Synthesis, antimicrobial activity, and molecular docking study of fluorine-substituted indole-based imidazolines

Article abstract of DOI:10.1007/s00044-018-2177-x

A series of 2- or 3-(4,5-dihydro-1H-imidazol-2-yl)-1H-indole derivatives were synthesized, characterized, and evaluated for their in vitro antibacterial and antifungal activities. Additionally, the synthesized compounds were docked into the II DNA gyrase B active site, and their predicted binding modes were inspected. Inhibitory activity were tested against two species of Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa), two species of Gram-positive bacteria (Staphylococcus aureus, Listeria monocytogenes) and two fungi (Candida albicans, Aspergillus niger) using the broth microdilution method. The fluorine-substituted 2-(2-imidazolyl)indole 2b was found to be the most potent antibacterial compound against E. coli and S. aureus strains (MIC value 80 μg/mL). Compounds showed better activity against Gram-positive bacteria compared to Gram-negative bacteria. The docking results predicted that the imidazoline-indole hybrid moiety bind to the active site protein ATP-binding pocket from E. coli and S. aureus with good interaction energy scores. The significant loss of antibacterial activity for some imidazoline-indole analogs could be attributed to several nonoptimal enzyme interactions, including poor hydrogen bonds provided by Asp73 (E. coli gyrase numbering) or Asp81 (S. aureus gyrase numbering) and an associated water molecule.

Full text of DOI:10.1007/s00044-018-2177-x

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-018-2177-x
ORIGINAL RESEARCH
Synthesis, antimicrobial activity, and molecular docking study of
fluorine-substituted indole-based imidazolines
Humberto L. Mendoza-Figueroa1,2 Maria Trinidad Serrano-Alva1,2 Gerardo Aparicio-Ozores3
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Gelacio Martínez-Gudiño1,2 Oscar R. Suárez-Castillo4 Nadia A. Pérez-Rojas1 Martha S. Morales-Ríos
1,2
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Received: 8 February 2018 / Accepted: 26 March 2018
© Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
A series of 2- or 3-(4,5-dihydro-1H-imidazol-2-yl)-1H-indole derivatives were synthesized, characterized, and evaluated for
their in vitro antibacterial and antifungal activities. Additionally, the synthesized compounds were docked into the II DNA
gyrase B active site, and their predicted binding modes were inspected. Inhibitory activity were tested against two species of
Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa), two species of Gram-positive bacteria (Staphylo-
coccus aureus, Listeria monocytogenes) and two fungi (Candida albicans, Aspergillus niger) using the broth microdilution
method. The fluorine-substituted 2-(2-imidazolyl)indole 2b was found to be the most potent antibacterial compound against
E. coli and S. aureus strains (MIC value 80 μg/mL). Compounds showed better activity against Gram-positive bacteria
compared to Gram-negative bacteria. The docking results predicted that the imidazoline-indole hybrid moiety bind to the
active site protein ATP-binding pocket from E. coli and S. aureus with good interaction energy scores. The significant loss of
antibacterial activity for some imidazoline-indole analogs could be attributed to several nonoptimal enzyme interactions,
including poor hydrogen bonds provided by Asp73 (E. coli gyrase numbering) or Asp81 (S. aureus gyrase numbering) and
an associated water molecule.
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Keywords Imidazole Indole Tautomers Antibacterial activity Docking studies Gyrase B
Introduction
several biologically active compounds such as tryptophan,
tryptamine, indoleacetic acid and is a subunity of many
terrestrial and marine alkaloids. In particular, fluorine-
containing indole derivatives have been described as phy-
siologically active compounds having applications such as
antiviral (Zoidis et al. 2016), antibacterial and antifungal
(Joshi and Joshi 2000; KumarMittal et al. 2011; Lei et al.
2011; Lee et al. 2012; Tejpal-Singh et al. 2016), and anti-
histamine (Fonquerna et al. 2004). No single factor
accounts for the beneficial pharmaceutical effects observed
with fluorine substitution, in part, because fluorine affects
many molecular properties that influence drug function and
efficacy, e.g., lipophilicity, bioavailability, chemical stabi-
lity, metabolic reactivity, and receptor binding (Smart
2001).
Owing to their widespread occurrence and pharmaceu-
tical potential, 5-membered heterocyclic scaffolds with
more than one heteroatom have been an attractive target for
organic and medicinal chemists. In particular, in recent
years, imidazoline scaffold is reported to be physiologically
and pharmacologically active in wide range of therapeutic
applications. It is therefore considered an important
Indole is one of the most ubiquitous heterocycles in the
microorganisms, plant, and animal kingdom; it is present in
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00044-018-2177-x) contains supplementary
material, which is available to authorized users.
* Martha S. Morales-Ríos
smorales@cinvestav.mx
1
Departamento de Química, Centro de Investigación y de Estudios
Avanzados del Instituto Politécnico Nacional, Apartado 14-740,
Mexico City 07000, Mexico
2
Programa de Posgrado en Farmacología, Centro de Investigación y
de Estudios Avanzados del Instituto Politécnico Nacional,
Apartado 14-740, Mexico City 07000, Mexico
3
Departamento de Microbiología, Escuela Nacional de Ciencias
Biológicas, Instituto Politécnico Nacional, Apartado 42-186,
Mexico City 11340, Mexico
4
Área Académica de Química, Universidad Autónoma del Estado
de Hidalgo, Mineral de la Reforma, Hidalgo 42184, Mexico

Medicinal Chemistry Research
pharmacophore of medicinal relevance. Various imidazo-
lines arylamino substituted (Servi et al. 2005) and hybrid
molecules containing indole-based imidazoline scaffolds
find use for the design of antimicrobial agents. Further,
imidazolines as well as indoles are utilized as pharmaco-
phores because of their favorable metabolic profile and
ability to engage in hydrogen bonding (Panchal et al. 2009).
The emergence and dissemination of Gram-positive and
Gram-negative bacteria that are resistant to almost all cur-
rently available antimicrobials are a major concern of public
health system worldwide (WHO 2014), triggering a new
wave of drug discovery efforts. One of the researched
therapeutic targets in the field of antibacterial discovery is
DNA Gyrase (Gyr), an essential enzyme for nucleic acids
synthesis and bacterial growth. This enzyme has no direct
counterpart in mammals and consequently it represents an
attractive antibacterial target. Structurally the enzyme has
two discrete subunits GyrA and GyrB. The role of GyrA
subunit is the breakage and reunion of the double DNA
strand, while the GyrB subunit possesses the ATPase
activity, providing a sufficient amount of energy for the
DNA supercoiling. The GyrB subunit is comprised of an N-
terminal domain (43 kDa) which contains the ATPase active
site and a C-terminal domain (47 kDa) involved in the
interaction with both the GyrA subunit and DNA. (Azam
et al. 2015; Bisacchi and Manchester 2015; Oblak et al.
2007).
Considering these perspectives, and in continuation of
our search for biologically active indoles (Morales-Ríos
et al. 2015) we decided to synthesize a series of indole-
based imidazoline scaffolds in search of antimicrobial
agents. A structure–activity relationship (SAR) study was
carried out in order to define the effects that substituents and
their position on the indole ring have on the antimicrobial
activity. In addition, based on the available 3D structure of
DNA GyrB and the structural similarity of these indole-
based imidazoline compounds with known GyrB inhibitors
(Bansal et al. 2014; Oblak et al. 2005; Zidar et al. 2015), we
also decided to explore the interaction of this hybrid phar-
macophore with the active site of DNA GyrB from E. coli
and S. aureus. Therefore, virtual screening was performed
using AutoDock Vina, with the aim to determine binding
affinity of the tested small molecules to the active site of the
target macromolecules and establishing template molecule.
Results and discussion
Chemistry
The synthesis of 2-imidazoline-indoles 2a, 2b, and 4a–4d is
shown in Scheme 1. Ester 1b was converted into the cor-
responding 2-acetonitrilindole 3a in a three-step procedure
by reduction with LiAlH4 to give alcohol 1c, which by
1
1
Scheme 1 Synthesis of
imidazoline-indoles 2a, 2b, and
4a–4d. a conc. H2SO4, OH
(CH2)2OH, reflux, 3 h; b
a
2
+
2
1a or 1b
2
LiAlH4/dry THF, room
temperature, 1 h; c BzCl/dry
THF, NEt3, room temperature
3 h; d DMSO, KCN, 65–70 °C,
4 h; e (CH2NH2)2, elemental S,
reflux, 2.5 h
1a: R1 = H, R2 = CO2Et
1b: R1 = F, R2 = CO2Et
1c: R1 = F, R2 = CH2OH
1d: R1 = F, R2 = CH2OCOPh
3a: R1 = F, R2 = CH2CN
2a: R1 = H
2b: R1 = F
b
c
d
'
4
4
5
'
3
3a
3
'
5
6
'
2
7a
2
'
1
7
8
1
3
4a
e
2
+
2
2
8
2
1
3a: R1 = F, R2 = H, 2-
3b: R1 = R2 = H, 3-
CN
CN
2
3c: R1 = H, R2 = Me, 3-
3d: R1 = F, R2 = H, 3-
CN
CN
4b: R1 = R2 = H
4c: R1 = H, R2 = Me
4d: R1 = F, R2 = H

Medicinal Chemistry Research
Fig. 1 Molecular structures of
1d (left) and 4c (right).
Displacement parameters are
drawn at the 30% probability
level
C5’
C4’
F5
N1’
C2’
C4
C5
C3a
O9
C3
N3’
C8
C3a
C11
C4
C6
C8
C3
C9
N1
C7a
C2
C12
C13
C7
C5
C6
C10
O8
C2
N1
C7a
C9
C15
C14
C7
Table 1 Selected bond distances (Å) bond angles (deg) and dihedral
angles (deg) of 4c
treatment with benzoyl chloride gave benzoate 1d followed
by nucleophilic displacement of the leaving group with
cyanide ion in 44% global yield. The preparation of 2-
substituted 2-imidazolines from different precursors is well
reported in literature (Crouch 2009; Han and Du 2009).
Accordingly, the preparation of 2-(2-imidazolyl)indoles 2a
and 2b was achieved by adopting a simple one-pot proce-
dure that involves the condensation of ethylenediamine
(EDA) with the corresponding ethyl indoly-2-carboxylate
(1a or 1b) using conc. H2SO4 as a cyclizing agent and
ethylenglycol as a reaction media in 75 and 44% yield,
respectively. Whereas, the synthesis of 2- and 3-(2-imida-
zolyl)methyl substituted indoles 4a–4d was carried out by
nucleophilic addition of EDA to the corresponding nitriles
3a–3d in the presence of catalytic amounts sulfur (Sawa
1968), in moderated yields (56–82%).
Bond distances
Bond angles and dihedral angles
C2′−N1′
C2′−N3′
C5′−N1′
C4′−N3′
C4′−C5′
C8−C2′
C8−C3
1.347(3)
1.274(2)
1.441(3)
1.478(3)
1.525(3)
1.498(3)
1.498(3)
C2′−N1′−C5′
C2′−N3′−C4′
N1′−C2′−N3′
C3−C8−C2′
N1′−C2′−N3′−C4′
N3′−C2′−N1′−C5′
C3−C8−C2′−N3′
109.32(19)
106.04(16)
116.59(18)
117.16(17)
2.35(0.23)
−4.40(0.25)
9.82(0.30)
organism. Although for bacteria, MIC is seen as a gold
standard, in practice it is a crude measure with limitations, it
is more an observation than a calculation.
Satisfactory spectral data were determined for all com-
pounds and crystals suitable for X-ray diffraction analysis
were obtained for benzoate 1d and imidazoline 4c (Fig. 1).
Equipopulated mixture of two identical imidazolyl tauto-
The MIC determination of the tested compounds (2a, 2b,
4a–4d) was carried out simultaneously in comparison with
gentamycin against Gram-positive (Staphylococcus aureus
ATCC 25923, Listeria monocytogenes ATCC 19115) and
Gram-negative bacteria (Escherichia coli ATCC 25922
Pseudomonas aeruginosa ATCC 27853) by broth micro-
dilution method. The antifungal activity was assayed
against the yeast Candida albicans (ATCC 10231) and the
mold Aspergillus niger ATCC 16404, using fluconazole as
standard. The results shown in Table 2 evidenced that
structural differences among the tested compounds influ-
ence the antimicrobial activity against bacteria. The MIC
tests revealed that 2b had MIC between 40 and 80 μg/mL
against the organisms Gram-positive. Similarly, 2a and 4a
had MIC between 80 and 160 μg/mL against the organisms
Gram-positive. A simple SAR analysis of the compounds
demonstrates that the substitution at the indole ring plays a
pivotal role in the microbial inhibition potentiality. Thus,
C2-substituted indoles with imidazolyl (2a and 2b) or
imidazolyl methylene group (4a) show better antibacterial
activity than 4b–4d where the indole ring is substituted at
the C3 position. Moreover, the presence of fluorine sub-
stitution in the indole ring (2b) improved the antimicrobial
1
mers was present in the H NMR spectra of 2-imidazoline-
indoles 2a, 2b, and 4a–4d, giving rise in all cases to an
average singlet for the four imidazoline C–H protons. In
contrast, X-ray structure determination of 4c showed that in
the crystal lattice (Fig. 1) this compound adopt a config-
uration in which the NH amino proton is located on N1′,
with no evidence of disorder of this proton between the two
nitrogens in the ring, as revealed by the electron-density
map close to N1′ and by the different pattern of bond dis-
tances and angles around N1′ and N3′ (Table 1).
Antibacterial activity
Potency measurements by microbiological assays provide
information about the bioactivity of a given compound
(Desroy et al. 2013). In an in vitro setting, the potency of an
antimicrobial agent may be assessed through an estimation
of the minimum inhibitory concentration (MIC) which is the
lowest concentration of drug that inhibits the growth of the

Medicinal Chemistry Research
potentiality, probably due to enhanced bacterial cell pene-
tration (Zhou et al. 2016) However, the activity was sig-
nificantly reduced by inclusion of a methylene linkage
between imidazoline and indole moieties, thus 2b exhibited
better activity than 4a at the same dose levels. The anti-
bacterial activity of these 2-imidazoline-indoles can be
displayed in the following sequence: 2a, 4a<2b, and were
more effective against Gram-positive organisms than
against Gram-negative. An insight into the antifungal
activity with respect to chemical structure revealed that
compounds 2a and 2b showed the best activity, and 2a was
the most active compound against A. niger with MIC
between 80 and 160 μg/mL (Table 2). Although the ana-
lyzed 2-imidazoline-indoles were substantially less potent
than standards, these compounds as scaffolds could be
valuable for the development of antimicrobial agents.
Molecular docking
Docking studies were performed for all synthetized com-
pounds in order to predict their affinity to bacterial protein
DNA GyrB, from E. coli and S. aureus. The binding modes
were predicted with a native water molecule located in the
immediate vicinity of ligands at the protein-binding site.
The least energy-binding mode for each ligand–protein
complex (the top ranked solution) was inspected for
potential interactions with the amino acids residues of the
ATP-binding site, particularly taking into account the
hydrogen bond interactions provided by the highly con-
served water molecule, W451 (PDB: 4DUH), coordinated
with Asp73, Gly77, and Thr165 residues (E. coli gyrase
numbering) or W424 (PDB: 5D6P) coordinated with
Asp81, Gly85, and Thr173 residues (S. aureus gyrase
numbering).
Analysis of the docking poses for all investigated 2-
imidazoline-indoles showed that they occupy the same area
of DNA gyrase B ATP-binding pocket (Lewis et al. 1996)
with good in silico interaction energy scores (Table 3). The
ligands were anchored to the receptor forming putative
hydrogen bonds with the conserved water molecule, except
in the case of 4a from E. coli and 4b and 4d from S. aureus
(see Figs. S17-S22 of Supporting Information for best
binding modes of all analyzed compounds for both
enzymes). Among the ligands virtually screened to the
DNA gyrase 2a and 2b emerged as lead compounds, in
accordance with the experimentally MIC obtained values.
The determined binding mode for compounds 2a and 2b is
Table 2 In vitro antimicrobial activity of compounds 2a, 2b, and 4a–
4d against Gram-positive, Gram-negative, and fungi pathogens
Compound
Test organism MIC (μg/mL)
Gram-positivea
Gram-
Fungic
negativeb
Sa
Lm
Ec
Pa
Ca
An
2a
160
80
160
80
320
160
320
>640
>640
640
0.98
–
320
>320
320
160
320
>320
320
>320
>320
–
2b
320
4a
160
>640
>640
640
<0.25
–
160
>640
>640
640
<0.25
–
320
>320
>320
>320
>320
–
4b
>320
>320
>320
<0.25
–
4c
4d
Gentamicin
Fluconazole
<3.5
56.3
aGram-positive bacteria: Sa Staphylococcus aureus ATCC 25923, Lm
Listeria monocytogenes ATCC 19115
bGram-negative bacteria: Ec Escherichia coli ATCC 25922, Pa
Pseudomonas aeruginosa ATCC 27853
cFungi: Ca Candida albicans ATCC 10231, An Aspergillus niger
ATCC 16404
Table 3 Binding free energy (ΔGbind), intramolecular hydrogen bonds (N-H⋯X) and hydrophobic contactsa in ligands 2a, 2b, and 4a–4d
comp. GyrB
ΔGbind (kcal/mol) N-H⋯X (d in Å)
Hydrophobic contacts
2a
2b
4a
4b
4c
4d
2a
2b
4a
4b
4c
4d
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
−7.4
−7.7
−7.5
−8.0
−8.1
−7.7
N1-H⋯W451 (2.94) N1′-H⋯Asp73 (2.80)
N1-H⋯W451 (2.94) N1′-H⋯Asp73 (2.80)
–
Val43, Ala47, Ile78, Val167
Val43, Ala47, Ile78, Val167
Val43, Ala47, Ile78, Ile94,
Val43, Ile78, Val120, Val167
Val43, Ile78, Ile94, Val120, Val167
Ile78, Val120, Val167
Ile51, Ile86, Ile175
N1′-H⋯W451 (3.16)
N1′-H⋯W451 (3.13)
N1′-H⋯W451 (3.19)
N1′-H⋯W424 (3.16)
S. aureus −6.4
S. aureus −6.7
S. aureus −7.4
S. aureus −6.7
S. aureus −6.9
S. aureus −7.0
N1-H⋯W424 (3.34) N1′-H⋯W424 (3.03)
N1′-H⋯W424 (3.07)
N1-H⋯Asp81 (2.93) N1-H⋯Ser55 (3.16) N1′-H⋯Glu58 (3.11) Ile51, Ile86, Ile175
Ile86, Ile175
Ile86, Ile175
N1′-H⋯W424 (3.14)
Ile51, Ile86, Ile102, Ile175
N1-H⋯Ser55 (3.12) N1-H⋯Asp81 (3.03) N1′-H⋯Glu58 (3.11) Ile51, Ile86, Ile175
aFrom molecular docking

Medicinal Chemistry Research
a
c
ALA47
b
d
ALA47
GLU50
GLU50
ASN46
LYS103
ASN46
LYS103
ASP73
W451
GLY77
ASP73
W451
GLY77
VAL43
VAL43
THR165
THR165
VAL167
VAL167
ASN54
ILE78
ILE78
GLU58
GLU58
W424
SER55
ASP81
SER55
ASP81
W424
GLY85
ASN54
ILE175
ILE51
GLY85
THR173
THR173
ILE175
ILE86
ILE86
Fig. 2 Best docking pose of 2a (a) and 2b (b) in the central part of the
E. coli GyrB ATP-binding pocket (PDB: 4DUH; W451). Best docking
pose of 2a (c) and 2b (d) in the central part of the S. aureus GyrB
ATP-binding pocket (PDB: 5D6P; W424). Proteins shown in cartoon
representation, ligands shown as sticks. Hydrogen bonds are indicated
as green dotted lines
depicted within the ATP-binding site in Fig. 2. Stereoview
of both docked complex shows primarily the formation of a
direct hydrogen bond between the hydrogen of the N1’
nitrogen of the imidazoline ring and the Asp73 residue,
while the hydrogen of the N1 nitrogen of the indole ring
interacts with the conserved W451 water molecule, and is
further coordinated with the Asp73, Gly77, and Thr165
residues of DNA gyrase (E. coli) (Fig. 2a, b). These two H-
bonds, of the length 2.8 and 2.94 Å, together form the most
critical, and seemingly indispensable, hydrogen bond
donor–acceptor interaction pattern representative for the
majority of DNA gyrase inhibitors (Bisacchi and Manche-
ster 2015). Apart from hydrogen bonding, the imidazoline
and indole rings of 2a and 2b forms hydrophobic interac-
tions with the residues Val43, Ala47, Ile78, and Val167, at
the hydrophobic pocket within the ATP-binding site. The
score values showed that the binding poses of 2a and 2b to
the active site of S. aureus GyrB were weaker than those of
E. coli GyrB by 1.0 kcal mol−1 (Table 3). These results can
be attributed to several nonoptimal ligand–enzyme inter-
actions, including bound Asp81 missing and that ligands are
less implicated in hydrophobic interactions (Fig. 2c, d).
containing heterocycle motives and defined the effects that
the relative position of imidazoline ring on the indole ring
have on antibacterial activity. Additionally, the SAR data
demonstrates the importance of a fluorine substituent in the
indole ring. Based on the available 3D structure of DNA
GyrB and the structural similarity of these hybrid com-
pounds with known GyrB inhibitors, the docking results
showed that the studied ligands reach the GyrB ATP-
binding site under hydrogen bond and hydrophobic inter-
actions, exhibiting a good in silico interaction energy
scores. We completed a successful search for the N-H
donors groups of the two pharmacophores, 2-imidazoline
and indole, that would achieved the key Asp73 (E.
coli)–water interaction.
Taken together, the results we obtained suggest that
hybrid 2-imidazoline-indoles may be considered as good
molecular templates for potential antibacterial agents and
can be used for further chemical modifications for
improving antibacterial activity.
Materials and methods
Chemistry
Conclusions
All reagent-grade chemicals were purchased from Sigma-
Aldrich Co. and were used as received. Melting points were
determined on an electrothermal instrument and are uncor-
rected. IR spectra were measured with a Perkin-Elmer 16
FPC FT infrared spectrophotometer. Low-resolution mass
spectra were conducted on a Varian CP 3800 GC spectro-
meter equipped with a selective mass Varian Saturn 2000
A series with 2-imidazoline-indole structural scaffold was
synthesized and characterized by spectral analysis. The
pharmacological study was undertaken to evaluate the
effects of substituents on the antibacterial activity. The SAR
revealed two ligands with increased potency for anti-
bacterial activity that incorporate N-H donor groups

Medicinal Chemistry Research
detector. MS analyses were obtained in the electron impact
(EI) mode at an ionizing voltage of 70 eV. High-resolution
mass spectra (HRMS) were recorded on an Agilent LCTOF
spectrometer at the UCR Mass Spectrometry Facility,
University of California, Riverside CA and on a Waters
Synapt G2 spectrometer at the Department of Chemistry
and Biochemistry, University of Colorado, Boulder, CO,
127.8 (d, J = 10.4 Hz, C-3a), 111.8 (d, J = 9.2 Hz, C-7),
111.3 (d, J = 26.5 Hz, C-6), 105.5 (d, J = 24.2 Hz, C-4),
104.1 (d, J = 4.6 Hz, C-3), 60.1 (CH2). GC-MS (EI, 70 eV):
m/z (%) = 269 (M+, 36), 148 (46), 147(40), 105 (100).
HRMS m/z calcd for C16H12FNO2+H: 270.0925; found:
270.0926.
1
USA. H and 13C NMR spectra were recorded on Varian
2-(4,5-Dihydro-1H-imidazol-2-yl)-1H-indole 2a
1
Mercury spectrometers (300 MHz for H and 75 MHz for
13C) in the indicated solvent. Chemical shifts are given in
ppm (δ) and are referenced to TMS as internal standard. J
values are quoted in Hertz, with the normal abbreviations (s,
singlet; d, doublet; t, triplet; q, quartet; br, broad; m, mul-
tiplet). All structural assignments were supported by
gHMBC, and gHSQC. Analytical thin-layer chromato-
graphy (TLC) was performed on silica gel 60 F254 coated
aluminum sheets (0.25 mm thickness) with a fluorescent
indicator. Visualization was accomplished with UV light
(254 nm) or on heating after spraying ceric ammonium
sulfate reagent as developing agent. Flash chromatography
was performed on silica gel 60 (230–400 mesh). Spectro-
scopic data of indole-2-carboxylic esters 1a and 1b (Koenig
et al. 2010), 5-fluoroindol-2-ylmethanol (1c) (Nishiyama
et al. 2016), and acetonitrilindoles 3b, 3c (Rivera-Becerril
et al. 2008), and 3d (Pérez-Alvarez et al. 1997) are in
agreement with literature data. Compounds 2a (Kou et al.
2013) and 3a (Spadoni et al. 2006) were synthesized as
indicated in Scheme 1.
A stirred mixture of carboxylic ester 1a (100 mg,
0.53 mmol), ethylenglycol (1.5 mL), EDA (0.35 mL), and
conc. H2SO4 (0.1 mL) was heated at reflux for 3 h until
disappearance of starting material (TLC). The reaction
mixture was then cooled, poured into cold water (15 mL),
and extracted with CHCl3 (3 × 30 mL). The combined
organic extracts were washed with brine (20 mL), dried over
anhydrous MgSO4, filtered, and concentrated under reduced
pressure. The solid product was collected and purified by
recrystallization from MeOH/H2O to afford 2a (74 mg,
0.4 mmol, 75%) as yellow crystals; mp 130–132 °C; Rf =
0.32 (MeOH/NEt3 95:5); IR (CHCl3) νmax 3445 (N-H),
1
1634 (C = N) cm−1; H NMR (CD3OD) δ: 7.57 (dm, J =
8.2 Hz, 1H, H-4), 7.41 (dm, J = 8.2 Hz, 1H, H-7), 7.20
(ddd, J = 8.2, 7.0, 1.2 Hz, 1H, H-6), 7.05 (ddd, J = 8.2, 7.0,
1.2 Hz, 1H, H-5), 6.95 (s, 1H, H-3), 3.75 (s, 4H, H-4′, H-
5′); 13C NMR (CD3OD) δ: 162.4 (C-2’), 139.4 (C-7a),
129.9 (C-3a), 129.4 (C-2), 125.8 (C-6), 123.6 (C-5), 122.0
(C-4), 113.6 (C-7), 106.4 (C-3), 50.1 (C-4′, C-5′); GC-MS
(EI, 70 eV): m/z (%) = 185 (M+, 100), 156 (21). HRMS m/z
calcd for C11H11N3+H: 186.1026; found: 186.1032.
(5-Fluoro-1H-indol-2-yl)methyl benzoate 1d
Triethyl amine (2.5 mL, 18 mmol) and benzoyl chloride
(1.1 mL, 9.5 mmol) were sequentially added via syringe to a
solution of alcohol 1c (1 g, 6.1 mmol) in anhydrous THF
(50 mL) at 0 °C under argon atmosphere. The reaction
mixture was allowed to warm at room temperature and
stirred for 3 h until disappearance of starting material
(TLC). The mixture was filtered and the filtrate was diluted
with a 10% aq solution of NaOH (5 mL) and brine (15 mL).
The mixture was extracted with EtOAc (2 × 25 mL), the
combined organic extracts were dried over anhydrous
MgSO4, filtered, and concentrated under reduced pressure.
The solid product was collected and purified by recrys-
tallization from CH2Cl2/hexane to afford 1d (1.27 g,
4.7 mmol, 78%) as pale yellow crystals; mp 147–148 °C; Rf
= 0.41 (EtOAc-hexane 2:8); IR (CHCl3) νmax 3440 (N-H),
1707 (C = O), cm−1; 1H NMR (CDCl3) δ: 8.75 (br s, 1H, N-
H), 8.06 (m, 2H, Ho), 7.57 (m, 1H, Hp), 7.44 (m, 2H, Hm),
7.26 (dd, partial overlap, 1H, H-7), 7.24 (dd, partial overlap,
1H, H-4), 6.94 (ddd, J = 8.8, 8.8, 2.9 Hz, 1H, H-6), 6.57 (d,
J = 1.8 Hz, 1H, H-3), 5.45 (s, 2H, CH2). 13C NMR (CDCl3)
δ: 167.8 (C = O), 157.9 (d, J = 233.8 Hz, C-5), 134.8 (C-2),
133.4 (Cp), 133.1 (C-7a), 129.8 (Co), 129.5 (Ci), 128.5 (Cm),
2-(4,5-Dihydro-1H-imidazol-2-yl)-5-fluoro-1H-indole 2b
A
stirred mixture of carboxylic ester 1b (200 mg,
0.97 mmol), ethylenglycol (2.4 mL) EDA (0.7 mL), and
conc. H2SO4 (0.2 mL) was heated at reflux for 3 h until
disappearance of starting material (TLC). The reaction
mixture was cooled and poured into cold water (15 mL).
The precipitated solid was filtered off, washed with cold
water, and crystallized from MeOH/H2O to afford 2b
(87 mg, 0.43 mmol, 44%) as yellowish solid; mp 254–256 °
C (lit. 254–256 °C, Morales-Ríos et al. 2015); (Rf = 0.39
(MeOH/NEt3 95:5); IR (KBr) νmax 3438 (N-H), 1630 (C =
N) cm−1; 1H NMR (CD3CO2D) δ: 7.55 (dd, J = 8.8, 4.7 Hz,
1H, H-7), 7.37 (s, 1H, H-3), 7.29 (dd, J = 9.4, 2.4 Hz, 1H,
H-4), 7.11 (ddd, J = 9.4, 9.4, 2.4 Hz, 1H, H-6), 4.00 (s, 4H,
H-4′, H-5′); 13C NMR (CD3CO2D) δ: 159.6 (C-2′), 159.4
(d, J = 237.2 Hz, C-5), 136.6 (C-7a), 128.0 (d, J = 11.0 Hz,
C-3a), 121.7 (C-2), 116.7 (d, J = 27.7 Hz, C-6), 115.5 (d, J
= 9.4 Hz, C-7), 111.4 (d, J = 2.5 Hz, C-3), 107.0 (d, J =
23.7 Hz, C-4), 45.7 (C-4′, C-5′). GC-MS (EI, 70 eV): m/z
(%) = 203 (M+, 100), 174 (34); HRMS m/z calcd for
C11H10FN3+H+: 204.0932; found: 204.0938.

Medicinal Chemistry Research
2-(5-Fluoro-1H-indol-2-yl)acetonitrile 3a
100.2 (d, J = 4.6 Hz, C-3), 49.3 (C-4′, C-5′), 28.3 (C-8);
GC-MS (EI, 70 eV): m/z (%) = 217 (M+, 100); HRMS m/z
calcd for C12H12FN3+H: 218.1088; found: 218.1081.
Potasium cyanide (0.95 g, 14.6 mmol) was added to a
solution of ester 1d (1 g, 3.7 mmol) in DMSO (23 mL) at
room temperature. The mixture was stirred at 65–70 °C for
4 h until disappearance of starting material (TLC). After
cooling, a 5% aq solution of Na2SO4 (10 mL) was added to
the crude followed by brine (15 mL), and the mixture was
extracted with EtOAc (3 × 30 mL). The combined organic
extracts were dried over anhydrous MgSO4, filtered, and
concentrated under reduced pressure. The residue was
purified by flash chromatography from EtOAc/hexane to
afford 3a (0.46 g, 2.6 mmol, 71%) as yellowish solid; mp
116–118 °C; Rf = 0.11 (EtOAc-hexane 2:8); IR (CHCl3)
3-((4,5-dihydro-1H-imidazol-2-yl)methyl)-1H-indole
4b Compound 4b (262 mg, 1.32 mmol, 82%) was
obtained from 3b (250 mg, 1.60 mmol): yellowish solid; mp
132–133 °C (lit. 132 °C, Dash et al. 2004); Rf = 0.31
(MeOH/NEt3 95:5); IR (CHCl3) νmax 3479 (N-H), 1624 (C
1
= N) cm−1; H NMR (CDCl3) δ: 9.07 (br s, 1H, N1-H),
7.53 (dm, J = 7.9 Hz, 1H, H-4), 7.33 (dm, J = 7.9 Hz, 1H,
H-7), 7.12 (ddd, J = 8.0, 7.0, 1.0 Hz, 1H, H-6), 7.04 (ddd, J
= 7.8, 7.0, 1.0 Hz, 1H, H-5), 6.96 (s, 1H, H-2), 3.67 (s, 2H,
H-8), 3.45 (s, 4H, H-4′, H-5′); 13C NMR (CDCl3) δ: 167.9
(C-2’), 136.4 (C-7a), 126.9 (C-3a), 123.5 (C-2), 121.7 (C-
6), 119.2 (C-5), 118.2 (C-4), 111.5 (C-7), 108.7 (C-3), 49.2
(C-4′, C-5′), 25.5 (C-8); GC-MS (EI, 70 eV): m/z (%) = 199
(M+, 55), 198 (100), 130 (58); HRMS m/z calcd for
C12H13N3+H: 200.1182; found: 200.1190.
1
νmax 3449 (N-H), 2258 (CN) cm−1; H NMR (CDCl3) δ:
8.30 (br s, 1H, N1-H), 7.27 (dd, J = 8.8, 4.1 Hz, 1H, H-7),
7.22 (dd, J = 9.4, 2.4 Hz, 1H, H-4), 6.96 (ddd, J = 9.4, 9.4,
2.4 Hz, 1H, H-6), 6.44 (dd, J = 2.4, 1.2 Hz, 1H, H-3), 3.91
(s, 2H, CH2). 13C NMR (CDCl3) δ: 158.2 (d, J = 235.0 Hz,
C-5), 133.0 (C-7a), 128.4 (d, J = 10.3 Hz, C-3a), 127.5 (C-
2), 116.1 (CN), 111.6 (d, J = 9.2 Hz, C-7), 111.1 (d, J =
26.5 Hz, C-6), 105.7 (d, J = 24.0 Hz, C-4), 102.8 (d, J =
4.6 Hz, C-3), 17.6 (CH2). GC-MS (EI, 70 eV): m/z (%) =
174 (M+, 100), 147 (40), 148 (36). HRMS m/z calcd for
C10H7FN2+H+: 175.0666; found: 175.0672.
3-((4,5-Dihydro-1H-imidazol-2-yl)methyl)-1-methyl-1H-
indole 4c Compound 4c (190 mg, 0.89 mmol, 56%) was
obtained from 3c (275 mg, 1.62 mmol): yellowish solid; mp
142–145 °C; Rf = 0.61 (MeOH/NEt3 95:5); IR (CHCl3) νmax
3433 (N-H), 1624 (C = N) cm−1; 1H NMR (CDCl3) δ: 7.63
(dm, J = 7.9, Hz, 1H, H-4), 7.31 (dm, J = 7.9 Hz, 1H, H-7),
7.25 (ddd, J = 8.1, 6.7, 1.2 Hz, 1H, H-6), 7.12 (ddd, J =
7.9, 6.7, 1.2 Hz, 1H, H-5), 7.00 (s, 1H, H-2), 3.76 (s, 5H, H-
8, Me), 3.55 (s, 4H, H-4′, H-5′); 13C NMR (CDCl3) δ: 167.3
(C-2’), 137.0 (C-7a), 127.6 (C-3a), 127.5 (C-2), 121.9 (C-
6), 119.3 (C-5), 118.8 (C-4), 109.3 (C-7), 108.5 (C-3), 49.9
(C-4′, C-5′), 32.6 (Me), 25.9 (C-8); GC-MS (EI, 70 eV): m/
z (%) = 214 (M++H, 12), 200 (100).
General procedure for the synthesis of imidazolyl-indoles
4a–4d
A stirred mixture of nitrile 3a–3d (1.6 mmol), EDA
(2.6 mL), and S (12.8 mg, 0.4 mmol) was heated at reflux
for 2–3 h until disappearance of starting material (TLC).
The reaction mixture was then cooled, poured into cold
water, and extracted with CHCl3 (3 × 20 mL). The com-
bined organic extracts were washed with brine (20 mL),
dried over anhydrous MgSO4, and filtered. The solvent was
removed under reduced pressure and the crude product was
collected and purified by recrystallization from acetone-
hexane (for 4b and 4c) or flash chromatography (EtOAc/
MeOH 4:6) and crystallization (for 4a and 4d).
3-((4,5-Dihydro-1H-imidazol-2-yl)methyl)-5-fluoro-1H-
indole 4d Compound 4d (199 mg, 0.91 mmol, 57%) was
obtained from 3d (280 mg, 1.61 mmol): yellowish solid; mp
118–120 °C (lit. 119–121 °C, Wysong and Whithe 1968); Rf
= 0.41 (MeOH/NEt3 95:5); IR (CHCl3) νmax 3487 (N-H),
1637(C = N) cm−1; 1H NMR (DMSO-d6) δ: 11.07 (br s, 1H,
N1-H); 7.32 (m, 1H, H-4), 7.30 (m, 1H, H-7), 7.27 (s, 1H, H-
2), 6.88 (ddd, J = 9.1, 9.1, 2.5 Hz, 1H, H-6), 3.38 (s, 4H, H-
4′, H-5′), 3.23 (s, 2H, H-8); 13C NMR (DMSO-d6) δ: 167.2
(C-2′), 156.9 (d, J = 230.5 Hz, C-5), 133.0 (C-7a), 127.5 (d,
J = 10.0 Hz, C-3a), 126.1 (C-2), 112.5 (d, J = 9.4 Hz, C-7),
109.6 (d, J = 4.4 Hz, C-3), 109.3 (d, J = 26.0 Hz, C-6), 103.5
(d, J = 23.3 Hz, H-4), 49.1 (C-4′, C-5′), 25.5 (C-8). GC-MS
(EI, 70 eV): m/z (%) = 218 (M++H, 100).
2-((4,5-Dihydro-1H-imidazol-2-yl)methyl)-5-fluoro-1H-
indole 4a Compound 4a (185 mg, 0.85 mmol, 53%) was
obtained from 3a (280 mg, 1.61 mmol): yellowish solid; mp
133–135 °C (lit. 133–135 °C, Morales-Ríos et al. 2015); Rf
= 0.19 (MeOH/NEt3 95:5); IR (CHCl3) νmax 3426 (N-H),
1628 (C = N) cm−1; 1H NMR (DMSO-d6) δ: 7.29 (dd, J =
8.8, 4.7 Hz, 1H, H-7), 7.16 (dd, J = 10.6, 2.4 Hz, 1H, H-4),
6.82 (m, 1H, H-6), 6.23 (s, 1H, H-3), 3.60 (s, 2H, H-8), 3.43
(s, 4H, H-4′, H-5′); 13C NMR (DMSO-d6) δ: 164.4 (C-2’),
156.9 (d, J = 230.4 Hz, C-5), 136.8 (C-2), 132.8 (C-7a),
128.4 (d, J = 10.4 Hz, C-3a), 111.8 (d, J = 10.4, C-7),
108.2 (d, J = 25.3 Hz, C-6), 103.9 (d, J = 23.0 Hz, C-4),
X-Ray diffraction analysis
The study was done on a Bruker-Nonius CAD4 dif-
fractometer using Cu Kα radiation (λ = 1.54184 Å). The

Medicinal Chemistry Research
data were collected in the ω-2θ scan mode. Unit cell
refinements were done using CAD4 Express v 2.0 software
and structures were solved by direct methods using the
SHELXS-97 program included in the WINGX v 1.64.05
package. The N-bond H atoms were located by difference
Fourier synthesis and refined freely. The non-hydrogen
atoms were treated anisotropically, and the hydrogen atoms,
included in the structure factor calculation, were refined
isotropically. Selected X-ray crystallographic data for
Molecular modeling
Ligand preparation
Three-dimensional models and subsequent conformational
search of designed compounds was carried out using
Spartan’14 (2013) (Wavefunction, Inc.). A Monte Carlo
random conformational search with the MMFF94 force-
field minimization was applied for compounds 2a, 2b, and
4a. The geometry optimization of the imidazoline-indoles
was carried out employing density functional theory-DFT
using Gaussian ‘09 program package (Frisch et al. 2009).
The calculations were performed with the initial geometry
generated from standard geometrical parameters and were
minimized without any constraint in the potential energy
surface with the B3LYP hybrid density functional adopting
the standard 6–31 G+(d,p) basis set.
compound 1d: C16H12FNO2, FW = 269.27 g mol−1
orthorhombic system, space group Pbn21, a = 5.88(2), b =
8.42(4), c = 26.43(1) Å, V = 1308.0(9) Å3, Z = 4, Dcalcd
1.37 g cm−3
3.08 < θ < 26.33, R = 6.3%, wR = 17.7%,
,
=
,
crystal dimension of 0.35 × 0.35 × 0.1 mm. Selected X-ray
crystallographic data for compound 4c: C13H15N3, FW =
213.28 g mol−1, monoclinic system, space group P21/c, a =
10.57(1), b = 5.19(2), c = 20.52(7) Å, β = 91.86(1)°, V =
1125.9(6) Å3, Z = 4, Dcalcd = 1.26 g cm−3, 4.18 < θ < 59.93,
R = 5.0%, wR = 14.4%, crystal dimension of 0.2 × 0.1 ×
0.08 mm. Cambridge Crystallographic Data Center (CCDC)
number contains crystallographic data for compounds 1d
(No. 1831291) and 4c (No. 1831290). These data can be
obtained free of charge from the Cambridge Crystal-
lographic Data Center via www.ccdc.cam.a-c.uk/data_
request/cif.
Docking studies
Molecular docking calculations were performed using
Autodock Vina (Trott and Olson 2010) with UCFS Chimera
as interface (Pettersen et al. 2004). The crystal structures of
DNA gyrase B ATP-binding domain from E. coli and S.
aureus were obtained from the Brookhaven Protein Data
Bank http://www.rcsb.org/pdb PDB entry: 4DUH (Brvar
et al. 2012) and 5D6P (Zhang et al. 2015), respectively.
LigPlot+ 1.4 (Laskowski and Swindells 2011) and Auto-
Docktools 1.5.6/AutoDock 4.2 (Morris et al. 2009) software
packages were employed for docking flexible ligands into
the rigid enzyme model for identification of hydrogen bonds
and hydrophobic interactions between residues at the active
site. For DNA gyrase B ATP-binding domain from E. coli
the grid size was set to 22 × 22 × 22 xyz points with grid
spacing of 0.35 Å and grid center was designated at
dimensions (x, y, z): 33.5, 6.22, 7.65. Whereas for S. aureus
the vales for gride size were 22 × 22 × 22 xyz points with
grid spacing of 0.35 Å and grid center (x, y, z): −6.7, 3.1,
−0.3. The genetic algorithm was used for optimal parameter
settings and the number of runs was set to 50. The para-
meters of docking procedure are as follows: population size
150, maximum number of energy assessments of 2.5 × 107
per cycle analysis, 100 mating receptor conformations were
generated for each ligand, the clusters remained a root mean
square deviation (RMSD) of 0.3 Å, while other parameters
were set at their default values.
Biological activity
In vitro inhibition of bacterial and fungal growth
MIC values were determined by the broth microdilution
method in 96-well plates (NCCLS 2003). Testing was
carried out in Mueller–Hinton broth and Sabouraud
dextrose broth for bacteria and fungi, respectively. The
final inoculum size was 5 × 104 CFU/mL for bacteria,
0.5 × 103 for C. albicans ATCC 10231 and 1 × 104 for A.
niger ATCC 16404. Test compounds were dissolved in
DMSO at an initial concentration of 10240 μg/mL, and
then were serially diluted in culture medium to 2.5 μg/
mL. The culture medium without test compounds was
considered as a negative control. Triplicate wells were
applied for each concentration of the individual test
materials. To ensure that the solvent had no effect on the
bacterial growth, a control was performed with the test
medium supplemented with DMSO at the same dilutions
as used in the experiments and DMSO had no effect on
the microorganisms in the concentrations studied. Anti-
microbial activity was determined after incubation for
24 h at 37 °C for bacteria and after incubation for 48 h at
35 °C for the fungi, using the tetrazolium dye MTT as
bacterial growth indicator.
The 3D rendering of complex enzyme–ligand and the
conformer with the best docking energy and interactions
with amino acid residues were analyzed and represented
using UCFS Chimera and AutoDocktools 1.5.6. The posi-
tion of the conserved water molecule (W451/W424) was

Medicinal Chemistry Research
obtained directly from the high-resolution X-ray crystal
structures (resolution ≤ 2.1 Å) of the DNA gyrase B ATP-
binding domain from E. coli (PDB: 4DUH) and S. aureus
(PDB: 5D6P). Redocking was performed in order to con-
firm the accuracy in pose prediction of the two protein
complexes. The predicted docking poses presented a RMSD
values of 1.0 Å (4DUH) and 2.2 Å (5D6P), when compared
to the experimental co-crystallized binding pose (Fig. S23
of Supporting Information).
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Acknowledgements The present work is partially supported by
CONACYT grant 179187 and Programa de Fortalecimiento Acadé-
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Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
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