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

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

Discovery of new azoles with potent activity against Candida spp. and

Candida albicans biofilms through virtual screening

Suat Sari^a*, Didem Kart^b, Naile Öztürk^c,d, F. Betül Kaynak^e, Melis Gencel^f, Gülce Taşkor^g, Arzu

Karakurt^h, Selma Saraç^a, Şebnem Eşsiz^e, Sevim Dalkara^a

^aHacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06100,

Ankara, Turkey

^bHacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology, 06100,

Ankara, Turkey

^cHacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Technology, 06100,

Ankara, Turkey

^dİnönü University, Faculty of Pharmacy, Department of Pharmaceutical Technology, 44280, Malatya,

Turkey (permanent address)

^eHacettepe University, Faculty of Engineering, Department of Physical Engineering, 06800, Ankara,

Turkey

^fKadir Has University, Faculty of Engineering and Natural Sciences, Department of Bioinformatics and

Genetics, 34083, Istanbul, Turkey

^gHacettepe University, Faculty of Pharmacy, Department of Basic Pharmaceutical Sciences, 06100,

Ankara, Turkey

^hİnönü 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

ACCEPTED MANUSCRIPT

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

N

Cl

N

O

N

O

N

O

N

OH

F

Cl

F

Cl

Miconazole

Cl

Oxiconazole

Fluconazole

Ketoconazole

N

O

N

O

R

N

F

N

R

O

N

O

N

O

N

O

OH

F

Cl

F

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 6^thaxial 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].

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 consensus^a

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

‐7.19

‐7.10

‐7.02

‐6.00

5s

5t

ACCEPTED MANUSCRIPT

^aCalculated 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.

ACCEPTED MANUSCRIPT

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.

ACCEPTED MANUSCRIPT

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 CH₂next 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 CH₂next 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 H⁴and H⁵of the imidazole. Imidazole’s H²resonated further downfield than H⁴

and H⁵and 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.

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.

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

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

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

5q

5r

5s

5t

6a

6b

6c

6d

1

8

16

32

256

1

2

4

8

256

128

256

1

128

4

8

16

256

128

256

1

256

64

128

4

256

4

6e

6f

6g

6h

6i

6j

6k

6l

6m

64

2

64

256

64

1

64

2

16

8

128

32

64

0.5

2

128

64

32

16

1

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

128

4

MBEC

256

128

64

5b

5c

5i

5j

5o

5p

5q

6c

6e

6g

6l

6m

256

64

>1024

256

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].

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.

ACCEPTED MANUSCRIPT

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 H^5,6, 4‐terphenyl H^{3,5,2',3',5',6',2",3",5",6"}),

8.01 (m, 1H, 4‐terphenyl H^4"), 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, J_AX= 8.4 Hz, J_AB= 4 Hz, 1H, OCH), 7.43‐7.54 (m, 5H, 2,4‐

dichlorophenyl H^5,6, 4‐phenylbenzoyl H^3'‐5'), 7.66 (s, 1H, imidazole H⁴), 7.74‐7.77 (m, 3H, 2,4‐

dichlorophenyl H³, 4‐phenylbenzoyl H^2',6'), 7.80 (s, 1H, imidazole H⁵), 7.84‐8.16 (m, 4H, 4‐

13

phenylbenzoyl H^2,3,5,6), 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, J_AX= 7.6 Hz, J_AB= 4 Hz, 1H, OCH), 7.50‐7.58 (m, 2H, 2,4‐dichlorophenyl

H^5,6), 7.66‐7.75 (m, 3H, 2‐naphthoyl H^4,6,7), 7.78 (d, J = 2 Hz, 2,4‐dichlorophenyl H³), 7.90 (s,

1H, imidazole H⁴), 8.05‐8.23 (m, 4H, 2‐naphthoyl H^1,3,5,8), 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 H^3,5), 7.42‐7.48 (m, 2H, 2,4‐dichlorophenyl H^5,6), 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 H^2,6), 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+)

ACCEPTED MANUSCRIPT

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 H^5,6, 4‐isopropylbenzoyl H^3,5), 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 H^2,6), 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, J_AX= 4.8 Hz, J_AY= 4.8 Hz, 4H, morpholine H^{3,3’,5,5’}), 3.71 (t, J_XA= 4.8 Hz, J_XB= 4.8 Hz,

4H, morpholine H^{2,2’,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 H^3,5), 7.39‐7.48 (m, 2H, 2,4‐dichlorophenyl H^5,6),

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 H^2,6), 9.24 (s, 1H, imidazole H²);

¹³C NMR (100 MHz, DMSO‐d₆): δ=46.66 (morpholine C^3,5), 50.68 (CHCH₂), 65.77 (morpholine

C^2,6), 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, J_AX= 7 Hz, J_AB= 4 Hz, 1H, OCH), 7.41‐7.50 (m,

2H, 2,4‐dichlorophenyl H^5,6), 7.58 (d, J = 8.8 Hz, 2H, 4‐tert‐butylbenzoyl H^3,5), 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 H^2,6), 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

H^4,5), 8.07 (d, J = 8.4 Hz, 2H, 4‐cyanobenzoyl H^3,5), 8.24 (d, J = 8.4 Hz, 2H, 4‐cyanobenzoyl

13

H^2,6), 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 H^5,6), 7.36‐7.54

(m, 4H, indole H^3,7, 2,4‐dichlorophenyl H^5,6), 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 H^5,6, cinnamoyl H^3'‐5'), 7.67 (s, 1H, imidazole H⁴), 7.73‐7.75 (m, 3H,

cinnamoyl H^2',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 C^4,5), 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 H^3,5), 7.47‐7.67 (m,

3H, 2,4‐dichlorophenyl H^5,6, imidazole H⁴), 7.71‐7.81 (m, 2H, 2,4‐dichlorophenyl H³,

imidazole H⁵), 7.98 (d, J = 8.4 Hz, 2H, 4‐ethylbenzoyl H^2,6), 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 H^4,5, 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

H^4,5, 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 H^5,6), 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 H^3,5), 7.39‐7.49 (m, 2H, 2,4‐dichloropheyl H^5,6), 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 H^2,6), 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, CH₂C₆H_5,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 H^3'‐5', imidazole H^4,5, 2,4‐dichlorobenzoyl

H⁶), 7.46‐7.72 (m, 4H, 2,4‐dichlorobenzoyl H^3,5, 4‐phenylbutanoyl H^2',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 H^4,5, 2,4‐dichlorophenyl H³), 9.20

(s, 1H, imidazole H²); ¹³C NMR (100 MHz, DMSO‐d₆): δ=25.18 (2C, cyclopentane C^3,4), 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 H^3'‐5'

,

imidazole H^4,5), 7.72 (d, J = 2 Hz, 1H, 2,4‐dichlorophenyl H³), 7.95‐7.97 (m, 2H, 3‐

benzoylpropanoyl H^2',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 H^5,6),

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 H^3,5), 8.30 (d, J = 8.4 Hz, 2H, 4‐

13

trifluoromethylbenzoyl H^2,6), 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 H^3"‐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 H^2",6"), 7.84 (d, J = 8.4 Hz, 2H, 4‐

terphenyl H^3',5'), 7.91 (d, J = 8.4 Hz, 2H, 4‐terphenyl H^2',6'), 7.99 (d, J = 8.8 Hz, 2H, 4‐terphenyl

H^3,5), 8.27 (d, J = 8.4 Hz, 2H, 4‐terphenyl H^2,6), 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 H^3,6, 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 H^4'), 7.52‐7.56 (m, 3H, 2,4‐dichlorophenyl H⁶, 4‐

phenylbenzoyl H^3',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 H^2',6'), 7.92 (d, J = 8.8 Hz, 2H, 4‐phenylbenzoyl H^3,5), 8.27

13

(d, J = 8.4 Hz, 2H, 4‐phenylbenzoyl H^3,5); 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 H^4,7,

imidazole H⁴), 7.68‐7.76 (m, 4H, 2,4‐dichlorophenyl H^3,5, indole H^4,7, 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 H^3,5), 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 H^2,6); 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 H^3,5),

7.52‐7.55 (m, 2H, 2,4‐dichlorophenyl H^5,6), 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 H^2,6), 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 H^3,5), 7.51 (d, J = 8 Hz, 2H, 4‐ethylbenzoyl

H^2,6), 7.66‐7.72 (m, 3H, 2,4‐dichlorophenyl H^5,6, 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),

Article Doi

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

DOI: 10.1016/j.ejmech.2019.06.083

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

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