New potent antifungal triazole alcohols containing N-benzylpiperazine carbodithioate moiety: Synthesis, in vitro evaluation and in silico study

Article abstract of DOI:10.1016/j.bioorg.2019.103060

A number of 1H-1,2,4-triazole alcohols containing N-(halobenzyl)piperazine carbodithioate moiety have been designed and synthesized as potent antifungal agents. In vitro bioassays against different Candida species including C. albicans, C. glabrata, C. parapsilosis, C. krusei, and C. tropicalis revealed that the N-(4-chlorobenzyl) derivative (6b) with MIC values of 0.063–0.5 μg/mL had the best profile of activity, being 4–32 times more potent than fluconazole. Docking simulation studies confirmed the better fitting of compound 6b in the active site of lanosterol 14α-demethylase (CYP51) enzyme, the main target of azole antifungals. Particularly, the potential of compound 6b against fluconazole-resistant isolates along with its minimal toxicity against human erythrocytes and HepG2 cells make this prototype compound as a good lead for discovery of potent and safe antifungal agents.

Full text of DOI:10.1016/j.bioorg.2019.103060

Accepted Manuscript
New potent antifungal triazole alcohols containing N-benzylpiperazine carbo-
dithioate moiety: Synthesis, in vitro evaluation and in silico study
Yaser Mahmoudi, Hamid Badali, Seyedeh Mahdieh Hashemi, Mahsa Ansari,
Hamed Fakhim, Marjan Fallah, Mohammad Shokrzadeh, Saeed Emami
PII:
S0045-2068(18)31331-2
https://doi.org/10.1016/j.bioorg.2019.103060
103060
DOI:
Article Number:
Reference:
YBIOO 103060
Bioorganic Chemistry
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Revised Date:
Accepted Date:
16 November 2018
26 May 2019
7 June 2019
Please cite this article as: Y. Mahmoudi, H. Badali, S. Mahdieh Hashemi, M. Ansari, H. Fakhim, M. Fallah, M.
Shokrzadeh, S. Emami, New potent antifungal triazole alcohols containing N-benzylpiperazine carbodithioate
moiety: Synthesis, in vitro evaluation and in silico study, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/
j.bioorg.2019.103060
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New potent antifungal triazole alcohols containing N-benzylpiperazine
carbodithioate moiety: Synthesis, in vitro evaluation and in silico study
Yaser Mahmoudi a, Hamid Badali b, Seyedeh Mahdieh Hashemi c, Mahsa Ansari d, Hamed
Fakhim e, Marjan Fallah f, Mohammad Shokrzadeh f, Saeed Emami c,*
a Student Research Committee, Faculty of Pharmacy, Mazandaran University of Medical Sciences,
Sari, Iran
b Department of Medical Mycology & Parasitology/ Invasive Fungi Research Center (IFRC), Faculty
of Medicine, Mazandaran University of Medical Sciences, Sari, Iran
c
Department of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Faculty of
Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran
d
Pharmaceutical Sciences Research Center, Student Research Committee, Faculty of Pharmacy,
Mazandaran University of Medical Sciences, Sari, Iran
e Department of Medical Parasitology & Mycology/Cellular and Molecular Research Center, Urmia
University of Medical Sciences, Urmia, Iran
f
Department of Toxicology and Pharmacology, Faculty of Pharmacy, Mazandaran University of
Medical Sciences, Sari, Iran
Abstract:
A
number of 1H-1,2,4-triazole alcohols containing N-(halobenzyl)piperazine
carbodithioate moiety have been designed and synthesized as potent antifungal agents. In vitro
bioassays against different Candida species including C. albicans, C. glabrata, C. parapsilosis, C.
krusei, and C. tropicalis revealed that the N-(4-chlorobenzyl) derivative (6b) with MIC values of
0.063-0.5 µg/mL had the best profile of activity, being 4-32 times more potent than fluconazole.
Docking simulation studies confirmed the better fitting of compound 6b in the active site of lanosterol
14α-demethylase (CYP51) enzyme, the main target of azole antifungals. Particularly, the potential of
compound 6b against fluconazole-resistant isolates along with its minimal toxicity against human
erythrocytes and HepG2 cells make this prototype compound as a good lead for discovery of potent
and safe antifungal agents.
Keywords: Azole antifungals; Antifungal activity; 1H-1,2,4-triazole, Lanosterol 14α-demethylase
*Correspondence: Saeed Emami, Department of Medicinal Chemistry, Faculty of Pharmacy,
Mazandaran University of Medical Sciences, Sari, Iran
E-mail: sdemami12@gmail.com; semami@mazums.ac.ir
Phone: 0098-11-33543082; Fax: 0098-11-33543084
1

1. Introduction
In recent years, epidemiological studies confirm that the infectious diseases can be caused by
bacteria, viruses, fungi or parasites, affecting millions of people worldwide and they are one
of the most common causes of morbidity and mortality [1]. These diseases generally can
happen to anyone and seem to be conveniently curable, but in particular in most susceptible
people with weakened immune system and those hospitalized with tumors, HIV/AIDS and
with other serious diseases are often fatal [2]. Excessive use of antimicrobial and
immunosuppressive agents leads to an increasing incidence of the multi-drug resistant
infections and mortality rates [3]. Hence, discovery of new more effective antimicrobials,
including antifungal agents is required [4]. Commonly used antifungal drugs are polyenes,
azoles, allylamines and echinocandins [5]. Among them, triazoles (such as fluconazole,
itraconazole, voriconazole, and posaconazole) are the most interesting class of antifungal
drugs because of their broad spectrum, high potency, and having a specific target enzyme [6].
Triazoles act by inhibiting the cytochrome P450 14α-demethylase (CYP51) enzyme. 14α-
demethylase is involved in the oxidative demethylation of lanosterol, as a key reaction in the
biosynthesis of ergosterol in the fungal cell membrane. The inhibition of ergosterol synthesis
leads to the prevention of fungal cell growth or fungal cell death [7].
Studies on the pharmacological properties of triazole antifungals reveal that they have more
advantages over imidazoles. The presence of 1,2,4-triazole ring (instead of imidazole) with
increased polarity resulted in improving solubility and decreasing the binding to plasma
proteins, as well as better specificity of these drugs for fungal enzymatic systems [6,8].
Among the triazole antifungals, fluconazole is the most widely used worldwide because of its
low toxicity, excellent safety profile, and linear pharmacokinetics. However, some drawbacks
of this drug have encouraged medicinal chemists for design and discovery of novel antifungal
agents based on the triazole alcohol structure of fluconazole [9]. Structurally, the difference
between newer azole antifungals and fluconazole mainly lies on the type of side chain
attached to the carbinol center of triazole alcohol scaffold. Accordingly, most of the recent
efforts aim to optimize this part of molecule, which can be well accommodated in the
hydrophobic pocket of target enzyme, 14α-demethylase [10].
On the other hand, carbodithioate (or dithiocarbamate) has been considered as a versatile
functional group in the bioactive compounds. In particular, carbodithioates have been used as
2

fungicide in agriculture extensively [11]. Recently, a series of azole-carbodithioate hybrids
have been synthesized and evaluated by Kumar and co-workers as vaginal anti-Candida
contraceptive agents [12]. Monti et al. investigated a number of N-mono- and N,N-
disubstituted carbodithioates against three β-carbonic anhydrases from the fungal pathogens,
Candida albicans, Candida glabrata, and Cryptococcus neoformans. Their results showed
that carbodithioates can strongly inhibit the beta-class fungal carbonic anhydrases at
subnanomolar to the micromolar range [13].
In continuation of our work on fluconazole modified antifungal agents [14-16], we designed a
new series of triazole alcohols containing N-benzylpiperazine carbodithioate moiety (Fig. 1).
Indeed, one triazole ring of fluconazole has been substituted by N-(halobenzyl)piperazine
carbodithioate scaffold. The (substituted)benzyl part of the designed molecules is found in
several azole antifungals including miconazole, econazole, oxiconazole, isoconazole and
sulconazole (Fig. 1). It has been postulated that the N-(halobenzyl)piperazine carbodithioate
side chain attached to the carbinol center of triazole alcohol can be well accommodated in the
hydrophobic pocket of target enzyme 14α-demethylase. Thus we report here synthesis,
antifungal activity and molecular docking of new triazole alcohols 6a-i containing N-
(halobenzyl)piperazine carbodithioate moiety.
2. Results and discussion
2.1. Chemistry
The target compounds 6a-i were synthesized following the synthetic route illustrated in
Scheme 1. The intermediate N-benzylpiperazine derivatives 3a-f were obtained by the
reaction of benzyl chloride derivatives 2a-f with the excess of piperazine in refluxing ethanol.
On the other hand, the oxirane derivatives 5a,b were prepared from the desired
phenacyltriazole (4a or 4b) in the presence of trimethylsulfoxonium iodide and NaOH 20%
in toluene. The final compounds 6a-i were synthesized by the sequential one-pot reaction of
N-benzylpiperazine derivatives 3a-f, carbon disulfide and oxiranes (5a or 5b) in the presence
of triethylamine (TEA) in ethanol. Accordingly, the in situ generated carbodithioate salt
underwent ring opening reaction with oxirane to afford desired carbodithioate alcohol 6. The
structural assignments of new compounds were based on their spectral data (IR, NMR, and
MS) as reported in the Experimental section.
3

2.2. Antifungal activity against fluconazole-susceptible and fluconazole-susceptible dose-
dependent Candida species
Clinically, Candida albicans is one of the major pathogens in immunecompromised patients.
Recent epidemiological investigations demonstrated that fungal infections shift towards non-
albicans Candida species with increasing resistant to the available antifungal agents. The
most prominent non-albicans species are C. glabrata, C. parapsilosis, C. krusei, and C.
tropicalis, which being responsible for more incidence of invasive candidiasis respect to C.
albicans [17,18]. Accordingly, the in vitro antifungal susceptibility testing of final
compounds 6a-i were performed against C. albicans and non-albicans species including C.
glabrata, C. parapsilosis, C. krusei, and C. tropicalis by using broth microdilution method
[19,20]. The obtained MIC (minimum inhibitory concentration) values of tested compounds
in comparison with fluconazole as standard antifungal drug are summarized in Table 1.
As observed in Table 1, all of tested compounds exhibited significant inhibitory activity
against fluconazole-susceptible and fluconazole-susceptible dose-dependent Candida species
(MICs ≤4 µg/mL). A survey on obtained results revealed that N-(4-chlorobenzyl) derivative
6b with MIC values of 0.063-0.5 µg/mL was the most potent compound. The antifungal
potency of this derivative was 4-32 times greater than that of the standard drug fluconazole.
Furthermore, the 3-chlorobenzyl analog 6a had a good profile of activity against both
albicans and non-albicans species of Candida.
The most of compounds listed in Table 1, contain 2,4-difluorophenyl as found in the
fluconazole structure. However, a limited series of 2,4-dichlorophenyl analog were also
synthesized and tested. The latter pharmacophoric part could be found in imidazole
antifungals i.e., miconazole and in triazole drugs such as terconazole and itraconazole. In
general, the obtained MICs in our study indicated that 2,4-difluorophenyl derivatives were
more potent than their dichlorophenyl analogs.
As mentioned in introduction, some imidazole antifungals possess mono- or di-
halosubstituted benzyl moiety as side chain. Thus in our SAR study, the attached benzyl
moiety to the piperazine ring was altered by mono- and dihalo-substitution. In general, the
mono-chlorobenzyl derivatives (6a and 6b) were inherently potent antifungal compounds.
4

However, replacement of chlorine with bromine in compound 6b resulted in compound 6f
with same or lower activity. Thus, 4-chloro is more preferred than 4-bromo substituent.
Introduction of second chlorine atom on benzyl group also had no positive effect as observed
with dichlorobenzyl derivatives 6c-e, 6h and 6i.
2.3. Antifungal activity against fluconazole-resistant Candida species
As resistance to the currently available antifungal agents such as fluconazole is emerging in
many of Candida species, thus the antifungal effect of all synthesized compounds was also
investigated against fluconazole-resistant species of Candida including C. albicans (n= 3), C.
parapsilosis (n= 1) and C. krusei (n= 2). The results were presented as MIC values in Table
2. The MIC values of fluconazole against C. albicans and C. krusei isolates were ≥64 µg/mL.
The representative compound 6b effectively inhibited the growth of C. albicans and C. krusei
isolates at the concentrations of 2-16 µg/mL. The MIC of this compound against C.
parapsilosis was 2 µg/mL, being significantly lower than that of fluconazole (MIC ≥ 8
µg/mL). The most potent compounds were 6d and 6f (MIC= 0.25 µg/mL), displaying 32-time
higher potency compared to fluconazole against C. parapsilosis. Moreover, the remaining
compounds were also significantly more potent than fluconazole against C. parapsilosis.
Overall, compound 6b showed good profile of activity against fluconazole-resistant species
of Candida.
2.4. Toxicity of compound 6b against erythrocytes
In order to check the safety profile of designed compounds, the toxicity of representative
compound 6b was evaluated against human erythrocytes by determining their hemolytic
activity. The compound was tested at different concentrations of 1, 10 and 50 µg/mL, in
comparison with standard antifungal agent fluconazole and positive control Triton X-100. As
observed in Fig. 2, while triton X-100 produced 100% hemolysis, only a limited hemolysis
occurred following exposure to compounds 6b at the higher concentration of 50 µg/mL.
Compound 6b as well as fluconazole showed no significant hemolytic activity at the
concentrations of 1 and 10 µg/mL compared to negative control. Therefore, the promising
5

compound 6b displayed minimal toxicity against human erythrocytes at the antifungal MIC
values.
2.5. Effect of compounds 6b on the viability of HepG2 cells
An important issue in the discovery of new antifungal agents is selectivity against fungi cells
and proper safety profile towards host cells. As reported previously, azole antifungals might
have liver toxicity [21,22]. Thus we next investigated the effect of selected compound 6b on
the viability of human hepatoma cells HepG2. The MTT assay result of compound 6b on
HepG2 cells was depicted in Fig. 3. As seen in Fig. 3, the viability of HepG2 cells for this
compound was affected less than 50% at the concentrations of 25, 50 and 100 µg/mL.
Accordingly, the IC50 value of test compound against HepG2 cell line was >100 µg/mL,
being similar to that of fluconazole. As our compound can inhibit the growth of Candida
species at concentrations ≤0.5 µg/mL, thus it has favorable safety profile.
2.6. Docking study
The key enzyme in the ergosterol biosynthetic pathway namely lanosterol 14α-demethylase
(CYP51) is the main target of azole antifungals [6]. The similar structure of our compounds
with azole antifungals prompted us to investigate their interactions with CYP51 enzyme in
comparison with fluconazole. Accordingly, the binding mode of the promising compound 6b
was studied by docking simulation. The structures of CYP51 enzymes originated from C.
glabrata and C. albicans (CGCYP51 and CACYP51, respectively) were used for
computational studies. Before simulation study of the target compound, we validated the
precision of our docking protocol. Towards this end, the cognate ligand itraconazole was
removed from the binding site and re-docked. We found a good fitting between the
localization of the itraconazole upon docking and that of crystal structure as evidenced from
the root mean square deviation values of 1.58 and 1.71 Å for CGCYP51 and CACYP51,
respectively.
Docking experiments were carried out on both (R)- and (S)-enantiomers of the selected
compound. Both stereoisomers interacted with CYP51 through a similar binding mode. As
shown in Fig. 4, (R)-6b binds to the active site of CGCYP51 with a binding energy of −10.17
6

kcal/mol and a coordinated bond forming distance 2.51 Å away from the heme iron. In the
case of CACYP51, the binding energy was −9.66 kcal/mol and the N4 atom of triazole
nucleus of the compound (R)-6b was coordinated to the heme iron of CACYP51 with a bond
distance of 2.31 Å. It should be noted that the binding energy and coordination bond distance
for fluconazole were −6.27 kcal/mol and 2.31Å, respectively. As depicted in Fig. 5, the
difluorophenyl moiety of the molecule (R)-6b is located in a hydrophobic pocket and
interacts with Phe228, Gly303, Ile304, Phe126, Ile131, Thr122 and Tyr132. The piperazinyl
part interacts with the surrounding hydrophobic amino acid residues including Leu376,
Phe380, Ile379, and Tyr118. The terminal 4-chlorobenzyl group binds through the
hydrophobic and van der Waals interactions with His377, Pro375 and Met508. The
carbodithioate and triazole moieties were found to be in contact with Leu121 and Thr311,
respectively. It would appear that the hydrophobic interaction with a larger hydrophobic side
chain residue of compound 6b is responsible for the more potent inhibitory activity of the
prototype compound respect to fluconazole.
3. Conclusion
We have designed and synthesized a number of triazole alcohol-based antifungals by
incorporating N-benzylpiperazine carbodithioate moiety into the phamacophoric framework
of fluconazole. In vitro bioassays against fluconazole-susceptible and fluconazole-susceptible
dose-dependent Candida species revealed that all compounds had potent activity against C.
albicans, C. glabrata, C. parapsilosis, C. krusei, and C. tropicalis, with MIC values ≤4
µg/mL. The N-(4-chlorobenzyl) derivative (6b), displaying MIC values of 0.063-0.5 µg/mL
was the best one. Its activity was 4-32 times higher than that of fluconazole. SAR analysis
demonstrated that the 2,4-difluorophenyl-carbinol is more favorable than 2,4-dichlorophenyl-
carbinol scaffold. Further evaluation against fluconazole-resistant isolates indicated that
compound 6b was active towards C. albicans, C. krusei and C. parapsilosis isolates,
displaying MIC values of 2-16 µg/mL. Toxicity assays against human erythrocytes and
HepG2 cells demonstrated the favorable therapeutic window of this prototype compound
comparable to standard drug fluconazole. Docking simulation studies confirmed the proper
accommodation of compound 6b in the active sites of lanosterol 14α-demethylase (CYP51)
7

enzymes originated from C. glabrata and C. albicans, being in accordance to the greater
activity of compound 6b respect to fluconazole.
4. Experimental
The intermediate compounds 4a,b and 5a,b were synthesized according to the literature
methods [14,23].
4.1. General procedure for the preparation of N-benzylpiperazine derivatives 3a-f
A solution of appropriate benzyl chloride derivative 2a-f (1.0 mmol) and piperazine (3.0
mmol) in ethanol (10 mL) was refluxed for 2 h. After composition of the benzyl chloride
derivative (checked by TLC, solvent n-hexane), and cooling of the reaction mixture, water
(10 mL) was added. Then, the mixture was extracted with ethyl acetate 3 times and the
organic layers were combined and dried (Na2SO4). After evaporation of the solvent under
reduced pressure, the desired product 3a-f was obtained which was use without further
purification.
4.2. General procedure for the preparation of compounds 6a–i
To a mixture of benzylpiperazine derivatives 3a-f (1 mmol) in ethanol (10 ml), triethylamine
(1 mmol) and carbon disulfide (3 mmol) was added and stirred in an ice bath for 2 h. After
consumption of starting materials 3a-f, oxiran derivative (5a or 5b, 1 mmol) was added and
the reaction mixture allowed to stir at room temperature for 24 h. After the completion of the
reaction, water (10 ml) was added and the mixture was left in refrigerator overnight. The
precipitated solid was collected and washed with water and then recrystallized from n-
hexane-diethyl ether to give pure compounds 6a–i.
4.2.1. 2-(2,4-Difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl 4-(3-
chlorobenzyl)piperazine-1-carbodithioate (6a)
8

Yield: 66%; mp: 116-118 °C; IR (νmax, cm-1): 3204, 3068, 2793, 2756, 1616, 1598, 1502,
1
1423, 1251, 1247, 1220, 1143, 1129, 966, 676, 526. H NMR (400 MHz, CDCl3) δ: 2.47-
2.58 (m, 4H, piperazine), 3.52 (s, 2H, CH2 benzylic), 3.85-3.98 (m, 2H, piperazine), 4.01 (d,
1H, J= 15.2 Hz, Ha), 4.29 (d, 1H, J= 15.2 Hz, Hb), 4.31-4.43 (m, 2H, piperazine), 4.68 (d, 1H,
J= 14.0 Hz, Hc), 4.72 (d, 1H, J= 14.4 Hz, Hd), 5.33 (s, 1H, OH), 6.8-6.88 (m, 2H, H-3 and H-
5), 7.17-7.21 (m, 1H, H-6'), 7.23-7.28 (m, 2H, H-4' and H-5'), 7.34 (d, 1H, J= 0.8 Hz, H-2'),
13
7.56-7.61 (m, 1H, H-6), 7.85 (s, 1H, triazole), 8.14 (s, 1H, triazole). C NMR (100 MHz,
DMSO-d6) δ: 46.60 (d, JC,F = 3.8 Hz, CH2-S), 50.17 (1C, piperazine), 52.32 (2C, piperazine),
52.99 (1C, piperazine), 56.81 (C-OH), 60.87 (CH2 benzylic), 73.75 (CH2-N), 104.56 (dd, JC,F
= 25.6 and 27.2 Hz, 2,4-F2Ph C-3), 111.33 (d, JC,F = 20.8 Hz, 2,4-F2Ph C-5), 125.42 (dd, JC,F
= 12.7 and 3.4 Hz, 2,4-F2Ph C-1), 127.57 (C-5′), 127.99 (C-6′), 129.00 (C-4′), 130.54 (d, JC,F
= 8.7 Hz, 2,4-F2Ph C-6), 130.58 (C-2′), 133.45 (C-3′), 140.79 (C-1′), 145.45 (C-5 triazole),
151.11 (C-3 triazole), 159.41 (dd, JC,F = 246.3 and 12.4 Hz, 2,4-F2Ph C-2), 162.40 (dd, JC,F
=
244.9 and 12.4 Hz, 2,4-F2Ph C-4), 195.09 (C=S). MS (m/z, %): 523 (M+, <1), 334 (8), 221
(10), 209 (36), 182 (8), 168 (13), 154 (37), 139 (13), 125 (100), 89 (17), 56 (6). Anal. Calcd
for C23H24ClF2N5OS2: C, 52.72; H, 4.62; N, 13.36. Found: C, 52.55; H, 4.63; N, 13.30.
4.2.2. 2-(2,4-Difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl 4-(4-
chlorobenzyl)piperazine-1-carbodithioate (6b)
Yield: 64%; mp: 134-136 °C; IR (νmax, cm-1): 3208, 3069, 2914, 1615, 1502, 1461, 1274,
1
1224, 1114, 1027, 1030, 966, 848, 676, 479. H NMR (400 MHz, CDCl3) δ: 2.47-2.55 (m,
4H, piperazine), 3.51 (s, 2H, CH2 benzylic), 3.85-3.98 (m, 2H, piperazine), 4.01 (d, 1H, J=
14.8 Hz, Ha), 4.29 (d, 1H, J= 15.2 Hz, Hb), 4.31-4.42 (m, 2H, piperazine), 4.67 (d, 1H, J=
14.0 Hz, Hc), 4.72 (d, 1H, J= 14.0 Hz, Hd), 5.32 (s, 1H, OH), 6.80-6.87 (m, 2H, H-3 and H-
5), 7.26 (d, 2H, J= 8.8 Hz, H-2' and H-6'), 7.32 (d, J= 8.4 Hz, 2H, H-3' and H-5'), 7.55-7.62
(m, 1H, H-6), 7.85 (s, 1H, triazole), 8.13 (s, 1H, triazole). 13C NMR (100 MHz, DMSO-d6) δ:
46.54 (CH2-S), 50.20 (1C, piperazine), 52.29 (2C, piperazine), 52.94 (1C, piperazine), 56.80
(C-OH), 61.57 (CH2 benzylic), 73.73 (CH2-N), 104.55 (dd, JC,F = 24.9 and 27.4 Hz, 2,4-F2Ph
C-3), 111.36 (d, JC,F = 20.3 Hz, 2,4-F2Ph C-5), 125.41 (dd, JC,F = 12.2 and 3.5 Hz, 2,4-F2Ph
C-1), 128.68 (C-3′ and C-5'), 130.44 (m, 2,4-F2Ph C-6), 131.17 (C-2' and C-6'), 132.11 (C-
4′), 137.12 (C-1′), 145.45 (C-5 triazole), 151.11 (C-3 triazole), 159.41 (dd, JC,F = 246.6 and
9

12.3 Hz, 2,4-F2Ph C-2), 162.39 (dd, JC,F = 245.1 and 12.6 Hz, 2,4-F2Ph C-4), 195.07 (C=S).
MS (m/z, %): 523 (M+, <1), 334 (8), 209 (31), 182 (8), 168 (9), 154 (18), 139 (16), 125
(100), 99 (6), 83 (34), 56 (14). Anal. Calcd for C23H24ClF2N5OS2: C, 52.72; H, 4.62; N,
13.36. Found: 52.91; H, 4.49; N, 13.55.
4.2.3. 2-(2,4-Difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl 4-(2,4-
dichlorobenzyl)piperazine-1-carbodithioate (6c)
Yield: 62%; mp: 113-115 °C; IR (νmax, cm-1): 3208, 3069, 2713, 1615, 1502, 1416, 1308,
1
1224; 1114, 1087, 1030, 966, 676, 514, 479. H NMR (400 MHz, CDCl3) δ: 2.54-2.63 (m,
4H, piperazine), 3.62 (s, 2H, CH2 benzylic), 3.86-3.99 (m, 2H, piperazine), 4.01 (d, 1H, J=
14.8 Hz, Ha), 4.29 (d, 1H, J= 14.8 Hz, Hb), 4.31-4.41 (m, 2H, piperazine), 4.68 (d, 1H, J=
14.4 Hz, Hc), 4.72 (d, 1H, J= 14.4 Hz, Hd), 5.32 (s, 1H, OH), 6.80-6.88 (m, 2H, H-3 and H-
5), 7.25 (dd, 1H, J= 8.2 and 2.0 Hz, H-5'), 7.39 (d, 1H, J= 8.4 Hz, H-6'), 7.40 (d, 1H, J= 2.0
Hz, H-3'), 7.56-7.62 (m, 1H, H-6), 7.85 (s, 1H, triazole), 8.13 (s, 1H, triazole). 13C NMR (100
MHz, DMSO-d6) δ: 46.60 (CH2-S), 50.14 (1C, piperazine), 52.41 (2C, piperazine), 53.08
(1C, piperazine), 56.76 (C-OH), 57.76 (CH2 benzylic), 73.70 (CH2-N), 104.56 (t, JC,F = 27.5
Hz, 2,4-F2Ph C-3), 111.35 (d, JC,F = 23.2 Hz, 2,4-F2Ph C-5), 125.41 (dd, JC,F = 13.0 and 3.8
Hz, 2,4-F2Ph C-1), 127.71 (C-5'), 129.22 (C-3'), 130.45 (m, 2,4-F2Ph C-6), 132.70 (C-6'),
132.88 (C-4′), 134.72 (C-1′), 134.79 (C-2'), 145.46 (C-5 triazole), 151.11 (C-3 triazole),
159.41 (dd, JC,F = 246.3 and 12.3 Hz, 2,4-F2Ph C-2), 162.40 (dd, JC,F = 244.6 and 12.7 Hz,
2,4-F2Ph C-4), 195.13 (C=S). MS (m/z, %): 557 (M+, <1), 404 (3), 243 (33), 221 (24), 202
(16), 188 (19), 173 (8), 159 (100), 141 (16), 127 (28), 101 (12), 86 (40), 70 (6), 56 (12). Anal.
Calcd for C23H23Cl2F2N5OS2: C, 49.46; H, 4.15; N, 12.54. Found: C, 49.64; H, 4.11; N,
12.70.
4.2.4. 2-(2,4-Difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl 4-(3,4-
dichlorobenzyl)piperazine-1-carbodithioate (6d)
Yield: 61%; mp: 127-130 °C; IR (νmax, cm-1): 3233, 2824, 1615, 1514, 1499, 1471, 1273,
1
1225, 1137, 1030, 964, 965, 818, 676, 535, 511, 459. H NMR (400 MHz, CDCl3) δ: 2.46-
2.60 (m, 4H, piperazine), 3.49 (s, 2H, CH2 benzylic), 3.84-3.98 (m, 2H, piperazine), 4.00 (d,
10

1H, J= 15.2 Hz, Ha), 4.28 (d, 1H, J= 15.2 Hz, Hb), 4.31-4.42 (m, 2H, piperazine), 4.67 (d, 1H,
J= 14.0 Hz, Hc), 4.72 (d, 1H, J= 14.4 Hz, Hd), 5.32 (s, 1H, OH), 6.79-6.88 (m, 2H, H-3 and
H-5), 7.16 (dd, 1H, J= 8.2 and 1.6 Hz, H-6'), 7.41 (d, 1H, J= 8.0 Hz, H-5'), 7.44 (d, 1H, J=
1.6 Hz, H-2'), 7.55-7.62 (m, 1H, H-6), 7.84 (s, 1H, triazole ), 8.13 (s, 1H, triazole). 13C NMR
(100 MHz, DMSO-d6): 45.94 (2C, piperazine and 1C, CH2-S), 46.96 (CH2 benzylic), 51.15
(1C, piperazine), 52.40 (1C, piperazine), 56.75 (C-OH), 73.66 (CH2-N), 104.57 (t, JC,F = 27.0
Hz, 2,4-F2Ph C-3), 111.39 (d, JC,F = 18.4 Hz, 2,4-F2Ph C-5), 125.37 (d, JC,F = 12.8 Hz, 2,4-
F2Ph C-1), 130.41 (C-6'), 130.46 (C-5'), 130.56 (C-2'), 131.17 (m, 2,4-F2Ph C-6), 131.21 (C-
4′), 131.61 (C-3'), 131.64 (C-1′), 145.54 (C-5 triazole), 151.12 (C-3 triazole), 159.41 (dd, JC,F
= 245.7 and 12.3 Hz, 2,4-F2Ph C-2), 162.41 (dd, JC,F = 245.1 and 12.6 Hz, 2,4-F2Ph C-4),
196.19 (C=S). MS (m/z, %): 557 (M+, <1), 221 (11), 202 (7), 187 (7), 159 (11), 141 (15), 127
(24), 101 (23), 86 (100), 72 (7), 58 (22). Anal. Calcd for C23H23Cl2F2N5OS2: C, 49.46; H,
4.15; N, 12.54. Found: C, 49.65; H, 4.22; N, 12.57.
4.2.5. 2-(2,4-Difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl 4-(2,6-
dichlorobenzyl)piperazine-1-carbodithioate (6e)
Yield: 69%; mp: 110-113 °C; IR (νmax, cm-1): 3696, 3662, 3239, 2920, 2810, 1616, 1597,
1498, 1435, 1272, 1227, 1136, 1102, 992, 964, 763, 673, 511. 1H NMR (400 MHz, CDCl3) δ:
2.61-2.71 (m, 4H, piperazine), 3.81 (s, 2H, CH2 benzylic), 3.86-3.92 (m, 2H, piperazine),
4.01 (d, 1H, J= 14.8 Hz, Ha), 4.22-4.38 (m, 2H, piperazine), 4.29 (d, 1H, J= 15.2 Hz, Hb),
4.68 (d, 1H, J= 14.0 Hz, Hc), 4.72 (d, 1H, J= 14.0 Hz, Hd), 5.35 (br s, 1H, OH), 6.80-6.88 (m,
2H, H-3 and H-5), 7.19 (t, 1H, J= 8.0 Hz, H-4'), 7.33 (d, 2H, J= 8.0 Hz, H-3' and H-5'), 7.56-
7.62 (m, 1H, H-6), 7.84 (s, 1H, triazole), 8.14 (s, 1H, triazole). 13C NMR (100 MHz, DMSO-
d6) δ: 46.62 (CH2-S), 50.30 (1C, piperazine), 51.54 (1C, piperazine), 52.49 (2C, piperazine),
55.83 (C-OH), 56.80 (CH2 benzylic), 73.76 (CH2-N), 104.55 (dd, JC,F = 26.7 and 25.7 Hz,
2,4-F2Ph C-3), 111.34 (d, JC,F = 19.2 Hz, 2,4-F2Ph C-5), 125.41 (dd, JC,F = 9.2 and 2.8 Hz,
2,4-F2Ph C-1), 129.14 (C-3' and C-5'), 130.47 (m, 2,4-F2Ph C-6), 130.58 (C-4'), 133.56 (C-
1′), 136.60 (C-2′ and C-6'), 145.46 (C-5 triazole), 151.11 (C-3 triazole), 159.42 (dd, JC,F
=
246.2 and 12.2 Hz, 2,4-F2Ph C-2), 162.40 (dd, JC,F = 245.00 and 12.4 Hz, 2,4-F2Ph C-4),
194.95 (C=S). MS (m/z, %): 557 (M+, <1), 404 (6), 243 (39), 221 (16), 202 (46), 188 (16),
11

159 (100), 139 (13), 123 (13), 89 (12), 71 (5), 56 (22). Anal. Calcd for C23H23Cl2F2N5OS2: C,
49.46; H, 4.15; N, 12.54. Found: C, 49.32; H, 3.99; N, 12.38.
4.2.6. 2-(2,4-Difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl 4-(4-
bromobenzyl)piperazine-1-carbodithioate (6f)
Yield: 63%; mp: 138-140 °C; IR (νmax, cm-1): 3696, 3239, 2920, 2910, 1597, 1616, 1498,
1435, 1272, 1227, 1136, 1102, 922, 964, 848, 703, 676. 1H NMR (400 MHz, CDCl3) δ: 2.45-
2.61 (m, 4H, piperazine), 3.49 (s, 2H, CH2 benzylic), 3.85-3.98 (br s, 2H, piperazine), 4.01
(d, 1H, J= 15.2 Hz, Ha), 4.29 (d, 1H, J= 15.2 Hz, Hb), 4.31-4.43 (m, 2H, piperazine), 4.67 (d,
1H, J= 14.0 Hz, Hc), 4.72 (d, 1H, J= 14.0 Hz, Hd), 5.33 (s, 1H, OH), 6.82-6.88 (m, 2H, H-3
and H-5), 7.20 (d, 2H, J= 8.4 Hz, H-2' and H-6'), 7.47 (d, 2H, J= 8.4 Hz, H-3' and H-5'),
13
7.56-7.62 (m, 1H, H-6), 7.85 (s, 1H, triazole), 8.14 (s, 1H, triazole). C NMR (100 MHz,
CDCl3) δ: 43.67 (d, JC,F = 4.0 Hz, CH2-S), 50.48 (1C, piperazine), 52.23 (2C, piperazine),
52.48 (1C, piperazine), 57.06 (d, JC,F = 4.9 Hz, C-OH), 61.62 (CH2 benzylic), 75.39 (d, JC,F
=
5.0 Hz, CH2-N), 104.24 (t, JC,F = 26.9 Hz, 2,4-F2Ph C-3), 111.59 (dd, JC,F = 20.5 and 3.2 Hz,
2,4-F2Ph C-5), 121.33 (C-4′), 124.66 (dd, JC,F = 13.9 and 3.5 Hz, 2,4-F2Ph C-1), 130.37 (dd,
JC,F = 9.5 and 5.5 Hz, 2,4-F2Ph C-6), 130.66 (C-2′ and C-6′), 131.57 ( C-3′ and C-5′), 136.29
(C-1′), 144.58 (C-5 triazole), 151.45 (C-3 triazole), 158.71 (dd, JC,F = 245.2 and 12.0 Hz, 2,4-
F2Ph C-2), 162.89 (dd, JC,F = 248.9 and 12.4 Hz, 2,4-F2Ph C-4), 197.65 (C=S). MS (m/z, %):
567 (M+, <1), 424 (10), 314 (3), 253 (45). 234 (12), 221 (47), 197 (23), 182 (61), 169 (100),
171 (98), 154 (20), 139 (40), 119 (30), 102 (9), 90 (45), 78 (81), 56 (94). Anal. Calcd for
C23H24BrF2N5OS2: C, 48.59; H, 4.26; N, 12.32. Found: C, 48.60; H, 4.37; N, 12.30.
4.2.7. 2-(2,4-Dichlorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl 4-(3-
chlorobenzyl)piperazine-1-carbodithioate (6g)
Yield: 53%; mp: 120-124 °C; IR (νmax, cm-1): 2925, 1515, 1469, 1421, 128, 1225, 1133,
1
1019, 598, 810. H NMR (400 MHz, DMSO-d6) δ: 2.80-3.42 (m, 4H, piperazine), 3.60-4.02
(m, 3H, piperazine), 4.05 (d, 1H, J= 13.2 Hz, Ha), 4.32 (s, 2H, CH2 benzylic), 4.38 (m, 1H,
piperazine), 4.40 (d, 1H, J= 13.2 Hz, Hb), 4.88 (d, 1H, J= 14.4 Hz, Hc), 5.10 (d, 1H, J= 14.4
Hz, Hd), 7.33 (dd, 1H, J= 8.8 and 2.0 Hz, H-5), 7.36-7.40 (m, 1H, H-6′), 7.47 (d, 1H, J= 8.8
12

Hz, H-6), 7.53 (t, 1H, J= 8.8 Hz, H-5′), 7.58 (d, 1H, J= 2.4 Hz, H-3), 7.61-7.76 (2H, m, H-2′,
13
and H-4′), 8.05 (s, 1H, triazole), 8.34 (s, 1H, triazole). C NMR (100 MHz, DMSO-d6) δ:
46.08 (CH2-S), 47.50 (1C, piperazine), 50.04 (2C, piperazine), 50.48 (1C, piperazine), 55.64
(C-OH), 57.80 (CH2 benzylic), 74.65 (CH2-N), 127.53 (C-5′), 128.85 (C-5), 129.25 (C-6′),
130.35 (C-4′), 131.41 (C-6), 131.69 (C-3), 133.73 (C-2′), 133.85 (C-2), 133.97 (C-4), 134.90
(C-3′), 137.69 (C-1′), 145.06 (C-1), 149.09 (C-5 triazole), 151.84 (C-3 triazole), 196.92
(C=S). MS (m/z, %): 555 (M+, <1), 407 (3), 314 (5), 270 (50), 252 (41), 235 (43), 219 (21),
203 (14), 187 (12), 160 (14), 128 (18), 98 (5), 83 (100), 64 (29). Anal. Calcd for
C23H24Cl3N5OS2: C, 49.60; H, 4.34; N, 12.57. Found: C, 49.49; H, 4.35; N, 12.50.
4.2.8. 4.2.9. 2-(2,4-Dichlorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl 4-(2,4-
dichlorobenzyl)piperazine-1-carbodithioate (6h)
Yield: 58%; mp: 100-104 °C; IR (νmax, cm-1): 3221, 2922, 1704, 1527, 1505, 1471, 1421,
1274, 1228, 1139, 1049, 995, 969, 806, 677, 454. 1H NMR (400 MHz, CDCl3) δ: 2.49-2.65
(m, 4H, piperazine), 3.47 (br s, 1H, piperazine), 3.62 (s, 1H, CH2 benzylic), 3.82-3.99 (m,
2H, piperazine), 4.29 (d, 1H, J= 14.8 Hz, Ha), 4.40 (br s, 1H, piperazine), 4.43 (d, 1H, J=
14.8 Hz, Hb), 4.88 (d, 1H, J= 14.0 Hz, Hc), 4.95 (d, 1H, J= 14.0 Hz, Hd), 5.48 (s, 1H, OH),
7.26 (d, 1H, J= 2.0 Hz, H-3), 7.23 (dd, 1H, J= 6.8 and 2.0 Hz, H-5′), 7.25 (dd, 1H, J= 6.4 and
2.0 Hz, H-5), 7.38 (d, 1H, J= 8.0 Hz, H-6′), 7.39-7.41 (m, 2H, H-3 and H-3′), 7.75 (d, 1H, J=
8.8 Hz, H-6), 7.85 (s , 1H, triazole ), 8.16 (s, 1H, triazole). 13C NMR (100 MHz, DMSO-d6):
45.56 (CH2-S), 51.40 (1C, piperazine), 52.19 (1C, piperazine), 52.42 (2C, piperazine), 55.46
(C-OH), 57.77 (CH2 benzylic), 74.92 (CH2-N), 127.42 (C-5′), 127.71 (C-5), 129.22 (C-6),
130.48 (C-3′), 131.40 (C-3), 131.73 (C-6′), 132.70 (C-2), 132.88 (C-4), 133.57 (C-4′), 134.73
(C-1′), 134.80 (C-2′), 138.06 (C-1), 145.53 (C-5 triazole), 151.11 (C-3 triazole), 195.08
(C=S). MS (m/z, %): 589 (M+, <1), 279 (7), 256 (40), 218 (73), 202 (48), 181 (23), 159
(100), 145 (5), 128 (25), 113 (9), 99 (13), 85 (15), 64 (42). Anal. Calcd for C23H23Cl4N5OS2:
C, 46.71; H, 3.92; N, 11.84. Found: C, 46.92; H, 3.85; N, 11.61.
4.2.9. 2-(2,4-Dichlorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl 4-(3,4-
dichlorobenzyl)piperazine-1-carbodithioate (6i)
13

Yield: 54 %; mp: 132-135 °C; IR (νmax, cm-1): 3380, 2543, 1708, 1556, 1467, 1416, 1373,
1
1252, 1210, 1090, 1016, 954, 808, 502, 484. H NMR (400 MHz, CDCl3) δ: 2.40-2.60 (m,
4H, piperazine), 3.49 (s, 2H, CH2 benzylic), 3.83-4.01 (m, 2H, piperazine), 4.23-4.30 (m, 1H,
piperazine), 4.28 (d, 1H, J= 15.2 Hz, Ha), 4.32-4.42 (m, 1H, piperazine), 4.43 (d, 1H, J= 15.2
Hz, Hb), 4.88 (d, 1H, J= 14.4 Hz, Hc), 4.95 (d, 1H, J= 14.4 Hz, Hd), 5.48 (s, 1H, OH), 7.16
(dd, 1H, J= 8.0 and 2.0 Hz, H-5′), 7.23 (dd, 1H, J= 8.8 and 2.0 Hz, H-5), 7.38-7.46 (m, 3H,
H-3, H-2′ and H-6′), 7.75 (d, 1H, J= 8.4 Hz, H-6) ,7.86 (s, 1H, triazole), 8.16 (s, 1H, triazole).
13C NMR (100 MHz, DMSO-d6) δ: 45.54 (CH2-S), 51.44 (1C, piperazine), 52.27 (2C,
piperazine), 52.88 (1C, piperazine), 55.44 (C-OH), 60.11 (CH2 benzylic), 74.89 (CH2-N),
127.42 (C-5), 129.60 (C-6), 130.07 (C-3′),130.47 (C-6′), 130.82 (C-5′), 130.88 (C-2′), 131.10
(C-3), 131.37 (C-4′), 131.72 (C-2), 133.57 (C-4), 138.05 (C-1′), 139.54 (C-1), 145.54 (C-5
triazole), 151.11 (C-3 triazole), 195.03 (C=S). MS (m/z, %): 589 (M+, <1), 252 (27), 218
(41), 202 (62), 181 (75), 159 (100), 123 (14), 101 (4), 85 (14), 56 (20). Anal. Calcd for
C23H23Cl4N5OS2: C, 46.71; H, 3.92; N, 11.84. Found: C, 46.69; H, 4.12; N, 11.88.
4.3. Antifungal susceptibility testing against different Candida species
The minimum inhibitory concentration (MIC) values of test compounds 6a-i and fluconazole
(Pfizer, Groton, CT, USA) were determined by micro-dilution method as recommended by
Clinical and Laboratory Standards Institute (CLSI) M27-A3 and M27-S4 documents [19,20].
The detailed procedure for MICs determination was included in the Supplementary Material.
4.4. Hemolysis assay
The hemolytic activity of selected compound 6b and fluconazole was evaluated using method
described by Carter et al. [24]. Fresh whole human blood was collected into dipotassium
EDTA-coated Vacutainer tubes and centrifuged at 500 g for 5 min. The red blood cell pellets
were washed in saline solution (two times) and then in phosphate-buffered saline (PBS). The
washed erythrocytes were re-diluted in PBS (1:25). On the other hand, different
concentrations of compounds were prepared in DMSO and an aliquot (10 µL) of each
concentration was added to diluted erythrocytes (190 µL) in a 96-well plate, giving final
concentrations of 1, 10 and 50 µg/mL in triplicate. DMSO and Triton X-100 (2% v/v) were
14

included as negative and positive controls, respectively. The plate was incubated at 37°C for
1 h and then cell suspensions were centrifuged at 500 g. After collecting each supernatant
(100 µL), their absorbance were measured at 451 nm. The percentage of hemolysis was
determined as follow:
% Hemolysis = [(A sample – A blank) /(A positive control)] × 100
4.5. Viability assay of HepG2 cells
HepG2 (Human hepatocarcinoma) cell line was purchased from Pasture Institute of Iran.
Viability of HepG2 cells was determined by MTT assay after exposure to test compound 6b
and fluconazole (at concentrations of 25, 50, 100, and 200 μg/mL). DMSO and cisplatin were
used as negative and positive controls, respectively. The detailed procedure for MTT assay
was included in the Supplementary Material.
4.6. Computational study
The X-ray crystal structures of CYP51 in complex with itraconazole (PDB ID: 5JLC for C.
glabrata and 5V5Z for C. albicans) were obtained from the RCSB Protein Data Bank.
AutoDockTools (ADT) 1.5.4 package was used for preparing all input files. Any
crystallographic water, if present was eliminated and hydrogens were added to the structure.
The 3D structure of the compound was constructed and subjected to energy minimization by
MOPAC Chem3D Ultra 8.0.3. Assigning appropriate charge and the heme cofactor and the
iron charges were carefully inspected. To define the active site for docking, the receptor-grid
was generated with grid box size of 60×60×60 with the spacing value of 0.375. The detailed
docking methodology has been described previously [15].
Acknowledgement
15

This work was supported by a grant from the Research Council of Mazandaran University of
Medical Sciences, Sari, Iran (Grant No. 552). This work was related to the Pharm.D. thesis of
YM (Faculty of Pharmacy, Mazandaran University of Medical Sciences).
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[24] G.P. Carter, J.R. Harjani, L. Li, N.P. Pitcher, Y. Nong, T.V. Riley, D.A. Williamson,
T.P. Stinear, J.B. Baell, B.P. Howden, 1,2,4-Oxadiazole antimicrobials act synergistically
with daptomycin and display rapid kill kinetics against MDR Enterococcus faecium.
Antimicrob. Chemother. 73 (2018) 1562–1569.
18

Captions:
Figure 1. Design of new antifungal triazole alcohols derived from fluconazole containing N-
benzylpiperazine carbodithioate moiety.
Figure 2. Hemolytic activity of compound 6b and fluconazole against human erythrocytes at
the concentrations of 1, 10 and 50 µg/mL. The negative control (CT) consisted of
erythrocytes treated with DMSO and the positive control consisted of erythrocytes treated
with Triton X-100 (TX, 2% v/v).
Figure 3. Cytotoxic activity of compound 6b and fluconazole against HepG2 cells at the
concentrations of 25, 50, 100 and 200 µg/mL.
Figure 4. 3D presentation of docked (R)-6b in the active site of CGCYP51 lanosterol 14α-
demethylase.
Figure 5. A) 3D presentation of docked (R)-6b in the active site of CACYP51 lanosterol
14α-demethylase; B) 2D presentation for the binding mode of (R)-6b with amino acids in the
active site of CACYP51 lanosterol 14α-demethylase.
19

Table 1. The MIC values (µg/mL) of compounds 6a-i against fluconazole-susceptible and
fluconazole-susceptible dose-dependent isolates of Candida species a.
S
OH
N
N
S
X
N
N
N
R
X
Compd
6a
X
F
F
F
F
F
F
R
C. a.
C. g.
(I)
C. g.
(II)
C. g.
(III)
C. g.
(IV)
C. p.
(I)
C. p.
(II)
C. p.
(III)
C. k.
C. t.
3-Cl
4-Cl
0.125 0.25
1
0.5
0.5
0.5
4
0.063 0.063 0.25
0.25
0.5
6b
0.5
0.063 0.5
0.5
4
0.063 0.063 0.063 0.125 0.5
6c
2,4-Cl2 0.125 0.5
8
1
2
4
4
4
2
4
0.5
0.5
2
4
0.5
1
6d
3,4-Cl2 0.5
2,6-Cl2 0.25
2
0.5
1
0.5
1
0.125 0.125 0.25
0.25
1
6e
0.25
0.063
0.5
1
0.125 0.25
0.25
1
6f
4-Br
0.25
0.25
2
1
0.125 0.063 0.25
0.5
0.25
2
1
6g
Cl 3-Cl
2
2
1
0.25
1
0.5
0.5
0.5
1
16
32
32
4
6h
Cl 2,4-Cl2 0.25
Cl 3,4-Cl2 0.5
2
4
1
6i
1
2
1
1
1
0.5
4
Fluconazole
1
2
4
2
0.5
0.5
a C. a.: C. albicans IFRC 194; C. g. (I): C. glabrata IFRC 339; C. g. (II): C. glabrata IFRC 1274; C. g. (III): C.
glabrata IFRC 1275; C. g. (IV): C. glabrata IFRC 1276; C. p. (I): C. parapsilosis IFRC 1269; C. p. (II): C.
parapsilosis IFRC 1270; C. p. (III): C. parapsilosis IFRC 1271; C. k.: C. krusei IFRC 1012; C. t.: C. tropicalis
IFRC1057.
20

Table 2. The MIC values (µg/mL) of compounds 6a-i against fluconazole-resistant isolates.
Compound
C. a.
C. a.
C. a.
C. p.
C. k.
C. k.
IFRC 1260
32
IFRC 1261
32
IFRC 1262
16
IFRC 84 IFRC 1280
IFRC 1281
32
6a
0.5
2
32
2
6b
16
32
32
32
16
64
64
64
≥64
16
32
32
32
32
64
64
64
≥64
8
16
32
32
32
32
64
64
64
≥64
6c
32
32
32
16
64
64
64
≥64
2
32
32
64
32
64
64
64
≥64
6d
0.25
1
6e
6f
0.25
0.5
1
6g
6h
6i
1
Fluconazole
≥8
Abbreviations: Candida albicans IFRC 1260; Candida albicans IFRC 1261; Candida albicans IFRC 1262;
Candida parapsilosis IFRC 84; Candida krusei IFRC 1280; Candida krusei IFRC 1281.
21

HO
N
S
N
N
N
R1
R3
N
N
N
F
S
R2
Carbodithioates
F
Fluconazole
Cl
Cl
R
O
Cl
Cl
Cl
O
O
Cl
S
N
N
N
N
N
N
N
N
N
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Oxiconazole
Sulconazole
Isoconazole
Econazole,
Miconazole
R= H
, R= Cl
S
OH
N
S
N
N
N
N
X
R
X
Designed compounds
Figure 1. Design of new antifungal triazole alcohols derived from fluconazole containing N-
benzylpiperazine carbodithioate moiety.
22

Figure 2. Hemolytic activity of compound 6b and fluconazole against human erythrocytes at
the concentrations of 1, 10 and 50 µg/mL. The negative control (CT) consisted of
erythrocytes treated with DMSO and the positive control consisted of erythrocytes treated
with Triton X-100 (TX, 2% v/v).
23

Figure 3. Cytotoxic activity of compound 6b and fluconazole against HepG2 cells at the
concentrations of 25, 50, 100 and 200 µg/mL.
24

Figure 4. 3D presentation of docked (R)-6b in the active site of CGCYP51 lanosterol 14α-
demethylase.
25

A
B
Figure 5. A) 3D presentation of docked (R)-6b in the active site of CACYP51 lanosterol
14α-demethylase; B) 2D presentation for the binding mode of (R)-6b with amino acids in the
active site of CACYP51 lanosterol 14α-demethylase.
26

N
O
X
Cl
N
N
HN
NH
+
R
X
4a,
4b,
X= F
X=Cl
2a-f
1
a
b
N
O
N
R
N
HN
N
X
X
3a-f
5a,
5b,
X= F
X=Cl
c
Hb
Hc
S
OH
Ha
S
Hd
3
2
4
N
N
1
N
N
X
2
N
5

R
1
3
4
X
6a-i
R= Cl, di-Cl, Br
X= F, Cl;
Scheme 1. Synthesis of the title compounds 6a–i. Reagents and conditions: a) EtOH, reflux,
2 h, b) TMSI, NaOH, toluene, 60 °C, 3 h; e) TEA, CS2, EtOH.
27

Graphical Abstract
S
HO
N
HO
N
S
N
N
R
N
N
N
N
N
X
N
N
F
6b:
X= F, R= 4-Cl
X
F
MICs: 0.063-0.5 g/mL
Active against fluconazole-resistant isolates
Fluconazole
MICs: 0.5-4 g/mL
Minimal toxicity against human erythrocytes and HepG2 cells
Lanosterol 14α-demethylase Inhibitor
28

Research Highlights
 Fluconazole analogs containing N-benzylpiperazine carbodithioate have been synthesized.
 All compounds showed potent activity against Candida spp. (MICs ≤4 µg/mL).
 Compound 6b (MICs = 0.063-0.5 µg/mL) was 4-32 times more potent than fluconazole.
 Compound 6b was active (MICs ≤ 16 µg/mL) against fluconazole-resistant isolates.
 Compound 6b had no significant toxicity on erythrocytes and HepG2 cells (at MIC levels).
29