Imidazole derivatives as antiparasitic agents and use of molecular modeling to investigate the structure–activity relationship

Article abstract of DOI:10.1007/s00436-020-06668-6

Toxoplasmosis is a common parasitic disease caused by Toxoplasma gondii. Limitations of available treatments motivate the search for better therapies for toxoplasmosis. In this study, we synthesized a series of new imidazole derivatives: bis-imidazoles (c

Full text of DOI:10.1007/s00436-020-06668-6

Parasitology Research
https://doi.org/10.1007/s00436-020-06668-6
TREATMENT AND PROPHYLAXIS - ORIGINAL PAPER
Imidazole derivatives as antiparasitic agents and use of molecular
modeling to investigate the structure–activity relationship
Oluyomi Stephen Adeyemi1,2 & Abiodun Omokehinde Eseola3,4 & Winfried Plass3 & Olubunmi Atolani5 & Tatsuki Sugi6
&
Yongmei Han6,7 & Gaber El-saber Batiha6,8 & Kentaro Kato6,9 & Oluwakemi Josephine Awakan10
&
Tomilola Debby Olaolu10 & Charles Obiora Nwonuma10 & Omokolade Alejolowo10 & Akinyomade Owolabi11
&
Damilare Rotimi10 & Omowumi Titilola Kayode10
Received: 4 September 2019 /Accepted: 15 March 2020
#
Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract
Toxoplasmosis is a common parasitic disease caused by Toxoplasma gondii. Limitations of available treatments motivate
the search for better therapies for toxoplasmosis. In this study, we synthesized a series of new imidazole derivatives: bis-
imidazoles (compounds 1–8), phenyl-substituted 1H-imidazoles (compounds 9–19), and thiopene-imidazoles (com-
pounds 20–26). All these compounds were assessed for in vitro potential to restrict the growth of T. gondii. To explore
the structure–activity relationships, molecular analyses and bioactivity prediction studies were performed using a stan-
dard molecular model. The in vitro results, in combination with the predictive model, revealed that the imidazole
derivatives have excellent selectivity activity against T. gondii versus the host cells. Of the 26 compounds screened,
five imidazole derivatives (compounds 10, 11, 18, 20, and 21) shared a specific structural moiety and exhibited signif-
icantly high selectivity (> 1176 to > 27,666) towards the parasite versus the host cells. These imidazole derivatives are
potential candidates for further studies. We show evidence that supports the antiparasitic action of the imidazole deriv-
atives. The findings are promising in that they reinforce the prospects of imidazole derivatives as alternative and
effective antiparasitic therapy as well as providing evidence for a probable biological mechanism.
.
.
.
Keywords Drug discovery Infectious diseases Medicinal biochemistry Medicinal chemistry
Section Editor: Xing-Quan Zhu
6
* Oluyomi Stephen Adeyemi
National Research Center for Protozoan Diseases, Obihiro University
oluyomi.stephen.adeyemi@tdtu.edu.vn
of Agriculture and Veterinary Medicine, Inada-cho,
Obihiro, Hokkaido 080-8555, Japan
7
8
9
1
Inner Mongolia University for the Nationalities College of Animal
Science and Technology, Tongliao, Inner Mongolia, China
Laboratory of Theoretical and Computational Biophysics, Ton Duc
Thang University, Ho Chi Minh City, Vietnam
Department of Pharmacology and Therapeutics Department, Faculty
of Veterinary Medicine, Damanhour University, Damanhour, Egypt
2
Faculty of Applied Sciences, Ton Duc Thang University, Ho Chi
Minh City, Vietnam
Laboratory of Sustainable Animal Environment, Graduate School of
Agricultural Science, Tohoku University, 232-3 Yomogida,
Naruko-onsen, Osaki, Miyagi 989-6711, Japan
3
Institute of Inorganic and Analytical Chemistry,
Friedrich-Schiller-Universität Jena, Humboldtstraße 8,
07743 Jena, Germany
10
11
Department of Biochemistry, Medicinal Biochemistry,
Nanomedicine & Toxicology Laboratory, Landmark University,
PMB 1001, Omu-Aran 251101, Nigeria
4
Department of Chemical Sciences, Redeemer’s University,
Ede, Nigeria
5
Department of Chemistry, University of Ilorin, PMB 1515,
Ilorin, Nigeria
Department of Microbiology, Landmark University, PMB 1001,
Omu-Aran 251101, Nigeria

Parasitol Res
Introduction
asmiconazole and ketoconazole. In light of these facts,
we synthesized a new series of imidazole derivatives that
could be grouped into bis-imidazoles, phenyl-substituted
IH-imidazoles, and thiopene-imidazoles. All compounds
were screened for in vitro potential to restrict the growth
of T. gondii as well as by in silico modeling for
bioactivity.
Toxoplasmosis, which is caused by an intracellular parasite
Toxoplasma gondii, an apicomplexan (Kamau et al. 2012), is
a common parasitic infection in nearly one third of the human
population. The life cycle of T. gondii is complex in that it
exists in three forms, sporozoite, bradyzoite, and tachyzoite.
Due to its low host specificity, T. gondii can infect a vast array
of hosts including animals and humans (Alday and Doggett
2017). The host becomes infected, usually through consump-
tion of either tissue cysts (bradyzoites) in undercooked meat
or oocysts (sporozoites) in feline fecal matter. T. gondii infec-
tions are usually asymptomatic in healthy individuals but can
lead to fatal outcomes in immune-compromised individuals
(Kamau et al. 2012).
Currently, antimalarial drugs or antibiotics are the treat-
ment of choice for toxoplasmosis. However, the available
therapies are fraught with shortcomings such as limited effi-
cacy and poor tolerance (Kamau et al. 2012). According to
some reports, the side effects of toxoplasmosis medication
caused cessation of treatment in up to 40% of patients
(Alday and Doggett 2017; Harrell and Carvounis 2014).
Furthermore, although the available therapies for toxoplasmo-
sis are effective against active infections, they do not eradicate
latent ones, and relapse is common in patients on suppressive
therapy (Alday and Doggett 2017; Harrell and Carvounis
2014). Consequently, toxoplasmosis presents a growing glob-
al health burden (Flegr et al. 2014), which is further reinforced
by the therapeutic shortcomings highlighted above. Therefore,
effective therapeutic agents for toxoplasmosis are urgently
required.
Materials and methods
General
Materials, reagents, and substrates for the experimental
syntheses and catalysis were commercially sourced as re-
agent grade and used as supplied. The synthesis and char-
acterization of compounds C-1–C-8, and C-16–C-26
followed established methodologies (Eseola et al. 2012,
2018, 2019; Lipinski et al. 1997). Impurities were removed
from the imidazole-based compounds by either re-
crystallization or silica gel column chromatography.
Elemental analyzers (Leco CHNS-932 or vario EL Cube)
were used to determine the elemental composition, while a
Bruker 400 MHz instrument was used to obtain 1H and 13
C
NMR spectra, using deuterated solvents as the internal
standard. A Bruker Equinox spectrophotometer equipped
with diamond ATR in the range of 4000–370 cm−1 was
used to obtain the IR spectra. The general synthesis of
the imidazole derivatives is shown in Scheme 1, while
further synthetic protocols are presented below.
Investigations have revealed that screening a vast array
of natural or synthetic compounds for potential bioactivity
can be an effective approach to the discovery of effective
therapies for parasitic diseases (Adeyemi et al. 2017a, b,
c, 2018a, b, c, 2019; Castro et al. 2006; Finlayson et al.
2004; Fonseca-Berzal et al. 2014; Grellier et al. 1999).
Imidazoles have been reported to possess antiparasitic
properties (Coura and Castro 2002; Flores-Holguín and
Glossman-Mitnik 2005). Additional investigations have
reported both antiparasitic and antimicrobial activity for
imidazole-based compounds (Adeyemi et al. 2017a;
Liesen et al. 2010). Moreover, imidazole rings are part
of many natural compounds including alkaloids, purine,
histidine, histamine, and nucleic acids, among others.
Furthermore, imidazole rings are part of the chemical
structure of antibiotics such as megazol (2-amino-5-(1-
methyl-5-nitro-2-imidazolyl)-1,3,4-thiadiazole), which
was first reported as an antimicrobial and later as an ex-
cellent trypanocide (Eseola et al. 2011). Other commer-
cially available therapeutic agents that are imidazole de-
rivatives include the antiprotozoan compounds metronida-
zole and benznidazole and the antifungal compounds
Synthetic protocols
Compound 9—2-(1,4,5-triphenyl-1H-imidazol-2-yl)phenol)
1.2 g of salicylaldehyde, 2.0 g of benzil, 1.3 g of aniline, and
2.2 g of ammonium acetate were refluxed in glacial acetic
acid. After a 3-h reflux, a whitish crystalline product was
formed in the flask, which was immediately filtered and
washed with water, and the product was dried at 40 °C to
obtain compound C-9. (3.3 g, 89%). Mp. 258 °C. Selected
IR peaks (ATR): ν 3063 m (C–H aromatic), 1588s (C=C or
C=N), 1482vs, 749vs, 692vs. 13C NMR (101 MHz, d6-
DMSO) δ 149.36, 149.14, 148.90, 148.11, 145.11, 134.32,
133.64, 131.81, 131.49, 130.28, 129.55, 129.17, 128.92,
127.37, 126.50, 121.21, 118.78, 118.11, 114.22, 113.16,
113.05, 111.68, 56.25, 56.22, 55.93. 13C NMR (101 MHz,
d6-DMSO) δ 157.76, 144.90, 137.03, 134.83, 133.64,
131.74, 131.22, 130.66, 130.09, 129.85, 129.73, 129.19,
129.12, 128.98, 128.92, 127.39, 127.26, 126.57, 118.59,
117.40, 114.32. MS: 388 (100.0%), 388, 267, 194, 165, 77.

Parasitol Res
Scheme 1 Representative
scheme for the synthesis of
imidazole derivatives
Compound 10—1-(1,4,5-triphenyl-1H-imidazol-2-yl)
naphthalen-2-ol)
128.94, 127.45, 126.55, 118.14, 117.34, 113.63, 111.04,
55.15. MS (EI) m/z 418 (M+, 100%): 418, 403 (–methyl),
209, 165, 77.
Benzil (1.20 g, 5.75 mmol), 2-hydroxynaphthalene-1-
carbaldehyde (0.99 g, 5.75 mmol), aniline (0.64 g~0.64 mL,
0.69 mmol), and ammonium acetate (1.33 g, 17.25 mmol)
were treated as in the preparation of C-9, but the purification
was carried out on a silica gel column using 100% chloroform
to remove impurities followed by washing with 100% ethyl
acetate to elute compound C-10 as colorless crystalline blocks
(1.34 g, yield = 53%). Mp. 269 °C. Selected IR peaks (ATR):
Compound 12—4-tert-butyl-2-(1-(4-methoxyphenyl)
-4,5-diphenyl-1H-imidazol-2-yl)phenol)
B e n z i l ( 1 . 0 g , 4 . 8 m m o l ) , 5 - t e r t - b u t y l - 2 -
hydroxybenzaldehyde (0.9 g, 4.8 mmol), p-methoxylaniline
(0.6 g, 4.8 mmol), ammonium acetate (1.1 g, 14.3 mmol),
and 15 mL of glacial acetic acid were reacted as for compound
9 to obtain compound C-12 (1.2 g, 52%). MP = 209 °C.
Selected IR peaks (KBR): ν 3450, 3024, 1606, 1250. 1H
NMR (400 MHz, d6-DMSO) δ 12.87 (s, 1H), 7.43 (d, J =
7.2 Hz, 2H), 7.39–7.27 (m, 9H), 7.25–7.18 (m, 2H), 7.00–
6.97 (m, 2H), 6.90 (d, J = 8.6 Hz, 1H), 6.75 (d, J = 2.4 Hz,
1H), 3.74 (s, 3H), 0.93 (s, 9H). 13C NMR (101 MHz, d6-
DMSO) δ 160.06, 155.71, 145.29, 140.39, 134.32, 133.53,
131.82, 131.49, 130.48, 130.12, 129.87, 129.22, 129.00,
128.94, 127.41, 126.49, 123.54, 116.94, 115.27, 112.75,
55.97, 33.88, 31.29. MS (EI) m/z 474 (M+, 100%): 474,
459, 237, 165, 77.
1
ν 3051 m, 1618 m, 1595s, 1493s, 690vs, 518s. H NMR
(400 MHz, CDCl3) δ 7.59 (t, J = 6.6 Hz, 3H), 7.45 (t, J =
8.5 Hz, 2H), 7.33–7.25 (m, 4H), 7.23–7.13 (m, 4H), 6.95 (q,
J = 6.9 Hz, 5H), 6.88 (d, J = 6.7 Hz, 2H), 6.73 (d, J = 8.8 Hz,
1H). 13C NMR (101 MHz, CDCl3): δ 155.14, 143.93, 136.88,
136.08, 133.71, 132.49, 131.18, 130.17, 130.10, 128.33,
128.21, 128.14, 127.75, 127.53, 127.28, 127.23, 126.82,
126.08, 124.22, 122.85, 119.39, 109.91. MS (EI) m/z 438
(M+, 100%): 438, 421, 361, 267. Anal. Calc. for
C31H22N2O: C, 84.91; H, 5.06; N, 6.39%. Found: C, 84.62;
H, 5.08; N, 6.40%.
Compound
Compound 13—2-(1-(4-methoxyphenyl)
11—4-methoxy-2-(1,4,5-triphenyl-1H-imidazol-2-yl)phenol)
-4,5-diphenyl-1H-imidazol-2-yl)phenol)
Benzil (1.0 g, 4.8 mmol), 2-hydroxy-5-methoxybenzaldehyde
(0.7 g, 4.8 mmol), aniline (0.4 g, 4.8 mmol), ammonium ace-
tate (1.1 g, 14.3 mmol), and 15 mL of glacial acetic acid were
reacted as for C-9 above. The resulting precipitate, compound
C-11, was filtered and purified by column chromatography
(1.1 g, yield = 49%). MP = 217 °C. Selected IR peaks
(KBR): ν 3430, 3057, 1595, 1224. 1H NMR (400 MHz, d6-
DMSO) δ 12.29 (s, 1H), 7.44 (d, J = 6.3 Hz, 7H), 7.34–7.27
(m, 7H), 7.38–7.25 (m, 7H), 7.23 (t, J = 7.3 Hz, 1H), 6.89 (d,
J = 8.9 Hz, 1H), 6.79 (dd, J = 8.9, 3.0 Hz, 1H), 6.20 (d, J =
3.0 Hz, 1H), 3.26 (s, 3H). 13C NMR (101 MHz, d6-DMSO) δ
151.82, 151.23, 144.65, 137.09, 134.78, 133.53, 131.77,
131.34, 130.01, 129.98, 129.84, 129.33, 129.25, 129.00,
Benzil (2.0 g, 9.5 mmol), salicylaldehyde (1.4 g,
9.5 mmol), p-methoxyaniline (1.2 g, 9.5 mmol), ammoni-
um acetate (2.2 g, 28.6 mmol), and 15 mL of glacial
acetic acid were reacted as for compound 9 and purified
by a 1:2:20 mixture of ethyl acetate/dichloromethane/n-
hexane over a silica gel column to obtain compound C-
13 (1.4 g, 34%). MP = 206 °C. Selected IR peaks (KBR):
ν 3061 m (C–H aromatic), 2843 m (methyl), 1604s (C=C
or C=N), 1583s (C=C or C=N), 1512vs, 1247vs, 696vs.
1H NMR (400 MHz, d6-DMSO) δ 12.84 (s, 1H), 7.44 (d,
J = 7.3 Hz, 2H), 7.35–7.26 (m, 9H), 7.23 (d, J = 7.2 Hz,
1H), 7.17 (dd, J = 11.4, 4.5 Hz, 1H), 6.97 (d, J = 7.8 Hz,
1H), 6.92 (d, J = 8.9 Hz, 2H), 6.71 (dd, J = 8.0, 1.4 Hz,

Parasitol Res
1H), 6.57 (dd, J = 11.2, 4.0 Hz, 1H), 3.74 (s, 1H). 13C
NMR (101 MHz, d6-DMSO) δ 159.77, 157.92, 145.10,
134.57, 133.65, 131.77, 131.56, 130.57, 130.30, 130.19,
129.59, 129.17, 128.98, 128.91, 127.36, 126.93, 126.54,
118.62, 117.49, 114.93, 114.19, 55.79. MS (EI) m/z 418
(M+, 100%): 418, 403 (–methyl), 373, 209, 165, 77.
Parasite
In this study, the T. gondii RH strain 2F (ATCC® 50839) was
used. To maintain the parasite, T. gondii was repeatedly pas-
saged in cultures of human foreskin fibroblast (HFF; ATCC®)
cells in a medium composed of Dulbecco’s modified Eagle’s
medium (DMEM; Nissui, Tokyo, Japan), 1% L-glutamine
(200 mM; Gibco, Fisher Scientific, UK), 10% (v/v) fetal bo-
vine serum (FBS; Gibco, Fisher Scientific, UK), and penicillin
and streptomycin (100 U/mL; Biowhittaker, UK). To purify
the parasite for the antiparasite assays, T. gondii-infected HFF
cells were lysed, filtered, and washed three times in fresh
medium.
Compound 14—2-methoxy-6-(1-(3,4-dimethoxyphenyl)
-4,5-diphenyl-1H-imidazol-2-yl)phenol)
Benzil (2.0 g, 9.5 mmol), 2-hydroxy-3-methoxybenzaldehyde
(1.5 g, 9.5 mmol), 3,4-dimethoxybenzenamine (1.6 g,
9.5 mmol), and ammonium acetate (2.2 g, 28.6 mmol) in
glacial acetic acid were reacted as for compound C-9 and
purified by 2:5 of ethyl acetate/n-hexane over a silica gel
column to obtain compound C-14 (3.57 g, 78%). MP =
203 °C. Selected IR peaks (KBR): ν 3064 m (C–H aromatic),
3011 m (C–H aromatic), 2939 m (methyl), 2831 m (methyl),
In vitro assays for inhibition of parasite growth
Imidazole derivatives were prepared in dimethyl sulfoxide
(DMSO, Sigma-Aldrich), to make stock solutions that were
screened for antiparasitic action at various concentrations be-
tween 0.01 and 10 μM.
1
1591s (C=C or C=N), 1512vs, 1244vs, 777vs. H NMR
(400 MHz, d6-DMSO) δ 12.86 (s, 1H), 7.47–7.42 (m, 2H),
7.35–7.27 (m, 7H), 7.22 (t, J = 7.3 Hz, 1H), 7.06 (d, J =
2.2 Hz, 1H), 6.95–6.85 (m, 3H), 6.55 (t, J = 8.1 Hz, 1H),
6.37 (dd, J = 8.2, 1.2 Hz, 1H), 3.80 (s, 3H), 3.74 (s, 3H),
3.60 (s, 3H). 13C NMR (101 MHz, d6-DMSO) δ 149.36,
149.14, 148.90, 148.11, 145.11, 134.32, 133.64, 131.81,
131.49, 130.28, 129.55, 129.17, 128.92, 127.37, 126.50,
121.21, 118.78, 118.11, 114.22, 113.16, 113.05, 111.68,
56.25, 56.22, 55.93. MS (EI) m/z 478 (M+, 100%): 478,
461, 449, 435, 165.
The in vitro assays for inhibition of parasite invasion
and growth were performed as described previously
(Adeyemi et al. 2017b). In brief, the test compounds in
the cell culture medium (as highlighted above) were
added to newly purified parasites in growing HFF cells,
while the medium lacking the test compounds served as
the negative drug control. After a 72-h incubation in an
atmosphere of 37 °C and 5% CO2, parasite viability was
determined by assaying for galactosidase activity using a
Promega Beta-Glo reagent kit (Madison, USA) and fol-
lowing the manufacturer’s instructions. The luminescence
signal was acquired on a Multi Detection System plate
reader (GloMax® Promega Japan). The final concentra-
tion of DMSO in the cultures was < 0.1% (v/v). To vali-
date the assay, pyrimethamine was included as reference
drug(s). The biological evaluation was in triplicate and
independently replicated thrice. The Nunc 96-well optical
bottom plate (Fisher Scientific, Pittsburgh, USA) was
used for the assay unless otherwise indicated.
Compound 15—2-(1-phenyl-1H-phenanthro[9,10-d]
imidazol-2-yl)phenol)
Phenanthrenequinone (2.1 g, 10.0 mmol), 2-
hydroxybenzaldehyde (2.2 g, 10.0 mmol), aniline (3.7 g,
40.0 mmol), and ammonium acetate (3.1 g, 40.0 mmol) in
glacial acetic acid were reacted as for compound C-9 and
purified by 2:5 of a dichloromethane/n-hexane mixture as el-
uent over a silica gel column to obtain compound C-15 (3.2 g,
83%). MP = 178 °C. Selected IR peaks (KBR): ν 3051 m (C–
H aromatic), 1584s (C=C or C=N), 1480vs, 753vs. 1H NMR
(400 MHz, d6-DMSO) δ 11.88 (s, 1H), 8.95 (s, 1H), 8.89 (d,
J = 8.4 Hz, 1H), 8.62 (dd, J = 7.9, 0.9 Hz, 1H), 7.80 (t, J =
7.3 Hz, 1H), 7.75–7.67 (m, 6H), 7.59–7.54 (m, 1H), 7.34 (t,
J = 7.4 Hz, 1H), 7.27–7.20 (m, 1H), 7.06–7.00 (m, 2H), 6.98–
6.92 (m, 1H), 6.70–6.61 (m, 1H). 13C NMR (101 MHz,
d6-DMSO) δ 157.86, 149.43, 138.53, 135.23, 131.41,
130.84, 130.81, 129.25, 129.17, 129.02, 128.18, 128.13,
127.29, 127.20, 126.42, 126.35, 125.87, 125.01, 124.23,
122.68, 122.31, 120.73, 118.71, 117.15, 115.65. MS (EI) m/
z 386 (M+, 100%): 386, 369, 266, 193, 178, 77.
The cytotoxic potential of imidazole derivatives
The HFF cells were cultured in DMEM as indicated
above. At confluence, the cells were sub-cultured into
96-well plates at a concentration of 1 × 105 cells per well.
After a 72-h incubation at 37 °C and 5% CO2, the imid-
azole compounds (between 0.01 and 10 μM) in the cell
culture medium were added, while the culture medium
lacking imidazole derivatives served as the negative drug
control. To validate the assay, staurosporine (1 μM, final
concentration) was used as a positive control drug. After a
72-h incubation, the colorimetric determination of cell vi-
ability (Promega CellTitre-Aqueous One kit; Madison,

Parasitol Res
USA) was at 490 nm on a microplate reader (MTP 500;
Corona Electric, Hitachinaka, Japan). The biological eval-
uation was carried out in triplicate and independently rep-
licated thrice.
Molecular modeling studies: descriptors and potential
bioactivities evaluation
To explore the antiparasitic potential of the synthesized
compounds, molecular modeling was used to evaluate
the physicochemical characteristics (structural profile)
and potential bioactivities (enzyme inhibition) using on-
line chemoinformatics tools (http://www.molinspiration.
com). The compounds were optimized on the Chemdraw
software (Lipinski et al. 2001) and the simplified
molecular-input line-entry system (SMILES) format fed
into the Molinspiration program. The structural profile
of compounds such as the partition coefficient (octanol/
water), total polar surface area, molecular weight, molec-
ular volume, the number of hydrogen bond acceptors/do-
nors, number of rotatable bonds, and degree of violation
of Lipinski’s rule (Lipinski 2004; Atolani et al. 2014) for
drug-likeness were evaluated following reported standard
procedures (Rocha-Roa et al. 2018; Flores-Holguín and
Glossman-Mitnik 2004). Likewise, the bioactivity score
based on ion channel modulation, potential as GPCR li-
gand, kinase inhibition, nuclear receptor interaction, pro-
tease inhibition, and enzyme inhibition were also calcu-
lated to give insight into the biological potential of the
compounds (Lipinski 2004; Atolani et al. 2014). The bio-
activity measurement is premised on Lipinski’s “rule of
five,” which evaluates the similarity of the molecular
properties and structural features of a molecule to those
of known drugs.
Immunofluorescence assays (IFA)
Invasion
The invasion assays were carried out as reported elsewhere
(Adeyemi et al. 2017b). In brief, purified parasites [1 × 105]
with the various imidazole-based compounds (at double IC50
for each compound) in a culture medium were added to HFF
monolayers growing on a coverslip. The medium only served
as negative drug control. After a 1-h incubation at 37 °C, the
cells were washed and stained on ice with polyclonal rabbit
anti-T. gondii antibodies [1:1000] (Bio-Rad Laboratories,
USA) in washing buffer (2% FCS-PBS) for 30 min.
Thereafter, the cells were fixed with 4% paraformaldehyde
in PBS and permeabilized with ice-cold methanol. Then, the
cells were primarily stained with anti-SAG1 antibodies
[1:1000] (Hytest, Finland), while Alexa Fluor 488 anti-
mouse antibody, Alexa Fluor 594 anti-rabbit antibody, and
DAPI [1:1000] were used for secondary staining.
Visualization of the stained cells was performed on a fluores-
cence microscope (Nikon Eclipse E400, Tokyo, Japan), with
at least 50 microscopic fields randomly selected. Rabbit anti-
T. gondii antibodies were used to detect the extracellular par-
asites, while anti-SAG1 antibodies were used to detect the
total parasites.
Ranking of compounds based on toxicological risks
Intracellular doubling
Furthermore, the imidazole compounds were ranked based on
toxicological risks prediction using PROTOX II modeling
(http://tox.charite.de/protox_II/index.php?site=compound_
input). The PROTOX II allows for sorting or ranking
according to possible toxicological risks, by comparing input
compounds with dataset compounds using the SMILES
codes.
Intracellular doubling assays were carried out as previously
reported (Adeyemi et al. 2017b). In brief, purified parasites
(1 × 105) were added to HFF monolayers growing on a cov-
erslip. After a 1-h incubation at 37 °C, the cells were washed
three times with culture medium to remove non-invaded par-
asites. Fresh culture containing imidazole-based compounds
was added while medium lacking imidazoles served as the
negative drug control. After 24-h incubation at 37 °C, the cells
were fixed with 4% paraformaldehyde in PBS and perme-
abilized with ice-cold methanol. Thereafter, a blocking buffer
(2% BSA in 0.1% Tween 20-PBS) was added to the cells and
after 1-h incubation at room temperature, the primary staining
was done with anti-SAG1 antibodies (1:1000) (Hytest,
Finland) in a blocking buffer, while the Alexa Fluor 594 goat
anti-mouse antibody and DAPI were used for secondary stain-
ing. The stained cells were viewed on a fluorescence micro-
scope (Nikon Eclipse E400, Tokyo, Japan). To count the
parasitophorous vacuoles and estimate the number of
tachyzoites per vacuole, at least 50 microscopic fields were
randomly viewed.
Statistical analysis
A one-way ANOVA (GraphPad, San Diego, CA, USA) was
used to analyze the results. The data are presented as the mean
standard error of mean (SEM). The Dunnet post hoc test was
used to compare the groups, and the mean values were signif-
icant at p < .05. The concentration that caused inhibition of
T. gondii growth or cellular toxicity by 50% (EC50 and/or
IC50) was estimated from a dose–response curve of the com-
pound concentrations versus the viability of either the parasite
or the host cell. The curve was fitted using a non-linear regres-
sion analysis.

Parasitol Res
Results and discussion
substituents. Furthermore, strong intra- and intermolecular
interactions are often observed for these azole molecules
via hydrogen bonding and pi–pi stacking interactions, as
was displayed by the dimeric pairing in the structure of
compound C-10 (Eseola et al. 2019).
Chemistry of imidazole derivatives
Scheme 1 shows the synthetic routes for the imidazole com-
pounds depicted in Scheme 2. The azole derivatives were
designed to allow for modifications based on electronic and
steric considerations. Within the bis-imidazoles, phenyl-
substituted 1H-imidazoles, or thiophene-imidazoles, the elec-
tronic differential was designed to allow for electron-releasing
or electron-withdrawing exchanges. Thus, the intermolecular
interaction potential of compounds is likely to differ
considerably.
The identities of the synthesized molecules were
established using mass spectrometry and/or elemental
analysis in addition to spectroscopic data (NMR and
FTIR). X-ray structural confirmations were also obtained
for representative members of each molecular group, as
presented in Fig. 1. Based on previous studies (Eseola
et al. 2012, 2018, 2019), it is important to mention that
molecules bearing unsubstituted imidazole protons pos-
sess notably different electronic characteristics compared
with their analogues that have phenyl or alkyl
Biological assays
The in vitro screening of synthetic or natural compounds
is hinged on the fact that the screening of a vast array of
compounds may allow for the identification of likely lead
candidates for neglected tropical diseases (Adeyemi et al.
2017a, b, c, 2018a, b, c, 2019). In this study, we synthe-
sized a new series of imidazole derivatives and evaluated
these compounds (Scheme 2) for anti-T. gondii properties.
The synthesis of the new series of imidazole derivatives
followed our recent report (Adeyemi et al. 2017a), which
showed that compounds containing an imidazole ring are
promising antiprotozoal agents. In the current study, the
antiparasite assays revealed that the imidazole derivatives
strongly restricted the growth of T. gondii (Fig. 2), with
the EC50 ranging between 0.003 and 16 μM (Table 1).
Meanwhile, the estimated selectivity index showed
Scheme 2 Imidazole compounds
according to structural grouping:
(a) bis-imidazoles, (b) imidazole-
phenols, and (c) thiophene-azoles

Parasitol Res
Fig. 1 Ortep structural plot for probe molecules C-7 (left), C-10 (middle), and C-25 (right). Some hydrogen atoms and co-crystallized solvent molecules
have been omitted for clarity
appreciable selective action (< 1- to > 27,666-fold activi-
ty) towards the parasite versus the host cell, suggesting
specific antiparasitic potential by the imidazole deriva-
tives. The imidazole derivatives significantly suppressed
the parasite growth with an average of ≥ 75%, ≥ 90%, and
≥ 91% for bis-imidazoles, phenyl-substituted 1H-imidaz-
oles, and thiopene-imidazoles, respectively (Table 2).
These antiparasitic activities by imidazole compounds
not only compared favorably with that of the reference
drug, pyrimethamine but also showed no apparent toxicity
to the host cells, even up to the highest concentration,
10 μM, used in this study. While most of the imidazole
compounds showed good selective action against the par-
asite versus the host cells, phenyl-substituted 1H-imidaz-
oles (C-10, C-11, C-12, C-14, and C-18) and thiopene-
imidazoles (C-20 and C-21) showed the most specific
action against the parasite. Taken together, our current
findings are consistent with studies that demonstrated that
compounds containing an imidazole ring possess
antiprotozoal properties (Coura and Castro 2002; Flores-
Holguín and Glossman-Mitnik 2005; Liesen et al. 2010).
In addition, Liesen, et al. (Berkelhammer and Asato 1969)
reported that a series of thiadiazole derivatives showed
promising anti-T. gondii activity when compared with a
standard drug, sulfadiazine. The findings indicate that
compounds containing imidazole rings are a potential
source of alternative antiparasitic agents. Furthermore, re-
ports have shown that imidazole-based compounds are
active against T. gondii likewise as other compounds
based on similar chemical scaffold (Dzitko et al. 2014;
Tessmann et al. 2017).
C-7, C-10, C-11, C-12, C-14, C-18, C-20, and C-21. The SI
was estimated as the ratio of the host toxicity (EC50) to para-
site toxicity (IC50).
Imidazole derivatives affect the invasion
and intracellular replication cycle in T. gondii
To preliminarily establish the mechanistic antiparasitic ac-
tion of the imidazole derivatives, we determined whether
the T. gondii lytic cycle was affected. The lytic cycle of
T. gondii is a repeated process of invasion, replication,
and egress. Therefore, we used IFA to determine whether
the imidazole derivatives affected the invasion and intra-
cellular doubling of the parasite. The data revealed a dra-
matic decrease in the capacity of the parasite to invade
monolayers of HFF cells by 80 to 40% (Fig. 3a).
Similarly, the imidazole derivatives appreciably decreased
the number of parasites per vacuole (Fig. 3b), resulting in
a > 70% reduction in average parasitic replication per vac-
uole (> 100 vacuoles counted per experiment) compared
with the control (Fig. 3c). Furthermore, imidazole deriva-
tives depleted the number of parasites per vacuole (Fig.
3d); < 8 parasites per vacuole for the imidazole deriva-
tives compared with the negative drug control, in which
the parasites per vacuole increased steadily.
Representative fluorescent images of the IF staining are
presented in Figs. 4 and 5. Taken together, our findings
established that the imidazole derivatives significantly in-
fluenced the invasion and replication processes of the
T. gondii lytic cycle, thus yielding an early perspective
about how imidazole derivatives restrict parasite growth.
Additionally, the depleted parasite vacuoles and parasites
per vacuole suggest that the imidazole derivatives arrested
the replication of T. gondii rather than slowed it. Also, it
is unlikely that the depletion in parasite vacuoles was a
result of the parasite egressing prematurely from the host
Moreover, several currently available therapeutic agents,
such as metronidazole, benznidazole, megazol, miconazole,
and ketoconazole, contain imidazole rings.
For further experiments, only imidazole compounds with
greater selectivity index ≥ 100 were selected. These include

Parasitol Res
Fig. 2 Dose–response curve after
a 72-h treatment with imidazoles.
Data are expressed as the mean
standard error of mean (SEM)
Bis-imidazoles
100
80
60
40
20
0
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
Pyrimethamine
-2
-1
0
1
mM
Phenyl-substituted 1H-imidazoles
and oxazoles
100
80
60
40
20
0
C-9
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
-2
-1
0
1
mM
C-19
Pyrimethamine
Thiopene-imidazoles
100
80
60
40
20
0
C-20
C-21
C-22
C-23
C-24
C-25
C-26
Pyrimethamine
-2
-1
0
1
mM
cells; the host cells were apparently intact and extracellu-
lar parasites were not observed. Taken together, the data
indicate that imidazole derivatives may be clearing the
parasites through an as yet unknown host-mediated
mechanism.
Structure–activity relationships: anti-T. gondii activity
Exploring the structure–activity relationship (SAR), the
electron-donating or electron-withdrawing substituents on
the phenyl moieties as well as the steric bulkiness due to the

Parasitol Res
Table 1 Estimation of EC50 and
selectivity index for imidazole
derivatives
Compounds
EC50 (anti-Toxoplasma gondii) μM
IC50 (host cells—HFF) μM
Selectivity
index (SI)
C-1
4.334
7.617
1.285
14.81
1.103
0.814
0.038
0.915
0.302
0.034
0.123
0.014
0.182
0.377
0.753
0.113
0.519
0.003
0.702
0.024
0.006
0.003
0.008
0.183
1.459
0.139
1.906
49
260
20
57
60
10
4
11
C-2
34
C-3
15
C-4
3
C-5
54
C-6
12
C-7
105
2
C-8
2
C-9
5
16
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
C-22
C-23
C-24
C-25
C-26
Pyrimethamine
40
105
36
0.1
254
ND
ND
ND
83
ND
85
10
ND
ND
20
72
2
1176
853
2571
1
673
ND
ND
ND
27,666
ND
3541
1666
ND
ND
109
49
14
10
5
The EC50 and/or IC50 were estimated from a dose–response curve of the compound concentrations versus the
viability of either the parasite or the host cell respectively. The curve was fitted using a non-linear regression
analysis on a GraphPad Prism 5
ND not determined
presence of aromatic rings may play a role in the differential
activity towards the parasite as well as towards the host cells.
While electronic effects could be considered to play a role in
the action of the tested compounds, the electron-donating or
electron-withdrawing capacity of the aromatic substituents did
not appear to dramatically alter the anti-T. gondii activity,
although it might have contributed to differential host cell
toxicity, thereby giving rise to the low or high selective anti-
parasitic action. Additionally, the hydrophobic nature of the
phenyl rings might have been responsible for differential ac-
tivity exhibited by the compounds.
The compounds C-1, C-2, C-3, C-4, and C-5 that bore one
hydrophobic o-CH3 on their central rings with large cyclic
hydrocarbon moieties apparently confer poorly specific anti-
T. gondii activity on the molecules (Table 1), probably due to
the high molecular weights of the compounds. This suspicion
was supported by the results obtained from the molecular
description analysis (Table 3). The bis-imidazoles apparently
had too high a partition coefficient (miLogP ˃ 5.0) and mo-
lecular weight (˃ 500), which might have limited their bio-
availability and absorption by the parasite membrane. A high
logP value is an indication of increased hydrophobicity, which
retards the penetration of the compound through the parasite
membrane, thereby reducing the concentration of compounds
reaching the target action site. The high carbon-to-hydrogen
ratio may also have contributed to reducing the antiparasitic
activities, but with increased toxicity to the host cells.
Increased hydrophobicity has been reported to reduce the bio-
activity of other compounds (Lavorato et al. 2017; Rutkowska
et al. 2017). The presence of the hydrophilic o–OCH3 groups
on C-2 seems not to have enhanced the antiparasitic activity
but affected the selectivity by increasing the toxicity to the

Parasitol Res
Table 2 Parasite inhibition and
host cell viability
Compounds
Parasite inhibition (%)a
Parasite average inhibition (%)
74.90
Host cell viability (%)b
C-1
64.30 2.68
49.12 4.29
84.48 0.65
37.58 0.37
85.73 1.39
96.07 0.64
87.65 2.58
94.29 0.89
92.53 0.58
87.77 2.59
87.57 5.03
97.01 4.91
91.77 6.49
91.86 7.35
88.52 4.84
89.48 2.27
92.83 5.46
92.60 1.01
83.72 5.48
94.50 3.81
92.26 7.10
92.02 7.94
93.09 5.96
93.72 8.83
83.59 6.17
85.78 1.06
108.84 9.66
109.51 14.06
110.57 7.98
107.76 6.56
106.30 6.48
103.31 2.30
6.30 1.35
C-2
C-3
C-4
C-5
C-6
C-7
C-8
122.86 11.93
97.90 3.39
C-9
90.51
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
C-22
C-23
C-24
C-25
C-26
96.65 2.30
128.40 23.54
102.31 11.85
117.92 17.96
90.71 1.06
137.62 23.01
149.81 16.53
104.41 6.90
146.42 4.26
108.41 13.90
104.77 3.55
124.90 5.75
121.16 12.88
114.98 7.74
89.02 1.44
90.71
110.19 3.33
104.82 10.11
a Parasite growth inhibition values from screening with RH-2F at 10 μM. DMSO control was calculated as 0%
inhibition, and 10 μM pyrimethamine control was calculated as 100% growth inhibition
b Host cell viability values from preliminary screening with HFF cells at 10 μM concentration. DMSO control was
calculated as 100% cell viability
host cells. C-3, an analog of C-1, was found to possess im-
proved selectivity to the parasite owing to the addition of two
hydrophobic phenyl groups.
with low selectivity, apparently as a result of its molecular size
and polarity. C-6, a very polar compound, was the compound
with the smallest molecular weight, molecular volume, and
partition coefficient (Table 3). Small molecules are renowned
for having greater biological activity. Moreover, phenolic
compounds are well reported as having strong interactions
with biological molecules because of their potential to easily
form phenoxide ions (Qasim et al. 2017). Therefore, the high
carbon-to-hydrogen ratio in C-7 to C-9 may partially account
for the selectivity observed. It is no surprise that C-7 had low
selectivity in that it is the only compound that violated as
many as three of Lipinski’s rules (Table 3).
On the other hand, the electron-donating capacity and re-
duced bulkiness of C-10 and C-11 may have enhanced their
selective action against the parasite versus the host cell when
compared with the others in the same group (C-12 to C-14).
The presence of an alkyl substituent in C-12 could have weak-
ened its electron-donating capacity, thus making it less reac-
tive than C-11 or C-14. These observations were consistent
Fused aromatic rings (phenanthrene moieties), which re-
duced the number of rotatable bonds to 2 (Table 2) and there-
by conferred increased rigidity in C-4, seem to have increased
host cell toxicity while decreasing selective activity towards
the parasite compared with C-1 or C-5. The addition of the
phenanthrene moiety might have conferred electron-
withdrawing capacity on the bis-imidazoles, and this may
have enhanced the compounds’ reactivity with the cellular
membrane or macromolecules. This is consistent with our
recent report (Flegr et al. 2014), which found that fusing aro-
matic rings increased bulkiness and enhanced host cytotoxic-
ity. C-4 and C-5 are structurally the same except for the addi-
tional phenyl groups symmetrically attached to two of the four
heteroatoms (nitrogen), thereby improving the flexibility of
the molecule for interactions with the parasite. The phenolic
derivative C-6 had significant anti-T. gondii activity although

Parasitol Res
b
a
Parasite intracellular replication
24 hpi
Parasite invasion rate
1 hpi
100
15
80
60
40
20
0
b
g
10
5
g
g
0
7
0
2
4
8
1
2
l
ol
7
2
1
2
1
11
1
1
1
20
r
o
C-
10
11
1
14
18
20
-
tr
C-
C-
C-
C-
C-
C-
C-
C-
nt
Co
C-
C-
C-
C
C-
C-
C-
n
o
C
c
d
Parasite per vacuole
24 hpi
Parasite intracellular replication
24 hpi
100
80
60
40
20
0
100
80
60
40
20
0
1
2
4
8
16
32
g
l
7
0
1
0
-2
7
1
2
4
8
o
1
12
14
18
21
10
20
21
rol
nt
Co
-1
-1
-1
-1
-1
tr
C-
C-
C
C-
C-
C-
C-
C
C-
C-
C
C
C
C
C-
C-
on
C
Fig. 3 Immunofluorescence (IF) staining of T. gondii-infected HFF cells
that were treated with imidazole derivatives. a Freshly purified parasite
suspension and imidazole derivatives were added to growing HFF mono-
layers on coverslips. Invasion was allowed for 1 h before IF staining. b
Average parasites per vacuole following treatment with imidazole deriv-
atives. Freshly purified parasite suspensions were added to growing HFF
monolayers on coverslips. Invasion was allowed for 1 h, after which the
infection medium was replaced with fresh medium containing imidazole
derivatives. IF staining of intracellular parasites was carried out after 24-
h. c Percentage replication of intracellular parasite. d Parasite numbers
based on 100 parasitophorus vacuoles after treatment with imidazole
derivatives for 24-h incubation. Data are expressed as the mean stan-
dard error of mean (SEM). Experiment was repeated three times indepen-
dently. β is significant at p < .001 while γ is significant at p < .0001
relative to the control
Fig. 4 Representative fluorescent images showing replicating tachyzoites (RH-2F strain) only at 24-h incubation with either culture medium only
(control) or imidazole derivatives

Parasitol Res
Fig. 5 Representative fluorescent images showing replicating tachyzoites (RH-2F strain) with nuclear staining (DAPI) at 24-h incubation. IF staining
was performed following treatment with imidazole derivatives. Each experiment was performed three times independently.
with the molecular descriptions and predicted bioactivities
(Tables 3 and 4). C-10 and C-11 were the only two
compounds that revealed potential activity as predicted ion
channel modulators with probable high activities as GPCR
Table 3 Molecular descriptors
for the synthesized compounds
Compounds ID
MiLogP
TPSA (Å2)
MW (Da)
nON
nOHNH
NoV
nRotB
MV (A3)
C-1
8.90
5.46
9.91
8.50
9.78
1.15
8.44
9.77
7.56
8.53
6.79
7.89
6.56
5.72
7.15
5.20
6.77
8.29
4.94
5.55
5.90
8.04
6.93
7.64
5.62
4.98
68.34
105.27
50.76
68.34
50.76
84.74
88.56
70.99
35.49
35.49
44.73
44.73
44.73
63.20
35.49
41.49
41.49
41.49
44.28
24.05
24.05
15.27
15.27
15.27
33.73
21.26
582.96
703.07
747.25
578.93
743.22
286.42
598.96
763.25
416.69
470.79
446.72
502.83
446.72
506.77
414.68
333.52
389.62
445.73
334.55
336.59
401.46
483.60
402.69
481.59
462.74
321.53
5
9
5
5
5
5
6
6
3
3
4
4
4
6
3
3
3
3
3
2
2
2
2
2
4
2
5
5
3
5
3
5
6
4
2
2
2
2
2
2
2
2
2
2
3
2
2
1
1
1
1
1
2
2
2
2
2
0
3
2
1
1
1
2
1
2
1
1
1
1
0
1
1
1
1
1
1
0
6
10
8
2
4
4
6
8
4
4
5
6
5
7
2
1
2
3
3
3
3
4
2
2
4
1
616.16
718.45
796.91
594.58
775.33
291.32
624.20
804.96
445.49
501.90
471.06
537.27
471.06
522.20
434.70
340.90
407.11
473.33
355.11
348.18
349.50
439.88
411.18
429.09
462.32
317.38
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
C-22
C-23
C-24
C-25
C-26
miLogP calculated partition coefficient, TPSA total polar surface area, MW molecular weight, nON number of
hydrogen bond acceptors, nOHNH number of hydrogen bond donors, noV number of violations, nrotb number of
rotatable bonds, MV mean molecular volume

Parasitol Res
Table 4 Predicted bioactivity potentials of the synthesized compounds
In addition, the introduction of the thiophene group (C-20
to C-22) induced a significant change in the selectivity against
T. gondii versus the host cells. Interestingly, bromination of
the thiophene at position C2 in C-21 seems to have had a
significant effect, in that the host cytotoxicity increased. It
may be that the introduction of bromine (Br) at position 2 of
the thiophene enhanced its activity against T. gondii but re-
duced its selectivity since C-20, which lacked the bromine
atom, showed lower host toxicity. Moreover, bromine is elec-
tronegative, and its presence might cause an electron-
withdrawing effect, thus making the compound more reactive.
Because bromine has a high electron-withdrawing potential,
many brominated (or halogenated) compounds are known to
be bioactive (Tessmann et al. 2017). C-23 to C-26, which had
close structural similarities, also had moderate antiparasitic
activities as well as similar selectivity indexes. This implies
that the structural modification of the side chains had little or
no effect on antiparasitic action. However, C-25 showed the
highest potential to be a GPCR ligand, proteinase, and enzyme
inhibitor of all the examined compounds.
It is interesting to observe that each of the five compounds
(C-10, C-11, C-18, C-20, and C-21) that exhibited significant
antiparasitic activity with high selectivity against the parasite
versus the host cells had only one violation of Lipinski’s rule
of drug-likeness. Additionally, these same compounds all had
positive enzyme inhibition values based on the predicted bio-
activity score. These five compounds only slightly violated
the partition coefficient threshold and complied with all the
other rules as indicated. The other compounds, which violated
other rules such as molecular weight, molecular size, total
polar surface area, number of rotatable bonds, and number
of hydrogen bond donors/acceptors, showed lower selective
activity against T. gondii versus the host cells. This implies
that only the partition coefficient threshold may be violated for
drug candidacy to be maintained. Potential enzyme inhibition
also seems to be a key factor to consider in identifying lead
candidates for early drug development for T. gondii therapy.
This discovery may guide future studies in designing drug
candidates for use against T. gondii.
Compounds ID GPCR ligand ICM KI
NRL PI
EI
C-1
0.09
− 0.52
− 1.10
0.10
− 1.10
0.25
0.08
− 1.25
0.28
0.29
0.30
0.28
0.29
0.38
0.29
0.19
0.16
0.16
0.30
0.15
0.08
0.12
0.29
0.13
0.41
0.16
− 0.36 − 0.25 − 0.15 0.17 − 0.06
− 1.41 − 1.11 − 1.08 − 0.21 − 0.86
− 2.22 − 1.84 − 1.87 − 0.66 − 1.53
− 0.34 − 0.28 − 0.16 0.13 − 0.04
− 2.20 − 1.86 − 1.88 − 0.69 − 1.51
0.00 − 0.21 − 0.05 0.16 0.27
− 0.45 − 0.27 − 0.17 0.16 − 0.09
− 2.44 − 2.02 − 2.05 − 0.79 − 1.70
0.17 − 0.01 0.03 0.30 0.20
0.20 − 0.03 0.08 0.27 0.22
0.20 − 0.02 0.05 0.37 0.23
0.15 − 0.12 0.01 0.30 0.21
0.18 − 0.02 0.03 0.32 0.21
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
C-22
C-23
C-24
C-25
C-26
0.19 0.11
0.11
0.42 0.28
0.21 − 0.06 0.01 0.25 0.24
0.13 − 0.18 0.28 0.19 0.31
0.01 − 0.29 0.27 0.12 0.31
0.03 − 0.29 0.26 0.12 0.29
0.17 − 0.01 0.05 0.28 0.22
− 0.04 − 0.21 − 0.19 0.19 0.09
− 0.11 − 0.28 − 0.26 0.09 0.02
− 0.07 − 0.21 − 0.24 0.14 0.03
− 0.07 − 0.24 − 0.31 0.33 0.24
− 0.04 − 0.26 − 0.25 0.08 0.07
0.09 − 0.12 − 0.15 0.47 0.35
− 0.23 − 0.45 − 0.12 0.26 0.33
ICM ion channel modulation, KI kinase inhibition, NRL nuclear receptor
ligand, PI protease inhibition, EI enzyme inhibition
ligands and enzyme inhibitors. Their selectivity could have
also been enhanced by their low total polar surface area
(TPSA) and moderate flexibilities (the degree of rotation of
the molecules). The low anti-T. gondii activity of C-9 with
great structural similarity to C-11, which had higher antipara-
sitic action and selectivity, cannot be explained yet. The mod-
erate antiparasitic activities of C-15 to C-17 may be due to the
fused phenanthrene rings (to the oxazole), which could facil-
itate dissociation of the heterocyclic ring, making the charged
electronegative atoms (O and N) more available for membrane
interactions via covalent and hydrogen bonds. Further, it ap-
pears that the presence of oxazole in C-18 greatly enhanced
the selective action against the parasite when compared with
the other phenyl-substituted 1H-imidazole compounds. In ad-
dition, the predicted nuclear receptor ligand and enzyme inhi-
bition potentials suggest a possible mode of action of the
compounds (Table 3). The low antiparasitic activity of C-19
may indicate that the stearic bulky groups shielded the imid-
azole ring from interacting with the parasite.
SAR and prediction of toxicological risks: molecular
modeling studies
The combination of the various structural features of a com-
pound is known to govern its bioactivity. The properties of a
molecule such as hydrophobicity, electronic distribution, hy-
drogen bonding characteristics, molecule size, and flexibility
determine the absorption (bioavailability), distribution, me-
tabolism (reactivity), and excretion (ADME) of that molecule
(Atolani et al. 2014; Rocha-Roa et al. 2018). Therefore, ap-
plying the online cheminformatics tool, Molinspiration,
afforded the determination of the properties related to follow-
ing Lipinski’s rule of five, according to which the optimum

Parasitol Res
properties are the partition coefficient (miLogP) of < 5.0, mo-
lecular weight ≤ 500, hydrogen bond acceptors 10, and donors
5. The rule was further expanded to include the number of
rotatable bonds (ten or fewer) and a total polar surface area
(TPSA) equal to or less than 140 Å2 as ideal parameters for
drug-likeness. Furthermore, compounds violating more than
one rule demonstrate a reduced oral bioavailability potential
(Flores-Holguín and Glossman-Mitnik 2004). The result of
molecular modeling studies (Tables 3 and 4) provided addi-
tional insights into the physicochemical features of the com-
pounds and buttressed the SAR in explaining the in vitro anti-
T. gondii studies.
Following Lipinski’s rules, of the class A series, the bis-
imidazoles, only compound C-6 with the smallest molecular
size and volume did not violate more than one rule (Table 3).
This may account for the generally low bioactivity score for
the class (Table 4). Of particular interest is compound C-7,
which had three violations (partition coefficient, molecular
weight, and number of hydrogen bond donors). This finding
correlates with an extremely low bioactivity score for C-7
(Table 3). In classes B and C, series C-9 to C-23, only
compounds C-12 and C-14 with a relatively high miLogP
(˃ 5) and molecular weight (˃ 500 Da) violated two
Lipinski’s rules, while the others violated either one or none.
Compounds C-6, C-9, C-10, C-11, C-12, C-13, C-14, C-15,
C-19, C-23, and C-25 showed high potentials as GPCR li-
gands, while of the entire series only C-10 and C-11 had a
significant predicted ion channel modulation potential
(Table 4). However, only C-16, C-17, and C-18 showed good
bioactivity scores as nuclear receptor compounds and high
scores as enzyme inhibitors. This could be the result of the
phenanthro[9,10-d] moiety, which was uniquely present in
their structures. C-14 and C-25 exhibited the highest potential
protease inhibition scores of 0.42 and 0.47, respectively. C-25
showed the highest potential on the basis of having GPCR
ligand and enzyme inhibitor scores of 0.41 and 0.35,
respectively.
In addition, the imidazole compounds were ranked based
on toxicological risks prediction using PROTOX II modeling.
The oral toxicity prediction results showed that majority of the
compounds belong to classes 3, 4, and 5 except C-26 which is
class 6 (Table 5). The prediction accuracy ranged between
Table 5 Sorting of imidazole
Compound IDs
Toxicity class
LD50a (mg/kg)
Average similarity
Prediction accuracy
compounds based on possible
toxicological risks using
PROTOX II
C-1
3
4
4
5
4
4
4
4
4
4
4
4
4
4
4
4
4
4
3
3
3
4
4
4
5
6
300
1190
2000
5000
1000
1000
2000
2000
2000
2000
2000
2000
2000
2000
595
70.09
100
69.26
100
C-2
C-3
62.09
61.18
60.58
44.76
68.47
52.07
52.80
52.46
53.54
53.76
55.58
50.66
61.77
76.53
74.23
72.32
76.15
54.57
50.06
40.46
50.45
46.37
48.26
79.22
67.38
68.07
68.07
54.26
68.07
67.38
67.38
67.38
67.38
67.38
67.38
67.38
68.07
69.26
69.26
69.26
69.26
67.38
67.38
52.26
67.38
54.26
54.26
69.26
C-4
C-5
C-6
C-7
C-8
C-9
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
C-22
C-23
C-24
C-25
C-26
1600
1600
1600
300
300
300
2000
770
1000
3420
10,000
a Oral toxicity

Parasitol Res
Table 6 Probability for toxicity endpoints by modeling on PROTOX II
HPTa
CCGa
IMMTa
MUTGa
CYTOXa
AhRa
MMPa
ARTa
PP53
a
Compound IDs
C-1
–
–
–
0.72
0.75
0.50
0.72
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
0.52
–
–
–
–
–
–
C-2
–
–
–
–
–
–
–
C-3
0.54
0.55
0.53
–
0.52
–
–
0.51
–
–
C-4
–
–
0.61
–
–
C-5
0.52
–
–
–
–
–
–
C-6
–
–
–
–
–
–
C-7
0.54
0.54
0.56
–
–
–
0.69
–
–
–
–
–
C-8
–
–
–
–
–
–
C-9
0.53
–
–
–
–
–
–
–
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
C-22
C-23
C-24
C-25
C-26
–
–
–
–
–
–
0.51
0.54
0.53
0.71
–
0.55
–
–
–
–
–
–
–
–
0.51
0.54
0.51
0.52
0.54
–
0.55
0.50
0.61
–
–
–
–
–
0.52
–
0.59
0.58
0.53
–
–
–
–
0.54
0.55
–
–
–
–
0.55
0.70
0.90
–
–
–
–
–
–
0.53
0.53
–
–
–
–
–
–
–
–
0.55
0.56
0.60
0.56
0.52
0.55
–
–
0.77
0.76
0.60
–
–
–
–
–
–
–
–
–
–
–
–
–
0.51
0.51
0.61
0.53
0.52
–
–
–
–
–
–
–
0.60
–
–
–
0.68
0.59
0.65
–
–
–
–
–
–
–
0.53
–
–
–
0.52
–
–
0.61
0.50
–
–
–
– means no detectable binding
HPT hepatoxicity, CCG carcinogenicity, IMMT immunotoxicity, MUTG mutagenicity, CYTOX cytotoxicity, AhR aryl hydrocarbon receptor, MMP
mitochondrial membrane potential, ART aromatase, PP53 phosphoprotein (tumor suppressor) p53
a Probability
52.26 and 100%, while the average similarity lies between
40.46 and 100%. For the toxicity endpoint modeling, the pre-
diction results showed that 65.39% (17) of the imidazole com-
pounds may be potentially hepatoxic, while 34.62% (9) have
carcinogenic tendency (Table 6). Also, of the 26 compounds,
6 (23.08%), 15 (57.69%), 4 (15.38%), 7 (26.92%), 2 (7.69%),
2 (7.69%) and 1 (3.85%) respectively were found to be
immunotoxic, mutagenic, cytotoxic, and modulator of AhR,
aromatase, MMP, and phosphoprotein P53. Meantime only C-
19 and C-26 showed binding to toxicity targets. C-19 binds
Table 7 Toxicity target binding prediction using PROTOX II
Compound IDs
Toxicity target
Average pharmacophore
fit (%)
Average ligand
similarity (%)
C-10
C-11
C-12
C-13
C-14
C-19
C-26
Prostaglandin G/H synthase I
Prostaglandin G/H synthase I
Prostaglandin G/H synthase I
Prostaglandin G/H synthase I
Prostaglandin G/H synthase I
Amine oxidase
0.00
0.00
80.44
75.1
0.00
75.52
78.95
72.41
0.00
0.00
0.00
26.84
31.94
Prostaglandin G/H synthase I
0.00

Parasitol Res
Adeyemi OS, Murata Y, Sugi T, Han Y, Kato K (2017c) Modulation of
host HIF-1α activity and the tryptophan pathway contributes to the
anti-Toxoplasma gondii potential of nanoparticles. Biochem
Biophys Rep 11:84–92
Adeyemi OS, Sugi T, Han Y, Kato K (2018a) Screening of chemical
compound libraries identified new anti-Toxoplasma gondii agents.
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Adeyemi OS, Murata Y, Sugi T, Han Y, Kato K (2018b) Exploring amino
acid-capped nanoparticles for selective anti-parasitic action and im-
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Metal nanoparticles show potential to restrict Trypanosoma growth.
Artif Cells Nanomed Biotechnol 46:S86–S94
amine oxidase with average pharmacophore fit of 26.84%,
while C-26 binds prostaglandin G/H synthase I (PGH1) with
a fit of 31.94% (Table 7). Both C-19 and C-26 had 0.00%
average ligand similarity in this regard. Additionally, C-10,
C-11, C-12, C-13, and C-14 had average ligand similarity
between 72.41 and 80.44% for PGH1 target binding.
Conclusion
We report the synthesis and biological potentials of imidazole
derivatives, a new class of potential anti-T. gondii compounds.
The substituents and their location impacted the bioactivity
and physicochemical properties. The combination of the struc-
tural features reported for the compounds is believed to gov-
ern the anti-T. gondii activities of the compounds studied. We
also confirmed that a small structural modification, as in the
oxazole derivative of C-16 to yield C-18, could lead to unex-
pected bioactivity and selectivity. The data herein depict
promising evidence warranting further exploration of imidaz-
ole derivatives, especially compounds C-10, C-11, C-12,
C-14, C-16, C-18, C-20, and C-21 with high selective activi-
ties towards the parasite versus host cell, as possible source of
alternative and effective anti-T. gondii agents. Further investi-
gations to determine the anti-T. gondii potential of imidazole
derivatives in a mouse model as well as the mode of antipar-
asitic action with the goal of developing new and effective
treatment strategies for acute and/or latent toxoplasmosis form
part of our future research efforts.
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Acknowledgments The authors would like to thank the Department for
Management of Science and Technology Development, Faculty of
Applied Science, Ton Duc Thang University, Ho Chi Minh City. The
JSPS Fellowship to OS Adeyemi is also acknowledged. Also, AO
Eseola appreciates the Alexander von Humboldt Foundation support
through the Georg Forster postdoctoral scholarship and the Redeemer’s
University for fellowship leave. Additionally, the financial support by the
Deutsche Forschungsgemeinschaft (DFG) is appreciated (PL 155/11, PL
155/12, and PL155/13). For editorial assistance, our appreciation goes to
Ed and Rhoda Perozzi and Carey Johnson of the Chemistry Department,
University of Kansas, USA.
Eseola AO, Görls H, Bangesh M, Plass W (2018) ESIPT-capable 2,6-
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Eseola AO, Görls H, Plass W (2019) Importance of monodentate mono-
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ambient temperature C C coupling: donor strengths and steric fea-
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Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interest.
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