p-Trifluoromethyl- and p-pentafluorothio-substituted curcuminoids of the 2,6-di[(E)-benzylidene)]cycloalkanone type: Syntheses and activities against Leishmania major and Toxoplasma gondii parasites

Article abstract of DOI:10.1016/j.bioorg.2021.105099

A series of the title curcuminoids with structural variance in the heteroatom of the cycloalkanone and the p-substituents of the phenyl rings were tested for their activities against Leishmania major and Toxoplasma gondii parasites. The majority of them showed high activities against both parasite forms with EC₅₀ values in the sub-micromolar concentration range. Bis(p-pentafluorothio)-substituted 3,5-di[(E)-benzylidene]piperidin-4-one 1b was not just noticeable antiparasitic, but also exhibited a considerable selectivity for L. major promastigotes over normal Vero cells. While derivatives differing only in the p-phenyl substituents being CF₃ or SF₅ showed similar antiparasitic activities, the cyclic ketone hub was more decisive both for the anti-parasitic activities and the selectivities for the parasites vs. normal cells. QSAR calculations confirmed the observed structure–activity relations and suggested structural variations for a further improvement of the antiparasitic activity. Docking studies based on DFT calculations revealed L. major pteridine reductase 1 as a likely molecular target protein of the title compounds.

Full text of DOI:10.1016/j.bioorg.2021.105099

Bioorganic Chemistry 114 (2021) 105099
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
p-Trifluoromethyl- and p-pentafluorothio-substituted curcuminoids of the
2,6-di[(E)-benzylidene)]cycloalkanone type: Syntheses and activities against
Leishmania major and Toxoplasma gondii parasites
,
,
,e
Ibrahim S. Al Nasra b, Riadh Hanachic, Ridha B. Saidc d, Seyfeddine Rahalid
,
Bahoueddine Tangoure, Siddig I. Abdelwahabf, Abdullah Farasanif g, Manal M. E. Tahah,
,
Anil Bidwaig, Waleed S. Kokob, Tariq A. Khani, Rainer Schobertj, Bernhard Biersackj
,
*
a Department of Biology, College of Science and Arts, Qassim University, Unaizah 51911, Saudi Arabia
b Department of Science Laboratories, College of Science and Arts, Qassim University, King Abdelaziz Road, Ar Rass 51921, Saudi Arabia
c
´
´
´
´
´
Laboratoire de Caracterisations, Applications et Modelisations des Materiaux, Faculte des Sciences de Tunis, Universite de Tunis El Manar, Tunis 2092, Tunisia
d Department of Chemistry, College of Science and Arts in Ar Rass, Qassim University, Ar Rass 51921, Saudi Arabia
e
´
IPEIEM, Research Unit on Fundamental Sciences and Didactics, Universite de Tunis El Manar, Tunis 2092, Tunisia
f Medical Research Center, Jazan University, Jazan 45142, Saudi Arabia
g College of Applied Medical Sciences, Jazan University, Jazan 45142, Saudi Arabia
h Substance Abuse Research Center, Jazan University, Jazan 45142, Saudi Arabia
i Department of Clinical Nutrition, College of Applied Health Sciences, Qassim University, Ar Rass 51921, Saudi Arabia
j
¨
Organic Chemistry Laboratory, University Bayreuth, Universitatsstrasse 30, 95440 Bayreuth, Germany
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of the title curcuminoids with structural variance in the heteroatom of the cycloalkanone and the p-
substituents of the phenyl rings were tested for their activities against Leishmania major and Toxoplasma gondii
parasites. The majority of them showed high activities against both parasite forms with EC50 values in the sub-
micromolar concentration range. Bis(p-pentafluorothio)-substituted 3,5-di[(E)-benzylidene]piperidin-4-one 1b
was not just noticeable antiparasitic, but also exhibited a considerable selectivity for L. major promastigotes over
normal Vero cells. While derivatives differing only in the p-phenyl substituents being CF3 or SF5 showed similar
antiparasitic activities, the cyclic ketone hub was more decisive both for the anti-parasitic activities and the
selectivities for the parasites vs. normal cells. QSAR calculations confirmed the observed structure–activity re-
lations and suggested structural variations for a further improvement of the antiparasitic activity. Docking
studies based on DFT calculations revealed L. major pteridine reductase 1 as a likely molecular target protein of
the title compounds.
Curcumin
Fluorine
Anti-parasitic drugs
Neglected tropical diseases
activity in some patients with pancreatic cancer when administered
orally as a monotherapy [7]. As a natural product of a tropical plant,
curcumin also has a great potential as a drug against infections and
tropical diseases [8,9]. It was tested against various protozoal parasites
and it showed activity against Leishmania, Plasmodia and Toxoplasma
1. Introduction
Curcumin
[1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-
3,5-dione] is the major biologically active constituent of turmeric, a
spice made from the roots of Curcuma longa which is widely applied in
Indian cuisine and traditional Indian medicine [1]. Curcumin has shown
significant anticancer activity by addressing various cellular targets
involved in cancer cell survival and metastasis. It was found to inhibit or
down-regulate NF-κB, COX-2, STAT-3, Akt, and MMP-2 [2–6]. A phase II
trial in patients suffering from advanced pancreas tumor diseases
revealed a high tolerability of curcumin by all patients, and its biological
species [10,11]. Curcumin and certain metal complexes of it displayed
activity against Leishmania major [12]. In addition, curcumin inhibited
glyoxalase 1 of Toxoplasma gondii and the proliferation of this parasite in
vitro [13]. As immune-compromised people are at risk of severe com-
plications when infected with the globally occurring Toxoplasma gondii
parasites (i.e., the causative agents of toxoplasmosis), new drugs for the
treatment of toxoplasmosis in such patients are necessary [14].
* Corresponding author.
E-mail addresses: bernhard.biersack@yahoo.com, bernhard.biersack@uni-bayreuth.de (B. Biersack).
https://doi.org/10.1016/j.bioorg.2021.105099
Received 2 April 2021; Received in revised form 21 May 2021; Accepted 15 June 2021
Available online 17 June 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

I.S. Al Nasr et al.
Bioorganic Chemistry 114 (2021) 105099
Abbreviations
MW
NTD
PDB
POL
Molecular weight
Neglected tropical disease
Protein data bank
Polarizability
AmB
ATO
CL
Amphotericin B
Atovaquone
Cutaneous leishmaniasis
Density functional theory
Fetal bovine serum
Hydraulic energy
PTR1A Pteridine reductase-1 subunit A
DFT
FBS
HE
QSAR
REF
RPMI
SAG
SI
Quantitative structure–activity relationship
Molecular refractivity
Roswell Park Memorial Institute
Surface area grid
HOMO Highest occupied molecular orbital
LGA
Lamarckian genetic algorithm
Lowest unoccupied molecular orbital
Mucocutaneous leishmaniasis
Multiple linear regression
Selectivity index
LUMO
MCL
MLR
TPSA
VL
Topological polar surface area
Visceral leishmaniasis
Leishmaniasis is categorized as a neglected tropical disease (NTD) and
clinically subdivided into visceral leishmaniasis (VL), cutaneous leish-
maniasis (CL), and mucocutaneous leishmaniasis (MCL). The CL form
brought about by various Leishmania species such as L. major, L. tropica,
L. mexicana, L. amazonensis etc., causes severe skin lesions. It is the most
prevalent leishmaniasis form responsible for up to 1 million, mostly
young, patients annually [15,16]. Although usually not lethal, CL leads
to stark and disfiguring skin lesions and to stigmatization of affected
persons [17,18]. CL patients are currently treated with pentavalent
antimonials, miltefosine, amphotericin or pentamidine [16]. Aside of
the general toxicity of clinically applied drugs such as antimonials,
another growing problem is the emergence of drug-resistant parasite
forms. Thus, new potent anti-parasitic drugs against leishmaniasis are
needed.
The efficacy of curcumin as a therapeutic agent for treating human
diseases is often hampered by its low bioavailability. Modified curcumin
derivatives were reported to have an improved bioavailability and as a
consequence a significantly enhanced in vitro and in vivo activity
compared with curcumin [19]. Fluorinated curcuminoids like EF24 have
been reported to be superior to curcumin in the treatment of various
cancers (Fig. 1) [20,21]. The monocarbonyl compound EF24 showed
improved metabolic stability and bioavailability data when compared
with curcumin [22]. Recently, we disclosed new curcuminoids of the
3,5-di[(E)-benzylidene]piperidin-4-one, or 2,6-di[(E)-benzylidene]cyclo-
hexanone, or (1E,4E)-1,5-diphenylpenta-1,4-dien-3-one type, which
showed improved anti-tumor activities when compared with EF24
[23,24]. Further modifications with a curtailed curcumin motif included
acryl amides, sulfonamides, dioxolanes and thiopyranones [25–27].
However, studies of the anti-parasitic activities of such curcuminoids are
rare.
Perfluorinated lipophilic and electron-withdrawing substituents
such as CF3 and SF5 groups are of particular interest as xenobiotic,
chemically stable mimics of negatively charged biomolecules. The SF5
Fig. 1. Structures of curcuminoids EF24, 1a–l, and 2a–o used in this study.
́
́
́
́
group was even labelled as super trifluoromethyl group [28]. The syn-
thesis of an 8-pentafluorothio analog of the antimalarial drug meflo-
quine is a prominent example that showed a higher antimalarial in vivo
activity and a longer half-life than mefloquine [28,29]. The enhancing
effect of SF5 substituents on the antitumoral activity of curcuminoids
was recently reported [23]. For trifluoromethyl derivatives, promising
activities were also observed against Leishmania parasites as well as an
inhibition of folate enzymes in cancer cells and of analogous pteridine
reductase enzymes of Leishmania and Trypanosoma parasites [30–34]. In
the present report, we disclose a series of new and some already known
curcuminoids bearing 4-trifluoromethyl or 4-pentafluorosulfanyl
substituted phenyl rings, and their promising activities against
L. major and T. gondii parasites (Fig. 1).
2. Results and discussion
EF24 and compounds 1a–l were prepared from the condensation
reaction of one equivalent of the corresponding cyclic ketone with two
equivalents of 4-trifluoromethylbenzaldehyde or 4-pentafluorothioben-
zaldehyde in MeOH under basic conditions (Scheme 1) [23,26]. Com-
pounds 2a–o were prepared from the corresponding aryl sulfonamides
of 4-piperidinone with 4-trifluoromethyl- or 4-pentafluorothiobenzalde-
hyde in ethanol under acidic conditions (Scheme 1). The compounds
1a–l and 2a–o were obtained as yellow solids in low to moderate yields
(30–70%). The structures of all test compounds are provided in detail in
Fig. 1. The presented curcuminoids are new except for the compounds
1a, 1c, 1e, 1f, 1h, 2a, and 2c, which were prepared according to liter-
ature procedures [23,26,27].
The curcuminoids 1a–l and 2a–o (Fig. 1) were initially tested for
2

I.S. Al Nasr et al.
Bioorganic Chemistry 114 (2021) 105099
Table 1
Inhibitory concentrations IC50 (in µM) of test compounds 1a–l and 2a–o when
applied to cells of the Vero (African green monkey kidney epithelial) cell line,
effective concentrations EC50 when applied to cells of Toxoplasma gondii.a
Amphotericin B (AmB, Vero) and atovaquone (ATO, Vero and T. gondii) were
applied as positive controls for the indicated cells.
Compd.
EC50 (T. gondii)
IC50 (Vero)
SI (Vero / T. gondii)b
1a
0.95
1.33
0.99
0.48
0.72
0.22
0.67
0.40
0.28
0.15
0.51
1.61
0.22
0.20
0.32
0.24
0.29
0.26
0.37
0.26
0.15
0.10
0.14
0.10
0.39
0.11
0.10
0.68
38.3c
–
1.41
2.28
1.88
1.14
1.89
0.41
0.77
0.70
1.10
0.81
0.68
0.53
0.27
0.27
0.39
0.41
0.36
0.29
0.33
0.31
0.12
0.07
0.11
0.06
0.46
0.15
0.09
1.22
–
1.48
1.71
1.90
2.38
2.63
1.86
1.15
1.75
3.93
5.40
1.33
0.33
1.23
1.35
1.22
1.71
1.24
1.12
0.89
1.19
0.80
0.70
0.79
0.60
1.18
1.36
0.90
1.79
–
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
Scheme 1. Reagents and conditions: (i) 4-CF3-C6H4CHO or 4-SF5-C6H4CHO,
NaOH, MeOH/H2O, r.t., 1 h, 31–70%; (ii) 4-CF3-C6H4CHO or 4-SF5-C6H4CHO,
conc. HCl, EtOH, reflux, 24 h, 30–41%.
EF24
Curcumin
AmB
ATO
7.7
–
0.07
9.5
136
their activity against T. gondii tachyzoites (Table 1). Of the compound 1
series, the acrylate 1f and the thiopyranones 1i and 1j showed the
highest activity against T. gondii, which was also slightly more sensitive
to these test compounds than Vero cells. Compound 1j also showed the
highest selectivity index (SI = 5.4) of this group of curcuminoids and it
was also more active and selective than EF24. Lower activities were
observed for compounds 1a–c and 1l, which were less active even than
EF24. The series of phenylsulfonamides 2 also comprised very active
derivatives against T. gondii such as the chlorophenyl derivatives 2i, 2j,
2n, and 2o, and the 3-fluorophenyl derivatives 2k and 2l which were
much more active than EF24 and almost as active as atovaquone (ATO).
However, these compounds showed no selectivity for parasites over
Vero cells. Nevertheless, all test compounds 1 and 2 were more active
than the lead curcumin [13].
a
Values are the means of at least three independent experiments (SD ± 15%).
They were obtained from concentration–response curves by calculating the
percentage of treated cells in comparison to untreated controls after 72 h. bSe-
lectivity index SI (IC50/EC50) was calculated from the corresponding IC50 values
for the Vero cells and the EC50 values against T. gondii. cValue was taken from
Goo et al. [13].
specificity for promastigotes vs. amastigotes) for the test compounds
also underline the high specificity of many tested curcuminoids for
promastigotes when compared with their activities against amastigotes.
Compared with the positive control amphotericin B (AmB), all tested
curcuminoids showed distinctly higher activities against the promasti-
gotes. The lowest SPI values (i.e., the highest specificities for promas-
tigotes) of 0.002–0.003 were observed for compounds 1b-d, 2e and 2m.
This is remarkable because in contrast to that, many approved anti-
leishmanial drugs display a high activity against amastigotes, and
other drug candidates show a higher activity against L. major amasti-
gotes than against promastigotes [35,36]. AmB with an SPI of 1.766 is
also more active against amastigotes than against promastigotes, how-
ever, compounds with SPI < 2 can be considered as active against both
promastigotes and amastigotes.
The activity of compounds 1a–l and 2a–o against L. major promas-
tigotes and amastigotes was also determined (Table 2). Among the series
of compounds 1a–l, high activities against promastigotes were observed
for piperidones 1a–f, thiopyranone 1i, and ketals 1k and 1l with 1f and
1l performin best (EC50 = 0.022–0.024 µM). These compounds are much
more active against L. major promastigotes than curcumin, which was
reported to have an EC50 value of 103.2 µM [12]. But in most cases, the
test compounds were much less active against the amastigotes. Only 1e
also showed a specific activity against the amastigotes. Compounds 1a–c
exhibited considerable selectivities for the promastigotes and the high-
est selectivity was observed for the new SF5-substituted compound 1b
(SI = 76). How far the SF5 group of 1b actually contributes to the pro-
longed half-life and increased activity of this compound in L. major in
vivo models, in analogy to the above mentioned anti-malarial 8-penta-
fluorothio-mefloquine, remains to be elucidated. Compounds 1f, 1i,
1k and 1l also showed distinct selectivities for the promastigotes.
Among the compounds of the 2a–o series, the highest activities were
observed for 2a, 2c, 2e–h, and 2 k–m. A considerable selectivity was
found for 2m (SI = 19.2). The determined specificity indices (SPI,
The dependence of the measured activities on the presence and in-
fluence of the CF3 or SF5 groups remained obscure. Sometimes, CF3
analogs were more active, yet very often there was no distinct difference
among compounds with the same central cyclic ketone fragment. In
contrast, the substitution pattern of the central cyclic ketone scaffold
was found to be decisive for high activity and selectivity or lack of ac-
tivity in certain cases. The sulfonamides 2a–o were generally more
active against T. gondii than EF24 and most of the curcuminoids of the
1a–l series. Only acrylate 1f and thiopyranones 1i and 1j reached the
activities of the sulphonamides here. It seems that a carbonamide or
sulfonamide function is crucial for high activity. The sulfur atoms of the
thiopyranones 1i and 1j might form H-bonds (as acceptors) or can be
3

I.S. Al Nasr et al.
Bioorganic Chemistry 114 (2021) 105099
Table 2
Effective concentrations EC50 (in µM) of test compounds 1a–l and 2a–o when applied to promastigotes and amastigotes of Leishmania major.a Amphotericin B (AmB)
was applied as a positive control.
Compd.
EC50 promastigotes
EC50 amastigotes
SPIb
SI Vero / promastigotesc
SI Vero / amastigotesc
1a
1b
1c
1d
1e
1f
0.037
0.030
0.040
0.041
0.035
0.022
0.103
0.158
0.047
0.110
0.030
0.024
0.022
0.066
0.030
0.111
0.024
0.026
0.023
0.025
0.072
0.093
0.023
0.022
0.024
0.126
0.110
0.28
3.16
13.7
17.2
13.0
0.72
2.79
8.60
11.6
2.10
2.02
2.43
1.90
2.00
7.79
2.48
2.05
9.97
3.01
3.69
3.21
10.1
2.28
3.69
2.19
10.2
1.45
8.01
19.9
0.47
0.012
0.002
0.002
0.003
0.049
0.008
0.012
0.014
0.022
0.054
0.012
0.013
0.011
0.008
0.012
0.054
0.002
0.009
0.006
0.008
0.007
0.041
0.006
0.010
0.002
0.087
0.014
0.014
1.766
38.1
76
0.45
0.17
0.11
0.09
2.63
0.15
0.09
0.06
0.52
0.40
0.28
0.28
0.14
0.04
0.16
0.20
0.04
0.10
0.09
0.10
0.01
0.03
0.03
0.03
0.05
0.1
47
27.8
54
18.6
7.5
1g
1h
1i
4.43
23.4
7.36
22.7
22.1
12.3
4.1
1j
1k
1l
2a
2b
2c
2d
2e
2f
13.0
3.69
15
11.2
14.4
12.4
1.67
0.75
4.79
2.72
19.2
1.19
0.82
4.36
9.6
2g
2h
2i
2j
2k
2l
2m
2n
2o
EF24
AmB
0.01
0.06
16.4
0.83
a
Values are the means of three experiments (SD ± 15%). They were obtained from concentration–response curves by calculating the percentage of treated cells in
comparison to untreated controls after 72 h. bSpecificity index SPI for promastigotes vs. amastigotes was calculated from the EC50 values of promastigotes and
amastigotes. cSelectivity index SI (IC50/EC50) was calculated from the corresponding IC50 values for the Vero cells (Table 1) and the EC50 values against L. major.
oxidised to sulfones in the cells, mimicking the sulfonamides of com-
pounds 2a–o. Concerning the arene substituents of compounds 2a–o, 3-
fluoro-, 4-chloro-, and 3,4-dichlorophenylsulfonamides led to the high-
est activities against T. gondii.
The following structure activity-relationships for the compounds
1a–l and 2a–o were identified from the results in L. major parasites: The
SF5 derivatives 1g, 1h and 1j showed relatively low activities against the
L. major promastigotes indicating a negative role for the SF5 group in
combination with N-acryloylpiperidone, cyclohexanone and thiopyr-
anone scaffolds. However, compound 1i, the CF3 analog of 1j, displayed
reasonable activity and selectivity, and also showed high activity against
T. gondii. High activity and considerable selectivity were identified for
compounds 1a–e indicating a crucial role of the unmodified piperidone
and the N-(m)ethylpiperidone scaffolds to effectuate L. major promas-
tigote selectivity (Fig. 2). Only the N-ethyl derivative 1e showed activity
at submicromolar concentrations against the L. major amastigotes which
is a hint at the possible positive role of more hydrophobic N-alkyl sub-
stituents for the future design of more active analogs against L. major
amastigotes. The phenylsulfonamide derivatives 2a–o, SF5 derivatives
2d and 2j were less active than their congeners, and the combination of
SF5 substitution with toluene- or 4-chlorosulfonamide central scaffolds
led to a reduced efficacy. In addition, 3,4-dichlorophenylsulfonamides
2n and 2o showed a distinctly lower activity than the other com-
pound 2 analogs. Some selectivity was observed for compounds 2g, 2h,
and 2m. Hence, a central 4-fluorophenyl or 3,4-difluorophenylsulfonyl
piperidone moiety apparently correlated with a better selectivity for
promastigotes while the 3-fluoro- and 4-chlorophenyl congeners 2i–l
showed only lesser selectivities.
In addition to Vero cells, the effects of selected test compounds on
macrophages were investigated in order to obtain more information
about the selectivity of the test compounds for the tested parasites
(Table 3). Compound 1b showed the lowest growth inhibitory activity
against macrophages of the tested compounds. 1b already showed the
Fig. 2. Structure-activity relationships of curcuminoids 1a–l and 2a–o.
4

I.S. Al Nasr et al.
Bioorganic Chemistry 114 (2021) 105099
- The cross-validity coefficient R2 (LOO) was used to estimate the
Table 3
CV
internal validity of the model. The threshold for approval is R2
Inhibitory concentrations IC50 (in µM) of selected test compounds 1 and 2 when
applied to macrophages and selectivity indices (SI) of the test compounds.
CV
(LOO) greater than 0.5 [41].
- For a positive external validation, the quadratic correlation coeffi-
cient R2ext should be greater than 0.6 [42].
- Significance P < 0.05.
Compd.
IC50
SI
SI
SI macrophages
/
macrophagesa
macrophages /
macrophages /
T. gondiib
amastigotesb
promastigotesb
1a
1b
1j
3.65
16.50
4.04
2.99
2.57
1.63
3.18
3.08
1.02
1.13
3.84
12.4
26.9
5.86
1.60
7.41
9.94
12.8
10.2
10.3
1.15
1.21
2.0
99
550
37
The compounds were divided into two groups for each experimental
activity: 21 compounds were used as a training set (internal validation)
and six compounds were used for the external test (external validation).
The measured and predicted activities and residual values are presented
in Table S2 (cf. SI).
1k
1l
1.23
1.35
0.82
1.29
1.5
100
107
74
2a
2c
2d
2l
106
28
The values of compounds denoted with boldfaced numbers were not
used to build the models nor for the external validation.
The following equations show meaningful results of association an-
alyses between the curcuminoid’s activities and their calculated
descriptors:
0.47
0.78
46
2n
8.97
a
Values are the means of three repeated experiments (SD ± 15%). They were
obtained from concentration–response curves by calculating the percentage of
treated cells in comparison to untreated controls after 72 h. bSelectivity index
(IC50/EC50) calculated from the corresponding IC50 values for the macrophages
and the EC50 values from T. gondii and L. major (pro- and amastigotes).
EC50(T.gondii) = 1.68421669 ꢀ 0.05004291*TPSA + 0.8481162*nON
+ 0.44750966*nOHNH ꢀ 0.35542755*nROTB
(1)
lowest activity against the Vero cells. Consequently, 1b has a high
selectivity for L. major promastigotes (SI = 550) and a considerable
selectivity for T. gondii cells (SI = 12.4). The highest selectivity for
T. gondii was observed for compound 1j (SI = 26.9). Due to the low
activity of the tested compounds against L. major amastigotes, the
selectivity for amastigotes is only marginal, compared with their activ-
ities against macrophages.
IC50(Vero) = 11.03542873 + 29.78379959*HOMO ꢀ 0.24482523*POL
+ 0.06420943*REF + 0.59274826*nOHNH
(2)
EC50(promastigotes) = ꢀ 0.22486011+0.044368*logPꢀ 0.00249733*TPSA
+0.01339477*nROTB
(3)
In order to establish a rationale for the observed structure–activity
relations, i.e., a mathematical connection between biological activity
expressed in terms of pIC50 and physicochemical and structural prop-
erties of the test compounds, we used a Quantitative Structure-Activity
Relationships (QSAR) approach [37]. This method implies a prediction
of the pharmacological activities of the test compounds by processing
their molecular parameters (a.k.a. descriptors), either measured or
calculated, e.g. via density functional theory (DFT), in a quantitative
model based on multiple linear regression (MLR). This combined QSAR-
DFT approach allows to build simple (less parameterized) models
[38–40]. First, the geometries of all compounds were optimized using
B3LYP with the 6–311 + G(d,p) basis set (cf. Supporting Information,
Figure S1). Based upon these geometries, the following descriptors were
calculated or deduced. HOMO and LUMO energies, the ctanol-water
partition coefficient (log10P), molar weight (MW), polarizability
(POL), surface area grid (SAG), hydraulic energy (HE), molecular
refractivity (REF), volume (V), topological polar surface area (TPSA),
number of atoms (nATOMS), hydrogen bond acceptors (nON, counting
all N and O atoms), hydrogen bond donor (nOHNH, counting the sum of
all NH and OH groups), number of rotatable bonds (nROTB), total
electronic energy E and dipolar moment µ (cf. SI, Table S1).
From Eq. (1) and Eq. (2), the coefficients of nOHNH are positive,
which indicate a promoter effect of weak bonding between curcuminoid
derivatives and parasite. The positive coefficient of the octanol–water
partition coefficient (logP) in Eq. (3) is in line with the increased bio-
logical activity of more lipophilic curcuminoid derivatives.
Overall, a good correlation between experimental data and predicted
biological activities was obtained (Fig. 3) with high values of R2cv (0.72,
0.84, and 0.60 for EC50 (T. gondii), IC50 (Vero), and EC50 (promasti-
gotes), respectively). The R2 values (0.85, 0.81, and 0.88 for EC50
ext
(T. gondii), IC50 (Vero), and EC50 (promastigotes), respectively) confirm
these results.
Protozoan parasites like Leishmania depend on exogenous pteridine
sources (folates and pterins). Upon uptake by the parasite, these pteri-
dines are reduced to bioactive tetrahydro-forms by parasite enzymes.
Pteridine reductase-1 (PTR1) of L. major is one of the enzymes involved
in parasitic pteridine reduction [43,44] and, thus, a suitable drug target
for treating L. major infections. We carried out docking experiments with
the curcuminoids 1i and EF24. The active site of the L. major PTR1
harbors crucial hydrogen donating amino acid residues (e.g., tyrosines),
which can interact with hydrogen acceptors such as fluorine of 1i and
EF24 [44]. All binding parameters of 1i and EF24 obtained after docking
with PTR1A are listed in Table 4. Estimated total free energy of binding
of the two ligands was ꢀ 8.12 ± 0.12 and ꢀ 9.16 ± 0.14 kcal/mol (values
are mean ± SD of 20 docking runs), respectively. The estimated Ki values
Table S1 shows that compound 2o has a large logP value of 8.73,
which corresponds to the large values of SAG (766.57 Å3), MW (736.48
g/mol), MR (144.23 Å3), V (1409.9 A3) and nATOMS (44) and the
smallest HOMO energy (-0.27187 a.u.). Compound 1a had the smallest
value of logP (5.66) which corresponds to the smallest values of SAG
(601.21 Å3), MW (411.35 g/mol), MR (99.41 Å3), Vol (1011.75 A3) and
nATOMS (29) and associates with the smallest dipolar moment (0.35
Debye). TPSA values were in the range of 17.07 – 63.69 Å2, which were
still within the acceptable range (<140 Å2) to have a high oral absorp-
tion. The number of rotatable bonds lay between 4 and 6 for all com-
pounds. 2f has the largest HE value (7.45 kcal/mol) while the smallest
HE value was calculated for 1d (1.63 kcal/mol).
were 1.02 μM and 0.18 μM, respectively (values calculated for lowest
energy docking run out of 20 runs). The total free energy of binding (and
hence the Ki) estimated for 1i and EF24 suggests a considerable affinity.
The two ligands appear to bind at separate sites of the enzyme
(Fig. 4). The strong inhibition by 1i can be explained by its binding to
the active site of the enzyme, which would interfere with substrate
binding (see below). In contrast, EF24 appears to bind outside the sub-
strate binding pocket. Therefore, in spite of strong binding, it may be
unable to block the substrate binding site, which would explain the
reduced experimental parasite growth inhibition by EF24.
For validation of the QSAR model, different statistical parameters
were used:
An analysis of docked complexes of the two compounds was done
using LigPlot + software. LigPlots are shown in Figures 5 and S2. LigPlot
analysis of the docked complexes of the ligands with PTR1A revealed
several significant interactions of the ligands with PTR1A. The ligands
5

I.S. Al Nasr et al.
Bioorganic Chemistry 114 (2021) 105099
Fig. 3. Generic leverage plot of experimental versus corresponding predicted activities {EC50 (T. gondii) (A), IC50 (Vero) (B) and EC50 (promastigotes) (C)} and
diagram of residual values {EC50 (T. gondii) (A‘), IC50 (Vero) (B‘) and EC50 (promastigotes) (C‘)}.
Table 4
Interaction energies and inhibitor constants (Ki) for the binding of 1i and EF24 to
pteridine reductase – A subunit (PTR1A) (values for the lowest energy confor-
mation from 20 docking runs).
Parameters
1i
EF24
vdW + H-bond + desolvation energy (kcal/mol)
Electrostatic energy (kcal/mol)
ꢀ 9.83
+0.16
0.00
ꢀ 8.51
ꢀ 1.58
ꢀ 0.61
+0.89
ꢀ 9.20
0.18
Final total internal energy (kcal/mol)
Torsional free energy (kcal/mol)
+1.49
ꢀ 8.18
1.02
Estimated free binding energy (kcal/mol)
Estimated inhibition constant Ki (µM, 298.15 K)
appear to bind in extended conformations and make extensive van der
Waals contacts with certain enzyme residues (Figures 5 and S2). 1i
established four H-bonds with active site residues of the protein. The
carbonyl group of 1i formed H-bonds with the carboxamide of the Asn-
109 side-chain and with the backbone NH of Gly-19. The interaction of
1i with the phenolic hydroxyl group of Tyr-194 occurred via one of its
CF3 groups while the opposite CF3 group interacted with the backbone
NH of His-38. Tyr-194 is crucial for the interaction with and the first-
step reduction of the pterin substrate to the dihydro form and builds a
catalytic triad with Lys-198 and Asp-181 in L. major PTR1 for this reason
[44]. Various known PTR1 inhibitors were shown to interact with these
residues and to block substrate binding to the active site, which
Fig. 4. Optimized structures of selected curcuminoids. Partial structure of
PTR1 subunit A (pdb file 1E92). Helices are shown in pink, beta structures in
yellow and bends in blue. Figure shows 1i (green) and EF24 (red) bound to
PTR1A. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
underlines the importance of the described H-bond interaction by 1i
[33,44,45]. In the case of the PTR1 inhibitor methotrexate, for instance,
the replacement of the oxo group of the pteridine ring of folic acid by an
6

I.S. Al Nasr et al.
Bioorganic Chemistry 114 (2021) 105099
applications of some of the tested curcuminoids for the treatment of
cutaneous leishmaniasis in in vivo models (i.e., L. major infection) appear
promising. Feasible combinations of the most active curcuminoids with
approved anti-parasitic drugs can be useful in order to optimize their
efficacy, and to reduce the minimal effective dosage and possible side-
effects in future animal studies.
4. Experimental
4.1. Chemistry
Compounds 1a, 1c, 1e, 1f, 1h, 2a, and 2c were prepared following
literature procedures and analyzed. The obtained analytical data was in
line with published data of these compounds [23–27]. Synthetic pro-
cedures and analytical data of the new derivatives are given below. All
starting compounds were purchased from the usual retailers and used
without further purification. Column chromatography: silica gel 60
(230–400 mesh). Melting points (uncorrected), Electrothermal 9100;
NMR spectra, Bruker Avance 300 spectrometer; chemical shifts are
given in parts per million (δ) downfield from tetramethylsilane as in-
ternal standard; Mass spectra, Thermo Finnigan MAT 8500 (EI).
Elemental analysis were carried out with an Elementar UNICUBE (Ele-
mentar Analysensysteme GmbH).
4.1.1. (3E,5E)-3,5-Bis-(4-pentafluorothiobenzylidene)-piperidine-4-one
(1b)
4-Piperidinone monohydrate hydrochloride (153 mg, 1.0 mmol) and
4-(pentafluorothio)benzaldehyde (464 mg, 2.0 mmol) were dissolved in
MeOH (10 mL) and NaOH (40 mg) in H2O (1 mL) was added. The re-
action mixture was stirred at room temperature for 2 h. The formed
precipitate was collected, washed with MeOH/H2O and dried in vac-
uum. Yield: 366 mg (0.70 mmol, 70%); yellow solid of m.p. 90–92 ◦C;
υmax(ATR)/cmꢀ 1 2936, 1679, 1617, 1598, 1496, 1410, 1325, 1302,
1253, 1206, 1181, 982, 821, 794, 733, 663; 1H NMR (300 MHz,
DMSO‑d6) δ 4.00 (4H, s), 7.61 (2H, s), 7.7–7.8 (4H, m), 7.9–8.0 (4H, m);
13C NMR (75.5 MHz, DMSO‑d6) δ 47.3, 56.6, 126.0, 131.0, 132.3, 136.8,
138.4, 152.3, 187.6; m/z (%) 527 (70) [M+], 499 (63), 247 (100), 231
(77), 115 (57); Anal. Calcd. for C19H15F10NOS2: C, 43.27; H, 2.87; N,
2.66. Found: C, 43.10; H, 2.93; N, 2.59.
Fig. 5. LigPlot of 1i interactions with residues of PTR1A. Ligand and residue
atoms are shown in CPK colors. Residues are numbered. Hydrogen bonds are
shown with green dotted lines and hydrophobic interactions with red dotted
rays. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
additional amino group in the methotrexate molecule is crucial for the
inhibitory activity of methotrexate based on the H-bond of this amino
group with Tyr-194 [44]. 1i also established extensive hydrophobic
contacts with a number of amino acids of the active site, namely Gly-13,
Ala-15, Lys-16, Arg-17, Leu-18, Tyr-37, Asp-181, Asn-109, Ala-110, Ser-
111, Val-180 and Pro-224. The hydrophobic interaction with Asp-181 of
the already mentioned catalytic triad (together with Tyr-194 and Lys-
198) is of particular interest. The reference compound EF24 formed
two H-bonds with Asp-181 and Tyr-283, respectively. The H-bond with
Tyr-283 was formed by one of the fluorines of the EF24 molecule while
the piperidone NH group established the second H-bond with the
carboxylate group of Asp-181. In addition, hydrophobic interactions of
EF24 were seen with Arg-17, Leu-18, Asp-181, Met-183, Gln-186, Leu-
188, Pro-224, Gly-225, Leu-226, Ser-227 and Tyr-283.
4.1.2. (3E,5E)-3,5-Bis-(4-trifluoromethylbenzylidene)-1-ethylpiperidine-4-
one (1d)
N-Ethylpiperidin-4-one (95 mg, 0.75 mmol) and 4-trifluoromethyl-
benzaldehyde (258 mg, 1.5 mmol) were dissolved in MeOH (10 mL)
and NaOH (40 mg, dissolved in H2O) was added. The reaction mixture
was stirred at room temperature for 1 h. The formed precipitate was
collected, washed with MeOH and dried in vacuum. Yield: 100 mg (0.23
mmol, 31%); yellow solid of m.p. 205–206 ◦C; υmax(ATR)/cmꢀ 1 2986,
2785, 1675, 1615, 1593, 1416, 1326, 1259, 1162, 1111, 1070, 1015,
990, 932, 844, 699; 1H NMR (300 MHz, CDCl3) δ 1.04 (3H, t, J = 7.1 Hz),
2.60 (2H, q, J = 7.1 Hz), 3.78 (4H, s), 7.48 (4H, d, J = 8.2 Hz), 7.67 (4H,
d, J = 8.2 Hz), 7.80 (2H, s); 13C NMR (75.5 MHz, CDCl3) δ 12.3, 51.3,
54.2, 122.1, 125.5–125.7 (m), 130.1, 130.3, 130.5, 130.9, 135.0, 138.6,
186.9; m/z (%) 439 (91) [M+], 411 (38), 252 (30), 226 (37), 173 (43),
145 (43), 97 (60), 83 (69), 69 (60), 61 (100), 43 (79); Anal. Calcd. for
3. Conclusions
C
23H19F6NO: C, 62.87; H, 4.36; N, 3.19. Found: C, 62.78; H, 4.29; N,
3.24.
Promising results were obtained from the evaluation of a series of
curcuminoids as potential drugs against pathogenic parasites such as
T. gondii and L. major, which can cause severe infections in humans. Both
high activities against and considerable selectivities for the parasites
were discovered. More detailed research about the mechanisms of action
is warranted in order to identify the reason for these peculiar effects. The
L. major pteridine reductase 1 might be a possible drug target as to
docking results. It is remarkable that the new SF5 derivative 1b showed
considerable selectivity for L. major promastigotes. Investigational
4.1.3. (3E,5E)-3,5-Bis-(4-pentafluorothiobenzylidene)-1-
acryloylpiperidone (1g)
1b (100 mg, 0.19 mmol) was suspended in acetone and treated with
acryloyl chloride (35 µL, 0.43 mmol). K2CO3 (197 mg, 1.43 mmol, dis-
solved in 2 mL H2O) was added and the reaction mixture was stirred at
room temperature for 24 h. Water (20 mL) was added and the formed
precipitate was collected, washed with water and dried in vacuum.
Yield: 68 mg (0.12 mmol, 63%); yellow solid of m.p. 99–100 ◦C;
7

I.S. Al Nasr et al.
Bioorganic Chemistry 114 (2021) 105099
υmax(ATR)/cmꢀ 1 2934, 1647, 1616, 1496, 1440, 1410, 1295, 1262,
1231, 1209, 1191, 1170, 1135, 1100, 976, 827, 799, 739, 665; 1H NMR
(300 MHz, CDCl3) δ 4.76 (4H, s), 5.6–5.7 (1H, m), 6.2–6.3 (2H, m),
7.4–7.6 (4H, m), 7.7–7.9 (6H, m); 13C NMR (75.5 MHz, CDCl3) δ 46.0,
125.7, 126.4, 126.7, 129.1, 129.7, 130.2, 133.5, 137.6, 154.1, 165.5,
185.8; m/z (%) 581 (82) [M+], 526 (30), 115 (25), 55 (100); Anal.
Calcd. for C22H17F10NO2S2: C, 45.44; H, 2.95; N, 2.41. Found: C, 45.32;
H, 2.99; N, 2.48.
mg (0.31 mmol, 31%); yellow solid of m.p. 195–197 C; νmax (ATR)/
◦
cmꢀ 1: 2956, 2899, 1670, 1614, 1583, 1496, 1408, 1343, 1321, 1294,
1265, 1240, 1188, 1154, 1096, 7044, 1008, 988, 947, 825, 803, 760,
732, 704, 664, 633; 1H NMR (300 MHz, DMSO‑d6): δ 3.1–3.2 (4H, m),
3.8–3.9 (4H, m), 7.7–7.8 (6H, m), 7.9–8.0 (4H, m); 13C NMR (75 MHz,
DMSO‑d6): δ 37.1, 64.3, 105.8, 122.2, 126.0, 130.9, 135.1, 135.5, 138.8,
152.2–152.6 (m), 187.1; m/z (%) 584 (100) [M+], 313 (21), 241 (19),
115 (55), 86 (23); Anal. Calcd. for C22H18F10O3S2: C, 45.21; H, 3.10.
Found: C, 45.12; H, 3.04.
4.1.4. (3E,5E)-3,5-Bis-(4-trifluoromethylbenzylidene)-
tetrahydrothiopyran-4-one (1i)
4.1.8. (3E,5E)-3,5-Bis-(4-pentafluorothiobenzylidene)-1-
Tetrahydro-4H-thiopyran-4-one (116 mg, 1 mmol) and 4-trifluoro-
methylbenzaldehyde (348 mg, 2.0 mmol) were dissolved in methanol
(10 mL) and NaOH (200 mg, dissolved in 2 mL H2O) was added. The
reaction mixture was stirred at room temperature for 1 h. The formed
precipitate was collected and dried in vacuum. Yield: 150 mg (0.35
mmol, 35%); yellow solid of m.p. 152–153 ◦C; υmax(ATR)/cmꢀ 1 2886,
1662, 1606, 1581, 1410, 1322, 1276, 1194, 1165, 1118, 1068, 1016,
983, 963, 918, 855, 834, 750, 732, 669, 632; 1H NMR (300 MHz,
DMSO‑d6) δ 4.00 (4H, s), 7.65 (2H, s), 7.24 (4H, d, J = 8.3 Hz), 7.82 (4H,
d, J = 8.3 Hz); 13C NMR (75.5 MHz, DMSO‑d6) δ 29.1, 122.2, 125.4,
125.8, 128.2, 128.6, 129.1, 129.4, 130.5, 130.8, 133.6, 136.2, 138.6,
156.0, 188.1; m/z (%) 428 (62) [M+], 359 (43), 215 (27), 184 (62), 147
(38), 115 (100); Anal. Calcd. for C21H14F6OS: C, 58.88; H, 3.29. Found:
C, 58.79; H, 3.21.
(benzenesulfonyl)-piperidone (2b)
1-(Benzolsulfonyl)-4-piperidinone (140 mg, 0.79 mmol) and 4-
(pentafluorothio)-benzaldehyde (348 mg, 1.5 mmol) were dissolved in
EtOH (10 mL) and conc. HCl (1 mL) was added. The reaction mixture
was refluxed for 24 h. After cooling down to room temperature, the
formed precipitate was collected, washed with EtOH and dried in vac-
uum. Yield: 160 mg (0.24 mmol, 30%); yellow solid of m.p. 237–238 ◦C;
υmax(ATR)/cmꢀ 1 1676, 1612, 1591, 1573, 1496, 1447, 1409, 1354,
1297, 1244, 1208, 1184, 1169, 1099, 1036, 983, 962, 933, 837, 824,
799, 762, 750, 732, 721, 694, 665; 1H NMR (300 MHz, DMSO‑d6) δ 4.67
(4H, s), 7.5–7.6 (6H, m), 7.6–7.8 (5H, m), 8.05 (4H, d, J = 8.9 Hz); 13
C
NMR (75.5 MHz, DMSO‑d6) δ 46.5, 126.3, 127.2, 129.5, 131.1, 132.9,
133.7, 134.9, 137.3, 137.8, 152.9, 183.9; m/z (%) 667 (26) [M+], 648
(6), 526 (100), 398 (22), 270 (12), 242 (27), 115 (24), 77 (37); Anal.
Calcd. for C25H19F10NO3S3: C, 44.98; H, 2.87; N, 2.10. Found: C, 45.05;
H, 2.92; N, 2.17.
4.1.5. (3E,5E)-3,5-Bis-(4-pentafluorothiobenzylidene)-
tetrahydrothiopyran-4-one (1j)
Tetrahydrothiopyran-4-one (31 mg, 0.27 mmol) and 4-(penta-
fluorothio)benzaldehyde (124 mg, 0.54 mmol) were dissolved in MeOH
(5 mL). NaOH (20 mg, 0.5 mmol) and H2O (1 mL) were added and the
reaction mixture was stirred at room temperature for 1 h. The formed
precipitate was collected, washed with MeOH/H2O and dried in vac-
uum. Yield: 45 mg (0.083 mmol, 31%); yellow solid of m.p. 180–181 ◦C;
υmax(ATR)/cmꢀ 1 1661, 1609, 1580, 1493, 1424, 1406, 1317, 1278,
4.1.9. (3E,5E)-3,5-Bis-(4-pentafluorothiobenzylidene)-1-(toluene-4-
sulfonyl)-piperidone (2d)
1-Tosyl-4-piperidinone (200 mg, 0.79 mmol) and 4-(penta-
fluorothio-)benzaldehyde (348 mg, 1.5 mmol) were dissolved in EtOH
(10 mL) and conc. HCl (1 mL) was added. The reaction mixture was
refluxed for 24 h. After cooling down to room temperature, the formed
precipitate was collected, washed with EtOH/H2O and dried in vacuum.
Yield: 195 mg (0.29 mmol, 37%); yellow solid of m.p. 275 ◦C;
υmax(ATR)/cmꢀ 1 2935, 1675, 1617, 1595, 1574, 1496, 1431, 1408,
1296, 1266, 1244, 1206, 1188, 1164, 1103, 1086, 1036, 987, 956, 937,
820, 793, 750, 729, 707, 692, 664, 635, 614, 597, 580; 1H NMR (300
MHz, CDCl3) δ 2.42 (3H, s), 4.52 (4H, s), 7.25 (2H, d, J = 8.1 Hz), 7.40
(4H, d, 8.8 Hz), 7.51 (2H, d, J = 8.1 Hz), 7.70 (2H, s), 7.84 (4H, d, J =
8.8 Hz); 13C NMR (75.5 MHz, CDCl3) δ 21.6, 46.9, 126.6, 127.6, 129.9,
130.1, 132.1, 134.0, 136.4, 137.4, 144.6, 184.2; m/z (%) 681 (18) [M+],
662 (6), 554 (3), 526 (100), 398 (17), 270 (17), 242 (29), 155 (14), 91
(44); Anal. Calcd. for C26H21F10NO3S3: C, 45.82; H, 3.11; N, 2.05.
Found: C, 45.72; H, 3.02; N, 2.11.
1
1199, 1185, 1139, 1101, 986, 922, 824, 811, 765, 741, 678, 658; H
NMR (300 MHz, CDCl3) δ 3.84 (4H, s), 7.45 (4H, d, J = 8.4 Hz), 7.7–7.9
(6H, m); 13C NMR (75.5 MHz, CDCl3) δ 29.9, 126.3, 126.4, 129.8, 129.9,
134.7, 135.8, 138.3, 153.2, 188.2; m/z (%) 544 (100) [M+], 417 (65),
242 (55), 147 (24), 115 (36); Anal. Calcd. for C19H14F10O2S3: C, 41.91;
H, 2.59. Found: C, 41.79; H, 2.66.
4.1.6. (7E,9E)-7,9-Bis-(4-trifluoromethylbenzylidene)-1,4-dioxaspiro[4.5]
decan-8-one (1k)
1,4-Cyclohexanedione monoethylene acetal (156 mg, 1 mmol) and
4-trifluoromethylbenzaldehyde (348 mg, 2.0 mmol) were dissolved in
methanol (10 mL) and NaOH (200 mg, dissolved in 2 mL H2O) was
added. The reaction mixture was stirred at room temperature for 1 h.
The formed precipitate was collected and dried in vacuum. Yield: 150
4.1.10. (3E,5E)-3,5-Bis-(4-trifluoromethylbenzylidene)-1-(1-
methoxybenzene-4-sulfonyl)-piperidone (2e)
◦
mg (0.32 mmol, 32%); yellow solid of m.p. 172–173 C; νmax (ATR)/
1-(4-Methoxybenzolsulfonyl)-4-piperidinone (213 mg, 0.79 mmol)
and 4-trifluoromethylbenzaldehyde (260 mg, 1.5 mmol) were dissolved
in EtOH (10 mL) and conc. HCl (1 mL) was added. The reaction mixture
was refluxed for 24 h. After cooling down to room temperature, the
formed precipitate was collected, washed with EtOH and dried in vac-
uum. Yield: 140 mg (0.24 mmol, 30%); yellow solid of m.p. 244–245 ◦C;
υmax(ATR)/cmꢀ 1 1675, 1614, 1594, 1576, 1499, 1468, 1442, 1414,
1341, 1321, 1300, 1263, 1243, 1160, 1138, 1124, 1111, 1091, 1068,
1037, 1015, 987, 969, 955, 941, 854, 839, 811, 799, 765, 743, 717, 692,
680, 640, 627, 618; 1H NMR (300 MHz, DMSO‑d6) δ 3.87 (3H, s), 4.63
(4H, s), 7.06 (2H, d, J = 9.0 Hz), 7.45 (2H, d, J = 9.0 Hz), 7.64 (2H, s),
7.71 (4H, d, J = 8.2 Hz), 7.89 (4H, d, J = 8.2 Hz); 13C NMR (75.5 MHz,
DMSO‑d6) δ 46.5, 55.8, 114.6, 122.2, 125.6–125.8 (m), 128.7, 129.1,
129.6, 129.9, 130.9, 132.6, 135.6, 138.0, 163.0, 184.0; m/z (%) 581
(11) [M+], 562 (8), 410 (100), 226 (11), 212 (28), 184 (28), 171 (42),
123 (15), 115 (25), 107 (24), 77 (23); Anal. Calcd. for C28H21F6NO4S: C,
57.83; H, 3.64; N, 2.41. Found: C, 57.76; H, 3.71; N, 2.46.
cmꢀ 1: 3047, 29645, 2896, 1677, 1615, 1587, 1414, 1324, 1267, 1232,
1195, 1154, 1113, 1068, 1043, 1015, 987, 948, 936, 920, 895, 850, 836,
822, 769, 736, 699, 638; 1H NMR (300 MHz, DMSO‑d6): δ 3.1–3.2 (4H,
m), 3.8–3.9 (4H, m), 7.7–7.9 (10H, m); 13C NMR (75 MHz, DMSO‑d6): δ
37.1, 64.3, 105.8, 122.2, 125.4, 125.8, 129.0, 130.8, 135.0, 135.8,
138.9, 187.2; m/z (%) 468 (100) [M+], 255 (22), 183 (54), 115 (88), 86
(27); Anal. Calcd. for C24H18F6O3: C, 61.54; H, 3.87. Found: C, 61.43; H,
3.95.
4.1.7. (7E,9E)-7,9-Bis-(4-pentafluorothiobenzylidene)-1,4-dioxaspiro
[4.5]decan-8-one (1l)
1,4-Cyclohexanedione monoethylene acetal (88 mg, 0.5 mmol) and
4-(pentafluorothio)benzaldehyde (232 mg, 1.0 mmol) were dissolved in
methanol (10 mL) and NaOH (200 mg, dissolved in 2 mL H2O) was
added. The reaction mixture was stirred at room temperature for 1 h.
The formed precipitate was collected and dried in vacuum. Yield: 180
8

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Bioorganic Chemistry 114 (2021) 105099
4.1.11. (3E,5E)-3,5-Bis-(4-pentafluorothiobenzylidene)-1-(1-
vacuum. Yield: 140 mg (0.24 mmol, 30%); yellow solid of m.p. 266 ◦C;
υmax(ATR)/cmꢀ 1 3094, 2822, 1674, 1614, 1584, 1478, 1453, 1413,
1367, 1319, 1300, 1264, 1237, 1188, 1169, 1140, 1113, 1094, 1068,
1039, 1013, 994, 967, 956, 937, 912, 861, 846, 825, 770, 755, 742, 736,
709, 699, 649, 636, 625; 1H NMR (300 MHz, CDCl3) δ 4.69 (4H, s),
7.5–7.6 (2H, m), 7.6–7.7 (4H, m), 7.72 (4H, d, J = 8.3 Hz), 7.89 (4H, d, J
= 8.3 Hz); 13C NMR (75.5 MHz, CDCl3) δ 47.0, 122.7, 126.1–126.3 (m),
129.7, 129.9, 130.1, 130.3, 130.5, 131.4, 132.8, 136.3, 136.7, 138.4,
139.1, 184.5; m/z (%) 587 (23) [M+], 585 (63), 516 (17), 410 (100),
381 (23), 340 (33), 212 (47), 184 (77), 115 (43), 111 (27); Anal. Calcd.
for C27H18ClF6NO3S: C, 55.35; H, 3.10; N, 2.39. Found: C, 55.29; H,
3.20; N, 2.45.
methoxybenzene-4-sulfonyl)-piperidone (2f)
1-(4-Methoxybenzolsulfonyl)-4-piperidinone (213 mg, 0.79 mmol)
and 4-(pentafluorothio)-benzaldehyde (348 mg, 1.5 mmol) were dis-
solved in EtOH (10 mL) and conc. HCl (1 mL) was added. The reaction
mixture was refluxed for 24 h. After cooling down to room temperature,
the formed precipitate was collected, washed with EtOH and dried in
vacuum. Yield: 165 mg (0.24 mmol, 30%); yellow solid of m.p.
298–299 ◦C; υmax(ATR)/cmꢀ 1 2944, 2908, 1674, 1616, 1595, 1575,
1497, 1466, 1437, 1409, 1312, 1265, 1245, 1207, 1189, 1160, 1145,
1102, 1091, 1025, 1036, 988, 954, 937, 824, 795, 751, 730, 692, 678,
665; 1H NMR (300 MHz, DMSO‑d6) δ 3.86 (3H. s), 4.63 (4H, s), 7.06 (2H,
d, J = 8.8 Hz), 7.46 (2H, d, J = 8.8 Hz), 7.61 (2H, s), 7.71 (4H, d, J = 8.8
Hz), 8.05 (4H, d, J = 8.8 Hz); 13C NMR (75.5 MHz, DMSO‑d6) δ 46.5,
55.8, 114.6, 126.2, 126.3, 128.6, 129.6, 131.1, 133.0, 134.9, 137.9,
152.6, 152.8, 163.1, 183.9; m/z (%) 697 (11) [M+], 678 (8), 526 (100),
398 (18), 270 (23), 242 (26), 171 (36), 115 (15), 107 (20); Anal. Calcd.
for C26H21F10NO4S3: C, 44.76; H, 3.03; N, 2.01. Found: C, 44.68; H, 2.97;
N, 2.07.
4.1.15. (3E,5E)-3,5-Bis-(4-pentafluorothiobenzylidene)-1-(1-
chlorobenzene-4-sulfonyl)-piperidone (2j)
1-(4-Chlorobenzolsulfonyl)-4-piperidinone (216 mg, 0.79 mmol)
and 4-(pentafluorothio)-benzaldehyde (348 mg, 1.5 mmol) were dis-
solved in EtOH (10 mL) and conc. HCl (1 mL) was added. The reaction
mixture was refluxed for 24 h. After cooling down to room temperature,
the formed precipitate was collected, washed with EtOH and dried in
vacuum. Yield: 170 mg (0.24 mmol, 30%); yellow solid of m.p. 270 ◦C;
υmax(ATR)/cmꢀ 1 2933, 1677, 1617, 1587, 1475, 1409, 1354, 1266,
1243, 1189, 1168, 1096, 1037, 1016, 987, 951, 938, 823, 795, 756, 728,
693, 666; 1H NMR (300 MHz, DMSO‑d6) δ 4.69 (4H, s), 7.5–7.6 (6H, m),
7.73 (4H, d, J = 8.8 Hz), 8.04 (4H, d, J = 8.8 Hz); 13C NMR (75.5 MHz,
DMSO‑d6) δ 46.5, 126.3, 129.2, 129.6, 131.2, 132.7, 135.1, 136.2,
137.8, 138.7, 152.9, 183.9; m/z (%) 703 (23) [M+], 701 (46) [M+], 575
(8), 527 (100), 399 (42), 270 (36), 242 (85), 115 (67), 111 (53); Anal.
Calcd. for C25H18ClF10NO3S3: C, 42.77; H, 2.58; N, 2.00. Found: C,
42.68; H, 2.66; N, 2.08.
4.1.12. (3E,5E)-3,5-Bis-(4-trifluoromethylbenzylidene)-1-(1-
fluorobenzene-4-sulfonyl)-piperidone (2g)
1-(4-Fluorobenzolsulfonyl)-4-piperidinone (203 mg, 0.79 mmol) and
4-trifluoromethylbenzaldehyde (260 mg, 1.5 mmol) were dissolved in
EtOH (10 mL) and conc. HCl (1 mL) was added. The reaction mixture
was refluxed for 24 h. After cooling down to room temperature, the
formed precipitate was collected, washed with EtOH and dried in vac-
uum. Yield: 150 mg (0.26 mmol, 33%); yellow solid of m.p. 288–289 ◦C;
υmax(ATR)/cmꢀ 1 3106, 3075, 3050, 1673, 1612, 1588, 1496, 1487,
1452, 1436, 1414, 1323, 1292, 1261, 1241, 1228, 1200, 1187, 1161,
1112, 1086, 1070, 1038, 1015, 969, 958, 951, 942, 918, 846, 837, 803,
765, 738, 710, 694, 674, 639, 617; 1H NMR (300 MHz, DMSO‑d6) δ 4.69
4.1.16. (3E,5E)-3,5-Bis-(4-trifluoromethylbenzylidene)-1-(1-
(4H, s), 7.3–7.5 (2H, m), 7.6–7.8 (8H, m), 7.89 (4H, d, J = 8.3 Hz); 13
C
fluorobenzene-3-sulfonyl)-piperidone (2k)
NMR (75.5 MHz, DMSO‑d6) δ 46.5, 116.5–116.8 (m), 122.2, 124.5,
125.6–125.8 (m), 129.1, 129.6, 129.8, 130.3, 130.4, 130.5, 130.9,
131.0, 131.3, 132.3, 133.8, 135.7, 137.9, 138.5, 163.1, 166.4, 183.9; m/
z (%) 569 (67) [M+], 550 (17), 500 (15), 410 (100), 381 (23), 340 (37),
212 (52), 184 (93), 159 (40), 115 (55), 95 (43); Anal. Calcd. for
C27H18F7NO3S: C, 56.94; H, 3.19; N, 2.46. Found: C, 57.00; H, 3.11; N,
2.51.
1-(3-Fluorobenzolsulfonyl)-4-piperidinone (203 mg, 0.79 mmol) and
4-trifluoromethylbenzaldehyde (260 mg, 1.5 mmol) were dissolved in
EtOH (10 mL) and conc. HCl (1 mL) was added. The reaction mixture
was refluxed for 24 h. After cooling down to room temperature and 24 h
in the refrigerator, the formed yellow crystals were collected, washed
with EtOH and dried in vacuum. Yield: 150 mg (0.26 mmol, 33%);
yellow solid of m.p. 257–258 C; υmax(ATR)/cmꢀ 1 1672, 1612, 1588,
◦
1478, 1437, 1412, 1356, 1324, 1265, 1242, 1227, 1188, 1161, 1124,
1069, 1038, 1016, 986, 952, 939, 886, 842, 807, 791, 766, 739, 703,
692, 679, 648, 625, 599; 1H NMR (300 MHz, DMSO‑d6) δ 4.7–4.8 (4H,
m), 7.2–7.4 (2H, m), 7.5–7.7 (4H, m), 7.7–7.8 (4H, m), 7.8–7.9 (4H, m);
13C NMR (75.5 MHz, DMSO‑d6) δ 46.4, 114.0–114.4 (m), 120.7–121.0
(m), 122.2, 123.4, 125.6–125.7 (m), 129.2, 129.6, 131.0, 131.9, 132.3,
135.7, 137.8, 139.5, 160.0, 163.3, 184.0; m/z (%) 569 (94) [M+], 550
(26), 500 (24), 410 (100), 381 (37), 358 (29), 340 (60), 226 (23), 212
(78), 184 (96), 159 (37), 115 (82), 95 (66); Anal. Calcd. for
4.1.13. (3E,5E)-3,5-Bis-(4-pentafluorothiobenzylidene)-1-(1-
fluorobenzene-4-sulfonyl)-piperidone (2h)
1-(4-Fluorobenzolsulfonyl)-4-piperidinone (203 mg, 0.79 mmol) and
4-(pentafluorothio)-benzaldehyde (348 mg, 1.5 mmol) were dissolved in
EtOH (10 mL) and conc. HCl (1 mL) was added. The reaction mixture
was refluxed for 20 h. After cooling down to room temperature, the
formed precipitate was collected, washed with EtOH and dried in vac-
◦
uum. Yield: 175 mg (0.26 mmol, 33%); yellow solid of m.p. 252 C;
υmax(ATR)/cmꢀ 1 1675, 1613, 1591, 1570, 1489, 1409, 1358, 1296,
1263, 1230, 1171, 1160, 1092, 1037, 981, 955, 933, 837, 823, 797, 750,
731, 712, 690, 675, 664, 636, 617, 599, 582; 1H NMR (300 MHz, CDCl3)
δ 4.58 (4H, s), 7.1–7.2 (2H, m), 7.41 (4H, d, J = 8.8 Hz), 7.6–7.7 (2H, m),
7.70 (2H, s), 7.85 (4H, d, J = 8.8 Hz); 13C NMR (75.5 MHz, DMSO‑d6) δ
46.5, 116.7, 116.9, 126.3, 130.6, 131.2, 132.7, 135.2, 137.8, 165.8,
183.9; m/z (%) 685 (66) [M+], 666 (12), 558 (12), 526 (100), 398 (25),
270 (22), 242 (51), 115 (32), 95 (28); Anal. Calcd. for C25H18F11NO3S3:
C, 43.80; H, 2.65; N, 2.04. Found: C, 43.71; H, 2.58; N, 2.11.
C27H18F7NO3S: C, 56.94; H, 3.19; N, 2.46. Found: C, 57.02; H, 3.13; N,
2.50.
4.1.17. (3E,5E)-3,5-Bis-(4-pentafluorothiobenzylidene)-1-(1-
fluorobenzene-3-sulfonyl)-piperidone (2l)
1-(3-Fluorobenzenesulfonyl)-4-piperidinone (203 mg, 0.79 mmol)
and 4-(pentafluorothio)-benzaldehyde (348 mg, 1.5 mmol) were dis-
solved in EtOH (10 mL) and conc. HCl (1 mL) was added. The reaction
mixture was refluxed for 20 h. After cooling down to room temperature,
the formed precipitate was collected, washed with EtOH and dried in
vacuum. Yield: 170 mg (0.25 mmol, 32%); yellow solid of m.p.
206–207 ◦C; υmax(ATR)/cmꢀ 1 2933, 1683, 1618, 1595, 1498, 1479,
1438, 1412, 1353, 1306, 1272, 1239, 1224, 1185, 1160, 1101, 1083,
1070, 1041, 1041, 981, 955, 937, 892, 832, 795, 749, 731, 703, 683,
663; 1H NMR (300 MHz, DMSO‑d6) δ 4.73 (4H, s), 7.3–7.4 (2H, m),
7.6–7.7 (4H, m), 7.7–7.8 (4H, m), 8.0–8.1 (4H, m); 13C NMR (75.5 MHz,
DMSO‑d6) δ 46.4, 114.5, 120.7, 123.6, 126.2–126.3 (m), 131.2, 131.9,
4.1.14. (3E,5E)-3,5-Bis-(4-trifluoromethylbenzylidene)-1-(1-
chlorobenzene-4-sulfonyl)-piperidone (2i)
1-(4-Chlorobenzolsulfonyl)-4-piperidinone (216 mg, 0.79 mmol)
and 4-trifluoromethylbenzaldehyde (260 mg, 1.5 mmol) were dissolved
in EtOH (10 mL) and conc. HCl (1 mL) was added. The reaction mixture
was refluxed for 24 h. After cooling down to room temperature, the
formed precipitate was collected, washed with EtOH and dried in
9

I.S. Al Nasr et al.
Bioorganic Chemistry 114 (2021) 105099
132.7, 135.0, 137.7, 139.4, 160.6–163.3 (m), 184.0; m/z (%) 685 (37)
[M+], 526 (100), 398 (26), 242 (45), 115 (31), 95 (33); Anal. Calcd. for
C25H18F11NO3S3: C, 43.80; H, 2.65; N, 2.04. Found: C, 43.70; H, 2.60; N,
2.09.
4.2. Leishmania major cell isolation, culture conditions, and assays
Promastigotes of L. major were isolated from a Saudi patient
(February 2016) and maintained at 26 ◦C in Schneider’s Drosophila
medium (Invitrogen, USA) containing 10% heat inactivated fetal bovine
serum (FBS, Invitrogen, USA) and antibiotics in a tissue culture flask
with weekly transfers. Promastigotes were cryopreserved in liquid ni-
trogen at concentrations of 3 × 106 parasite/mL. Virulent L. major
parasites were maintained by passing in female BALB/c mice by
injecting hind footpads with 1x106 stationary-phase promastigotes. l.
major amastigotes were isolated from the mice after 8 weeks. Isolated
amastigotes were converted to promastigotes by cultivation at 26 ◦C in
Schneider’s medium supplemented with antibiotics and 10% FBS.
Amastigote-derived promastigotes, which had undergone less than five
in vitro passages, were used for infection. BALB/c mice (male and female
individuals) were obtained from Pharmaceutical College, King Saud
University, Kingdom of Saudi Arabia, and maintained in specific
pathogen-free facilities. The handling of the laboratory animals followed
the instructions and rules of the committee of research ethics, Deanship
of Scientific Research, Qassim University, permission number 20–03-20.
L. major promastigotes from logarithmic-phase were cultured in
phenol red-free RPMI 1640 medium (Invitrogen, USA) with 10% FBS
and suspended on 96-wells plates to yield 106 cells mLꢀ 1 (200 µL/well)
after counting by a hemocytometer. Compounds were added to the
wells, obtaining final concentrations of 50, 25, 12.5, 6.25, 3.13, 1.65,
and 0.75 µg mLꢀ 1. Negative controls contained cultures with DMSO
(1%) devoid of test compound and positive control wells contained
cultures with decreasing concentrations of amphotericin B (50, 25, 12.5,
6.25, 3.13, 1.65, 0.75 µg mLꢀ 1) as active reference compound. After
4.1.18. (3E,5E)-3,5-Bis-(4-trifluoromethylbenzylidene)-1-(1,2-
difluorobenzene-4-sulfonyl)-piperidone (2m)
1-(3,4-Difluorobenzolsulfonyl)-4-piperidinone (217 mg, 0.79 mmol)
and 4-trifluoromethylbenzaldehyde (260 mg, 1.5 mmol) were dissolved
in EtOH (10 mL) and conc. HCl (1 mL) was added. The reaction mixture
was refluxed for 24 h. After cooling down to room temperature and 24 h
in the refrigerator, the formed yellow crystals were collected, washed
with EtOH and dried in vacuum. Yield: 140 mg (0.24 mmol, 30%);
yellow solid of m.p. 251–252 C; υmax(ATR)/cmꢀ 1 1672, 1611, 1586,
◦
1506, 1414, 1353, 1327, 1278, 1262, 1243, 1213, 1187, 1158, 1116,
1072, 1038, 1016, 985, 952, 943, 914, 887, 843, 828, 805, 776, 766,
739, 708, 699, 677, 644, 624; 1H NMR (300 MHz, DMSO‑d6) δ 4.7–4.8
(4H, m), 7.4–7.5 (1H, m), 7.6–7.8 (8H, m), 7.8–7.9 (4H, m); 13C NMR
(75.5 MHz, DMSO‑d6) δ 46.5, 117.1–117.4 (m), 118.9–119.1 (m), 122.2,
125.4–125.7 (m), 129.2–129.6 (m), 131.0, 132.3, 134.6, 135.8, 137.8,
150.8, 184.1; m/z (%) 587 (87) [M+], 568 (22), 518 (25), 410 (100),
381 (23), 340 (35), 212 (47), 184 (95), 115 (49), 100 (37); Anal. Calcd.
for C27H17F8NO3S: C, 55.20; H, 2.92; N, 2.38. Found: C, 55.11; H, 2.86;
N, 2.30.
4.1.19. (3E,5E)-3,5-Bis-(4-trifluoromethylbenzylidene)-1-(1,2-
dichlorobenzene-4-sulfonyl)-piperidone (2n)
1-(3,4-Dichlorobenzenesulfonyl)-4-piperidinone (243 mg, 0.79
mmol) and 4-trifluoromethylbenzaldehyde (261 mg, 1.5 mmol) were
dissolved in EtOH (10 mL) and conc. HCl (1 mL) was added. The reaction
mixture was refluxed for 20 h. After cooling down to room temperature,
the reaction mixture was kept in a refrigerator at 4 ◦C for 24 h, the
formed precipitate was collected, washed with EtOH and dried in vac-
uum. Yield: 200 mg (0.32 mmol, 41%); yellow solid of m.p. 201–203 ◦C;
υmax(ATR)/cmꢀ 1 3098, 1678, 1614, 1590, 1455, 1412, 1351, 1319,
1372, 1351, 1319, 1258, 1241, 1199, 1165, 1124, 1109, 1069, 1031,
1015, 982, 966, 956, 928, 891, 846, 821, 800, 766, 739, 703, 686, 654,
630; 1H NMR (300 MHz, DMSO‑d6) δ 4.78 (4H, s), 7.4–7.5 (1H, m),
7.6–7.7 (3H, m), 7.7–7.8 (4H, m), 7.8–8.0 (5H, m); 13C NMR (75.5 MHz,
DMSO‑d6) δ 46.5, 122.2, 125.7, 127.3, 128.8, 128.9, 129.2, 129.4,
129.6, 131.1, 131.8, 132.1, 132.4, 135.9, 136.8, 137.7, 137.9, 183.9; m/
z (%) 621 (17) [M+], 619 (32) [M+], 602 (3), 600 (4), 552 (6), 550 (8),
410 (100), 381 (12), 340 (21), 212 (28), 184 (48), 145 (35), 115 (45);
Anal. Calcd. for C27H17Cl2F6NO3S: C, 52.27; H, 2.76; N, 2.26. Found: C,
52.19; H, 2.69; N, 2.20.
◦
incubation at 26 C for 72 h, the number of viable promastigotes was
assessed by colorimetric method (tetrazolium salt colorimetric assay,
MTT). The formed colored formazan was isolated and solubilized by
addition of a detergent solution. The samples were analysed by using an
ELISA reader at 570 nm. EC50 values were calculated from three inde-
pendent experiments [46].
For evaluation of the activity against amastigotes in macrophages,
peritoneal macrophages were collected from female BALB/c mice (6–8
weeks of age) by aspiration. 5 × 104 cells/well were placed into 96-wells
plates containing phenol red-free RPMI 1640 medium with 10% FBS and
◦
were incubated to promote cell adhesion at 37 C for 4 h in 5% CO2
atmosphere. Thereafter, the medium was discarded and the cells were
washed with phosphate buffered saline (PBS). L. major promastigotes
solution (200 µL at a ratio of 10 promastigotes : 1 macrophage in RPMI
1640 medium with 10% FBS) was added to each well and the plates were
incubated for 24 h at 37 ◦C in humidified 5% CO2 atmosphere to enable
macrophage infection and amastigote differentiation. The infected cells
were washed three times with PBS to remove the free promastigotes and
overlaid with fresh phenol red-free RPMI 1640 medium containing test
compounds (50, 25, 12.5, 6.25, 3.13, 1.65, and 0.75 µg mLꢀ 1) where-
upon the cells were incubated at 37 ◦C for 72 h in humidified 5% CO2
atmosphere. Cultures solely containing DMSO (1%) were used as
negative controls while wells containing cultures with decreasing con-
centrations of amphotericin B (reference compound, 50, 25, 12.5, 6.25,
3.13, 1.65, and 0.75 µg mLꢀ 1) were used as positive control. The per-
centage of infected macrophages was evaluated microscopically after
the removal of the medium, washing, fixation, and Giemsa staining of
the cells. Calculated EC50 values were obtained from three independent
experiments [46].
4.1.20. (3E,5E)-3,5-Bis-(4-pentafluorothiobenzylidene)-1-(1,2-
dichlorobenzene-4-sulfonyl)-piperidone (2o)
1-(3,4-Dichlorobenzenesulfonyl)-4-piperidinone (243 mg, 0.79
mmol) and 4-(pentafluorothio)-benzaldehyde (348 mg, 1.5 mmol) were
dissolved in EtOH (10 mL) and conc. HCl (1 mL) was added. The reaction
mixture was refluxed for 20 h. After cooling down to room temperature,
the formed precipitate was collected and washed with EtOH. The yellow
precipitate was recrystallized from EtOH. Yield: 190 mg (0.26 mmol,
33%); yellow solid of m.p. 225–226 C; υmax(ATR)/cmꢀ 1 1675, 1615,
◦
1589, 1495, 1450, 1434, 1407, 1374, 1357, 1292, 1265, 1243, 1188,
1171, 1141, 1097, 1035, 987, 950, 940, 891, 821, 792, 750, 728, 702,
692, 678, 666, 653, 627; 1H NMR (300 MHz, DMSO‑d6) δ 4.78 (4H, s),
7.49 (1H, d, J = 8.5 Hz), 7.61 (2H, s), 7.7–7.8 (5H, m), 7.84 (1H, d, J =
8.5 Hz), 8.0–8.1 (4H, m); 13C NMR (75.5 MHz, DMSO‑d6) δ 46.5, 126.3,
127.3, 129.0, 131.2, 132.4, 132.5, 135.2, 136.8, 137.6, 137.9, 152.9,
183.8; m/z (%) 737 (17) [M+], 735 (24) [M+], 718 (3), 716 (4), 610 (4),
608 (6), 526 (100), 398 (31), 270 (25), 242 (58), 145 (43), 115 (68);
Anal. Calcd. for C25H17Cl2F10NO3S3: C, 40.77; H, 2.33; N, 1.90. Found:
C, 40.70; H, 2.39; N, 1.85.
4.3. Toxoplasma gondii cell line, culture conditions, and assay
Serial passages of cells of the Vero cell line (ATCC® CCL81™, USA)
were applied for the cultivation of T. gondii tachyzoites of the RH strain
(a gift from Dr. Saeed El-Ashram, State Key Laboratory for Agro-
biotechnology, China Agricultural University, Bejing, China). Vero cells
were cultured by using complete RPMI 1640 medium with heat-
10

I.S. Al Nasr et al.
Bioorganic Chemistry 114 (2021) 105099
inactivated 10% FBS in a humidified 5% CO2 atmosphere at 37 ◦C. 96-
Well plates (5 × 103 cells/well in 200 µL RPMI 1640 medium) were
used for the cultivation of the Vero cells and then incubated at 37 ◦C and
5% CO2 for one day, followed by removal of medium and washing the
cells with PBS. Then, RPMI 1640 medium containing 2% FBS and
T. gondii tachyzoites (RH strain) was given to the cells at a ratio of 5
(parasite) : 1 (Vero cells). After incubation at 37 ◦C and 5% CO2 for 5 h,
cells were washed with PBS and then treated as described below.
Control: RPMI 1640 medium containing DMSO (1%)
charges were assigned to all atoms. In applying torsions in ligands all
rotatable bonds were rotated. The ligands were made flexible and the
protein was kept rigid. PDBQT files (file format that contains partial
charges and torsion records along with atom coordinates) were written
for the protein and each ligand and were used as input files for docking
experiments.
Standard docking procedures for a rigid protein and a flexible ligand
were used as per user guide for AutoDock.4.2. Briefly, using AutoGrid a
grid of 60x60x60 points in ×, y, and z directions was built with grid
points spaced at 0.375 Å. Electrostatic maps were calculated by using a
distance dependent function of dielectric constants. All other parameters
were set as default. Docking simulations were performed by employing
Lamarckian Genetic Algorithm (LGA). The implementation of LGA
included the creation of an initial population of 150 individuals, for
which random torsions were applied. In each docking run, a maximum
of 2,500,000 energy evaluations was performed. For either of the two
ligands, at least 20 such runs were performed. The best binding modes
for each ligand were analyzed with LigPlot+, ADT and RasMol (Roger
Sayle) programs [57].
Experimental: Medium + compounds (dissolved in DMSO) (50, 25,
12.5, 6.25, 3.13, 1.65, and 0.75 µg mLꢀ 1).
After incubation at 37 ◦C and 5% CO2 for 72 h, the cells were washed
with PBS, fixed in 10% formalin and stained with 1% toluidine blue.
Examination of the cells and determination of the infection index
(number of cells infected from 200 cells tested) of T. gondii were carried
out with an inverted photomicroscope. The following equation was used
for the calculation of the inhibition in %:
Inhibition (%) = (I Control - I Experimental)/(I Control) × 100
́
́
́
́
where, I Control refers to the infection index of untreated cells and I
́
́
́
́
Experimental refers to the infection index of cells treated with test
compounds.
Declaration of Competing Interest
Then effects of test compounds on parasite growth were expressed as
EC50 (effective concentration at 50%) values. EC50 values were obtained
from three independent experiments [46,47].
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
4.4. In vitro cytotoxicity assay
Acknowledgements
MTT assays were carried out for a cytotoxicity evaluation of the test
compounds. Briefly, Vero cells were cultured in 96-well plates (5 × 103
cells/well/200 µL) for 24 h in RPMI 1640 medium with 10% FBS and 5%
CO2 at 37 ◦C. Cells were washed with PBS, followed by treatment with
test compounds for 72 h at varying concentrations (50, 25, 12.5, 6.25,
3.13, 1.65, and 0.75 µg mLꢀ 1) in 10% FBS medium. Cells treated solely
with medium in 2% FBS were used as negative control. The supernatant
was discarded and 50 µL RPMI 1640 medium containing 14 µL MTT (5
mg mLꢀ 1) was added and the cells were incubated for 4 h. The super-
natant was removed again and 150 µL DMSO was added in order to
dissolve the formed formazan. A FLUOstar OPTIMA spectrophotometer
was applied for colorimetric analysis (λ = 540 nm). The cytotoxicity was
expressed by IC50 values (concentration which caused a 50% reduction
in viable cells). IC50 values were calculated from three independent
experiments [46,48].
We are grateful to the College of Applied Health Sciences at Ar Rass,
Qassim University.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.105099.
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