Imidazolylchromanones containing non-benzylic oxime ethers: Synthesis and molecular modeling study of new azole antifungals selective against Cryptococcus gattii

Article abstract of DOI:10.1016/j.ejmech.2014.02.019

A series of imidazolylchromanone oximes containing phenoxyethyl ether moiety, as found in omoconazole, were synthesized and evaluated against yeasts (Candida albicans and Cryptococcus gattii) and filamentous fungi (Aspergillus fumigatus and Exophiala derm

Full text of DOI:10.1016/j.ejmech.2014.02.019

European Journal of Medicinal Chemistry 76 (2014) 264e273
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Preliminary communication
Imidazolylchromanones containing non-benzylic oxime ethers:
Synthesis and molecular modeling study of new azole antifungals
selective against Cryptococcus gattii
Mojtaba Babazadeh-Qazijahani a, Hamid Badali b, Hamid Irannejad c,
,
Mohammad Hosein Afsarian b, Saeed Emami c
*
a Student Research Committee, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran
b Department of Medical Mycology & Parasitology/Invasive Fungi Research Center (IFRC), Faculty of Medicine, Mazandaran University of Medical Sciences,
Sari, Iran
c Department of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Faculty of Pharmacy, Mazandaran University of Medical Sciences,
Sari, Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of imidazolylchromanone oximes containing phenoxyethyl ether moiety, as found in omoco-
nazole, were synthesized and evaluated against yeasts (Candida albicans and Cryptococcus gattii) and
filamentous fungi (Aspergillus fumigatus and Exophiala dermatitidis). Although the title compounds
showed marginal activity against filamentous fungi but all of them exhibited potent activity against C.
Received 4 October 2013
Received in revised form
6 January 2014
Accepted 8 February 2014
Available online 11 February 2014
gattii (MIC values ꢀ4
mg/mL). Among them, (3-chlorophenoxy)ethyl analog 7c with MIC value of 0.5 mg/
mL was the most potent compound. Further molecular docking studies provided a better insight into the
binding of designed compounds within the homology modeled active site of CnCYP51 (Cryptococcus
CYP51-14a-demethylase).
Keywords:
Antifungal activity
Azole antifungals
Cryptococcus
Ó 2014 Elsevier Masson SAS. All rights reserved.
Chroman-4-one
Imidazole
Lanosterol 14a-demethylase
1. Introduction
commonly to the brain and skin [4e6]. Two predominant varieties
are recognized for C. neoformans: var. neoformans and var. gattii [7].
C. gattii has recently received widespread attention owing to out-
breaks in British Columbia, Canada and the US Pacific Northwest,
and is believed to be clinically more virulent than C. neoformans [8].
Infections with C. gattii in patients can be severe and often fatal if
untreated [1].
Many drugs are available for the treatment of systemic or su-
perficial fungal infections, but only a limited number of them are
effective in the treatment of cryptococcal infections [9]. The poly-
ene drugs such as amphotericin B and azole antifungals such as
fluconazole are the most widely used drugs to treat cryptococcosis.
Severe side effects such as nephrotoxicity limit the clinical useful-
ness of polyene drugs. Azole antifungals are generally considered as
Fungal infections cause a persistent burden on human and an-
imal health, plants and agricultural economy [1]. In human, many
fungal infections are caused by opportunistic pathogenic fungi that
may be endogenous flora or acquired from the environment. The
incidence of fungal infections has significantly increased in recent
years. The vast majority of fungal infections are due to Candida,
Aspergillus and Cryptococcus species, especially in all categories of
immunocompromised patients [2]. The immunocompromised pa-
tients including cancer patients receiving chemotherapy, organ
transplant recipients and patients with AIDS are prone to fungal
infections [3].
Cryptococcus neoformans is encapsulated basidiomycetous yeast
that infects pulmonary organs and can disseminate widely, most
fungistatic agents and act by inhibiting lanosterol 14a-demethylase
in the ergosterol biosynthetic pathway [10]. The emergence of
resistance and fungistatic rather than fungicidal activities represent
limitations of current azole antifungals. Moreover, the pharmaco-
kinetic deficiencies of azole may have a profound effect on the
* Corresponding author.
E-mail address: sd_emami@yahoo.com (S. Emami).
http://dx.doi.org/10.1016/j.ejmech.2014.02.019
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

M. Babazadeh-Qazijahani et al. / European Journal of Medicinal Chemistry 76 (2014) 264e273
265
apparent drug resistance of azoles. For example, oral itraconazole
may be less effective in the treatment of cryptococcal meningitis
because of its variable oral absorption and poor cerebrospinal fluid
levels [11]. Therefore, there remains an urgent need for finding new
antifungal agents with proper safety and pharmacokinetics to
overcome this situation and develop effective therapies especially
against cryptococcal infections.
In our quest to develop new azole antifungal agents, recently we
have designed 3-imidazolylchromanone oxime ethers (Fig. 1,
structure A) as conformationally constrained analogs of oxiconazole
[12]. Oxiconazole and most of imidazole antifungals such as mi-
conazole, econazole, isoconazole, sulconazole and fenticonazole
contain benzyl ether side chains (Fig. 1). Besides azole antifungals
with benzyl ether side chains, omoconazole is a distinct azole anti-
fungal with non-benzylic side chain namely 2-(4-chlorophenoxy)
ethyl ether. In continuation of our research program on azole anti-
fungals, we focused our modifications mainly on the side chains of
imidazolylchromanone oxime ethers, and attached 2-phenoxyethyl
ether moiety tothe imidazolylchromanone oxime scaffold instead of
benzyl ether residue. Thus, we report here, synthesis and antifungal
activity of imidazolylchromanone O-(phenoxyethyl)oxime ethers
(Fig. 1, structure B).
hydrochloride in methanol to afford the corresponding oxime 3.
Reaction of 3-bromo-oxime derivative 3 with imidazole in DMF
offered 3-imidazolyl-4-chromanone-(E)-oxime (4) [12]. On the
other hand, phenol derivatives 5 were refluxed with 1-bromo-2-
chloroethane in butan-2-one and converted to the corresponding
phenoxyethyl chloride derivatives 6. In the final step, (E)-oxime 4
was reacted with phenoxyethyl chloride derivatives 6 in the pres-
ence of NaH in DMF to give (E)-oxime ether derivatives 7.
The structures of final compounds 7aef were fully characterized
by IR, 1H NMR, 13C NMR and MS spectral data. Representatively, the
spectral studies of compound 7a are discussed here. In the IR
spectrum of compound 7a, a medium band due to the C]N of oxime
moiety was appeared at 1601 cmꢁ1. In the 1H NMR, the CH2CH2 unit
of phenoxyethyl moiety displayed two triplet signals at 4.32 and
4.54 ppm with coupling constants of 4.4 Hz. The hydrogens located
on the C-2 position of chroman ring were appeared at 4.70 and
5.01 ppm as doublets of doublets. The geminal coupling constant of
the latter hydrogens was 12.0 Hz. The observed peak at 5.64 ppm
was attributed to the H-3 of chroman ring. The resonances of ortho
and para hydrogens of phenoxy group and H-6 and H-8 of chroman
ring were occurred in the range of 6.90e7.18 ppm. The H-3 and H-5
of phenoxy group showed absorption at 7.28 ppm. A triplet at
7.46 ppm is attributed to the H-7 of chroman structure. The H-5 of
chroman ring was appeared downfield at 8.57 ppm due to the
deshielding of (E)-oxime functionality. The hydrogens of imidazole
ring were observed at 7.68, 7.72 and 9.22 ppm. Since the compound
7a was prepared as nitrate salt, thus the downfield shift of the latter
protons is due to the protonation of imidazole ring.
2. Chemistry
The synthesis of target compounds imidazolylchromanone O-
(phenoxyethyl)oxime ethers 7, starting from 4-chromanone (1) and
phenol derivatives 5 is outlined in Scheme 1. Firstly, 4-chromanone
(1) was brominated with copper (II) bromide to give 3-bromo-4-
chromanone (2). Compound 2 was reacted with hydroxylamine
In the 13C NMR of compound 7a, the aliphatic carbons of chro-
man core (C-2 and C-3) showed upfield signals at 74.0 and
Fig. 1. Structures of representative azole antifungals containing benzyl ether side chain, omoconazole as azole antifungal bearing 2-(4-chlorophenoxy)ethyl ether fragment, and
designed 3-imidazolylchromanone oxime ethers (structure B) as non-benzylic analogs of structure A.

266
M. Babazadeh-Qazijahani et al. / European Journal of Medicinal Chemistry 76 (2014) 264e273
O
O
O
O
NOH
Br
Br
a
b
O
c
1
3
2
HO
N
Cl
N
N
d
O
OH
R
R
O
4
5
6a-f
e,f
6a, 7a: R= H
O
R
6b, 7b: R= 4-Cl
6c, 7c: R= 3-Cl
6d, 7d: R= 4-F
6e, 7e: R = 3-F
.HNO3
O
N
N
5
4
N
4a
8a
3
6
7
2
6f, 7f: R= 2,4-Cl2
O1
8
7a-f
Scheme 1. Synthesis of imidazolylchromanone O-(phenoxyethyl) oxime ethers 7. Reagents and conditions: (a) copper (II) bromide, CHCl3-EtOAc, reflux; (b) hydroxylamine hy-
drochloride, MeOH, rt; (c) imidazole, DMF; (d) 1-bromo-2-chloroethane, butan-2-one, reflux; (e) NaH, DMF; (f) propan-2-ol, 65% HNO3.
55.7 ppm, while the unsaturated C]N carbon (C-4) showed ab-
sorption at downfield region of 158.3 ppm. The signals related to
the aromatic carbons of chroman ring were located at 113.5, 117.6,
120.7, 131.4, 133.2 and 141.7 ppm. The resonances of imidazole
carbons C-2, C-4 and C-5 were occurred at 135.3, 121.8 and 121.0,
fumigatus and Exophiala dermatitidis) were evaluated in vitro. The
minimal inhibitory concentrations (MICs) of test compounds were
determined by the micro-broth dilution method in 96-well
microplates according to the CLSI methods for yeasts and fila-
mentous fungi [13,14]. Briefly, compounds were dissolved in DMSO
and serially diluted in growth medium. The inoculum suspension
was added to each well and incubated at 35 ꢂC. MIC was defined as
the minimum inhibitory concentration of test compound which
resulted in total inhibition of the fungal growth. All susceptibility
testing was performed in triplicate.
respectively. The peaks at 65.8 and 67.1 were assigned for OCH2
-
CH2O unit. The phenoxy group showed four signals at 114.5, 120.5,
129.5, and 154.8 ppm.
The mass spectral data of compound 7a provided further evi-
dence of its structure. The molecular ion peak was observed at the
expected m/z value of 349.
The (E)-configuration of compounds 7aef was readily estab-
lished by 1H NMR spectral data. Particularly, proximity of the oxime
oxygen to the C-5 hydrogen of chroman ring in (E)-isomers result in
4. Results and discussion
4.1. Antifungal activity
downfield shift of the H-5 (d H-5 > 8.5 ppm) respect to that of (Z)-
oxime ethers (
d
H-5 ꢀ 7.9 ppm) [12]. The investigation of 1H NMR
The MIC values of target compounds 7aef in comparison with
those of parent 3-imidazolyl-4-chromanone oxime (4) and standard
drugs fluconazole and itraconazole were summarized in Table 1.
Generally, the parent oxime 4 showed no activity against all fungal
species. Furthermore, all title compounds 7aef were inactive
spectral data revealed that the chemical shifts of C-5 proton in
oxime ethers 7aef were in the range of 8.53e8.57 ppm. Thus, the
orientation of the oxime ether moiety of compounds 7aef was
assigned to be of (E)-geometry.
The compounds 7aef possess a chiral center at C-3 position of
chroman ring. Substantially, the bromination of 4-chromanone (1)
at C-3 position is not stereoselective. Thus, the originated in-
termediates 2e4 were as a mixture of enantiomers and as well the
target compounds 7aef were obtained as a racemic mixture.
against C. albicans (MIC values >64
pounds exhibited potent activity against C. gattii (MIC values ¼ 0.5e
g/mL). The 3-chloro analog 7c with MIC value of 0.5 g/mL was
the most potent compound. The MIC values of compounds against
A. fumigatus and E. dermatitidis revealed that all compounds
exhibited marginal activity. It should be noted that standard drug
fluconazole was inactive against filamentous fungi.
mg/mL). In contrast, all com-
4
m
m
3. Biology
By comparing the MIC values of title compounds 7aef and their
parent oxime 4, it could be concluded that the phenoxyethyl ether
scaffold is essential for antifungal activity. Although halogen
The antifungal activity of target compounds against yeasts
(Candida albicans and C. gattii) and filamentous fungi (Aspergillus

M. Babazadeh-Qazijahani et al. / European Journal of Medicinal Chemistry 76 (2014) 264e273
267
Table 1
Mycobacterium tuberculosis (MtCYP51). The crystal structure of
MtCYP51 complexed with 4-phenylimidazole (PDB code 1E9X) was
retrieved from the Brookhaven Protein Data Bank (www.rcsb.org)
and used as a template for homology modeling of C. neoformans
The minimum inhibitory concentrations (MICs,
different fungi.
mg/mL) of test compounds against
O
serotype A CYP51-14
a-demethylase (CnCYP51, SWISS-Prot acces-
R
sion number Q870D1).
HO
O
.HNO3
N
N
N
O
As shown in Fig. 2, the sequence of CnCYP51 with that of
MtCYP51 was aligned using SWISS-MODEL [18]. The first 57 resi-
dues at the N-terminus of CnCYP51 were deleted since, MtCYP51
lacks the N-terminal transmembrane domain found in fungal
CYP51 proteins and the 57 N-terminal residues of CnCYP51 were
assumed to constitute the membrane binding region and had no
counterpart in the catalytic domain.
N
O
N
N
4
7a-f
The obtained 3D model of CnCYP51 built by SWISS-MODEL was
subjected to energy minimization using Gaussian98 with Amber96
force field in order to get a stable low energy conformation. After
energy minimization, 100 ps molecular dynamic simulations were
performed at constant temperature (298 K) and pressure
(P ¼ 1 atm) using an integration time step of 2 fs with Amber96
force field in an implicit solvent model. OpenMM 5.0 (Simbios, an
NIH National Center for Physics Based Simulation of Biological
Structures) was used for molecular dynamic (MD) simulations [19].
The root mean square deviation (RMSD) of heavy atoms of protein
as a function of time was recorded during simulation which shows
the relative structural drift or stability of the structure
(Supplementary material). The potential energy of the CnCYP51
model decreased substantially and was stabilized after 100 ps dy-
namic simulations. The structure of the CnCYP51 model deviated
rapidly from the initial structure within the first 60 ps of the MD
simulation. This increase in the RMSD is due to the optimization of
the interactions within the protein structure. After about 100 ps,
the total RMSD of the CnCYP51 model was stabilized at around
Compound
R
C. albicans
C. gattii
A. fumigatus
E. dermatitidis
4
7a
7b
7c
7d
7e
7f
e
>64
>64
>64
>64
>64
>64
>64
2
>64
2
4
0.5
4
2
>64
32
64
32
64
64
64
>64
4
>128
128
64
64
128
64
128
>64
0.5
H
4-Cl
3-Cl
4-F
3-F
2,4-Cl2
2
Fluconazole
Itraconazole
>64
0.5
0.25
substitution had positive effect in some cases depends on its po-
sition, but could not significantly improve antifungal activity.
However, 3-chloro analog 7c was 4e8 folds more potent than other
halo-substituted congeners against C. gattii.
As mentioned above, amphotericin B and fluconazole are the
most widely used drugs to treat cryptococcosis. There are few re-
ports concerning the in vitro antifungal susceptibility of C. gattii.
Assessment of antifungal susceptibility for fluconazole and
amphotericin B by Silva et al. have been demonstrated that the
ꢀ
1.9 A, suggesting that this simulation time is sufficient for stabi-
fluconazole had MICs ranged from 1 to 64
amphotericin B were low, ranging from 0.12 to 0.5
m
g/mL, while the MICs for
g/mL [15].
lizing a fully relaxed model.
m
Ramachandran plot was obtained to assess the stereochemical
quality and the geometrical accuracy of the modeled protein
(Fig. 3). Ramachandran plot is an interesting parameter for the
In our study, since the designed compounds were structurally
related to azole antifungals, thus we tested them side-by-side to the
standard azole drugs, fluconazole and itraconazole. The suscepti-
bility testing of designed compounds against different fungi strains
revealed that C. gattii is more susceptible. Particularly, our data
showed that all new compounds tested possess low MIC values
against C. gattii. The results of our antifungal activity studies,
together with the selectivity displayed by our compounds against
C. gattii, introduces new lead compounds in the development of
novel antifungal drugs for treatment of cryptococcal infections.
Cryptococcal infections and specially infections caused by C. gattii
are life-threatening complications for immunocompromised hosts,
producing fatal cryptococcosis in patients who have undergone
organ transplants and being the main cause of fatal meningoen-
cephalitis in AIDS patients. Thus the designed compounds acting
against this fungal pathogen are highly desirable for further in vivo
and clinical studies.
assessment of protein quality by the means of 4/j-angle distribu-
tion of the protein structure after dynamic simulation. For the
CnCYP51 model, 90.5% of the residues were in the favored regions,
6.6% in the allowed regions and 2.9% in disallowed regions. Thus, a
total of 97.1% of the residues of the modeled CnCYP51 structure
after MD simulation were in the two core (favored and allowed)
regions. These data indicated that the backbone dihedral angles 4
and
j in the model were reasonable. The residues in disallowed
regions are found far away from the active site (Fig. 3).
In order to clarify the binding mode of the newly synthesized
azoles in the active site of CnCYP51 and to provide more straight-
forward information into the interactions between ligand and
enzyme for further rational drug design, molecular docking was
performed using AutoDock 4.2 [20]. Energy minimization and ge-
ometry optimization was done on the ligand structures prior to any
docking calculation. Itraconazole as a reference drug was docked
into the active site of modeled CnCYP51 (Supplementary material).
As shown, triazole ring is pointed towards the heme plane and a ring
4.2. Docking studies
We also accomplished a preliminary study in order to predict
how the newly synthesized compounds might interact with the
nitrogen atom coordinating the iron ion. The coordinated triazole
ꢀ
nitrogen atom is 3.1
A
distant from the heme iron. 2,4-
target enzyme lanosterol 14
a
-demethylase of cryptococci through
Dichlorophenyl group of itraconazole makes van der Waals and
hydrophobic interactions with Tyr74, Met259, Phe82 and Thr78. The
successive phenyl-piperazine-phenyl rings are located in a lipo-
philic part of active site constituted mainly by hydrophobic side
chains of Tyr333, Phe183 and His263. Triazolone ring is situated in a
polar region and hydrogen bonded to the polar side chains of Glu177,
Asp180 and Glu262. The ending isobutyl chain makes non-polar
interactions with Leu144, Val473 and Ser266. Since itraconazole
computer molecular modeling. It is well known that in the absence
of crystal structure of related enzyme, homology modeling is a
valuable tool for gaining insight into the interaction between in-
hibitor and P450-dependent enzymes [16,17].
Herein, we constructed the model of cryptococcal lanosterol14
demethylase by homology modeling on the basis of the reported
crystal structure of cytochrome P450 14- -sterol demethylase of
a-
a

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M. Babazadeh-Qazijahani et al. / European Journal of Medicinal Chemistry 76 (2014) 264e273
Fig. 2. Alignment of the amino acid sequences of cytochrome P450 14-a-sterol demethylase of Cryptococcus neoformans and Mycobacterium tuberculosis.
has a very long side chain, the terminal isobutyl group reached the
entrance of substrate accessing channel to the active site.
As discussed above, compound 7c bearing (3-chlorophenoxy)
ethyl moiety attached to the (E)-oxime, was the most potent
compound against C. gattii. Thus, this compound was considered
for further molecular and computational analysis. Figs. 4 and 5
show the binding mode of the two enantiomeric forms (R and S)
of compound 7c in the CnCYP51 active site. The chroman

M. Babazadeh-Qazijahani et al. / European Journal of Medicinal Chemistry 76 (2014) 264e273
269
5. Conclusion
In summary, we described a series of imidazolylchromanones
containing (phenoxyethyl)-oxime ether moiety as new azole anti-
fungals selective against C. gattii. All synthesized compounds 7aef
exhibited potent activity against C. gattii. Among them, (3-
chlorophenoxy)ethyl analog 7c with MIC value of 0.5 mg/mL was
the most potent compound. Our molecular docking studies pro-
vided a better insight into the binding of designed compounds
within the homology modeled active site of CnCYP51.
6. Experimental
6.1. Chemistry
All starting materials and solvents were purchased from Merck
Company and used without further purification. The intermediates
2-phenoxyethyl chloride (6a) and 2-(4-chlorophenoxy)ethyl chlo-
ride (6b) were commercially available materials and purchased
from Merck and SigmaeAldrich companies, respectively. The key
intermediate oxime 4 was prepared as previously described
method [12]. The progress of the reactions was checked by thin-
layer chromatography (TLC) using silica gel 60 F254 plastic sheets
(Merck). UV light (254 nm) or iodine vapor were used for visuali-
zation. Yields are based on isolated products and were not opti-
mized. Melting points were determined in open glass capillaries
using Bibby Stuart Scientific SMP3 apparatus (Stuart Scientific,
Stone, UK) and are uncorrected. The IR spectra were recorded on a
PerkinElmer FT-IR spectrophotometer. The NMR spectra were ob-
tained using a Bruker 500 spectrometer. In the interpretation of
Fig. 3. Ramachandran plot for the last frame of the molecular dynamic simulation of
the modeled CnCYP51. Amino acids in the favored regions are in green, in the allowed
regions in yellow and in the disallowed regions are represented in red signs. (For
interpretation of the references to color in this figure legend, the reader is referred to
the web version of this article.)
framework and the imidazole ring of one enantiomer are super-
posed on those of the other enantiomer and occupy exactly the
same regions in the active site with surrounding amino acids Tyr74,
Tyr333, Ala73 and Thr75. However, 3-chlorophenyl group is ori-
ented differently in the (R) and (S)-enantiomers. In the (R)-enan-
tiomer of 7c, it resides as 2,4-dichlorophenyl part of itraconazole
with the same interacting amino acids. The 3-chlorophenyl side
chain of (S)-enantiomer has directed towards Tyr20 and Ser331.
The imine nitrogen atom in both enantiomers is protonated in
physiological environment and hydrogen bonded to the imidazole
NMR data, chemical shifts are expressed as
d (ppm) with tetra-
methylsilane (TMS) as internal standard. The multiplicities of sig-
nals are reported as follows: s, singlet; d, doublet; t, triplet; q,
quartet; br, broad; m, multiplet, and the coupling constants (J) are
expressed in Hertz (Hz).
6.1.1. General procedure for the synthesis of compounds 6cef
A mixture of substituted phenol (10 mmol) and potassium car-
bonate (15 mmol) in butan-2-one (25 mL) was refluxed for 0.5 h.
Then, 1-bromo-2-chloroethane (30 mmol) was added to the reac-
tion mixture and refluxing was continued for 10 h. After completion
of the reaction, water (40 mL) was added and the mixture was
extracted with diethyl ether several times. The organic phase was
washed (10% NaOH solution and water, respectively) and dried
(Na2SO4). The solvent and the excess of 1-bromo-2-chloroethane
were evaporated under reduced pressure to give compounds 6ce
f as oil.
ꢀ
ring of His263 (3.7 A). The N-3 nitrogen atom of imidazole in both
enantiomers is coordinated to the heme iron with appropriate
ꢀ
ꢀ
distance (3.1 A for (S)-enantiomer and 2.8 A for (R)-enantiomer,
respectively).
To rationalize the experimental MIC values and to explain the
significant potency differences between the inactive compound 4
and target compound 7c and also itraconazole as a potent drug
against C. neoformans, the intermolecular energies including van
der Waals, hydrogen bonds, desolvation and electrostatic energies
as well as estimated free energy of binding (
D
G, kcal/mol) and
6.1.1.1. 2-(3-Chlorophenoxy)ethyl chloride (6c). Yield 75%; 1H NMR
estimated inhibition constant (Ki, M) were calculated and are
summarized in Table 2. As shown, enantiomeric forms of 7c have
lower intermolecular energy, free energy of binding and inhibition
constant of fungal lanosterol 14a-demethylase than inactive com-
pound 4. There is not a great difference between the enantiomeric
forms of 7c in the estimated energy values, although (S)-enan-
tiomer shows a bit more potency in inhibiting the fungal enzyme.
The lowest binding energy and inhibition constant of itraconazole
m
(500 MHz, CDCl3)
d
: 3.82 (t, J ¼ 5.4 Hz, 2H, CH2), 4.23 (t, J ¼ 5.4 Hz,
2H, CH2), 6.82 (d, J ¼ 7.5 Hz, 1H, H-6), 6.93 (s, 1H, H-2), 6.98 (d,
J ¼ 7.5 Hz, 1H, H-4), 7.22 (t, J ¼ 7.5 Hz, 1H, H-5).
6.1.1.2. 2-(4-Fluorophenoxy)ethyl chloride (6d). Yield 68%; 1H NMR
(500 MHz, CDCl3)
2H, CH2), 6.86e7.03 (m, 4H, phenyl H).
d: 3.81 (t, J ¼ 5.4 Hz, 2H, CH2), 4.20 (t, J ¼ 5.4 Hz,
(
D
G ¼ ꢁ7.72 kcal/mol, Ki ¼ 2.2
m
M) demonstrate its high capability
6.1.1.3. 2-(3-Fluorophenoxy)ethyl chloride (6e). Yield 70%; 1H NMR
to inhibit lanosterol 14 -demethylase from C. neoformans. Indeed,
a
(500 MHz, CDCl3)
d
: 3.83 (t, J ¼ 5.5 Hz, 2H, CH2), 4.23 (t, J ¼ 5.5 Hz, 2H,
itraconazole bearing a long side chain forms additional van der
Waals contacts and hydrogen bonds with the active site of CnCYP51
(intermolecular energy ¼ ꢁ10.24 kcal/mol). As concluded, there is
high correlation between the estimated inhibition constants
calculated by docking and scoring of compounds in the active site of
CnCYP51 and the measured MIC values.
CH2), 6.62e6.74 (m, 3H, H-2, H-4 and H-6), 7.15e7.30 (m, 1H, H-5).
6.1.1.4. 2-(2,4-Dichlorophenoxy)ethyl chloride (6f). Yield 72%; 1H
NMR (500 MHz, CDCl3)
d: 3.86 (t, J ¼ 5.4 Hz, 2H, CH2), 4.27 (t,
J ¼ 5.4 Hz, 2H, CH2), 6.88 (d, J ¼ 8.5 Hz, 1H, H-6), 7.20 (d, J ¼ 8.5 Hz,
1H, H-5), 7.40 (s, 1H, H-3).

270
M. Babazadeh-Qazijahani et al. / European Journal of Medicinal Chemistry 76 (2014) 264e273
ꢀ
Fig. 4. (A) Binding mode of R-7c in the active site of CnCYP51. For clarity only amino acids within 7 A distant from the docked ligand are shown. (B) 2D map of R-7c in the active site
of CnCYP51.
6.1.2. General procedure for the synthesis of compounds 7aef
solution of 3-imidazolyl-4-chromanone-(E)-oxime (4,
overnight. The separated viscous oil was dissolved in propan-2-ol
(2 mL) and treated with 65% HNO3 (0.07 mL) and the mixture
was left in a fridge to form nitrate salts. The precipitated crystals
were separated and wash with cold propan-2-ol to give pure
compound 7.
A
0.75 mmol) in dry DMF (1.5 mL) was mixed with a suspension of
NaH (18 mg, 0.75 mmol) in dry DMF (1.5 mL). The reaction mixture
was stirred at room temperature for 0.5 h. Then, a solution of
phenoxyethyl chloride derivative 6 (0.825 mmol) in dry DMF (1 mL)
was added gradually and stirring was continued at room temper-
ature for 2 days. After completion of the reaction, the reaction
mixture was poured into water (20 mL) and left in refrigerator
6.1.2.1. (ꢃ)-(E)-2,3-Dihydro-3-(1H-imidazol-1-yl)-4H-1-benzopyran-
4-one O-(2-phenoxy ethyl) oxime nitrate (7a). Yield 75%; mp 113e
114 ꢂC; IR (KBr, cmꢁ1 nmax: 1601 (C]NO). 1H NMR (500 MHz,
)

M. Babazadeh-Qazijahani et al. / European Journal of Medicinal Chemistry 76 (2014) 264e273
271
ꢀ
Fig. 5. (A) Binding mode of S-7c in the active site of CnCYP51. For clarity only amino acids within 7 A distant from the docked ligand are shown. (B) 2D map of S-7c in the active site
of CnCYP51.
DMSO-d6)
d
: 4.32 (t, J ¼ 4.4 Hz, 2H, CH2), 4.54 (t, J ¼ 4.4 Hz, 2H, CH2),
imidazole H-2), 14.73 (br s, 1H, NHþ). 13C NMR (125 MHz, DMSO-d6)
d: 55.7 (C-3, chroman), 65.8 (CH2), 67.1 (CH2), 74.0 (C-2, chroman),
113.5 (C-4a, chroman), 114.5 (C-2 and C-6, phenyl), 117.6 (C-8,
chroman), 120.5 (C-4, phenyl), 120.7 (C-6, chroman), 121.0 (C-5,
imidazole), 121.8 (C-4, imidazole), 129.5 (C-3 and C-5, phenyl), 131.4
(C-5, chroman), 133.2 (C-7, chroman), 135.3 (C-2, imidazole), 141.7
(C-8a, chroman), 154.8 (C-1, phenyl), 158.3 (C-4, chroman). MS (m/z,
4.70 (dd, J ¼ 12.0 and 3.0 Hz, 1H, chroman H-2a), 5.01 (dd, J ¼ 12.0
and 3.6 Hz, 1H, chroman H-2b), 5.64 (dd, J ¼ 3.6 and 3.0 Hz, 1H,
chroman H-3), 6.90e7.18 (m, 5H, phenyl H-2, H-4 and H-6, chroman
H-6 and H-8), 7.28 (t, J ¼ 7.3 Hz, 2H, phenyl H-3 and H-5), 7.46 (t,
J ¼ 7.5 Hz, 1H, chroman H-7), 7.68 (s, 1H, imidazole H-4), 7.72 (s, 1H,
imidazole H-5), 8.57 (d, J ¼ 8.0 Hz, 1H, chroman H-5), 9.22 (br s, 1H,

272
M. Babazadeh-Qazijahani et al. / European Journal of Medicinal Chemistry 76 (2014) 264e273
Table 2
Estimated energies (
DG, kcal/mol) and inhibition constant (Ki,
m
M) calculated by AutoDock.
Compound
Intermolecular energy
Estimated binding
Estimated inhibition
energy (
DG)
constant (Ki)
VdW þ H-bond þ desolv.
Electrostatic
energy
energy
(S)-7c
(R)-7c
(S)-4
(R)-4
ꢁ8.27
ꢁ7.85
ꢁ5.53
ꢁ6.19
ꢁ10.24
ꢁ0.13
ꢁ0.24
ꢁ0.24
ꢁ0.03
ꢁ0.17
ꢁ6.61
ꢁ6.3
14.28
24.26
161.66
92.24
2.2
ꢁ5.17
ꢁ5.5
Itraconazole
ꢁ7.72
%): 349 (Mþ, 37), 256 (68), 229 (75), 171 (31), 161 (52), 146 (72), 95
(100), 77 (97), 69 (61). Anal. Calcd for C20H20N4O6: C, 58.25; H, 4.89;
N, 13.59. Found: C, 58.02; H, 4.94; N, 13.59.
4.71 (dd, J ¼ 12.5 and 3.0 Hz, 1H, chroman H-2a), 5.01 (dd, J ¼ 12.5
and 3.6 Hz, 1H, chroman H-2b), 5.64 (dd, J ¼ 3.6 and 3.0 Hz, 1H,
chroman H-3), 6.97e7.01 (m, 2H, chroman H-6 and H-8), 7.06e7.15
(m, 4H, phenyl H), 7.47 (dt, J ¼ 8.1 and 1.3 Hz, 1H, chroman H-7), 7.68
(t, J ¼ 1.6 Hz, 1H, imidazole H-4), 7.72 (t, J ¼ 1.6 Hz, 1H, imidazole H-
5), 8.55 (dd, J ¼ 8.1 and 1.3 Hz, 1H, chroman H-5), 9.18 (s, 1H, imid-
6.1.2.2. (ꢃ)-(E)-2,3-Dihydro-3-(1H-imidazol-1-yl)-4H-1-benzopyran-
4-one O-[2-(4-chloro phenoxy) ethyl] oxime nitrate (7b). Yield 63%;
mp 102e103 ꢂC; IR (KBr, cmꢁ1
)
nmax: 1603 (C]NO). 1H NMR
azole H-2), 14.50 (br s, 1H, NHþ). 13C NMR (100 MHz, DMSO-d6)
d:
(500 MHz, DMSO-d6)
d
: 4.32 (t, J ¼ 4.3 Hz, 2H, CH2), 4.53 (t,
56.2 (C-3, chroman), 67.1 (CH2), 67.6 (CH2), 74.5 (C-2, chroman),114.0
(C-4a, chroman), 116.3 (d, JC,F ¼ 9.7 Hz, C-3 and C-5, phenyl), 116.4 (d,
JC,F ¼ 5.1 Hz, C-2 and C-6, phenyl), 118.1 (C-8, chroman), 121.1 (C-6,
chroman), 121.5 (C-5, imidazole), 122.3 (C-4, imidazole), 131.9 (C-5,
chroman), 133.7 (C-7, chroman), 135.8 (C-2, imidazole), 142.3 (C-8a,
chroman), 155.1 (d, JC,F ¼ 1.5 Hz, C-1, phenyl), 155.3 (C-4, chroman),
157.1 (d, JC,F ¼ 234.5 Hz, C-4, phenyl). MS (m/z, %): 367 (Mþ, 4), 296
(82), 269 (12), 229 (10), 161 (24), 123 (100), 111 (16), 95 (61), 69 (12).
Anal. Calcd for C20H19FN4O6: C, 55.81; H, 4.45; N, 13.02. Found: C,
56.08; H, 4.66; N, 12.94.
J ¼ 4.3 Hz, 2H, CH2), 4.69 (dd, J ¼ 11.0 and 3.0 Hz, 1H, chroman H-2a),
5.00 (dd, J ¼ 11.0 and 3.6 Hz,1H, chroman H-2b), 5.63 (dd, J ¼ 3.6 and
3.0 Hz, 1H, chroman H-3), 6.95e7.15 (m, 4H, chroman H-6 and H-8,
phenyl H-2 and H-6), 7.30 (d, J ¼ 7.6 Hz, 2H, phenyl H-3 and H-5),
7.46 (t, J ¼ 7.5 Hz, 1H, chroman H-7), 7.68 (s, 1H, imidazole H-4), 7.72
(s, 1H, imidazole H-5), 8.53 (d, J ¼ 8.0 Hz, 1H, chroman H-5), 9.20 (s,
1H, imidazole H-2). 13C NMR (125 MHz, DMSO-d6)
d: 55.7 (C-3,
chroman), 66.3 (CH2), 67.1 (CH2), 73.9 (C-2, chroman), 113.5 (C-4a,
chroman), 116.3 (C-2 and C-6, phenyl), 117.6 (C-8, chroman), 120.5
(C-6, chroman), 120.9 (C-5, imidazole), 121.8 (C-4, imidazole), 124.5
(C-4, phenyl), 129.2 (C-3 and C-5, phenyl), 131.4 (C-5, chroman),
133.2 (C-7, chroman), 135.2 (C-2, imidazole), 141.8 (C-8a, chroman),
154.8 (C-1, phenyl), 157.1 (C-4, chroman). MS (m/z, %): 383 (Mþ, 8),
315 (16), 278 (35), 256 (28), 229 (32), 212 (41), 188 (27), 161 (100),
144 (40), 128 (74), 111 (90), 95 (57), 75 (60), 58 (67). Anal. Calcd for
6.1.2.5. (ꢃ)-(E)-2,3-Dihydro-3-(1H-imidazol-1-yl)-4H-1-benzopyran-
4-one O-[2-(3-fluoro phenoxy) ethyl] oxime nitrate (7e). Yield 84%;
mp 116e117 ꢂC; IR (KBr, cmꢁ1 nmax: 1597 (C]NO). 1H NMR
)
(500 MHz, DMSO-d6)
d: 4.35 (t, J ¼ 4.4 Hz, 2H, CH2), 4.54 (t,
J ¼ 4.4 Hz, 2H, CH2), 4.70 (dd, J ¼ 12.5 and 3.0 Hz,1H, chroman H-2a),
5.00 (dd, J ¼ 12.5 and 3.6 Hz, 1H, chroman H-2b), 5.63 (dd, J ¼ 3.6
and 3.0 Hz,1H, chroman H-3), 6.7e6.88 (m, 3H, chroman H-6 and H-
8, and phenyl H-2), 7.05e7.11 (m, 2H, phenyl H-6 and H-4), 7.31 (q,
J ¼ 7.2 Hz, 1H, phenyl H-5), 7.46 (dt, J ¼ 8.2 and 1.5 Hz, 1H, chroman
H-7), 7.68 (t, J ¼ 1.6 Hz, 1H, imidazole H-4), 7.71 (t, 1H, J ¼ 1.6 Hz,
imidazole H-5), 8.55 (dd, J ¼ 8.1 and 1.5 Hz, 1H, chroman H-5), 9.17
(s, 1H, imidazole H-2), 14.60 (br s, 1H, NHþ). 13C NMR (100 MHz,
C20H19ClN4O6: C, 53.76; H, 4.29; N,12.54. Found: C, 54.07; H, 4.22; N,
12.61.
6.1.2.3. (ꢃ)-(E)-2,3-Dihydro-3-(1H-imidazol-1-yl)-4H-1-benzopyran-
4-one O-[2-(3-chloro phenoxy) ethyl]oxime nitrate(7c). Yield 45%; mp
108e109 ꢂC; IR (KBr, cmꢁ1 nmax: 1598 (C]NO). 1H NMR (500 MHz,
)
DMSO-d6)
d: 4.37 (t, J ¼ 4.4 Hz, 2H, CH2), 4.54 (t, J ¼ 4.4 Hz, 2H, CH2),
4.7 (dd, J ¼ 12.5 and 3.0 Hz,1H, chroman H-2a), 5.01 (dd, J ¼ 12.5 and
3.6 Hz, 1H, chroman H-2b), 5.63 (dd, J ¼ 3.6 and 3.0 Hz, 1H, chroman
H-3), 6.96 (dd, J ¼ 8.3 and 1.9 Hz, 1H, chroman H-8), 7.00e7.11 (m,
4H, chroman H-6, phenyl H-2, H-4 and H-6), 7.30 (t, J ¼ 8.2 Hz, 1H,
phenyl H-5), 7.46 (dt, J ¼ 8.3 and 1.4 Hz, 1H, chroman H-7), 7.68 (t,
J ¼ 1.5 Hz, 1H, imidazole H-4), 7.71 (t, J ¼ 1.5 Hz, 1H, imidazole H-5),
8.54 (dd, J ¼ 8.0 and 1.4 Hz, 1H, chroman H-5), 9.17 (s, 1H, imidazole
DMSO-d6) d: 56.2 (C-3, chroman), 66.9 (CH2), 67.6 (CH2), 74.4 (C-2,
chroman), 102.6 (d, JC,F ¼ 24.8 Hz, C-2, phenyl), 107. 9 (d,
JC,F ¼ 21.1 Hz, C-4, phenyl), 111.5 (d, JC,F ¼ 2.6 Hz, C-6, phenyl), 113.9
(C-4a, chroman), 118.1 (C-8, chroman), 121.1 (C-6, chroman), 121.5
(C-5, imidazole), 122.3 (C-4, imidazole), 131.2 (d, JC,F ¼ 10.1 Hz, C-5,
phenyl), 131.9 (C-5, chroman), 133.8 (C-7, chroman), 135.8 (C-2,
imidazole), 142.3 (C-8a, chroman), 155.3 (C-4, chroman), 160.3 (d,
JC,F ¼ 11.1 Hz, C-1, phenyl), 163.4 (d, JC,F ¼ 241.5 Hz, C-3, phenyl). MS
(m/z, %): 367 (Mþ,14), 366 (15), 337 (18), 256 (14), 224 (80),197 (40),
180 (61), 152 (40), 126 (41), 95 (100), 69 (33). Anal. Calcd for
H-2), 14.50 (br s, 1H, NHþ). 13C NMR (100 MHz, DMSO-d6)
d: 56.2 (C-
3, chroman), 66.9 (CH2), 67.6 (CH2), 74.4 (C-2, chroman), 113.9 (C-4a,
chroman), 114.3 (C-6, phenyl), 115.0 (C-2, phenyl), 118.1 (C-8, chro-
man), 121.1 (C-6, chroman), 121.3 (C-5, imidazole), 121.5 (C-4, imid-
azole), 122.3 (C-4, phenyl), 131.4 (C-5, phenyl), 131.9 (C-5, chroman),
133.8 (C-7, chroman), 134.2 (C-3, phenyl), 135.8 (C-2, imidazole),
142.3 (C-8a, chroman), 155.3 (C-1, phenyl), 159.8 (C-4, chroman). MS
(m/z, %): 383 (Mþ, 2), 373 (77), 348 (22), 316 (22), 289 (19), 254 (20),
229 (21), 161 (55), 135 (100), 111 (33), 78 (42), 51 (20). Anal. Calcd for
C20H19FN4O6: C, 55.81; H, 4.45; N, 13.02. Found: C, 55.83; H, 4.53; N,
13.25.
6.1.2.6. (ꢃ)-(E)-2,3-Dihydro-3-(1H-imidazol-1-yl)-4H-1-benzopyran-
4-one O-[2-(2,4-dichloro phenoxy)ethyl] oxime nitrate (7f).
Yield 53%; mp 84e85 ꢂC; IR (KBr, cmꢁ1 nmax: 1606 (C]NO). 1H NMR
)
C
20H19ClN4O6: C, 53.76; H, 4.29; N, 12.54. Found: C, 53.70; H, 4.31; N,
(500 MHz, DMSO-d6)
d
: 4.43 (t, J ¼ 4.2 Hz, 2H, CH2), 4.56 (t,
12.60.
J ¼ 4.2 Hz, 2H, CH2), 4.69 (dd, J ¼ 12.5 and 3.0 Hz,1H, chroman H-2a),
5.00 (dd, J ¼ 12.5 and 3.4 Hz, 1H, chroman H-2b), 5.63 (dd, J ¼ 3.4
and 3.0 Hz, 1H, chroman H-3), 7.00e7.12 (m, 2H, chroman H-6 and
H-8), 7.23 (d, J ¼ 8.9 Hz,1H, phenyl H-6), 7.36 (dd, J ¼ 8.9 and 2.5 Hz,
1H, phenyl H-5), 7.47 (dt, J ¼ 8.5 and 1.3 Hz, 1H, chroman H-7), 7.56
(d, J ¼ 2.5 Hz,1H, phenyl H-3), 7.68 (s,1H, imidazole H-4), 7.72 (s,1H,
6.1.2.4. (ꢃ)-(E)-2,3-Dihydro-3-(1H-imidazol-1-yl)-4H-1-benzopyran-
4-one O-[2-(4-fluoro phenoxy) ethyl]oximenitrate (7d). Yield 44%; mp
120e121 ꢂC; IR (KBr, cmꢁ1 nmax: 1604 (C]NO). 1H NMR (500 MHz,
)
DMSO-d6)
d: 4.31 (t, J ¼ 4.5 Hz, 2H, CH2), 4.53 (t, J ¼ 4.5 Hz, 2H, CH2),

M. Babazadeh-Qazijahani et al. / European Journal of Medicinal Chemistry 76 (2014) 264e273
273
imidazole H-5), 8.57 (dd, J ¼ 8.0 and 1.3 Hz, 1H, chroman H-5), 9.19
Qazijahani (Faculty of Pharmacy, Mazandaran University of Medical
Sciences).
(s, 1H, imidazole H-2), 14.50 (br s, 1H, NHþ). 13C NMR (100 MHz,
DMSO-d6) d: 56.1 (C-3, chroman), 67.7 (CH2), 68.1 (CH2), 74.2 (C-2,
chroman), 113.9 (C-4a, chroman), 116.0 (C-6, phenyl), 118.1 (C-8,
chroman), 121.2 (C-6, chroman), 121.4 (C-5, imidazole), 122.2 (C-4,
imidazole), 123.0 (C-2, phenyl), 125.2 (C-5, phenyl), 128.6 (C-4,
phenyl), 129.8 (C-3, phenyl), 131.9 (C-5, chroman), 133.7 (C-7, chro-
man), 135.8 (C-2, imidazole), 142.5 (C-8a, chroman), 153.3 (C-1,
phenyl), 155.3 (C-4, chroman). MS (m/z, %): 418 (Mþ, 43), 382 (100),
256 (11), 212 (11), 188 (10), 161 (10), 146 (12), 94 (17), 67 (17). Anal.
Calcd for C20H18Cl2N4O6: C, 49.91; H, 3.77; N, 11.64. Found: C, 50.08;
H, 3.70; N, 11.76.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.02.019.
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The Lamarckian genetic algorithm (LGA) was used as a search
engine. The active site was defined using AutoGrid 4.2. The grid size
ꢀ
was set to 22 ꢄ 22 ꢄ 22 points with a grid spacing of 1.0 A centered
on the mass of original ligand in the crystal structure complex. A
maximum number of energy evaluations were set to 2,500,000. For
each of the 10 independent runs, a maximum number of 27,000
LGA operations were generated on a single population of 150 in-
dividuals. Operator weights for crossover, mutation, and elitism
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Acknowledgments
This work was supported by a grant from the Research Council
of Mazandaran University of Medical Sciences, Sari, Iran. A part of
this work was related to the Pharm.D thesis of Mojtaba Babazadeh-