Novel 1,3-thiazolidin-4-one derivatives as promising anti- Candida agents endowed with anti-oxidant and chelating properties

Article abstract of DOI:10.3109/14756366.2016.1156103

Pursuing our recent outcomes regarding the antifungal activity of N-substituted 1,3-thiazolidin-4-ones, we synthesized thirty-six new derivatives introducing aliphatic, cycloaliphatic and heteroaromatic moieties at N1-hydrazine connected with C2 position of the thiazolidinone nucleus and functionalizing the lactam nitrogen with differently substituted (NO₂, NH₂, Cl and F) benzyl groups. These compounds were tested to evaluate their minimum inhibitory concentration (MIC) against several clinical Candida spp. with respect to topical and systemic reference drugs (clotrimazole, fluconazole, ketoconazole, miconazole, tioconazole, amphotericin B). Moreover, anti-oxidant properties were also evaluated by using different protocols including free radical scavenging (DPPH and ABTS), reducing power (CUPRAC and FRAP), metal chelating and phosphomolybdenum assays. Moreover, for the most active derivatives we assessed the toxicity (CC₅₀) against Hep2 human cells in order to characterize them as multi-target agents for fungal infections.

Full text of DOI:10.3109/14756366.2016.1156103

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Identification of new anti-Candida compounds by
ligand-based pharmacophore virtual screening
Maria Concetta Gidaro, Stefano Alcaro, Daniela Secci, Daniela Rivanera,
Adriano Mollica, Mariangela Agamennone, Letizia Giampietro & Simone
Carradori
To cite this article: Maria Concetta Gidaro, Stefano Alcaro, Daniela Secci, Daniela
Rivanera, Adriano Mollica, Mariangela Agamennone, Letizia Giampietro & Simone
Carradori (2016): Identification of new anti-Candida compounds by ligand-based
pharmacophore virtual screening, Journal of Enzyme Inhibition and Medicinal Chemistry, DOI:
10.3109/14756366.2016.1156103
To link to this article: http://dx.doi.org/10.3109/14756366.2016.1156103
View supplementary material
Published online: 17 Mar 2016.
Submit your article to this journal
Article views: 6
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=ienz20
Download by: [University of California Santa Barbara]
Date: 19 March 2016, At: 06:20

http://informahealthcare.com/enz
ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–4
2016 Taylor & Francis. DOI: 10.3109/14756366.2016.1156103
!
SHORT COMMUNICATION
Identification of new anti-Candida compounds by ligand-based
pharmacophore virtual screening
Maria Concetta Gidaro1, Stefano Alcaro1, Daniela Secci2, Daniela Rivanera3, Adriano Mollica4,
Mariangela Agamennone4, Letizia Giampietro4, and Simone Carradori4
1
`
Dipartimento Di Scienze Della Salute, Universita ‘‘Magna Graecia’’ Di Catanzaro, Campus Universitario ‘‘S. Venuta’’, Catanzaro, Italy,
2 3
Dipartimento Di Chimica E Tecnologie Del Farmaco, Sapienza Universita Di Roma, Rome, Italy, Dipartimento Di Sanita Pubblica E Malattie
`
`
4
Infettive, Sapienza University of Rome, Rome, Italy, and Department of Pharmacy, ‘‘G. D’Annunzio’’ University of Chieti-Pescara, Chieti, Italy
Abstract
Keywords
Candida albicans represents the most prevalent microbial population in mucosal and systemic
infections, usually confined to severely immunocompromised people. Considering the increase
of resistant strains and the demand for new antifungal drugs endowed with innovative
mechanism of action, we performed a ligand-based virtual screening in order to identify new
anti-Candida compounds. Starting from a large library of natural/semisynthetic products and
several published synthesized compounds, three coumarin derivatives were discovered in silico
as new hit compounds and submitted to the in vitro assay in order to confirm their predicted
biological activity.
Candida, coumarin, ligand-based approach,
(thiazol-2-yl)hydrazines, virtual screening
History
Received 28 December 2015
Revised 10 February 2016
Accepted 10 February 2016
Published online 11 March 2016
Introduction
reported in other works as potent anti-Candida spp. compounds
endowed with low cytotoxicity and synergy with clotrimazole2,10–
12, without a determinate molecular target, to generate a ligand-
based pharmacophore model with the purpose to virtually screen a
library of natural/semisynthetic products and to identify new
potential antifungal drugs. Some 2-thiazolylhydrazine scaffold
derivatives were also already13 studied for their selective human
monoamine oxidase inhibitory effects by computational methods.
The frequency of Candida infections has increased in recent years
and Sanguinetti et al.1 recently reviewed the mechanisms of
resistance. Clinically, azoles are first-line agents in treating fungal
infections; however, there is an emergent demand for the
discovery of new antifungal agents to open the possibility of a
novel therapeutic approach2. As regards natural products, in
literature there are several studies investigating the promising
antimicrobial activity of chemical compounds content in essential
oils and medicinal plants, also traditionally used for the treatment
Ligand-based approach
of various health disorders3–5
.
The 109 structurally similar molecules were ranked according to
Both structure and ligand-based virtual screening (SBVS and their MIC values, expressed in mg/mL against several clinical
LBVS) represent alternative and innovative approaches for iden- isolates of Candida albicans, as follows: compounds with MIC
tifying new compounds, but, until now, in the field of anti-Candida, values ꢀ0.25 mg/mL were classified as ‘‘active’’, while com-
computational techniques were used only to explain the structure– pounds with MIC equal to the value of 256 mg/mL were
activity relationships, to design synthetic derivatives for the lead considered as ‘‘inactive’’. Ligand-based pharmacophore modeling
optimization and to display hypothetic mechanisms of action6–8
.
usually requires the ‘‘Training-Set’’ molecules, generally the most
LBVS often relies on 3D pharmacophore model that is a set of active, that are used for the pharmacophore creation and the
interactions, or chemical features, aligned in three-dimensional ‘‘Test-Set’’ ligands, active and inactive, used to verify the
space and developed by two possible approaches: one based on the resulting pharmacophore hypothesis. Due to the fact that the anti-
known X-ray or NMR structure of the receptor, and the other one Candida activity of most of the compounds (without asterisk in
starting from a set of ligands that are supposed to bind to the same Table S1) was evaluated using a mixture of E and Z isomers and
unknown target9. Therefore, we exploited our synthetic library of the biological activity is usually due to one of the isomers, the
(4-aryl-thiazol-2-yl)hydrazine derivatives (Table S1), previously molecules 14, 33, 36 and 61 (Figure 1) (with asterisk in Table S1
because without isomers) were used as ‘‘Training Set’’ for the
pharmacophore creation, into the Ligand Scout’s Ligand-Based
Modeling Perspective version 3.19. In order to verify the resulting
Address for correspondence: Dr Maria Concetta Gidaro, Dipartimento di
`
Scienze della Salute, Universita ‘‘Magna Græcia’’ di Catanzaro, Campus
pharmacophore hypothesis, compounds 1–3, 12–15, 33–41, 44
and 59–64 were selected as ‘‘Test-Set’’.
All 2D molecules were built into the graphic interface Maestro
¨
of the Scrodinger Suite 2014 , and saved as .mol2 files, then
converted in the .lbd format by the ‘‘Idbgen’’ tool of LigandScout.
‘‘S. Venuta’’, Viale Europa, 88100 Catanzaro, Italy. Tel: +39 0961
3694197. E-mail: mgidaro@unicz.it
Dr Simone Carradori, Department of Pharmacy, ‘‘G. D’Annunzio’’
University of Chieti-Pescara, Via dei Vestini 31, 66100 Chieti, Italy. Tel:
+39 0871 3554583. E-mail: simone.carradori@unich.it
14

2
M. C. Gidaro et al.
J Enzyme Inhib Med Chem, Early Online: 1–4
Figure 1. 2D chemical structures of the
‘‘active’’ compounds 14, 33, 36 and 61,
with MIC values of 0.25 mg/mL, used as
‘‘Training-Set’’ into the LigandScout’s
Ligand-Based Modeling Perspective, in order
to generate the pharmacophore model for the
LBVS.
Ligand conformations were generated using the default settings of
OMEGA best, which produced a maximum of 500 conformations
per molecule. Finally, LigandScout interpolated common pharma-
cophore features for a configurable number of best alignment
solutions. We observed that shared and merged features were
located in the same spatial positions, in particular, 10 pharma-
cophore hypotheses were found with the feature tolerance scale of
1.0 and ranked using the pharmacophore fit and atom overlap as
scoring functions. The chemical functionalities of the selected
model are all matched by the chemical groups of the ‘‘Training-
Set’’ molecules, except the aliphatic portion (Figure 2): the
thiazole nucleus is located inside an aromatic ring and a
hydrophobic feature (AR-HF1), moreover, it fits a region of
hydrogen bond acceptor (HBA1) by the nitrogen atom; the phenyl
portion at C4 of the thiazole is located inside the second aromatic
ring and another hydrophobic feature (AR-HF2), while the
substituents at the para position of this ring map an additional
HF; finally, the hydrazone linker defines a region of hydrogen
bond donor (HBD) and the second site of HBA2. The best model
(Figure 2) was validated to prospectively evaluate its performance
in a VS approach. To this aim, E and Z isomers of all molecules in
Table S1, except those with the asterisk, were included for the
construction of two libraries (‘‘active’’ and ‘‘inactive’’ com-
pounds). Therefore, 42 ‘‘active’’ compounds were used to
generate 2100 decoys (1:50), downloaded from the DUD-E
site15 and then added to the 21 ‘‘inactive’’ for a total of 2121
compounds. The screening process of the built libraries and the
receiver-operating curve (ROC) analysis showed a good discrim-
ination of the pharmacophore model; in fact, 34 hits (32 ‘‘active’’
and 2 ‘‘inactive’’) were found with a sensitivity of about 78% and
the AUC value of 88.0% (Figure 3).
Figure 2. The ‘‘Training-Set’’ compounds, rendered as stick with the
carbon color indicated in brackets, 14 (blue), 33 (cyan), 36 (red) and 61
(grey) in the best alignment solution, mapped to the different chemical
features of the final pharmacophore hypothesis. The feature dictionary
used is the following: aromatic ring and hydrophobic features (AR-HF1
and AR-HF2) displayed as grey toroids into the yellow spheres, hydrogen
bond acceptors (HBA1 and HBA2) defined as red spheres, hydrophobic
feature (HF) as yellow sphere and hydrogen bond donor (HBD) as green
arrow. All non-carbon atoms are colored according to atom types. (For
the colored version of this Figure, please refer to the online article.).
The LBVS was performed into the LigandScout’s Screening
Perspective using the validated pharmacophore model displayed
in Figure 2. A library of 152 056 natural compounds was
downloaded from the Zinc Database site16, using all entries of the
Natural Products Catalogs, while 105 490 natural/semi-synthetic
compounds were available from our in house database. Therefore
257 546 ligands were prepared as follows: (i) the software
Canvas17 was used to remove the duplicates and to select 237 950
compounds with drug-like properties; (ii) the 3D structures were
¨
generated by the ‘‘LigPrep’’ utility of the Scrodinger package
201418; (iii) tautomeric forms and protonated states at the pH
range 5–9 were also considered, ending up with a library of
238 377 molecules, further converted in the .ldb format using the
same protocol previously reported for the ‘‘active’’ and
‘‘inactive’’ libraries. The LBVS permitted to early identify three
virtual hit compounds, belonging to the coumarin scaffold, with
the corresponding ZINC codes 72325331, 72325699 and
72325788 (Figure 4). The potential anti-Candida compounds,
only results of the LBVS study, displayed the same alignment,
except for the different aliphatic portion, with appropriate
chemical functions for a good fit into the six features of the
pharmacophore hypothesis. In fact, for each of them, the
coumarin portion fulfils the AR-HF2 and the HF features; the
Figure 3. Into the LigandScout, the automated generation of the ROC
curve for performance assessments, demonstrated the good discrimination
of the selected pharmacophore model. In fact, 34 hits (32 ‘‘active’’ and 2
‘‘inactive’’) were found, starting from a library of 2163 compounds, with
a sensitivity of about 78% and the AUC value of 88.0%.
pyridine ring maps the AR-HF1 and, in addition, the HBA1 region
by the nitrogen atom; finally, the amide group is located within
the HBA2 feature, by the carbonylic portion, and within the HBD
by the protonated nitrogen atom (Figure 4). In order to verify a
possible therapeutic use of these bioactive molecules, the

DOI: 10.3109/14756366.2016.1156103
Identification of new anti-Candida compounds
3
Figure 4. 2D chemical structures of the coumarin derivatives, with the respective ZINC codes, identified as new potential C. albicans inhibitors, by the
LBVS performed from a large library of natural/semisynthetic compounds. The 3 new hits, on the right of the figure, are rendered as stick with
the carbon color indicated in brackets, ZINC 72325331 (green), 72325699 (violet), 72325788 (grey). They are in the best alignment solution, mapped to
the different chemical features of the pharmacophore hypothesis described in the previous figure. All non-carbon atoms are colored according to atom
types. (For the colored version of this Figure, please refer to the online article.).
chemico-physical properties prediction was carried out by Canvas
and reported in Table S2, concluding that all compounds have a
Table 1. Minimal inhibitory concentration (MIC) of
the coumarin derivatives, displayed as ZINC codes,
and reference compound (fluconazole) against six
clinical strains of Candida albicans.
drug-like profile19–21
.
Antifungal activity
Compound
C. albicans (MIC mg/mL)
The antifungal activity of the three coumarins, purchased by
MolPort with purity of 92–95%, was evaluated against six clinical
fungal isolates of Candida albicans and compared with topical
and systemic reference drug fluconazole (Table 1) by using the
same experimental protocols carried out for our synthetic library
of (4-aryl-thiazol-2-yl)hydrazine derivatives. The used clinical
isolates were recently collected from specimens of patients at the
‘‘Azienda Policlinico Umberto I’’ (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 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 recom-
mended by the NCCLS. Briefly, 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 control (medium only). The plates
were incubated for 24 h at 37 ꢁC. The MIC for all isolates was
defined as the lowest concentration of antifungal agents that
completely inhibited the growth of the organism, as detected by
unaided eye22,23. From these data it was possible to extrapolate
that these new compounds are active antifungal agents endowed
with the same anti-Candida efficacy of Fluconazole in vitro.
72325331
72325699
72325788
Fluconazole
2-8
2-4
2-4
2-4
experimental efforts on, starting from a large database of natural
and semisynthetic ligands. In particular, by a comparison between
the ‘‘Training Set’’ molecules, used to generate the ligand-based
pharmacophore model, and the three coumarin hit compounds
identified by our LBVS study, we observed the importance of a
shared heterocycle with a HBA (e.g. thiazole and pyridine) as
central feature linked to HBD/acceptor sites (e.g. hydrazone and
carbonylic moiety) and to aromatic/hydrophobic features (e.g.
phenyl and coumarin portions). In conclusion, our study repre-
sents the first example of a successful application of a LBVS
approach to identify promising anti-Candida compounds. The
ligand-based pharmacophore hypothesis generated in silico, and
validated by the experimental results, may be used for future
virtual screening studies starting from in house libraries of
natural, semisynthetic and synthetic compounds. Further investi-
gations will also focus on the identification of the macromolecular
target and more potent anti-Candida agents.
Declaration of interest
The authors report no declaration of interest. This work was
supported by Interregional Research Center for Food Safety and
Health, the Magna Græcia University of Catanzaro (MIUR PON
a3_00359) and local grants from University ‘‘G. d’Annunzio’’ of
Chieti (Dr. S. Carradori 2015).
Results and discussion
References
The selected coumarin scaffold displayed good biological activity
confirming that the aliphatic chain (methyl, isopropyl and
isobutyl) at the lateral amidic portion was not crucial for the in
vitro anti-Candida efficacy. Actually, in literature, several works
¨
1. Sanguinetti M, Posteraro B, Lass-Florl C. Antifungal drug resistance
among Candida species: mechanisms and clinical impact. Mycoses
2015;58:2–13.
2. Carradori S, Secci D, Bolasco A, et al. Synthesis and cytotoxicity of
novel (thiazol-2-yl)hydrazine derivatives as promising anti-Candida
agents. Eur J Med Chem 2013;65:102–11.
3. (a) Vandal J, Abou-Zaid MM, Ferroni G, Leduc LG. Antimicrobial
activity of natural products from the flora of Northern Ontario,
Canada. Pharm Biol 2015;53:800–6. (b) De Monte C, Bizzarri B,
Gidaro MC, et al. Bioactive compounds of Crocus sativus L. and
their semi-synthetic derivatives as promising anti-Helicobacter
demonstrate the anti-fungal activity of coumarin derivatives24–29
.
In particular, for the methyl and isopropyl ZINC compounds, we
found a reference that previously reported the antimicrobial
activity30, confirming the good ability of the pharmacophore
hypothesis to early retrieve anti-Candida compounds. In fact, our
computational studies permitted to identify in silico, with reduced
time and costs, the most promising candidates to focus the

4
M. C. Gidaro et al.
J Enzyme Inhib Med Chem, Early Online: 1–4
¨
pylori, anti-malarial and anti-leishmanial agents. J Enzyme Inhib 14. Maestro, version 9.7. New York, NY: Schrodinger, LLC; 2014.
Med Chem 2015;30:1027–33.
15. Mysinger MM, Carchia M, Irwin JJ, Shoichet BK. Directory of
useful decoys, enhanced (DUD-E): better ligands and decoys for
better benchmarking. J Med Chem 2012;55:6582–94.
16. Irwin JJ, Shoichet BK. ZINC – a free database of commercially
available compounds for virtual screening. J Chem Inf Model 2005;
45:177–82.
4. Cavaleiro C, Salgueiro L, Gonc¸alves MJ, et al. Antifungal activity of
the essential oil of Angelica major against Candida, Cryptococcus,
Aspergillus and dermatophyte species. J Nat Med 2015;69:241–8.
5. Cannas S, Molicotti P, Usai D, et al. Antifungal, anti-biofilm and
adhesion activity of the essential oil of Myrtus communis L. against
Candida species. Nat Prod Res 2014;28:2173–7.
¨
17. Canvas, version 2.2. New York, NY: Schrodinger, LLC; 2014.
¨
6. Rani N, Kumar P, Singh R, Sharma A. Molecular docking evaluation 18. LigPrep, version 2.9. New York, NY: Schrodinger, LLC; 2014.
of imidazole analogues as potent Candida albicans 14a-demethylase 19. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. Experimental
inhibitors. Curr Comput Aided Drug Des 2015;11:8–20.
and computational approaches to estimate solubility and permeabil-
ity in drug discovery and development settings. Adv Drug Deliv Rev
2001;46:3–26.
7. Jiang Z, Gu J, Wang C, et al. Design, synthesis and antifungal
activity of novel triazole derivatives containing substituted 1,2,3-
triazole-piperdine side chains. Eur J Med Chem 2014;82:490–7.
8. Xiao ZP, Wang XD, Wang PF, et al. Design, synthesis, and
evaluation of novel fluoroquinolone-flavonoid hybrids as potent
20. Veber D, Johnson S, Cheng H, et al. Molecular properties that
influence the oral bioavailability of drug candidates. J Med Chem
2002;45:2615–23.
antibiotics against drug-resistant microorganisms. Eur J Med Chem 21. Walters W, Murcko M. Prediction of ‘drug-likeness’. Adv Drug
2014;80:92–100. Deliv Rev 2002;54:255–71.
9. Wolber G, Langer T. LigandScout: 3-D pharmacophores derived 22. National Committee for Clinical Laboratory Standard. Reference
from protein-bound ligands and their use as virtual screening filters.
J Chem Inf Model 2005;45:160–9.
10. Secci D, Bizzarri B, Bolasco A, et al. Synthesis, anti-Candida
method for broth dilution antifungal susceptibility testing of yeasts;
approved standard. 3rd ed. DM27-A3 vol. 28, nꢁ 14. Wayne, PA:
National Committee for Clinical Laboratory Standard; 2008.
activity, and cytotoxicity of new (4-(4-iodophenyl)thiazol-2- 23. De Monte C, Carradori S, Bizzarri B, et al. Anti-Candida activity
yl)hydrazine derivatives. Eur J Med Chem 2012;53:246–53.
11. Chimenti F, Bizzarri B, Bolasco A, et al. Synthesis and biological
and cytotoxicity of a large library of new N-substituted-1,3-
thiazolidin-4-one derivatives. Eur J Med Chem 2016;107:82–96.
evaluation of novel 2,4-disubstituted-1,3-thiazoles as anti-Candida 24. Shi Y, Zhou CH. Synthesis and evaluation of a class of new
spp. agents. Eur J Med Chem 2011;46:378–82.
coumarin triazole derivatives as potential antimicrobial agents.
Bioorg Med Chem Lett 2011;21:956–60.
activity of 2-thiazolylhydrazone derivatives compared with the 25. Singh LK, Priyanka SV, Katiyar D. Design, synthesis and biological
12. Chimenti F, Bizzarri B, Maccioni E, et al. Synthesis and in vitro
activity of clotrimazole against clinical isolates of Candida spp. evaluation of some new coumarin derivatives as potential anti-
Bioorg Med Chem Lett 2007;17:4635–40. microbial agents. Med Chem 2015;11:128–34.
13. (a) Chimenti F, Bolasco A, Secci D, et al. Investigations on the 26. Damu GL, Cui SF, Peng XM, et al. Synthesis and bioactive
2-thiazolylhydrazyne scaffold: synthesis and molecular modeling of evaluation of a novel series of coumarinazoles. Bioorg Med Chem
selective human monoamine oxidase inhibitors. Bioorg Med Chem Lett 2014;24:3605–8.
Lett 2010;18:5715–23. (b) D’Ascenzio M, Carradori S, Secci D, 27. Arshad A, Osman H, Bagley MC, et al. Synthesis and antimicrobial
et al. Identification of the stereochemical requirements in the 4-aryl-
2-cycloalkylidenhydrazinylthiazole scaffold for the design of select-
ive human monoamine oxidase B inhibitors. Bioorg Med Chem 28. Mladenovic M, Vukovic N, Sukdolak S, Solujic S. Design of novel
properties of some new thiazolyl coumarin derivatives. Eur J Med
Chem 2011;46:3788–94.
´
´
´
2014;22:2887–95. (c) Chimenti P, Petzer A, Carradori S, et al. 4-hydroxy-chromene-2-one derivatives as antimicrobial agents.
Exploring 4-substituted-2-thiazolylhydrazones from 2-, 3-, and 4- Molecules 2010;15:4294–308.
acetylpyridine as selective and reversible hMAO-B inhibitors. Eur J 29. Chimenti F, Carradori S, Secci D, et al. Synthesis and biological
Med Chem 2013;66:221–7. (d) Chimenti F, Secci D, Bolasco A, evaluation of novel conjugated coumarin–thiazole systems.
et al. Synthesis, stereochemical separation, and biological evaluation J Heterocl Chem 2009;46:575–8.
of selective inhibitors of human MAO-B: 1-(4-arylthiazol-2-yl)-2- 30. Chai X, Yu S, Wang X, et al. Synthesis and antifungal activity of
(3-methylcyclohexylidene)hydrazines.
6516–20.
J
Med Chem 2010;53:
novel 7-O-substituted pyridyl-4-methyl coumarin derivatives. Med
Chem Res 2013;22:4654–62.
Supplementary material available online