d, J ¼ 6.4 Hz, 3H, CH3), 1.28e1.36 (m, 1H,
cyclopentane), 1.59e1.64 (m, 1H, cyclopentane), 1.79e1.85 (m, 1H,
cyclopentane), 2.03e2.07 (m, 1H, cyclopentane), 2.26e2.36 (m, 1H,
cyclopentane), 2.47e2.60 (m, 2H, cyclopentane), 3.82 (s, 2H, CH2,
thiazolidinone), 5.45 (s, 2H, ArCH2), 7.41e7.45 (t, 1H, Ar), 7.50e7.61
(m, 3H, Ar), 7.80e7.82 (d, J ¼ 8.4 Hz, 1H, Ar), 7.87e7.89 (d, J ¼ 8.0 Hz,
1H, Ar), 8.33e8.35 (d, J ¼ 8.4 Hz,1H, Ar). 13C NMR (100 MHz, CDCl3):
7.1.30. 3-(Naphthalen-1-ylmethyl)-2-(2-(pentan-3-ylidene)
hydrazono)thiazolidin-4-one (4B)
Orange oil, 67% yield; 1H NMR (400 MHz, CDCl3):
d 0.87e0.91 (t,
3H, CH3), 1.16e1.20 (t, 3H, CH3), 2.31e2.39 (q, 4H, 2 ꢂ CH2), 3.79 (s,
2H, CH2, thiazolidinone), 5.48 (s, 2H, ArCH2), 7.43e7.47 (t, 1H, Ar),
7.51e7.61 (m, 3H, Ar), 7.81e7.83 (d, J ¼ 8.4 Hz, 1H, Ar), 7.89e7.91 (d,
J ¼ 8.0 Hz, 1H, Ar), 8.33e8.35 (d, J ¼ 8.0 Hz, 1H, Ar).
d
17.0, 22.5, 30.4, 32.3, 33.8, 39.6, 44.5, 123.7, 125.1, 125.6, 125.7,
126.3, 126.4, 127.4, 128.4, 128.5, 128.7, 128.8, 130.8, 131.4, 133.7,
159.3, 172.1, 181.3.

C. De Monte et al. / European Journal of Medicinal Chemistry 107 (2016) 82e96
93
7.1.38. 2-(2-(3-Methylcyclopentylidene)hydrazono)-3-
1H, cyclohexane), 3.82 (s, 2H, CH2, thiazolidinone), 5.47 (s, 2H,
ArCH2), 7.41e7.45 (t, 1H, Ar), 7.50e7.57 (m, 3H, Ar), 7.80e7.82 (d,
J ¼ 8.0 Hz, 1H, Ar), 7.88e7.90 (d, J ¼ 8.0 Hz, 1H, Ar), 8.30e8.32 (d,
(naphthalen-1-ylmethyl)thiazolidin-4-one (12B)
Red oil, 64% yield; 1H NMR (400 MHz, CDCl3):
d 1.05e1.08 (d, 3H,
CH3), 1.27e1.35 (m, 4H, cyclopentane), 2.25e2.34 (m, 1H, cyclo-
pentane), 2.46e2.66 (m, 2H, cyclopentane), 3.84 (s, 2H, CH2, thia-
zolidinone), 5.45 (s, 2H, ArCH2), 7.41e7.45 (t, 1H, Ar), 7.48e7.57 (m,
3H, Ar), 7.81e7.83 (d, J ¼ 8.0 Hz, 1H, Ar), 7.88e7.90 (d, J ¼ 8.0 Hz, 1H,
Ar), 8.28e8.34 (m, 1H, Ar).
J ¼ 8.0 Hz, 1H, Ar). 13C NMR (100 MHz, CDCl3):
d 26.0, 26.1, 30.2,
32.3, 44.5, 46.8, 123.6, 125.1, 125.7, 126.3, 126.9, 128.4, 128.7, 130.8,
131.3, 133.7, 159.5, 172.1, 172.6.
7.1.45. 2-(2-(Furan-2-ylmethylene)hydrazono)-3-(naphthalen-1-
ylmethyl)thiazolidin-4-one (19B)
7.1.39. 2-(2-Cyclohexylidenehydrazono)-3-(naphthalen-1-
ylmethyl)thiazolidin-4-one (13B)
Orange powder, mp 145e150 ꢀC, 84% yield; 1H NMR (400 MHz,
CDCl3):
d 3.91 (s, 2H, CH2, thiazolidinone), 5.53 (s, 2H, ArCH2),
Yellow powder, mp 148e150 ꢀC, 91% yield; 1H NMR (400 MHz,
6.51e6.55 (m, 1H, furan), 6.82e6.83 (d, J ¼ 3.2 Hz, 1H, furan),
7.42e7.49 (m, 2H, Ar þ furan), 7.52e7.61 (m, 3H Ar), 7.81e7.83 (d,
J ¼ 8.0 Hz,1H, Ar), 7.89e7.91 (d, J ¼ 7.6 Hz,1H, Ar), 8.21 (s,1H, CH]),
8.29e8.31 (d, J ¼ 8.4 Hz, 1H, Ar).
CDCl3):
d 1.57e1.62 (m, 3H, cyclohexane), 1.71e1.74 (m, 2H, cyclo-
hexane), 2.20 (s, 1H, cyclohexane), 2.35e2.38 (t, 2H, cyclohexane),
2.48e2.51 (t, 2H, cyclohexane), 3.83 (s, 2H, CH2, thiazolidinone),
5.47 (s, 2H, ArCH2), 7.40e7.44 (t, 1H, Ar), 7.50e7.58 (m, 3H, Ar),
7.80e7.82 (d, J ¼ 8.0 Hz, 1H, Ar), 7.88e7.90 (d, J ¼ 7.6 Hz, 1H, Ar),
8.30e8.32 (d, J ¼ 8.0 Hz, 1H, Ar).
7.1.46. 2-(2-(1-(Furan-2-yl)ethylidene)hydrazono)-3-(naphthalen-
1-ylmethyl)thiazolidin-4-one (20B)
Orange powder, mp 169e171 ꢀC, 89% yield; 1H NMR (400 MHz,
7.1.40. 2-(2-(2-Methylcyclohexylidene)hydrazono)-3-(naphthalen-
1-ylmethyl)thiazolidin-4-one (14B)
CDCl3): d 2.23 (s, 3H, CH3), 3.88 (s, 2H, CH2, thiazolidinone), 5.53 (s,
2H, ArCH2), 6.48 (bs, 1H, furan), 6.85e6.86 (d, J ¼ 3.6 Hz, 1H, furan),
7.40e7.46 (t, 1H, Ar), 7.51e7.60 (m, 4H, Ar þ furan), 7.81e7.83 (d,
J ¼ 7.6 Hz, 1H, Ar), 7.88e7.90 (d, J ¼ 8.4 Hz, 1H, Ar), 8.32e8.34 (d,
J ¼ 8.4 Hz, 1H, Ar).
Yellow powder, mp 132e133 ꢀC, 96% yield; 1H NMR (400 MHz,
CDCl3):
d
1.22e1.23 (d, J ¼ 6.4 Hz, 3H, CH3), 1.28e1.44 (m, 2H,
cyclohexane), 1.47e1.53 (m, 1H, cyclohexane), 1.74e1.76 (m, 2H,
cyclohexane), 1.87e1.95 (m, 2H, cyclohexane), 2.37e2.42 (m, 1H,
cyclohexane), 3.05e3.08 (m, 1H, cyclohexane), 3.81 (s, 2H, CH2,
thiazolidinone), 5.47 (s, 2H, ArCH2), 7.40e7.44 (t, 1H, Ar), 7.49e7.57
(m, 3H, Ar), 7.79e7.81 (d, J ¼ 8.0 Hz, 1H, Ar), 7.87e7.89 (d, J ¼ 7.6 Hz,
1H, Ar), 8.31e8.33 (d, J ¼ 7.6 Hz, 1H, Ar).
7.1.47. 3-(Naphthalen-1-ylmethyl)-2-(2-(1-(thiophen-2-yl)
ethylidene)hydrazono)thiazolidin-4-one (21B)
Orange powder, mp 137e143 ꢀC, 83% yield; 1H NMR (400 MHz,
CDCl3): d 2.32 (s, 3H, CH3), 3.87 (s, 2H, CH2, thiazolidinone), 5.51 (s,
2H, ArCH2), 7.04e7.07 (m, 1H, thiophene), 7.36e7.38 (m, 2H, thio-
phene), 7.42e7.46 (t, 1H, Ar), 7.51e7.58 (m, 3H, Ar), 7.81e7.83 (d,
J ¼ 8.0 Hz, 1H, Ar), 7.89e7.91 (d, J ¼ 8.0 Hz, 1H, Ar), 8.29e8.35 (m,
1H, Ar).
7.1.41. 2-(2-(4-Methylcyclohexylidene)hydrazono)-3-(naphthalen-
1-ylmethyl)thiazolidin-4-one (15B)
Brown oil, 60% yield; 1H NMR (400 MHz, CDCl3):
d 1.22e1.23 (d,
3H, CH3), 1.28e1.39 (m, 1H, cyclohexane), 1.46e1.58 (m, 1H, cyclo-
hexane), 1.86e2.20 (m, 4H, cyclohexane), 2.48e2.56 (m, 1H,
cyclohexane), 2.79e2.82 (m, 1H, cyclohexane), 3.44e3.48 (m, 1H,
cyclohexane), 4.11 (s, 2H, CH2, thiazolidinone), 5.74 (s, 2H, ArCH2),
7.68e7.72 (t, 1H, Ar), 7.77e7.85 (m, 3H, Ar), 8.07e8.09 (d, J ¼ 8.4 Hz,
1H, Ar), 8.15e8.17 (d, J ¼ 7.6 Hz, 1H, Ar), 8.58e8.60 (d, J ¼ 8.4 Hz, 1H,
Ar).
7.1.48. 3-(Naphthalen-1-ylmethyl)-2-(2-(1-phenylethylidene)
hydrazono)thiazolidin-4-one (22B)
Orange powder, mp 155e156 ꢀC, 87% yield; 1H NMR (400 MHz,
CDCl3): d 2.34 (s, 3H, CH3), 3.88 (s, 2H, CH2, thiazolidinone), 5.56 (s,
2H, ArCH2), 7.40e7.47 (m, 4H, Ar), 7.52e7.61 (m, 3H, Ar), 7.82e7.91
(m, 4H, Ar), 8.34e8.36 (d, J ¼ 8.4 Hz, 1H, Ar). 13C NMR (100 MHz,
CDCl3):
d 15.2, 32.5, 44.7, 123.6, 125.1, 125.7, 126.4, 126.8, 126.8,
7.1.42. 2-(2-Cycloheptylidenehydrazono)-3-(naphthalen-1-
128.3, 128.5, 128.8, 130.0, 130.7, 131.3, 133.7, 137.8, 162.0, 163.2,
172.2.
ylmethyl)thiazolidin-4-one (16B)
Brown oil, 65% yield; 1H NMR (400 MHz, CDCl3):
d 0.89e0.98 (m,
3H, cycloheptane), 1.22e1.33 (m, 1H, cycloheptane), 1.34e1.37 (m,
3H, cycloheptane), 1.62e1.76 (m, 4H, cycloheptane), 2.45e2.48 (m,
1H, cycloheptane), 3.95 (s, 2H, CH2, thiazolidinone), 5.62 (s, 2H,
ArCH2), 7.41e7.63 (m, 4H, Ar), 7.86e7.90 (m, 1H, Ar), 8.11e8.13 (m,
1H, Ar), 8.39e8.41 (m, 1H, Ar).
7.1.49. 3-(Naphthalen-1-ylmethyl)-2-(2-(pyridin-3-ylmethylene)
hydrazono)thiazolidin-4-one (23B)
Orange powder, mp 71e73 ꢀC, 90% yield; 1H NMR (400 MHz,
CDCl3):
d 3.93 (s, 2H, CH2, thiazolidinone), 5.55 (s, 2H, ArCH2),
7.31e7.61 (m, 5H, Ar), 7.82e7.92 (m, 2H, Ar), 8.14e8.16 (m, 1H, Ar),
8.20e8.25 (m, 1H, Ar), 8.39e8.40 (m, 1H, Ar), 8.65e8.67 (m, 1H, Ar),
8.89 (bs, 1H, CH]).
7.1.43. 2-(2-Cyclooctylidenehydrazono)-3-(naphthalen-1-
ylmethyl)thiazolidin-4-one (17B)
Orange oil, 67% yield; 1H NMR (400 MHz, CDCl3):
d
1.16e1.46 (m,
7.1.50. 3-(Naphthalen-1-ylmethyl)-2-(2-(1-(pyridin-2-yl)
ethylidene)hydrazono)thiazolidin-4-one (24B)
5H, cyclooctane), 1.53e1.61 (m, 3H, cyclooctane), 1.69e1.80 (m, 2H,
cyclooctane), 2.27e2.39 (m, 2H, cyclooctane), 2.41e2.48 (m, 2H,
cyclooctane), 3.83 (s, 2H, CH2, thiazolidinone), 5.45 (s, 2H, ArCH2),
7.40e7.44 (t, 1H, Ar), 7.50e7.60 (m, 3H, Ar), 7.80e7.82 (d, J ¼ 8.0 Hz,
1H, Ar), 7.88e7.90 (d, J ¼ 8.0 Hz,1H, Ar), 8.29e8.31 (d, J ¼ 8.0 Hz,1H,
Ar).
Yellow powder, mp 125e126 ꢀC, 92% yield; 1H NMR (400 MHz,
CDCl3): d 2.47 (s, 3H, CH3), 3.88 (s, 2H, CH2, thiazolidinone), 5.57 (s,
2H, ArCH2), 7.44e7.66 (m, 5H, Ar), 7.70e7.75 (m, 1H, Ar), 7.83e7.91
(m, 2H, Ar), 8.18e8.21 (m, 1H, Ar), 8.33e8.40 (m, 1H, Ar), 8.63 (bs,
1H, Ar).
7.1.44. 2-(2-(1-Cyclohexylethylidene)hydrazono)-3-(naphthalen-1-
ylmethyl)thiazolidin-4-one (18B)
7.1.51. 2-(2-(Benzo[d][1,3]dioxol-5-ylmethylene)hydrazono)-3-
(naphthalen-1-ylmethyl)thiazolidin-4-one (25B)
White powder, mp 116e121 ꢀC, 85% yield; 1H NMR (400 MHz,
Yellow powder, mp 128e130 ꢀC, 85% yield; 1H NMR (400 MHz,
CDCl3):
d
1.20e1.40 (m, 6H, cyclohexane), 1.69e1.72 (m, 1H, cyclo-
DMSO-d6):
d 4.18 (s, 2H, CH2, thiazolidinone), 5.41 (s, 2H, ArCH2),
hexane), 1.81 (bs, 3H, cyclohexane), 1.85 (s, 3H, CH3), 2.20e2.26 (m,
6.09 (s, 2H, CH2, benzodioxole), 6.98e7.00 (d, J ¼ 8.0 Hz, 1H, Ar),

94
C. De Monte et al. / European Journal of Medicinal Chemistry 107 (2016) 82e96
7.20e7.22 (m, 1H, Ar), 7.26e7.29 (m, 2H, Ar), 7.42e7.49 (m, 1H, Ar),
7.59e7.64 (m, 2H, Ar), 7.87e7.88 (m, 1H, Ar), 7.98e8.00 (m, 1H, Ar),
8.21e8.30 (m, 2H, Ar þ CH]).
7.1.60. 2-((2-(2-(Heptan-3-ylidene)hydrazono)-4-oxothiazolidin-
3-yl)methyl)isoindoline-1,3-dione (8C)
White powder, mp 70e74 ꢀC, 86% yield; 1H NMR (400 MHz,
CDCl3):
d 0.82e0.93 (m, 2H, CH2, 3H, CH3), 1.08e1.12 (m, 1H, CH2),
1.30e1.37 (m, 3H, CH3), 1.51e1.55 (m, 1H, CH2), 2.24e2.32 (m, 2H,
CH2), 2.36e2.44 (m, 2H, CH2), 3.82 (s, 2H, CH2, thiazolidinone), 5.71
(s, 2H, ArCH2), 7.73e75 (m, 2H, Ar), 7.85e7.86 (m, 2H, Ar).
7.1.52. 3-(Naphthalen-1-ylmethyl)-2-(2-(naphthalen-1-
ylmethylene)hydrazono)thiazolidin-4-one (26B)
Orange powder, mp 185e190 ꢀC, 93% yield; 1H NMR (400 MHz,
CDCl3):
d 3.96 (s, 2H, CH2, thiazolidinone), 5.61 (s, 2H, ArCH2),
7.1.61. 2-((2-(2-(Octan-2-ylidene)hydrazono)-4-oxothiazolidin-3-
yl)methyl)isoindoline-1,3-dione (9C)
7.45e7.47 (m, 1H, Ar), 7.549e7.58 (m, 4H, Ar), 7.61e7.65 (m, 2H, Ar),
7.83e7.85 (m, 1H, Ar), 7.91e7.99 (m, 4H, Ar), 8.30e8.36 (m, 1H, Ar),
8.85e8.92 (m, 1H, Ar), 9.00 (s, 1H, CH]).
Brown powder, mp 65e70 ꢀC, 81% yield; 1H NMR (400 MHz,
CDCl3):
d 0.89e0.91 (m, 4H, CH2, 3H, CH3), 1.53e1.62 (m, 3H, CH2),
1.93 (s, 3H, CH3), 2.24e2.35 (m, 2H, CH2), 2.44e2.47 (m, 1H, CH2),
3.83 (s, 2H, CH2, thiazolidinone), 5.72 (s, 2H, ArCH2), 7.70e76 (m,
2H, Ar), 7.86e7.88 (m, 2H, Ar).
7.1.53. 2-((2-(2-(Propan-2-ylidene)hydrazono)-4-oxothiazolidin-3-
yl)methyl)isoindoline-1,3-dione (1C)
Yellow powder, mp 119e124 ꢀC, 99% yield; 1H NMR (400 MHz,
DMSO-d6):
d 1.79 (s, 3H, CH3), 1.88 (s, 3H, CH3), 3.95 (s, 2H, CH2,
7.1.62. 2-((2-(2-Cyclopentylidenehydrazono)-4-oxothiazolidin-3-
yl)methyl)isoindoline-1,3-dione (10C)
thiazolidinone), 5.52 (s, 2H, ArCH2), 7.73e8.31 (m, 4H, Ar).
Pink powder, mp 169e175 ꢀC, 85% yield; 1H NMR (400 MHz,
7.1.54. 2-((2-(2-(Butan-2-ylidene)hydrazono)-4-oxothiazolidin-3-
yl)methyl)isoindoline-1,3-dione (2C)
DMSO-d6):
d 1.62 (bs, 4H, cyclopentane), 2.15 (bs, 2H, cyclo-
pentane), 2.29 (bs, 2H, cyclopentane), 3.98 (s, 2H, CH2, thiazolidi-
none), 5.51 (s, 2H, ArCH2), 7.84e7.93 (m, 4H, Ar).
White powder, mp 135e142 ꢀC, 87% yield; 1H NMR (400 MHz,
CDCl3): d 1.08e1.12 (t, 3H, CH3), 2.20 (s, 3H, CH3), 2.27e2.31 (q, 2H,
CH2), 3.83 (s, 2H, CH2, thiazolidinone), 5.73 (s, 2H, ArCH2), 7.76 (bs,
2H, Ar), 7.86e7.87 (m, 2H, Ar).
7.1.63. 2-((2-(2-(2-Methylcyclopentylidene)hydrazono)-4-
oxothiazolidin-3-yl)methyl)isoindoline-1,3-dione (11C)
White powder, mp 158e162 ꢀC, 83% yield; 1H NMR (400 MHz,
7.1.55. 2-((2-(2-(Pentan-2-ylidene)hydrazono)-4-oxothiazolidin-3-
yl)methyl)isoindoline-1,3-dione (3C)
CDCl3):
d
1.14e1.17 (d, J ¼ 10 Hz, 3H, CH3), 1.28e1.31 (m, 2H,
cyclopentane), 1.83 (bs, 1H, cyclopentane), 1.98e2.02 (m, 1H,
cyclopentane), 2.25e2.30 (m, 1H, cyclopentane), 2.48e2.54 (m, 2H,
cyclopentane), 3.83 (s, 2H, CH2, thiazolidinone), 5.71 (s, 2H, ArCH2),
7.76e7.77 (m, 2H, Ar), 7.86e7.88 (m, 2H, Ar). 13C NMR (100 MHz,
White powder, mp 125e126 ꢀC, 84% yield; 1H NMR (400 MHz,
CDCl3): d 0.92e0.95 (t, 3H, CH3), 1.59e1.61 (m, 2H, CH2), 1.93 (s, 3H,
CH3), 2.23e2.27 (t, 2H, CH2), 3.83 (s, 2H, CH2, thiazolidinone), 5.73
(s, 2H, ArCH2), 7.75e7.76 (m, 2H, Ar), 7.86e7.87 (m, 2H, Ar). 13C NMR
CDCl3):
d 16.9, 22.5, 30.3, 32.5, 33.7, 39.6, 45.1, 123.6, 131.7, 134.3,
(100 MHz, CDCl3):
d 13.8, 14.0, 17.5, 19.6, 19.8, 22.9, 32.2, 34.1, 40.6,
157.9, 166.7, 171.0, 182.3.
45.0, 45.1, 123.6, 131.7, 134.3, 158.0, 166.7, 170.0, 171.1.
7.1.64. 2-((2-(2-(3-Methylcyclopentylidene)hydrazono)-4-
oxothiazolidin-3-yl)methyl)isoindoline-1,3-dione (12C)
White powder, mp 174e175 ꢀC, 77% yield; 1H NMR (400 MHz,
7.1.56. 2-((2-(2-(Pentan-3-ylidene)hydrazono)-4-oxothiazolidin-3-
yl)methyl)isoindoline-1,3-dione (4C)
White powder, mp 155e156 ꢀC, 89% yield; 1H NMR (400 MHz,
CDCl3):
d 1.31e1.32 (d, 3H, CH3), 1.55e1.58 (m, 1H, cyclopentane),
CDCl3):
d 0.87e0.91 (t, 3H, CH3), 1.09e1.12 (t, 3H, CH3), 2.28e2.34
2.20 (bs, 1H, cyclopentane), 2.29e2.35 (m, 3H, cyclopentane),
2.84e2.87 (m, 2H, cyclopentane), 4.12 (s, 2H, CH2, thiazolidinone),
5.97 (s, 2H, ArCH2), 8.02e8.04 (m, 2H, Ar), 8.13e8.15 (m, 2H, Ar).
(q, 2H, CH2), 2.38e2.44 (q, 2H, CH2), 3.83 (s, 2H, CH2, thiazolidi-
none), 5.73 (s, 2H, ArCH2), 7.74e7.76 (m, 2H, Ar), 7.86e7.88 (m, 2H,
Ar).
7.1.65. 2-((2-(2-Cyclohexylidenehydrazono)-4-oxothiazolidin-3-yl)
methyl)isoindoline-1,3-dione (13C)
7.1.57. 2-((2-(2-(Hex-5-en-2-ylidene)hydrazono)-4-oxothiazolidin-
3-yl)methyl)isoindoline-1,3-dione (5C)
Yellow powder, mp 184e189 ꢀC, 80% yield; 1H NMR (400 MHz,
White powder, mp 133e135 ꢀC, 80% yield; 1H NMR (400 MHz,
CDCl3):
d 1.52 (bs, 4H, cyclohexane), 1.68e1.69 (m, 2H, cyclo-
CDCl3):
d
1.94 (s, 3H, CH3), 2.33e2.39 (m, 4H, 2 ꢂ CH2), 3.83 (s, 2H,
hexane), 2.29e2.32 (t, 2H, cyclohexane), 2.55e2.58 (t, 2H, cyclo-
hexane), 3.84 (s, 2H, CH2, thiazolidinone), 5.72 (s, 2H, ArCH2),
7.74e7.76 (m, 2H, Ar), 7.85e7.87 (m, 2H, Ar).
CH2, thiazolidinone), 4.96e4.98 (m, 1H, ]CH2), 5.02e5.07 (m, 1H,
]CH2), 5.73 (s, 2H, ArCH2), 5.81e5.87 (m, 1H, CH]), 7.74e7.76 (m,
2H, Ar), 7.86e7.88 (m, 2H, Ar).
7.1.66. 2-((2-(2-(3-Methylcyclohexylidene)hydrazono)-4-
oxothiazolidin-3-yl)methyl)isoindoline-1,3-dione (14C)
Yellow powder, mp 183e184 ꢀC, 86% yield; 1H NMR (400 MHz,
7.1.58. 2-((2-(2-(4-Methylpentan-2-ylidene)hydrazono)-4-
oxothiazolidin-3-yl)methyl)isoindoline-1,3-dione (6C)
Yellow powder, mp 66e67 ꢀC, 75% yield; 1H NMR (400 MHz,
CDCl3):
d
1.10e1.11 (d, J ¼ 6.4 Hz, 3H, CH3), 1.26e1.32 (m, 2H,
DMSO-d6):
d
0.91e0.92 (d, J ¼ 6.8 Hz, 6H, 2 ꢂ CH3), 1.91 (s, 3H, CH3),
cyclohexane), 1.49e1.54 (m, 1H, cyclohexane), 1.71e1.77 (m, 2H,
cyclohexane), 1.88e1.95 (m, 2H, cyclohexane), 2.32e2.37 (m, 1H,
cyclohexane), 3.18e3.22 (m, 1H, cyclohexane), 3.82 (s, 2H, CH2,
thiazolidinone), 5.73 (s, 2H, ArCH2), 7.73e7.75 (m, 2H, Ar),
7.85e7.87 (m, 2H, Ar).
1.94e2.01 (m, 1H, CH), 2.14e2.16 (d, J ¼ 7.2 Hz, 2H, CH2), 3.82 (s, 2H,
CH2, thiazolidinone), 5.73 (s, 2H, ArCH2), 7.74e7.80 (m, 2H, Ar),
7.86e7.88 (m, 2H, Ar).
7.1.59. 2-((2-(2-(Heptan-2-ylidene)hydrazono)-4-oxothiazolidin-
3-yl)methyl)isoindoline-1,3-dione (7C)
7.1.67. 2-((2-(2-(4-Methylcyclohexylidene)hydrazono)-4-
Grey powder, mp 84e85 ꢀC, 85% yield; 1H NMR (400 MHz,
oxothiazolidin-3-yl)methyl)isoindoline-1,3-dione (15C)
DMSO-d6):
d
1.19e1.29 (m, 8H, CH2), 1.92 (s, 3H, CH3), 2.20e2.28 (t,
Orange oil, 67% yield; 1H NMR (400 MHz, CDCl3):
d 0.87e0.88
3H, CH3), 3.78 (s, 2H, CH2, thiazolidinone), 5.53 (s, 2H, ArCH2),
7.88e7.89 (m, 4H, Ar).
(m, 3H, CH3), 1.26e1.30 (t, 1H, cyclohexane), 1.37e1.39 (m, 2H,
cyclohexane), 1.79e1.92 (m, 3H, cyclohexane), 2.16e2.24 (m, 1H,

C. De Monte et al. / European Journal of Medicinal Chemistry 107 (2016) 82e96
95
cyclohexane), 2.45e2.48 (m, 1H, cyclohexane), 3.31e3.35 (m, 1H,
cyclohexane), 3.84 (s, 2H, CH2, thiazolidinone), 5.72 (s, 2H, ArCH2),
7.74e7.76 (m, 2H, Ar), 7.85e7.87 (m, 2H, Ar).
8.03e8.08 (m, 1H, Ar), 8.28e8.29 (m, 1H, Ar), 8.57e8.58 (m, 1H, Ar),
8.81 (s, 1H, CH]).
7.1.76. 2-((2-(2-(1-(Pyridin-2-yl)ethylidene)hydrazono)-4-
oxothiazolidin-3-yl)methyl)isoindoline-1,3-dione (24C)
White powder, mp 181e184 ꢀC, 94% yield; 1H NMR (400 MHz,
7.1.68. 2-((2-(2-Cycloheptylidenehydrazono)-4-oxothiazolidin-3-
yl)methyl)isoindoline-1,3-dione (16C)
Yellow powder, mp 118e119 ꢀC, 81% yield; 1H NMR (400 MHz,
CDCl3): d 2.45 (s, 3H, CH3), 3.90 (s, 2H, CH2, thiazolidinone), 5.82 (s,
CDCl3):
d 1.38e1.54 (m, 8H, cycloheptane), 1.65 (bs, 1H, cyclo-
2H, ArCH2), 7.70e7.75 (m, 3H, Ar), 7.89e7.90 (m, 2H, Ar), 8.12e8.17
(m, 2H, Ar), 8.61e8.62 (m, 1H, Ar).
heptane), 2.46e2.53 (m, 3H, cycloheptane), 3.83 (s, 2H, CH2, thia-
zolidinone), 5.72 (s, 2H, ArCH2), 7.74e7.76 (m, 2H, Ar), 7.86e7.88
(m, 2H, Ar).
7.1.77. 2-((2-(2-((Benzo[d][1,3]dioxol-6-yl)methylene)hydrazono)-
4-oxothiazolidin-3-yl)methyl)isoindoline-1,3-dione (25C)
Yellow powder, mp 125e130 ꢀC, 85% yield; 1H NMR (400 MHz,
7.1.69. 2-((2-(2-Cyclooctylidenehydrazono)-4-oxothiazolidin-3-yl)
methyl)isoindoline-1,3-dione (17C)
CDCl3):
d 3.88 (s, 2H, CH2, thiazolidinone), 5.75 (s, 2H, ArCH2), 5.99
Red oil, 65% yield; 1H NMR (400 MHz, CDCl3):
d 1.33e1.49 (m,
(s, CH2, benzodioxole), 6.81e6.83 (m, 1H, Ar), 7.10e7.13 (m, 1H, Ar),
7.37e7.38 (m, 1H, Ar), 7.74e7.77 (m, 2H, Ar), 7.89e7.91 (m, 2H, Ar),
8.22e8.23 (m, 1H, CH]).
8H, cyclooctane), 2.37e2.42 (m, 3H, cyclooctane), 2.44e2.53 (m,
3H, cyclooctane), 3.84 (s, 2H, CH2, thiazolidinone), 5.73 (s, 2H,
ArCH2), 7.74e7.76 (m, 2H, Ar), 7.86e7.88 (m, 2H, Ar).
7.1.78. 2-((2-(2-((Naphthalen-1-yl)methylene)hydrazono)-4-
oxothiazolidin-3-yl)methyl)isoindoline-1,3-dione (26C)
7.1.70. 2-((2-(2-(1-Cyclohexylethylidene)hydrazono)-4-
oxothiazolidin-3-yl)methyl)isoindoline-1,3-dione (18C)
Yellow powder, mp 153e154 ꢀC, 80% yield; 1H NMR (400 MHz,
Yellow powder, mp 146e147 ꢀC, 90% yield; 1H NMR (400 MHz,
CDCl3):
d 3.94 (s, 2H, CH2, thiazolidinone), 5.82 (s, 2H, ArCH2),
CDCl3):
d 1.23e1.40 (m, 3H cyclohexane), 1.59e1.67 (m, 1H, cyclo-
7.51e7.57 (m, 2H, Ar), 7.72e7.82 (m, 3H, Ar þ CH]), 7.85e7.98 (m,
hexane), 1.70e1.87 (m, 4H, cyclohexane), 1.91 (s, 3H, CH3),
2.16e2.21 (m, 1H, cyclohexane), 2.44e2.52 (m, 2H, cyclohexane),
4.13 (s, 2H, CH2, thiazolidinone), 5.72 (s, 2H, ArCH2), 7.75e7.77 (m,
2H, Ar), 7.85e7.87 (m, 2H, Ar).
5H, Ar), 8.85e8.96 (m, 2H, Ar).
7.2. Antibacterial activity
Microtiter plates containing serial dilutions of each compound
7.1.71. 2-((2-(2-((Furan-2-yl)methylene)hydrazono)-4-
oxothiazolidin-3-yl)methyl)isoindoline-1,3-dione (19C)
Yellow powder, mp 154e155 ꢀC, 91% yield; 1H NMR (400 MHz,
ranging from 256 to 0.5
m
g/mL were inoculated with each organism
L final volume;
to yield the appropriate density (105/mL) in a 100
m
each plate included positive controls (bacteria without the com-
pound), and a negative control (medium only). The plates were
incubated for 18e22 h at 35 ꢀC. The minimum 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.
CDCl3):
d 3.90 (s, 2H, CH2, thiazolidinone), 5.75 (s, 2H, ArCH2), 6.50
(bs, 1H, furan), 6.82e6.83 (m, 1H, furan), 7.56 (bs, 1H, furan),
7.75e7.77 (m, 2H, Ar), 7.88e7.90 (m, 2H, Ar), 8.16e8.17 (m, 1H,
CH]).
7.1.72. 2-((2-(2-(1-(Furan-2-yl)ethylidene)hydrazono)-4-
oxothiazolidin-3-yl)methyl)isoindoline-1,3-dione (20C)
Yellow powder, mp 208e211 ꢀC, 82% yield; 1H NMR (400 MHz,
7.3. Antifungal activity
DMSO-d6): d 2.18 (s, 3H, CH3), 3.73 (s, 2H, CH2, thiazolidinone), 5.58
(s, 2H, ArCH2), 6.58e6.60 (m, 1H, furan), 6.96e6.98 (m, 1H, furan),
7.81 (bs, 1H, furan), 7.83e7.90 (m, 4H, Ar).
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 agents that completely inhibited the growth of the or-
ganism, as detected by unaided eye.
7.1.73. 2-((2-(2-(1-(Thiophen-2-yl)ethylidene)hydrazono)-4-
oxothiazolidin-3-yl)methyl)isoindoline-1,3-dione (21C)
Orange powder, mp 165e170 ꢀC, 84% yield; 1H NMR (400 MHz,
CDCl3): d 2.37 (s, 3H, CH3), 3.87 (s, 2H, CH2, thiazolidinone), 5.78 (s,
2H, ArCH2), 7.02e7.04 (m, 1H, thiophene), 7.34e7.37 (m, 2H, thio-
phene), 7.74e7.76 (m, 2H, Ar), 7.87e7.89 (m, 2H, Ar).
7.4. Cytotoxicity assay
7.1.74. 2-((2-(2-(1-Phenylethylidene)hydrazono)-4-oxothiazolidin-
3-yl)methyl)isoindoline-1,3-dione (22C)
The Hep2 cell line, delivered from a human epidermoid carci-
noma 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-Sapienza University of Rome). Cells were
cultured in Dulbecco's modified eagle media (DMEM), supple-
mented with 10% fetal bovine serum (FBS; Gibco, USA) and 1%
streptomycinepenicillin. Cell lines were maintained as adherent
type cultures under humidified 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 solu-
tions of the investigated compounds were prepared in sterile DMSO
and successive dilutions were made in culture medium; the per-
centage of DMSO present in culture medium never exceeded 0.5%.
White powder, mp 185e186 ꢀC, 89% yield; 1H NMR (400 MHz,
DMSO-d6):
(s, 2H, ArCH2), 7.39 (bs, 3H, Ar), 7.75e7.77 (m, 2H, Ar), 7.82e7.89 (m,
4H, Ar). 13C NMR (100 MHz, CDCl3):
15.1, 32.3, 45.2, 123.6, 126.7,
d 2.39 (s, 3H, CH3), 3.89 (s, 2H, CH2, thiazolidinone), 5.80
d
128.3, 130.0, 131.7, 134.4, 137.8, 163.9, 166.8, 171.1.
7.1.75. 2-((2-(2-((Pyridin-3-yl)methylene)hydrazono)-4-
oxothiazolidin-3-yl)methyl)isoindoline-1,3-dione (23C)
Yellow powder, mp 189e190 ꢀC, 82% yield; 1H NMR (400 MHz,
CDCl3):
d 3.87 (s, 2H, CH2, thiazolidinone), 5.71 (s, 2H, ArCH2),
7.27e7.30 (m, 1H, Ar), 7.69e7.72 (m, 2H, Ar), 7.82e7.84 (m, 2H, Ar),

96
C. De Monte et al. / European Journal of Medicinal Chemistry 107 (2016) 82e96
Hep2 cells in the exponential phase of growth (1 ꢂ 105/mL) were
seeded into 24-well microplate and incubated for 24 h with five
Appendix A. Supplementary data
different concentrations of the compounds (0.05e100
m
g/mL).
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.10.048.
Some plates containing cells alone or cells and DMSO represented
the negative controls, whereas cells incubated with 1 mM natrium
nitroprusside represented the positive one. After incubation time,
cells were mechanically scraped off from the plates, resuspended in
fresh medium and incubated for 30 min with gentle shaking at
37 ꢀC in atmosphere of 5% CO2 to recover from the eventual stress.
An aliquot was then diluted (1:1) with a solution 0.4% Trypan blue
stain. After few minutes at room temperature, cells were counted
under an optical microscope in a Thoma hemocytometer chamber
by two different operators. On the basis that Trypan blue is a vital
dye [28] and can enter and interact with the cells unless the plas-
matic membrane is damaged, blue stained cells were considered as
having died. Values are expressed as % of viable cells. Cell viability
in control samples was always 97e98%.
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Conflict of interest
[25] C. De Monte, B. Bizzarri, M.C. Gidaro, S. Carradori, A. Mollica, G. Luisi,
A. Granese, S. Alcaro, G. Costa, N. Basilico, S. Parapini, M.M. Scaltrito, C. Masia,
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The authors state that they have no conflict of interest and have
received no payment in preparation of this manuscript.
Acknowledgements
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This work was supported by grants from Sapienza University of
Rome to Prof. Bruna Bizzarri (Progetto d'Ateneo 2015) and Filas
(research project nꢀ ASR2, Regione Lazio, Italy).
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