Design and synthesis of new drugs inhibitors of Candida albicans hyphae and biofilm formation by upregulating the expression of TUP1 transcription repressor gene

Article abstract of DOI:10.1016/j.ejps.2020.105327

Candida albicans is a common human fungal pathogen that causes disease ranging from superficial to lethal infections. C. albicans grows as budding yeast which can transform into hyphae in response to various environmental or biological stimuli. Although b

Full text of DOI:10.1016/j.ejps.2020.105327

Journal Pre-proof
Design and Synthesis of New Drugs Inhibitors of Candida albicans
Hyphae and Biofilm Formation by Upregulating the Expression of
TUP1 Transcription Repressor Gene
Rania Hamdy , Sameh S.M. Soliman , Abrar I. Alsaadi ,
Bahgat Fayed , Alshaima Hamoda , Samia A. Elseginy ,
Mohamed I. Husseiny , Ashraf S. Ibrahim
PII:
S0928-0987(20)30116-0
DOI:
Reference:
https://doi.org/10.1016/j.ejps.2020.105327
PHASCI 105327
To appear in:
European Journal of Pharmaceutical Sciences
Received date:
Revised date:
Accepted date:
3 January 2020
21 March 2020
27 March 2020
Please cite this article as: Rania Hamdy , Sameh S.M. Soliman , Abrar I. Alsaadi , Bahgat Fayed ,
Alshaima Hamoda , Samia A. Elseginy , Mohamed I. Husseiny , Ashraf S. Ibrahim , Design and
Synthesis of New Drugs Inhibitors of Candida albicans Hyphae and Biofilm Formation by Upregu-
lating the Expression of TUP1 Transcription Repressor Gene, European Journal of Pharmaceutical
Sciences (2020), doi: https://doi.org/10.1016/j.ejps.2020.105327
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier B.V.

Design and Synthesis of New Drugs Inhibitors of Candida albicans Hyphae
and Biofilm Formation by Upregulating the Expression of TUP1
Transcription Repressor Gene
Rania Hamdya,b#, Sameh S. M. Solimana,b # *, Abrar I. Alsaadic, Bahgat Fayedc,d, Alshaima
Hamodaa,e, Samia A. Elseginyf,g, Mohamed I. Husseinyb,h and Ashraf S. Ibrahimc,i *
aSharjah Institute for Medical Research, and College of Pharmacy, University of Sharjah, P.O.
Box 27272, Sharjah, UAE
bFaculty of Pharmacy, Zagazig University, Zagazig 44519, Egypt
cDivision of Infectious Diseases, The Lundquist Institute for Biomedical Innovations, Harbor-
University of California Los Angeles (UCLA) Medical Center, Torrance, CA 90509, USA
dChemistry of Natural and Microbial Product Department, National Research Centre, Cairo,
Egypt
eDepartment of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut, Egypt
fGreen Chemistry Department, Chemical Industries Research Division, National Research
Center P.O. Box 12622, Egypt
gMolecular Modeling Lab., Biochemistry School, Bristol University, Bristol, Uk.
hDepartment of Translational Research & Cellular Therapeutics, Beckman Research Institute of
City of Hope, Duarte, CA 91010-3000, USA
iDavid Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA
#Equal contribution: Rania Hamdy and Sameh S. M. Soliman
*Correspondence: Sameh Soliman
Department of Medicinal Chemistry, College of Pharmacy, University of Sharjah, Sharjah, UAE,
27272, Phone: +971650 574 72, ssoliman@sharjah.ac.ae
or
Ashraf Ibrahim
Division of Infectious Diseases, The Lundquist Institute for Biomedical Innovations, Harbor-
UCLA, Torrance, CA 90509, USA, ibrahim@lundquist.org
1

Abstract
Candida albicans is a common human fungal pathogen that causes disease ranging from
superficial to lethal infections. C. albicans grows as budding yeast which can transform into
hyphae in response to various environmental or biological stimuli. Although both forms have
been associated with virulence, the hyphae form is responsible for the formation of multi-drug
resistance biofilm. Here, new compounds were designed to selectively inhibit C. albicans hyphae
formation without affecting human cells to afford sufficient safety. The newly designed 5-[3-
substitued-4-(4-substituedbenzyloxy)-benzylidene]-2-thioxo-thiazolidin-4-one derivatives,
named SR, showed very specific and effective inhibition activity against C. albicans hyphae
formation. SR compounds caused hyphae inhibition activity at concentrations 10-40 fold lower
than the concentration required to inhibit Candida yeast and bacterial growths. The anti-hyphae
inhibition activities of SR compounds were via activation of the hyphae transcription repressor
gene, TUP1. Correlation studies between the expression of TUP1 gene and the activity of SR
compounds confirmed that the anti-C. albicans activities of SR compounds were via inhibition
of hyphae formation. The newly designed SR compounds showed 10-40% haemolytic activity
on human erythrocytes when compared to 100% haemolysis by 0.1% triton employed as positive
control. Furthermore, theoretical prediction of absorption, distribution, metabolism, excretion,
and toxicity (ADMET) of SR compounds confirmed their safety, efficient metabolism and
possible oral bioavailability. With the minimal toxicity and significant activity of the newly-
designed SR compounds, a future optimization of pharmaceutical formulation may develop a
promising inhibitor of hyphal formation not only for C. albicans but also for other TUP1-
dependent dimorphic fungal infections.
2

Keywords: Candida albicans; TUP1 gene; Hyphae; Biofilm; Rhodanine; Thiazolidine-one
1. Introduction
C. albicans mostly causes superficial easily treated infections (Sydnor and Perl, 2011).
However, it can hematogenously disseminate to become life-threatening particularly with
immunocompromised patients or those receiving broad spectrum antibiotic treatments (Edwards
et al., 1978; Elewski, 1998). In fact, C. albicans is one of the most common causes of healthcare-
associated bloodstream infections (Geffers and Gastmeier, 2011; Wenzel and Gennings, 2005).
C. albicans can switch between two major forms, yeast and hyphal forms (Brand, 2012). The
switch from yeast to hyphae is recognized as one of the most virulent traits of C. albicans
(Brand, 2012), since it is associated with the ability of the fungus to invade host tissues and
form drug-resistance biofilms. Compared to yeast forms, Candida biofilms are 30 to 2000 times
more resistant to various antifungal drugs (Hawser and Douglas, 1995; Seddiki et al., 2015). The
mechanisms of resistance developed by Candida biofilms are very complicated. These
mechanisms can include changes in critical gene expression, changes in the phenotype and
formation of extracellular polymers; that affect the influx of antifungal agents (Douglas, 2003).
Therefore, the majority of antifungal drugs shows limited anti-biofilm activity (Touil et al.,
2020).
Biofilms are architecturally complex structures consisting of basal yeast cells and poly-
layers of hyphae encapsulated in extracellular matrix (Choi et al., 2016). Thus, biofilms are
inherently resistant to most antifungal treatments, provide haven for C. albicans from host
defenses and constitute a reservoir for recurrent infection (Cavalheiro and Teixeira, 2018;
3

LaFleur et al., 2006). Consequently, biofilm structures are often the cause of failed antifungal
treatment, and indeed are associated with greater mortality of patients with candidemia
(Blankenship and Mitchell, 2006; Tumbarello et al., 2007). The contribution of biofilm
formation to C. albicans pathogenicity and drug resistance emphasizes the need for new anti-
hyphae agents that can inhibit Candida biofilm formation and hence prevents recalcitrant
infection.
Quorum sensing (QS) molecules accumulated in the extracellular Candida environment
can affect the morphological characteristics of the pathogen, forcing the formation of hyphae and
thereby favoring biofilm formation and resistance to antimicrobial agents. The best characterized
QS molecule involved in C. albicans biofilm formation is farnesol, which suppresses filament
formation and reduces biofilm size (Deveau and Hogan, 2011). Taken in consideration the
specific behaviour of QS molecules, we have been able to design new compounds with farnesol-
like function, despite of structural differences.
It has been reported that thiazolidinedione derivatives (Figure 1A) exert significant
inhibitory activities against C. albicans hyphal growth (Chauhan et al., 2012). Similarly,
rhodanine derivatives showed inhibitory effects on C. albicans biofilm (Orchard et al., 2004),
bacterial biofilm (AbdelKhalek et al., 2016) and numerous fungal targets including mannosyl
transferase 1 (PMT1) (Feldman et al., 2014). Furthermore, aromatic compounds such as 1,2-
benzisothiazolinone (Alex et al., 2012) and pyridine-rhodanine (Orchard et al., 2004) derivatives
(Figure 1A) showed potent anti-Candida activities. Inspired by all these facts, we designed a
hybrid structure made of rhodanine analogues coupled with a side chain made of two fused
aromatic structures containing lipophilic group with electron withdrawing substitutions in order
to generate high potent anti-Candida activities (Figure 1B).
4

ADMET studies play a critical role to predict the successful discovery and development
of a drug candidate whereas it provids important information that significantly reduces the time
and cost required (Di et al., 2018). ADMET score used to predict the drug likeness of a
compound. It is estimated that almost 50% of the drug candidates failed because of unaccepted
efficacy and inappropriate ADMET properties at the therapeutic dose.(Di et al., 2018).
2. Material and methods
2.1. General Chemistry
Most chemicals and solvents employed in chemical synthesis were of analytical grade
and, when necessary, were purified and dried by standard methods. Reactions were monitored by
thin-layer chromatography (TLC) using pre-coated silica gel plates (kiesel gel 60 F254, BDH),
and spots were visualized under UV light (254nm). Melting points were determined using a
Gallenkamp melting point apparatus and are uncorrected. Column chromatography was
performed with Merck silica gel 60 (40-60 M). 1HNMR and 13CNMR spectra were recorded on
a Bruker spectrometer at 500 MHz. Chemical shifts were expressed in parts per million (ppm)
relative to tetramethylsilane, and coupling constant (J) values were represented in hertz (Hz) and
the signals are designated as follows: s, singlet; d, doublet; t, triplet; m, multiplet. Mass
spectroscopic data were obtained through Electrospray ionization (ESI) mass spectrum. Detailed
synthesis of SR compounds can be found at Supplementary Data file.
In order to define the structural geometry of the compound SR7, the relative stability of
the isomers was optimized by MOE software. Energy minimization and different descriptors
5

were calculated including the heat of formation (HF), potential energy (E), angle bend energy (E-
ang), Dipole moment (DM) and the Total energy.
2.2. Organisms and growth conditions
Collections of bacterial and fungal strains were obtained from the laboratory of Dr.
Ibrahim at Harbor-UCLA medical center, Torrance, California. The bacterial strains used in this
study were the Gram-negative bacterium Escherichia coli and the Gram-Positive bacterium
methicillin-resistant Staphylococcus aureus (MRSA) LAC300. All bacterial strains were cultured
on sterile Luria-Bertani medium (LB). The fungal strain used in this study was C. albicans
SC5314. C. albicans was inoculated into sterile yeast potato dextrose broth (YPD) and incubated
for 24h at 37oC (This was used as an initial culture for all studies described), and incubated in
two different media, Yeast Nitrogen Base (YNB) broth or Roswell Park Memorial Institute
medium (RPMI-1640). Colistin, vancomycin and Ketoconazole were purchased from (Sigma-
Aldrich).
2.3. Determination of the antimicrobial activity
The antimicrobial activity of compounds SR1-7 on agar plates, liquid broth media and
MIC were measured according to modified Clinical and Laboratory Standards Institute (CLSI)
and as described by Soliman et al., 2017 (Soliman et al., 2017a; Soliman et al., 2017b). Briefly,
0.1mL containing 105 CFU/mL was spread on Luria-Bertani (LB) agar plates. The plates were
then incubated at 37°C for 34h with filter discs (8mm diameter) saturated with different dilutions
of the compounds (1- 400μg/mL).The inhibition zones (mm) were measured by determining the
diameter of the clear area. Similarly, the activity in liquid media was measured by incubating the
aforementioned concentrations of the compounds into LB broth media inoculated with 105
6

CFU/mL in 96-well microplates at 37°C for 24h. The turbidity representing the microbial growth
was measured by microplate reader (DYNEX technologies) at OD600. The MIC was the lowest
concentration of the compound that prevented microbial growth (showed no turbidity). At low
MIC values, the concentration was repeated with increasing/ decreasing the concentrations by 1
μg/mL at a time until the MIC was identified. At high MIC values, the concentration was
repeated with increasing/ decreasing the concentrations by 5 μg/mL at a time until the MIC was
identified. Each test was performed in triplicate. Ketoconazole, colistin and vancomycin were
employed as positive controls against C. albicans, E. coli and S. aureus bacteria, respectively.
Cultures without the compounds or antimicrobials were employed as negative control.
2.4. Inhibition of Candida biofilm formation
The MICs for cells forming biofilm were determined by microtiter plate assay as described
previously by Soliman et al., 2017(Soliman et al., 2017a; Soliman et al., 2017b). Briefly, 96-well
polystyrene microtiter plate was seeded with 200μL of RPMI-1640 containing 106 C. albicans
cells in the presence of the compound of interest at concentration range of 1-100μg/mL. After
24h of incubation at 37°C, the biofilms were washed and the fungal viability was analyzed using
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Sigma) and the final
absorbance was measured at 540nm. The assay was performed in triplicate and repeated three
times. The MIC of the compounds causing 100% inhibition of C. albicans biofilm formation was
determined by measuring the metabolic activity of biofilm compared to solvent control.
2.5. Quantitative real time RT-PCR
To quantify the expression of C. albicans hyphae-specific genes (Table 1), Candida cells
were grown in either yeast (YNB) or hyphae/biofilm (RPMI 1640 media) growing conditions as
7

described previously in the presence of compounds SR1-7 at10µg/mL. The cultures were
incubated in 6-well plates for 24h at 37oC. For yeast growing conditions, the cultures were
collected separately, centrifuged and the pellets were washed by PBS prior to RNA extraction,
cDNA synthesis and gene expression analysis. For hyphae growing conditions, the cultures were
centrifuged, and the isolated hyphae are washed with PBS. RNA extraction was performed using
RibopureTM RNA purification kit (ThermoFisher) following the manufacturer’s manual.
Contaminating genomic DNA was removed from RNA samples by treatment with 4μL of Turbo-
DNaseI (Invitrogen) for 30 min at room temperature. DNase was then removed using DNase
inactivation reagent (Invitrogen). First-strand cDNA synthesis was performed using the
Retroscript first-strand synthesis kit (Ambion). The amplification efficiency was determined by
serial dilution experiments, and the resulting efficiency coefficient was used for the
quantification of the products (Pfaffl, 2001). Gene expression was analyzed by an ABI Prism
7000 Sequence Detection System (Applied Biosystems) using the Power SYBR green PCR
master mix (Applied Biosystems). PCR conditions were 10 min at 90°C and 40 cycles of 15s at
95°C and 1min at 60°C. Single PCR products were confirmed with the heat dissociation protocol
at the end of the PCR cycles. The amount of gene expression was normalized to 18S
rRNA(Morschhäuser et al., 2007) and then quantified using the 2(−ΔΔC(T)) method(Livak and
Schmittgen, 2001). All reactions were performed in triplicate, and the mixture included a
negative no-reverse transcription (RT) control in which reverse transcriptase was omitted.
2.6. Correlation between anti-C. albicans activities of the compounds and TUP1 gene
expression
In order to determine how strong the relationship was between TUP1 gene expression and
either the anti-yeast or anti-hyphae activities of the synthesised SR compounds, a formula was
8

generated using Graph Pad 5.0 to produce what is referred to as the coefficient value(Zar,
1984). A Pearson's correlation attempts to draw a line of best fit through the data of two
variables (TUP1 gene expression and anti-C. albicans activities of the compounds), where each
row represented a different compound, the X column represented the TUP1 gene expression and
the Y columns represented the anti-C. albicans activities of the compound against either yeast or
hyphae forms. The Pearson correlation coefficient, r, indicates how far away all these data points
are to this line of best fit. A value of -1 is representing a perfect negative linear relationship,
while +1 for a perfect positive linear relationship.
2.7. Quantitative structure–activity relationship (QSAR) studies
To explore the correlation between the 3D structures of the training set (SR1-7) and their
biological activities, a predictive 3D QSAR model was built. Comparative Molecular Field
Analysis (CoMFA) was used to build the 3D QSAR model using Py-CoMFA 3D QSAR service.
CoMFA calculates steric and electrostatic properties of each molecule on 3D cubic lattice with
grids pacing of 2OA in x, y, and z directions using the Tripos force field (Clark et al., 1989). A
sp3 carbon atom probe with a Vander Waals radius of 1.52OA and a charge of þ1.0 was used to
generate the steric and electrostatic (Coulombic potential) field with distance-dependent
dielectric at each lattice point. The steric and electrostatic energy values were truncated at 30.0
Kcal/mol. The minimum sigma (column filtering) was set to 0.05Kcal/mol. Equal weights for
CoMFA were assigned to steric and electrostatic fields using CoMFA STD scaling option. The
regression analysis was carried out using the partial least squares (PLS) analysis and the cross
validation was carried with leave-one-out (LOO) method(Bush and NachbarJr., 1993) in which
one compound is removed from the data set and its activity is predicted using the model derived
from the rest of the molecules in the dataset.
9

2.8. In silico pharmacokinetics and toxicological properties of SR compounds
The physicochemical and pharmacokinetics properties of the synthesized SR compounds
were examined using theoretical prediction of absorption, distribution, metabolism, excretion,
toxicity (ADMET) qualitative models. The compounds were built in 3D structures using MOE
software and saved as sdf files. The compounds were then submitted to admetSAR software,
available at (https://www. lmmd.ecust. edu.cn/admetsar1/ predict/) (Yang et al., 2018). The
parameters generated by the software evaluated the pharmacokinetics characteristics of the
compounds. The parameters evaluated in the pharmacokinetics study included human intestinal
absorption (HIA), cytochrome binding, biodegradation, acute oral toxicity, carcinogenicity, rat
acute toxicity and aqueous solubility (Ramos et al., 2019).
2.9. Cytotoxicity assay
The cytotoxic assay of SR compounds was measured by quantifying the amount of
hemoglobin released by lysis of human erythrocytes as previously described (Soliman et al.,
2017a; Soliman et al., 2017b). Briefly, fresh erythrocytes were re-suspended to 3 × 107cells/mL
in DPBS followed by incubation with the compounds in round-bottomed 96-well plates in 200
μL final volume. Washing buffer and 0.1% Triton X-100 were used as negative and positive
controls, respectively. The plate was incubated at 37°C for 30 min, and the intact cells were
precipitated by centrifugation at 500g for 10min at 4°C and the hemoglobin released was
measured by absorbance at 405nm. The absorbance values for each sample were subtracted from
the absorbance of the washing buffer-treated cells and the hemolytic activity (%) was calculated.
The experiment was conducted in triplicate.
10

Blood samples were collected after obtaining a signed informed consent from healthy
volunteers under an approved Los Angeles Biomedical Research Institute IRB protocol #11671.
2.9. Statistical analysis
The data was graphed using Graph Pad5.0 for Windows (GraphPad Software, La Jolla,
CA, USA). The statistical significance was analyzed using one-way or two-way analysis of
variance (ANOVA) using either Bonferroni’s multiple comparisons test or Dunn’s Multiple
Comparison Test. P-value <0.05 was considered significant.
3. Results
3.1. Chemistry of the hybrid SR compounds
The synthetic route of 5-[3-substitued-4-(4-substituedbenzyloxy)-benzylidene]-2-thioxo-
thiazolidin-4-one (SR) compounds is described in Scheme 1 (Figure 2). The intermediate 3-
substituted-4-(4-substituedbenzyloxy) benzaldehyde (R1-7) was synthesized by coupling
substituted-benzyl chloride (a-c) with 3-substituted-4-hydroxybenzaldehyde in the presence of
K2CO3 and KI in acetonitrile. The obtained compounds (R1-7) were then reacted with rhodanine
in the presence of β-alanine and glacial acetic acid via Knoevenagel condensation reactions in
order to provides 5-[3-substitued-4-(4-substituedbenzyloxy)-benzylidene]-2-thioxo-thiazolidin-
4-one derivatives, SR1-7 (Figure 2).
To predict the isomerisation (Z or E) of the compound, the molecular structure of Z and E
isomers were compared (Supplementary Figure 1) and the energy minimization parameters
11

were predicted (Supplementary Table 1). The heat of formation of Z-isomer (-164.316) less
than that of E-isomer (-161.005), and the total energy of Z-isomer is less than of E-isomer. The
results revealed that the most stable structure among both isomers was Z-isomer determined by
the total energy together with the heat of formation energy and other parameters identified in
Supplementary Table1. This result was in consistent with previously reported isomerisation of
rhodanine derivative with exocyclic double bond (Bataille et al., 2017; Fan et al., 2010); whereas
Z-isomer was the major thermodynamically stable diastereomers. Furthermore, it has been
reported that the main product obtained from rhodanine reaction with aromatic aldehyde is Z-
isomer (Kaczor et al., 2019).
3.2. Inhibitory activities of SR compounds on Candida hyphae formation
The effect of the newly-synthesized compounds, named SR1-7 (Figure 2), on C.
albicans growing either in yeast- or hyphae-forming conditions were tested separately at
different concentrations including 1, 10, 50, 100 µg/mL. All tested concentrations showed weak
inhibition activities against C. albicans yeast (Figure 3A), while all compounds except
compounds SR6 showed significant inhibition activities (60-100%) against Candida hyphae in
particular at concentrations 50 and 100 µg/mL (Figure 3B). At the lower concentration of 10
µg/mL, all compounds showed inhibition of yeast at 10-20%, whereas compounds SR1-3 and 5
showed 60 to 80% hyphae inhibition. Furthermore, compounds SR4 and 6 showed ~ 50%
hyphae inhibitory activities, while compound SR7 showed 90% inhibition activity (Figure 3C).
These results indicated that SR compounds were more effective against C. albicans hyphae
formation compared to their activity against yeast (two-way ANOVA, p<0.0001). Concordant
with these results, the minimum inhibitory concentration of the compounds that resulted in 100%
killing (MIC100) against C. albicans yeast ranged from 125 µg/mL for compounds SR6 and 7;
12

200 µg/mL for compounds SR2, 4 and 5; to 400 µg/mL for compounds SR1 and 3 (Table 2). In
contrast, the MIC100 that resulted in almost complete inhibition of biofilm formation (i.e. hyphal
formation) ranged between 10-20 µg/mL (Figure 4 and Table 2). Finally, the MIC100 of the
tested compounds ranged from 125-400 µg/mL against E. coli and >400 µg/mL against
Staphylococcus aureus (Table 2). Collectively, these results confirmed that the newly designed
compounds (SR) are C. albicans hyphae-specific inhibitors.
3.3. The hyphae inhibitory activities of SR compounds was via TUP1 activation
To define the mechanism by which the SR inhibitors exerted their effect on C. albicans
hyphae, we studied the effects of SR 1-7 on the expression of genes that are known to either
control C. albicans yeast/hyphal formation or are expressed in hyphae. The analyzed genes
included those associated with: 1) adhesion/invasion/biofilm formation such as HWP1 (hyphae
cell wall protein), ALS3 (agglutinin-like sequence), EAP1 (extracellular adhesion protein); 2) the
transcription activator UME6 responsible for hyphae formation; and 3) the transcription
repressors of Candida filamentation (TUP1 and NRG1). At the yeast growth condition, all
compounds showed significant increase in the expression level of TUP1 gene (Figure 5).
Compounds SR4, 5, 6, and 7 caused 3-4 fold increase in the expression of TUP1 gene compared
to the expression level of 18S rRNA. Furthermore, all compounds except SR6 caused 2-fold
increase in the expression of NRG1. In contrast, all other tested genes were not significantly
changed. At the hyphae growth condition, all tested compounds caused ~ 4-5-fold increase in the
expression level of TUP1 gene, while other genes were significantly not affected compared to
the expression level of 18S rRNA gene (Figure 5). Moreover, a correlation studies between
TUP1 gene expression levels and C. albicans inhibition activities by SR compounds either at
yeast or hyphae growing conditions was conducted (Figure 6). TUP1 gene expression showed
13

strong direct correlation with hyphae inhibition activities (r2 = 0.85) but not with yeast inhibition
activity (r2 = 0.02) (Figure 6). The results from these studies indicated that SR compounds
primarily exert their inhibitory effect on C. albicans hyphae formation through activation of the
TUP1 hyphae repressor expression. Further, these studies revealed that SR7 is the best candidate
for further development of prevention of C. albicans hyphal growth and biofilm formation.
3.4. QSAR modelling predicted the activity of compound SR7
The relation of SR compounds structure to their concentration required for 50%
inhibition [pIC50 (-log MIC50)] against C. albicans was also calculated using Comparative
Molecular Field Analysis (CoMFA) steric and electrostatic interactions of each molecule, Partial
Least square (PLS) and Leave-one-out (LOO) analyses. The results showed that the squared
correlation coefficient (r2) was 0.992 and the cross-validated correlation coefficient (q2) was
0.50, while the standard deviation error of calculation (SDEC) was 0.021 and the standard
deviation error of prediction (SDEP) was 0.165 (Table 3). The experimental activities of the
compounds on yeast and hyphae were listed in Table 4. The correlation between the
experimental activities and the predictive activities was 0.02 (Supplementary Figure 1). This
correlation suggests the reliability and effectiveness of the established 3D QSAR model and its
effectiveness in designing novel C. albicans inhibitors.
CoMFA steric contour maps were represented in (Figure 7A). The steric interactions
were illustrated by green and yellow contours, while electrostatic interactions were denoted by
red and blue contours (Figure 7B). A large green contour (region A) and small green contour
(region B) were found near the plane of the 3-CF3 substituent of phenyl ring (R1) and OCH3
group in R2 positions (compound SR7), respectively (Figure 7A). This result suggested that
bulky substituent were preferred in these two regions and indicated that they were important for
14

the activity, thus explaining the highest activity of compound SR7. In contrast, a yellow contour
(region C) was located around the 4-CH3 substituent of phenyl ring (R1) of compound SR1,
indicating that groups with low steric factor were favoured in this region to increase the activity
and therefore explaining the decreased activity of compound SR1 (Figure 7A). Blue-colored
(region A) and red-colored (region B) contours represented the regions where the positively- and
negatively-charged groups enhanced the activity, respectively (Figure 7B). These results
indicated that the electron-deficient substituent at 3-position of phenyl ring (R1) were curial for
the activity, and electron-rich groups near 4-position (R1) of the phenyl ring were preferred in
this position.
In conclusion, the rhodanine and 3- trifluroromethyl-phenyl were employed as privilege
structure, which occupied the same position in 3D QSAR results (Figure 7). The QSAR model
also suggested that other points were important in designing novel compounds with potent
activity (Cruz et al., 2019). Furthermore, CoMFA study revealed that positively-charged bulky
groups were favored at position number 3 of the phenyl ring (R1) and less steric, negatively-
charged groups were favored at position number 4 of the phenyl ring (R1). Furthermore, bulky
group at R2 position was favored for the activity.
3.5. SR compounds have limited toxicity and efficient metabolism
The cytotoxic activities of the compounds were examined by testing their hemolytic
activities using human erythrocytes. Interestingly all compounds were significantly less toxic
when compared to hemolysis caused by 0.1% triton (one-way ANOVA, p = 0.0001).
Specifically, at >200 µg/mL, a concentration that is 20x the MIC100 required to inhibit hyphal
formation, only 10-40% erythrocyte hemolysis was detected versus 100% hemolysis caused by
15

0.1% triton (Figure 8). Furthermore, the physicochemical properties of the synthesized SR
compounds were examined using theoretical prediction of absorption, distribution,
metabolism, excretion, toxicity (ADMET) qualitative models via admetSAR server
(https://www. lmmd.ecust. edu.cn/admetsar1/ predict/) (Yang et al., 2018). For instance, human
intestinal absorption (HIA), cytochrome binding, biodegradation, acute oral toxicity,
carcinogenicity, rat acute toxicity and aqueous solubility were calculated (Supplementary Table
1). The calculations indicated that all compounds showed good intestinal absorption, good water
solubility, and no inhibition of cytochromes. Hence, good metabolism and no toxicity of these
compounds are predicted (Supplementary Table 1). To investigate the absorption of the drug,
total polar surface area (TPSA) of rhodanine derivatives were calculated. The TPSA of the
compounds understudy was less than 140Å which indicates the compounds are likely to be orally
bio-available (Shimada et al., 2014).
4. Discussion
With the growing threat due to the multi-drug resistance of Candida pathogen mainly
because of its hyphae form, since it can generate the most-difficult to eradicate biofilms, it is
urgent to develop novel compounds with novel mechanism of action. Based on the reported
Candida inhibition activities of several synthesized thiazolidinedione derivatives (Kagan et al.,
2014) including in particular the rhodanine isosteres, 1,2-benzisothiazolinone (Alex et al.,
2012), rhodanine privileged structures were selected for this study (Figure 1). A novel series of
hybrid compounds (SR1-7) were designed and developed that possessing a rohdanine nucleus
and lipophilic aromatic side chain with different electron withdrawing substituent to improve the
16

cellular penetration along with the hydrophilic polar methoxy and phenoxy groups. The novel
compounds showed enhanced physiochemical properties and selectivity against C. albicans
hyphae formation.
Our results showed that the newly-synthesized compounds (SR1-7) are inhibitors of C.
albicans hyphae formation by activation of the hyphae repressor factor, TUP1. TUP1 is a critical
transcription repressor that inhibits Candida hyphae formation by repressing the expression of
hyphae-specific genes including CPH1 and UME6 (Braun et al., 2000; Braun and Johnson,
1997). Thus, up-regulation of TUP1 by SR compounds significantly contributed to the inhibition
of C. albicans hyphal formation. However, we did not detect any significant changes in the
expression of genes involved in C. albicans yeast/hyphal switch, adhesion or those expressed
during hyphae growth including HWP1, ALS3, EAP1, and UME6, that is in accordance to
previously reported data by (Braun et al., 2000). Although the thiazoldinedione derivatives
reported before showed variable expression activities on C. albicans hyphae-associated genes
(Feldman et al., 2014), the newly-designed rhodanines were more selectively affecting TUP1
expression. The results indicate that SR compounds activated the expression of TUP1 repressor
which in turn inhibited Candida hyphae formation without affecting C. albicans hyphae effector
genes (Figure 9).
The transcriptional repressor, TUP1 and the signalling molecule farnesol are both capable
of negatively regulating the yeast to hyphae conversion. Specifically, it has been shown that
TUP1 are up-regulated in C. albicans biofilms exposed to farnesol (Cao et al., 2005; Han et al.,
2012). Therefore, we hypothesized that the mechanism of action of SR compounds is similar to
farnesol, despite the fact that they have completely different structures. Our data are consistent
with the critical role of TUP1 in response to farnesol in C. albicans (Kebaara et al., 2008).
17

Furthermore, it has been shown that 10-40 times the hyphae inhibitory concentrations of SR
compounds were required to inhibit either Candida yeast or bacteria which is in accordance with
the selective inhibitory effect of farnesol to C. albicans hyphae formation but not the yeast form
(Nickerson et al., 2006; Polke et al., 2018; Ramage et al., 2002). Both results confirmed the
selective inhibitory effect of SR compounds on C. albicans hyphae formation.
TUP1 is known to function with the DNA binding proteins NRG1 to repress the
expression of hypha-specific genes (Kebaara et al., 2008; Lu et al., 2014). However, our data
indicated that SR compounds caused no significant increase in the expression of NRG1,
indicating that the major effect of these inhibitors are on TUP1 rather than NRG1. In contrast,
UME6, a key filament-specific activator, known to be down regulated by TUP1 was not affected
by SR compounds, indicative of a more likely signalling mechanism of hyphae activities by
TUP1.
Targeting the expression of TUP1 gene provides two important advantages over the
currently available drugs, specificity and safety. To the best of our knowledge, there are no drugs
developed against C. albicans hyphae formation by selectively affecting TUP1 gene expression
without affecting the other hyphae-associated genes including ALS3, EAP1, HWP1, NRG1 and
UME6. Our results showed also that activation of TUP1 gene alone was able to inhibit C.
albicans hyphae formation. TUP1 gene does not exist in human and hence afford superior safety
when compared to other available drugs, since most fungal genes (eukaryotes) are shared with
human genome (Galagan et al., 2005). In addition to the limited number of available antifungal
drugs including pyrimidine analogs, polyenes, azoles, and echinocandins, they all have different
mechanisms of actions and none of them affects the expression of TUP1 gene (Juvvadi et al.,
2017). Furthermore, they all developed resistance and showed toxicities to human.
18

During infection, it is reported that C. albicans morphologies have distinct interactions
with the host cells and activation of TUP1 results in significant reduction in C. albicans
virulence, since yeast forms can be easily cleared from tissues by host defenses much more
efficiently than hyphae (Cleary et al., 2016). Our study indicated that the specificity and minimal
toxicity of the newly-designed compounds can be of special value particularly against C.
albicans resistance forms (biofilm). Furthermore, the use of the new rhodanine derivatives in
combination with available anti-fungal drugs may be considered as promising strategy to
enhance the treatment outcome of C. albicans infection.
QSAR has been widely used in drug discovery and lead optimization that effectively
reduces the cost of experimental evaluation of untested chemicals and enhances the selection of
best candidate compound (Tong et al., 2005). Although the reliability and importance of the
QSAR results obtained in this study, the limitations associated with this study can include the
number of derivatives with variable substitutions need to be increased, since the accuracy and
confidence of a model’s prediction depend on the size and diversity of the chemical structures.
Furthermore, running a QSAR analysis on TUP1 gene expression will improve the model’s
prediction. Therefore, a future study may be conducted in order to increase the number of
derivatives with diverse substitutions in relation to TUP1 gene expression.
5. Conclusion
We have designed and synthesized a series of rhodanine derivatives possessing
thiazolidine core and aromatic side chain targeting TUP1 transcription repressor and hence
inhibited C. albicans hyphae formation. Substitutions of R1 and R2 with 3-CF3 and OCH3,
19

respectively followed by 4-CH3 and OCH3 enhanced the hyphae inhibitory activities of the
compounds. Furthermore, the compounds showed no hemolytic activities at ~20 times the
concentrations showed hyphae inhibitory activities. We propose that the newly-designed
compounds are candidates for the effective use of adjunctive therapy with currently used
antifungal agents to ameliorate infections due to C. albicans. Moreover, the newly-designed
compounds, in particular compounds SR7, 1 and 3, may be future new quorum signalling
candidates that serve as promising lead drugs for not only C. albicans infection but also other
TUP1-dependent dimorphic fungal infections.
Acknowledgements
This work was supported by grants from University of Sharjah, U.A.E. to S.S. and by a Public
Health Service grants R01AI063503 and R01AI141202 to A.S.I.
Declaration of Competing Interest
The University of Sharjah has filed intellectual property rights concerning SR compounds.
Supplementary data
Detailed stepwise synthesis procedures were explained in Supplementary Data file available at
JAC Online. Supplementary Figure 1 and Table 1 can be found in Supplementary data.
20

Spectroscopy analysis of SR compounds including Mass, 1HNMR and 13CNMR spectra can be
found in Appendix A.
References
AbdelKhalek, A., Ashby, C.R., Jr., Patel, B.A., Talele, T.T., Seleem, M.N., 2016. In vitro
antibacterial activity of rhodanine derivatives against pathogenic clinical isolates. PLOS
ONE 11, e0164227.
Alex, D., Gay-Andrieu, F., May, J., Thampi, L., Dou, D., Mooney, A., Groutas, W., Calderone,
R., 2012. Amino acid-derived 1,2-benzisothiazolinone derivatives as novel small-molecule
antifungal inhibitors: Identification of potential genetic targets. Antimicrob Agents
Chemother 56, 4630-4639.
Bataille, C.J., Brennan, M.B., Byrne, S., Davies, S.G., Durbin, M., Fedorov, O., Huber, K.V.,
Jones, A.M., Knapp, S., Liu, G., 2017. Thiazolidine derivatives as potent and selective
inhibitors of the PIM kinase family. Bioorganic & medicinal chemistry 25, 2657-2665.
Blankenship, J.R., Mitchell, A.P., 2006. How to build a biofilm: A fungal perspective. Curr Opin
Microbiol 9, 588-594.
Brand, A., 2012. Hyphal growth in human fungal pathogens and its role in virulence. Int J
Microbiol 2012.
Braun, B.R., Head, W.S., Wang, M.X., Johnson, A.D., 2000. Identification and characterization
of TUP1-regulated genes in Candida albicans. Genetics 156, 31-44.
Braun, B.R., Johnson, A.D., 1997. Control of filament formation in Candida albicans by the
transcriptional repressor TUP1. Science 277, 105-109.
Bush, B.L., NachbarJr., R.B., 1993. Sample-distance partial least squares: PLS optimized for
many variables, with application to CoMFA. J Comput Aided Mol Des 7, 587–619.
21

Cao, Y.-Y., Cao, Y.-B., Xu, Z., Ying, K., Li, Y., Xie, Y., Zhu, Z.-Y., Chen, W.-S., Jiang, Y.-Y.,
2005. cDNA microarray analysis of differential gene expression in Candida albicans
biofilm exposed to farnesol. Antimicrob Agents Chemother 49, 584-589.
Cavalheiro, M., Teixeira, M.C., 2018. Candida biofilms: Threats, challenges, and promising
strategies. Front Med 5, 28.
Chauhan, K., Sharma, M., Singh, P., Kumar, V., Shukla, P.K., Siddiqi, M.I., Chauhan, P.M.S.,
2012. Discovery of a new class of dithiocarbamates and rhodanine scaffolds as potent
antifungal agents: Synthesis, biology and molecular docking. MedChemComm 3, 1104-
1110.
Choi, J.Y., Kwak, Y.G., Yoo, H., Lee, S.-O., Kim, H.B., Han, S.H., Choi, H.J., Kim, H.Y., Kim,
S.R., Kim, T.H., 2016. Trends in the distribution and antimicrobial susceptibility of
causative pathogens of device-associated infection in Korean intensive care units from
2006 to 2013: Results from the Korean Nosocomial Infections Surveillance System
(KONIS). J Hosp Infect 92, 363-371.
Clark, M., Cramer III, R.D., Van Opdenbosch, N., 1989. Validation of the general purpose tripos
5.2 force field. J Comput Chem 10, 982-1012.
Cleary, I.A., Reinhard, S.M., Lazzell, A.L., Monteagudo, C., Thomas, D.P., Lopez-Ribot, J.L.,
Saville, S.P., 2016. Examination of the pathogenic potential of Candida albicans
filamentous cells in an animal model of haematogenously disseminated candidiasis. FEMS
Yeast Res 16, fow011-fow011.
Cruz, J.N., Costa, J.F., Khayat, A.S., Kuca, K., Barros, C.A., Neto, A., 2019. Molecular
dynamics simulation and binding free energy studies of novel leads belonging to the
22

benzofuran class inhibitors of Mycobacterium tuberculosis polyketide synthase 13. J
Biomol Struct Dyn 37, 1616-1627.
Deveau, A., Hogan, D., 2011. Linking quorum sensing regulation and biofilm formation by
Candida albicans. Methods Mol Biol 692, 219–233.
Di, P., Guan, L., Sun, L., Li, W., Tang, Y., Liu, G., Yang, H., Cai, Y., 2018. ADMET-score—a
comprehensive scoring function for evaluation of chemical drug-likeness. Med Chem
Comm 10, 148-157.
Douglas, L.J., 2003. Candida biofilms and their role in infection. Trends Microbiol 11, 30-36.
Edwards, J.E., Jr, Lehrer, R.I., Stiehm, E., Fischer, T.J., Young, L.S., 1978. Severe candidal
infections: Clinical perspective, immune defense mechanisms, and current concepts of
therapy. Ann Intern Med 89, 91-106.
Elewski, B.E., 1998. Onychomycosis: Pathogenesis, diagnosis, and management. Clin Microbiol
Rev 11, 415-429.
Fan, C., Clay, M.D., Deyholos, M.K., Vederas, J.C., 2010. Exploration of inhibitors for
diaminopimelate aminotransferase. Bioorganic & medicinal chemistry 18, 2141-2151.
Feldman, M., Al-Quntar, A., Polacheck, I., Friedman, M., Steinberg, D., 2014. Therapeutic
potential of thiazolidinedione-8 as an antibiofilm agent against Candida albicans. PLOS
ONE 9, e93225.
Galagan, J.E., Henn, M.R., Ma, L.-J., Cuomo, C.A., Birren, B., 2005. Genomics of the fungal
kingdom: Insights into eukaryotic biology. Genome Res 15, 1620-1631.
Geffers, C., Gastmeier, P., 2011. Nosocomial infections and multidrug-resistant organisms in
germany: Epidemiological data from kiss (the hospital infection surveillance system).
Dtsch Arztebl Int 108, 87-93.
23

Han, T.-L., Cannon, R.D., Villas-Bôas, S.G., 2012. The metabolic response of Candida albicans
to farnesol under hyphae-inducing conditions. FEMS Yeast Res 12, 879-889.
Hawser, S.P., Douglas, L.J., 1995. Resistance of Candida albicans biofilms to antifungal agents
in vitro. Antimicrob Agents Chemother 39, 2128-2131.
Holcombe, L.J., McAlester, G., Munro, C.A., Enjalbert, B., Brown, A.J.P., Gow, N.A.R., Ding,
C., Butler, G., apos, Gara, F., Morrissey, J.P., 2010. Pseudomonas aeruginosa secreted
factors impair biofilm development in Candida albicans. Microbiology 156, 1476-1486.
https://www.3d-qsar.com/pycomfa/.
Juvvadi, P.R., Lee, S.C., Heitman, J., Steinbach, W.J., 2017. Calcineurin in fungal virulence and
drug resistance: Prospects for harnessing targeted inhibition of calcineurin for an antifungal
therapeutic approach. Virulence 8, 186-197.
Kaczor, A., Witek, K., Podlewska, S., Czekajewska, J., Lubelska, A., Żesławska, E., Nitek, W.,
Latacz, G., Alibert, S., Karczewska, E., 2019. 5-Arylideneimidazolones with Amine at
Position 3 as Potential Antibiotic Adjuvants against Multidrug Resistant Bacteria.
Molecules 24, 438.
Kagan, S., Jabbour, A., Sionov, E., Alquntar, A.A., Steinberg, D., Srebnik, M., Nir-Paz, R.,
Weiss, A., Polacheck, I., 2014. Anti-Candida albicans biofilm effect of novel heterocyclic
compounds. J Antimicrob Chemother 69, 416-427.
Kebaara, B.W., Langford, M.L., Navarathna, D.H.M.L.P., Dumitru, R., Nickerson, K.W., Atkin,
A.L., 2008. Candida albicans Tup1 is involved in farnesol-mediated inhibition of
filamentous-growth induction. Eukaryot Cell 7, 980-987.
LaFleur, M.D., Kumamoto, C.A., Lewis, K., 2006. Candida albicans biofilms produce
antifungal-tolerant persister cells. Antimicrob Agents Chemother 50, 3839-3846.
24

Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time
quantitative PCR and the 2−ΔΔCT method. Methods 25, 402-408.
Lu, Y., Su, C., Unoje, O., Liu, H., 2014. Quorum sensing controls hyphal initiation in Candida
albicans through Ubr1-mediated protein degradation. Proc Natl Acad Sci USA 111, 1975-
1980.
Morschhäuser, J., Barker, K.S., Liu, T.T., Blaß-Warmuth, J., Homayouni, R., Rogers, P.D.,
2007. The transcription factor Mrr1p controls expression of the MDR1 efflux pump and
mediates multidrug resistance in Candida albicans. PLoS Pathog 3, e164.
Nickerson, K.W., Atkin, A.L., Hornby, J.M., 2006. Quorum sensing in dimorphic fungi:
Farnesol and beyond. Appl Environ Microbiol 72, 3805-3813.
O'Connor, L., Caplice, N., Coleman, D.C., Sullivan, D.J., Moran, G.P., 2010. Differential
filamentation of Candida albicans and Candida dubliniensis is governed by nutrient
regulation of UME6 expression. Eukaryot Cell 9, 1383-1397.
Orchard, M.G., Neuss, J.C., Galley, C.M.S., Carr, A., Porter, D.W., Smith, P., Scopes, D.I.C.,
Haydon, D., Vousden, K., Stubberfield, C.R., Young, K., Page, M., 2004. Rhodanine-3-
acetic acid derivatives as inhibitors of fungal protein mannosyl transferase 1 (PMT1).
Bioorganic Med Chem Lett 14, 3975-3978.
Pfaffl, M.W., 2001. A new mathematical model for relative quantification in real-time RT–PCR.
Nucleic Acids Res 29, e45-e45.
Polke, M., Leonhardt, I., Kurzai, O., Jacobsen, I.D., 2018. Farnesol signalling in Candida
albicans–more than just communication. Crit Rev Microbiol 44, 230-243.
25

Ramage, G., Saville, S.P., Wickes, B.L., López-Ribot, J.L., 2002. Inhibition of Candida albicans
biofilm formation by farnesol, a quorum-sensing molecule. Appl Environ Microbiol 68,
5459-5463.
Ramos, R.S., Macêdo, W.J., Costa, J.S., da Silva, C.H.d.P., Rosa, J.M., da Cruz, J.N., de
Oliveira, M.S., de Aguiar Andrade, E.H., e Silva, R.B., Souto, R.N., 2019. Potential
inhibitors of the enzyme acetylcholinesterase and juvenile hormone with insecticidal
activity: study of the binding mode via docking and molecular dynamics simulations. J
Biomol Struct Dyn, 1-23.
Samaranayake, Y.H., Cheung, B.P.K., Yau, J.Y.Y., Yeung, S.K.W., Samaranayake, L.P., 2013.
Human serum promotes Candida albicans biofilm growth and virulence gene expression
on silicone biomaterial. PLOS ONE 8, e62902.
Seddiki, S., Boucherit-Otmani, Z., Boucherit, K., Kunkel, D., 2015. Fungal infectivities of
implanted catheters due to Candida sp. Biofilms formation and resistance. J Mycol Med
25, 130-135.
Shimada, C., Uesawa, Y., Ishihara, M., Kagaya, H., Kanamoto, T., Terakubo, S., Nakashima, H.,
Takao, K., Miyashiro, T., Sugita, Y., Sakagami, H., 2014. Quantitative structure–
cytotoxicity relationship of piperic acid amides. Anticancer Res 34, 4877-4884.
Soliman, S., Alsaadi, A., Youssef, E., Khitrov, G., Noreddin, A., Husseiny, M., Ibrahim, A.,
2017a. Calli essential oils synergize with lawsone against multidrug resistant pathogens.
Molecules 22, 2223.
Soliman, S.S.M., Semreen, M.H., El-Keblawy, A.A., Abdullah, A., Uppuluri, P., Ibrahim, A.S.,
2017b. Assessment of herbal drugs for promising anti-Candida activity. BMC
Complement Altern Med 17, 257.
26

Sydnor, E.R.M., Perl, T.M., 2011. Hospital epidemiology and infection control in acute-care
settings. Clin Microbiol Rev 24, 141-173.
Tong, W., Hong, H., Xie, Q., Shi, L., Fang, H., Perkins, R., 2005. Assessing QSAR limitations-A
regulatory perspective. Current Computer-Aided Drug Design 1, 195-205.
Touil, H.F.Z., Boucherit, K., Boucherit-Otmani, Z., Kohder, G., Madkour, M., Soliman, S.S.M.,
2020. Optimum inhibition of amphotericin-B-resistant Candida albicans strain in single-
and mixed-species biofilms by Candida and non-Candida terpenoids. Biomolecules 10,
342.
Tumbarello, M., Posteraro, B., Trecarichi, E.M., Fiori, B., Rossi, M., Porta, R., de Gaetano
Donati, K., La Sorda, M., Spanu, T., Fadda, G., Cauda, R., Sanguinetti, M., 2007. Biofilm
production by Candida species and inadequate antifungal therapy as predictors of mortality
for patients with candidemia. J Clin Microbiol 45, 1843-1850.
Uppuluri, P., Dinakaran, H., Thomas, D.P., Chaturvedi, A.K., Lopez-Ribot, J.L., 2009.
Characteristics of Candida albicans biofilms grown in a synthetic urine medium. J Clin
Microbiol 47, 4078-4083.
Wenzel, R.P., Gennings, C., 2005. Bloodstream infections due to Candida species in the
intensive care unit: Identifying especially high-risk patients to determine prevention
strategies. Clin Infect Dis 41, S389-S393.
Yang, H., Lou, C., Sun, L., Li, J., Cai, Y., Wang, Z., Li, W., Liu, G., Tang, Y., 2018. admetSAR
2.0: web-service for prediction and optimization of chemical ADMET properties.
Bioinformatics, bty707-bty707.
Zar, J., 1984. Chapter 18 of in: Zar, J. (Ed.), Biostatistical Analysis, 2nd ed, Prentice-Hall.
27

Figure legends
Figure 1. The rational design of anti-Candida hyphae compounds based on rhodanine
scaffold. (A) Structures of thizolidinedione, pyridine-rhodanine and 1,2-benzisothiazolinone
derivatives. (B) Proposed rhodanine-phenolic hybrid derivatives as novel anti-Candida hyphae
compounds.
28

Figure 2. Synthesis and structure of 5-[3-Substitued-4-(4-substituedbenzyloxy)-benzylidene]-
2-thioxo-thiazolidin-4-one compounds. (A) Synthesis scheme of 5-[3-Substitued-4-(4-
substituedbenzyloxy)-benzylidene]-2-thioxo-thiazolidin-4-one derivatives. Reagents and
conditions: i. K2CO3, KI, acetonitrile, overnight stirring under nitrogen; ii. β-alanine, glacial
acetic acid, 100oC, 3 h; R1. CH3, 3CF3, 4CF3; R2. Cl, Br, OCH3. (B) 2D structure of SR7.
29