Synthesis, structural studies and molecular modelling of a novel imidazoline derivative with antifungal activity

Article abstract of DOI:10.3390/molecules200814761

Six novel imidazoline derivatives were synthesized and tested in antifungal assays. One of the compounds, N-cyclohexyl-2-imino-3-(4-nitrophenyl)imidazolidine-1-carboxamide showed moderate activity against several clinical strains of Candida albicans. Its structure was solved by X-ray crystallography and its mode of action was deduced using molecular modelling. It was found to be similar to that of fluconazole. The potential for further optimization including SAR of the compound is briefly discussed.

Full text of DOI:10.3390/molecules200814761

Molecules 2015, 20, 14761-14776; doi:10.3390/molecules200814761
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis, Structural Studies and Molecular Modelling of a
Novel Imidazoline Derivative with Antifungal Activity
Tomasz M. Wróbel 1,*, Urszula Kosikowska 2, Agnieszka A. Kaczor 1,3, Sylwia Andrzejczuk 2,
Zbigniew Karczmarzyk 4, Waldemar Wysocki 4, Zofia Urbańczyk-Lipkowska 5, Maja Morawiak 5
and Dariusz Matosiuk 1
1
Department of Synthesis and Chemical Technology of Pharmaceutical Substances,
Medical University of Lublin, Chodzki 4a, 20-093 Lublin, Poland;
E-Mails: agnieszka.kaczor@umlub.pl (A.A.K.); dariusz.matosiuk@umlub.pl (D.M.)
2
Department of Pharmaceutical Microbiology, Medical University of Lublin,
Chodzki 1, 20-093 Lublin, Poland; E-Mails: urszula.kosikowska@umlub.pl (U.K.);
sylwia.andrzejczuk@umlub.pl (S.A.)
3
School of Pharmacy, University of Eastern Finland, Yliopistonranta 1, P.O. Box 1627,
FI-70211 Kuopio, Finland
4
Department of Chemistry, Siedlce University of Natural Sciences and Humanities, 3 Maja 54,
08-110 Siedlce, Poland; E-Mails: kar@uph.edu.pl (Z.K.); wwysocki@uph.edu.pl (W.W.)
5
Institute of Organic Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warszawa,
Poland; E-Mails: zofia.lipkowska@icho.edu.pl (Z.U.-L.); maja.morawiak@icho.edu.pl (M.M.)
* Author to whom correspondence should be addressed; E-Mail: tomasz.wrobel@umlub.pl;
Tel.: +48-81-448-7273; Fax: +48-81-448-7272.
Academic Editor: Jean Jacques Vanden Eynde
Received: 14 July 2015 / Accepted: 10 August 2015 / Published: 13 August 2015
Abstract: Six novel imidazoline derivatives were synthesized and tested in antifungal assays.
One of the compounds, N-cyclohexyl-2-imino-3-(4-nitrophenyl)imidazolidine-1-carboxamide
showed moderate activity against several clinical strains of Candida albicans. Its structure
was solved by X-ray crystallography and its mode of action was deduced using molecular
modelling. It was found to be similar to that of fluconazole. The potential for further
optimization including SAR of the compound is briefly discussed.
Keywords: antifungal; Candida albicans; X-ray analysis; 14-α-sterol demethylase; molecular
modelling; synthesis

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1. Introduction
Candida spp. encompasses a class of fungi that includes over 150 species. It exists harmlessly in the
body and is a component of the human mycobiome. The mycobiome, which refers to the fungal biota, by
interacting with other biomes, as well as with the host, contributes to the progression of fungus-associated
diseases and plays an important role in health and disease [1,2]. Fungi are also pathogens that can
affect different part of the body. They commonly infect immunocompromised patients, those undergoing
chemotherapy, infected with HIV or on immunosuppressive medicines after transplantation [3]. Many
serious infections also happen as a result of the development of a fungal biofilm [4]. Moreover fungi
cause infections of the skin, hair and nails which are source of substantial discomfort for patients [5,6].
Invasive fungal infections (IFIs) are the most serious type in which brain, heart, blood or other important
parts of the body are infected [7,8]. These present the biggest challenge especially in the light of increased
occurrence over the past decades and the urgent need for more effective drugs [9]. It is clear that more
basic research on new antifungal drugs is needed with emphasis on structural biology, homology modelling
and virtual imaging to drive discovery [10].
On the other hand protection of natural microflora, mainly intestinal microbiota should not be neglected.
Microorganisms colonizing the human gut are responsible for crucial functions to our health such as
elimination of potentially toxic substances or preventing uncontrolled growth of pathogens [11]. It was
recently demonstrated that treatment with cyclophosphamide, a drug commonly used in chemotherapy
benefits from certain bacteria from the intestinal flora by stimulating fresh immune defense mechanisms [12].
During the course of another investigation [13] we studied novel urea derivatives with antiproliferative
properties. Having accounted for their cytotoxicity and consequently the need for protection of the
natural microbiological flora we deemed it necessary to check how they influence survival of different
microorganisms. We were particularly interested in assessing antifungal activity because of the similarity
to imidazole drugs currently in clinical use.
2. Results and Discussion
2.1. Chemistry
The compounds presented in this manuscript were synthesized as outlined in Scheme 1. The synthesis
started with commercially available 1-chloro-4-nitrobenzene (1) which when refluxed with ethylenediamine
gave very good yield of N1-(4-nitrophenyl)ethane-1,2-diamine (2). This product was cyclized using
cyanogen bromide to afford 1-(4-nitrophenyl)imidazolidin-2-imine (3). The last step involved reacting 3
with a variety of isocyanates (Table 1). In most cases a mixture of two regioisomers was obtained, with
compounds 4 being the major products. This is probably due to steric hindrance and greater nucleophilicity
of the ring nitrogen. This corresponds well with a previous report indicating delocalization of electrons
over guanidinium fragment and shortening of the bond between carbon and the ring nitrogen [14]. The
obtained regioisomers were virtually indistinguishable by NMR spectroscopy due to their very similar
1H-NMR spectra. Additionally, the lack of expected correlation between methylene hydrogens and
carbonyl carbon in compounds 4 made this vital clue unavailable. This was confirmed in compound 4f,
whose structure was solved by X-ray crystallography and the measured HMBC spectra did not reveal
H4-C7 coupling even using a range of 3J setting (Figure 1). The position of N-H signals which are most

Molecules 2015, 20
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1
downfield in the H-NMR spectra may be characteristic for compounds 4. These signals were shifted
beyond or close to 10 ppm, but no consistent pattern was revealed in relation to compounds 5. However,
taking into consideration the fact that these are of poor diagnostic value we relied on X-ray data for
absolute certainty (Figure 2). Compounds 5 can exist in two main tautomeric forms, as imidazolidine or as
imidazoline derivatives, however X-ray analysis of another reaction product where R = phenyl suggests
preference of the former in the solid state [13].
Table 1. Isocyanates and substituents in target compounds.
Compound
R
a
Cl
b
Cl
Cl
H2
C
c
H2
C
C
H2
d
H2
C
e
f
OCH3
NH
N
NH2
Cl
HN
N
N
(i)
(ii)
NH2
NH
NO2
NO2
O2N
O2N
1
2
3
(iii)
O
R
N
NH
N
N
H
O
O
N
N
N
NH
N
N
H
+
R
R
N
H
N
H
O2N
O2N
O2N
4a-f
5a-f
Scheme 1. Synthetic sequence. Reagents and conditions: (i) NH2CH2CH2NH2, reflux; (ii) 1.
BrCN, iPrOH, reflux; 2. NaOH; (iii) RNCO, rt.

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NO2
N
N
NH
NH
H
O
X
Figure 1. HMBC spectrum of compound 4f shows absence of expected three-bond correlation
between methylene hydrogens H4 and carbonyl carbon C7.
Figure 2. A view of the molecule 4f with the atom labelling. Non-H atoms are represented
by displacement ellipsoids at the 30% probability level.
2.2. Microbiology
Compound 4f at 1000 mg/L concentration showed potential activity against some Candida species.
On the basis of the MIC values it was shown that compound 4f had strain-dependent activity against
both reference strains of Candida albicans (MIC = 250–1000 mg/L) and C. parapsilosis (MIC = 500 mg/L).

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Antifungal activity against clinical isolates of C. albicans was within MIC values ranged from 0.98
to ≥1000 mg/L (Table 2). Clotrimazole used as a reference compound had higher activity against the
reference strains (MIC = 1.95–3.9 mg/L) of Candida spp. and against clinical isolates of C. albicans
(MIC = 0.06–7.8 mg/L). The activity assessed on the basis of MIC50 = 250 mg/L and MIC90 = 1000 mg/L
values for 4f was lower in comparison to clotrimazole activity with MIC50 = 0.24 mg/L and
MIC90 = 1.95 mg/L against C. albicans clinical isolates.
Table 2. Activity of 4f and clotrimazole against clinical strains of Candida albicans detected
on the basis of minimal inhibitory concentration (MIC, mg/L) values.
4f
Clotrimazole
MIC Value
No. of Strains
No. of Strains
% of Strains MIC50 MIC90
% of Strains MIC50 MIC90
(n = 41)
(n = 31)
0.015
0.03
0.06
0.12
0.24
0.49
0.98
1.95
3.9
0 (0.0)
0
0
3
5
2
3
5
5
3
2
1
2
0
0
0
0
0
0
0
0
9.7
16.1
6.5
0
0
0
0
0
1
1
1
2
1
2
3
7
5
10
7
1
0
0
9.7
0
16.1
16.1
9.7
0
2.4
2.4
2.4
6.5
3.2
250
1000
0.24
1.95
7.82
15.63
31.25
62.5
125
4.9
2.4
6.5
0
4.9
0
7.3
0
17.1
12.2
24.4
17.1
2.4
0
250
0
500
0
1000
>1000
0
0
2.3. X-ray Analysis
A detailed X-ray analysis of compound 4f was performed in order to confirm the synthesis pathway
and identification of its tautomeric form in the solid state. Compound 4f crystallizes as isopropanol
solvate with one solvent molecule in the asymmetric unit (Figure 3). The bond lengths and angles
are within the normal ranges [15] and are comparable to those found in closely related structures
1-(3-chlorophenyl)-3-(1-p-tolylimidazolidin-2-ylidene)urea [16] and 2-(methoxyimino)-N-((4-methyl-
phenyl)-sulfonyl)imidazolidine-1-carboxamide monohydrate [17]. The bond length of C7=O8, 1.229(3) Å
is characteristic of that for the normal C=O double bond (1.230(7) Å in ureas [15]). This value and the
positions of the H atoms in the vicinity of the N6 and N9 atoms in the difference electron-density map
indicate that compound 4f exists in the N6-imine/O8-keto tautomeric form in the crystalline state. The
guanidine-urea moiety of 4f is slightly distorted from planarity with maximum deviation from its mean
plane of 0.0661(14) Å for the N1 atom.

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Figure 3. Unit-cell packing of the compound 4f. H atoms not involved in hydrogen bonding
have been excluded.
The conformation of this system is stabilized by an intramolecular N9–H9···N6 hydrogen bond
(Table 3). The nitro group in p-nitrophenyl substituent is almost coplanar with the benzene ring which
is shown by the torsion angle C23–C24–N27–O28 of 11.1(4)o, while the benzene and imidazoline rings
are twisted of each other with the dihedral angle of 31.79(15)°. The imidazoline ring adopts an envelope
conformation, with puckering parameters of Q = 0.252(3) Å and φ = 140.4(7)° [18]. The cyclohexane
ring is disordered in two chair conformations A and B (Figure 3), which is confirmed by the puckering
parameters of Q = 0.536(9) Å and θ = 8.7(9)° for ring A and Q = 0.57(2) Å and θ = 15.1(2)° for ring B.
Table 3. Hydrogen bonding geometry (Å, °) for compound 4f.
D–H···A
D–H
0.86(4)
0.93
H···A
2.57(4)
2.56
D···A
D–H···A
139(3)
164
Symmetry Codes
i = x, y, z
N6–H6···O44A
C22–H22···O44A
N9–H9···N6
3.263(11)
3.462(15)
2.692(3)
ii = 1 − x, 1 − y, −z
0.94(4)
1.88(4)
143(4)
The gauche conformation of the cyclohexane ring with respect to the urea moiety is described by
the torsion angle C7–N9–C31–C36 of 87.1(5) and 106.3(9)° for rings A and B respectively. In the crystal
structure, the molecules of 4f interact with each other by van der Waals forces but in addition each of the
these molecules is linked with two molecules of isopropanol solvent via the intermolecular N6–H6···O44A
and C22–H22···O44A hydrogen bonds (Table 3, Figure 4).

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Figure 4. Molecular interactions of 4a and 4f with cytochrome P450 14-α-sterol demethylase
(homology model) from Candida albicans. (A) comparison of binding poses of fluconazole
(yellow carbon atoms), inactive 4a (cyan carbon atoms) and active 4f (magenta carbon atoms).
Protein shown in cartoon representation, ligands shown as sticks. Non-polar hydrogen atoms
omitted for clarity; (B) 2D map of binding site for 4f; (C) details of binding site of 4f. Ligand
and protein shown as sticks with magenta and grey carbon atoms, respectively. Non-polar
hydrogen atoms omitted for clarity; (D) 2D map of the binding site of 4a; (E) details of binding
site of 4a. Ligand and protein shown as sticks with cyan and grey carbon atoms, respectively.
Hydrogen bonds shown as red dashed lines. Non-polar hydrogen atoms omitted for clarity.
2.4. Molecular Modelling
It is a well-known fact that imidazole derivatives, such as fluconazole exert their pharmacological
activity through inhibition of enzyme cytochrome P450 14-α-sterol demethylase from C. albicans. Thus,

Molecules 2015, 20
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this mechanism of action has already been suggested for similar compounds despite a lack of experimental
verification of this hypothesis [19].
As the crystal structure of cytochrome P450 14-α-sterol demethylase from C. albicans is not available,
the homology model of this enzyme was constructed using cytochrome P450 14-α-sterol demethylase
from Mycobacterium tuberculosis in complex with fluconazole (PDB ID: 1EA1) [20] as a template with
sequence identity 28.97%. The model is similar to earlier published models [21–23].
Molecular docking allowed to conclude that the binding pocket occupied by 4f partially overlaps with
the one occupied by fluconazole. The overview of potential inhibitor-enzyme complex is shown in
Figure 4A. It can be seen that the position of the active compound 4f is different than this of the inactive
compound 4a. The details of the binding pocket are presented in Figure 4B,C for 4f and in Figure 4D,E
for 4a. Similarly for fluconazole in the respective crystal structure of the template, no hydrogen bond
interactions were found for 4f in the binding site of the enzyme. Except for Thr311 and Ser312, only
non-polar amino acids are present in the enzyme binding site, including aromatic Phe145, Phe463 and
Phe475 as well as aliphatic Ala149, Leu150, Leu204, Ile304, Gly307, Gly308, Cys370, Met374,
Leu376, Ile379, Pro462, Gly464, Gly472 and Ala476. The stability of the inhibitor-enzyme complex
was confirmed in molecular dynamics simulations. The complex of compound 4f with cytochrome P450
14-α-sterol demethylase was stable during 100 ns simulations as indicated by decreasing value of complex
potential energy (Figure 5A) and complex RMSD (Figure 5B). The stability of selected potential inhibitor
binding pose is confirmed by ligand RMSD value below 3 Å Figure 5C). RMSD values were measured
in comparison to initial position.
Figure 5. Cont.

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Figure 5. Molecular dynamics simulations results for 4f in complex with P450 14-α-sterol
demethylase (homology model) from Candida albicans. (A) changes of potential energy for
inhibitor-enzyme complex during 100 ns simulations; (B) changes in complex RMSD during
100 ns simulations; (C) changes in ligand RMSD during 100 ns simulations.
3. Experimental Section
3.1. Synthesis
All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without further
purification unless stated otherwise. NMR spectra were recorded on Fourier 300 and AVANCE III
600 MHz spectrometers (Bruker, Fallanden, Switzerland). Solvents for NMR spectroscopy were obtained
from Armar Chemicals (Dottingen, Switzerland). Abbreviations used in NMR spectra s- singlet, d- doublet,
t- triplet, q- quarter, m- multiplet, br- broad. MS spectra were acquired on a Bruker microTOF-Q II
spectrometer. TLC analysis was carried out on HPTLC Silica gel 60 F254 plates (Merck, Darmstadt,
Germany) prewashed with methanol.
N1-(4-Nitrophenyl)ethane-1,2-diamine (2). 1-Chloro-4-nitrobenzene (1, 10 g, 0.06 mol) and ethylenediamine
(40 g, 0.6 mol) were refluxed together for 90 min. Excess ethylenediamine was distilled and the remaining
residue was dissolved in hot water, filtered while hot and allowed to cool. Crystals formed very quickly.
They were filtered, washed with water and air dried. Thus 9.6 g (83%) of orange solid was obtained. All
1
analytical data corresponds to that found in the literature [24] m.p. 148–150 °C. H-NMR (300 MHz,
DMSO-d6) δ 7.89–8.07 (m, 2H), 7.30 (br t, J = 4.66 Hz, 1H), 6.56–6.72 (m, 2H), 3.13 (q, J = 6.08 Hz,
2H), 2.73 (t, J = 6.29 Hz, 2H), 1.60 (brs, 2H); 13C-NMR (75 MHz, DMSO-d6) δ 155.2, 135.9, 126.7, 111.1,
46.5, 41.1.
1-(4-Nitrophenyl)imidazolidin-2-imine (3). Cyanogen bromide (3.18 g, 30 mmol) dissolved in iPrOH
(50 mL) was added dropwise to N-(4-nitrophenyl)ethane-1,2-diamine (2, 5.44 g, 30 mmol) suspended
in iPrOH (50 mL). During addition the amine went into solution and colour turned orange-brown. The
reaction was heated under reflux for 90 min and then the solvent was removed on a rotavap. The yellow
residue was recrystallized from EtOH to afford 7.3 g (85%) of yellow solid as a hydrobromide salt. The
salt was converted to a free base form by dissolving it in warm water, adding to 5% NaOH (2.5 eq) and
filtering the resulting precipitate. Free base 1H-NMR (300 MHz, DMSO-d6) δ 8.09–8.18 (m, 2H), 7.96–8.07

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(m, 2H), 6.06 (brs, 2H), 3.81–3.94 (m, 2H), 3.35–3.43 (m, 2H); 13C-NMR (75 MHz, DMSO-d6) δ 158.5,
148.2, 140.0, 124.9, 117.1, 47.5, 40.0.
General Procedure to Obtain Compounds 4
To a stirred mixture of 1 eq. of 1-(4-nitrophenyl)imidazolidin-2-imine (3) in dichloromethane (DCM),
tetrahydrofuran (THF) or acetonitrile (ACN) there was added a solution of 1 eq. of an isocyanate in
the same solvent. The mixture was stirred for 2 h at room temperature and resulting precipitate filtered
(reaction mixture becomes clear as soon as the isocyanate is added and then starts turning cloudy and
opaque as it progresses) or the solvent was evaporated.
N-(3-Chlorophenyl)-2-imino-3-(4-nitrophenyl)imidazolidine-1-carboxamide (4a). THF was used as solvent.
The formed precipitate was filtered and washed with THF. 1H-NMR (600 MHz, CDCl3) δ = 12.28 (brs,
1H), 8.39–8.36 (m, 2H), 7.69 (t, J = 2.1 Hz, 1H), 7.41 (ddd, J = 0.8, 2.1, 8.2 Hz, 1H), 7.39–7.36 (m, 2H),
7.25 (t, J = 8.0 Hz, 1H), 7.06 (ddd, J = 0.8, 2.0, 8.0 Hz, 1H), 6.46 (brs, 1H), 4.16–4.11 (m, 2H), 3.99–3.91
(m, 2H); 13C-NMR (151 MHz, CDCl3) δ = 152.48, 151.24, 144.93, 144.18, 139.59, 134.60, 129.91, 125.81,
123.41, 120.78, 119.67, 117.60, 44.75, 40.66; HRMS (ESI) [M + H]+ calc. 360.0858 exp. 360.0855.
N-(3,4-Dichlorophenyl)-2-imino-3-(4-nitrophenyl)imidazolidine-1-carboxamide (4b). THF was used as
1
solvent. The formed precipitate was filtered and washed with THF. H-NMR (600 MHz, CDCl3)
δ = 12.35 (brs, 1H), 8.41–8.36 (m, 2H), 7.81 (d, J = 2.6 Hz, 1H), 7.40–7.36 (m, 4H), 6.46 (brs, 1H),
4.15–4.11 (m, 2H), 3.98–3.93 (m, 2H); 13C-NMR (151 MHz, CDCl3) δ = 152.51, 151.22, 144.86, 144.30,
138.02, 132.72, 130.47, 126.43, 125.87, 121.21, 120.88, 118.87, 44.81, 40.68; HRMS (ESI) [M + H]+
calc. 394.0468 exp. 394.0468.
N-Benzyl-2-imino-3-(4-nitrophenyl)imidazolidine-1-carboxamide (4c). THF was used as solvent and
removed on a rotavap when the reaction was finished. The residue was recrystallized repeatedly from
iPrOH. 1H-NMR (600 MHz, CDCl3) δ = 10.10 (brs, 1H), 8.33 (d, J = 9.1 Hz, 2H), 7.42–7.24 (m, 7H), 6.30
(s, 1H), 4.57 (d, J = 5.8 Hz, 2H), 4.12–4.04 (m, 2H), 3.94–3.85 (m, 2H); 13C-NMR (151 MHz, CDCl3)
δ = 152.48, 151.24, 144.93, 144.18, 139.59, 134.60, 129.91, 125.81, 123.41, 120.78, 119.67, 117.60,
44.75, 40.66; HRMS (ESI) [M + H]+ calc. 340.1404 exp. 340.1401.
2-Imino-3-(4-nitrophenyl)-N-(2-phenylethyl)imidazolidine-1-carboxamide (4d). ACN was used as solvent
and removed on a rotavap when the reaction was finished. The residue was recrystallized repeatedly
1
from iPrOH. H-NMR (600 MHz, CDCl3) δ = 9.71 (brs, 1H), 8.37–8.28 (m, 2H), 7.36–7.20 (m, 7H),
6.23 (s, 1H), 4.08–4.00 (m, 2H), 3.91–3.83 (m, 2H), 3.65–3.57 (m, 2H), 2.91 (t, J = 7.3 Hz, 2H);
13C-NMR (151 MHz, CDCl3) δ = 154.04, 152.56, 145.36, 143.75, 139.29, 128.83, 128.48, 126.34,
125.69, 120.40, 44.91, 41.58, 40.72, 36.29; HRMS (ESI) [M + H]+ calc. 354.1561 exp. 354.1558.
2-Imino-N-[(4-methoxyphenyl)methyl]-3-(4-nitrophenyl)imidazolidine-1-carboxamide (4e). DCM was
used as solvent and removed on a rotavap when the reaction was finished. The residue was recrystallized
repeatedly from iPrOH. 1H-NMR (600 MHz, CDCl3) δ = 10.01 (brs, 1H), 8.36–8.28 (m, 2H), 7.35–7.29
(m, 4H), 6.93–6.85 (m, 2H), 6.27 (brs, 1H), 4.49 (d, J = 5.6 Hz, 2H), 4.09–4.03 (m, 2H), 3.90–3.86 (m,
13
2H), 3.81 (s, 3H); C-NMR (151 MHz, CDCl3) δ = 158.80, 154.04, 152.58, 145.32, 143.76, 131.11,

Molecules 2015, 20
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128.83, 125.68, 120.40, 113.99, 55.31, 44.92, 43.43, 40.78; HRMS (APCI) [M + H]+ calc. 370.1510
exp. 370.1514.
N-Cyclohexyl-2-imino-3-(4-nitrophenyl)imidazolidine-1-carboxamide (4f). THF was used as solvent.
The reaction mixture was filtered through a plug of silica and eluted with fresh THF. The eluant was
stripped of solvent and recrystallized repeatedly from iPrOH. 1H-NMR (600 MHz, CDCl3) δ = 9.64 (br
d, J = 6.9 Hz, 1H), 8.35–8.31 (m, 2H), 7.33 (d, J = 9.0 Hz, 2H), 6.27 (brs, 1H), 4.07–4.02 (m, 2H), 3.90–3.85
(m, 2H), 3.84–3.76 (m, 1H), 2.00–1.91 (m, 2H), 1.76–1.68 (m, 2H), 1.63–1.56 (m, 1H), 1.47–1.38 (m,
2H), 1.38–1.23 (m, 3H); 13C-NMR (151 MHz, CDCl3) δ = 153.14, 152.73, 145.43, 143.71, 125.68, 120.35,
48.65, 44.90, 40.73, 33.15, 25.71, 24.59; HRMS (ESI) [M + H]+ calc. 332.1717 exp. 332.1713.
3.2. Microbiology
The antimicrobial activity of six tested compounds was screened using the reference strains of bacteria
and fungi. The reference strains of Gram-positive bacteria from American Type Culture Collection (ATCC;
LGC Standards, London, UK) included Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis
ATCC 12228, Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 10876, Micrococcus luteus ATCC
10240, while from MICROBANK collection (National Institute of Hygiene—NIH, Warsaw, Poland)—
Staphylococcus aureus MICROBANK 14001 (MRSA—Methicillin Resistant Staphylococcus aureus) were
used. The panel of Gram-negative bacteria contained Escherichia coli ATCC 25922, Klebsiella pneumoniae
ATCC 13883, Proteus mirabilis ATCC 12453, Pseudomonas aeruginosa ATCC 9027. Moreover, the
yeasts belonging to Candida spp.—3 reference strains (C. albicans ATCC 2091, C. albicans ATCC 10231,
and C. parapsilosis ATCC 22019) and 41 clinical isolates of C. albicans from the Department of
Pharmaceutical Microbiology of Medical University of Lublin collection were used.
Antimicrobial activity was detected according to the method described previously [25] using microbial
suspensions in sterile 0.85% NaCl standardized with an optical density of 0.5 McFarland standard—
150 × 106 CFU/mL. All stock solutions of the tested compounds were prepared in DMSO (Sigma-Aldrich).
Mueller-Hinton agar buffered at pH 7.2 (for bacteria) or Mueller-Hinton agar supplemented with 2%
glucose and buffered at pH 5.6 (for fungi) was used for examination of antibacterial or antifungal activity.
Ciprofloxacin and clotrimazole (both from Sigma-Aldrich Co.) were used as reference antibacterial and
antifungal agents, respectively.
Initially, the in vitro antimicrobial potencies of all tested compounds were screened using the agar
dilution method on the basis of the microbial growth inhibition on the agar medium to which the tested
compounds were added at concentrations of 1000 mg/L. 10 μL of each microbial suspension was put onto
the prepared solid media. The plates were pre-incubated at room temperature for 1–1.5 h and then were
incubated at 35 °C for 18 h for bacteria and for 24–48 h for yeasts. The medium with DMSO at the final
concentration and without the tested compounds served as a negative control; no microbial growth
inhibition was observed.
Subsequently, for the compound 4f showing an inhibitory effect on the visible growth of some tested
bacteria and yeasts, MIC values were estimated using the broth microdilution technique, according to
the Clinical and Laboratory Standards Institute (CLSI) standards for bacteria and yeasts, with some
modification described previously [25]. The MIC values, defined as the lowest concentration of a compound

Molecules 2015, 20
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that prevents visible growth of the tested microorganisms, were determined after 24 h by comparison
with the microbial growth in control (compound free) medium.
The microdilution technique was developed using sterile 96-well microplates (Nunc, Roskilde,
Denmark), which were inoculated with 2 µL of the microbial suspension (0.5 McFarland standard) put
into 198 µL of Mueller Hinton broth without (for bacteria) or with 2% glucose (for fungi) and buffered
at pH 7.2 or at pH 5.6, respectively. The two-fold dilution of the tested compound at final concentration
from 0.0019 to 1000 mg/L was added to the broth medium. The medium without the tested compound was
used as negative control. The optical density (OD600) measurements were determined spectrophotometrically
(ELx800 microreader, BioTek Instruments, Inc., Winooski, VT, USA) after incubation at 35 °C for 24 h.
The blank control wells with two-fold dilution of each of the tested compounds added to broth medium (total
volume—200 µL) without the bacteria or yeast suspension were incubated under the same conditions.
3.3. X-ray Analysis
Straw-yellow needle-like crystals, suitable for X-ray diffraction analysis, were obtained from an
isopropanol solution upon slow evaporation at room temperature. X-ray data of 4f was collected on a
Bruker SMART APEX II CCD diffractometer using CuKα (λ = 1.54178 Å) radiation; crystal sizes
0.26 × 0.25 × 0.18 mm, ω scans, T = 142 K, absorption correction: multi-scan SADABS [26],
Tmin/Tmax = 0.835/0.883. The structure was solved by direct methods using SHELXS-2013/1 [27] and
refined by full-matrix least-squares with SHELXL-2014/7 [27]. The C32, C33, C35 and C36 atoms in
the cyclohexane ring and the C41, C43 and O44 atoms of the isopropanol molecule were disordered over
two positions with a ratio of occupancy of 0.63(1):0.37(1). The N-bond H atoms were located by
difference Fourier synthesis and refined freely. The remaining H atoms were positioned geometrically and
treated as riding on their parent C atoms with C–H distances of 0.98 Å (methine), 0.97 Å (methylene),
0.96 Å (methyl), 0.93 Å (aromatic) and 0.82 Å (hydroxyl). All H atoms were refined with isotropic
displacement parameters taken as 1.5 times those of the respective parent atoms. All calculations
were performed using WinGX v. 2014.1 package [28]. CCDC 1412468 contains the supplementary
crystallographic data for this paper. These data can be obtained free of charge from The Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk.
1
Crystal data of 4f: C16H21N5O3 C3H7OH, M = 364.44, triclinic, space group P , a = 9.0739(3),
b = 9.5028(3), c = 14.0775(4) Å, α = 78.537(2), β = 71.537(2), γ = 65.896(2)°, V = 1047.61(6) Å3, Z = 2,
Dcalc = 1.241 gcm−3, F(000) = 420, μ (CuKα) = 0.727 mm−1, T = 142 K, 6787 measured reflections
(θ range 5.12–66.58°), 3265 unique reflections (Rint = 0.0388) final R = 0.0576, wR = 0.1772, S = 1.063
for 2336 reflections with I > 2σ(I).
3.4. Molecular Modelling
The X-ray structure of cytochrome P450 14-α-sterol demethylase from Mycobacterium tuberculosis
in complex with fluconazole (PDB ID: 1EA1) [20] was used as a template. Sequence alignment was
performed with Muscle software [29]. Homology modelling was carried out using Modeller v.9.14 [30].
A hundred homology models of cytochrome P450 14-α-sterol demethylase from Candida albicans in
complex with fluconazole were generated, and subsequently assessed by Modeller objective function and
Discrete Optimized Protein Energy profiles [31]. The best model was subjected to quality assessments

Molecules 2015, 20
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using the Schrödinger software suite [32] tool for Ramachandran plots. Chimera software v. 1.5.3 [33]
was used for visualization of results.
Molecular docking was performed using Glide from the Schrödinger software suite [32]. The grid file
was generated indicating fluconazole as a reference ligand. The structure of 4a was modelled using
LigPrep protocol from Schrödinger software suite. Crystal structure of 4f was used as input ligand
conformation. Molecular docking of compound 4a and 4f was performed using the SP (standard precision)
protocol of Glide. 50 poses were generated for the ligand. PyMol v. 0.99 [34] was used for visualization
of results.
Molecular dynamics studies of the ligand-receptor complex was performed using Desmond
v. 3.0.3.1 [35]. The complex was hydrated and ions were added to neutralize protein charges and then to
the concentration of 0.15 M NaCl. The complex was minimized and subjected to MD first in the NVT
ensemble for 1 ns and then in NPT ensemble for 20 ns with the restrictions on the protein backbone in
each case. The production run was performed in NPT ensemble with no restrictions for 100 ns. Analysis
of molecular dynamics simulations was performed with the Schrödinger software suite tools [32].
4. Conclusions
Six novel imidazoline derivatives were successfully synthesized and examined for their antifungal
activity. All compounds showed no activity in tested assays except compound 4f which exhibited
strain-depending activity against C. albicans.
The Candida fungi, mainly C. albicans, can be opportunistic etiological factors of invasive or
non-invasive infections. These yeasts pose the greatest threat especially in immunocompromised and/or
hospitalized patients with serious underlying diseases. The search for new compounds active against
opportunistic fungi is important because agents available today are characterized by their high toxicity,
many side effects, undesired drug interactions, limited routes of administration and very varied activity.
Compound 4f with antifungal activity against Candida spp. therefore possesses potential for further
optimization especially since it constitutes a novel structure. Molecular modelling suggested a similar
mode of action to that of fluconazole. It can be envisioned that by replacing the saturated imidazoline
ring with aromatic imidazole activity could be enhanced. Furthermore the presence of the cyclohexyl
substituent is likely to be the key element for the antifungal activity of this compound, as compounds
without the cyclohexyl functionality were inactive. Any further modification would need to take into
account fact that the majority of amino acids in the binding site are non-polar. This suggests incorporating
structural features that lack possibility to form hydrogen bonds.
Acknowledgments
The paper was developed using equipment purchased within the project “The equipment of innovative
laboratories doing research on new medicines used in the therapy of civilization and neoplastic diseases”
within the Operational Program Development of Eastern Poland 2007–2013, Priority Axis I modern
Economy, operations I.3 Innovation promotion. Some of the calculations were performed under a
computational grant by Interdisciplinary Center for Mathematical and Computational Modelling (ICM),
Warsaw, Poland, grant number G30-18 and under resources and licenses by CSC, Finland. Many thanks
to Scott Lowe for proof reading the manuscript.

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Author Contributions
T.M.W. prepared the manuscript, designed the research, performed synthesis and spectral analysis;
U.K. and S.A. performed microbiological assays; A.A.K. performed molecular modelling; Z.K., W.W.,
Z.U.-L. and M.M. performed X-ray analysis, D.M. provided scientific support.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compound 4f are available from the authors.
© 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
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(http://creativecommons.org/licenses/by/4.0/).