Synthesis, Biological evaluation and molecular docking of certain sulfones as potential nonazole antifungal agents

Article abstract of DOI:10.3390/molecules21010114

We reported herein the synthesis, antifungal activity, docking and in silico ADME prediction studies of four novel series of sulfones 6a-f, 8a-c, 10a-f and 12a-c. All the newly synthesized sulfones were tested against four strains of Candida (including fluconazole-resistant Candida), two strains of Aspergillus, two dermatophytic fungi (Trichophytons mentagrophyte and Microsporum canis) and Syncephalastrum sp. with fluconazole as a reference drug. In general, compounds 8a and 10b showed selective and potent anticandidal activity (MIC: 0.19-0.81 μM) relative to fluconazole (MIC = 1.00M). Furthermore, 10e and 12a elicited a remarkable and selective antifungal activity against Aspergillus sp. and the dermatophytic fungi (MIC: 0.16-0.79 μM) relative to fluconazole (MIC: 2-2.6 μM). Moreover, the docking results of the sulfones 6a, 8a, 10a and 10b at the active site of CYT P450 14-sterol demethylase showed a comparable binding interaction (interaction Energy = -34.87 to -42.43 kcal/mol) with that of fluconazole (IE =40.37 kcal/mol).

Full text of DOI:10.3390/molecules21010114

molecules
Article
Synthesis, Biological Evaluation and Molecular
Docking of Certain Sulfones as Potential Nonazole
Antifungal Agents
Mohamed Fares 1,*, Mohamed A. Said 1,†, Muhammad A. Alsherbiny 2,†, Radwa A. Eladwy 3,†
,
Hadia Almahli 1,‡, Marwa M. Abdel-Aziz 4, Hazem A. Ghabbour 5,6, Wagdy M. Eldehna 1 and
Hatem A. Abdel-Aziz 7,
*
Received: 23 December 2015 ; Accepted: 13 January 2016 ; Published: 20 January 2016
Academic Editor: Maria Emília Sousa
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian University, Badr City,
Cairo 11829, Egypt; madyyy_@hotmail.com (M.A.S.); hadia.almahli@yahoo.com (H.A.);
wagdy2000@gmail.com (W.M.E.)
2
3
4
5
Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, Cairo 11562, Egypt;
muhammad.alsherbiny@pharma.cu.edu.eg
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Egyptian Russian University, Badr City,
Cairo 11829, Egypt; radwa.eladwy22@gmail.com
The Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo 11759, Egypt;
marwa2rcmb@yahoo.com
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P. O. Box 2457,
Riyadh 11451, Saudi Arabia; ghabbourh@yahoo.com
Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
Department of Applied Organic Chemistry, National Research Center, Dokki, Giza 12622, Egypt
Correspondence: ph.fares@yahoo.com (M.F.); hatem_741@yahoo.com (H.A.A.-A.);
Tel.: +20-2-2864-3342 (M.F.); +20-2-3337-1635 (H.A.A.-A.)
6
7
*
†
‡
These authors contributed equally to this work.
Present address: Department of Chemistry, University of Oxford, Oxford OX1 3TA, UK.
Abstract: We reported herein the synthesis, antifungal activity, docking and in silico ADME prediction
studies of four novel series of sulfones 6a 8a 10a and 12a . All the newly synthesized
–
f
,
–
c
,
–
f
–c
sulfones were tested against four strains of Candida (including fluconazole-resistant Candida), two
strains of Aspergillus, two dermatophytic fungi (Trichophytons mentagrophyte and Microsporum canis)
and Syncephalastrum sp. with fluconazole as a reference drug. In general, compounds 8a and
10b showed selective and potent anticandidal activity (MIC: 0.19–0.81
(MIC = 1.00 M). Furthermore, 10e and 12a elicited a remarkable and selective antifungal activity
against Aspergillus sp. and the dermatophytic fungi (MIC: 0.16–0.79 M) relative to fluconazole (MIC:
2–2.6 M). Moreover, the docking results of the sulfones 6a 8a 10a and 10b at the active site of CYT
P450 14 -sterol demethylase showed a comparable binding interaction (interaction Energy = 34.87
µM) relative to fluconazole
µ
µ
µ
,
,
α
´
to ´42.43 kcal/mol) with that of fluconazole (IE = ´40.37 kcal/mol).
Keywords: synthesis; sulfone; antifungal; anticandidal; docking
1. Introduction
In recent decades, fungal infection is considered as an escalating public health problem and causes
a continuous and serious threat to humans and animals [ ]. The increasing incidence of fungal
infections is mainly due to evolution of the resistance to antifungal agents and the growing number
of immunocompromised patients [ ]. In addition, mutation of the fungal species and the selection
1–3
4,5
Molecules 2016, 21, 114; doi:10.3390/molecules21010114
www.mdpi.com/journal/molecules

Molecules 2016, 21, 114
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pressure caused by the overuse of antifungal agents in humans and animals over the past 75 years are
considered the most crucial reasons of resistance [ ]. Moreover, the spread of antifungals’ resistance all
6
over the world arises as a consequence of inter species gene transmission, poor sanitation conditions
and the growing prevalence of nosocomial infection [6].
Invasive fungal infections (IFIs) and dermatomycoses have increased significantly since the
second half of the 20th century mainly because of the large number of individuals with higher
vulnerability such as neonates, cancer patients, those facing long hospital stays or infected with
acquired immunodeficiency virus [7,8]. Surveillance data indicated that there are fundamentally
three opportunistic species responsible for the IFIs; Candida albicans, Cryptococcus neoformans and
Aspergillus fumigatus. Furthermore, the epidemiology of IFIs has shifted towards non-albicans Candida,
non-fumigatus Aspergillus [7], this shift caused by the use of millions of tons of the antifungal
prophylaxis therapy. Regarding the candidiasis, C. albicans was the most common species, accounting
for about more than half of all cases (54%) followed by C. glabrata (19%), C. parapsilosis (11%), C. tropicalis
(11%), and C. krusei (2%) [9]. Generally, one fifth of the population at any given time are infected with
dermatophytes, which are fungi causing localized infection of the hair, nail and stratum corneum.
Trichophyton, Microsporum and Epidermophyton are the most abundant genera of dermatophytes that
account for the majority of dermatomycoses [10,11].
Unfortunately, there is a limited arsenal of the antifungal chemotherapies including polyenes
like amphotericin B and azoles such as voriconazole and fluconazole [12
approved drugs may present some undesirable side effects such as drug interaction, bioavailability
and hepatotoxicity [14 15]. Azole antifungals VI (Figure 1) exert their effect via the inhibition of
cytochrome P450 dependent enzyme lanosterol 14 -demethylase (Cyp 51). Cyp 51 is an important
,13]. Furthermore, these
,
I–
α
enzyme required for the synthesis of ergosterol from its precursor lanosterol. Ergosterol is a vital
component of the fungal cell membrane [16]. In addition, with extensive use and prolonged therapy of
the antimycotic azoles on different Candida species cells, the fungal cells adapt to be eventually azole
resistant [17].
Figure 1. Structures of antifungal drugs
and 12a–c.
I–VII and the novel entitled compounds 6a–f, 8a–c, 10a–f
Fluconazole
Candida species but has less activity against C. glabrata and no apparent activity against C. krusei [18].
Fluconazole and voriconazole VI are the drugs of choice for the treatment of severe systemic
mycoses and invasive aspergillosis, respectively [19 20]. On the other hand, because of the similarity
V, the first member of this class, demonstrates an excellent activity against most
V
,

Molecules 2016, 21, 114
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between mammalian and fungal cells in the composition of cell membrane, azoles are not entirely
selective to fungi [12]. They are reported to be hepatotoxic due to the fact that they undergo hepatic
metabolism. Consequently, it is recommended that patients who are administering any of this class
should be carefully monitored for hepatotoxicity [16,21]. Although the azole ring is the most important
pharmacophore for the antimycotic activity of this class, it is responsible too for the undesirable
hepatotoxicity side effect [19]. Therefore, owing to the limited success of azoles, due to resistance and
toxicity, there is urgent need to develop new antifungal agents that combine safety with antifungal
efficacy against the most common fungi and against fluconazole-resistant strains.
Aryl sulfones are reported to possess strong antifungal activity [22–25]. Curti et al. [22]
proved that the aryl sulfone derivative VII exhibited antifungal activity against Candida tropicalis.
Further investigation on this interesting scaffold was done by our research group and resulted in the
discovery of the broad spectrum anticandidal agent 8a (R = H) [25]. In the present work, it was thought
worthwhile to extend and optimize our previous investigation regarding the antifungal activity of
the phenyl sulfone derivatives via modifications of the linker type and substitution of ring C of 8a
(
R = H) with the prime aim to obtain a more potent and safe antifungal agent especially against drug
resistant strains.
In view of the facts mentioned above and as a part of our ongoing effort to prepare
anti-microbial agents [26 28], herein we present the design and synthesis of a focused library
of novel aryl sulfone analogs 6a 8a 10a and 12a and evaluation of their anti-fungal
–
–
f,
–
c
,
–
f
–c
activity against different invasive fungi (fluconazole-resistant Candida albicans, Candida albicans,
Candida tropicalis, Candida parapsilosis; Aspergillus fumigatus and Aspergillus niger) and dermatophytic
Fungi (Trichophytons mentagrophytes and Microsporum canis).
2. Results and Discussion
2.1. Chemistry
1-(4-Chlorophenyl)-2-(phenylsulfonyl)ethanone
all the new targeted sulfone derivatives (Schemes 1 and 2). Furthermore, X-ray single crystal
analysis for compound gave an absolute confirmation for the structure. CCDC 1430111 contains the
3 [22] was used as a starting material to obtain
3
supplementary crystallographic data for this paper (see Tables S1–S3 for details) (Figure 2). These data
can be obtained free of charge via http://www.crystallography.net/search.html. Diazotization of
aromatic amines 4a
which subsequently coupled with the active methylene of the phenyl sulfone derivative
presence of sodium acetate trihydrate and ethanol to afford the corresponding hydrazones 6a–f [29].
1H-NMR spectra of the hydrazone derivatives 6a
showed D2O exchangeable singlet signals in
the regions 11.46–11.77 and 12.30–12.44 ppm attributable to the resonating NH group, in addition
to the disappearance of the singlet signal of methylene group of . The phenyl sulfone was further
reacted with phenylhydrazine derivatives 7a in ethanol at ambient temperature to afford the title
compounds 8a
(Scheme 1). The 1H-NMR of the hydrazones 8b and 8c exhibited the exchangeable
signal of the NH group which appeared at 9.86 and 9.87 ppm and the signal of CH2 protons appeared
5.14 and 5.15 ppm, whereas the 13C-NMR of 8c revealed the signal of CH2 carbon at
–
f
with hydrochloric acid and sodium nitrite afforded the diazonium salts 5a
–f
3
in the
–f
δ
3
3
–
c
–c
δ
as singlet at
δ
δ 70.30 ppm.
In order to obtain more structural variations of the main phenyl sulfone scaffold to explore
and understand more about the structure activity relationship, the starting sulfone was
3
reacted with the highly reactive N,N-dimethylformamide dimethyl acetal (DMFDMA) to obtain
1-(4-chlorophenyl)-3-(dimethylamino)-2-(phenylsulfonyl)prop-2-en-1-one 9 [30]. The structure of the
1
enaminone derivative
9
was confirmed using different spectroscopic techniques. H-NMR revealed
the disappearance of the methylene group singlet signal and the presence of the methine group signal
at
δ
8.05 ppm, while the two methyl groups protons appeared at
δ
3.09 ppm. Moreover, 13C-NMR of
δ
105.49 ppm and the signal of the sp3 methyl groups
9
showed the signal of the methine sp2 carbon at
at δ 41.73 ppm.

Molecules 2016, 21, 114
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The enaminone derivative
9
reacted with aniline derivatives 4a
–f
and afforded 1-(4-chlorophenyl)-
3-(arylamino)-2-(phenylsulfonyl)prop-2-en-1-ones 10a–f. The latter phenyl sulfone derivatives are
assumed to be formed by the nucleophilic substitution of the anilines 4a–f on the activated double
bond in the enaminones 9 followed by elimination of the dimethylamine.
The structures of the reaction products were assigned based on their elemental analyses and
spectral data. Moreover, the 1H-NMR spectrum of the compounds 10a
revealed the presence of
the two doublet signals of the two configurations of enaminone (CH = N signal) at 7.88–8.07 and
–
f
δ
8.02–8.55 ppm. Interestingly, upon deuteration, the doublet signals disappeared in line with the
appearance of singlet peak. Similarly, the exchangeable peak of the NH group appeared as doublet,
while, upon adding D2O it is disappeared completely.
The synthesis of the pyrazole derivatives 12a
–
c
was achieved via the reaction of compound
9 with
phenyl hydrazine derivatives 7a . Furthermore, the compounds 12a–c
–
c
showed characteristic 1H-NMR
signals in the region δ 8.34–8.37 ppm corresponding to the pyrazole proton and disappearance of the
methine and methyl protons signals that are present in the enamine derivative 9 [31].
Scheme 1. Synthesis of Target compounds 6a
(ii) CH3COONa/EtOH/0–5 ˝C; (iii) EtOH/Catalytic amount of glacial acetic acid/reflux 6 h.
–f and 8a–c. Reagents and conditions: (i
) NaNO2/HCl/0–5 ˝C;
Scheme 2. Synthesis of Target compounds 10a
–f and 12a–c Reagents and conditions: (i) Neat
reaction/reflux 4 h; (ii) Glacial acetic acid/reflux 2 h; (iii) EtOH/reflux 6 h.

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Figure 2. An ORTEP diagram of final X-ray structure of compound 3.
2.2. Biological Data
2.2.1. In Vitro Antifungal Activities
The antifungal activity was performed at The Regional Center for Mycology and Biotechnology
(RCMB), Al-Azhar University, Cairo, Egypt.
As depicted in Tables 1 and 2 target compounds 6a–f, 8a–c, 9, 10a–f, 12a–c and fluconazole,
as antifungal reference drug, were evaluated in vitro for their antifungal activity by inhibition zone
technique and minimum inhibitory concentration (MIC) against fluconazole-resistant Candida albicans
(RCMB 05036, CaR), Candida albicans (RCMB 05079, Ca), Candida tropicalis (RCMB 05142, Ct),
Candida parapsilosis (RCMB 05324, Cp), Aspergillus fumigatus (RCMB 02568, Af), Aspergillus niger
(RCMB 02542, An), Trichophytons mentagrophyte (RCMB 09285, Tm), Microsporum canis (RCMB 09321,
Mc) and Syncephalastrum racemosum (RCMB 05922). All the compounds showed no activity against
Syncephalastrum racemosum. However, data in Tables 1 and 2 revealed that the unsubstituted hydrazone
and unsubstituted aniline phenyl sulfone counterparts 6a and 10a, respectively, showed significant
broad antifungal activity against most of the tested species.
Observing the results, we could deduce some valuable data about the structure activity correlation
of the tested phenyl sulfone derivatives. Firstly, we explored that the substitution of ring C has
an impact of the antimycotic spectrum and extent of inhibition. For example, among all the first series
compounds 6a
with broad and good activity against all the invasive and dermatophytic fungi (MIC = 1.57–3.14
relative to fluconazole (MIC = 1.00–2.60 M). However, substitution with any other group abolished
–
f
, incorporation of
α
-unsubstituted phenyl hydrazone group led to compound 6a
µM)
µ
the activity, which may be attributed to solubility problems. Similarly, bioisosteric replacement of N
of the hydrazone group with CH in 6a led to 10a counterpart with broad spectrum activity against
most of the tested compounds (MIC = 1.57–3.14
substituent at the 4-position, showed an excellent activity and selectivity against the tested Candida sp.
(MIC = 0.19 M against Ca, Ct and
M) relative to fluconazole (no activity against CaR and MIC = 1
Cp). Furthermore, grafting bromine, compound 10e, shifted the activity towards the Aspergillus sp.
and dermatophytic fungi (MIC = 0.16–0.66 M) relative to the reference drug (MIC = 2.00–2.60 M).
µM). However, compound 10b, bearing fluorine
µ
µ
µ
µ
Conversely, substitution with bulky electron withdrawing groups such as 2,4-difluoro (10c), 4-Cl (10d
and 4-NO2 (10f) abolished the antifungal activity.
)
On the other hand, among the second series compounds, 8a
hydrazone derivative displayed selective and potent activity against all the Candida sp.
(MIC = 0.20–0.81 M against Ca, Ct and
M) relative to fluconazole (no activity against CaR and MIC = 1
Cp). Further substitution with 4-chloro or 4-bromo resulted in loss of the activity. Rigidification of the
hydrazone group of 8a into pyrazole ring resulted in 12a analogs. Incorporation of unsubstituted
phenyl group to the pyrazole derivative, compound 12a, showed a remarkable activity being 13-fold,
–c, the unsubstituted phenyl
µ
µ
–
c
–c

Molecules 2016, 21, 114
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2.5-fold, 2.5-fold and 10-fold more active the fluconazole against A. fumigatus, A. niger, T. mentagrophyte
and M. Canis, respectively. Substitution with 4-chloro 12b or 4-bromo 12c led to complete loss of
activity, which again may be due to solubility problems.
Interestingly, we identified two new broad spectrum antifungal agents, 6a and 10a, while two
phenyl sulfone counterparts, 8a and 10b, possessed strong inhibition of three Candida sp. (C. albicans,
C. tropicalis and C. parapsilosis), three compounds, 6a, 8a and 10b, were shown to fight against
fluconazole-resistant Candida albicans and two novel agents had excellent anti-aspergillosis and
anti-dermatophytic activity, 10e and 12a. However, further iterative cycles of optimization of ring A
and ring B of the active compounds 6a
, 8a and 10b are ongoing in our lab for lead identification and
development with strong activity against fluconazole-resistant species.
Table 1. Mean zone of inhibition in mm
on a range of environmental and clinically pathogenic microorganisms using (125
of tested samples.
˘
Standard deviation beyond well diameter (6 mm) produced
µg/mL) concentration
Invasive Fungi
Dermatophytic Fungi
Comp.
CaR
Ca
Ct
Cp
Af
An
Tm
Mc
6a
8a
10a
10b
10e
12a
Flu
14.2 ˘ 0.58 12.4 ˘ 0.63 11.5 ˘ 0.58 12.4 ˘ 0.63 11.2 ˘ 0.63 11.9 ˘ 0.63
9.3 ˘ 0.63
10.2 ˘ 0.63
18.2 ˘ 0.72 20.3 ˘ 0.63 19.4 ˘ 0.58 20.6 ˘ 0.63
NA
12.3 ˘ 0.63 14.1 ˘ 0.63 15.2 ˘ 0.58 10.6 ˘ 0.72
NA
NA
NA
9.3 ˘ 0.58
NA
NA
NA
10.1 ˘ 0.63 12.1 ˘ 0.58
19.6 ˘ 0.63 20.9 ˘ 0.72 21.2 ˘ 0.45 22.4 ˘ 0.58
NA NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
20.3 ˘ 0.63 18.2 ˘ 0.63 17.1 ˘ 0.63 18.9 ˘ 0.63
19.3 ˘ 0.72 18.1 ˘ 0.58 18.2 ˘ 0.72 20.1 ˘ 0.58
16.6 ˘ 0.58 15.4 ˘ 0.58 17.2 ˘ 0.72 19.6 ˘ 0.58 16.3 ˘ 0.63 13.6 ˘ 0.72 15.4 ˘ 0.63
Only the compounds with I.Z > 9 mm are mentioned. NA: No Activity (I.Z < 9 mm). The screening organisms,
Invasive Fungi: fluconazole resistant Candida albicans (RCMB 05036, CaR), Candida albicans (RCMB 05079, Ca),
Candida tropicalis (RCMB 05142, Ct), Candida parapsilosis (RCMB 05324, Cp); Aspergillus fumigatus (RCMB 02568,
Af) and Aspergillus niger (RCMB 02542, An). Dermatophytic Fungi: Trichophytons mentagrophytes (RCMB 09285,
Tm) and Microsporum canis (RCMB 09321, Mc); Flu: Fluconazole.
Table 2. Antimicrobial activity as MICs (µM) of fluconazole and synthesized compounds against tested
microorganisms and selectivity index (SI) of the synthesized compounds.
Invasive Fungi
Dermatophytic Fungi
PC3
IC50
Comp.
CaR
Ca
Ct
Cp
Af
An
Tm
Mc
1.57
1.57
1.57
1.57
1.57
1.57
3.14
(12.38)
1.57
(24.75)
6a
8a
38.86
37.36
35.14
(24.75) (24.75) (24.75) (24.75) (24.75) (24.75)
0.81
0.20
0.41
0.20
NA
1.57
NA
3.14
NA
NA
(46.12) (>100) (91.12) (>100)
NA
0.19
1.57
1.57
1.57
1.57
(22.38)
1.57
(22.38)
10a
10b
10e
(22.38) (22.38) (22.38) (22.38) (11.19)
0.19
0.19
0.19
NA
0.16
NA
0.66
NA
NA
37.09
38.73
(>100) (>100) (>100) (>100)
0.66
(58.68)
0.66
(58.68)
NA
NA
NA
NA
(>100) (58.68)
0.20
(>100)
0.79
(36.6)
0.79
(36.60)
0.20
(>100)
12a
NA
NA
NA
1
NA
1
NA
1
28.90
Fluconazole
2.6
2
2
2

Molecules 2016, 21, 114
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2.2.2. In Vitro Cytotoxicity
In vitro cytotoxicity of the most active compounds 6a
,
8a,
10a
,
10b
,
10e and 12a was evaluated
against human prostate cancer PC-3 cells using sulforhodamine B (SRB) colorimetric assay. The IC50
values obtained for these compounds are listed in Table 2. Moreover, selectivity index (SI) is used
to estimate the therapeutic effect of a drug and to identify drug candidates for further studies. SI of
each compound was determined as the ratio of IC50 to MIC (Table 2). It was reported that new drugs
candidates must have SI equal or higher than 10 [32]. In this study, all the tested compounds could be
considered as promising new antifungal drug candidates with SI > 11.19.
2.3. Computational Study
2.3.1. Docking
To further understand the mechanism of action of the synthesized compounds, molecular
modeling and docking studies of 6a
, 8a, 10a and 10b were performed on the X-ray crystal structure of
cytochrome P450 14 -sterol demethylase from Mycobacterium tuberculosis (Mycobacterium P450 DM)
α
and co-crystallized fluconazole (PDB code: 1EA1) using Discovery Studio 4/CDOCKER protocol
(Accelrys Software Inc., San Diego, CA, USA). It is well known that fluconazole exerts its antifungal
activity by binding to Cyp-P450 DM enzyme. The nitrogen of azole ring of fluconazole coordinated to
the heme iron, while the azole ring itself in the fluconazole structure is positioned almost perpendicular
to the porphyrin plane (cofactor) [33]. Moreover, the 2,4-difluorophenyl forms a pi-cation interaction
with Arg96 and the other triazole ring formed a pi-pi hydrophobic interaction with Tyr76 and Phe78
(Figure 3) [33].
Figure 3. Cont.

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Figure 3. 3D representations of the binding patterns of fluconazole, 6a, 8a, 10a and 10b into the binding
site cytochrome P450 14α-sterol demethylase (PDB code 1EA1).
In general, data obtained from docking shows that all the docked hits can interact with porphyrin
plane and occupy the binding site of the enzyme with strong binding interaction energy ranging from
´34.873 to
´
42.431 kcal/mol relative to fluconazole ( 40.374 kcal/mol) (Table 3). As illustrated in
´
Figure 3, in case of compounds 6a, ring B forms a pi-cation interaction with the heme iron, while
in compound 8a the oxygen of sulfone moiety is perpendicular to the porphyrin plane heme ion.
The p-chlorophenyl (ring A) of 6a and 8a forms a hydrophobic interaction (pi-pi interaction) with
Phe78, Met79, and Leu321 and almost occupies the same hydrophobic pocket. Similarly, ring C of
6a and 8a extends into the unoccupied hydrophobic region above the heme ring and showed good
van der Waals interactions with amino acids Met99, Leu100, Ala256. The carbonyl group of the
hydrazone derivative 6a and the sulfone group form a hydrogen bond with the side chain of Arg96,
while the amino acid formed a pi-cation interaction with fluconazole.
Table 3. Docking scores of the most active compounds and fluconazole.
Compound
CDOCKER Interaction Energy (kcal/mol)
6a
8a
10a
´42.431
´36.214
´34.873
´39.229
´40.374
10b
Fluconazole
The docking result revealed that the phenyl sulfone group, the p-chlorophenyl group and the
aniline side chain of 10a and 10b can bind in the same manner to the active site of CYP51. Ring B forms
a pi-cation interaction with heme ion and pi-pi interaction with the pyrrole rings of porphrin. Ring A
of 10a and 10b forms hydrophobic and van der Waals interactions with surrounding hydrophobic
residues such as Phe83, Leu100, and Ala256. It was observed that the anilino group (ring C) of 10a
occupies the hydrophobic pocket above the heme ring and interacts with Phe78, Met79 and Leu321.
The p-fluoro anilino group of 10b affords more hydrophobic interaction points with the hydrophobic
pocket and forms a hydrophobic interaction with Phe78, Met79, His259 Met433 and Leu321. Finally, the
sulfone group forms a hydrogen bond with the side chain of Arg96.
It was observed that compounds 10a and 10b were oriented in the binding groove of enzyme
in such a fashion that favors the possibility of pi-pi interaction of the three benzene rings with
the hydrophobic amino acid residues of the binding site of the enzyme (Figure 4). Overlaying the
bioactive conformers of 10a and 10b on fluconazole showed that both the counterparts extend with the
4-chlorophenyl and anilino fragments into hydrophobic pockets above the porphyrin ring. Our future

Molecules 2016, 21, 114
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study will be directed towards optimizing the biology and modeling results in order to obtain a more
active compound.
Figure 4. Overlay of compounds 10a and 10b and fluconazole (yellow) showing the hydrophobic
surface in the groove of cytochrome P450 14α-sterol demethylase.
2.3.2. ADME and Molecular Property Prediction
The ADME of the phenyl sulfone counterparts 6a–f, 8a–c, 9, 10a–f and 12a–c was predicted via
a theoretical kinetic study performed by means of the Discovery Studio software (Figure 5 and Table 4).
Both AlogP98 and PSA (polar surface area) descriptors were calculated to evaluate the lipophilicity and
polar surface area. Also, solubility, absorption and CYP2D inhibition levels were predicted. Most of
the compounds showed extremely low solubility except 9, 10a, 10b and 10f. Finally, all members were
predicted to be CYP2D non-inhibitors. It is noteworthy that careful study of the molecular properties
suggested that the inactivity of most of the substituted derivatives may be due to the high lipophilicity
and low solubility.
Table 4. Computer aided ADME and molecular property prediction of the sulfone derivatives.
Solubility
Level c
Absorption
Level d
CYP2D6
Num_H
Bond Donor
Num_H Bond
Acceptor
Molecular Rotatable
Comp. AlogP98 a
PSA b
Probability e
Weight
Bonds
6a
6b
6c
6d
6e
6f
8a
8b
8c
5.21
5.42
5.63
5.88
5.96
5.11
5.14
5.80
5.88
3.20
4.51
5.34
5.55
5.80
5.88
5.03
5.63
5.80
6.38
76.04
76.04
76.04
76.04
76.04
118.86
58.74
58.74
58.74
55.25
64.71
64.71
64.71
64.71
64.71
107.54
51.21
51.21
51.21
1
1
1
1
1
1
1
1
1
2
2
2
1
1
1
2
1
1
1
0
1
1
1
1
2
0
1
1
0
0
0
0
0
0
1
0
1
1
0.02
0
0
0.02
0.02
0
0
0
0
0
0.05
0
0
0.04
0.02
0.02
0
1
1
1
1
1
1
1
1
1
0
1
1
1
1
1
1
0
0
0
5
5
5
5
5
8
4
4
4
4
4
4
4
4
4
7
4
4
4
398.86
416.85
434.84
433.31
477.76
443.86
384.88
419.32
463.78
349.83
397.88
415.87
433.86
432.32
476.77
442.87
394.87
429.32
473.77
6
6
6
6
6
7
6
6
6
5
6
6
6
6
6
7
4
4
4
9
10a
10b
10c
10d
10e
10f
12a
12b
12c
0
0
a
Lipophilicity descriptor; b Polar surface area; c Solubility level. (0 = extremely low, 1 = very low but possible,
2 = low, 3 = good, 4 = optimal); d Absorption level. (0 = good, 1 = moderate, 2 = low, 3 = very low); e CYP2D6
Probability: 0–0.5 = non inhibitor; 0.5–1 = inhibitor.

Molecules 2016, 21, 114
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Figure 5. Computer aided solubility prediction of the sulfone derivatives (Solubility parameter.
(0~´2 = optimal, ´2~´4 = good, ´4~´6 = low, ´6~´8 = very low).
3. Materials and Methods
3.1. Chemistry
Melting points were measured with a Stuart apparatus (Bibby Scientific Limited, Staffordshire,
United Kingdom) and were uncorrected. The NMR spectra were recorded by Varian Gemini-300BB
1
300 MHz FT-NMR spectrometers (Varian Inc., Palo Alto, CA, USA). H and 13C spectra were run
at 300 and 75 MHz, respectively, in deuterated dimethyl sulfoxide (DMSO-d6). Chemical shifts (δH
)
are reported relative to TMS as internal standard. All coupling constant (J) values are given in hertz.
Chemical shifts (δC) are reported relative to DMSO-d6 as internal standards. The abbreviations used
are as follows: s, singlet; d, doublet; m, multiplet. Microanalyses were carried out using Perkin Elmer
PE 2400 CHN Elemental Analyzer (Akron, OH, USA) and the results were within
˘
0.4%. Analytical
thin layer chromatography (TLC) on silica gel plates containing UV indicator was employed routinely
to follow the course of reactions and to check the purity of products. All reagents and solvents were
purified and dried by standard techniques.
3.1.1. Synthesis of (1-(Aryl)-2-(2-(4-fluorophenyl)hydrazono)-2-(phenylsulfonyl)ethanones 6a–f
To a solution of the appropriate amine 4a–f (1 mL), hydrochloric acid (15 mL) was added.
A solution of sodium nitrite (0.07 g, 1 mmol) in water (10 mL) was then gradually added with
stirring. The resulting solution was added gradually within 30 min to a stirred cold solution (0–5 ˝C)
of sulfone
3 (1 mmol) and sodium acetate trihydrate (0.26 g, 20 mmol) in ethanol (50 mL) and then left
˝
for 8 h in a refrigerator (4 C). The resulting solid was collected by filtration, washed thoroughly with
water, and dried to give the respective crude hydrazones which recrystallized from ethanol to give the
corresponding hydrazones 6a–f, respectively.
1-(4-Chlorophenyl)-2-(2-phenylhydrazono)-2-(phenylsulfonyl)ethanone (6a): Yellow powder (yield 71%),
˝
1
m.p. 170 C; H-NMR (DMSO-d6)
7.63–7.83 (m, 4H, Ar-H), 7.88 (d, J = 7.8 Hz, 2H, Ar-H), 7.95 (d, J = 8.1 Hz, 2H, Ar-H), 8.19 (d, J = 7.8 Hz
2H, Ar-H), 11.46 and 12.31 (s, 1H, D2O exchangable, -NH); 13C-NMR (DMSO-d6)
ppm: 114.37, 116.45,
δ ppm: 7.01–7.26 (m, 2H, Ar-H), 7.57 (d, J = 8.1 Hz, 2H, Ar-H),
,
δ
127.68, 128.05, 129.34, 130.86, 131.65, 134.05, 137.27, 139.19, 139.76, 141.55, 142.43, 188.10; Anal. Calcd.
for C20H15ClN2O3S (398.86): C, 60.22; H, 3.79; N, 7.02; Found C, 60.41; H, 3.70; N, 7.32.
1-(4-Chlorophen˝yl)-2-(2-(4-flurophenyl)hydrazono)-2-(phenylsulfonyl)ethanone (6b): Yellow powder (yield
74%); m.p. 181 C, 1H-NMR (DMSO-d6)
δ ppm: 7.11 (d, J = 8.7 Hz, 2H, Ar-H), 7.23–7.29 (m, 1H, Ar-H),
7.54 (d, J = 8.7 Hz, 2H, Ar-H), 7.67 (d, J = 7.5 Hz, 2H, Ar-H), 7.75 (dd, J = 7.5, 14.9, 2H, Ar-H), 7.89 (d,
J = 8.1 Hz, 2H, Ar-H), 8.18 (d, J = 8.1 Hz, 2H, Ar-H), 11.49 and 12.33 (s, 1H, D2O exchangable, -NH);
Anal. Calcd. for C20H14ClFN2O3S (416.85): C, 57.63; H, 3.39; N, 6.72; Found C, 57.94; H, 3.45; N, 6.52.

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1-(4-Chlorophenyl)-2-(2-(2,4-diflurophenyl)hydrazono)-2-(phenylsulfonyl)ethanone (6c): Yellow powder
(yield 80%), m.p. 177 ˝C; 1H-NMR (DMSO-d6)
ppm: 7.08–7.28 (m, 3H, Ar-H), 7.53 (d, J = 8.7 Hz
δ
,
2H, Ar-H), 7.66 (d, J = 8.1 Hz, 2H, Ar-H), 7.77 (s, 1H, Ar-H), 7.79 (d, J = 8.4 Hz, 1H, Ar-H), 7.90 (d,
J = 7.5 Hz, 1H, Ar-H), 8.16 (d, J = 7.5 Hz, 2H, Ar-H), 11.46 and 12.33 (s, 1H, D2O exchangable, -NH);
13C-NMR (DMSO-d6)
δ ppm: 117.84, 118.00, 125.07, 128.06, 129.28, 130.85, 131.70, 134.69, 135.14, 137.54,
138.75, 139.49, 139.73, 184.91; Anal. Calcd. for C20H13ClF2N2O3S (434.84): C, 55.24; H, 3.01; N, 6.44;
Found C, 55.02; H, 2.83; N, 6.59.
1-(4-Chlorophenyl)-2-(2-(4-chlorophenyl)hydrazono)-2-(phenylsulfonyl)ethanone (6d): Yellow powder (yield
˝
85%), m.p. 213 C; 1H-NMR (DMSO-d6)
δ ppm: 7.07 (d, J = 8.7 Hz, 2H, Ar-H), 7.33 (m, 1H, Ar-H), 7.45
(d, J = 8.7 Hz, 2H, Ar-H), 7.68 (d, J = 6.3 Hz, 2H, Ar-H), 7.75 (dd, J = 7.5, 6.3 Hz, 2H, Ar-H), 7.90 (d,
J = 7.5 Hz, 2H, Ar-H), 8.19 (d, J = 7.5 Hz, 2H, Ar-H), 11.47 and 12.31 (s, 1H, D2O exchangable, -NH);
Anal. Calcd. for C20H14Cl2N2O3S (433.30): C, 55.44; H, 3.26; N, 6.47; Found C, 55.63; H, 3.39; N, 6.31.
1-(4-Chlorophenyl)-2-(2-(4-bromophenyl)hydrazono)-2-(phenylsulfonyl)ethanone (6e): Yellow powder (yield
87%), m.p. 232 ˝C; 1H-NMR (DMSO-d6)
δ ppm: 7.01 (d, J = 8.7 Hz, 2H, Ar-H), 7.30 (d, J = 8.7 Hz,
2H, Ar-H), 7.54 (m, 1H, Ar-H), 7.68 (d, J = 7.8 Hz, 2H, Ar-H), ), 7.75 (dd, J = 7.8, 6.3 Hz, 2H, Ar-H),
7.90 (d, J = 7.5 Hz, 2H, Ar-H), 8.19 (d, J = 7.5 Hz, 2H, Ar-H), 11.47 and 12.30 (s, 1H, D2O exchangable,
-NH); 13C-NMR 1H-NMR (DMSO-d6)
δ ppm: 114.9, 116.31, 118.33, 128.10, 129.58, 131.67, 132.14, 133.79,
135.35, 137.45, 139.819, 139.99, 141.04, 187.99; Anal. Calcd. for C20H14BrClN2O3S (477.76): C, 50.28;
H, 2.95; N, 5.86; Found C, 50.43; H, 3.10; N, 5.93.
1-(4-Chlorophenyl)-2-(2-(4-nitrophenyl)hydrazono)-2-(phenylsulfonyl)ethanone (6f): Orange powder (yield
60%), m.p. 216 ˝C; 1H-NMR (DMSO-d6)
δ ppm: 7.20 (d, J = 9.0 Hz, 2H, Ar-H), 7.54 (d, J = 9.0 Hz, 2H,
Ar-H), 7.69–7.79 (m, 1H, Ar-H), 7.82 (d, J = 8.1 Hz, 2H, Ar-H), 7.93 (d, J = 8.1 Hz, 2H, Ar-H), 8.16 (d,
J = 9.0 Hz, 2H, Ar-H), 8.24–8.29 (m, 1H, Ar-H), 11.77 and 12.44 (s, 1H, D2O exchangable, -NH); Anal.
Calcd. for C20H14ClN3O5S (443.86): C, 54.12; H, 3.18; N, 9.47; Found C, 54.29; H, 3.36; N, 9.64.
3.1.2. Synthesis of 1-(Aryl)-2-(1-(4-chlorophenyl)-2-(phenylsulfonyl)ethylidene)hydrazines 8a–c
To a stirred solution of the corresponding phenyl hydrazide 7a–c (5 mmol) in absolute ethanol
(20 mL), the sulfone derivative (5 mmol) and catalytic amount of glacial acetic acid were added.
3
The reaction mixture was heated under reflux for 6 h. The precipitate formed was collected by
filtration while hot, washed with hot ethanol, dried and crystallized from ethanol/DMF to afford
compounds 8a–c.
1-(Phenyl)-2-(1-(4-chlorophenyl)-2-(phenylsulfonyl)ethylidene)hydrazine (8a): [25].
1-(4-Chlorophenyl)-2-(1-(4-chlorophenyl)-2-(phenylsulfonyl)ethylidene)hydrazine (8b): Yellow powder (yield
˝
85%), m.p. 205 C; 1H-NMR (DMSO-d6)
δ ppm: 5.14 (s, 2H, -CH2), 7.09 (d, J = 9.0 Hz, 2H, Ar-H), 7.25
(d, J = 9.0 Hz, 2H, Ar-H), 7.30 (d, J = 8.7 Hz, 2H, Ar-H), 7.48 (t, J = 7.5, 1H, Ar-H), 7.57–7.65 (m, 2H,
Ar-H), 7.69 (d, J = 8.7 Hz, 2H, Ar-H), 7.84 (d, J = 7.5 Hz, 2H, Ar-H), 9.86 (s, 1H, D2O exchangable, -NH);
Anal. Calcd. for C20H16Cl2N2O2S (419.32): C, 57.29; H, 3.85; N, 6.68; Found C, 57.50; H, 4.04; N, 6.83.
1-(4-Bromophenyl)-2-(1-(4-chlorophenyl)-2-(phenylsulfonyl)ethylidene)hydrazine (8c): Yellow powder (yield
˝
1
88%), m.p. 216 C; H-NMR (DMSO-d6)
δ ppm: 5.15 (s, 2H, -CH2), 7.04 (d, J = 8.7 Hz, 2H, Ar-H),
7.30 (d, J = 8.7 Hz, 2H, Ar-H), 7.37 (d, J = 8.7 Hz, 2H, Ar-H), 7.48 (t, J = 7.2, 1H, Ar-H), 7.57 (m, 2H,
Ar-H), 7.69 (d, J = 8.7 Hz, 2H, Ar-H), 7.83 (d, J = 7.2 Hz, 2H, Ar-H), 9.87 (s, 1H, D2O exchangable, -NH);
13C-NMR (DMSO-d6)
δ ppm: 70.30, 111.04, 114.97, 127.34, 137.47, 128.07, 129.26, 130.47, 130.72, 131.73,
132.15, 136.21, 139.12, 134.77; Anal. Calcd. for C20H16BrClN2O2S (463.77): C, 51.80; H, 3.48; N, 6.04;
Found C, 52.06; H, 3.71; N, 6.26.

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3.1.3. Synthesis 1-(4-Chlorophenyl)-3-(dimethylamino)-2-(phenylsulfonyl)prop-2-en-1-one (9)
A mixture of sulfone 3 (10 mmol) and dimethylformamide-dimethylacetal (DMF-DMA) (12 mmol)
was refluxed for 4 h. The residue was triturated with ether and the resulting yellow needles were
collected by filtration, washed thoroughly with ether, dried and finally recrystallized from ethanol to
˝
9
. yellow powder (yield 75%), m.p. 190 C; 1H-NMR (DMSO-d6)
afford the corresponding enaminone
ppm: 3.09 (s, 6H, N(CH3)2), 7.46–7.63 (m, 7H, Ar-H), 7.73 (d, J = 7.2 Hz, 2H, Ar-H), 8.05 (s, 1H, = CH-);
13C-NMR (DMSO-d6)
ppm: 41.73, 105.49, 126.52, 128.63, 130.14, 131.86, 125.15, 136.89, 128.99, 144.24,
δ
δ
155.19, 187.67; Anal. Calcd. for C17H16ClNO3S (349.83): C, 58.37; H, 4.61; N, 4.00; Found C, 58.60;
H, 4.75; N, 4.15.
3.1.4. Synthesis of 3-(Aryl amino)-1-(4-chlorophenyl)-2-(phenylsulfonyl)prop-2-en-1-one 10a–f
A mixture of enaminone
(25 mL) was refluxed for 2 h, then left to cool. The solid product was filtered off, washed with water,
dried and finally recrystallized from ethanol to afford the corresponding products 10a , respectively.
9 (5 mmol) and the appropriate aniline 4a–f (5 mmol) in glacial acetic acid
–
f
1-(4-Chlorophenyl)-3-(phenylamino)-2-(phenylsulfonyl)prop-2-en-1-one (10a): White powder (yield 77%),
˝
m.p. 210 C; 1H-NMR (DMSO-d6)
(d, J = 6.9 Hz, 2H, Ar-H), 8.08 (d, J = 7.5 Hz, 2H, Ar-H), 8.00 and 8.55 (d, 1H, = CH-), 10.45 and 11.11 (s,
1H, D2O exchangable, -NH); 13C-NMR (DMSO-d6)
ppm:109.65, 111.58, 118.53, 125.34, 126.98, 128.67,
δ ppm: 7.29–7.52 (m, 8H, Ar-H), 7.58 (d, J = 6.9 Hz, 2H, Ar-H), 7.65
δ
130.64, 132.42, 135.87, 137.36, 139.20, 142.64, 149.83, 151.03, 187.89; Anal. Calcd. for C21H16ClNO3S
(397.87): C, 63.39; H, 4.05; N, 3.52; Found C, 63.60; H, 4.23; N, 3.69.
1-(4-Chlorophenyl)-3-((4-flurophenyl)amino)-2-(phenylsulfonyl)prop-2-en-1-one (10b): White powder (yield
˝
78%), m.p. 219 C; 1H-NMR (DMSO-d6)
δ ppm: 7.22–7.38 (m, 4H, Ar-H), 7.57 (d, J = 9.3 Hz, 2H, Ar-H),
7.63 (d, J = 6.6 Hz, 2H, Ar-H), 7.71–7.76 (m, 1H, Ar-H), 7.88 (d, J = 8.7 Hz, 2H, Ar-H), 7.95 (d, J = 8.7 Hz,
2H, Ar-H), 8.07 and 8.47 (d, 1H, = CH-), 10.46 and 11.11 (s, 1H, D2O exchangable, -NH); Anal. Calcd.
for C21H15ClFNO3S (415.87): C, 60.65; H, 3.64; N, 3.37; Found C, 60.86; H, 3.93; N, 3.20.
1-(4-Chlorophenyl)-3-(˝(2,4-diflurophenyl)amino)-2-(phenylsulfonyl)prop-2-en-1-one (10c): White powder
(yield 73%), m.p. 213 C; 1H-NMR (DMSO-d6)
δ ppm: 7.21 (d, J = 8.4 Hz, 1H, Ar-H), 7.34 (d, J = 8.7 Hz,
2H, Ar-H), 7.75 (s, 1H, Ar-H), 7.57 (d, J = 9.9 Hz, 2H, Ar-H), 7.63 (d, J = 9.9 Hz, 2H, Ar-H), 7.65–7.76
(m, 2H, Ar-H), 7.88 (d, J = 8.4 Hz, 1H, Ar-H), 7.95–8.02 (m, 1H, Ar-H), 8.06 and 8.54 (d, 1H, = CH-),
10.40 and 11.33 (s, 1H, D2O exchangable, -NH); 13C-NMR (DMSO-d6)
δ ppm: 110.27, 112.28, 127.12,
127.92, 128.72, 128.87, 130.86, 133.94, 134.36, 136.49, 137.07, 139.19, 142.41, 152.10, 188.09; Anal. Calcd.
for C21H14ClF2NO3S (433.85): C, 58.14; H, 3.25; N, 3.23; Found C, 58.46; H, 3.42; N, 3.00.
1-(4-Chlorophenyl)-3-((4-chlorophenyl)amino)-2-(phenylsulfonyl)prop-2-en-1-one (10d): White powder (yield
80%), m.p. 225 ˝C; 1H-NMR (DMSO-d6)
δ
ppm: 7.36–7.51 (m, 8H, Ar-H), 7.57–7.70 (m, 3H, Ar-H),
7.94–8.00 (m, 2H, Ar-H), 7.88 and 8.48 (d, 1H, = CH-), 10.46 and 10.93 (s, 1H, D2O exchangable, -NH);
13C-NMR (DMSO-d6)
ppm: 111.96, 120.45, 127.24, 127.95, 128.67, 128.87, 129.20, 130.33, 132.58, 136.14,
δ
137.38, 138.52, 142.81, 149.26, 189.78; Anal. Calcd. for C21H15Cl2NO3S (432.32): C, 58.34; H, 3.50;
N, 3.24; Found C, 3.26; H, 3.75; N, 3.39.
1-(4-Chlorophenyl)-3-((4-bromophenyl)amino)-2-(phenylsulfonyl)prop-2-en-1-one (10e): White powder (yield
82%), m.p. 235 ˝C; 1H-NMR (DMSO-d6)
δ ppm: 7.36–7.70 (m, 11H, Ar-H), 8.07 (d, J = 7.2 Hz, 2H,
Ar-H), 7.95 and 8.47 (d, 1H, = CH-), 10.44 and 10.94 (s, 1H, D2O exchangable, -NH); Anal. Calcd. for
C21H14BrClNO3S (476.77): C, 52.90; H, 3.17; N, 2.94; Found C, 53.12; H, 3.39; N, 3.17.
1-(4-Chlorophenyl)-3-((4-nitrophenyl)amino)-2-(phenylsulfonyl)prop-2-en-1-one (10f): Buff powder (yield
63%), m.p. 237 ˝C; 1H-NMR (DMSO-d6)
δ ppm: 7.53–7.76 (m, 7H, Ar-H), 7.88–7.97 (m, 6H, Ar-H),
7.95 and 8.47 (d, 1H, = CH-), 10.46 and 10.96 (s, 1H, D2O exchangable, -NH); Anal. Calcd. for
C21H15ClN2O5S (442.87): C, 56.95; H, 3.41; N, 6.33; Found C, 57.03; H, 3.72; N, 6.56.

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3.1.5. Synthesis of 1-(4-Aryl)-5-(4-chlorophenyl)-4-(phenylsulfonyl)-1H-pyrazole 12a–c
A mixture of enaminone
9
(5 mmol) and the appropriate phenyl hydrazine 7a
–
c
(5 mmol) in
absolute ethanol (30 mL) was refluxed for 6 h, then left to cool. The precipitated product was collected
by filtration, washed with ethanol and dried. Recrystallization from ethanol afforded pyrazole
derivatives 12a–c, respectively.
˝
5-(4-Chlorophenyl)-1-phenyl-4-(phenylsulfonyl)-1H-pyrazole (12a): White powder (yield 70%), m.p. 215 C;
1H-NMR (DMSO-d6)
ppm: 7.09 (d, J = 8.7 Hz, 2H, Ar-H), 7.17 (d, J = 8.4 Hz, 2H, Ar-H), 7.21–7.32 (m,
δ
3 H, Ar-H), 7.40 (d, J = 8.4 Hz, 2H, Ar-H), 7.51 (d, J = 7.2 Hz, 2H, Ar-H), 7.58–7.67 (m, 1H, Ar-H), 7.68
(d, J = 7.2 Hz, 2H, Ar-H), 8.34 (s, 1H, pyrazole-H); Anal. Calcd. for C21H15ClN2O2S (394.87): C, 63.87;
H, 3.83; N, 7.09; Found C, 63.99; H, 4.03; N, 7.01.
1,5-Bis-(4-chlorophenyl)-4-(phenylsulfonyl)-1H-pyrazole (12b): White powder (yield 84%), m.p. 225 ˝C;
1H-NMR (DMSO-d6)
δ ppm: 7.20 (d, J = 7.8 Hz, 2H, Ar-H), 7.29 (d, J = 7.8 Hz, 2H, Ar-H), 7.35–7.48
(m, 4H, Ar-H), 7.51 (d, J = 7.8 Hz, 2H, Ar-H), 7.60–7.76 (m, 3H, Ar-H), 8.36 (s, 1H, pyrazole-H); Anal.
Calcd. for C21H14Cl2N2O2S (429.32): C, 58.75; H, 3.29; N, 6.53; Found C, 58.94; H, 3.46; N, 6.26.
1-(4-Bromophenyl)-5-(4-chlorophenyl)-4-(phenylsulfonyl)-1H-pyrazole (12c): White powder (yield 84%),
˝
m.p. 245 C; 1H-NMR (DMSO-d6)
δ ppm: 7.20 (d, J = 4.2 Hz, 2H, Ar-H), 7.23 (d, J = 4.2 Hz, 2H, Ar-H),
7.30–7.40 (m, 2H, Ar-H), 7.44 (d, J = 6.3 Hz, 2H, Ar-H), 7.47–7.72 (m, 5H, Ar-H), 8.37 (s, 1H, pyrazole-H);
Anal. Calcd. for C21H14BrClN2O2S (473.77): C, 53.24; H, 2.98; N, 5.91; Found C, 53.40; H, 3.15; N, 6.15.
3.2. Biological Evaluation
3.2.1. Antifungal Activity
All strains were provided from culture collection of the Regional Center for Mycology and
Biotechnology (RCMB), Al-Azhar University, Cairo, Egypt. Antifungal activities were expressed as
the diameter of inhibition zones; agar well diffusion method was used. Holes (1 cm diameter) were
digger in the agar using sterile cork borer in sterile malt agar plates for fungi, which had previously
been uniformly seeded with tested microorganisms. The holes were filled by fungal filtrates (100 µL).
˝
˝
Plates were left in a cooled incubator at 4 C for one hour for diffusion and then incubated at 28 C for
tested fungi. Inhibition zones developed due to active antimicrobial metabolites were measured after
48 h of incubation for fungi. Fluconazole was used as antifungal positive control. The experiment was
performed in triplicate and the average zone of inhibition was calculated [27].
3.2.2. In Vitro Cytotoxic Activity
PC-3 human prostate cancer cells was grown in RPMI-1640 and supplemented with 10% heat
˝
inactivated FBS, 50 units/mL of penicillin and 50 g/mL of streptomycin and maintained at 37 C
in a humidified atmosphere containing 5% CO2. The cells were maintained as “monolayer culture”
by serial subculturing. Cytotoxicity was determined using SRB method as previously described by
Skehan et al. [34,35]. Exponentially growing cells were collected using 0.25% Trypsin-EDTA and seeded
in 96-well plates at 1000–2000 cells/well in DMEM supplemented medium. After 24 h, cells were
incubated for 48 h with various concentrations of the tested compounds. Following 48 h of treatment,
˝
the cells will be fixed with 10% trichloroacetic acid for 1 h at 4 C. Wells were stained for 10 min
at room temperature with 0.4% SRB dissolved in 1% acetic acid. The plates were air dried for 24 h
and the dye was solubilized with Tris-HCl for 5 min on a shaker at 1600 rpm. The optical density
(OD) of each well was measured spectrophotometrically at 564 nm with an ELISA microplate reader
(ChroMate-4300, FL, USA). The IC50 values were calculated according to the equation for Boltzman
sigmoidal concentration-response curve using the nonlinear regression fitting models (Graph Pad,
Prism Version 5, GraphPad Software, Inc., San Diego, CA, USA). The results reported are means of at

Molecules 2016, 21, 114
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least three separate experiments. Statistical differences were analyzed according to one way ANOVA
test wherein the differences were considered to be significant at p < 0.05.
3.3. Docking
The molecular docking of the tested compounds was performed using Discovery Studio
4/CDOCKER protocol (Accelrys Software Inc.). The protein crystallographic structure of
Mycobacterium tuberculosis (Mycobacterium P450 DM) and co-crystallized fluconazole (PDB code:
1EA1) was downloaded from the Protein Data Bank (PDB). The protein was prepared for docking
process according to the standard protein preparation procedure integrated in Accelry’s discovery
studio 4 and prepared by prepare protein protocol. Fluconazole, 6a, 8a, 10a and 10b were drawn
as a database and prepared by prepare ligand protocol to generate 3D structure and refine using
CHARMM force field with full potential. Docking simulations were run using CDOCKER protocol
where a maximum bad orientations was 800 and orientation vdW energy threshold was 300. Simulated
annealing simulation would be then carried out consisting of a heating phase 700 K with 2000 steps and
a cooling phase back to 5000 steps. The binding energy was calculated as a score to rank the docking
poses. The top 10 docking poses would be finally saved. Docking poses were ranked according to
their –CDOCKER interaction energy, and the top poses were chosen for analysis of interactions for
each compound.
4. Conclusions
In an extension of our ongoing efforts towards developing potent antifungal agents, we
developed a methodology for the synthesis of four novel target sulfone series, 6a
–f, 8a–c,
10a and 12a . All the newly prepared phenyl sulfones were screened against invasive fungi,
–
f
–c
namely, Candida species (fluconazole-resistant Candida albicans, Candida albicans, Candida tropicalis,
Candida parapsilosis) and Asperigillus species (Aspergillus fumigatus, Aspergillus niger), dermatophytic
fungi, namely Trichophytons mentagrophyte and Microsporum canis and the non-dermatophytic fungi
Syncephalastrum racemosum. Fluconazole was used as a reference drug for the antifungal screening.
The active compounds 6a, 8a, 10a, 10b, 10e and 12a showed no remarkable cytotoxicity against PC-3
cancer cell line with a selectivity index >11.19. The results evidenced that the p-fluoro phenyl aniline
derivative 10b exhibited excellent activity against all the candida species and was five times more
potent than fluconazole on C. albicans, C. tropicalis and C. Parapsilosis.
Overall, compounds 8a and 10b could be considered as good lead candidates for further
optimization of new potent anticandidal agents. Moreover, the unsubstituted phenyl hydrazine
6a and the substituted aniline 10a elicited broad spectrum activity against most of the tested organisms.
On the other hand, the p-bromo aniline derivative 10e and the pyrazole derivative 12a could also act
as a starting point in the process of discovery of new anti-aspergillosis and anti-dermatophytic agents.
A docking study of the most active derivatives 6a, 8a, 10a and 12b was performed on Cyp-P450 DM
enzyme (PDB code: 1EA1) and presented a rationale for the activity of these compounds. Furthermore,
a theoretical kinetic study was established to predict the ADME of the active derivatives. Finally, the
work also described an X-ray study of the starting 1-(4-chlorophenyl)-2-(phenylsulfonyl)ethanone 3.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/
1/114/s1.
Acknowledgments: Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian University,
Cairo, Egypt, is highly appreciated for supporting this research. National Research Center, Dokki, Giza, Egypt, is
highly appreciated for supporting this research. The authors would like to extend their sincere appreciation to
the Deanship of Scientific Research at King Saud University for its funding of this research through the Research
Group Project No. RGP-1436-038.
Author Contributions: H.A.A. and M.F. formulated the research idea and participated in the preparation of
manuscript; M.F., M.A.S., M.A.A., H.A.G., H.A. and W.M.E. carried out the experimental, interpreted the data
and prepared the manuscript; M.M.A. and R.A.E. performed the biological screening. All authors have read and
approved the final manuscript.

Molecules 2016, 21, 114
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Conflicts of Interest: The authors declare no conflict of interest.
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