Symmetrical and unsymmetrical substituted 2,5-diarylidene cyclohexanones as anti-parasitic compounds

Article abstract of DOI:10.1016/j.ejmech.2018.06.031

Symmetrical and unsymmetrical bis-aryl-α,β-unsaturated ketones were synthesized in moderate to excellent yield by treating cyclohexanone with various aldehydes. Dimethylammonium dimethylcarbamate (DIMCARB) was used as both catalyst and reaction medium for the synthesis of monoarylidenes cycloadduct intermediates, which was further used to produce diarylidene cyclohexanones. All the 34 compounds synthesized were evaluated for their anti-proliferative activity, particularly against promastigote of Leishmania amazonensis, epimastigoteand trypomastigoteof Trypanosoma cruzi. Eighteen compounds displayed anti-leishmanial activity against promastigotes of L. amazonensis with IC₅₀ values ranging from 2.8 to 10 μM. In addition, two compounds exhibited significant antitrypanosomal activity against epimastigotes of T. cruzi with IC₅₀ values of 5.2 ± 0.8 and 3.0 ± 0.0 μM, while five compounds exhibited activity from 15.0 ± 1.4 to 30.2 ± 1.8 μM against trypomastigote of T. cruzi. Moreover, all compounds were more selective against the parasites than the epithelial cells. The unsymmetrical compounds 16, 28, 30 and 33 can be considered as favorable anti-parasitic lead molecule having IC₅₀ and EC₅₀ values in the low-micromolar range, better than the reference drug benznidazole, and low cytotoxicity against Vero cells. The potent compounds were screened in silico against 17 enzymes of T. cruzi and best scoring were found against Dihydroorotate Dehydrogenase.

Full text of DOI:10.1016/j.ejmech.2018.06.031

Accepted Manuscript
Symmetrical and unsymmetrical substituted 2,5-diarylidene cyclohexanones as anti-
parasitic compounds
Zia Ud Din, Marilia Almeida Trapp, Lívia Soman de Medeiros, Danielle Lazarin-Bidóia,
Francielle Pelegrin Garcia, Francieli Peron, Celso Vataru Nakamura, Ihosvany Camps
Rodríguez, Abdul Wadood, Edson Rodrigues-Filho
PII:
S0223-5234(18)30522-1
10.1016/j.ejmech.2018.06.031
EJMECH 10501
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 September 2017
Revised Date: 7 March 2018
Accepted Date: 12 June 2018
Please cite this article as: Z.U. Din, M.A. Trapp, Lí. Soman de Medeiros, D. Lazarin-Bidóia, F.P.
Garcia, F. Peron, C.V. Nakamura, I.C. Rodríguez, A. Wadood, E. Rodrigues-Filho, Symmetrical and
unsymmetrical substituted 2,5-diarylidene cyclohexanones as anti-parasitic compounds, European
Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.06.031.
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ACCEPTED MANUSCRIPT
O
R'
R
1 - 34
Screening against T. Cruzi
and L. amazonesis
O
OCH3
OCH3
O
OCH3
H3CO
H3CO
NO2
16
28
OCH3
O
OCH3
O
H3CO
OCH3
OCH3
H3CO
OCH3
OCH3
33
30
OCH3

ACCEPTED MANUSCRIPT
Symmetrical and unsymmetrical substituted 2,5-diarylidene cyclohexanones as anti-
parasitic compounds
Zia Ud Dina,e*, Marilia Almeida Trappa, Lívia Soman de Medeirosa, Danielle Lazarin-Bidóiab,
Francielle Pelegrin Garciab, Francieli Peronb, Celso Vataru Nakamurab, Ihosvany Camps
Rodríguezc Abdul Wadoodd Edson Rodrigues-Filhoa*
,
,
aLaBioMMi, Departament of Chemistry, Federal University of São Carlos, CP 676, 13.565-
905, São Carlos, SP, Brazil.
bLaboratório de Inovação Tecnológica no Desenvolvimento de Fármacos e Cosméticos,
Universidade Estadual de Maringá, Av. Colombo 5790, 87020-900, Bloco B-08, Maringá,
PR, Brazil.
cLaboratório de Modelagem Computacional - LaModel, Instituto de Ciências Exatas - ICEx,
Universidade Federal de Alfenas - UNIFAL-MG, Av. Jovino Fernandes Sales s/n, Bairro
Santa Clara, 37130-000. Alfenas. MG. Brazil.
dDepartment of Biochemistry, Abdul Wali Khan University Mardan, Mardan-23200, Pakistan.
eDepartment of Chemistry, Woman University Swabi, Swabi-23340, KP, Pakistan.

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Abstract:
Symmetrical and unsymmetrical bis-aryl-α,β-unsaturated ketones were synthesized in
moderate to excellent yield by treating cyclohexanone with various aldehydes.
Dimethylammonium dimethylcarbamate (DIMCARB) was used as both catalyst and reaction
medium for the synthesis of monoarylidenes cycloadduct intermediates, which was further
used to produce diarylidene cyclohexanones. All the 34 compounds synthesized were
evaluated for their anti-proliferative activity, particularly against promastigote of Leishmania
amazonensis, epimastigoteand trypomastigoteof Trypanosoma cruzi. Eighteen compounds
displayed anti-leishmanial activity against promastigotes of L. amazonensis with IC50 values
ranging from 2.8 to 10 µM. In addition, two compounds exhibited significant
antitrypanosomal activity against epimastigotes of T. cruzi with IC50 values of 5.2 ± 0.8 and
3.0 ± 0.0 µM, while five compounds exhibited activity from 15.0 ± 1.4 to 30.2 ± 1.8 µM
against trypomastigote of T. cruzi. Moreover, all compounds were more selective against the
parasites than the epithelial cells. The unsymmetrical compounds 16, 28, 30 and 33 can be
considered as favorable anti-parasitic lead molecule having IC50 and EC50 values in the low-
micromolar range, better than the reference drug benznidazole, and low cytotoxicity against
Vero cells. The potent compounds were screened in silico against 17 enzymes of T. cruzi and
best scoring were found against Dihydroorotate Dehydrogenase.
E-mail: zia.ufscar@gmail.com; edinho@pq.cnpq.br
1. Introduction
Chagas disease and leishmaniasis are parasitic diseases caused by protozoan Trypanasoma
cruzi and Leishmania sp respectively. Chagas disease is found mainly in Latin America, but
approximately 6 to 7 million people are estimated to be infected worldwide. Among them up
to 30% of chronically infected people develop cardiac alterations and up to 10% develop
digestive and neurological alteration [1]. The parasite infection leads to sudden death or heart
failure by the destruction of the heart muscle and nervous system [1]. Leishmaniasis affects
the poorest people on the planet, estimated 1.3 million new cases and 20,000 to 30,000 deaths
occur annually in 98 countries [2]. In the absence of suitable medication, it has a mortality
rate of almost 100% independent of the immunological position of the patient [3-5].
Both diseases are fatal and recently are spread to other countries from Latin American due to
global exchange and travelling [1]. The drugs available in the market are inadequate due to

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extreme toxicity, cost and ineffectiveness [6,7]. The two drugs nifurtimox (launched in1970)
and benznidazole (launched in 1974) used to treat Chagas disease are not much sufficient due
to long treatment courses, adverse effects and its ineffectiveness in late-stage patients [8,9].
Pentavalent antimonials drugs for leishmanisis, which were developed in 1940s, present high
toxicity and low efficiency. Pentamidine presents several side effects, including renal and
hepatic toxicity, hypotension, pancreatitis, and cardiac abnormalities [10]. Amphotericin B
and its formulations are quite active for visceral leishmaniasis (VL). However, it is very
costly and toxic [11]. Until now, no specific vaccine is appropriate for human use [12].
Therefore, there is an urgent need for the development of new, effective, inexpensive, and
harmless drugs to cure these parasitic diseases [9].
Bis-(arylmethylidene)-cycloalkanones have been found to display a great potential
applications, such as anti-inflammatory [13], anti-tumor [14], anti-oxidant [15-17], anti-
tubercular [18], cytotoxic [19,20], anti-fertility [21], and antifungal [22]. It has been proposed
that the activity of this class of compounds is related to their ability to act as Michael
acceptors with nucleophilic biological macromolecules, especially nucleic acids and enzymes
[23]. Further investigation using curcuminoids, which resembles the bis-aryl-α,β-unsaturated
ketone structures, have shown that the two aromatic moieties might be important for potential
protein-ligand binding activity. Thus these molecules have become a rational design of
potential inhibitor (ligand) of targeting protein [24].
Recently Bragaet al. have shown that symmetrical bis-(arylmethylidene)-cycloalkanones
(having exactly the same aryl group at both sides of the ketone) have great potential to treat
parasitic infections [25]. So, in the light of these findings, we aimed to synthesize symmetrical
and unsymmetrical bis-(arylmethylidene)-cyclohexanones, with different substitutions at the
aromatic rings, in order to evaluate the in vitro antileishmanial and antitrypanosomal
activities. To the best of our knowledge, this is the first evaluation of unsymmetrical bis-
(arylmethylidene)-cyclohexanones analogues as antileishmanial and anti-trypanosomal
agents. Inspired by the potent curcumin activities against many diseases, we started the
synthesis of unsymmetrical bis-(arylmethylidene)-cyclohexanones with methoxyl substituted
aldehydes [26].
Bis-aryl α,β-unsaturated cyclohexanones comprise the class of compounds having
cyclohexanone attached to two aryl system at both β-positions of two exocyclic C=C
conjugated with the C=O. The classical aldol condensation catalyzed by acid or base failed to
produce unsymmetrical bis-(arylmethylidene)-cyclohexanones, because it leads to the

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formation of symmetrical products [27]. In the present work these moieties were synthesized
via a two-steps reaction; the first step involved running a DIMCARB-catalyzed reaction of
cyclohexanone with anisaldehyde or substituted benzaldehydes to form monoarylidene
cycloadduct [28]; while the second step involved the reaction of monoarylidene cycloadduct
with various aldehyde under base-catalyzed conditions to produce unsymmetrical
diarylalkanone. DIMCARB was recovered by distillative dissociation-reassociation in
vacuum or under an atmosphere of CO2.
The antileishmanial and antitrypanosomal activity, as well as their cytotoxicity against non-
infected Vero cells (kidney epithelial cells from African monkey), were evaluated for all
synthesized compounds. Using docking studies, efforts were made to discover the best
possible target for potent compounds. Among seventeen evaluated target, the best binding
performance were got against Dihydroorotate dehydrogenase (3W88).
2. Results and Discussion
2.1. Chemistry
Several methodologies have been reported for the synthesis of symmetrical diarylalkanones,
ranging from use of alkaline mediums [29-31] to Al2O3 catalytic reaction under microwaves
[32]. In the present study we have applied acid-catalysed reactions in order to synthesize
symmetrical diarylalkanones (1-11) (Scheme 1). The reactions were carried out at room
temperature and all the starting materials were consumed in reaction times, with no recovery
of unreacted starting materials. The products were isolated from reaction solutions by re-
crystallization from ethanol and characterized mainly by NMR and MS. A good guide NMR
signals is a broad singlet at δH 7.50-7.80, which corresponds to the β-carbonyl hydrogen’s.
13
Aromatic signals appear at δH 6.00-7.50 depending on the nature of substituent attached. C
NMR spectra show C=O signals around δC 190 and aromatic signals at δC 100-170 depending
on the substituent attached to it (see supplementary materials).

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Scheme 1: Synthesis of symmetrical bis-(arylmethylidene)-cycloalkanones 1-11.
Braga et al. synthesized symmetrical alkanone by reacting two equivalent of the appropriate
aldehyde with ketone in basic media [25]. In the whole manuscript only symmetrical products
with their anti-parasitic study were reported. In our study we produced different symmetrical
compounds, as well as extended the synthesis and application to unsymmetrical alkanones.
The challenge has been to control the reaction to obtain monoarylidene product, which can
further be utilized as an intermediate for the preparation of unsymmetrical diarylalkanone.
This is because it is difficult to control the reaction at monoarylidende by using common acid
or base catalysts. This path usually goes to symmetrical diarylidene products. We have made
an attempt to control the reaction at monoarylidene step by using amine based ionic liquid
DIMCARB. DIMCARB can slowdown the aldol reaction at monoarylidene step producing
compounds 12 and 13 (Scheme 2).

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Scheme 2: Synthesis of unsymmetrical diarylalkanones12-34 with catalysis by DIMCARB.
Highlighted compounds were the most active ones.
The monoarylidenecy cloadducts (12 and 13, Scheme 2) were synthesized by the reaction of
cyclohexanone respectively with anisaldehyde and 2,3-dimethoxybenzaldehyde in the

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presence of DIMCARB as a catalyst in good yield. The use of ionic liquid DIMCARB helps
to control the reaction at one side by amine based catalysis process. It is an interesting
methodology for obtaining monoaldol product, and further functionalization of
monoarylidene intermediate by second step leading to unsymmetrical bis-(arylmethylidene)-
cycloalkanones. For the synthesis of unsymmetrical analogues (14-34), first the reaction was
carried on one alpha carbon (compounds 12 and 13) and the second step was optimized for
further reaction (Scheme 2). The compounds can be obtained in a crystalline nature by simple
and rapid steps in good to excellent yield from readily available starting materials. All
compounds were characterized using spectroscopic data.
1H NMR spectra of compound 12 and 13 show four specific signals of four methylene groups
in the shielded region at δH 1.7 - 2.9, which is the evidence that among five methylene of
cyclohexanone one is involved in the reaction, whereas one =CH signal was shown at δH 7.67
as singlet. Additionally, two aromatic signals having integration for four protons were
detected at δH 7.41 and 6.93, which confirm the aromatic moiety has added to cyclohexanone.
13C NMR spectrum of 12 and 13 showed four signals at δC 23.2 - 55.3, and six peaks from δC
113.9 - 1559.9 for eight carbons, with two of these peaks having double intensity for aromatic
carbons. The typical peak at δC 201.5 is due to carbonyl carbon. The spectra of all compounds
14-34 are similar except some signals in aromatic regions due to different substitution at
different position on second benzene ring. The presence of two broad singlet at deshielded
region (δH 7.00 and 8.00), clearly showed two =CH signals which confirm that the second
reaction did occur.
The reaction of 12 and 13 with various aldehydes gave products 14-34 in 72-94 % after re-
crystallization from ethanol. All these reactions were carried in basic medium in ethanol, and
their progress was monitored by TLC. The reaction time varied for each product and
depended on the nature and reactivity of the aldehyde used. Thus, as expected, electron
donating groups at various positions in the benzene rings increases the reaction time, while
electron withdrawing groups decreases reaction time.
2.2. Biological Evaluation
Initially, the in vitro antiprotozoal activities of the synthesized compounds were assayed
against promastigotes of L. amazonensis, the extracellular form in the insect host, and, against
epimastigote and trypomastigote forms of T. cruzi, found in the insect host and mammalian

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bloodstream, respectively. The inhibitory concentrations values are summarized in Table 1.
The symmetrical bisarylakanones 1, 3-5, 7, 8 and 11 displayed in vitro trypanocidal activity
with IC50 values ranging from 9.5 to 30 µM. The most potent compound among symmetrical
bisarylalkanones is 11, having methoxyl substituent on both aromatic rings A and B.
Monoarylidene intermediate 13 and 14 does not show significant activity, while the
introduction of second aryl group significantly changes its pharmacological potential.
Fourteen unsymmetrical bisarylalkanones (16, 18, 19, 21-25, 27, and 30-34) displayed in vitro
trypanocidal activity with IC50 values ranging from 3.3 to 30 µM (Table 1). Compounds 16
and 30 exhibited trypanocidal activity against epimastigote form with IC50 values of 5.2 ± 0.8
and 3.0 ± 0.0 µM respectively, while against promastigote form of L. amazonensis the IC50
were 8.9 ± 0.7 and 2.6 ± 0.4µM respectively. The compounds 16 and 30 were the most potent
in the whole series. In addition, our compounds were evaluated against Vero cells. The results
showed that the active compounds were more toxic against the parasites than for the cell
tested (Table 1).
It seems that there is a correlation between leishmanicidal and trypanocidal activity. All
compounds active against T. cruzi also showed some activity against L. amazonensis. The
potent activity of compounds 16 and 30 may be due to the electron donating effect of
methoxyl substitution on aromatic ring like that present in curcumin, a natural compound
having two aryl groups attached to the ends of an unsaturated C7 carbon-chain that resembles,
in this sense, the compounds described here. Recently it was investigated that methoxyl group
in curcuminoid analogues play an important role in the biological activity by binding with
amino acids arginine, histidine, lysine, and leucine in the active site of enzymes [33]. Singh
and Misra also investigated through distance mapping of active site that methoxyl is crucial
for interaction with catalytic residues of thioredoxin [34]. These literature data corroborate
our results for the most active substances.
It was observed that substitution has a great impact upon activity. The -N(CH3)2 and -NO2
groups at para position on aromatic ring B of compounds 15 and 17 diminishes activity.
Chlorine substitution at para position positively impacts the activity. Thus, compound 19
shows IC50 21.6 µM, greater than compound 18 having chlorine at ortho position. The
number of chlorine on aromatic ring B also affects the activity. That is why compound 21,
having two chlorine atoms, show better activity than 18 and 19. The fluorine atom has
different effect on activity than chlorine. The fluorine substitution at ortho position is more
significant than at para position. The difference arises due to the size of the halogens, since
fluorine has smaller size than chlorine so affect the inhibition differently. Bromine at meta

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position was found significant than para position in compounds 20 and 24. In the whole series
the potent compounds are 11, 16 and 30, having methoxyl substitutions on aromatic rings A
and B. In overall, the electron donating groups attached to aromatic ring increases the activity,
while electron withdrawing group decreases it. These result corroborate the study reported by
Aanandhi and coworker [35] and also Suryawanshiand and his collogues [36], that also
showed the importance of methoxyl substituent for antiparasitic activity. In overall, the
electron donating groups attached to aromatic ring increases the activity, while electron
withdrawing group decreases it.
The mechanism of drugs acting against parasites have long been studied by other researchers
with different approaches [37]. We studied how the parasite biochemistry deal with the drugs
tested, considering in vitro inhibition of different enzymes of T. cruzi like arginase, cruzain,
trypanothione reductase and GAPDH, but our compounds showed no activity against these
targets (results not shown here). Later during other studies, we realized that our compounds
could be good Michael acceptors due the dienone system. We also found in our previous
study that enzymes having nucleophilic centers for Michael reaction chances, like those
cysteine-containing ones, may be the target. When compounds enter to the parasite, increase
of reactive oxygen and nitrogen species occurred, while thiol level decreases and induces
oxidative stress, which damage internal cell structure, and causes parasite death [38].
Table 1: Antiparasitic and cytotoxicitic activities of cycloalkanones 1-34, curcumin,
benznidazole and amphotericin B.
a
b
c
Compounds
Epimastigote Trypomastigote
Promastigote
L.amazonesis
IC50 (µM)
Vero cells
CC50 (µM)
SIe-v
SIt-v
SIp-v
T. cruzi
IC50(µM)
T. cruzi
EC50 (µM)
Average ± SD
Average ± SD
Average ± SD
Average± SD
18.4 ± 0.2
63.6 ± 2.0
16.2 ± 4.5
29.3 ± 6.8
24.7 ± 2.8
>100± 0.0
76.2 ± 2.2
>100± 0.0
85.3 ± 3.7
49.0 ± 2.8
68.7 ± 5.3
78.0 ± 15.5
2.8 ± 0.2
33.3 ± 3.1
3.6 ± 0.4
4.2 ± 0.3
4.3 ± 0.4
22.0 ± 0.6
40.6 ± 4.4
177.9 ± 12.3
32.1 ± 1.7
2.2
2.8
2.0
2.0
1.4
-
0.5
-
14.5
5.3
1
2
3
4
5
6
0.4
1.2
0.5
3.3
8.9
58.0 ± 5.3
13.8
8.0
34.4 ± 1.5
256.7 ± 23.6
11.7

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23.6 ± 2
27.8 ± 1.1
83.9 ± 5.4
33.3 ± 4.7
9.5 ± 0.6
27.0 ± 2.8
36.2 ± 3.1
93.5 ± 2.1
63.2 ± 1.7
69.5±9.2
4.1 ± 0.1
4.6 ± 0.0
4.9 ± 0.8
2.3 ± 0.4
17.0 ± 3.3
54.0 ± 4.2
>100± 0.0
11.8 ± 1.4
91.0 ± 7.1
8.9 ± 0.7
>100 ± 0.0
13.0 ± 1.4
17.8 ± 2.5
17.3 ± 2.4
5.2 ± 0.1
10.8 ± 3.1
9.0 ± 1.4
5.7 ± 0.8
16.7 ± 0.5
20.1 ± 3.0
4.7 ± 0.0
3.9 ± 0.5
19.8 ± 2.7
2.6 ± 0.4
4.8 ± 0.4
10.0 ± 0.0
4.5 ± 1.1
3.7 ± 0.5
>100± 0.0
24.9 ± 3.1
30.6 ± 4.2
573.7 ± 37.2
35.9 ± 0.6
155.3 ± 19.7
193.6 ± 6.5
720.0 ± 28.3
290.6 ± 9.4
392.3 ± 7.7
401.2 ± 8.8
435.0 ± 5.0
237.6 ± 6.8
401.2 ± 8.8
250.0 ± 2.7
231.9 ± 9.7
378.1 ± 6.6
269.5 ± 8.3
30.0 ± 3.9
33.5 ± 0.2
31.5 ± 1.8
33.2 ± 0.6
<10 ± 0.0
1.1
1.1
6.8
1.1
16.3
3.5
-
0.9
0.8
6.1
0.6
2.2
-
6.1
6.7
7
8
117.1
15.6
9.1
9
10
11
54.8 ± 3.3
>100± 0.0
20.6 ± 1.7
>100 ± 0.0
5.2 ± 0.8
>100 ± 0.0
>100± 0.0
>100 ± 0.0
>100 ± 0.0
27.4 ± 3.6
>100 ± 0.0
>100 ± 0.0
>100 ± 0.0
>100 ± 0.0
54.2 ± 0.9
>100 ± 0.0
>100 ± 0.0
>100 ± 0.0
60.0 ± 14.1
77.0 ± 2.8
70.2 ± 6.1
27.7 ± 3.8
72.0 ± 5.7
30.2 ± 1.8
>100± 0.0
82.0 ± 11.3
23.8 ± 5.4
35.0 ± 4.2
15.0 ± 1.4
3.6
12
-
-
13
14.1
-
-
24.6
4.3
14
-
15
77.2
-
14.6
-
45.1
-
16
>100 ± 0.0
26.6 ± 4.0
21.6 ± 0.4
55.0 ± 3.0
20.5 ± 3.7
21.0 ± 3.0
13.6 ± 2.5
20.2 ± 3.3
25.8 ± 1.1
35.0 ± 8.9
24.4 ± 1.6
33.3 ± 4.7
33.0 ± 1.4
3.0 ± 0.0
17
8.9
18.6
4.5
11.3
18.0
19.8
1.5
1.3
0.9
1.4
-
-
18.3
22.5
14.5
44.6
35.0
29.9
5.3
18
-
19
-
20
4.3
-
21
22
-
23
-
24
0.6
0.4
0.5
-
2.0
25
1.6
26
7.1
27
-
28
31.5 ± 1.8
29.3 ± 4.8
120.0 ± 1.0
177.5 ± 17.2
36.1 ± 1.6
160.1 ± 14.2
36.8 ± 0.6
1.0
9.8
4.5
6.8
1.3
6.1
0.5
0.4
1.0
-
1.6
29
11.3
25.0
17.8
8.0
30
26.5 ± 0.2
26.1 ± 1.5
28.5 ± 3.3
26.2 ± 1.7
67.3 ± 1.8
31
2.2
1.5
4.6
2.5
32
33
43.3
-
34
35- Curcumin

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Benznidazole
6.5±0.7
34.5 ± 7.6
Amphotericin B
0.06 ± 0.00
IC50: inhibitory concentration growth of 50% values of epimastigote and promastigote forms.
EC50: concentration that lyses 50% of trypomastigote forms.
CC50: cytotoxic concentration corresponding to 50% inhibition of Vero cells growth.
f
e Selectivity index (CC50(Vero cells)/IC50(epimastigote)). Selectivity index (CC50(Vero cells)/IC50(trypomastigote)).
g Selectivity index (CC50(Vero cells)/IC50(promastigote)). Highlighted values indicate the potent compounds and positive
control.
2.3. Optimization of ligands structures
The structures of compounds 1-34 were optimized to find the geometries with lower potential
energy by using force field Ghemical (best force field), by advanced semantic chemical editor
software Avogadro 1.1.1 [39]. The optimization calculations resulted in possible best
conformations and best stable structures of compounds. The lowest energy conformation of
each compound was used for docking with the target chosen among those available.
2.4. Studies on molecular orbitals by computational analysis
The highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital
(LUMO) for compounds 1-34 were analyzed by Gaussian [40]. The energy of HOMO is the
capacity of a compound to lose electron easily, and consider equal to the ionization potential
in general case. The energy of LUMO describes the capability of compounds to gain
electrons. Therefore, the negative sign of the energy of LUMO is taken as electron affinity.
High or low electronic density in a molecule is useful to have idea on how they interact with
receptors. Thus we can first estimate that the symmetrical bisaryl cyclohexenones (1-11) will
have the same electronic density in both aryl groups, which will not be differentiated by
receptors. In its turn, compounds 14-34 contain different HUMO-LUMO contributions for the
aryl groups, and this may be important for their bioactivities.
For instance, Fig. 1 succinctly illustrates the frontier orbitals of the global minimum
configurations for 1 and for two unsymmetrical alkanones (14 and 17). In the case of the
compound 1 the HOMO has dominating contributions equally from both benzene rings A and
B, and from the adjacent C=C bond, with little contribution from cyclohexanone system.
Compounds 14-34 possessing different moieties on ring B and tend to increase or decrease the
electronic density on ring B. Compound 14 has ring B with no substitution, so here HUMO
has a major contribution from ring A (which is para-methoxyl substituted) and adjacent C=C

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bond, and a little from C=O bond. In case of compound 17 the LUMO has contributing effect
from benzene ring B having –NO2 substitution, a strong withdrawing group. In case of
compounds 15 and 16 electron donating groups has attached to aromatic ring B, so HUMO
dominating contribution shift to aromatic ring B with a little from adjacent C=C bond. LUMO
in this case has exclusive contribution from aromatic ring A, adjacent C=C, cyclohexanone
system and C=O bond. In case of compounds 17-24 the electron withdrawing substituents are
attached to aromatic ring B, due to which it withdraw electronic density. So HUMO has
dominating contribution from ring A and from adjacent C=C bond and the C=O bond,
whereas LUMO has exclusive contribution from aromatic ring B and adjacent double bond.
By comparing the electronic density on potent compounds 16, 30 and 33 with other
compounds it was confirmed that there is maximum electronic density on aromatic ring A and
B on these compounds. The substitution on aromatic ring A and B are electron donors which
tend to increase electronic density. This may be one of the possible reasons that greater
electronic density on aromatic rings A and B, which come by electron donor substitution,
increases the activity of compound. Moreover the methoxyl substitution also provides
possibility of H-bonding with amino acids residue in enzymes, which is another reason of the
potency of compounds 16, 30 and 33 potency.

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Fig. 1: Electronic distributions for compound 1, 14 and 17. Semi-empirical calculations were
performed using the Gaussian software package with the semi empirical CAM-B3LYP/6-
31G(d,p) method and full geometric optimization.
2.5. Molecular docking
The biochemical target identification of a group of compounds with experimentally
determined biological activities is a very challenging task. In fact, the observed in vivo effects
can involve several biochemical targets simultaneously. Together with virtual screening
methodologies to identify lead compounds for a specific target, the availability of a great
number of proteins 3D structures has stimulated the development of in silico approaches to
search for the targets of objective compounds, a process called target fishing or reverse
docking [41,42]. The approach is useful particularly in the case where purified target enzymes
are not present in sufficient amount to apply experimental procedures, such as in the case of
the many parasites associated with neglected diseases. We performed molecular docking
calculations (using Molecular Operating Environment - MOE) as a way to implement small-
scale target fishing for our active compounds against T. cruzi enzymes. The chosen enzymes
listed in Table 2 are all somehow involved in the parasite metabolism. To improve the
chances of identifying the best docking solution for each enzyme, 30 docking conformations
for each enzyme were generated, and the conformation with the best docking score was
selected for further analysis. MOE docking scores are dimensionless, but the scale of the
score provide a guide as to how good a ligand pose is - the more negative the score for a
specific function, the better the docking result is likely to be.
Table 2. Trypanosoma cruzi enzymes used for target fishing.
S. No
PDB Code
Name of enzyme
Resolution
1
1BZL
Trypanothionereductase
2.4 Å
2
3
1MXH
1QXS
Pteridinereductase
2.2 Å
Glyceraldehyde-3-phosphate dehydrogenase
2.75 Å
4
5
6
1S0I
Trans-sialidase
1.6 Å
2.0 Å
1.95 Å
1SUX
1YHL
Triosephosphateisomerase
Farnesyldiphosphate synthase

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7
3BWC
3IRM
3K7S
3QV9
3W88
4CK8
4EM5
4XUI
4YRT
5BRD
5CUU
Spermidine synthase
2.3 Å
2.1 Å
1.9 Å
2.1 Å
8
Dihydrofolatereductase
5-phosphate isomerase
Pyruvate kinase
9
10
11
12
13
14
15
16
17
Dihydroorotate dehydrogenase (TcDHODH) 1.4 Å
Sterol 14-alpha demethylase
Glucose-6-P dehydrogenase
Cruzain
2.62 Å
3.35 Å
2.51 Å
2.05 Å
2.4 Å
Histidyl-tRNAsynthetase
Glucokinase
Pyrophosphatase
2.96 Å
Although there is a limitation of scoring functions to produce data that correlate with activity
data, the docking programs have demonstrated the ability to identify active compounds from a
pharmaceutically relevant pool of decoy compounds [43]. Based on IC50 data (Table 1), it can
be observed that the most active compounds against T. cruzi were 16, 28, 30 and 33.
Therefore, our analysis was focused on identifying enzyme (drug target) for which these most
active compounds showed an improved binding interaction in comparison with the remaining
enzymes, as measured by docking score (Table 2). As seen in Table 3, compounds 16, 28, 30
and 33 showed the best docking scores with T. cruzi dihydroorate dehydrogenase
(TcDHODH, PDB Code 3W88) as the target when compared to other targets. DHODH is an
enzyme that involves in the fourth step in the de novo biosynthesis of pyrimidines,
heterocyclic compounds essential for the synthesis of RNA and DNA. In a previous study, it
has been shown that a DHODH-knockout T.cruzi was not viable [44]. The differences in
biochemical properties between human and T. cruzi DHODHs as well as its essentiality for
the parasite make TcDHODH a promising drug target.

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Table 3. Docking score of compounds 16, 28, 30 and 33 against seventeen enzymes of T.
cruzi.
Enzyme PDB
ID
Compound 16
-13.9056
-20.2915
-16.2497
-18.8073
-13.2700
-9.9409
Compound 28
-12.8686
-21.9722
-14.1808
-17.1618
-13,6693
-8.0821
Compound 30
-13.8855
-18.1101
-15.6557
-19.7537
-14.9573
-7.4040
Compound 33
-11-1378
-20.1764
-14.5520
-22.1932
-14.1644
-57894
1BZL
1MXH
1QXS
1S0I
1SUX
1YHL
3BWC
3IRM
3K7S
3QV9
3W88
4CK8
4EM5
4XUI
4YRT
5BRD
5CUU
-18.0294
-19.7652
-16.6753
-14.7900
-26.4188
-19.9470
-18.4714
-18.4546
-16.8554
-18.1645
-15.8612
-9.7091
-174.3698
-19.2920
-17.0384
-15.9803
-24.8778
-19.4188
-15.1450
-22.1103
-13.9806
-16.3138
-16.8763
-14.7174
-17.5422
-17.5026
-16.1825
-22.5645
-20.5645
-18.9369
-22.4522
-10.7520
-19.5546
-15.6087
-20.3865
-14.1393
-9.5311
-23.2683
-15.8778
-17.4246
-17.8522
-12.7979
-16.4464
-13.4085
Bold values indicate the four highest docking scores.
After identification of TcDHODH enzyme as the most promising target of our synthesized
compounds against T. cruzi, we compared the interaction profiles of the best pose obtained for

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the most active compound (16) with the co-crystalized ligand (PDB code 3W88). It can be
observed in Fig. 2 that compound 16 showed about similar interaction with the important
active site residues (Gly70, Lys43, Asp53, Asn67 and Lys194) of the enzyme as was observed
for co-crystalized ligand. The docking conformation of compound 16 showed that four of the
oxygen atoms formed polar interaction with active site residues Asn53, Gly70, Asn67 and
Asn194 of the enzyme. One of the phenyl rings of the compound established an arene-cation
interaction with the active site residue Lys43 of the enzyme. Furthermore, several
hydrophobic interactions were observed with hydrophobic amino acids (Ala19, Met69 and
Pro131 etc.) present in the active site of TcDHODH enzyme
Fig. 2: Binding mode of the most active compound (16) and co-crystalized ligand in the
active site of T. cruzi DHODH enzyme. The golden and pink color represents the most active
compound and co-crystalized ligand respectively.
3. Conclusions
A series of symmetrical and unsymmetrical substituted diarylidene cyclohexanone has been
synthesized and studied as antiparasitic compounds, showing promising moieties, including
compounds active at low micro molar concentrations against the parasites T. cruzi and L.
amazonensis. Based on their antiparasitic activity compounds 16, 28, 30 and 33 are the best
candidates for further studies for the development of novel antiparasitic drugs. Docking
analysis shows that dihydroorotate dehydrogenase (3W88) may be one of the possible targets

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for these compounds. In overall, it is apparent from our results that the molecular asymmetry
in this class of compounds is a very important element for their bioactivities.
4. Experimental
4.1. General
All chemicals were purchased from Organics, Sigma-Aldrich, Acros Chemicals and Fisher
Scientific Ltd and used without further purification. The deuterated solvents of Apolo were
used for the NMR analysis. Thin layer chromatography was performed with precoated silica
gel G-25-UV254 plates and detection was carried out at 254 nm under UV, and by vanillin in
methanol/H2SO4 solution. Mass spectra (ESI-HRMS) were measured by using a Thermo
Scientific LTQ Orbitrap XL spectrometer operated at a resolution of 60,000 FWHM, in
positive mode, m/z 90-800. HRMS-MS data were performed by CID.1H and13C NMR data
were acquired on a Brüker AVANCE 400 operating at 400.15 MHz and 100.62 MHz,
respectively.CDCl3 was used as solvent and tetramethylsilane (TMS) as internal reference.
Compounds 1-34 were dissolved in organic solvents at about 10 mg.mL-1 each and shifted
into a 5-mm NMR tube. Chemical shifts (δ ppm) were measured with accuracy of 0.01 (1H)
and 0.1 ppm (13C). The UV-Vis spectra were recorded on a Perkin Elmer Lambda 25
spectrophotometer using 1 cm optical length quartz cuvettes at 25 °C and methanol as solvent.
4.2. Chemistry
4.2.1. 2,6-di((E)-benzylidene)cyclohexan-1-one (01)
A mixture of cyclohexanone (5 g, 51mmol, 5.31 mL), and benzaldehyde (5.406 g, 51mmole,
5.198 mL) were taken in 100 mL two-necked round bottom flask. Dry HCl gas was passed in
the content of the flask until it was saturated and red coloration start appearing. The reaction
mixture was stirred for 3 h. The crude product was diluted with toluene and washed with
NaHSO3 solution. The organic layer was separated, dried with anhydrous Na2SO4 and
evaporated in vacuum. The crude product was collected and recrystallized from ethanol, to
1
give the pure product. Percent yield: 86 %; Mp: 118-119 °C; H NMR (400 MHz, CDCl3) δ
7.82 (s, 1H), 7.47 (d, J = 7.4 Hz, 2H), 7.44 - 7.37 (m, 2H), 7.37 - 7.31 (m, 1H), 2.94 (td, J =
13
6.6, 2.0 Hz, 2H), 1.86 - 1.73 (m, 1H); C NMR (400 MHz, CDCl3) δ 190.50 (1C), 137.06

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(2C), 136.31 (1C), 136.09 (1C), 130.49 (4C), 129.36 (2C), 128.70 (2C), 128.50 (4C), 28.57
(2C), 23.13 (1C); HRMS-ESI(+): calcd 275.1430; found 275.1432.
4.2.2. (2E,6E)-2,6-bis(4-methoxybenzylidene)cyclohexanone (02)
1
Percent yield: 86 %; H NMR (400 MHz, CDCl3) δ 7.82 (s, 1H), 7.47 (d, J = 7.4 Hz, 2H),
7.44 - 7.37 (m, 2H), 7.37 - 7.31 (m, 1H), 2.94 (td, J = 6.6, 2.0 Hz, 2H), 1.86 - 1.73 (m, 1H);
13C NMR (400 MHz, CDCl3) δ 190.38 (1C), 160.04 (2C), 136.66 (2C), 134.47 (2C), 132.37
(4C), 128.88 (2C), 114.03 (4C), 55.46 (2C), 28.66 (2C), 23.17 (1C); HRMS-ESI(+): calcd
335.4083; found 335.1129.
4.2.3. (2E,6E)-2,6-bis(4-chlorobenzylidene)cyclohexanone (03)
Percent yield: 82 %; 1H NMR (400 MHz, CDCl3) δ 7.72 (s, 2H), 7.45 – 7.30 (m, 8H), 2.94 –
2.79 (m, 4H), 1.88 – 1.72 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 189.96 (1C), 136.53 (2C),
135.89 (2C), 134.73 (2C), 134.41 (2C), 131.71 (4C), 128.80 (4C), 28.49 (2C), 22.92 (1C).;
HRMS-ESI(Na): calcd 365.0578; found 365.0463.
4.2.4. (2E,6E)-2,6-bis(4-bromobenzylidene)cyclohexanone (04)
1
Percent yield: 80 %; H NMR (400 MHz, CDCl3) δ 7.70 (s, 2H), 7.53 (d, J = 8.4 Hz, 4H),
13
7.31 (d, J = 8.4 Hz, 4H), 2.87 (td, J = 6.5, 2.1 Hz, 4H), 1.87 – 1.70 (m, 2H); C NMR (101
MHz, CDCl3) δ 190.01 (1C), 136.66 (2C), 135.98 (2C), 134.87 (2C), 131.94 (4C), 131.79
(4C), 123.09 (2C), 28.51 (2C), 22.93 (1C); HRMS-ESI(Na): calcd 452.9568; found 452.9449.
4.2.5. (2E,6E)-2,6-bis(4-fluorobenzylidene)cyclohexanone (05)
1
Percent yield: 79 %; H NMR (400 MHz, CDCl3) δ 7.75 (s, 2H), 7.44 (dd, J = 8.7, 5.5 Hz,
4H), 7.09 (t, J = 8.7 Hz, 4H), 3.05 – 2.71 (m, 4H), 1.88 – 1.70 (m, 2H); 13C NMR (101 MHz,
CDCl3) δ 190.12 (1C), 164.02 (2C), 161.54 (2C), 135.91 (4C), 132.27 (4C), 132.34
(4C),115.75 (4C), 115.53 (4C), 28.46 (2C), 23.01 (1C).; HRMS-ESI(Na): calcd 333.1169;
found 333.1059.
4.2.6. (2E,6E)-2,6-bis(benzo[d][1,3]dioxol-5-ylmethylene)cyclohexanone (06)
Percent yield: 85 %; 1H NMR (400 MHz, CDCl3) δ 7.70 (s, 2H), 7.05 – 6.94 (m, 4H), 6.85 (d,
J = 7.8 Hz, 2H), 6.00 (s, 4H), 2.90 (td, J = 6.7, 2.0 Hz, 4H), 1.80 (dt, J = 12.4, 6.2 Hz, 2H);
13C NMR (101 MHz, CDCl3) δ 190.11 (1C), 148.02 (2C), 147.72 (2C), 136.73 (2C), 134.67
(2C), 130.21 (2C), 125.85 (2C), 110.05 (2C), 108.44 (2C), 101.38 (2C), 28.52 (2C), 22.95
(1C); HRMS-ESI(Na): calcd 385.1154; found 275.1047.

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4.2.7. (2E,6E)-2,6-bis(2,3-dimethoxybenzylidene)cyclohexanone (07)
Percent yield: 78 %; 1H NMR (400 MHz, CDCl3) δ 7.93 (s, 2H), 7.05 (t, J = 8.0 Hz, 2H), 6.95
– 6.88 (m, 4H), 3.87 (s, 6H), 3.81 (s, 6H), 2.79 (td, J = 6.5, 1.9 Hz, 4H), 1.83 – 1.63 (m, 2H);
13C NMR (101 MHz, CDCl3) δ 190.44 (1C), 152.94 (1C), 148.42 (1C), 137.62 (1C), 132.56
(4), 130.53 (1C), 123.55 (4C), 122.24 (4C), 112.86 (4C), 61.23 (2C), 55.94 (2C), 28.82 (2C),
23.39 (1C); HRMS-ESI(Na): calcd 417.1780; found 417.1662.
4.2.8. (2E,6E)-2,6-bis(2-chlorobenzylidene)cyclohexanone (08)
1
Percent yield: 77 %; H NMR (400 MHz, CDCl3) δ 7.93 (s, 2H), 7.45 (dt, J = 7.5, 3.9 Hz,
2H), 7.42 – 7.32 (m, 2H), 7.32 – 7.23 (m, 4H), 2.79 (td, J = 6.5, 1.9 Hz, 4H), 1.85 – 1.66 (m,
2H); 13C NMR (101 MHz, CDCl3) δ 189.84 (1C), 137.86 (1C), 135.12 (1C), 134.50 (1C),
134.20 (3C), 130.62 (2C), 129.78 (2C), 126.38 (2C), 28.49 (2C), 23.27 (1C); HRMS-
ESI(Na): calcd 365.0578; found 365.0467.
4.2.9. (2E,6E)-2,6-bis(2,6-dichlorobenzylidene)cyclohexanone (09)
1
Percent yield: 78 %; H NMR (400 MHz, CDCl3) δ 7.61 (s, 2H), 7.36 (d, J = 8.1 Hz, 4H),
7.26 – 7.15 (m, 2H), 2.47 (td, J = 6.6, 1.7 Hz, 4H), 1.82 – 1.66 (m, 2H); 13C NMR (101 MHz,
CDCl3) δ 188.14 (1C), 140.23 (1C), 134.57 (2C), 134.40 (1C), 132.36 (2C), 129.51 (2C),
128.04 (4C), 28.29 (2C), 22.02 (1C); HRMS-ESI(+): calcd 432.9799; found 432.9680.
4.2.10. (2E,6E)-2,6-bis(3-chlorobenzylidene)cyclohexanone (10)
Percent yield: 80 %; 1H NMR (400 MHz, CDCl3) δ 7.63 (s, 2H), 7.52 (s, 2H), 7.43 – 7.37 (m,
2H), 7.30 (d, J = 7.8 Hz, 2H), 7.20 (d, J = 7.5 Hz, 2H), 2.83 (td, J = 6.6, 2.0 Hz, 4H), 1.74 (dt,
J = 12.5, 6.3 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 137.93 (1C), 137.09 (2C), 135.53 (2C),
132.81 (2C), 131.53 (2C), 129.94 (2C), 128.92 (2C), 122.51 (2C), 28.31 (2C), 22.78 (1C);
HRMS-ESI(Na): calcd 452.9568; found 452.9444.
4.2.11. (2E,6E)-2,6-bis(3,4,5-trimethoxybenzylidene)cyclohexanone (11)
1
Percent yield: 76 %; H NMR (400 MHz, CDCl3) δ 7.74 (s, 2H), 6.72 (s, 4H), 3.90 (s, 6H),
3.90 (s, 12H), 3.05 – 2.88 (m, 4H), 1.92 – 1.76 (m, 2H); 13C NMR (101 MHz, CDCl3) δ
190.06 (1C), 153.09 (4C), 138.88 (2C), 137.23 (2C), 135.49 (2C), 131.53 (2C), 107.98 (4),
61.06 (2C), 56.30 (4C), 28.54 (2C), 23.08 (1C); HRMS-ESI(+): calcd 477.1992; found
477.1873.

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4.2.12. (E)-2-(4-methoxybenzylidene)cyclohexan-1-one (12)
A mixture of cyclohexanone (1.07 g, 10.95mmol), anisaldehyde (1 g, 7.3 mmol) and
DIMCARB (10 mL) was taken in 100 mL round bottom flask and stirred for 10 h with
vigorous stirring. The DIMCARB was removed from reaction mixture by rotary evaporator at
60 °C. The crude product was extracted 3 times with ether and 1 molar HCl solution. The
solvent was evaporated by vacuum and the crude product was forwarded for column
1
chromatography to get pure 12. Percent yield: 87 %; Mp: 70-73 °C; H NMR (400 MHz,
CDCl3) δ 7.52 (s, 1H), 7.42 (d, J = 8.8 Hz, 2H), 6.94 (d, J = 8.8 Hz, 2H), 3.86 (s, 3H), 2.87
(td, J = 6.4, 2.0 Hz, 2H), 2.55 (t, J = 6.7 Hz, 2H), 1.94 (dt, J = 13.0, 6.6 Hz, 2H), 1.84 – 1.75
(m, 2H); 13C NMR (400 MHz, CDCl3) δ 201.5, 159.9, 135.8, 134.4, 132.3, 128.3, 113.9, 55.3,
40.2, 28.9, 23.9, 23.3; HRMS- ESI(+): calcd 217.1223; found 217.1222.
4.2.13. (E)-2-(2,3-dimethoxybenzylidene)cyclohexanone (13)
Percent yield: 85 %; 1H NMR (400 MHz, CDCl3) δ 7.58 (t, J = 2.0 Hz, 1H), 6.92 (dd, J = 8.2,
1.4 Hz, 1CH), 6.89 – 6.84 (m, 2H), 3.83 (s, 3H), 3.81 (s, 3H), 2.84 – 2.75 (m, 2H), 2.75 – 2.69
(m, 2H), 2.55 (t, J = 6.7 Hz, 2H), 2.46 – 2.32 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 202.01
(1C), 152.79 (1C), 138.16 (1C), 130.75 (1C), 124.40 (1C), 123.41 (1C), 122.11 (1C), 119.28
(1C), 112.75 (1C), 60.97 (1C), 55.82 (1C), 40.57 (1C), 29.16 (1C), 24.01 (1C), 23.69 (1C);
HRMS- ESI(+): calcd 247.1256; found 247.1316.
4.2.14. 2-((E)-benzylidene)-6-((E)-4-methoxybenzylidene)cyclohexan-1-one (14)
A Solution of 12 (200 mg, 0.943mmole), and benzaldehyde (100 mg, 0.943mmole) in ethanol
(5 mL) was stirred for 5 min at room temperature and a sodium hydroxide solution in ethanol
(4 mL, 50 mmole) was added and stirring continued for six hours; the solvent evaporated in
vacuum and the residue was dissolved in ethyl acetate. Extraction with a NaHSO3 solution,
drying with Na2SO4, and concentration in vacuum, the crude product, which was collected as
yellow precipitate and further purified by column chromatography and recrystallized from
ethanol, gave pure compound 14. Percent yield: 85 %; Mp: 103-104 °C; 1H NMR (400 MHz,
CDCl3) δ 7.81 (d, J = 7.8 Hz, 2H), 7.48 (d, J = 8.8 Hz, 4H), 7.42 (dd, J = 7.3, 5.8 Hz, 2H),
7.38 - 7.32 (m, 1H), 6.96 (d, J = 8.8 Hz, 2H), 3.86 (s, 3H), 3.00 - 2.87 (m, 4H), 1.87 - 1.75 (m,
13
2H); C NMR (400 MHz, CDCl3) δ 190.7, 160.5, 137.4, 136.9, 136,8, 134.6, 132.8, 132.7,
130.8, 129.1, 128.9, 128.8, 114.4, 55.8, 29.0, 28.8, 23.5; HRMS-ESI(+): calcd 305.1536;
found 305.1534.

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4.2.15. -((E)-4-(dimethylamino)benzylidene)-6-((E)-4-methoxybenzylidene)cyclohexan-1-one
(15)
Percent yield: 82 %; Mp: 164-168 °C;1H NMR (400 MHz, CDCl3) δ 7.82 - 7.68 (m, 5H), 7.52
- 7.39 (m, 10H), 6.93 (d, J = 8.8 Hz, 6H), 6.81 (s, 4H), 3.84 (s, 8H), 3.03 (s, 12H), 2.97 - 2.87
13
(m, 10H), 1.87 - 1.74 (m, 5H); ); C NMR (400 MHz, CDCl3) δ 189.9, 159.6, 137.6, 137.5,
136.5, 135.9, 134.7, 134.4, 132.6, 132.3, 132.2, 128.9, 113.9, 55.3, 28.7, 28.5, 23.0; HRMS-
ESI(+): calcd348.1958; found 348.1960.
4.2.16.
2-((E)-2,3-dimethoxybenzylidene)-6-((E)-4-methoxybenzylidene)cyclohexan-1-one
(16)
1
Percent yield: 80 %; Mp: 126-127 °C; H NMR (400 MHz, CDCl3) δ 7.94 (s, 1H), 7.80 (s,
1H), 7.48 (d, J = 8.7 Hz, 2H), 7.08 (t, J = 8.0 Hz, 1H), 6.99 - 6.90 (m, 4H), 3.90 (s, 3H), 3.87
(s, 3H), 3.84 (s, 3H), 2.94 (dd, J = 8.6, 3.7 Hz, 2H), 2.85 - 2.76 (m, 2H), 1.85 - 1.71 (m, 2H);
13C NMR (400 MHz, CDCl3) δ 190.4, 160.1, 153.0, 148.4, 137.8, 137.3, 134.4, 132.5, 132.1,
130.7, 128.9, 123.6, 122.3, 114.0, 112.8, 61.2, 56.0, 55.5, 28.9, 28.6, 23.3; HRMS-ESI(+):
calcd 365.1746; found 365.1747.
4.2.17. 2-((E)-4-methoxybenzylidene)-6-((E)-4-nitrobenzylidene)cyclohexan-1-one (17)
Percent yield: 89 %; Mp: 193-196 °C; 1H NMR (400 MHz, CDCl3) δ 8.25 (d, J = 8.7 Hz, 6H),
7.79 (s, 3H), 7.77 (s, 3H), 7.57 (d, J = 8.7 Hz, 6H), 7.47 (d, J = 8.7 Hz, 6H), 6.95 (d, J = 8.7
Hz, 7H), 3.85 (s, 9H), 2.99 - 2.93 (m, 6H), 2.93 - 2.86 (m, 6H), 1.83 (dt, J = 12.4, 6.3 Hz,
13
7H); C NMR (400 MHz, CDCl3) δ 189.7, 160.3, 147.1, 142.7, 139.5, 138.2, 133.5, 132.5,
130.7, 128.4, 123.6, 114.0, 55.4, 28.5, 28.4, 22.8; HRMS-ESI(+): calcd 350.1387; found
350.1388.
4.2.18. 2-((E)-2-chlorobenzylidene)-6-((E)-4-methoxybenzylidene)cyclohexan-1-one (18)
Percent yield: 80 %; Mp: 120-121 °C; H NMR (400 MHz, CDCl3) δ 7.90 (s, 1H), 7.82 (s,
1
1H), 7.48 (d, J = 11.5 Hz, 2H), 7.45 (dd, J = 5.3, 3.9 Hz, 1H), 7.36 - 7.32 (m, 1H), 7.31 - 7.25
(m, 2H), 6.96 (d, J = 8.8 Hz, 2H), 3.87 (s, 3H), 2.95 (t, J = 6.3 Hz, 2H), 2.77 (t, J = 6.1 Hz,
13
2H), 1.86 – 1.76 (m, 2H); C NMR (400 MHz, CDCl3) δ 189.9, 160.0, 138.1, 137.6, 134.9,
134.6, 133.9, 133.1, 132.4, 130.5, 129.7, 129.3, 128.6, 126.2, 113.9, 55.3, 28.7, 28.1, 23.1;
HRMS-ESI(+): calcd 339.1146; found 339.1148.
4.2.19. 2-((E)-4-chlorobenzylidene)-6-((E)-4-methoxybenzylidene)cyclohexan-1-one (19)

ACCEPTED MANUSCRIPT
Percent yield: 86 %; Mp: 146-149 °C; 1H NMR (400 MHz, CDCl3) δ 7.77 (t, J = 1.9 Hz, 1H),
7.72 (t, J = 2.1 Hz, 1H), 7.46 (d, J = 8.7 Hz, 2H), 7.37 (dd, J = 5.3, 2.3 Hz, 4H), 6.94 (d, J =
8.8 Hz, 2H), 3.85 (s, 3H), 2.96 - 2.90 (m, 2H), 2.90 - 2.85 (m, 2H), 1.86 - 1.75 (m, 2H);13C
NMR (400 MHz, CDCl3) δ 190.1, 160.1, 137.3, 136.8, 135.1, 134.5, 134.4, 133.9, 132.4,
131.5, 128.6, 113.9, 55.4, 28.5, 28.4, 22.9; HRMS-ESI(+): calcd 339.1146; found 339.1141.
4.2.20. 2-((E)-4-bromobenzylidene)-6-((E)-4-methoxybenzylidene)cyclohexan-1-one (20)
1
Percent yield: 82 %; Mp: 156-158 °C; H NMR (400 MHz, CDCl3) δ 7.77 (s, 1H), 7.69 (s,
1H), 7.52 (d, J = 8.5 Hz, 2H), 7.46 (d, J = 8.7 Hz, 2H), 7.31 (d, J = 8.4 Hz, 2H), 6.94 (d, J =
8.8 Hz, 2H), 3.84 (s, 3H), 2.92 (t, J = 6.1 Hz, 2H), 2.89 - 2.82 (m, 2H), 1.84 - 1.76 (m, 2H);
13C NMR (400 MHz, CDCl3) δ 190.1, 160.2, 137.5, 137.0, 135.2, 135.1, 134.0, 132.5, 131.9,
131.7, 128.7, 122.8, 114.1, 55.5, 28.7, 28.5, 23.0; HRMS-ESI(+): calcd 383.0641; found
383.0640.
4.2.21. 2-((E)-2,6-dichlorobenzylidene)-6-((E)-4-methoxybenzylidene)cyclohexan-1-one (21)
Percent yield: 88 %; Mp: 88-93 °C; 1H NMR (400 MHz, CDCl3) δ 7.75 (s, 1H), 7.47 (s, 1H),
7.40 (d, J = 8.7 Hz, 2H), 7.34 - 7.24 (m, 3H), 7.15 - 7.10 (m, 1H), 6.87 (d, J = 8.9 Hz, 2H),
3.78 (s, 3H), 2.86 (td, J = 6.4, 2.0 Hz, 2H), 2.44 - 2.27 (m, 2H), 1.71 (dt, J = 12.5, 6.3 Hz,
13
2H); C NMR (400 MHz, CDCl3) δ 189.1, 160.2, 140.5, 138.1, 134.5, 134.5, 133.8, 132.5,
130.9, 129.9, 129.2, 128.9, 128.6, 127.9, 113.9, 55.4, 28.8, 28.0, 22.5; HRMS-ESI(+): calcd
373.0757; found 373.0759.
4.2.22. 2-((E)-4-fluorobenzylidene)-6-((E)-4-methoxybenzylidene)cyclohexan-1-one (22)
1
Percent yield: 94%; Mp: 124-125 °C; H NMR (400 MHz, CDCl3) δ 7.77 (s, 1H), 7.74 (s,
1H), 7.50 - 7.40 (m, 4H), 7.09 (t, J = 8.7 Hz, 2H), 6.94 (d, J = 8.8 Hz, 2H), 3.85 (s, 3H), 2.96 -
2.91 (m, 2H), 2.91 - 2.85 (m, 2H), 1.89 - 1.72 (m, 2H); 13C NMR (400 MHz, CDCl3) δ 190.3,
163.9, 160.2, 137.3, 136.2, 135.5, 134.2, 132.5, 132.4, 132.3, 128.8, 115.7, 115.5, 114.1,
55.5,28.7, 28.5, 23.1; HRMS-ESI(+): calcd 323.1442; found 323.1439.
4.2.23. 2-((E)-2-fluorobenzylidene)-6-((E)-4-methoxybenzylidene)cyclohexan-1-one (23)
1
Percent yield: 80 %; Mp: 115-116 °C; H NMR (400 MHz, CDCl3) δ 7.81 (s, 1H), 7.78 (s,
1H), 7.46 (d, J = 8.8 Hz, 2H), 7.40 - 7.28 (m, 2H), 7.13 (dt, J = 17.4, 8.0 Hz, 2H), 6.94 (d, J =
8.8 Hz, 2H), 3.85 (s, 3H), 2.93 (t, J = 5.3 Hz, 2H), 2.82 - 2.75 (m, 2H), 1.83 - 1.74 (m,

ACCEPTED MANUSCRIPT
2H);13C NMR (400 MHz, CDCl3) δ 189.9, 162.2, 160.2, 138.7, 137.6, 134.1, 132.5, 130.9,
130.4, 130.3, 129.1, 128.8, 123.9, 115.9, 115.8, 114.1, 55.5, 28.9, 28.5, 23.1; HRMS-ESI(+):
calcd 323.1442; found 323.1440.
4.2.24. 2-((E)-3-bromobenzylidene)-6-((E)-4-methoxybenzylidene)cyclohexan-1-one (24)
Percent yield: 81 %; Mp: 123-124 °C; 1H NMR (400 MHz, CDCl3) δ 7.80 (t, J = 2.0 Hz, 1H),
7.71 (t, J = 2.0 Hz, 1H), 7.61 (t, J = 1.7 Hz, 1H), 7.48 (dd, J = 8.6, 2.8 Hz, 3H), 7.38 (d, J =
7.8 Hz, 1H), 7.30 (d, J = 7.8 Hz, 1H), 6.97 (d, J = 8.8 Hz, 2H), 2.98 - 2.93 (m, 2H), 2.93 -
2.88 (m, 2H), 1.90 - 1.76 (m, 2H); 13C NMR (400 MHz, CDCl3) δ 190.1, 160.3, 138.3,137.7,
137.6, 134.8, 134.0, 132.9, 132.5, 131.4, 130.0, 128.9, 128.7, 122.6, 114.1, 55.5, 28.7, 28.4,
23.1; HRMS-ESI(+): calcd 383.0641; found 383.0643.
4.2.25. (2E,6E)-2-benzylidene-6-(2,3-dimethoxybenzylidene)cyclohexanone (25)
Percent yield: 81 %; 1H NMR (400 MHz, CDCl3) δ 7.94 (s, 1H), 7.81 (s, 1H), 7.50 – 7.43 (m,
2H), 7.43 – 7.37 (m, 2H), 7.35 – 7.30 (m, 1H), 7.08 – 7.04 (m, 1H), 6.94 – 6.90 (m, 2H), 3.88
(s, 3H), 3.82 (s, 3H), 2.96 – 2.89 (m, 2H), 2.84 – 2.77 (m, 2H), 1.81 – 1.71 (m, 2H); 13C NMR
(101 MHz, CDCl3) δ 190.38 (1C), 152.87 (1C), 148.33 (1C), 137.47 (1C), 137.05 (1C),
136.33 (1C), 136.05 (1C), 132.43 (1C), 130.39 (2C), 128.48 (2C), 123.49 (1C), 122.15 (1C),
112.78 (1C), 61.14 (1C), 55.86 (1C), 28.59 (2C), 23.18 (2C); HRMS-ESI(Na): calcd
357.1569; found 383.1488.
4.2.26. (2E,6E)-2-(4-bromobenzylidene)-6-(2,3-dimethoxybenzylidene)cyclohexanone (26)
Percent yield: 82 %; 1H NMR (400 MHz, CDCl3) δ 7.93 (s, 2H), 7.06 (t, J = 8.0 Hz, 2H), 6.96
– 6.88 (m, 5H), 3.96 – 3.85 (m, 3H), 3.82 (s, 3H), 2.80 (td, J = 6.6, 1.9 Hz, 4H), 1.82 – 1.65
(m, 2H); 13C NMR (101 MHz, CDCl3) δ 190.26 (1C), 152.97 (1C), 148.43 (1C), 137.35 (1C),
136.99 (1C), 135.77 (1C), 135.00 (1C), 132.86 (1C), 131.98 (2C), 131.81 (2C), 130.37 (1C),
123.61 (1C), 122.90 (1C), 122.21 (1C), 112.96 (1C), 61.25 (1C), 55.96 (1C), 28.54 (2C),
23.16 (1C); HRMS-ESI(Na): calcd 435.0674; found 435.0553.
4.2.27. (2E,6E)-2-(2,3-dimethoxybenzylidene)-6-(4-fluorobenzylidene)cyclohexanone (27)
1
Percent yield: 87 %; H NMR (400 MHz, CDCl3) δ 7.93 (s, 1H), 7.75 (s, 1H), 7.44 (dd, J =
8.6, 5.5 Hz, 2H), 7.13 – 7.00 (m, 3H), 6.92 (d, J = 7.9 Hz, 2H), 3.87 (s, 3H), 3.81 (s, 3H), 2.88
(t, J= 6.1 Hz, 2H), 2.79 (t, J = 6.1 Hz, 2H), 1.82 – 1.69 (m, 2H); 13C NMR (101 MHz, CDCl3)

ACCEPTED MANUSCRIPT
δ 190.27 (1C), 163.95 (1C), 161.46 (1C), 152.95 (1C), 148.41 (1C), 137.41 (1C), 135.96
(1C), 132.61 (2C), 132.40 (1C), 130.41 (1C), 123.58 (1C), 122.20 (1C), 115.68 (2C), 115.46
(1C), 112.91 (1C), 61.21 (1C), 55.94 (1C), 28.61 (1C), 23.19 (1C); HRMS-ESI(Na): calcd
375.1475; found 375.1364.
4.2.28. (2E,6E)-2-(2,3-dimethoxybenzylidene)-6-(4-nitrobenzylidene)cyclohexanone (28)
1
Percent yield: 92 %; H NMR (400 MHz, CDCl3) δ 7.93 (s, 1H), 7.75 (s, 1H), 7.44 (dd, J =
8.6, 5.5 Hz, 2H), 7.13 – 7.00 (m, 3H), 6.92 (d, J = 7.9 Hz, 2H), 3.87 (s, 3H), 3.81 (s, 3H), 2.88
(t, J = 6.1 Hz, 2H), 2.79 (t, J = 6.1 Hz, 2H), 1.82 – 1.69 (m, 2H); 13C NMR (101 MHz,
CDCl3) δ 190.27 (1C), 163.95 (1C), 161.46 (1C), 152.95 (1C), 148.41 (1C), 137.41 (1C),
135.96 (1C), 132.61 (2C), 132.40 (1C), 130.41 (1C), 123.58 (1C), 122.20 (1C), 115.68 (2C),
115.46 (1C), 112.91 (1C), 61.21 (1C), 55.94 (1C), 28.61 (1C), 23.19 (1C); HRMS-ESI(Na):
calcd 402.1420; found 402.1307.
4.2.29. (2E,6E)-2-(3-bromobenzylidene)-6-(2,3-dimethoxybenzylidene)cyclohexanone (29)
1
Percent yield: 79 %; H NMR (400 MHz, CDCl3) δ 7.86 (s, 1H), 7.62 (s, 1H), 7.51 (s, 1H),
7.41 – 7.35 (m, 1H), 7.29 (d, J = 7.8 Hz, 1H), 7.20 (t, J = 7.8 Hz, 1H), 6.99 (dd, J = 8.3, 7.5
Hz, 1H), 6.85 (d, J = 8.1 Hz, 2H), 3.81 (s, 3H), 3.75 (s, 3H), 2.87 – 2.77 (m, 2H), 2.77 – 2.67
(m, 2H), 1.69 (dt, J = 12.5, 6.3 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 190.17 (1C), 153.00
(1C), 148.47 (1C), 138.26 (1C), 137.63 (1C), 137.32 (1C), 135.30 (1C), 132.94 (1C), 131.48
(1C), 130.38 (1C), 130.01 (1C), 129.03 (1C), 123.62 (1C), 122.57 (1C), 122.23 (1C), 113.00
(1C), 61.28 (1C), 55.98 (1C), 28.63 (1C), 23.18 (1C); HRMS-ESI(Na): calcd 435.0674; found
435.0547.
4.2.30. (2E,6E)-2-(2,3-dimethoxybenzylidene)-6-(3,4,5-trimethoxybenzylidene)cyclohexanone
(30)
Percent yield: 80 %; 1H NMR (400 MHz, CDCl3) δ 7.92 (s, 1H), 7.73 (s, 1H), 7.12 – 7.00 (m,
1H), 6.92 (d, J = 8.0 Hz, 2H), 6.71 (s, 2H), 3.89 (s, 3H), 3.88 (s, 9H), 3.85 – 3.72 (m, 3H),
3.01 – 2.87 (m, 2H), 2.85 – 2.71 (m, 2H), 1.86 – 1.69 (m, 2H); 13C NMR (101 MHz, CDCl3) δ
190.27 (1C), 153.06 (1C), 148.42 (1C), 137.47 (1C), 135.75 (1C), 132.57 (1C), 131.68 (1C),
130.53 (1C), 123.62 (1C), 122.26 (1C), 112.88 (1C), 108.00 (2C), 61.18 (1C), 56.33 (1C),
55.99 (1C), 28.87 (1C), 28.56 (1C), 23.30 (1C); HRMS-ESI(Na): calcd 447.1886; found
447.1761.

ACCEPTED MANUSCRIPT
4.2.31. (2E,6E)-2-(2,3-dimethoxybenzylidene)-6-(2-methoxy-4- nitrobenzylidene)
cyclohexanone (31)
Percent yield: 83 %; 1H NMR (400 MHz, CDCl3) δ 7.99 – 7.80 (m, 3H), 7.75 (d, J = 1.9 Hz,
2H), 7.42 (d, J = 8.4 Hz, 2H), 7.11 – 6.86 (m, 1H), 4.62 – 3.17 (m, 9H), 3.00 – 2.56 (m, 4H),
13
1.79 (dt, J = 12.5, 6.2 Hz, 2H); C NMR (101 MHz, CDCl3) δ 189.54 (1C), 158.67 (1C),
152.98 (1C), 148.76 (1C), 139.27 (1C), 138.82 (1C), 137.22 (1C), 133.38 (1C), 132.07 (1C),
131.76 (1C), 131.03 (1C), 130.37 (1C), 130.20 – 129.86 (1C), 123.61 (1C), 122.19 (1C),
115.38 (1C), 113.07 (1C), 105.69 (1C), 61.26 (1C), 56.65 – 56.46 (1C), 56.11 (1C), 28.75
(1C), 23.15 (1C); HRMS-ESI(Na): calcd 432.1525; found 383.1404.
4.2.32. (2E,6E)-2-(3-(benzo[d][1,3]dioxol-5-yl)-2-methylpropylidene)-6-(2,3-
dimethoxybenzylidene)cyclohexanone (32)
Percent yield: 78 %; 1H NMR (400 MHz, CDCl3) δ 7.84 (s, 1H), 7.03 (t, J = 8.0 Hz, 1H), 6.97
– 6.80 (m, 2H), 6.80 – 6.66 (m, 2H), 6.67 – 6.53 (m, 2H), 5.89 (s, 2H), 3.86 (s, 3H), 3.79 (s,
3H), 2.75 – 2.61 (m, 2H), 2.57 (d, J = 7.1 Hz, 2H), 2.52 – 2.40 (m, 1H), 2.34 – 2.20 (m, 1H),
13
1.73 – 1.48 (m, 2H), 1.05 (d, J = 6.5 Hz, 3H); C NMR (101 MHz, CDCl3) δ 190.03 (1C),
152.92 (1C), 148.34 (1C), 147.48 (1C), 145.83 (1C), 145.42 (1C), 137.60 (1C), 135.00 (1C),
134.01 (1C), 132.13 (1C), 130.55 (1C), 123.51 (1C), 122.18 (1C), 112.75 (1C), 109.53 (1C),
108.09 (1C), 100.82 (1C), 61.17 (1C), 55.93 (1C), 42.70 (1C), 35.26 (1C), 28.46 (1C), 26.44
(1C), 22.83 (1C), 19.68 (1C); HRMS-ESI(Na): calcd 443.1937; found 443.1817.
4.2.33. (2E,6E)-2-(2,3-dimethoxybenzylidene)-6-(3,4-dimethoxybenzylidene)cyclohexanone
(33)
1
Percent yield: 81 %; H NMR (400 MHz, CDCl3) δ 7.84 (s, 1H), 7.68 (s, 1H), 7.03 (dd, J =
8.4, 1.8 Hz, 1H), 6.98 (d, J = 8.0 Hz, 1H), 6.96 – 6.92 (m, 1H), 6.83 (dd, J = 8.3, 5.0 Hz, 3H),
3.82 (d, J = 3.1 Hz, 6H), 3.79 (s, 3H), 3.73 (s, 3H), 2.86 (t, J = 6.9 Hz, 2H), 2.70 (dd, J = 8.6,
13
3.4 Hz, 2H), 1.76 – 1.61 (m, 2H); C NMR (101 MHz, CDCl3) δ 190.20 (1C), 152.89 (1C),
149.70 (1C), 148.68 (1C), 148.31 (1C), 137.48 (1C), 134.54 (1C), 132.20 (1C), 130.49 (1C),
129.01 (1C), 124.11 (1C), 123.52 (1C), 122.18 (1C), 113.77 (1C), 112.75 (1C), 110.94 (1C),
61.15 (1C), 55.92 (1C), 28.82 (1C), 28.45 (1C), 23.20 (1C); HRMS-ESI(+): calcd 417.1780;
found 417.1666.
4.2.34. (2E,6E)-2-(2,6-dichlorobenzylidene)-6-(2,3-dimethoxybenzylidene)cyclohexanone (34)

ACCEPTED MANUSCRIPT
Percent yield: 83 %; 1H NMR (400 MHz, CDCl3) δ 8.00 (s, 1H), 7.57 (s, 1H), 7.35 (d, J = 8.1
Hz, 2H), 7.24 – 7.18 (m, 1H), 7.11 – 7.05 (m, 1H), 6.95 (d, J = 8.0 Hz, 2H), 3.94 – 3.87 (m,
3H), 3.85 (s, 3H), 2.82 (td, J = 6.7, 1.8 Hz, 2H), 2.51 – 2.41 (m, 2H), 1.81 – 1.65 (m, 2H); 13C
NMR (101 MHz, CDCl3) δ 189.18 (1C), 152.93 (1C), 148.47 (1C), 140.56 (1C), 137.18 (1C),
134.52 (1C), 133.49 (2C), 131.42 (1C), 130.31 (2C), 129.38 (1C), 127.98 (1C), 123.56 (1C),
122.20 (1C), 113.06 (1C), 61.25 (1C), 55.94 (1C), 28.85 (1C), 28.28 (1C), 22.69 (1C);
HRMS-ESI(Na): calcd 425.0789; found 425.0676.
4.3. Biological activity
4.3.1. Parasites and cells
Promastigote forms of L. amazonensis (MHOM/BR/Josefa) were maintained at 25
°CinWarren’s medium (brain heart infusion plus haemin and folic acid) pH 7.0, supplemented
with 10% Fetal Bovine Serum (FBS, Gibco Invitrogen, Grand Island, NY, USA).
Epimastigote forms of T. cruzi (Y strain) were maintained at 28 °C in liver infusion tryptose
medium (LIT) supplemented with 10% inactivated FBS and trypomastigote forms were
obtained from the supernatant of a monolayer of infected LLCMK2 cells (epithelial cells from
the kidney of the monkey Macaca mulatta) in DMEM supplemented with 10% FBS at 37 °C
in a humidified 5% CO2 atmosphere.
4.3.2. Antiproliferative assay
The effects of synthesized compounds were evaluated in promastigotes of L. amazonensis and
epimastigotes of T. cruzi. The inoculum (1
106 cells/mL) was introduced into 24-well plate
X
containing the compounds dissolved in dimethylsulfoxide (DMSO)and Warren’s medium or
LIT in several concentrations(1.0 - 100.0 µM). The final concentration of DMSO did not
exceed1%. Cell grown was determined by counting the parasites with Neubauer
hemocytometer after incubation for 72 h at 25°C for L. Amazonensis and 96 h at 28°Cfor T.
cruzi. The results were expressed as percentage of inhibition in relation to the control
cultured. The 50% inhibitory concentration (IC50) was determined by logarithm regression
analysis of the data obtained.
4.3.3. Viability of trypomastigote forms of T. cruzi

ACCEPTED MANUSCRIPT
The tissue-culture-derived trypomastigote forms (1
X
107 cells/mL) were resuspended in
DMEM and added in duplicate to 96-wellmicroplates in presence of different concentrations
of the compounds (1.0 - 100.0 µM). Parasites were incubated for 24 h at 37 °C in a 5% CO2
atmosphere. The results were obtained by observing motility, allowing the determination of
the viability of the parasites, using the Pizzi-Brener method[45]. The EC50 value (i.e., the
concentration that lyses 50% of the parasites) was then calculated.
4.4. Molecular docking
To computationally identify target for these newly synthesized compounds, target fishing
(reverse docking) was carried out using the MOE-Dock as the docking software implemented
in MOE (www.chemcomp.com). From the protein data bank (www.rcsb.org), 17 complex X-
ray crystallographic structures of T. cruzi enzymes were retrieved (Table 2). Before docking
these protein structures were checked for missing atoms, bonds and contacts. All the water
molecules were removed from all the protein structures. The energies of the retrieved protein
molecules were minimized after the 3D protonation using the default parameters of MOE
energy minimization algorithm (gradient: 0.05, Force Field: MMFF94X).
The three dimensional coordinates of the synthesized compounds were constructed using
MOE software. The geometrically optimized compounds structures were used for the docking
into the active sites of the retrieved T. cruzi enzymes. Default MOE docking parameters were
used for the present docking studies. The top ranked pose of each compound against each
enzyme was selected on the basis of docking score (S) for further analysis.
Acknowledgments
The authors are grateful to Fundação de Amparo à Pesquisa do Estado de São Paulo
(FAPESP, Proc. Num. 2010/11384-6), Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq), and Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior
(CAPES) for financial support and Third World Academy of Science (TWAS) for Ph.D.
fellowship.
Supplementary data
Supplementary data associated with this article can be found in the online version, at
…

ACCEPTED MANUSCRIPT
References
[1] WHO, Chagas disease (American trypanosomiasis), 2015.
http://www.who.int/mediacentre/factsheets/fs340/en/.
[2] F. sheet N°340, World Health Organization, WHO. March (2014).
http://www.who.int/mediacentre/factsheets/fs340/en/.
[3] Grogl M, Thomason TN, Franke ED, Drug Resistance in Leishmaniasis: Its Implication
in Systemic Chemotherapy of Cutaneous and Mucocutaneous Disease, Am J Trop Med
Hyg. 47 (1992) 117–126. http://www.ajtmh.org/content/47/1/117.full.pdf+html.
[4] D. Kumar, A. Kulshrestha, R. Singh, P. Salotra, In vitro susceptibility of field isolates
of Leishmania donovani to miltefosine and amphotericin B: Correlation with sodium
antimony gluconate susceptibility and implications for treatment in areas of
endemicity, Antimicrob. Agents Chemother. 53 (2009) 835–838.
doi:10.1128/AAC.01233-08.
[5] Gandapur AS, Tanoli ZM, Raj ME., Clinical presentation and management of visceral
leishmaniasis., J Ayub Med Coll Abbottabad. 17 (2005) 51–53.
http://www.ncbi.nlm.nih.gov/pubmed/16599036.
[6] Croft SL, Yardley V, Chemotherapy of Leishmaniasis, Curr. Pharm. Des. 8 (2002)
319–342. doi:10.2174/1381612023396258.
[7] S.L. Croft, G.H. Coombs, Leishmaniasis– current chemotherapy and recent advances in
the search for novel drugs, Trends Parasitol. 19 (2003) 502–508.
doi:10.1016/j.pt.2003.09.008.
[8] Nwaka S, Ridley RG, Virtual drug discovery and development for neglected diseases
through public–private partnerships, Nat. Rev. Drug Discov. 2 (2003) 919–928.
doi:10.1038/nrd1230.
[9] . Pink R, Hudson A, Mouriès MA, Bendig M, Opportunities and Challenges in
Antiparasitic Drug Discovery, Nat. Rev. 4 (2005) 727–740.
http://www.nature.com/nrd/journal/v4/n9/pdf/nrd1824.pdf.
[10] Rybniker J, Goede V, Mertens J, Ortmann M, Kulas W, Kochanek M, Benzing T,
Arribas JR, Fätkenheuer G., Treatment of visceral leishmaniasis with intravenous
pentamidine and oral fluconazole in an HIV-positive patient with chronic renal failure--
a case report and brief review of the literature., Int. J. Infect. Dis. 14 (2010) e522-5.
doi:10.1016/j.ijid.2009.06.010.
[11] L.H. Freitas-Junior, E. Chatelain, H.A. Kim, J.L. Siqueira-Neto, Visceral leishmaniasis
treatment: What do we have, what do we need and how to deliver it?, Int. J. Parasitol.
Drugs Drug Resist. 2 (2012) 11–9. doi:10.1016/j.ijpddr.2012.01.003.
[12] J.M. Mutiso, J.C. Macharia, M.M. Gicheru, A review of adjuvants for Leishmania
vaccine candidates., J. Biomed. Res. 24 (2010) 16–25. doi:10.1016/S1674-
8301(10)60004-8.
[13] L.N. Lee KH, Aziz AB, Syahida A, Abas F, Shaari K, Israf DA, Synthesis and
biological evaluation of curcumin-like diarylpentanoid analogues for anti-
inflammatory, antioxidant and anti-tyrosinase activities., Eur. J. Med. Chem. 44 (2009)
3195–200. doi:10.1016/j.ejmech.2009.03.020.
[14] Inayama S, Mamoto K, Shibata T, Hirose T, Structure and antitumor activity relation of
2-arylidene-4-cyclopentene-1,3-diones and 2-arylideneindan-1,3-diones, J. Med. Chem.
19 (1976) 433–436. doi:10.1021/jm00225a022.