Discovery of new azoles with potent activity against Candida spp. and Candida albicans biofilms through virtual screening

Article abstract of DOI:10.1016/j.ejmech.2019.06.083

Systemic candidiasis is a rampant bloodstream infection of Candida spp. and C. albicans is the major pathogen isolated from infected humans. Azoles, the most common class of antifungals which suffer from increasing resistance, and especially intrinsically resistant non-albicans Candida (NAC) species, act by inhibiting fungal lanosterol 14α-demethylase (CYP51). In this study we identified a number of azole compounds in 1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethanol/ethanone oxime ester structure through virtual screening using consensus scoring approach, synthesized and tested them for their antifungal properties. We reached several hits with potent activity against azole-susceptible and azole-resistant Candida spp. as well as biofilms of C. albicans. 5i's minimum inhibitor concentration (MIC) was 0.125 μg/ml against C. albicans, 0.5 μg/ml against C. krusei and 1 μg/ml against azole-resistant C. tropicalis isolate. Considering the MIC values of fluconazole against these fungi (0.5, 32 and 512 μg/ml, respectively), 5i emerged as a highly potent derivative. The minimum biofilm inhibitor concentration (MBIC) of 5c, 5j, and 5p were 0.5 μg/ml (and 5i was 2 μg/ml) against C. albicans biofilms, lower than that of amphotericin B (4 μg/ml), a first-line antifungal with antibiofilm activity. In addition, the active compounds showed neglectable toxicity to human monocytic cell line. We further analyzed the docking poses of the active compounds in C. albicans CYP51 (CACYP51) homology model catalytic site and identified molecular interactions in agreement with those of known azoles with fungal CYP51s and mutagenesis studies of CACYP51. We observed the stability of CACYP51 in complex with 5i in molecular dynamics simulations.

Full text of DOI:10.1016/j.ejmech.2019.06.083

Accepted Manuscript
Discovery of new azoles with potent activity against Candida spp. and Candida
albicans biofilms through virtual screening
Suat Sari, Didem Kart, Naile Öztürk, F. Betül Kaynak, Melis Gencel, Gülce Taşkor,
Arzu Karakurt, Selma Saraç, Şebnem Eşsiz, Sevim Dalkara
PII:
S0223-5234(19)30610-5
DOI:
https://doi.org/10.1016/j.ejmech.2019.06.083
EJMECH 11486
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 May 2019
Revised Date: 18 June 2019
Accepted Date: 28 June 2019
Please cite this article as: S. Sari, D. Kart, N. Öztürk, F.Betü. Kaynak, M. Gencel, Gü. Taşkor, A.
Karakurt, S. Saraç, Ş. Eşsiz, S. Dalkara, Discovery of new azoles with potent activity against Candida
spp. and Candida albicans biofilms through virtual screening, European Journal of Medicinal Chemistry
(2019), doi: https://doi.org/10.1016/j.ejmech.2019.06.083.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Discovery of new azoles with potent activity against Candida spp. and
Candida albicans biofilms through virtual screening
Suat Saria*, Didem Kartb, Naile Öztürkc,d, F. Betül Kaynake, Melis Gencelf, Gülce Taşkorg, Arzu
Karakurth, Selma Saraça, Şebnem Eşsize, Sevim Dalkaraa
a Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06100,
Ankara, Turkey
b Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology, 06100,
Ankara, Turkey
c Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Technology, 06100,
Ankara, Turkey
d İnönü University, Faculty of Pharmacy, Department of Pharmaceutical Technology, 44280, Malatya,
Turkey (permanent address)
e Hacettepe University, Faculty of Engineering, Department of Physical Engineering, 06800, Ankara,
Turkey
f Kadir Has University, Faculty of Engineering and Natural Sciences, Department of Bioinformatics and
Genetics, 34083, Istanbul, Turkey
g Hacettepe University, Faculty of Pharmacy, Department of Basic Pharmaceutical Sciences, 06100,
Ankara, Turkey
h İnönü University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 44280, Malatya,
Turkey
Declarations of interest: none
Acknowledgments
This study was funded by grants from The Scientific and Technological Research Council of
Turkey (TÜBITAK) (115S387) and Hacettepe University Scientific Research Projects
Coordination Unit (014 D09 301 001‐703, TPT‐2015‐6794, TPT‐2018‐16666).
* Correspondence: Suat Sari, PhD
Hacettepe University Faculty of Pharmacy
Department of Pharmaceutical Chemistry
06100 Sihhiye Ankara/TURKEY
Tel.: + 90 312 305 18 72
Fax: + 90 312 305 32 72
e‐mail: suat.sari@hacettepe.edu.tr
suat1039@gmail.com

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Abstract
Systemic candidiasis is a rampant bloodstream infection of Candida spp. and C. albicans is
the major pathogen isolated from infected humans. Azoles, the most common class of
antifungals which suffer from increasing resistance, and especially intrinsically resistant non‐
albicans Candida (NAC) species, act by inhibiting fungal lanosterol 14α‐demethylase (CYP51).
In this study we identified a number of azole compounds in 1‐(2,4‐dichlorophenyl)‐2‐(1H‐
imidazol‐1‐yl)ethanol/ethanone oxime ester structure through virtual screening using
consensus scoring approach, synthesized and tested them for their antifungal properties.
We reached several hits with potent activity against azole‐susceptible and azole‐resistant
Candida spp. as well as biofilms of C. albicans. 5i’s minimum inhibitor concentration (MIC)
was 0.125 μg/ml against C. albicans, 0.5 μg/ml against C. krusei and 1 μg/ml against azole‐
resistant C. tropicalis isolate. Considering the MIC values of fluconazole against these fungi
(0.5, 32 and 512 μg/ml, respectively), 5i emerged as a highly potent derivative. The minimum
biofilm inhibitor concentration (MBIC) of 5c, 5j, and 5p were 0.5 μg/ml (and 5i was 2 μg/ml)
against C. albicans biofilms, lower than that of amphotericin B (4 μg/ml), a first‐line
antifungal with antibiofilm activity. In addition, the active compounds showed neglectable
toxicity to human monocytic cell line. We further analyzed the docking poses of the active
compounds in C. albicans CYP51 (CACYP51) homology model catalytic site and identified
molecular interactions in agreement with those of known azoles with fungal CYP51s and
mutagenesis studies of CACYP51. We observed the stability of CACYP51 in complex with 5i in
molecular dynamics simulations.
Keywords: consensus scoring; azoles; Candida albicans; biofilm; cytotoxicity; molecular
docking; molecular dynamics simulations

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1.
Introduction
Systemic candidiasis is a major public health issue, especially with immune‐suppressed cases
reaching high mortality rates. The members of the genus Candida are the most frequently
recovered from human fungal infection and Candida albicans, so far, is the leading pathogen
identified in nosocomial candidiasis [1]. In addition to increasing drug‐resistant strains of C.
albicans, emergence of non‐albicans Candida spp. (NAC) complicate the treatment of
mycoses [2]. C. tropicalis is among the NACs that show reduced susceptibility to first‐line
antifungals reportedly leading to breakthrough fungemia among high‐risk patients [3, 4].
Also, C. krusei is known to be intrinsically resistant to a number of azoles including
fluconazole [5]. One of the several mechanisms of therapy‐resistance is formation of
biofilms, which are complex microorganism colonies enclosed in an exopolysaccharide
matrix on biotic and non‐biotic surfaces. Persistent biofilms make fungi much less
susceptible to antifungal drugs compared to their planktonic forms for a number of reasons
[6‐8]. Therefore it is essential to design new molecules effective against resistant fungi as
well as fungal biofilms.
Azoles are commonly preferred in fungal infections for their advantages such as broad
spectrum of activity, well‐tolerability, and oral availability (Fig. 1), however their wide usage
brings about the issue of resistance [9]. Azoles act through inhibition of lanosterol 14α‐
demethylase (CYP51), a ubiquitous cytochrome P450 enzyme that plays a key role in the
biological synthetic cascade of ergosterol, a fungal sterol included in membrane structure
[10]. The emergence of experimental fungal CYP51 structures accelerated the efforts
towards rational design of new azoles [11‐16]. Also merging molecular modelling studies
with mutational studies lead to identification of key molecular determinants for CYP51
inhibition to better design new hits [11, 12, 17].

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O
Cl
Cl
Cl
N
N
N
N
Cl
N
N
O
N
N
N
N
O
N
O
O
O
N
N
N
N
OH
F
Cl
Cl
F
Cl
Cl
Miconazole
Cl
Cl
Oxiconazole
Fluconazole
Ketoconazole
N
N
N
O
N
O
R
N
N
N
N
N
F
N
R
O
N
N
N
N
O
O
O
N
O
N
N
N
O
OH
F
Cl
Cl
Cl
Cl
F
Cl
Cl
5a-t
Itraconazole
Voriconazole
6a-r
Fig. 1. Molecular structure of azole antifungals from different generations and the title
compounds
In this study we present rational design of a set of miconazole and oxiconazole analogues
(5a‐t and 6a-r) through virtual screening, their synthesis and biological activity studies (Fig.
1). We obtained hit molecules with activity against azole‐resistant C. tropicalis as well as
inhibition of C. albicans biofilms. The active compounds showed neglectable cytotoxicity.
Also, in‐depth analysis of C. albicans CYP51 (CACYP51) inhibition via molecular modelling
studies provided valuable insights.
2.
Results and discussion
2.1. Identification of the compounds for synthesis through virtual screening
2.1.1. Virtual library generation and physicochemical properties filtering
Azole antifungals feature a number of pharmacophores: an azole group, an aromatic ring,
and a "tail" group attached to an alkylene bridge between the two via various linker groups.
In our study, we constructed an azole scaffold with imidazole as the azole ring, 2,4‐
dichlorophenyl as the aromatic ring, either alcohol ester or oxime ester as the linker
functionalities for the tail defined as R in Fig. 1. We, then, envisaged a virtual library of more
than 200 compounds by modifying the tail considering the commercially available synthetic
building blocks, i.e. carboxylic acids, for this position. The virtual library was created by 3D‐
modelling the envisaged compounds with proper tautomeric and ionization forms and

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enantiomers, and geometry optimization, and subjected to a drug‐likeness filtering to
eliminate the candidates with poor physicochemical properties such as molecular weight
(MW), number of rotatable bonds (RB), hydrogen bond donor and acceptor counts (HD and
HA), octanol/water partition coefficient (LogP), and total polar surface area (PSA) [18‐20].
Compounds with more than two "Lipinski’s rule of 5" violations were phased out and the
remaining 146 compounds were selected for the next step.
2.1.2. Molecular docking and consensus scoring
The eukaryotic CYP51 is composed of a catalytic domain which contains a heme co‐factor at
the bottom of the catalytic site. An anchor domain attached to the catalytic domain tethers
the enzyme to endoplasmic reticulum (ER) membrane. A narrow, hydrophobic entry
channel, reaching from a location close to where the two domains connect and the ER
membrane, leads to the catalytic cavity and heme. According to the crystallographic data
azoles occupy this catalytic domain with a common conformation in the following way: the
azole group interacts with the heme making the 6th axial coordination with heme iron
through one of the nitrogen atoms, the aromatic ring fits in a cavity between the heme and
the protein in hydrophobic contacts and the tail, which includes H bond donors and
acceptors in addition to hydrophobic groups, occupies the entry channel (Fig. 2) [21, 22].
<Figure 2>
Fig. 2. The catalytic site of Saccharomyces cerevisiae CYP51 (protein is showed as rippons
and the binding site molecular surface is rendered) with the co‐crystallized ligand
itraconazole (green sticks) interacting with heme cofactor (gray sticks) and heme iron
(orange sphere).
Although the azole ring‐heme interaction is key to this binding, the interactions of the tail
with this gorge determine the tightness of this binding [23]. In this respect we docked the
remaining 146 compounds from the previous step to the catalytic site of CACYP51 homology
model using AutoDock (v4.2) [24] and Glide (2018‐1: Schrödinger, LLC, NY, 2018) [25‐27]. We
determined the docking scores of each compound from the two software out of the best
poses identified upon visual inspection in comparison with the available crystallographic
data. The compounds were then ranked as two separate groups, alcohol ester and oxime
ester derivatives, according to their consensus scores, which, simply, is the average of the

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scores from AutoDock and Glide (See Table S1 of Supporting Information for the structures
of the docked compounds and full results of the virtual screening study). Consensus scoring,
which combines multiple scoring functions in binding affinity estimation, reportedly leads to
higher hit‐rates in virtual library screening studies by eliminating false positives since
different scoring functions may bias certain interaction terms [28, 29]. Since AutoDock and
Glide yield docking scores of compatible units we preferred the rank‐by‐number strategy, in
which we simply ranked the compounds according to the mean values of the scores from
each. Other strategies are rank‐by‐rank, in which the compounds are ranked by their mean
rank values obtained from each scoring function instead of docking score, and the rank‐by‐
vote strategy, in which the compounds are assigned a number for each scoring function
according to the percentage they are ranked in each scoring function and then ranked
according to the mean value of these numbers [28]. Compounds with top consensus score
among the two groups were selected for synthesis (Table 1). Some of the top‐scoring
compounds were skipped in the synthesis step due to no or very low yield through the
common synthetic protocol described below (see Table S1 of Supporting Information for
details).
Table 1. Virtual screening scores (kcal/mol) of the selected compounds.
Comp. AutoDock Glide consensusa
Comp. AutoDock Glide consensusa
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
‐11.79
‐10.48
‐9.41
‐8.36
‐8.99
‐9.09
‐9.27
‐8.14
‐8.24
‐8.85
‐8.63
‐8.35
‐8.24
‐8.15
‐8.06
‐8.28
‐7.97
‐8.10
‐8.13
‐8.11
‐5.40
‐6.34
‐6.42
‐6.91
‐6.23
‐6.11
‐5.89
‐6.78
‐6.65
‐6.03
‐5.84
‐6.08
‐6.06
‐6.08
‐6.16
‐5.88
‐6.11
‐5.63
‐5.44
‐5.42
‐8.60
‐8.41
‐7.92
‐7.64
‐7.61
‐7.60
‐7.58
‐7.46
‐7.45
‐7.44
‐7.24
‐7.22
‐7.15
‐7.12
‐7.11
‐7.08
‐7.04
‐6.87
‐6.79
‐6.77
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
‐12.56
‐10.14
‐10.5
‐8.87
‐9.68
‐8.97
‐8.63
‐8.70
‐8.63
‐8.69
‐8.53
‐8.64
‐9.04
‐8.55
‐9.12
‐8.58
‐8.26
‐7.94
‐5.77
‐6.59
‐5.84
‐6.98
‐6.01
‐6.14
‐6.46
‐6.33
‐6.34
‐6.19
‐6.27
‐6.06
‐5.39
‐5.88
‐5.26
‐5.62
‐5.77
‐6.05
‐9.17
‐8.37
‐8.17
‐7.93
‐7.85
‐7.56
‐7.55
‐7.52
‐7.49
‐7.44
‐7.40
‐7.35
‐7.22
‐7.22
‐7.19
‐7.10
‐7.02
‐6.00
5s
5t

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a Calculated by the formula: (AutoDock Score + Glide score)/2
2.2. Synthesis of the selected compounds
The synthetic procedure for the selected compounds is outlined in Scheme 1. Synthesis of
5g, 5j, 5n, and 5r was previously reported [30, 31]. Starting from 2,2',4'‐
trichloroacetophenone (1) we obtained 1‐(2,4‐dichlorophenyl)‐2‐(1H‐imidazol‐1‐yl)ethanone
(2) by N‐alkylation of imidazole with 1 in dimethylformamide (DMF). 2 was reduced using
sodium borohydride (NaBH4) methanol (MeOH) to yield 1‐(2,4‐dichlorophenyl)‐2‐(1H‐
imidazol‐1‐yl)ethanol (3) and converted to its oxime derivative 1‐(2,4‐dichlorophenyl)‐2‐(1H‐
imidazol‐1‐yl)ethanone oxime (4) using first hydroxylamine hydrochloride (NH2OH.HCl) at
basic medium and refluxing in ethanol (EtOH) then at basic medium in water. The title
compounds (5a-t and 6a-r) were afforded through esterification of 3 and 4 with proper
carboxylic acids in the presence of 4‐dimethylaminopyridine (DMAP), a transacylation
catalyst, and N,N'‐dicyclohexylcarbodiimide (DCC), a coupling agent, in dry dichloromethane
(DCM). 2, 3, 5b-t, 6a, 6b, 6d, 6f-h, 6j, 6k, 6m, 6o, and 6r were converted to their HCl salts
using gaseous HCl (gHCl) to improve purity and solubility. Structures and purity of the
compounds were confirmed via LC‐MS and NMR spectra and elemental analysis.

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Scheme 1. Synthesis of the selected compounds. Reagents and conditions: imidazole, DMF, 0‐5 °C (i); NaBH4, MeOH, 0‐5 °C (ii); NH2OH.HCl,
EtOH,
pH
14,
ref.,
conc.
HCl,
H2O,
pH
5
(iii);
RCOOH,
DCC,
DMAP,
DCM,
0‐5
°C,
gHCl.

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The HPLC chromatograms of the compounds showed a pure single peak whose mass
spectrum included molecular ion peaks with [M+2]+ and [M+4]+ chlorine isotopes and their
1
sodium adduct peaks. H NMR spectra of the alcohol ester derivatives featured a multiplet
between 4.5‐5.0 ppm for the protons of CH2 next to imidazole and a doublet of doublet at
around 6.5 ppm for the CH proton of the alcohol root, which gave a multiplet signal for some
compounds. The CH2 next to the imidazole of the oxime ester derivatives was observed as a
singlet around 6 ppm. The protons of 2,4‐dichlorophenyl ring were observed in the aromatic
region of the spectrum, usually overlapped with other aromatic protons of the tail groups as
well as with H4 and H5 of the imidazole. Imidazole’s H2 resonated further downfield than H4
and H5 and the deshielding effect of the HCl salt was very obvious for this proton which
resonated much further downfield, at around 9.2 ppm, compared to that of the derivatives
without HCl observed at 8.1‐8.4 ppm range. The protons of the tail groups were observed at
chemical shifts corresponding to the functional groups they belong and the integration
13
values of the signals were proportionate to the number of protons they represent. The C
NMR spectra were also in agreement with the structural composition of the compounds. The
carbonyl carbons of the alcohol ester and oxime ester functions were observed at 160‐164
ppm range and the oxime C=N‐O carbon of the oxime ester resonated around 160 ppm (See
Supporting Information for details).
2.3. X ray crystallography studies
According to the ORTEP [32] views, 4 is in Z configuration (Fig. 3), which is in accordance with
our previous studies with oximes and their derivatives [33‐35]. This finding supports the
possibility that the title compounds in oxime ester derivatives are in the configuration, too.
The packing of 4 is composed of Z isomers only, showing that 4 was obtained as Z isomer
(Fig. 4). Molecular structure of 5o was resolved as R enantiomer. In the crystal pack,
however, consecutively aligned R and S isomers of 5o interact with each other through non‐
classical H bonds as expected since 5a-t were synthesized as racemates through nonselective
method. In both compounds, the 2,4‐dichlorobenzene and imidazole rings are separately
planar. The 2,4‐dichlorobenzene and imidazole rings of 5o are almost in the same plane and
the deviation from the planarity is 2.12°. The dihedral angles between the imidazole ring and
the 4‐methoxybenzene ring and the 2,4‐dichlorobenzene ring of 5o are 77.16° and 75.60°,
respectively. For 4, the dihedral angle between 2,4‐dichlorobenzene and imidazole rings is

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54.38°. The crystal structures 4 and 5o are stabilized by intermolecular and intramolecular
hydrogen bonds. There are also C−Cl…π interactions in their crystal structures. For more
details see Table S3 and Table S4 of Supporting Information.
<Figure 3>
Fig. 3. ORTEP3 view of 4 (A) and 5o (B) showing the atom‐numbering scheme. Displacement
ellipsoids are drawn at 50% (A) and 35% (B) probability level.
<Figure 4>
Fig. 4. Hydrogen bonding interactions of 4 (A) and 5o (B), the unit cell packing of 4 (C, view
direction is along [100]), and 5o (D, view direction is along [010]).
2.4. Candidae susceptibility to the synthesized compounds
We identified the minimum inhibitor concentration (MIC) values of the synthesized
compounds against the ATCC strains of C. albicans, C. parapsilosis, and C. krusei. Some of the
active compounds were also tested against an azole‐resistant clinical isolate of C. tropicalis
(Table 2). MIC is the minimum concentration of material that inhibits visual growth of a
given microorganism. Many compounds were found active against C. albicans at
concentrations better than or comparable to fluconazole. Especially, 5b, 5i, 5j, and 6l stood
out as derivatives with MIC values better than or equal to fluconazole. Several compounds
were also active against C. krusei, a NAC known to be resistant against many azoles, at very
low MICs. 5c, 5i, 5j, and 5o were found highly potent against the azole‐resistant C. tropicalis
isolate. MIC values of 5g, 5j, 5n, and 5r against C. albicans were previously reported as 10, 2,
300, and >300 µg/ml, respectively [30, 31]. The efficacy of 5j was thus confirmed.
Table 2. MIC values (µg/ml) of the compounds against Candida spp.
C. albicans
C. krusei
C. parapsilosis
C. tropicalis
Compound
ATCC 90028
ATCC 6258
ATCC 90018
isolate
2
3
4
5a
5b
5c
5d
5e
5f
128
8
32
256
0.25
1
8
256
16
256
128
256
256
1
2
28
128
256
32
4
32
256
0.25
1
16
128
32
256
512
1

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5g
5h
5i
5j
5k
5l
5m
5n
128
8
0.125
0.5
256
8
8
128
2
256
64
0.5
1
256
32
128
256
16
8
128
4
0.25
1
256
4
16
128
1
16
1
4
16
5o
5p
4
32
8
2
2
5q
5r
5s
5t
6a
6b
6c
6d
1
8
16
32
32
256
256
256
1
2
4
8
256
128
256
1
128
4
8
16
256
128
256
1
256
256
64
128
4
256
4
6e
6f
6g
6h
6i
6j
6k
6l
6m
64
2
64
64
256
64
64
64
1
64
2
16
8
128
128
32
64
0.5
2
128
64
32
16
1
64
64
4
2
6n
6o
6p
6q
6r
128
16
64
32
128
0.5
256
32
64
32
256
32
256
16
64
32
256
0.5
Fluconazole
512
Biofilm formation is one of the common drug resistance mechanisms of fungi. Among the
most active derivatives, twelve were tested against mature biofilms of C. albicans, the
minimum biofilm inhibitor and the minimum biofilm eradicator concentration (MBIC and
MBEC) values were determined for each of the twelve compounds (Table 3). Most of the
tested compounds (5b, 5c, 5i, 5j, 5o, 5p, 6g, and 6l) better inhibited biofilm formation than
amphotericin B, a first‐line antifungal drug known for its efficacy against fungal biofilms. The
MBIC of 5j against C. albicans biofilms was reported as 2 µg/ml [31]. Biofilm eradicator
potential of the compounds were low, like amphotericin B, however the MBEC values of 5j
and 5p were promising.

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Table 3. MBIC and MBEC values (µg/ml) of the selected compounds against C. albicans
biofilms.
Compound
MBIC
2
0.5
2
0.5
1
0.5
32
16
128
2
2
128
4
MBEC
256
256
128
64
5b
5c
5i
5j
5o
5p
5q
6c
6e
6g
6l
6m
256
64
>1024
256
256
256
512
512
256
Amphotericin B
From the antifungal susceptibility assays, derivatives 5c, 5i, 5j, and 5o emerged as the most
potent compounds with activity against ATCC strains of C. albicans and other NACS, against
an azole‐resistant C. tropicalis isolate, and C. albicans biofilms at the same time (Table 2 and
Table 3). The alcohol ester library was apparently more active than the oxime ester library,
which includes potent derivative such as 6c, 6g, 6l, and 6m. All these active derivatives bear
a benzene ring on the tail. 4‐Biphenyl, cinnamyl, indol‐2‐yl, 4‐methoxyphenyl, and 2‐
naphthyl emerged as the most useful substitutions for the tail group. However the most
potent compound against C. albicans, 5i, is the only one with a hydrogen bond donor. The
most potent derivatives against the C. tropicalis isolate was again the alcohol ester
derivatives, 5c, 5i, 5j, and 5o, which bear 2‐naphthyl, indol‐2‐yl, cinnamyl, and 4‐
methoxyphenyl, respectively. Interestingly, compounds with potent activity against the ATCC
Candida strains such as 5b and 6c, which bear 4‐biphenyl tail, were almost ineffective against
this isolate.
2.5. Cytotoxicity evaluation
Selectivity of antimicrobial chemotherapeutic agents towards the microorganism and their
safety to the host is crucial. For the in vitro cytotoxicity evaluation of the selected six active
compounds (5b, 5c, 5i, 5j, 6c, and 6l) cell viability of U937 human monocytic cells in the
presence of the compounds after 24 and 48 hours were determined in comparison with the
control (Fig. 5A and Fig. 5B). The compounds did not display considerable toxicity at their

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active concentration ranges at any time points, although 5b lowered the viability
percentages below 80% at concentrations ≥ 8 µg/ml.
Fig. 5. The effect of 5b, 5c, 5i, 5j, 6c, and 6l on cell viability of human monocytic cell line for
24 h (A) and 48 h (B).
2.6. Drug-like chemical space evaluation
The calculated molecular descriptors of the title compounds, in general, were within the
recommended ranges derived from the known drug‐like chemical space, although there
were exceptions. The number of rotatable bonds, molecular weight, hydrogen bond donor
and acceptor counts, and polar surface area were found within the limit values. The
calculated LogP of 5a and 6a were too high, their aqueous solubility was also below the ideal
limit, which probably was the reason for these compounds to be inactive. These compounds

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violated the Lipinski’s rule of five for having molecular weight greater than 500 g/mol and
Log P value higher than 5 (See Table S2 of Supporting Information for the full data of the
calculated descriptors).
2.7. Molecular modelling of CACYP51 inhibition by the active compounds
The binding modes of the active compounds obtained from AutoDock and Glide fulfilled the
molecular determinants identified for CYP51 inhibition by azoles as described above (Fig. 6).
The imidazole ring of the compounds was in T‐shaped π‐π interaction with the heme while
the nitrogen at the second position of imidazole was in axial coordination with the heme
iron. The 2,4‐dichlorophenyl group was in the hydrophobic pocket just above the heme
surrounded by residues Phe126, Ile131, Tyr132, and Gly303, and the tail occupied the entry
channel (See Supporting Information for details). 5i’s NH of indole at the tail donates a
hydrogen bond to Ser378 backbone oxygen in the binding mode from AutoDock, but to
Met508 in the binding mode from Glide. The compound also engages in a π‐π interaction
with Tyr132 according to AutoDock. Other residues that contact with the tail are Tyr118,
Leu121, Thr122, Pro230, Leu376, Ile379, Phe380, and Val509 in both binding modes. Most of
these residues cited as key residues for the enzyme activity in mutagenesis studies and their
mutants were reportedly associated with decreased susceptibility to azoles [36].
<Figure 6>
Fig. 6. Superimposition of the binding modes of 5i from AutoDock (light green) and Glide
(dark green) in CACYP51 catalytic site (A), and their 2D interaction diagrams (B and C,
respectively). Ligands and heme are represented as sticks, heme iron as blue CPK, and
protein surface is rendered in color according to the electrostatic potential.
We further evaluated the 5i‐CACYP51 complex in terms of dynamic evolution and stability
using molecular dynamics (MD) simulations. We selected the binding mode form AutoDock
for this purpose due to its suitability to the available crystal structures, especially considering
the orientation of imidazole regarding the heme, the distance between heme iron and N2, as
well as the orientation of the 2,4‐dichlorophenyl ring in the hydrophobic cleft. We compared
the trajectories of water solvated 5i‐bound CACYP51 and the ligand free (apo) CACYP51
systems. The Cα atoms RMSD and total energy plots indicate higher stability for the 5i‐
bound system (Fig. 7A and Fig. 7B). The RMS fluctuation (RMSF) values for the residues also

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show that most of the residues fluctuated more in the apo form (Fig. 7C). Phe228, Pro230,
Gly307, and Met508 were the most fluctuating binding site residues in the absence of 5i.
Fig. 7. Plots showing apo and 5i‐bound CACYP51's Cα RMSD values (A) and the total energy
of the protein over time (B) and the average RMS fluctuations for each residue (C) (High
fluctuating binding site residues are indicated).

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We also monitored the hydrogen bond distance between the indole NH and Ser378 carbonyl
oxygen as well as the distance between the 2,4‐dichlorophenyl and Tyr132 side chain, which
were in π‐π stacks (Fig. 8). Both interactions maintained at around 4 Å although the
hydrogen bond distance showed certain fluctuations.
Fig. 8. Plots showing the deviation of the distance of the hydrogen bond between 5i’s indole
NH hydrogen and Ser378 (A) and the distance of the π‐π interaction between 5i’s 2,4‐
dichlorophenyl and Tyr132 side chain (B).
3.
Conclusion
In pursuit for ideally effective and safe antifungals in azole structure we took on a rational
design study using virtual screening method and consensus scoring approach. The selected
compounds were synthesized and tested against Candida spp. Fungi. We reached highly
potent derivatives including 5i with a MIC of 0.125 μg/ml against C. albicans, 0.5 μg/ml
against C. krusei, 0.25 against C. parapsilosis, and 1 μg/ml against a fluconazole‐resistant C.

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tropicalis isolate. In addition 5b, 5c, 5i, 5j, 5o, 5p, 6g, and 6l were better at inhibiting C.
albicans biofilms than amphotericin B, an antifungal drug with antibiofilm activity. Inhibiting
fungal biofilms, which account for drug resistance of some fungi among other mechanisms
and raises the inhibitor concentrations of first‐line antifungals by orders compared to their
MIC values against the planktonic forms, is a key feature of our compounds. Also, some of
these compounds were tested and found safe for human monocytes at their active
concentrations. Therefore, it is suggestable that our virtual screening study with rank‐by‐
number consensus scoring strategy, which took advantage of two different scoring
functions, paid off well with several hits and promising derivatives against drug‐resistant
fungi.
The binding modes and interactions of the active compounds from both AutoDock and Glide
were in good agreement with the experimental and theoretical data. From the MD
simulations we were able to deduce that CACYP51 was more stable with the presence of 5i
in its catalytic site with two key interactions, a hydrogen bond with Ser378 and π‐π
interaction with Tyr132 side chain.
This study yielded potent antifungal hits with excellent activity profile and future prospects
for further optimization as leads due to favorable calculated descriptors putting them within
the drug‐like chemical space. With strong in silico evidence to their proposed mechanism of
action we are now trying to design better CACYP51 inhibitors referring to the structure of 5i.
4.
Materials and Methods
4.1. Molecular modelling and virtual screening
The virtual library was created using 2D Sketcher and MacroModel (2018‐1: Schrödinger,
LLC, NY, 2018) of Maestro (2018‐1: Schrödinger, LLC, NY, 2018). The ligands were then
optimized using conjugate gradients method and OPLS_2005 force field [37]. Possible
tautomeric and ionization states (pH: 7±2) and enantiomers for each ligand were modelled
using LigPrep of Maestro. The molecular descriptors were calculated using QikProp (2018‐1:
Schrödinger, LLC, NY, 2018).
The homology modelling of CACYP51 was previously described [11]. In brief, it was created
according to comparative modelling approach on MODELLER (v9.18) [38] using the pairwise
amino acid sequence alignment of CACYP51 and S. cerevisiae CYP51 and the crystal structure

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of the latter (PDB ID: 5EQB [21]) as template. The co‐crystallized ligand, itraconazole in the
active site of the template was included in the CACYP51 homology model. For grid
generation the central coordinates of CACYP51 catalytic site (19.42 10.25 17.44) was
selected and each dimension of the grid box was set to 20 Å. Each ligand was docked 50
times with full flexibility to the active site grid using AutoDock and Glide. On AutoDock
Lamarckian genetic algorithm was selected with medium exhaustiveness, on Glide standard
precision was selected. The poses were ranked according to the consensus scores calculated
as the average of Autodock score and Glide score of the best poses for each ligand which
were selected upon visual evaluation of the results.
For the MD simulations study, the ligand‐free (apo) and 5i‐bound CACYP51 models were
created and solvated in a water box with a 5‐Å layer of water on each face using VMD
(v1.9.2) [39], which finally consisted of around 60000 atoms. CHARMM36 force‐field was
used for the protein and solvent with CMAP corrections, CHARMM General Force‐Field
(v3.1) via cgenff.paramchem.org server (v1.0) was used for the ligands, and water molecules
were modelled using TIP3P water model [40‐45]. Particle mesh Ewald (PME) summation was
used with grid sizes 114, 103, and 84 and full updates at every 2 fs [46]. Harmonic potential
constraints (5 kcal/mol*Å2) were applied on the backbone atoms of the membrane‐
embedded residues, Fe2+ of heme, and S− of heme‐coordinating cysteine and heme was
patched to keep planar. Following an initial 100‐step force field minimization, the systems
were run for 20 ns at constant temperature (310 K) and pressure (1 atm) (NPT ensemble)
with integration time step set to 2 fs, non‐bonded cut‐off starting at 10 Å, and pair list set to
14 Å using NAMD (v2.10) [47]. SHAKE algorithm [48] was used for hydrogens, and the
coordinates were saved every 500 steps.
4.2. Chemistry
The chemicals used in this study were obtained from commercial suppliers. Thin layer
chromatography (TLC) was performed using Merck Kieselgel 60 F254 as stationary phase and
chloroform‐methanol (90:10) as mobile phase to monitor the reactions; the TLC plate spots
were inspected under 254 nm UV light. Melting points (mp) were recorded on a Thomas‐
1
Hoover capillary melting point apparatus (USA) and uncorrected. H NMR (400 MHz) and 13C
NMR (100 MHz) spectra were obtained using Varian Mercury 400 FT (USA) NMR
spectrometer. LC‐MS spectra were recorded with Micromass ZQ mass spectrometer (USA)

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connected to Waters Alliance HPLC (USA) with electrospray ionization (ESI+) method and
MassLynx 4.1 software. Elemental analyses were performed by LECO 932 CHNS elemental
analysis apparatus (USA) and the results are reported as percentages (%). The chemical shifts
of the compounds in the NMR spectra are reported as δ (ppm) values using
tetramethylsilane as internal reference. The splitting patterns are described as s (singlet), d
(doublet), t (triplet), q (quartet), m (multiplet), and dd (doublet of doublet).
4.2.1. Synthesis of the compounds
The title compounds were synthesized as outlined in Scheme 1. Imidazole was N‐alkylated
with commercially obtained 2,2',4‐trichloroacetophenone (1) to yield 1‐(2,4‐dichlorophenyl)‐
2‐(1H‐imidazol‐1‐yl)ethanone (2), which was reduced to 1‐(2,4‐dichlorophenyl)‐2‐(1H‐
imidazol‐1‐yl)ethanol (3) with sodium borohydride (NaBH4) and converted to 1‐(2,4‐
dichlorophenyl)‐2‐(1H‐imidazol‐1‐yl)ethanone oxime (4) with hydroxylamine hydrochloride
(NH2OH.HCl) applying literature methods (see Supporting Information for details) [49, 50].
5a-t and 6a-r were afforded by Steglich esterification of 3 and 4 with various carboxylic acids
[51].
A
mixture of N,N'‐dicyclohexylcarbodiimide (DCC) (1 mmol) and 4‐
dimethylaminopyridine (DMAP) (0.07 mmol) in dichloromethane (DCM) was added dropwise
to a mixture of 3 or 4 (1 mmol) and the proper carboxylic acid (1 mmol) in DCM at 0‐5 °C.
The mixture was stirred for 0.5 h at 0‐5 °C then for an additional 3‐6 h at room temperature.
The resulting precipitate was filtered off and the filtrate was evaporated to dryness. The
residue of 5a-t was purified via column chromatography (chloroform‐methanol 90:10) and
that of 6a-r via crystallization from diethyl ether‐methanol. The compounds except 5a, 6c,
6e, 6i, 6l‐n, 6p, and 6p were converted to their HCl salts using ethereal solution of gaseous
HCl (gHCl). The structure and purity of the compounds were confirmed via NMR, LC‐MS, and
elemental analyses.
1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl 4-terphenyl-4-carboxylate (5a): White
1
powder (0.26 g, 47.8% yield); m.p.: 224‐25.5 °C; H NMR (400 MHz, DMSO‐d6): δ=4.62‐4.73
(m, 2H, CH2), 6.44 (dd, J1 = 6.8 Hz, J2 = 4 Hz, 1H, CH), 7.07 (s, 1H, imidazole H4), 7.31 (s, 1H,
imidazole H5), 7,37‐7.93 (m, 14H, 2,4‐dichlorophenyl H5,6, 4‐terphenyl H3,5,2',3',5',6',2",3",5",6"),
8.01 (m, 1H, 4‐terphenyl H4"), 8.14 (s, 1H, 2,4‐dichlorophenyl H3), 8.16 (s, 1H, imidazole H2).
13C NMR (100 MHz, DMSO‐d6): δ=49.12 (CH2), 71.39 (OCH), 120.64, 126.52 (2C), 126.85 (2C),
127.22 (2C), 127.35, 127.46 (2C), 127.61, 127.88, 128.88 (2C), 129.01, 130.08 (2C), 132.44,

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133.45 (2C), 133.97, 137.43 (2C), 137.53, 139.24, 140.14, 144.71, 164.05 (CO); MS (ESI+)
m/z: 535 [M+Na]+ (100%), 513 [M+H]+; Anal. calcd. for C30H22Cl2N2O2.H2O: C 67.80, H 4.55, N
5.27, found: C 67.59, H 4.12, N 5.44.
1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl 4-phenylbenzoate hydrochloride (5b): Off‐
1
white powder (0.25 g, 52.0% yield); m.p.: 218‐21 °C; H NMR (400 MHz, DMSO‐d6): δ=4.83‐
4.94 (m, 2H, CH2), 6.53 (dd, JAX = 8.4 Hz, JAB = 4 Hz, 1H, OCH), 7.43‐7.54 (m, 5H, 2,4‐
dichlorophenyl H5,6, 4‐phenylbenzoyl H3'‐5'), 7.66 (s, 1H, imidazole H4), 7.74‐7.77 (m, 3H, 2,4‐
dichlorophenyl H3, 4‐phenylbenzoyl H2',6'), 7.80 (s, 1H, imidazole H5), 7.84‐8.16 (m, 4H, 4‐
13
phenylbenzoyl H2,3,5,6), 9.21 (s, 1H, imidazole H2); C NMR (100 MHz, DMSO‐d6): δ=50.51
(CH2), 70.69 (OCH), 119.88, 122.88, 126.99 (2C), 127.04 (2C), 127.10, 128.10, 128.50, 129.06
(3C), 129.24, 130.21 (2C), 132.65, 132.85, 134.32, 136.20, 138.62, 145.42, 164.02 (CO); MS
(ESI+) m/z: 440 [M+4]+, 439 [M+2+H]+, 437 [M+H]+, 150 (100%); Anal. calcd. for
C24H19Cl3N2O2: C 60.84, H 4.04, N 5.91, found: C 60.73, H 4.17, N 6.21.
1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl 2-naphthoate hydrochloride (5c): White
1
powder (0.34 g, 75.4% yield); m.p.: 226‐8 °C; H NMR (400 MHz, DMSO‐d6): δ=4.90‐5.03 (m,
2H, CH2), 6.59 (dd, JAX = 7.6 Hz, JAB = 4 Hz, 1H, OCH), 7.50‐7.58 (m, 2H, 2,4‐dichlorophenyl
H5,6), 7.66‐7.75 (m, 3H, 2‐naphthoyl H4,6,7), 7.78 (d, J = 2 Hz, 2,4‐dichlorophenyl H3), 7.90 (s,
1H, imidazole H4), 8.05‐8.23 (m, 4H, 2‐naphthoyl H1,3,5,8), 8.83 (s, 1H, imidazole H5), 9.41 (s,
1H, imidazole H2); 13C NMR (100 MHz, DMSO‐d6): δ=50.67 (CH2), 70.84 (OCH), 119.87,
123.02, 124.75, 125.62, 127.12, 127.74, 128.16, 128.57, 129.01, 129.19, 129.29, 129.49,
131.38, 132.02, 132.73, 132.92, 134.38, 135.33, 136.34, 164.39 (CO); MS (ESI+) m/z: 414
[M+4]+, 413 [M+2+H]+, 411 [M+H]+, 155 (100%); Anal. calcd. for C22H17Cl3N2O2: C 59.02, H
3.83, N 6.26, found: C 58.77, H 3.85, N 6.34.
1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl 4-methylthiobenzoate hydrochloride (5d):
1
Pale yellow powder (0.17 g, 37.9% yield); m.p.: 231‐3 °C; H NMR (400 MHz, DMSO‐d6):
δ=2.52 (s, 3H, CH3), 4.79‐4.92 (m, 2H, CH2), 6.45‐6.48 (q, 1H, CHO), 7.37 (d, J = 8.4 Hz, 2H, 4‐
methylthiobenzoyl H3,5), 7.42‐7.48 (m, 2H, 2,4‐dichlorophenyl H5,6), 7.66 (s, 1H, imidazole
H4), 7.72 (d, J = 2 Hz, 1H, 2,4‐dichlorophenyl H3), 7.79 (s, 1H, imidazole H5), 7.95 (d, J = 8.4 Hz,
13
2H, 4‐methylthiobenzoyl H2,6), 9.27 (s, 1H, imidazole H2); C NMR (100 MHz, DMSO‐d6):
δ=13.91 (CH3), 50.57 (CH2), 70.55 (CHO), 119.84, 122.93, 124.12, 125.01 (2C), 128.12,
129.10, 129.27, 129.92 (2C), 132.68, 132.95, 134.34, 136.20, 146.68, 163.97 (CO); MS (ESI+)

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m/z: 411 [M+4+H]+, 410 [M+4]+, 408 [M+2]+ (100%); Anal. calcd. for C19H17Cl3N2O2S.H2O: C
48.06, H 3.82, N 8.85, found: C 48.08, H 3.85, N 8.86.
1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl 4-isopropylbenzoate hydrochloride (5e):
1
White powder (0.20 g, 45.6% yield); m.p.: 199‐200 °C; H NMR (400 MHz, DMSO‐d6): δ=1.20
(d, J = 7.2 Hz, 6H, CH3), 2.93‐2.99 (m, 1H, CH(CH3)2), 4.78‐4.86 (m, 2H, CH2), 6.45‐6.48 (q, 1H,
CHO), 7.40‐7.48 (m, 4H, 2,4‐dichlorophenyl H5,6, 4‐isopropylbenzoyl H3,5), 7.65 (s, 1H,
imidazole H4), 7.74 (d, J = 2 Hz, 1H, 2,4‐dichlorophenyl H3), 7.78 (s, 1H, imidazole H5), 7.97 (d,
J = 8 Hz, 2H, 4‐isopropylbenzoyl H2,6), 9.21 (s, 1H, imidazole H2); 13C NMR (100 MHz, DMSO‐
d6): δ=23.41 (2C, CH3), 33.52 (CH(CH3)2), 50.55 (CH2), 70.51 (CHO), 119.91, 122.91, 126.02,
126.88 (2C), 128.11, 129.03, 129.26, 129.77 (2C), 132.65, 132.97, 134.31, 136.22, 155.07,
164.13 (CO); MS (ESI+) m/z: 407 [M+4+H]+, 406 [M+4]+, 404 [M+2]+ (100%); Anal. calcd. for
C21H21Cl3N2O2: C 57.35, H 4.81, N 6.37, found: C 57.27, H 5.06, N 6.45.
1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl 4-morpholinobenzoate hydrochloride (5f):
1
Pale yellow powder (0.12 g, 25.7% yield); m.p.: 260‐2 °C; H NMR (400 MHz, DMSO‐d6):
δ=3.28 (t, JAX = 4.8 Hz, JAY = 4.8 Hz, 4H, morpholine H3,3’,5,5’), 3.71 (t, JXA = 4.8 Hz, JXB = 4.8 Hz,
4H, morpholine H2,2’,6,6’), 4.76‐4.88 (m, 2H, CH2), 6.42 (dd, J1 = 7.2 Hz, J2 = 4.0 Hz, 1H, CH),
6.98 (d, J = 9.2 Hz, 2H, 4‐morpholinobenzoyl H3,5), 7.39‐7.48 (m, 2H, 2,4‐dichlorophenyl H5,6),
7.66 (s, 1H, imidazole H4), 7.71 (d, J = 2.4 Hz, 1H, 2,4‐dichlorophenyl H3), 7.77 (s, 1H,
imidazole H5), 7.87 (d, J = 9.2 Hz, 2H, 4‐morpholinobenzoyl H2,6), 9.24 (s, 1H, imidazole H2);
13C NMR (100 MHz, DMSO‐d6): δ=46.66 (morpholine C3,5), 50.68 (CHCH2), 65.77 (morpholine
C2,6), 69.89 (OCH), 113.16 (2C), 116.87, 119.78, 122.91, 128.08, 128.98, 129.24, 131.20 (2C),
132.60, 133.30, 134.21, 136.15, 154.55, 164.00 (CO); MS (ESI+) m/z: 504 [M+Na]+ (100%),
482 [M+H]+; Anal. calcd. for C22H22Cl3N3O3: C 54.73, H 4.59, N 8.70, found: C 54.45, H 4.49, N
8.78.
1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl 4-tert-butylbenzoate hydrochloride (5g):
1
White powder (0.09 g, 20.0% yield); m.p.: 187‐90 °C; H NMR (400 MHz, DMSO‐d6): δ=1.31
(s, 9H, CH3), 4.81‐4.92 (m, 2H, CH2), 6.53 (dd, JAX = 7 Hz, JAB = 4 Hz, 1H, OCH), 7.41‐7.50 (m,
2H, 2,4‐dichlorophenyl H5,6), 7.58 (d, J = 8.8 Hz, 2H, 4‐tert‐butylbenzoyl H3,5), 7.68 (s, 1H,
imidazole H4), 7.77 (d, J = 2 Hz, 1H, 2,4‐dichlorophenyl H3), 7.80 (s, 1H, imidazole H5), 8.00 (d,
13
J = 8.8 Hz, 2H, 4‐tert‐butylbenzoyl H2,6), 9.23 (s, 1H, imidazole H2); C NMR (100 MHz,
DMSO‐d6): δ=31.20 (3C, CH3), 35.39 (C(CH3)3), 51.04 (CH2), 70.99 (OCH), 120.39, 123.40,

ACCEPTED MANUSCRIPT
126.14, 126.22 (2C), 128.59, 129.49, 129.75, 129.99 (2C), 133.13, 133.45, 134.80, 136.70,
157.70, 164.58 (CO); MS (ESI+) m/z: 420 [M+4]+, 419 [M+2+H]+, 417 [M+H]+ (100%); Anal.
calcd. for C22H23Cl3N2O2.1/2H2O: C 57.10, H 5.23, N 6.05, found: C 57.39, H 5.32; N, 6.52.
1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl 4-cyanobenzoate hydrochloride (5h):
1
White powder (0.16 g, 39.0% yield); m.p.: 188‐90 °C; H NMR (400 MHz, DMSO‐d6): δ=4.84‐
4.98 (m, 2H, CH2), 6.51‐6.54 (q, 1H, CHO), 7.50‐7.82 (m, 5H, 2,4‐dichlorophenyl, imidazole
H4,5), 8.07 (d, J = 8.4 Hz, 2H, 4‐cyanobenzoyl H3,5), 8.24 (d, J = 8.4 Hz, 2H, 4‐cyanobenzoyl
13
H2,6), 9.29 (s, 1H, imidazole H2); C NMR (100 MHz, DMSO‐d6): δ=50.49 (CHCH2), 71.48
(CHO), 116.11 (C≡N), 117.94, 119.94, 122.94, 128.15, 129.23, 129.31, 130.27 (2C), 132.33,
132.49, 132.72, 132.91 (2C), 134.50, 136.30, 163.16 (CO); MS (ESI+) m/z: 390 [M+4+H]+, 389
[M+4]+, 387 [M+2]+ (100%); Anal. calcd. for C19H14Cl3N3O2.H2O: C 51.78, H 3.66, N 9.53,
found: C 51.37, H 3.56, N 9.58.
1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl 1H-indole-2-carboxylate hydrochloride
(5i): Pale yellow powder (0.19 g, 41.5% yield); m.p.: 209‐11 °C; 1H NMR (400 MHz, DMSO‐d6):
δ=4.82‐4.90 (m, 2H, CHCH2), 6.47‐6.50 (m, 1H, OCH), 7.08‐7.32 (m, 2H, indole H5,6), 7.36‐7.54
(m, 4H, indole H3,7, 2,4‐dichlorophenyl H5,6), 7.67‐7.69 (m, 2H, imidazole H4, indole H4), 7.75
(d, J = 2 Hz, 1H, 2,4‐dichlorophenyl H3), 7.87 (s, 1H, imidazole H5), 9.41 (s, 1H, imidazole H2),
13
12.25 (d, J = 1.6 Hz, 1H, indole NH); C NMR (100 MHz, DMSO‐d6): δ=50.70 (CH2), 70.39
(OCH), 109.34, 112.75, 119.87, 120.46, 122.18, 123.02, 125.23, 125.73, 126.60, 128.15,
129.10, 129.26, 132.58, 132.92, 134.37, 136.44, 137.76, 159.46 (CO); MS (ESI+) m/z: 403
[M+4]+, 402 [M+2+H]+, 400 [M+H]+ (100%); Anal. calcd. for C20H16Cl3N3O2.2/3H2O: C 53.53, H
3.89, N 9.36, found: C 53.37, H 3.87, N 9.44.
1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl 3-phenylprop-2-enoate hydrochloride (5j):
1
Off‐white powder (0.21 g, 50.0% yield); m.p.: 183‐5 °C; H NMR (400 MHz, DMSO‐d6):
δ=4.76‐4.78 (m, 2H, CH2), 6.35‐6.37 (m, 1H, OCH), 6.73 (d, J = 16 Hz, 1H, COCH), 7.30‐7.48
(m, 5H, 2,4‐dichlorophenyl H5,6, cinnamoyl H3'‐5'), 7.67 (s, 1H, imidazole H4), 7.73‐7.75 (m, 3H,
cinnamoyl H2',6', 2,4‐dichlorophenyl H3), 7.77 (s, 1H, imidazole H5), 9.19 (s, 1H, imidazole H2);
13C NMR (100 MHz, DMSO‐d6): δ=50.69 (CH2), 69.90 (OCH), 116.84 (COCH), 119.83, 123.07,
128.05, 128.60, 128.92, 128.97, 129.26, 130.92, 132.63, 132.93, 133.78, 134.26 (16C,
benzene, imidazole C4,5), 136.31 (CHC6H5), 146.21 (imidazole C2), 164.60 (CO); MS (ESI+) m/z:

ACCEPTED MANUSCRIPT
409 [M+Na]+ (100%), 389 [M+2+H]+, 387 [M+H]+; Anal. calcd. for C20H17Cl3N2O2.2/3H2O: C
55.13, H 4.24, N 6.43, found: C 54.75, H 4.39, N 6.89.
1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl 4-ethylbenzoate hydrochloride (5k): Off‐
1
white powder (0.23 g, 53.5% yield); m.p.: 198‐9 °C; H NMR (400 MHz, DMSO‐d6): δ=1.19 (t,
J1 = 7.6 Hz, J2 = 7.2 Hz, 3H, CH3), 2.66‐2.71 (q, 2H, CH2CH3), 4.82‐4.96 (m, 2H, CHCH2), 6.50
(dd, J1 = 7.6 Hz, J2 = 4.0 Hz, 1H, CH), 7.38 (d, J = 8.4 Hz, 2H, 4‐ethylbenzoyl H3,5), 7.47‐7.67 (m,
3H, 2,4‐dichlorophenyl H5,6, imidazole H4), 7.71‐7.81 (m, 2H, 2,4‐dichlorophenyl H3,
imidazole H5), 7.98 (d, J = 8.4 Hz, 2H, 4‐ethylbenzoyl H2,6), 9.31 (s, 1H, imidazole H2); 13C NMR
(100 MHz, DMSO‐d6): δ=15.15 (CH3), 28.20 (CH2CH3), 51.56 (CHCH2), 70.52 (OCH), 119.80,
122.93, 125.87, 128.12, 128.31 (2C), 129.09, 129.25, 129.74 (2C), 132.68, 132.98, 134.32,
136.19, 150.61, 164.19 (CO); MS (ESI+) m/z: 411 [M+Na]+ (100%), 389 [M+H]+; Anal. calcd.
for C20H19Cl3N2O2: C 56.43, H 4.50, N 6.58, found: C 56.72, H 4.38, N 6.97.
1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl cyclohexanecarboxylate hydrochloride
1
(5l): Off‐white powder (0.20 g, 48.4% yield); m.p.: 149‐150.5 °C; H NMR (400 MHz, DMSO‐
d6): δ=1.14‐2.39 (m, 11H, cyclohexane), 4.68 (d, J = 6.0 Hz, CHCH2N), 6.23 (t, J1 = 5.6 Hz, J2 =
5.6 Hz, 1H, CHO), 7.28 (d, J = 8.8 Hz, 1H, 2,4‐dichlorophenyl H6), 7.48 (dd, J1 = 8.8 Hz, J2 = 2
Hz, 1H, 2,4‐dichlorophenyl H5), 7.66‐7.72 (m, 3H, imidazole H4,5, 2,4‐dichlorophenyl H3), 9.12
(s, 1H, imidazole H2); 13C NMR (100 MHz, DMSO‐d6): δ=24.57 (cyclohexane C3), 24.64
(cyclohexane C5), 25.11 (cyclohexane C4), 28.09 (cyclohexane C2), 28.23 (cyclohexane C6),
41.69 (cyclohexane C1), 50.51 (CHCH2N), 69.41 (CHO), 119.80, 122.87, 128.03, 128.89,
129.20, 132.67, 132.97, 134.21, 136.14, 173.39 (CO); MS (ESI+) m/z: 372 [M+4+H]+, 370
[M+2+H]+, 368 [M+H]+ (100%); Anal. calcd. for C18H21Cl3N2O2: C 53.55, H 5.24, N 6.94, found:
C 53.14, H 5.49, N 7.06.
1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl 4-(tert-butoxycarbonylamino)butanoate
1
hydrochloride (5m): White powder (0.17 g, 35.5% yield); m.p.: 136‐8 °C; H NMR (400 MHz,
DMSO‐d6): δ=1.35 (s, 9H, CH3), 1.55‐1.60 (m, 2H, COCH2CH2), 2.38 (t, J1 = 7.6 Hz, J2 = 7.2 Hz,
COCH2), 2.84‐2.89 (q, 2H, CH2NH), 4.67‐4.69 (m, 2H, CHCH2), 6.25 (t, J1 = 5.6 Hz, J2 = 5.2 Hz,
1H, OCH), 6.83 (t, J1 = 5.6 Hz, J2 = 5.6 Hz, 1H, NH), 7.26 (d, J = 8.4 Hz, 1H, 2,4‐dichlorophenyl
H6), 7.44 (dd, J1 = 8.4 Hz, J2 = 2 Hz, 1H, 2,4‐dichlorophenyl H5), 7.63‐7.71 (m, 3H, imidazole
H4,5, 2,4‐dichlorophenyl H3), 9.06 (s, 1H, imidazole H2); 13C NMR (100 MHz, DMSO‐d6):
δ=24.50 (COCH2CH2), 28.14 (3C, CH3), 30.50 (COCH2), 39.90 (CH2NH), 50.50 (CHCH2), 69.50

ACCEPTED MANUSCRIPT
(OCH), 77.42 (C(CH3)3), 119.81, 122.85, 127.87, 128.89, 129.12, 132.51, 132.74, 134.12,
136.10, 155.53 (NHCO), 171.23 (2C, COCH2, NHCO); MS (ESI+) m/z: 464 [M+Na]+ (100%), 444
[M+2+H]+, 442 [M+H]+; Anal. calcd. for C20H26Cl3N3O4: C 50.17, H 5.47, N 8.78, found: C
49.78, H 5.69, N 8.92.
1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl 2,4-dichlorobenzoate hydrochloride (5n):
1
Pale yellow powder (0.30 g, 63.5%); m.p.: 140‐2 °C; H NMR (400 MHz, DMSO‐d6): δ=4.82‐
4.94 (m, 2H, CH2), 6.52 (dd, J1 = 7.2 Hz, J2 = 4 Hz, 1H, OCH), 7.47‐7.53 (m, 2H, 2,4‐
dichlorophenyl H5,6), 7.62 (dd, J1 = 8.4 Hz, J2 = 2 Hz, 1H, 2,4‐dichlorobenzoyl H5), 7.65 (s, 1H,
imidazole H4), 7.74 (s, 1H, imidazole H5), 7.76 (d, J = 2 Hz, 1H, 2,4‐dichlorobenzoyl H3), 7.80
(d, J = 2 Hz, 1H, 2,4‐dichlorophenyl H3), 8.06 (d, J = 8.4 Hz, 1H, 2,4‐dichlorobenzoyl H6), 9.20
13
(s, 1H, imidazole H2); C NMR (100 MHz, DMSO‐d6): δ=50.39 (CH2), 71.49 (CH), 120.10,
122.84, 126.80, 127.82, 128.15, 129.30 (2C), 130.71, 132.41, 132.87, 133.28, 133.94, 134.51,
136.28, 138.16, 162.31 (CO); MS (ESI+) m/z: 457 [M+6+Na]+, 455 [M+4+Na]+, 453 [M+2+Na]+
(100%), 451 [M+Na]+; Anal. calcd. for C18H13Cl5N2O21/2H2O: C 45.46, H 2.97, N 5.89, found: C
45.19, H 2.76, N 6.24.
1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl 4-methoxybenzoate hydrochloride (5o):
Pale yellow powder (0.22 g, 51.2% yield); m.p.: 176 °C; 1H NMR (400 MHz, DMSO‐d6): δ=2.48
(s, 3H, CH3), 4.77‐4.88 (m, 2H CHCH2), 6.43‐6.46 (q, 1H, CHO), 7.06 (d, J = 8.8 Hz, 2H, 4‐
methoxybenzoyl H3,5), 7.39‐7.49 (m, 2H, 2,4‐dichloropheyl H5,6), 7.64 (s, 1H, imidazole H4),
7.73 (d, J = 2 Hz, 1H, 2,4‐dichlorophenyl H3), 7.76 (s, 1H, imidazole H5), 8.00 (d, J = 9.2 Hz, 2H,
4‐methoxybenzoyl H2,6), 9.18 (s, 1H, imidazole H2); 13C NMR (100 MHz, DMSO‐d6): δ=50.60
(CHCH2), 55.64 (CH3), 70.34 (CHO), 114.22 (2C), 119.90, 120.46, 122.90, 128.11, 129.04,
129.25, 131.79 (2C), 132.64, 133.08, 134.29, 136.21, 163.74, 163.85 (CO); MS (ESI+) m/z: 395
[M+4+H]+, 394 [M+4]+, 392 [M+2]+ (100%); Anal. calcd. for C19H17Cl3N2O3.H2O: C 51.20, H
4.30, N 6.28, found: C 51.37, H 4.39, N 6.40.
1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl 4-phenylbutanoate hydrochloride (5p):
1
Off‐white powder (0.11 g, 26.0% yield); m.p.: 148‐50 °C; H NMR (400 MHz, DMSO‐d6):
δ=1.72‐1.80 (m, 2H, CH2CH2C6H5), 2.38 (t, J1 = 7.6 Hz, J2 = 7.2 Hz, 2H, COCH2), 2.50‐2.52 (m,
2H, CH2C6H5, overlaps with DMSO), 4.68 (d, J = 6 Hz, 2H, CHCH2), 6.26 (t, J1 = 5.6 Hz, J2 = 5.6
Hz, 1H, OCH), 7.11‐7.29 (m, 6H, 4‐phenylbutanoyl H3'‐5', imidazole H4,5, 2,4‐dichlorobenzoyl
H6), 7.46‐7.72 (m, 4H, 2,4‐dichlorobenzoyl H3,5, 4‐phenylbutanoyl H2',6'), 9.11 (s, 1H,

ACCEPTED MANUSCRIPT
13
imidazole H2); C NMR (100 MHz, DMSO‐d6): δ=25.79 (CH2CH2C6H5), 32.57 (COCH2), 34.09
(CH2C6H5), 50.23 (CHCH2), 69.55 (OCH), 119.77, 122.86, 125.85, 127.95, 128.21 (2C), 128.28
(2C), 128.99, 129.18, 132.63, 132.81, 134.23, 136.12, 141.07, 171.31 (CO); MS (ESI+) m/z:
425 [M+Na]+ (100%), 406 [M+4]+, 405 [M+2+H]+, 403 [M+H]+; Anal. calcd. for C21H21Cl3N2O2:
C 57.36, H 4.81, N 6.37, found: C 57.07, H 5.03, N 6.59.
1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl cyclopentanecarboxylate hydrochloride
(5q): Off‐white powder (0.20 g, 51.0% yield); m.p.: 188‐90 °C; 1H NMR (400 MHz, DMSO‐d6):
δ=1.53‐1.84 (m, 9H, cyclopentane), 4.72 (d, J = 5.6 Hz, 2H, CHCH2N), 6.25 (t, J1 = 5.6 Hz, J2 =
6.4 Hz, 1H, CHO), 7.30 (d, J = 8.4 Hz, 1H, 2,4‐dichlorophenyl H6), 7.50 (dd, J1 = 8.4 Hz, J2 = 2
Hz, 1H, 2,4‐dichlorophenyl H5), 7.68‐7.72 (m, 3H, imidazole H4,5, 2,4‐dichlorophenyl H3), 9.20
(s, 1H, imidazole H2); 13C NMR (100 MHz, DMSO‐d6): δ=25.18 (2C, cyclopentane C3,4), 29.07
(cyclopentane C2), 29.24 (cyclopentane C5), 42.70 (cyclopentane C1), 50.52 (CHCH2N), 69.53
(CHO), 119.72, 122.89, 128.04, 128.86, 129.22, 132.68, 132.97, 134.22, 136.13, 174.10 (CO);
MS (ESI+) m/z: 357 [M+4+H]+, 356 [M+4]+, 354 [M+2]+ (100%); Anal. calcd. for C17H19Cl3N2O2:
C 52.39, H 4.91, N 7.19, found: C 52.52, H 5.19, N 7.41.
1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl 4-nitrobenzoate hydrochloride (5r): Yellow
1
powder (0.22 g, 50.5% yield); m.p.: 218 °C; H NMR (400 MHz, DMSO‐d6): δ=4.83‐4.97 (m,
2H, CH2), 6.50‐6.53 (q, 1H, CHO), 7.48‐7.75 (m, 4H, 2,4‐dichlorophenyl, imidazole H4), 7.80 (s,
1H, imidazole H5), 8.29‐8.35 (m, 4H, 4‐nitrobenzoyl), 9.28 (s, 1H, imidazole H2); 13C NMR (100
MHz, DMSO‐d6): δ=50.48 (CH2), 71.60 (CHO), 119.92, 122.96, 123.90 (2C), 128.15, 129.24,
129.32, 131.15 (2C), 132.44, 132.75, 133.79, 134.52, 136.30, 150.63, 162.90 (CO); MS (ESI+)
m/z: 409 [M+4]+, 408 [M+2+H]+, 406 [M+H]+ (100%); Anal. calcd. for C21H21Cl3N2O2.1/2H2O: C
47.86, H 3.35, N 9.30, found: C 47.87, H 3.28, N 9.44.
1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl 3-benzoylpropanoate hydrochloride (5s):
1
Off‐white powder (0.30 g, 66.0% yield); m.p.: 159‐61 °C; H NMR (400 MHz, DMSO‐d6):
δ=2.75‐2.78 (m, 2H, COCH2), 3.31‐3.35 (q, 2H, CH2COC6H5), 4.70 (d, J = 5.6 Hz, CHCH2), 6.27
(t, J1 = 5.6 Hz, J2 = 5.2 Hz, 1H, OCH), 7.32 (d, J = 8.4 Hz, 1H, 2,4‐dichlorophenyl H6), 7.46 (dd, J1
= 8.4 Hz, J2 = 2 Hz, 1H, 2,4‐dichlorophenyl H5), 7.52‐7.69 (m, 5H, 3‐benzoylpropanoyl H3'‐5'
,
imidazole H4,5), 7.72 (d, J = 2 Hz, 1H, 2,4‐dichlorophenyl H3), 7.95‐7.97 (m, 2H, 3‐
benzoylpropanoyl H2',6'), 9.12 (s, 1H, imidazole H2); 13C NMR (100 MHz, DMSO‐d6): δ=27.70
(COCH2), 32.87 (CH2COC6H5), 50.54 (CHCH2), 69.74 (OCH), 119.73, 122.81, 127.80 (3C),

ACCEPTED MANUSCRIPT
128.65 (2C), 129.05, 129.06, 132.48, 132.65, 133.28, 134.15, 136.04, 136.08, 171.06 (OCO),
198.05 (COC6H5); MS (ESI+) m/z: 439 [M+Na]+ (100%), 420 [M+4]+, 419 [M+2+H]+, 417
[M+H]+; Anal. calcd. for C21H19Cl3N2O3: C 55.59, H 4.22, N 6.17, found: C 55.18, H 4.37, N
6.47.
1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl 4-trifluoromethylbenzoate hydrochloride
(5t): Pale yellow powder (0.19 g, 40.8% yield); m.p.: 187‐90 °C; 1H NMR (400 MHz, DMSO‐d6):
δ=4.87‐5.01 (m, 2H, CH2), 6.54‐6.57 (q, 1H, CHO), 7.49‐7.55 (m, 2H, 2,4‐dichlorophenyl H5,6),
7.69 (s, 1H, imidazole H4), 7.77 (d, J = 2 Hz, 1H, 2,4‐dichlorophenyl H3), 7.85 (s, 1H, imidazole
H5), 7.95 (d, J = 8.4 Hz, 2H, 4‐trifluoromethylbenzoyl H3,5), 8.30 (d, J = 8.4 Hz, 2H, 4‐
13
trifluoromethylbenzoyl H2,6), 9.34 (s, 1H, imidazole H2); C NMR (100 MHz, DMSO‐d6):
δ=50.49 (CH2), 71.36 (CHO), 119.86, 122.24, 122.97, 124.95, 125.85, 125.89, 128.14, 129.25,
129.30, 130.54, 132.17, 132.55, 132.75, 133.24, 133.56, 134.48, 136.27, 163.26 (CO); MS
(ESI+) m/z: 432 [M+4]+, 431 [M+2+H]+, 429 [M+H]+ (100%); Anal. calcd. for C19H13Cl3F3N2O2: C
49.00, H 3.03, N 6.02, found: C 48.65, H 3.23, N 6.07.
1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethanone
O-(4-terphenylcarbonyl)
oxime
1
hydrochloride (6a): Pale yellow powder (0.07 g, 13.0% yield); m.p.: 265‐8 °C; H NMR (400
MHz, DMSO‐d6): δ=6.12 (s, 2H, CH2), 7.38‐7.52 (m, 4H, 4‐terphenyl H3"‐5", imidazole H4), 7.56
(dd, J1 = 8 Hz, J2 = 2 Hz, 1H, 2,4‐dichlorophenyl H5), 7.61 (s, 1H, imidazole H5), 7.68 (d, J = 8.4
Hz, 1H, 2,4‐dichlorophenyl H6), 7.74‐7.76 (m, 2H, 4‐terphenyl H2",6"), 7.84 (d, J = 8.4 Hz, 2H, 4‐
terphenyl H3',5'), 7.91 (d, J = 8.4 Hz, 2H, 4‐terphenyl H2',6'), 7.99 (d, J = 8.8 Hz, 2H, 4‐terphenyl
H3,5), 8.27 (d, J = 8.4 Hz, 2H, 4‐terphenyl H2,6), 9.11 (s, 1H, imidazole H2); MS (ESI+) m/z: 529
[M+4]+, 528 [M+2+H]+, 526 [M+H]+ (100%); Anal. calcd. for C30H22Cl3N3O2.H2O: C 62.03, H,
4.16, N 7.23, found: C 62.04, H 4.10, N 7.44.
1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethanone
O-(2-naphthoate)
oxime
1
hydrochloride (6b): White powder (0.16 g, 33.8% yield); m.p.: 157‐9 °C; H NMR (400 MHz,
DMSO‐d6): δ=5.80 (s, 2H, CH2), 7.56 (dd, J1 = 8.4 Hz, J2 = 1.6 Hz, 1H, 2,4‐dichlorophenyl H5),
7.60‐7.70 (m, 10H, naphthoyl, 2,4‐dichlorophenyl H3,6, imidazole H4), 8.27 (s, 1H, imidazole
H2), 9.35 (s, 1H, imidazole H2); 13C NMR (100 MHz, DMSO‐d6): δ=50.40 (CH2), 120.14, 123.10,
123.80, 124.50, 127.37, 128.12, 128.50, 128.88, 129.18 (3C), 129.22 (2C), 130.34, 130.91,
131.88, 135.25, 136.02, 136.61, 159.42 (CNO), 161.88 (CO); MS (ESI+) m/z: 446 [M+Na]+

ACCEPTED MANUSCRIPT
(100%), 426 [M+2+H]+, 424 [M+H]+; Anal. calcd. for C22H16Cl3N3O2.1/2H2O: C 56.25, H 3.65, N
8.95, found: C 56.66, H 3.39, N 9.29.
1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethanone O-(4-phenylbenzoyl) oxime (6c): Off‐
white powder (0.23 g, 50.0% yield); m.p.: 129‐31 °C; 1H NMR (400 MHz, DMSO‐d6): δ=5.82 (s,
2H, CH2), 6.74 (s, 1H, imidazole H4), 7.07 (s, 1H, imidazole H5), 7.46‐7.49 (m, 2H, 2,4‐
dichlorophenyl H5, 4‐phenylbenzoyl H4'), 7.52‐7.56 (m, 3H, 2,4‐dichlorophenyl H6, 4‐
phenylbenzoyl H3',5'), 7.63 (s, 1H, imidazole H2), 7.68 (d, J = 2 Hz, 1H, 2,4‐dichlorophenyl H3),
7.79‐7.81 (m, 2H, 4‐phenylbenzoyl H2',6'), 7.92 (d, J = 8.8 Hz, 2H, 4‐phenylbenzoyl H3,5), 8.27
13
(d, J = 8.4 Hz, 2H, 4‐phenylbenzoyl H3,5); C NMR (100 MHz, DMSO‐d6): δ=44.94 (CH2),
120.24, 126.46, 127.05 (3C), 127.18 (2C), 127.28, 128.40, 128.59, 128.85, 129.12 (2C),
129.63, 130.36 (2C), 132.12, 132.86, 135.42, 138.20, 145.55, 162.12 (CNO), 163.40 (CO); MS
(ESI+) m/z: 453 [M+4]+, 452 [M+2+H]+, 450 [M+H]+, 69 (100%); Anal. calcd. for C24H17Cl2N3O2:
C 64.01, H 3.81, N 9.33, found: C 63.82, H 3.60, N 9.39.
1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethanone
O-(1H-indole-2-carbonyl)
oxime
1
hydrochloride (6d): White powder (0.19 g, 43.0% yield); m.p.: 149‐50.5 °C; H NMR (400
MHz, DMSO‐d6): δ=6.20 (s, 2H, CH2), 7.15 (t, J1 = 7.6 Hz, J2 = 7.6 Hz, 1H, indole H5), 7.35 (t, J1
= 7.6 Hz, J2 = 7.6 Hz, 1H, indole H6), 7.55‐7.59 (m, 4H, 2,4‐dichlorophenyl H5, indole H4,7,
imidazole H4), 7.68‐7.76 (m, 4H, 2,4‐dichlorophenyl H3,5, indole H4,7, imidazole H5), 9.28 (s,
13
1H, imidazole H2), 12.42 (s, 1H, indole NH); C NMR (100 MHz, DMSO‐d6): δ=47.40 (CH2),
109.96, 112.83, 120.17, 120.64, 122.28, 123.00, 124.10, 125.50, 126.63, 127.81, 128.81,
129.18, 132.72, 132.85, 136.10, 136.73, 138.12, 157.57 (CNO), 160.27 (CO); MS (ESI+) m/z:
418 [M+4+H]+, 416 [M+2+H]+, 414 [M+H]+ (100%); Anal. calcd. for C20H15Cl3N4O2: C 53.41, H
3.36, N 12.46, found: C 52.99, H 3.54, N 12.20.
1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethanone O-(4-tert-butylbenzoyl) oxime (6e):
White powder (0.10 g, 22.7% yield); m.p.: 103‐4 °C; 1H NMR (400 MHz, DMSO‐d6): δ=1.32 (s,
9H, CH3) 5.77 (s, 2H, CH2), 6.77 (s, 1H, imidazole H4), 7.07 (s, 1H, imidazole H5), 7.45 (dd, J1 =
8.4 Hz, J2 = 2 Hz, 1H, 2,4‐dichlorophenyl H5), 7.51 (d, J = 8 Hz, 1H, 2,4‐dichlorophenyl H6),
7.62 (d, J = 8.8 Hz, 2H, 4‐tert‐butylbenzoyl H3,5), 7.66 (d, J = 2 Hz, 1H, 2,4‐dichlorophenyl H3),
7.68 (s, 1H, imidazole H2), 8.09 (d, J = 8 Hz, 2H, 4‐tert‐butylbenzoyl H2,6); 13C NMR (100 MHz,
DMSO‐d6): δ=30.74 (3C, CH3), 34.97 (C(CH3)3), 45.03 (CH2), 120.38, 124.95, 125.89 (2C),
127.32, 127.96, 128.87, 129.62 (3C), 132.18, 132.86, 135.43, 138.12, 157.37, 162.18 (CNO),

ACCEPTED MANUSCRIPT
163.11 (CO); MS (ESI+) m/z: 433 [M+4]+, 432 [M+2+H]+, 430 [M+H]+ (100%); Anal. calcd. for
C22H21Cl2N3O2.1/2H2O: C 60.15, H 5.04, N 9.56, found: C 59.90, H 4.98, N 9.68.
1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethanone
O-(2,4-dichlorobenzoyl)
oxime
1
hydrochloride (6f): White powder (0.23 g, 48.5% yield); m.p.: 135‐7 °C; H NMR (400 MHz,
DMSO‐d6): δ=6.03 (s, 2H, CH2), 7.52 (s, 1H, imidazole H4), 7.56 (dd, J1 = 8 Hz, J2 = 2 Hz, 1H,
2,4‐dichlorophenyl H5), 7.61 (s, 1H, imidazole H5), 7.67‐7.70 (m, 2H, 2,4‐dichlorophenyl H6,
2,4‐dichlorobenzoyl H5), 7.73 (d, J = 2 Hz, 1H, 2,4‐dichlorophenyl H3), 7.92 (d, J = 2 Hz, 1H,
2,4‐dichlorobenzoyl H3), 8.16 (d, J = 8.4 Hz, 1H, 2,4‐dichlorobenzoyl H6), 9.23 (s, 1H,
imidazole H2); 13C NMR (100 MHz, DMSO‐d6): δ=47.48 (CH2), 120.04, 122.94, 126.34, 127.82,
127.92, 128.44, 129.18, 130.79, 132.64, 132.71, 133.29, 133.90, 136.20, 136.76, 138.40,
160.46 (CNO), 161.58 (CO); MS (ESI+) m/z: 468 [M+4+Na]+, 466 [M+2+Na]+, 464 [M+Na]+,
153 (100%); Anal. calcd. for C18H12Cl5N3O2.H2O: C 43.45, H 2.84, N 8.45, found: C 43.11, H
2.69, N 8.89.
1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethanone
O-(4-methylthiobenzoyl)
oxime
1
hydrochloride (6g): Pale yellow powder (0.20 g, 43.1% yield); m.p.: 122‐3 °C; H NMR (400
MHz, DMSO‐d6): δ=2.56 (CH3), 6.11 (CH2), 7.44 (d, J = 8 Hz, 2H, 4‐methylthiobenzoyl H3,5),
7.52‐7.55 (m, 2H, 2,4‐dichlorophenyl H5,6), 7.63 (s, 1H, imidazole H4), 7.68‐7.71 (m, 2H, 2,4‐
dichlorophenyl H3, imidazole H5), 8.06 (d, J = 8.4 Hz, 2H, 4‐methylthiobenzoyl H2,6), 9.25 (s,
1H, imidazole H2); 13C NMR (100 MHz, DMSO‐d6): δ=13.89 (CH3), 47.35 (CH2), 120.01, 122.96,
122.99, 125.12 (2C), 127.76, 128.76, 129.12, 130.04 (2C), 132.70, 132.78, 136.05, 136.67,
147.14, 160.72 (CNO), 161.80 (CO); MS (ESI+) m/z: 423 [M+4]+, 422 [M+2+H]+, 420 [M+H]+
(100%); Anal. calcd. for C19H16Cl3N3O2S.H2O: C 48.06, H 3.82, N 8.85, found: C 48.08, H 3.85,
N 8.86.
1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethanone
O-(4-ethylbenzoyl)
oxime
1
hydrochloride (6h): Pale yellow powder (0.21 g, 48.4% yield); m.p.: 159‐61 °C; H NMR (400
MHz, DMSO‐d6): δ=1.12 (t, J1 = 7.6 Hz, J2 = 7.6 Hz, 3H, CH3), 2.58‐2.64 (q, 2H, CH2CH3), 5.69
(s, 2H, CH2N), 7.31 (d, J = 7.6 Hz, 2H, 4‐ethylbenzoyl H3,5), 7.51 (d, J = 8 Hz, 2H, 4‐ethylbenzoyl
H2,6), 7.66‐7.72 (m, 3H, 2,4‐dichlorophenyl H5,6, imidazole H4), 7.76 (s, 1H, imidazole H5), 7.87
(d, J = 2 Hz, 1H, 2,4‐dichlorophenyl H3), 9.24 (s, 1H, imidazole H2); 13C NMR (100 MHz, DMSO‐
d6): 14.94 (CH3), 28.13 (CH2CH3), 50.43 (CH2N), 120.07, 123.08, 124.65, 128.06, 128.47,
128.52 (2C), 129.03 (2C), 129.16, 130.25, 131.79, 135.92, 136.56, 150.80, 158.96 (CNO),