Essential oil-based design and development of novel anti-Candida azoles formulation

Article abstract of DOI:10.3390/molecules25061463

Candida is the most common fungal class, causing both superficial and invasive diseases in humans. Although Candida albicans is the most common cause of fungal infections in humans, C. auris is a new emergent serious pathogen causing complications similar to those of C. albicans. Both C. albicans and C. auris are associated with high mortality rates, mainly because of their multidrug-resistance patterns against most available antifungal drugs. Although several compounds were designed against C. albicans, very few or none were tested on C. auris. Therefore, it is urgent to develop novel effective antifungal drugs that can accommodate not only C. albicans, but also other Candida spp., particularly newly emergent one, including C. auris. Inspired by the significant broad-spectrum antifungal activities of the essential oil cuminaldehyde and the reported wide incorporation of azoles in the antifungal drugs, a series of compounds (UoST1-11) was designed and developed. The new compounds were designed to overcome the toxicity of the aldehyde group of cuminaldehyde and benefit from the antifungal selectivity of azoles. The new developed UoST compounds showed significant anti-Candida activities against both Candida species. The best candidate compound, UoST5, was further formulated into polymeric nanoparticles (NPs). The new formula, UoST5-NPs, showed similar activities to the nanoparticles-free drug, while providing only 25% release after 24 h, maintainng prolonged activity up to 48 h and affording no toxicity. In conclusion, new azole formulations with significantly enhanced activities against C. albicans and C. auris, while maintaining prolonged action and no toxicities at lower concentrations, were developed.

Full text of DOI:10.3390/molecules25061463

molecules
Article
Essential Oil-Based Design and Development of
Novel Anti-Candida Azoles Formulation
Rania Hamdy 1,2, Bahgat Fayed 1,3, Alshaimaa M. Hamoda 1,4, Mutasem Rawas-Qalaji 1,5
,
Mohamed Haider 1,5,6 and Sameh S. M. Soliman 1,5,
*
1
Research Institute for Medical and Health Sciences, University of Sharjah, P.O. Box, Sharjah 27272, UAE;
r.badawy@cuca.ae (R.H.); bfayed@sharjah.ac.ae (B.F.); alshaimaahamoda@gmail.com (A.M.H.);
mqalaji@sharjah.ac.ae (M.R.-Q.); mhaider@sharjah.ac.ae (M.H.)
2
3
4
5
6
Department of Medicinal Chemistry, Faculty of Pharmacy, Zagazig University, Zagazig 44519, Egypt
Chemistry of Natural and Microbial Product Department, National Research Centre, Cairo 12622, Egypt
Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut 71515, Egypt
College of Pharmacy, University of Sharjah, P.O. Box, Sharjah 27272, UAE
Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Cairo 11562,
Egypt
*
Correspondence: ssoliman@sharjah.ac.ae; Tel.: +97-10-6505-7472
Academic Editors: Valtcho Jeliazkov, Murray B. Isman, Farid Chemat, Vassya Bankova
and Niko Radulovic´
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 29 February 2020; Accepted: 20 March 2020; Published: 24 March 2020
Abstract: Candida is the most common fungal class, causing both superficial and invasive diseases
in humans. Although Candida albicans is the most common cause of fungal infections in humans,
C. auris is a new emergent serious pathogen causing complications similar to those of C. albicans.
Both C. albicans and C. auris are associated with high mortality rates, mainly because of their
multidrug-resistance patterns against most available antifungal drugs. Although several compounds
were designed against C. albicans, very few or none were tested on C. auris. Therefore, it is urgent
to develop novel effective antifungal drugs that can accommodate not only C. albicans, but also
other Candida spp., particularly newly emergent one, including C. auris. Inspired by the significant
broad-spectrum antifungal activities of the essential oil cuminaldehyde and the reported wide
incorporation of azoles in the antifungal drugs, a series of compounds (UoST1-11) was designed
and developed. The new compounds were designed to overcome the toxicity of the aldehyde group
of cuminaldehyde and benefit from the antifungal selectivity of azoles. The new developed UoST
compounds showed significant anti-Candida activities against both Candida species. The best candidate
compound, UoST5, was further formulated into polymeric nanoparticles (NPs). The new formula,
UoST5-NPs, showed similar activities to the nanoparticles-free drug, while providing only 25%
release after 24 h, maintainng prolonged activity up to 48 h and affording no toxicity. In conclusion,
new azole formulations with significantly enhanced activities against C. albicans and C. auris, while
maintaining prolonged action and no toxicities at lower concentrations, were developed.
Keywords: Candida albicans; Candida auris; anti-fungal; nanoparticles; azoles; essential oil
1. Introduction
Candida species are the most common cause of fungal infections in humans. Although Candida can
cause treatable superficial infections [
1], they can also generate serious health problems, particularly to
immunocompromised patients [ ]. C. auris is a newly-emerging Candida species [4,5
2
,
3
]. Both C. albicans
and C. auris are considered as serious global health threats mainly because of their multidrug-resistance
patterns associated with high mortality rates that can reach 60% [6–8].
Molecules 2020, 25, 1463; doi:10.3390/molecules25061463
www.mdpi.com/journal/molecules

Molecules 2020, 25, 1463
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The most common antifungal drugs present in the market [
9] can be classified into polyene
macrolides (amphotericin B and nystatin) that lead to alteration in the fungal membrane, azole
derivatives (ketoconazole, fluconazole, itraconazole, and voriconazole) that inhibit 14 -lanosterol
α
demethylase (CYP51A1), a key enzyme in ergosterol biosynthesis, DNA and RNA synthesis inhibitors
(flucytosine), and echinocandins (casofungin and micafungin) that inhibit 1,3-β-glucan synthase.
Drug resistance among Candida species is one of the greatest challenges for the clinical success
of antifungal agents. The mechanisms of Candida resistance against antifungal drugs can include
up-regulation of the target enzyme, alteration in the target site of the enzyme, increase in drug
efflux, permeability barriers associated with biofilm formation, and development of mutations in
the ERG3 gene that result in cell membranes devoid of ergosterol [10]. Furthermore, the emerging
threats of multidrug-resistant C. auris are also alarming. Therefore, there is a growing demand for
the development of novel anti-Candida agents to overcome the different mechanisms of resistance
to the currently available drugs. The aim of this study was thus to develop novel antifungal drugs
effective against not only C. albicans but ones that can also accommodate other Candida spp. including
in particular the newly emergent C. auris.
Recently, we have reported that cuminaldehyde essential oil isolated from Calligonum comosum
plant shows excellent antifungal activities [11,12]. Despite its excellent antifungal activities, nevertheless
it showed poor bioavailability since it is lipid-soluble and displays very high toxicity to mammalian
cells due to its aldehyde nature. Therefore, we have designed a series of novel compounds via structural
modification of the natural scaffold by replacing the aldehyde group with a 1,2,4-triazole ring carrying
a substituted benzylsulfanyl moiety [13], presumably, to generate new derivatives with enhanced
physicochemical properties and safety profiles. The newly designed azoles were formulated into
polymeric NPs in order to enhance the compounds’ bioavailability, reduce the toxicity and prolong
the activities.
Formulation of antifungal drugs into polymeric NPs is one of the approaches that have been
utilized to overcome poor drug solubility for hydrophobic antifungal drugs It provides an appropriate
time for the drug to reach infected sites, and enhance drug release for an extended time to improve
patient compliance [14]. Additionally, polymeric NPs can protect the loaded drug from degradation
by the acidic pH in the stomach and from liver enzymes after oral administration, therefore,
enhancing their bioavailability and absorption compared to the free drug [15,16]. In the present
work, poly(lactic-co-glycolic acid) (PLGA) has been employed to encapsulate the new UOST5 triazole
derivatives into NPs. The mentioned polymer was selected as it is biocompatible and has been
approved by the FDA, in addition to the ability to control the drug release rate by adjusting the PLGA
characters such as molecular weight and glycolic acid/lactic acid molar ratio [17]. Hence one can
eventually control the degradation rate.
2. Results
2.1. Rational Sesign of UoST1-11, Novel Anti-Candida Compounds Based on an Essential Oil Natural Product
Scaffold
Cuminaldehyde, a natural essential oil, was used as lead structure to develop novel safe and
effective anti-Candida agents. Inspired by the selective antifungal activity of azoles and the reported
antifungal activities of cuminaldehyde [11], a series of UoST compounds was designed and synthesized
(Figure 1).
The toxic aldehyde group of cuminaldehyde was replaced by a 1,2,4-triazole ring followed by
the introduction of the 3-trifluoromethylbenzylsulfanyl moiety to selectively enhance the antifungal
activity and reduce the toxicity. The synthesis of target compounds (UoST1-11) was performed as
described in Scheme 1.

Molecules 2020, 25, 1463
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R1
N
CHO
N
N
N
R2
N
S
N
HO
N
R
R
Sulconazole
Voriconazole
Cuminaldehyde
R
Structural modification of cuminaldehyde
to examine the importance of isoproyl
group a series of tert-butyl group was
synthezied
Replacing the aldehyde group with
1,2,4-triazole ring with reported antifungal
activity to decrease toxicity and improve
phsiochemical properties
N
N
R1
N
S
The introduction of 3-trifluoro-methyl-
benzylsulfanyl moeity to enhance the
activity
CF3
Figure 1. Rational design and synthesis of UoST compounds.
COCl
CHO
COOH
CONHNH
2
ii
i
iii
CNS
R
R
(1)
R
(2-3)
R
(6-7)
iv
(4-5)
R1
8(a-k)
R
1
N
N
HN
SH
v
N
HN
NH
O
S
S (1-11)
Compound
UOST1
UOST2
UOST3
UOST4
UOST5
UOST6
UOST7
UOST8
UOST9
UOST10
UOST11
R
R1
Isopropyl 4-Cl
Tert-butyl
R
Cl
H
vi
R (1-11)
Tert-butyl 3-Cl
Tert-butyl 4-CF3
Isopropyl 3-Br
CF
3
N
N
CF
R
3
N
Tert-butyl 4-OCH3
Tert-butyl 4-CH3
Isopropyl 4-OCH3
Tert-butyl 3-Br
Isopropyl 4-F
S
R
1
UOST (1-11)
Tert-butyl 4-F
Scheme 1. Synthesis of UoST compounds. Reagents and conditions: (
ii) oxalyl chloride/anhydrous DCM/DMF; (iii) NH2NH2 H2O/Ethanol; (iv) ethanol/reflux 3 h; (
NaOH/reflux for 3 h; (vi) KOH-ethanol-12 h/stirring.
i
) NaOH-I2/TBHP, 70 ◦C, 10–16 h;
) 2N
(
·
v

Molecules 2020, 25, 1463
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Initially, cuminaldehyde (
1) was oxidized to cumnic acid (2) by iodine, NaOH and TBHP as
catalyst in moderate yield. The acids
2, 3 were then converted to acid chlorides 4, 5 using oxalyl
chloride, DCM and DMF under nitrogen at 0 ◦C. The acid hydrazide derivatives 6, 7 were obtained in
high yields by refluxing the acid chlorides with hydrazine hydrate [18]. The hydrazides derivatives 6,
7
were treated with substituted phenyl isothiocyanates 8a
thiosemicarbazides R (1 11), which were subsequently converted to triazole thiols S (1–11) using 2N
NaOH. The target compounds UoST1 11 were formed in moderate yields (~70%) by nucleophilic
–k for the synthesis of the corresponding
–
–
substitution reactions between triazole thiols S (1–11) and 3-trifluorobenzyl chloride.
2.2. Selective Inhibition Activities of UoST Compounds Against Candida Spp.
The anti-Candida activities of the developed compounds UoST1
and C. auris. The results obtained indicated that compounds UoST5
variable inhibition activities on the growth of both C. albicans (Figure 2A) and C. auris (Figure 2B) at
different concentrations (2, 5, 15, 30, 50 and 100 g/mL) when compared to negative control. Compound
UoST5 showed persistent and significant promising activities on both Candida spp. when compared to
other compounds (two-way ANOVA, P < 0.0001, Figure 2) and with the lowest MIC50 (2 g/mL) on C.
–
11 were tested against C. albicans
, 7, 8
and 11 caused significant
µ
µ
auris when compared to amphotericin B, employed as positive control (Table 1). On the other hand,
compound UoST7 showed selective inhibition activities on C. auris but not C. albicans (Figure 2).
Figure 2. Anti-Candida activities of UoST compounds. (
and 11 compounds at different concentrations (2, 5, 15, 30, 50, 100
by UoST5 and 11 compounds at different concentrations (2, 5, 15, 30, 50, 100 µ
A
) C. albicans growth inhibition by UoST5
g/mL). ( ) C. auris growth inhibition
g/mL). Candida at 104
, 8
µ
B
,
7,
8
CFU/mL was grown in 96 well microplate containing YPD broth. The plates were incubated at 37 ◦C for
24 h prior to measurement at 600 nm using plate reader. Cultures without compounds were employed
as negative control. The data was analyzed using two-way ANOVA and statistical significance was
calculated with Bonferroni’s multiple comparisons test and significance level indicated by asterisks (* P
< 0.05; ** P < 0.01: *** P < 0.001). The data display the mean ± standard error (SEM) of three replicas.
Table 1. MIC50 of UoST compounds against C. auris.
Compound
MIC50 (µg/mL)
UOST 5
UOST 7
2
5
UOST 8
UOST 11
15
3
Amphotericin B
0.3
2.3. Formulation and Characterization of UoST5-Loaded NPs
Compound UoST5 was formulated into PLGA NPs using the emulsion/evaporation method.
The size and shape of the developed NPs were analyzed by a Zeta analyzer and scanning electron

Molecules 2020, 25, 1463
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microscope (SEM), respectively. The particle size and size distribution were evaluated by a Zetasizer
and found to be 199.5 0.035 nm with narrow size distribution (0.098 0.016) (Figure 3A). Additionally,
the particle surface charge (Zeta potential) was 1.58 0.25 mV (Figure 3B). The surface morphology
±
±
−
±
and topography of UoST5-loaded NPs showed well-defined spherical shaped NPs with smooth
surfaces without any cracks (Figure 3C). The amount of drug entrapped was calculated directly by
dissolving the NPs in DCM. The drug release was measured by UV/VIS spectrophotometer and the
entrapment efficiency was calculated to be 67.2%.
Figure 3. Characterization of nanoparticles.
measurement using a Malvern Instruments Zetasizer Nano ZS. NPs samples were diluted with distilled
water and the polydispersity index ( ) and zeta potential ( ) were determined using the Dynamic light
scattering (DLS) mode and Laser Doppler Velocimetry (LDV) mode, respectively. The measurement
was performed in triplicate. ( ) SEM micrographs of the developed nanoparticles. The NPs samples
after complete dryness were placed on standard carbon tape and coated with gold prior to measurement.
) Release pattern of UoST5-loaded NPs. UoST5-NPs were placed into dialysis membrane (MWCO =
(A,B) Particle size distribution and zeta potential
A
B
C
(D
12 kDa) containing 1ml of release media (0.9% NaCl and isopropanol (85:15), then immersed in 10 mL
of release media and incubated in shaking water bath at 37 ◦C and 50 rpm.
The release of UoST5 from PLGA NPs was determined within 10 days by incubating certain
amount of UoST5-NPs in a release media at 37 ◦C followed by measurement at different time intervals.
The release pattern was biphasic with an initial burst release of 19.79% after 4 h (Figure 3D). This was
followed by a gradual release as the cumulative release percentage reached 25.039% after 24 h and
92.9% after 6 days and stayed at the same percentage for another 4 days (Figure 3D).
2.4. NP Formulation Enhanced and Prolonged the Anti-Candida Activities of UoST5
The bioactivity of UoST5-loaded NPs on C. auris was compared to the free drug (suspended in
water or dissolved in DMSO). The results showed that UoST5-NPs (suspended in water) achieved
significantly higher activity than when the compound was suspended in water but lower than when
dissolved in DMSO (two-way ANOVA, P < 0.0001, Figure 4). The anti-Candida activities of UoST5-NPs
were comparable to the compound in DMSO (Figure 4), while afforded no toxicity to mammalian cells
at six times the MIC50 concentrations (Data not shown). Drug-free NPs, employed as control, showed
no effects on Candida or mammalian cells.

Molecules 2020, 25, 1463
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Figure 4. Anti-Candida activity of UoST5-Loaded NPs compared to free synthesized compound.
The anti-Candida auris activity of UoST5-NPs in water was compared to UoST5 in water and DMSO at
different concentrations (3.125, 6.25, 12.5, 25, 50 and 100 µ
g/mL). C. auris at 104 CFU/mL was grown in 96
well microplate containing YPD broth. The plates were incubated at 37 ◦C for 24 h prior to measurement
at 600 nm using a plate reader. Cultures without compounds were employed as negative control. The
data display the mean of the percentage of the survival rate of mammalian cells
the mean. A P-value < 0.05 was considered as significant. The level of significance was indicated by
asterisks (** P < 0.01: *** P < 0.001)
±
standard error of
The anti-Candida activities of UoST5-loaded NPs were compared to NPs-free drug dissolved
in DMSO at concentrations range 3.12–100 µg/mL within 48 h (Figure 5). Both the free drug and
NPs formulations exhibited significant growth inhibition activity on both C. albicans and C. auris
with different magnitudes depending on the drug concentrations and time of incubation. In the
case of C. albicans, the inhibition effect of UoST5-NPs after 24 h incubation was 75.46% at 100 µg/mL
which is significantly lower than the effect of free drug (two-way ANOVA, P < 0.0001, Figure 5A).
The inhibition was further enhanced after 48 h to reach 91.8% similarity to the effect of free drug
(Figure 5B). At 3.12
(40% and 19%, two-way ANOVA, P < 0.0001) after 24 h and 48 h, respectively. A similar pattern was
obtained in the case of C. auris (Figure 5C,D). UoST5-NPs at 100 and 3.12 g/ml after 24 h showed
74.34% and 30.96% inhibition activity, respectively. Unlike the effect on C. albicans, the inhibition
effect of UoST5-NPs was slightly reduced after 48 h to reach 60.2% and 15.96% at 100 and 3.12 g/mL,
µg/mL, the inhibition effect was 30.58%, and 39.3% when compared to free drug
µ
µ
respectively (Figure 5C,D). Comparing the anti-Candida activities of UoST5-loaded NPs to free UoST5
drug at the same concentration indicated that UoST5-loaded NPs showed lower inhibition activity at
24 h and higher inhibition activity at 48 h (Figure 5), although the released amount of the drug from the
NPs was only 25%; thus providing a significant increase in activity. Collectively, we have developed
novel triazole derivatives effective against both C. albicans and C. auris and its formulation in NPs
enhanced and prolonged the activities of the drug.

Molecules 2020, 25, 1463
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B
A
C
D
Figure 5. Anti-Candida activities of UoST5-NPs compared to free UoST5 at (
) represent the effects of the drugs on C. albicans, while ( ) represent the effects of the drugs on C.
auris. Candida growth inhibition was measured at different concentrations (3.125, 6.25, 12.5, 25, 50 and
100
g/mL). Candida at 104 CFU/mL was grown in 96 well microplate containing YPD broth. The plates
A,C) 24 h and (B,D) 48 h.
(A
,
B
C,D
µ
were incubated at 37 ◦C for 24 h prior to measurement at 600 nm using plate reader. Cultures without
compounds were employed as negative control in order to measure percent inhibition. The data was
analyzed using two-way ANOVA and statistical significance was calculated with Bonferroni’s multiple
comparisons test and significance level indicated by asterisks (* P < 0.05; ** P < 0.01: *** P < 0.001).
The data display the mean ± standard error (SEM) of three replicates.
3. Discussion
Candida spp. are responsible for 75–88% of all fungal infections in the US, causing an extra annual
health cost of $1.7 billion [19]. The general resistance of Candida spp. to most available antifungal
drugs [20] and the emergence of multidrug resistant C. auris [21] make urgent the need to develop
novel effective anti-Candida drugs. On the other hand, most compounds synthesized nowadays
are developed against C. albicans, while very few are tested against C. auris. Thus, we proposed to
develop novel azoles derivatives with effective antifungal activities against Candida spp. including
in particular C. auris. Azole derivatives show great potential activities against Candida infections.
Chandrika et al. developed 12 novel azole derivatives that showed significant antifungal activities
against different types of Candida spp., but none of them was C. auris [22]. Similarly, Shrestha et al.,
successfully developed some novel alkylated-azoles that showed great activities against 13 clinically
fungal isolates, but C. auris was not one of them [23]. Here, we have developed a series of novel
S-alkylated azole derivatives (compounds UoST1–11) by cyclization reactions of thiosemicarbazide
intermediates R(1–11) followed by S-alkylation in alkaline media.
Generally, 1,2,4-triazole-3-thiones have attracted considerable attention because of their broad
biological activities [24]. Some of our developed compounds (compounds UoST5, 7, 8 and 11) showed
variable promising activities against both C. albicans and C. auris. The variability of the activity
is attributed to the variation in the substitution on the newly developed structures including the
isopropyl or tert-butyl substituents at the C-4 position of the phenyl ring (R) and the substituted phenyl
attached to the N-4 position of the triazole ring (R1) carrying 3-Cl, 4-Cl, 4-CH3, 4-OCH3, 4-F, 3-Br or
unsubstituted phenyl. Looking at the chemical structures of the most active compounds UoST5
, 7,
8
and 11 that showed significant anti-Candida activity, it can be declared that 3-Br (UoST5), 4-OCH3

Molecules 2020, 25, 1463
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(UoST8) substitution on R1 with an isopropyl substituent on R, and the tert-butyl substituent on R
with 4-F on R1 (UoST11) are active against both Candida spp., while the substitution with 4-CH3 on R1
with tert-butyl on R (UoST7) was significantly selective against C. auris.
Compared to the positive control, UoST5 was selected as candidate for further development
mainly because of its significant anti-Candida activities against both Candida spp., its low MIC50 value
and its safety on mammalian cells. Like 90% of the molecules in the discovery pipeline, UoST5
showed limited aqueous solubility that consequently leads to poor bioavailability [25]. In view of
the foregoing, we have enhanced the bioavailability of UoST5 by encapsulation into PLGA-NPs.
The single emulsion solvent evaporation method was employed for UoST5-NPs fabrication [26].
The aforementioned fabrication method of hydrophobic drugs affords simplicity with the ability to
scale up [26
the same method [14
main advantages of encapsulating hydrophobic drugs into PLGA-NPs. This can be attributed to the
hydrophobic nature of the biodegradable polymer (PLGA) employed [15 26]. Furthermore, a relatively
–
28]. The obtained drug entrapment efficiency of 67.2% is similar to other reports using
,
29]. The relatively higher entrapment efficiency reported here is one of the
,
uniform particle distribution was obtained as the polydispersity index value was 0.098. The relatively
low particle charge obtained was also favorable since it increases the contact and interaction of the
NPs with the Candida outer membrane. Generally, Candida carry negative surface charge; thus it was
essential to develop NPs that carry either neutral or low negative charge to avoid any repulsion forces
that may prevent the attachment of NPs to the Candida surface [30,31]. The release of UoST5 from the
NPs followed the common biphasic pattern with an initial burst release followed by sustained release
of the drug over 6 days. The initial burst release of the drug is mainly related to the fraction of the drug
located and adsorbed on the surface of the NPs that has been reported and described elsewhere [32,33].
The sustained release is relevant to the amount of the drug encapsulated and the degradation rate of
PLGA employed.
The bioactivity of UoST5-NPs compared to the free UoST5 can be explained by the UoST5-NPs’
release pattern. Although 25% of the entrapped drug was released from the NPs after 24 h, the
inhibition effects on C. albicans and C. auris were 62.8–91.7% of the inhibition effect obtained by the free
drug (100% in direct contact with the Candida cells). Interestingly, UoST5 release after 48 h reached up
to 32.95%, while exerting similar or higher activities on Candida cells when compared to 100% free drug.
Initially, this confirms that the method and reagents used to encapsulate UoST5 did not modify or
degrade the UoST5 molecule as the drug conserved its bioactivity. Additionally, as the PLGA-NPs free
of UoST5 has no inhibitory effect on Candida, the enhancement of the antifungal activity of UoST-NPs
can be related to the ability of the NPs to adhere and attach to the outer surface of Candida cells.
Therefore, it provides direct contact and reduces the loss of the drug. The ability of the NPs to adhere
and even infiltrate to fungal cells was previously studied and explained [14,34]. A model represents
the synthesis of NPs, attachment to Candida cells and delivery of UoST5 is described in Figure 6.
Essential oils including cuminaldehyde are known for their promising antimicrobial activities [35],
while inducing toxic effects on human cells [11]. Therefore, extraction and purification of essential oil
followed by few synthetic steps in order to optimize its selective antimicrobial activities and provide
no toxicity will be of great value for both human and market uses.

Molecules 2020, 25, 1463
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O
CAN DIDA
CELLS
CUM I N ALDEHYDE
N
N
CF
3
N
S
R
N
N
C
F3
N
N
CF3
R
1
N
N
R
CF3
N
S
N
S
R
N
S
R
R1
N
N
N
N
CF3
C
F3
R1
N
S
R
N
S
R
N
N
C
F3
R1
N
N
R1
C
F
3R1
N
N
S
R
N
S
R
N
C
F3
N
N
R1
R
N
1 S
R
CF3
R
N
R1
N
N
N
C
F3
CF3
N
S
R
N
S
R
R1
N
N
N
S
CF3
R
N
N
N
S
R
C
F3
R
N
N
R1
N
S
CF3
N
N
R
C
F
3
N
N
R
N
S
N
S
C
F3
R1
N
S
R
R
1
N
N
R1
C
F
3
R1
R
N
S
R1
N
N
CF3
R1
N
N
R
1
C
F
N
N
3
C
F3
N
S
R
N
S
R
R
N
S
N
N
N
C
F
3
N
N
S
N
R
CF3
R
1
R
1
N
N
N
N
C
F3
CF
3
N
N
S
N
R
1
N
R
N
R1
CF3
N
N
N
N
S
C
F3
C
F
3
CF3
R
N
S
N
S
R
N
S
N
N
S
R
R
N
N
R
1
C
F3
R
N
N
N
S
R
N
S
N
R
1
C
F
3
N
N
R
1
N
R
1
R
C
F3
CF3
R
N
S
N
S
N
S
R
R1
R
1
N
C
N
F3
N
N
C
F3
R
1
R
N
S
R
1
N
S
R
1
R
N
R1
C
F
3
R
R
1
N
S
N
N
N
R
1
C
F3
N
S
N
N
R
C
F
3
R1
R1
N
N
R
F3
N
S
N
CF3
N
N
C
C
F
3
N
S
R
1
R
R
N
S
N
R
N
S
N
N
C
F3
R
1
DRUG
ENCAPSULATION
PLGA
Polymer
R
N
S
R
1
N
N
N
N
R
1
R
1
CF
C
3
F3
N
C
F3
R
R
N
N
S
N
S
N
S
R
1
R
R
1
R
1
N
CF3
N
N
R
1
N
N
C
F3
C
F
3
R
N
S
R
N
S
R
N
S
N
N
N
N
C
F
3
CF
3
N
N
C
C
F3
N
S
N
N
R
N
N
S
R
1
R
R
C
F
3
N
R
R
N
R
1
S
R
1
CF
3
UoST-5
R
N
S
N
S
N
R
1
R
1
1
N
N
R
CF3
F3
R
1
N
S
R
F3
R
N
S
N
N
N
C
R
N
S
N
N
R
1
C
F
N
N
R
1
3
CF
3
N
C
N
S
F3R
N
S
R
1
R
R
N
S
N
N
C
F3
R
N
S
N
N
R
1
C
F3
R
1
N
N
R
1
R
N
S
C
F3
N
N
R
1
R
N
S
C
F3
R
N
S
R
1
N
N
C
F3
N
N
C
F3
R
1
R
N
S
N
S
DEATH
R
R
1
N
N
C
F3
R
1
N
N
R
1
N
S
C
F3
DELIVERY
R
N
S
R
N
N
R
1
C
F3
R
1
R
N
S
N
N
C
N
F3
R
N
S
R
1
N
N
N
C
F3
C
F3
R
N
S
R1
R
N
S
N
N
C
F3
R
1
R
N
S
N
N
R
1
C
F3
N
S
R
R
1
R
1
N
N
CF
3
N
S
R
R
1
Figure 6. Graphical representation summarizes the synthesis, formulation and proposed mechanism of
attachment and delivery of UoST5-NPs.
4. Materials and Methods
4.1. Materials
Most chemicals and solvents were of analytical grade and, when necessary, were purified and
dried by standard methods. Poly (D,L-lactic-co-glycolic acid; PLGA) (50/50, M.W. 24–38 kDa), polyvinyl
alcohol (PVA), with molecular weight of MW 13,000–23,000, 87–89% hydrolyzed were purchased from
Sigma-Aldrich (St. Louis, MO, USA). Dichloromethane (DCM) was purchased from Merck (Darmstadt,
Germany).
4.2. General Information
Reactions were monitored by thin-layer chromatography (TLC) using pre-coated silica gel plates
(Kieselgel 60 F254, BDH, Taufkirchen, Germany), and spots were visualized under UV light (254 nm).
Melting points (mp) were determined using a Gallenkamp melting point apparatus (London, UK).
1H-NMR and 13C-NMR spectra were recorded at 500/125 MHz on a Bruker spectrometer (Karlsruhe,
Germany). 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 were designated as follows:
s, singlet; d, doublet; t, triplet; m, multiplet. Mass spectroscopic data were obtained using electrospray
ionization (ESI) mass spectroscopy (Bruker Daltonics mass spectrometer, Bremen, Germany).
4.2.1. Synthesis of 4-Isopropyl Benzoic Acid (Cuminic Acid) (2)
Cuminic acid (2) was prepared by the oxidation of cuminaldehyde (1) following a reported
procedure [36]. Cuminaldehyde (10 mmol) in water was mixed with iodine and NaOH (10 mmol) and
1
TBHP (20 mmol) as catalyst [36]. Yield 62%. H-NMR (DMSO-d6):
δ 1.21 (d, 6H, 2CH3 of isopropyl),
2.90 (m, 1H, CH of isopropyl), 7.34 (d, 2H, J = 8, ArH), 7.52 (d, 2H, J = 7.5, ArH).
4.2.2. General Procedure for the Synthesis of 4-Substituted Benzoyl Chlorides 4, 5
Oxalyl chloride (12 mmol) was added dropwise to a mixture of the appropriate 4-substituted
benzoic acid 2, 3 (10 mmol), in anhydrous DCM (30 mL) and anhydrous DMF (10 µL) under nitrogen
at 0 ◦C. The reaction mixture was stirred at room temperature for sn additional 3 h until the reaction
was completed. The reaction mixture was evaporated under vacuum and the oily residue was used
without further purification for the acid hydrazide preparation.

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4.2.3. General Procedure for the Synthesis of 4-Substituted Benzoic Acid Hydrazides 6, 7
The synthesis of 4-substituted benzoic acid hydrazides was performed as previously described [18].
To a solution of ethyl 4-substituted benzoyl chloride (10 mmol) in ethanol (10 mL), excess hydrazine
4, 5
monohydrate (5 mL) was added. The reaction mixture was refluxed for 24 h, and left to cool to room
temperature. The formed precipitate was collected by filtration, washed with water followed by cold
ethanol to remove excess hydrazine and left to dry to give the corresponding acid hydrazide.
1
4-Isopropyl-benzoic acid hydrazide (
6
). Yield 85%, H-NMR (DMSO-d6):
δ
1.21 (d, 6H, 2 CH3 of
isopropyl), 2.93 (m, 1H, CH of isopropyl), 4.45 (s, 2H, NH2), 7.31 (d, 2H, J = 8, ArH), 7.54 (d, 2H, J = 6.5,
ArH), 9.67 (s, 1H, NH).
4-tert-Butyl-benzoic acid hydrazide (7 δ 1.29 (s, 9H, tert-butyl),
). Yield 87%, 1H-NMR (DMSO-d6):
4.45 (s, 2H, NH2), 7.45 (d, 2H, J = 8.5, ArH), 7.75 (d, 2H, J = 6.5, ArH), 9.68 (s, 1H, NH).
4.2.4. General Procedure for Preparation of 4-Substituted Benzoyl-N-Substituted Phenyl
Thiosemicarbazides R(1–10)
The 4-substituted benzoyl-N-substituted phenyl thiosemicarbazide
as previously described [18]. To a solution of 4-substituted benzoic acid hydrazide 6, 7 (10 mmol) in
ethanol (30 mL) a solution of substituted phenyl isothiocyanate 8a (10 mmol) in ethanol (20 mL) was
R derivatives were synthesized
–
k
added with continuous stirring. The reaction mixture was refluxed for 3 h. After being cooled to room
temperature, the precipitate formed was collected by filtration, washed with cold ethanol (30 mL) to
give the corresponding thiosemicarbazide R(1–10), Yield 79%.
4-Isopropyl-benzoyl-N-(4-chlorophenyl) hydrazine carbothioamide (R1). Yield 75%, 1H-NMR
(DMSO-d6):
δ 1.21 (d, 6H, 2CH3 of isopropyl), 2.95 (m, 1H, CH of isopropyl), 7.38 (d, 4H, J = 8,
ArH), 7.48 (d, 2H, J = 6.5, ArH), 9.88 (d, 2H, J = 8, ArH), 9.7 (s, 2H, NH), 10.47 (s, 1H, NH).
4-tert-Butyl-benzoyl-N-(phenyl) hydrazine carbothioamide (R2). Yield 70%, 1H-NMR (DMSO-d6):
δ
1.31 (s, 9H, tert-butyl), 7.15 (t, 1H, J = 7.5, ArH), 7.32 (t, 2H, J= 7.5, ArH), 7.34 (d, 2H, J = 6.5, ArH), 7.52
(d, 2H, J = 2, ArH), 7.90 (d, 2H, J = 8, ArH), 9.6 (s, 1H, NH), 9.76 (s, 1H, NH), 10.46 (s, 1H, NH).
4-tert-Butyl-benzoyl-N-(3-chlorophenyl) hydrazine carbothioamide (R3). Yield 64%, 1H NMR
(DMSO-d6):
δ 1.31 (s, 9H, tert-butyl), 7.2 (d, 1H, J = 7.5, ArH), 7.35 (t, 1H, J = 8, ArH), 7.48 (d,
1H, J = 7, ArH), 7.53 (d, 2H, J = 7, ArH), 7.61 (s, 1H, ArH), 7.90 (d, 2H, J = 8.5, ArH), 9.83 (s, 1H, NH),
9.89 (s, 1H, NH), 10.50 (s, 1H, NH).
4-tert-Butyl-benzoyl-N-(4-trifluoromethylphenyl) hydrazine carbothioamide (R4). Yield 62%, 1H-NMR
(DMSO-d6):
δ 1.31 (s, 9H, tert-butyl), 7.52 (d, 2H, J = 7, ArH), 7.68 (d, 2H, J = 8.5, ArH), 7.76 (d, 2H, J =
2, ArH), 7.90 (d, 2H, J = 8.5, ArH), 9.96 (s, 2H, NH), 10.53 (s, 1H, NH).
4-Isopropyl-benzoyl-N-(3-bromophenyl) hydrazine carbothioamide (R5). Yield 70%, 1H-NMR
(DMSO-d6):
δ 1.23 (d, 6H, 2 CH3 of isopropyl), 2.94 (m, 1H, CH of isopropyl), 7.31 (m, 2H, J =
8, ArH), 7.32 (d, 1H, J = 4, ArH), 7.39 (d, 1H, J = 8.5, ArH), 7.52 (d, 1H, J = 8.5, ArH), 7.72 (s, 1H, ArH),
7.89 (d, 2H, J = 8, ArH), 9.83 (s, 1H, NH), 9.87 (s, 1H, NH), 10.49 (s, 1H, NH).
4-tert-Butyl-benzoyl-N-(4-methoxyphenyl) hydrazine carbothioamide (R6). Yield 64%, 1H-NMR
(DMSO-d6):
δ 1.31 (s, 9H, tert-butyl), 3.75 (s, 3H, OCH3), 6.89 (d, 2H, J = 9, ArH), 7.28 (d, 2H, J
= 6.5, ArH), 7.50 (d, 2H, J = 4.5, ArH), 7.89 (d, 2H, J = 6, ArH), 9.58 (s, 1H, NH), 9.66 (s, 1H, NH), 10.43
(s, 1H, NH).
4-tert-Butyl-benzoyl-N-(p-tolyl) hydrazine carbothioamide (R7). Yield 69%, 1H-NMR (DMSO-d6):
δ
1.31 (s, 9H, tert-butyl), 2.28 (s, 3H, CH3), 7.12 (d, 2H, J = 8.5 ArH), 7.31 (d, 2H, J = 6.5, ArH), 7.51 (d, 2H,
J = 8.5, ArH), 7.89 (d, 2H, J = 8, ArH), 9.62 (s, 1H, NH), 9.69 (s, 1H, NH), 10.44 (s, 1H, NH).
4-Isopropyl-benzoyl-N-(3-methoxyphenyl) hydrazine carbothioamide (R8). Yield 51%, 1H-NMR
(DMSO-d6):
δ 1.12 (d, 6H, 2 CH3 of isopropyl), 2.93 (m, 1H, CH of isopropyl), 3.68 (s, 3H, OCH3), 6.72
(dd, 1H, J= 8, ArH), 7.02 (d, 1H, J = 8, ArH), 7.10 (s, 1H, ArH), 7.10 (t, 1H, J = 8.5, ArH), 7.24 (d, 2H, J =
8, ArH), 7.85 (d, 2H, J = 8, ArH), 9.65 (s, 2H, NH), 10.43 (s, 1H, NH).
4-tert-Butyl-benzoyl-N-(3-bromophenyl) hydrazine carbothioamide (R9). Yield 64%, 1H-NMR
(DMSO-d6):
δ 1.31 (s, 9H, tert-butyl), 6.64 (dd, 2H, J = 6.5, ArH), 6.98 (d, 1H, J = 6, ArH), 7.29

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(d, 2H, J = 8.5, ArH), 7.48 (d, 1H, J= 8, ArH), 7.59 (d, 1H, J = 7, ArH), 7.82 (s, 1H, ArH), 9.68 (s, 1H, NH),
9.86 (s, 1H, NH), 10.50 (s, 1H, NH).
4-Isopropyl-benzoyl-N-(4-fluorophenyl) hydrazine carbothioamide (R10). Yield 63 %, 1H-NMR
(DMSO-d6):
δ 1.23 (d, 6H, 2 CH3 of isopropyl), 2.97 (m, 1H, CH of isopropyl), 7.37 (dd, 2H, J =
8, ArH), 7.68 (d, 2H, J = 8.5, ArH), 7.74 (d, 2H, J = 6.5, ArH), 7.88 (d, 2H, J = 8, ArH), 9.74 (s, 1H, NH),
9.77 (s, 1H, NH), 10.45 (s, 1H, NH).
4-tert-Butyl-benzoyl-N-(4-fluorophenyl) hydrazine carbothioamide (R11). Yield 64%, 1H-NMR
(DMSO-d6):
δ 1.31 (s, 9H, tert-butyl), 7.16 (t, 2H, J = 9, ArH), 7.42 (d, 2H, J = 7, ArH), 7.53 (d,
2H, J = 5.5, ArH), 7.90 (d, 2H, J = 8.5, ArH), 9.73 (s, 1H, NH), 9.77 (s, 1H, NH), 10.47 (s, 1H, NH).
4.2.5. General Procedure for Synthesis of Triazole Thiols S(1–11)
A solution of 4-substituted benzoyl-N-substituted phenyl thiosemicarbazides (10 mmol) R(1–11)
in 2 N NaOH (20 mL) were refluxed for 3 h. After cooling to room temperature, water was added and
the mixture was carefully neutralized with dilute HCl. The precipitate formed was filtered, dried and
re-crystallized from ethanol to give the corresponding triazole thiol, Yield 86%.
4-(4-Chlorophenyl)-5-(4-isopropylphenyl)-4H-[1,2,4]triazole-3-thiol (S1).
(DMSO-d6): 1.16 (d, 6H, 2CH3 of isopropyl), 2.87 (m, 1H, CH of isopropyl), 7.2 (d, 4H, J = 9,
ArH), 7.42 (d, 2H, J = 5.5, ArH), 7.58 (d, 2H, J = 5.5, ArH), 14.10 (s, 1H, SH).
5-(4-tert-Butylphenyl)-4-phenyl-4H-[1,2,4]triazole-3-thiol (S2). Yield 85%, 1H-NMR (DMSO-d6):
Yield 76%, 1H-NMR
δ
δ
1.22 (s, 9H, tert-butyl), 7.22 (d, 2H, J = 8.5, ArH), 7.36 (m, 4H, J = 9.5, ArH), 7.53 (m, 3H, J = 10, ArH),
14.09 (s, 1H, SH).
5-(4-tert-Butylphenyl)-4-(3-chlorophenyl)-4H-[1,2,4]triazole-3-thiol (S3), Yield 73%, 1H-NMR
(DMSO-d6):
δ 1.23 (s, 9H, tert-butyl), 7.24 (d, 2H, J = 6, ArH), 7.38 (d, 2H, J = 5.5, ArH), 7.64
(d, 2H, J = 8.5, ArH), 7.92 (d, 2H, J = 8, ArH), 14.14 (s, 1H, SH).
5-(4-tert-Butylphenyl)-4-(4-trifluoromethylphenyl)-4H-[1,2,4]triazole-3-thiol (S4). Yield 72%, 1H-NMR
(DMSO-d6):
δ 1.23 (s, 9H, tert-butyl), 7.25 (d, 2H, J = 9.5, ArH), 7.34 (d, 1H, J = 6.5, ArH), 7.39 (d, 2H, J
= 6.5, ArH),7.55 (t, 1H, J = 8, ArH), 7.60 (m, 2H, J = 9, ArH), 14.20 (s, 1H, SH).
5-(4-Isopropylphenyl)-4-(4-trifluoromethylphenyl)-4H-[1,2,4]triazole-3-thiol (S5). Yield 72%, 1H-NMR
(DMSO-d6):
δ 1.21 (d, 6H, 2 CH3 of isopropyl), 2.94 (m, 1H, CH of isopropyl), 7.3 (m, 4H, J = 12, ArH),
7.46 (d, 2H, J = 7.5, ArH), 7.85 (d, 2H, J = 8, ArH), 14.20 (s, 1H, SH).
5-(4-tert-Butylphenyl)-4-(4-methoxyphenyl)-4H-[1,2,4]triazole-3-thiol (S6). Yield 65%, 1H-NMR
(DMSO-d6):
δ 1.22 (s, 9H, tert-butyl), 3.76 (s, 3H, OCH3), 7.24 (d, 2H, J = 6.5, ArH), 7.32 (d, 2H,
J = 8.5, ArH), 7.43 (t, 1H, J = 9, ArH) 7.54 (d, 2H, J = 5, ArH), 7.67 (s, 1H, ArH), 14.0 (s, 1H, SH).
5-(4-tert-Butylphenyl)-4-p-tolyl-4H-[1,2,4]triazole-3-thiol (S7). Yield 68%, 1H-NMR (DMSO-d6):
δ
1.22 (s, 9H, tert-butyl), 2.37 (s, 3H, CH3), 7.24 (m, 4H, J = 8.25, ArH), 7.32 (d, 2H, J = 8, ArH), 7.37 (d,
2H, J = 8.5, ArH), 14.0 (s, 1H, SH).
5-(4-Isopropylphenyl)-4-(3-methoxyphenyl)-4H-[1,2,4]triazole-3-thiol (S8). Yield 68%, 1H-NMR
(DMSO-d6):
δ 1.12 (d, 6H, 2 CH3 of isopropyl), 2.82 (m, 1H, CH of isopropyl), 3.70 (s, 3H, OCH3), 6.8
(d, 1H, J = 6.5, ArH), 6.91 (t, 1H, J = 4.5, ArH), 7.06 (dd, 1H, J = 8.5, ArH), 7.20 (d, 2H, J = 8, ArH), 7.25
(d, 2H, J = 8, ArH), 7.39 (t, 1H, J = 9.5, ArH), 14.09 (s, 1H, SH).
5-(4-tert-Butylphenyl)-4-(3-bromophenyl)-4H-[1,2,4]triazole-3-thiol (S9). Yield 55%, 1H-NMR
(DMSO-d6):
δ 1.22 (s, 9H, tert-butyl), 7.21 (d, 1H, J = 9, ArH), 7.33 (t, 1H, J = 8, ArH), 7.32 (d,
1H, J = 4, ArH), 7.60 (d, 2H, J = 8.5, ArH), 7.83 (d, 2H, J = 8.5, ArH), 7.91 (s, 1H, ArH), 14.0 (s, 1H, SH).
4-(4-Fluorophenyl)-5-(4-isopropylphenyl)-4H-[1,2,4]triazole-3-thiol (S10). Yield 64%, 1H-NMR
(DMSO-d6):
δ 1.16 (d, 6H, 2 CH3 of isopropyl), 2.87 (m, 1H, CH of isopropyl),), 7.2 (m, 4H, J =
9, ArH), 7.42 (d, 2H, J = 5.5, ArH), 7.58 (d, 2H, J = 5.5, ArH), 14.10 (s, 1H, SH).
5-(4-tert-Butylphenyl)-4-(4-fluorophenyl)-4H-[1,2,4]triazole-3-thiol (S11). Yield 59%, 1H-NMR
(DMSO-d6):
δ 1.22 (s, 9H, tert-butyl), 7.25 (d, 2H, J = 6.5, ArH), 7.36 (m, 4H, J = 12, ArH), 7.44
(m, 2H, J = 12, ArH), 14.11 (s, 1H, SH).

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4.2.6. General Procedure for S-Alkylated Triazole Thiols UoST 1–11
To a solution of triazole thiol S (1-11) (10 mmol) in 70 % ethanol containing KOH (10 mmol), a
solution of 3-trifluorobenzyl chloride (10 mmol) in ethanol was added dropwise. The reaction mixture
was left with stirring at room temperature for 16 h. The formed precipitate was collected by filtration
and re-crystallized from ethanol to give the pure compounds UoST 1–11.
4-(4-Chlorophenyl)-3-(4-isopropylphenyl)-5-(3-trifluoromethylbenzylsulfanyl)-4H-[1,2,4]triazole
(
UoST1). Yield 60%, mp: 210–212 ◦C, 1H-NMR (DMSO-d6):
δ 1.15 (d, 6H, 2 CH3 of isopropyl), 2.85 (m,
1H, CH of isopropyl), 4.44 (s, 2H, CH2), 7.23 (d, 2H, J = 8.5, ArH), 7.25 (d, 2H, J = 2.5, ArH), 7.26 (d, 2H,
J = 2, ArH), 7.54 (m, 2H, J = 9.5, ArH), 7.56 (d, 1H, J = 2, ArH), 7.62 (m, 2H, J = 9, ArH), 7.64 (s, 1H,
ArH). 13C-NMR (DMSO-d6): δ 23.71, 33.39, 36.19, 123.97, 124.38, 124.41, 125.35, 125.68, 125.71, 126.89,
128.13, 129.19, 129.44, 129.76, 129.77, 130.14, 132.89, 133.33, 134.90, 139.17, 150.69, 151.05, 154.75. MS
analysis for C25H21ClF3N3S: Calcd mass: 487.97, found (m/z, ES+): 488.27
3-(4-tert-Butylphenyl)-4-phenyl-5-(3-trifluoromethylbenzylsulfanyl)-4H-[1,2,4]triazole
Yield 70%, mp: 198–200 ◦C. 1H-NMR (DMSO-d6):
1.22 (s, 9H, tert-butyl), 4.46 (s, 2H, CH2), 7.24 (m,
3H, J = 8.25, ArH), 7.34 (d, 2H, J = 8, ArH), 7.51 (m, 4H, J = 8.5, ArH), 7.63 (t, 2H, J = 9.5, ArH), 7.70 (s,
1H, ArH). 13C-NMR (DMSO-d6):
30.95, 34.62, 35.90, 120.97, 123.13, 123.81, 124.29, 125.50, 125. 56,
(UoST2).
δ
δ
125.63, 125.67, 127.67, 127.77, 128.86, 129.12, 129.37, 129.65, 130.25, 133.28, 133.97, 139.18, 151.12, 152.69,
154.53. MS analysis for C26H24F3N3S: Calcd mass: 467.16, found (m/z, ES+): 468.23.
3-(4-tert-Butylphenyl)-4-(3-chlorophenyl)-5-(3-trifluoromethylbenzylsulfanyl)-4H-[1,2,4]triazole
(
UoST3). Yield 70%, mp: 230–232 ◦C, 1H-NMR (DMSO-d6):
δ 1.23 (s, 9H, tert-butyl), 4.45 (s, 2H, CH2),
7.21 (dd, 1H, J = 2, ArH), 7.28 (dd, 2H, J = 2, ArH), 7.38 (dd, 2H, J = 2.5, ArH), 7.43 (t, 1H, J = 4, ArH),
7.54 (m, 2H, J = 4, ArH), 7.60 (m, 1H, J = 3, ArH), 7.63 (m, 2H, J = 5, ArH), 7.66 (s, 1H, ArH). 13C-NMR
(DMSO-d6):
δ 30.93, 34.64, 36.20, 123.10, 123.59, 124.28, 124.30, 125.27, 125.58, 125.61, 125.64, 126.83,
127.69, 127.84, 129.11, 129.37, 131.56, 133.24, 133.98, 135.29, 139.17, 150.92, 152.84, 154.46. MS analysis
for C26H23ClF3N3S: Calcd mass: 501.13, found (m/z, ES+): 502.25.
3-(4-tert-Butylphenyl)-5-(3-trifluoromethylbenzylsulfanyl)-4-(4-trifluoromethylphenyl)-4H-[1,2,4]triazole
(
UoST4). Yield 70%, mp: 224–226 ◦C, 1H-NMR (DMSO-d6):
δ
1.24 (s, 9H, tert-butyl), 4.43 (s, 2H, CH2),
7.26 (dd, 2H, J = 7.5, ArH), 7.39 (dd, 2H, J = 7.5, ArH), 7.48 (d, 2H, J = 9, ArH), 7.53 (m, 1H, J = 8.5,
ArH), 7.62 (m, 2H, J = 8, ArH), 7.65 (s, 1H, ArH), 7.87 (d, 2H, J = 8.5, ArH). 13C-NMR (DMSO-d6):
δ
30.827, 34.54, 36.12, 123.44, 124.156, 124.18, 125.56, 126.99, 127.02, 127.65, 128.83, 128.98, 129.23, 129.49,
129.97, 130.22, 133.10, 137.50, 138.99, 150.64, 152.71, 154.33. MS analysis for C27H23F6N3S: Calcd mass:
535.15, found (m/z, ES+): 536.28.
4-(3-Bromophenyl)-3-(4-isopropylphenyl)-5-(3-trifluoromethylbenzylsulfanyl)-4H-[1,2,4]triazole
(
UoST5). Yield 60%, mp: 201–203 ◦C, 1H-NMR (DMSO-d6):
δ 1.15 (d, 6H, 2 CH3 of isopropyl), 2.84 (m,
1H, CH of isopropyl), 4.5 (s, 2H, CH2), 7.24 (m, 5H, J = 9.5, ArH), 7.43 (t, 1H, J = 8, ArH), 7.54 (t, 1H, J
= 9, ArH), 7.57 (t, 1H, J = 7.5, ArH), 7.62 (d, 2H, J = 10, ArH), 7.67 (s, 1H, ArH), 7.73 (dd, 1H, J = 8,
ArH). 13C-NMR (DMSO-d6): δ 23.51, 33.19, 36.05, 122.03, 123.00, 123.98, 124.11, 125.49, 126.63, 127.08,
127.87, 127.89, 129.21, 129.52, 130.60, 133.16, 135.28, 139.09, 150.03, 151.11, 154.40. MS analysis for
C25H21BrF3N3S: Calcd mass: 532.04, found (m/z, ES+): 532.01
3-(4-tert-Butylphenyl)-4-(4-methoxyphenyl)-5-(3-trifluoromethylbenzylsulfanyl)-4H-[1,2,4]triazole
◦
1
(
UoST6). Yield 47%, mp: 204–206 C, H-NMR (DMSO-d6):
OCH3), 4.44 (s, 2H, CH2), 7.02 (d, 2H, J = 9, ArH), 7.13 (d, 2H, J = 8.5, ArH), 7.29 (d, 2H, J = 8.5, ArH),
7.36 (d, 2H, J = 8.5, ArH), 7.55 (t, 1H, J = 8, ArH), 7.65 (m, 3H, J = 11, ArH). 13C-NMR (DMSO-d6):
δ 1.21 (s, 9H, tert-butyl), 3.79 (s, 3H,
δ
30.02, 32.54, 36.12, 55.52, 123.14, 124.56, 124.86, 126.56, 126.99, 127.22, 127.75, 128.23, 129.00, 129.23,
129.54, 129.67, 131.22, 132.10, 136.45, 139.39, 150.64, 151.72, 154.33. MS analysis for C27H26F3N3OS:
Calcd mass: 497.17, found (m/z, ES+): 498.30.
3-(4-tert-Butylphenyl)-4-p-tolyl-5-(3-trifluoromethylbenzylsulfanyl)-4H-[1,2,4]triazole (UoST7). Yield
47%, mp: 184–186 ◦C, 1H-NMR (DMSO-d6):
δ
1.22 (s, 9H, tert-butyl), 2.36 (s, 3H, CH3), 4.45 (s, 2H,
CH2), 7.11 (d, 2H, J = 6.5, ArH), 7.29 (m, 4H, J = 9, ArH), 7.36 (d, 2H, J = 4.5, ArH), 7.54 (t, 1H, J = 7.5,
ArH), 7.63 (m, 2H, J = 8.5, ArH), 7.68 (s, 1H, ArH). 13C-NMR (DMSO-d6):
20.86, 30.96, 34.62, 35.79,
δ

Molecules 2020, 25, 1463
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123.13, 123.87, 124.24, 124.26, 125.57, 125.61, 125.64, 127.49, 127.55, 128.83, 129.09, 129.34, 129.63, 130.52,
131.38, 133.27, 139.97, 151.29, 152.64, 154.51. MS analysis for C27H26F3N3S: Calcd mass: 481.18, found
(m/z, ES+): 482.22.
3-(4-Isopropylphenyl)-4-(4-methoxyphenyl)-5-(3-trifluoromethylbenzylsulfanyl)-4H-[1,2,4]triazole
(
UoST8). Yield 47%, mp: 214–216 ◦C,1H-NMR (DMSO-d6):
δ 1.13 (d, 6H, 2 CH3 of isopropyl), 2.85 (m,
1H, CH of isopropyl), 3.75 (s, 3H, OCH3), 4.45 (s, 2H, CH2), 6.74 (dd, 1H, J = 8.5, ArH), 6.90 (t, 1H, J =
6.5, ArH), 7.09 (dd, 1H, J = 6.5, ArH), 7.21 (d, 2H, J = 5, ArH), 7.29 (d, 2H, J = 5, ArH), 7.38 (t, 1H, J =
7.5, ArH), 7.54 (t, 1H, J = 7.5, ArH), 7.61 (d, 1H, J = 8.5, ArH), 7.67 (d, 1H, J = 7, ArH), 7.72 (s, 1H,
ArH).13C-NMR (DMSO-d6):
δ 23.51, 33.18, 35.66, 55.52, 113.61, 115.44, 119.58, 124.08, 124.11, 125.52,
125.55, 126.52, 127.69, 128.95, 129.20, 129.45, 130.69, 133.15, 134.91, 139.13, 150.20, 154.35, 159.94. MS
analysis for C26H24F3N3OS: Calcd mass: 483.16, found (m/z, ES+): 484.23.
3-(4-Isopropylphenyl)-4-(4-fluorophenyl)-5-(3-trifluoromethylbenzylsulfanyl)-4H-[1,2,4]triazole
(
UoST10). Yield 56%, mp: 203–205 ◦C, 1H-NMR (DMSO-d6 ):
δ 1.23 (d, 6H, 2 CH3 of isopropyl), 2.97
(m, 1H, CH of isopropyl), 4.95 (s, 2H, CH2), 7.23 (d, 2H, J = 8.5, ArH), 7.38 (m, 3H, J = 10, ArH), 7.54 (d,
2H, J = 7.5, ArH), 7.67 (d, 2H, J = 8, ArH), 7.72 (d, 1H, J = 5, ArH), 7.87 (d, 2H, J = 5, ArH).13C-NMR
(DMSO-d6):
δ 23.02, 33.19, 36.04, 122.52, 123.88, 124.41, 125.45, 125.58, 125.66, 126.89, 127.88, 128.13,
129.19, 129.44, 130.14, 132.89, 135.27, 139.17, 150.69, 151.05, 154.39. MS analysis for C25H21F4N3OS:
Calcd mass: 471.14, found (m/z, ES+): 472.04
3-(4-tert-Butylphenyl)-4-(4-fluorophenyl)-5-(3-trifluoromethylbenzylsulfanyl)-4H-[1,2,4]triazole
(
UoST11). Yield 65%, mp: 223–225 ◦C, 1H-NMR (DMSO-d6 ):
δ
1.23 (s, 9H, tert-butyl), 4.45 (s, 2H,
CH2), 7.26 (d, 2H, J = 6.5, ArH), 7.34 (m, 6H, J = 9.5, ArH), 7.54 (t, 1H, J = 8, ArH), 7.63 (m, 2H, J =
7.5, ArH), 7.68 (s, 1H, ArH). 13C-NMR (DMSO-d6):
30.67, 34.51, 35.75, 116.79 116.98, 123.67, 124.11,
δ
125.46, 125.51, 125.54, 127.49, 128.96, 129.20, 129.49, 130.13, 130.24, 133.16, 139.08, 151.08, 152.52, 161.38.
MS analysis for C26H23F4N3S: Calcd mass: 485.18, found (m/z, ES+): 486.22.
4.3. Organisms and Growth Conditions
The fungal strains used in this study were C. albicans (SC5314) and C. auris clinical isolate (obtained
from the Centers for Disease Control and Prevention (CDC), Atlanta, GA, USA, South Asian clade,
bronchoalveolar lavage]. Each fungal strain was inoculated into sterile Yeast Potato Dextrose broth
(YPD) and incubated for 24 h at 37 ◦C.
4.4. Determination of the Antifungal Activity
The anti-Candida activities of UoST1
on agar plates and liquid broth media according to modified Clinical and Laboratory Standards Institute
(CLSI) and as described by Soliman et al. [12
37]. Briefly, 0.1 mL containing 104 CFU/mL was spread on
Sabouraud dextrose agar (SDA) plates. The plates were then incubated at 37 ◦C for 24 h with filter discs
(8 mm diameter) saturated with different dilutions of the compounds (1–100 g/mL). The inhibition
–11 compounds and the UoST5-loaded NPs were measured
,
µ
zones (mm) were measured by determining the diameter of clear area. Similarly, the activity in liquid
media was measured by incubating the aforementioned concentrations o◦f the compounds into YPD
broth media inoculated with 104 CFU/mL in 96-well microplates at 37 C for 48 h. The turbidity
representing the microbial growth was measured by microplate reader (LT-4500, Labtech, Pocklington,
York, UK) at OD600. Each test was performed in triplicate. Amphotricin-B (Cat#46006, Sigma-Aldrich)
was employed as positive control. Cultures containing drug-free NPs, and cultures containing YPD
broth media without the compounds or antimicrobials were employed as negative controls.
4.5. Preparation of UoST5-Loaded NPs
Polymeric NPs were prepared by single emulsion/evaporation method [26]. Fifty
µg of PLGA and
5
µg of UoST5 were mixed in 2 mL of DCM. The organic solution was then added to 20 mL aqueous
solution containing 2.5% PVA and emulsified by sonication for three successive cycles of 1 min/cycle at
30% voltage efficiency over an ice bath. The formed emulsion was stirred at 1000 rpm for 1 h at room

Molecules 2020, 25, 1463
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temperature under vacuum until complete evaporation of DCM. The produced NPs were separated by
centrifugation at 13,000 rpm for 40 min. The NPs were washed twice by chilled water and stored at
−20 ◦C.
4.6. Characterization of UoST5-Loaded NPs
Entrapment Efficiency
For the appropriate determination of the entrapment efficiency of the drug (UoST5), an efficient
assay was developed based on the UV/VIS spectrophotometery absorbance of the drug. The maximum
absorbance (ňmax) of UoST5 was determined by UV/VIS spectrophotometer (SYNERGY H1, Biotek Ltd,
Winoosk, VT, USA) and the selected ňmax (350 nm) was validated based on the selectivity, linearity and
quantification limit. A calibration curve was developed for UoST5 in DCM (Supplementary Figure
S1). The prepared NPs were mixed with DCM in order to completely dissolve the entrapped drug.
The amount of entrapped drug was measured at 350 nm and the percentage of entrapment efficiency
was calculated using the following formula (Equation (1)):
Entrapment efficiency % = (Amount of drug entrapped/Total amount of drug used) × 100 (1)
4.7. Topology and Particle Size Measurements
The morphological characteristics of the UoST5-loaded NPs were evaluated using a scanning
electron microscope (JSM-633OF; JEOL Ltd, Tokyo, Japan). NPs samples were suspended in distilled
water and a drop of the solution was placed on a clean slide cover and left to dry under vacuum.
The dried sample was mounted on carbon tape and sputter-coated with gold. The gold-coated samples
were scanned and photomicrographs were taken at an acceleration voltage of 3 kV. The effective
diameter, the size distribution, and zeta-potential of the developed NPs were measured by a Zetasizer
(Malvern, Cambridge, UK). NPs were diluted with distilled water and the effective diameter a◦nd
polydispersity index were determined using the Dynamic Light Scattering (DLS) mode at 25 C.
The Laser Doppler Velocimetry (LDV) mode was employed to measure the zeta potential (mV).
4.8. In-Vitro Drug Release Study
Certain weights of NPs equivalent to 2.1 mg of UoST5 were suspended in dialysis membrane
(MWCO = 12 kDa). The membranes were then immersed in 10 mL release media consisting of 0.9%
◦
NaCl: isopropanol (85:15) followed by incubation in water bath at 37 C with shaking at 50 rpm.
Samples were taken at different time intervals and the concentrations of UoST5 were determined.
The cumulative percentage release (%) was calculated according to the following formula (Equation (2)):
The cumulative percentage release (%) = [Volume of sample
(2)
withdrawn (ml)/Bath volume (v)] × P (t − 1) + Pt
where Pt = percentage release at time (t) and P (t − 1) = percentage release previous to t.
4.9. Cell Toxicity Assay Using MTT Staining
The cell viability was performed using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium
bromide (MTT) assay as described before [38]. Briefly, 96-well micro-plates were seeded with
normal fibroblast cells (F180) (4000 cells per well) for 24 h. The best candidates UoST5
, 7, 8, 11 at 3
µ
g/mL and UoST5-NPs at 3, 6.25, 12.5 g/mL were suspended in DMEM media and filter sterilized
µ
prior to application on seeded cells. The color change was measured using a Multiskan Go instrument
(Thermo Fisher Scientific, Japan) at 570 nm. Each experiment was repeated six times. Cell viability
was calculated, using the following formula (Equation (3)):
% of living cells = (OD experimental)/(OD control) × 100
(3)

Molecules 2020, 25, 1463
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where % of cell death was calculated by subtracting the living cells from the total number of cells [39].
4.10. Statistical Analysis
The data was collected and graphed using GraphPad Prism (5.04, GraphPad Inc., La Jolla, CA,
USA). The data were analyzed by two-way analysis of variance (ANOVA) using Bonferroni’s Multiple
Comparison Test. A P-value < 0.05 was considered as significant.
5. Conclusions
In conclusion, we have developed some novel azole derivatives by replacing the toxic aldehyde
group of cuminaldehyde, a natural essential oil, with 1,2,4-triazole rings and the introduction of
a 3-trifluoromethylbenzylsulfanyl moiety to selectively enhance the antifungal activity and reduce
the toxicity. The anti-Candida activity of the best candidate UoST5 was enhanced and prolonged
by encapsulation in polymeric NPs. The formulated drug showed significant anti-Candida activities
against C. albicans and C. auris, known multidrug-resistant human pathogens. The superior and
prolonged activities were proposed to be due to the nature of the polymer employed since it can
afford favorable adherence characteristics to Candida cells and hence can provide direct contact while
establishing continuous release.
Supplementary Materials: The following are available online. Supplementary Figure S1 and spectroscopy date
of compound UoST5 including mass, 1H-NMR and 13C-NMR spectra.
Author Contributions: R.H. synthesized the compounds and helped run the anti-Candida and toxicity activity
assays. B.F. developed the nanoparticles and studied their characteristics and activities. A.M.H. helped run the
anti-Candida and toxicity activity assays. M.H. and M.R.-Q. provided the polymer and advice on the development
of nanoparticles. S.S.M.S. designed and planned the manuscript. R.H., B.F. and S.S.M.S. interpreted the data, wrote
the first draft and the final version of the manuscript. All others helped in editing the manuscript. All authors
have read and agreed to the published version of the manuscript.
Funding: This work was fully supported by grants from University of Sharjah, U.A.E. to S.S. and partially
supported by an Al Jalila Foundation Research Fund grant (AJF201777) to M.H.
Conflicts of Interest: The authors declare there is no competing interest.
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2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
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