Unsymmetrical 1,5-diaryl-3-oxo-1,4-pentadienyls and their evaluation as antiparasitic agents

Article abstract of DOI:10.1016/j.bmc.2013.12.020

In this work the synthesis and antiparasitical activity of new 1,5-diaryl-3-oxo-1,4-pentadienyl derivatives are described First, compounds 1a, 1b, 1c and 1d were prepared by acid-catalyzed aldol reaction between 2-butanone and benzaldehyde, anisaldehyde, p-N,N-dimethylaminobenzaldehyde and p-nitrobenzaldehyde Reacting each of the methyl ketones 1a, 1b, 1c and 1d with the p-substituted benzaldehydes under basic-catalyzed aldol reaction, we further prepared compounds 2a-2p All twenty compounds were evaluated for antiproliferative activity, particularly for promastigote of Leishmania amazonensis and epimastigote of Trypanosoma cruzi All compounds showed good activity while nitro compounds 2i and 2k showed inhibition activity at a few μM

Full text of DOI:10.1016/j.bmc.2013.12.020

Bioorganic & Medicinal Chemistry 22 (2014) 1121–1127
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Unsymmetrical 1,5-diaryl-3-oxo-1,4-pentadienyls and their
evaluation as antiparasitic agents
Zia Ud Din a, Taicia Pacheco Fill a, Francisco Favaro de Assis b, Danielle Lazarin-Bidóia c, Vanessa Kaplum c,
Francielle Pelegrin Garcia c, Celso Vataru Nakamura c, Kleber Thiago de Oliveira b, Edson Rodrigues-Filho a,
⇑
a LaBioMMi, Departamento de Química, Universidade Federal de São Carlos, CP 676, 13.565-905 São Carlos, SP, Brazil
b LAQBO, Departamento de Química, Universidade Federal de São Carlos, CP 676, 13.565-905 São Carlos, SP, Brazil
c Laboratório de Inovação Tecnológica no Desenvolvimento de Fármacos e Cosméticos, Universidade Estadual de Maringá, Av. Colombo 5790,
87020-900, Bloco B-08 Maringá, PR, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
In this work the synthesis and antiparasitical activity of new 1,5-diaryl-3-oxo-1,4-pentadienyl deriva-
tives are described. First, compounds 1a, 1b, 1c and 1d were prepared by acid-catalyzed aldol reaction
between 2-butanone and benzaldehyde, anisaldehyde, p-N,N-dimethylaminobenzaldehyde and p-nitro-
benzaldehyde. Reacting each of the methyl ketones 1a, 1b, 1c and 1d with the p-substituted benzalde-
hydes under basic-catalyzed aldol reaction, we further prepared compounds 2a–2p. All twenty
compounds were evaluated for antiproliferative activity, particularly for promastigote of Leishmania ama-
zonensis and epimastigote of Trypanosoma cruzi. All compounds showed good activity while nitro com-
Received 6 November 2013
Revised 3 December 2013
Accepted 12 December 2013
Available online 23 December 2013
Keywords:
Dibenzalacetone
Chalcone analogues
Curcumin analogues
1,5-Diaryl-3-oxo-1,4-pentadienyl
Leishmania
pounds 2i and 2k showed inhibition activity at a few lM.
Ó 2013 Elsevier Ltd. All rights reserved.
Trypanosoma
1. Introduction
Leishmania amazonensis and Trypanosoma cruzi, the etiologic agents
that causes ‘Leishmaniosis’ and ‘Chagas disease’, respectivelly.
These two diseases affect, according to the World Health Organiza-
tion, 12 million of people in 88 countries, and 350 million are at
risk of acquiring this infection.17 Additionally, Chagas disease is
considered a serious public health problem that affects approxi-
mately 10 million people in Latin America. The incidence of this
disease has been estimated to include 300,000 new cases per year,
and approximately 10,000 people die from this infection annu-
ally.18–20 In view of the lack of safe medication and the serious side
effects caused by the use of available chemotherapy,20 there is a
need of new drugs for the treatment of these diseases. In the past
decade, chalcones and related compounds emerged as a new class
of antitrypanosomatids agents.21–26 Thus, due to their structural
similarities with chalcones and curcuminoids we attempted to
synthesize some dibenzylidene derivatives. The search for fascinat-
ing pharmacologically active molecular building blocks, based on
diverse structural features, easy synthetic routes and desired func-
tionalities have attracted our attention to synthesize dibenzylide-
neacetones systems.
The 1,5-diarylpentanoid dibenzylideneacetone (Fig. 1) is the
parent of a class of compounds having an acyclic dienone attached
to aryl groups in both b-positions. These structures resemble those
of the curcuminoids (1,7-diarylheptanes) and the chalcones
(1,3-diarylpropanes), which are very important bioactive natural
compounds found in many plant species. Accompanying these
structural similarities, synthetic chalcones and related compounds1
have shown biological activities such as antitumor,2 anticancer
and antioxidant,3 antifungal,4,5 antimitotic,6 chemoprotective,7
anti-inflammatory,8,9 antimicrobial,10 anti-nociceptive,11 antibac-
terial,12 antimalarial.13,14 In addition, dibenzylideneacetone poten-
tiates TRAIL-induced apoptosis by down-regulation of cell survival
proteins and up-regulation of death receptors through activation of
ROS and CHOP mediated pathways.15 The good bio-availability of
some dibenzylideneacetone and their derivatives, which is re-
quired for bioactivities16, as well as their mode of cross linking,
has raised the interest of chemists in their synthesis.
Aher et al. have shown that dibenzylideneacetone have good
potential to inhibit some parasites growth.14 These findings moti-
vated us to investigate the activity of similar compounds against
2. Synthesis plan
Dibenzylideneacetones (Fig. 1) can be formed by the direct
reaction of benzaldehyde (A1) with acetone (K1) using basic or acid
⇑
Corresponding author. Tel.: +55 16 3351 8053; fax: +55 16 3351 8350.
E-mail address: edinho@pq.cnpq.br (E. Rodrigues-Filho).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.12.020

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Z. Ud Din et al. / Bioorg. Med. Chem. 22 (2014) 1121–1127
Acetone (K1)
O
O
OH
CH3O
OCH3
OH
HO
Benzaldehyde (A1)
Benzaldehyde (A1)
Curcumin
Dibenzylideneacetone:
(1E,4E)-1,5-diphenylpenta-1,4-dien-3-one
O
Chalcone
Figure 1. Basic structure of a dibenzylideneacetone and their natural congeners curcumin and chalcone.
catalysis.27 Acid catalysts used for cross–aldol condensation reac-
tion include sulfuric, hydrochloric,28,29 and Lewis30–32 acids. Gener-
ally, aldol condensation can be carried at room temperature or
ethanol under reflux condition.33,34 Dibenzylideneacetones and
chalcones usually are more easily synthetized in one step by aldol
condensation under basic catalysis. This procedure works very well
for symmetric ketones and a variety of substituted aldehydes.
However, only a few studies used non-symmetric ketones and dif-
ferent aldehydes to be attached to the ends of the alkyl chain
spacer.16,35 These non-symmetric 1,5-diaryl-3-oxo-1,4-pentadie-
nyls can be formed in two steps.35 Methyl–alkyl ketones are good
for aldol condensation because of the easy regiochemistry control.
Therefore, these ketones react with an appropriate aldehyde in
acidic medium (thermodynamic enol formation), and then the
dehydrated aldol is isolated (Scheme 1). The second aldehyde is
then added dropwise in cold ethanol solution, stirred, and the
non-symmetric 1,5-diaryl-3-oxo-1,4-pentadienyl is formed
(Scheme 1). Using this methodology35 and combining different
aldehydes with ketones, it is possible to generate a great molecular
diversity, creating a library of compounds with 1,5-diarylpentane
basic structures, which are still to be explored as to their
bioactivities.
Thus, herein is reported a new series of dibenzylideneacetones,
which have been prepared according Scheme 1, by combination of
four p-substituted aldehydes (A1–A4) with butanone (K2) resulting
in the four 4-aryl-3-methylbutenones 1a-1d [R1 = H (A1) or OCH3
(A2) or NO2 (A3) or N(CH3)2 (A4), respectivelly; R2 = CH3], which
were individually further condensed with the same aldehydes,
resulting in the sixteen compounds 2a–2p (An(1–4)K2Am(1–4)).
The antiproliferative activities of the twenty-synthetized com-
pounds were evaluated, particularly in promastigotes of L. amazon-
ensis and epimastigotes and trypomastigotes of T. cruzi.
3. Results and discussion
3.1. Chemistry
The reaction of benzaldehyde (A1) and three p-substituted
benzaldehydes (A2–A4) with butanone, using gaseous HCl as cata-
lyst as outlined in Scheme 1, produced the 4-aryl-3-methylbute-
none 1a–1d, which were isolated and characterized by
spectroscopic data. These reactions were performed at room tem-
perature by stirring the reaction mixture and passing dry gaseous
HCl until the reaction mixture turned to red. Stirring continued un-
til the completion of starting materials, which was monitored by
TLC. The reaction was very clear giving yields of 48–61% of
1a–1d, which were analyzed by NMR as follows: compound
1 showed two characteristic signals of two methyl groups in the
shielded region at dH 2.06 and 2.47, whereas one @CHsignal was
displayed at dH 7.39 as singlet; additionally three aromatic signals
having integration for five proton were detected at dH 7.32, 7.41
and 7.52. 13C NMR spectra of 1 showed signals for two methyl
groups at dC 13.0 and 25.9, and six peaks from dC 128–139 for eight
carbons, with two of these peaks having double intensity for
aromatic carbons. The characteristic peak at dC 200.3 is due to car-
bonyl carbon. The 1H NMR spectra of compounds 1a, 1b and 1c are
similar each other. Only compound 1b showed an extra signal at dH
3.85 due to OCH3 substitution on benzene, and a signal at dH 3.02
for compound 1d due to N(CH3)2 substitution in benzene ring. By
reacting each of the intermediary compounds 1a–1d systemati-
cally with benzaldehyde (A1), anisaldehyde (A2), N,N-dimethyl-
aminobenzaldehyde (A3) and nitrobenzaldehyde (A4) the
respective sixteen dibenzylidene ketones 2a–2p were produced
in 45–93% yield after re-crystallization from ethanol. All these
reactions were carried in basic medium in ethanol, and their
O
O
H
O
O
O
1
5
R3
R2
3
H
2
R2
4
A
B
R2
R1
R1
R3
R1
NaOH in Ethanol
Am(1-4)
Dry HCl
An(1-4)
K2 (R2 = CH3)
2a-2p (An(1-4)K2Am(1-4)
)
1a-1d (An(1-4)K2)
Scheme 1. Reaction conditions for the synthesis of the assayed compounds, using p-substituted aldehydes A1 (R1,R3 = H), A2 (R1,R3 = OCH3), A3 (R1,R3 = NO2) and A4
(R1,R3 = N(CH3)2) and butanone (K2, R2 = CH3) resulting in 1a–1d, respectivelly; and further reaction of each enone 1a–1d with same aldehydes to give the collection
An(1–4)K2Am(1–4) (2a–2p).

Z. Ud Din et al. / Bioorg. Med. Chem. 22 (2014) 1121–1127
1123
progress was monitored by TLC. The reaction time varied for each
AnK2Am product and depended on the nature and reactivity of the
aldehyde used. Thus, as expected for aldol condensations, electron
donators (amine and methyl–ether) at para-position in the ben-
zene rings decreases the reaction time, while nitrobenzaldehyde
reacted very fast. The aldol-dehydrated products are preferably E
regeoisomers but Z isomers were also obtained in 1–5%. The struc-
tures of these new compounds were determined by 1H and 13C
NMR, HMQC and HMBC spectra. Compounds 2a–2d were
synthesized from 1a, by treating it with benzaldehyde, anisalde-
hyde, nitrobenzaldehyde and N,N-dimethylaminobenzaldehyde,
respectivelly, in basic conditions in ethanol. The presence of a pair
of doublets at dH 7.53 and 7.69, with a coupling constant of c.a.
16 Hz, typical for a trans two spins system, clearly confirms that
the aldol condensation with the second aldehyde did occur. Also,
extra signals in the aromatic region were noted in the 1H spectra
and the signal for methyl ketone group disappeared, showing that
an aromatic ring had been added and the methyl group had been
substituted to a significant extent. The major peaks in the NMR
spectra of these compounds are almost the same except from an
additional signal in compounds 2a, 2b and 2d. Compound 2a has
benzene ring with no substituent, while in compound 2b the para
hydrogen of benzene is substituted by a OCH3 group, showing a
singlet at dH 3.85 ppm in 1H NMR, and a peak at dC 55.4 ppm in
13C NMR. Compound 2d shows an extra signal at dH 3.06 due to
N(CH3)2, and 2c is prompt recognized by the strong deshielding
effect caused by NO2 group to the ortho hydrogen’s (dH 8.26). Com-
pounds 2e–2p were similarly synthesized from compound 1b–1d,
so the spectroscopic study is comparable to that of compounds 2a–
2d, showing approximately the same signals shifts as shown in the
compounds derived from 1. The structures of all compounds were
further identified using mass spectrometry and UV–vis analysis.
epimastigotes of T. cruzi were 2d, 2i, 2j, and 2k, exhibiting an IC50
value (inhibitory concentration for 50% of the parasites) of 13.3,
3.5, 10.0, and 1.8 lM, respectively (Table 1). It is important to note
that, for both forms epimastigote and trypomastigote, the activities
of compounds 2k and 2i (EC50, effective concentration for 50% of
the parasites, 15.4 and 17.5
l
M, respectively) were found greater
M and EC50
than the positive control benznidazole (IC50 of 6.5
l
34.5).36 Considering the evaluation of activity against promastig-
otes of L. amazonensis it was observed that substances 2c, 2g, 2i,
2l and 2k are the most active, with IC50 values of 11.6, 14.8, 13.4,
12.0 and 3.4
for L. amazonensis, has an IC50 of 0.06
our compounds, although its toxicity is high (3.74
l
M, respectively. Amphotericin B, the reference drug
M, which is better than
M).37
l
l
The literature concerning antiparasitary activities of curcumi-
noids and chalcones reports results with IC50 values around those
we found in the present work.26,38 Symmetrical dibenzylideneke-
tones made using cyclic ketones gave antileishmanial activity with
lower IC50 values.39 A recent study showed that both electron
withdraw or donator groups at the aryl part of symmetrical diben-
zylideneacetones, affect positively the bioactivity compared with
nonsubstituted compounds.40 Our results, using the series of asym-
metrical dibenzylydeneketone compounds (Table 1), shows that
the strong electron-attractor NO2 substituent in A3 is the most
responsible for the great activity observed. Compounds 2c
(A1K2A3) and 2i (A3K2A1) differ only in the aromatic ring where
the NO2 is attached, but their bioactivity is significantly different.
This result indicates that introducing the electron-attracting group
during the first reaction results in better activity of the final com-
pounds. Thus, nitro compounds 2i (A3K2A1) and 2k (A3K2A3)
were found to be the most active antiparasitics in the three assays
performed (Table 1), and indeed compound 2k, with two NO2
groups, is the most active amongst the twenty tested substances.
These results parallels those recently published by others research-
ers, which found that nitroreductase enzymes, present in
Leishmania danovani but not in the mammal cells, may activate
NO2-containing drugs into a more active form.41
3.2. Biological evaluation
The synthesized compounds 1a–1d and 2a–2p were evaluated
against promastigote forms of L. amazonensis and epimastigote
and trypomastigote forms of T. cruzi. The intermediate compounds
AnK2 (1a–1d) were found inactive in the bioassays, indicating that
the second aldol reaction is important since it introduces groups
that contribute to bioactivity. The more active compounds against
The mechanism by which drugs act against parasites have been
studied by many groups around the world.41,42 Many of these stud-
ies suggest that drugs can bind to nucleic acids and cell wall com-
ponents, or inhibit enzymes activity in the parasite. Along with the
presence of p-NO2 group in our compounds, that putatively can be
Table 1
Antiparasitary and citotoxicity activities measured for of compounds 1a–1d and 2a–2p
Compounds
Epimastigote IC50
Average SD
(
lM)
Trypomastigote EC50
Average SD
(l
M)
Promastigote IC50
Average SD
(lM)
LLCMK2 CC50
Average SD
(l
M)
M. J774A1 CC50
Average SD
(lM)
1a (A1K2)
1b (A2K2)
1c (A3K2)
1d (A4K2)
>100
>100
82.2 7.42
>100
>100
>100
>100
>100
71.7 2.33
>100
65.0 7.07
>100
73.3 4.67
>100
>100
>100
>100
>100
>100
687.5 17.68
280.0 28.28
742.5 81.32
240.0 14.14
70.5 16.26
160.0 14.14
34.5 7.78
51.0 11.31
26.0 1.41
20.2 3.96
54.0 8.49
34.1 1.75
30.0 6.56
36.2 4.35
47.3 4.16
36.6 1.15
43.0 4.24
24.7 2.52
28.5 0.71
34.3 5.13
966.6 57.74
10.0 0.00
266.0
12.73
150.0 0.00
33.6 5.28
93.0 9.90
43.3 4.16
53.0 4.24
44.0 0.00
75.0 7.07
20.1 6.80
411.0 55.15
264.3 3.25
172.1 15.77
32.5 1.20
25.9 9.50
191.0 12.73
285.1 81.81
167.0 8.49
314.3 0.00
614.7 115.2
2a (A1K2A1)
2b (A1K2A2)
2c (A1K2A3)
2d (A1K2A4)
2e (A2K2A1)
2f (A2K2A2)
2g (A2K2A3)
2h (A2K2A4)
2i (A3K2A1)
2j (A3K2A2)
2k (A3K2A3)
2l (A3K2A4)
2m (A4K2A1)
2n (A4K2A2)
2o (A4K2A3)
2p (A4K2A4)
Benznidazole
Amphotericin B
15.9 1.34
14.3 5.13
16.9 0.00
13.3 2.90
16.7 1.41
23.6 2.33
25.2 4.60
21.3 0.00
3.5 0.35
10.0 0.00
1.8 0.21
14.0 0.00
24.1 2.62
>100
15.3 0.35
20.4 3.32
11.6 1.70
20.0 2.83
22.3 1.77
21.8 0.28
14.8 1.77
18.0 0.00
13.4 1.98
15.5 1.70
3.4 0.07
12.0 0.71
21.5 0.71
20.1 1.48
>100
>100
17.5 2.05
73.3 0.00
15.4 4.10
>100
78.3 2.40
>100
>100
>100
34.5 7.6
>100
25.1 1.27
6.5 0.7
20.5 2.12
0.06 0.00
3.7 0.31

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Z. Ud Din et al. / Bioorg. Med. Chem. 22 (2014) 1121–1127
O
1
5
3
4
2
A
B
CH3
HH
O2N
NO2
2k
2k’
2k’’
2k’’’
Figure 2. Structural features of compound 2k. 2k0-2k000 are conformers generated from geometry computational optimization, showing that ring-A gets out of the plane
formed of ring-B and C-3 - C-5, due steric hindrance caused by the presence CH3 group close to Ar-H in ring-A.
reduced/activated by parasite reductases, our nitrobenzylideneke-
tones 2i and 2k may be good electrophiles to bind some parasite
cells components. These molecules were studied using the semi
empirical quantum-mechanical AM1 parameterization,43 in order
to see possible binding points. The molecular geometries that came
out from these calculations (see 2k0 to 2k000 in Fig. 2) shows that the
presence of the methyl group at C-2 of the 1,4-pentadienyl-3-one
potency. It is clear that the presence of the p-nitrobenzene group
at the methyl side strongly contributes to antiparasitic activity.
5. Experimental
5.1. Chemistry
chain disturbs the first enone
p system (1-en-3-one), while the
All chemicals were purchased from Organics, Sigma–Aldrich,
Acros Chemicals and Fisher Scientific Ltd and used without further
purification. The deuterated solvents from Apolo were used for the
NMR analysis. Thin layer chromatography (TLC) was performed
with precoated silica gel G-25-UV254 plates and was carried out
at 254 nm under UV, and by ceric sulphate in 10% H2SO4 solution.
High-resolution mass spectral data (ESI-HRMS) were acquired
using a Thermo Scientific LTQ Orbitrap XL spectrometer. 1H NMR,
13C NMR and 2D experiments (gHSQC (1H/1H), gHMBC (1H/13C)
and NOESY) were performed 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. Com-
pounds 1a–1d and 2a–2p were dissolved in organic solvents at
about 10 mg mLꢀ1 each and transferred into a 5-mm NMR tube.
Chemical shifts (d ppm) were measured with accuracy of 0.01
(1H) and 0.1 ppm (13C). The UV–vis spectra were recorded on a Per-
kin Elmer Lambda 25 spectrophotometer using 1 cm optical length
quartz cuvettes at 25 °C and chloroform as solvent.
second part, formed by the ring-B bound to C-5 and the 4-en-3-
one, is planar with an almost perfect parallelism of the p-orbitals.
As a result, carbon C-1 contains only small electron density com-
pared with C-5. These observations may have some relationship
with our bioactivity results, which showed that electron attractor
group present in the ring-A connected to C-1 contributes for great-
er bioactivity. Probably C-1 is a good electrophilic center to bind
parasite cell components.
In addition, we also evaluated the cytotoxicity of the com-
pounds against LLCMK2 cells (kidney epithelial cells from Macaca
mulatta) and macrophages J774A1. The results showed that the ac-
tive substances were more toxic against the parasites than for the
cell lines tested. Finally, our results demonstrated that compounds
2i and 2k proved to be the most active against this trypanosomat-
ids and may become promising compounds for treatment of leish-
maniasis and Chagas disease.
4. Conclusions
5.1.1. (E)-3-Methyl-4-phenylbut-3-en-2-one (1a)
Benzaldehyde (10 g, 90 mmol) and 2-butanone (1.36 g, 1.69 mL,
18.9 mmol) were taken in a 50 mL two–necked round bottom flask.
Dry HCl gas was passed in the content of the flask until it was sat-
urated and red coloration appeared. The reaction mixture was stir-
red for 8 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 res-
idue was distilled under reduced pressure to give pure compound,
which was solidified by keeping in a refrigerator for 2 days.
The reaction of 2-butanone with benzaldehyde, anisaldehyde,
N,N-dimethylaminobenzaldehyde and nitrobenzaldehyde gave 1a,
1b, 1c and 1d, which on subsequent coupling with the same alde-
hydes yielded the respective benzylidenedienones 2a–2p. These
compounds were investigated for antiproliferative against prom-
astigotes of L. amazonensis and epimastigotes and tripomastigotes
of T. cruzi, and some compounds were shown to have good repro-
ducible activity, while compounds 2i and 2k showed enhanced

Z. Ud Din et al. / Bioorg. Med. Chem. 22 (2014) 1121–1127
1125
Percent yield: 52%; mp: 34–35 °C; 1H NMR (400 MHz, CDCl3) dH
5.1.7. (1E,4E)-2-Methyl-5-(4-nitrophenyl)-1-phenylpenta-1,4-
dien-3-one (2c)
7.52 (d, 1H, J = 4 Hz, ArH), 7.41 (m, 3H, Ar H), 7.39 (m, 1H,
CH@CCH3), 7.32 (m, 1H, ArH), 2.47 (s, 3H, COCH3), 2.06 (d,
J = 1.6 Hz, 3H, CH@CCH3); 13C NMR (100 MHz, CDCl3) dC 129.7,
200.3, 139.7, 137.8, 135.9, 128.6, 128.5, 25.9, 12.9; HRMS ESI(+):
calcd for C11H13O (M+H)+ 161.0961, found 161.0962; UV–vis kmax
(nm) in CH2Cl2: 278.
Percent yield: 50.5%; mp: 138–140 °C; 1H NMR (400 MHz,
CDCl3) dH 8.26 (d, J = 8 Hz, 2H, ArH), 7.75 (d, J = 12 Hz, 2H, ArH),
7.69 (d, J = 16 Hz, 1H, @CH), 7.62 (s, 1H, @CH), 7.53 (d, J = 16 Hz,
1H, @CH), 7.43–7.50 (m, 5H, ArH), 7.34 (t, 1H, ArH), 2.21 (d,
J = 1.6 Hz, 3H, @CCH3); 13C NMR (100 MHz, CDCl3) dC 191.8,
148.4, 141.4, 1403.2, 139.9, 138.4, 135.6, 134.63, 129.8, 128.8,
125.7, 124.2, 124.0, 13.7; HRMS ESI(+): calcd for C18H16NO3
(M+H)+ 294.1125, found 294.1138; UV–vis kmax (nm) in CH2Cl2:
300.
5.1.2. (E)-4-(4-Methoxyphenyl)-3-methylbut-3-en-2-one (1b)
Percent yield: 67%; mp: Oil; 1H NMR (400 MHz, CDCl3) dH 7.47
(s, 1H, @CH), 7.40 (d, J = 12 Hz, 2H, ArH), 6.93 (d, J = 12 Hz, 2H,
ArH), 3.85 (s, 3H, OCH3), 2.45 (s, 3H, @CCH3), 2.07 (d, J = 1.6 Hz,
3H, COCH3); 13C NMR (100 MHz, CDCl3) dC 131.6, 200.2, 159.9,
139.6, 135.8, 128.4, 114.0, 55.3, 25.8, 12.9; HRMS ESI(+): calcd
for C12H15O2 (M+H)+ 191.1067, found 191.1072; UV–vis kmax
(nm) in CH2Cl2: 308.
5.1.8. (1E,4E)-5-(4-Dimethylamino)phenyl)-2-methyl-1-
phenylpenta-1,4-dien-3-one (2d)
Percent yield: 68%; mp: 91–92 °C; 1H NMR (400 MHz, CDCl3) dH
7.68 (d, J = 16Hz, 1H, @CH), 7.52 (d, J = 8Hz, 2H, ArH, 7.40 (m, 4H,
ArH), 7.31 (m, 1H, @CH), 7.19 (d, J = 16Hz, 1H, @CH), 6.68 (d,
J = 8Hz, 2H, ArH), 3.04 (s, 6H, N(CH3)2, 2.19 (d, J = 1.6 Hz, 2H,
@CCH3); 13C NMR (100 MHz, CDCl3) dC 193.1, 151.9, 144.55,
138.9, 137.0, 136.4, 130.1, 129.7, 128.4, 128.2, 122.9, 116.9,
111.7, 40.17, 14.1; HRMS ESI(+): calcd for C20H22NO (M+H)+
292.1696, found 292.1710; UV–vis kmax (nm) in CH2Cl2: 414.
5.1.3. (E)-3-Methyl-4-(4-nitrophenyl)but-3-en-2-one (1c)
Percent yield: 61%; mp: 84–85 °C; 1H NMR (400 MHz, CDCl3) dH
8.27 (d, J = 8 Hz, 2H, ArH), 7.55 (d, J = 8 Hz, 2H, ArH), 7.52 (s, 1H,
@CH), 2.49 (s, 3H, COCH3), 2.06 (d, J = 1.6 Hz, 3H, @CCH3); 13C
NMR (100 MHz, CDCl3) dC 199.6, 147.3, 142.5, 140.6, 136.5, 130.2,
123.7, 26.0, 13.2; HRMS ESI(+): calcd for C11H11NO3 (M+H)+
206.0812, found 206.0825; UV–vis kmax (nm) in CH2Cl2: 300.
5.1.9. (1E,4E)-1-(4-Methoxyphenyl)-2-methyl-5-phenylpenta-
1,4-dien-3-one (2e)
5.1.4. (E)-4-(Dimethylamino)phenyl)-3-methylbut-3-en-2-one
(1d)
Percent yield: 51%; mp: 57–58 °C; 1H NMR (400 MHz, CDCl3) dH
7.67 (d, J = 12 Hz, 1H, @CH), 7.61 (m, 2H, ArH), 7.56 (s, 1H, CH3-
C@CH), 7.45 (m, 3H, ArH), 7.39 (m, 3H, @CHand ArH), 6.95 (d,
J = 8 Hz, 2H, ArH), 3.85 (s, 3H, OCH3), 2.21 (d, J = 1.6 Hz, 3H, @C–
CH3); 13C NMR (100 MHz, CDCl3) dC 192.6, 159.9, 143.0, 138.8,
136.6, 135.3, 131.7, 130.08, 128.9, 128.5, 128.2, 122.1, 114.0,
55.4, 13.8; HRMS ESI(+): calcd for C19H19O2 (M+H)+ 279.1380,
found 279.1388; UV–vis kmax (nm) in CH2Cl2: 335.
Percent yield: 48%; mp: 122–123 °C; 1H NMR (400 MHz, CDCl3)
dH 7.45 (s, 1H, @CH), 7.41 (d, J = 8Hz, 2H, ArH), 6.71 (d, J = 12Hz, 2H,
ArH), 3.02 (s, 6H, N(CH3)2, 2.44 (s, 3H, COCH3), 2.10 (d, J = 4 Hz, 3H,
@CCH3); 13C NMR (100 MHz, CDCl3) dC 200.2, 150.5, 140.7, 133.4,
131.9, 123.6, 111.7, 40.2, 25.71, 13.0; HRMS ESI(+): calcd for
C
13H17NO (M+H)+ 204.1383, found 204.1385; UV–vis kmax (nm)
in CH2Cl2: 366.
5.1.10. (1E,4E)-1,5-Bis(4-methoxyphenyl)-2-methypenta-1,4-
dien-3-one (2f)
5.1.5. (1E,4E)-2-Methyl-1,5-diphenylpenta-1,4-dien-3-one (2a)
A Solution of A1K2 (1a, 50 mg, 3.13 mmole), and benzaldehyde
(39.8 mg, 3.75 mmole) 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 stirring continued for seven 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 com-
pound 2a. Percent yield: 51.4%; mp: 52–53 °C; 1H NMR
(400 MHz, CDCl3) dH 7.67 (d, J = 16 Hz, 1H, Ar-CH), 7.59 (m, 1H,
ArH), 7.56 (m, 2H, ArH), 7.40 (m, 4H, ArH), 7.34 (s, 1H, Ar-CH),
7.28 (d, J = 8 Hz, 1H, COCH), 6.92 (d, J = 8 Hz, 2H, ArH), 3.85 (s,
3H, OCH3), 2.19 (d, J = 1.6 Hz, 3H, @C–CH3); 13C NMR (100 MHz,
CDCl3) dC 192.9, 161.4, 143.4, 138.7, 138.1, 133.3, 130.0, 129.7,
128.4, 127.9, 119.7, 114.4, 55.41, 13.9; HRMS ESI(+): calcd for
Percent yield: 91%; mp: 86–88 °C; 1H NMR (400 MHz, CDCl3) dH
7.65 (d, J = 16, 1H, @CH), 7.56 (d, J = 12 Hz, 2H, ArH), 7.53 (s, 1H,
CH3C@CH), 7.44 (d, J = 8Hz, 2H, ArH), 7.30 (d, J = 12 Hz, 1H, @CH),
6.95 (d, J = 8 Hz, 2H, ArH), 6.92 (d, J = 8 Hz, 2H, ArH), 3.85 (d,
J = 4 Hz, 6H, OCH3), 2.20 (d, J = 1.6 Hz, 3H, @C–CH3); 13C NMR
(100 MHz, CDCl3) dC 13.9, 55.4, 114.0, 114.3, 119.8, 128.0, 128.7,
129.9, 131.6, 136.8, 138.1 (1C, @C), 159.8, 161.3, 192.7; HRMS
ESI(+): calcd for C20H21O3 (M+H)+ 309.1485, found 309.1494;
UV–vis kmax (nm) in CH2Cl2: 348.
5.1.11. (1E,4E)-1-(4-Methoxyphenyl)-2-methyl-5-(4-
nitrophenyl)penta-1,4-dien-3-one (2g)
Percent yield: 69.3%; mp: 145–146 °C; 1H NMR (400 MHz,
CDCl3) dH 8.25 (d, J = 12 Hz, 2H, ArH), 7.74 (d, J = 12 Hz, 2H, ArH),
7.66 (d, J = 16 Hz, 1H, @CH), 7.59 (s, 1H, @CH), 7.54 (d, J = 16 Hz,
1H, @CH), 7.47 (d, J = 8 Hz, 2H, ArH), 6.97 (d, J = 8Hz, 2H, ArH),
3.87 (s, 3H, OCH3), 2.22 (d, J = 1.6 Hz, 3H, @CCH3); 13C NMR
(100 MHz, CDCl3) dC 191.6, 160.2, 148.3, 141.5, 140.0, 139.8,
136.4, 131.83, 128.7, 128.2, 125.8, 124.2, 114.1, 55.4, 13.3; HRMS
ESI(+): calcd for C19H18NO4 (M+H)+ 324.1230, found 324.12449;
UV–vis kmax (nm) in CH2Cl2: 348.
C
18H17O (M+H)+ 249.1274, found 249.1283; UV–vis kmax (nm) in
CH2Cl2: 313.
5.1.6. (1E,4E)-5-(4-Methoxyphenyl)-2-methyl-1-phenylpenta-
1,4-dien-3-one (2b)
Percent yield: 57%; mp: 115–117 °C; 1H NMR (400 MHz,
CDCl3) dH 7.67 (d, J = 16 Hz, 1H, CH3OAr–CH), 7.59 (m, 1H, ArH),
7.56 (m, 2H, ArH), 7.40 (m, 4H, ArH), 7.34 (s, 1H, Ar-CH), 7.28
(d, J = 8 Hz, 1H, COCH), 6.92 (d, J = 8 Hz, 2H, ArH), 3.85 (s, 3H,
OCH3), 2.19 (d, J = 1.6 Hz, 3H, @C–CH3); 13C NMR (100 MHz,
CDCl3) dC 192.9, 161.4, 143.38, 138.7, 138.1, 133.3, 130.0, 129.7,
128.4, 127.9, 119.7, 114.4, 55.4, 13.9; HRMS ESI(+): calcd for
5.1.12. (1E,4E)-5-(4-(Dimethylamino)phenyl)-1-(4-
methoxyphenyl)-2-methypenta-1,4-dien-3-one (2h)
Percent yield: 93%; mp: 68–70 °C; 1H NMR (400 MHz, CDCl3) dH
7.66 (d, J = 12 Hz, 1H, @CH), 7.50 (s, 1H, @CH), 7.49 (d, J = 8 Hz, 2H,
ArH), 7.41 (d, J = 12 Hz, 2H, ArH), 7.20 (d, J = 16 Hz, 1H, @CH), 6.92
(d, J = 8 Hz, 2H, @CH), 6.65 (d, J = 8Hz, 2H, ArH), 3.82 (s, 3H, OCH3),
2.99 (s, 6H, N(CH3)2, 2.19 (d, J = 1.6 Hz, 3H, @CCH3); 13C NMR
(100 MHz, CDCl3) dC 192.9, 159.7, 151.8, 144.1, 137.0, 137.0,
C
19H18O2 (M+H)+ 279.1380, found 279.1388; UV–vis kmax (nm)
in CH2Cl2: 340.

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Z. Ud Din et al. / Bioorg. Med. Chem. 22 (2014) 1121–1127
131.52, 130.1, 128.9, 123.0, 116.9, 113.9, 111.7, 55.33, 40.1, 14.1;
HRMS ESI(+): calcd for
21H23NO2 (M+H)+ 322.1802, found
322.1811; UV–vis kmax (nm) in CH2Cl2: 413.
@CH), 6.91 (d, J = 8 Hz, 2H, ArH), 6.73 (d, J = 8 Hz, 2H, ArH),
3.85(s, 3H, OCH3), 3.03 (s, 6H, N(CH3)2), 2.23 (d, J = 4 Hz, 3H,
@CCH3); 13C NMR (100 MHz, CDCl3) dC 192.5, 161.1, 150.5, 142.1,
139.5, 134.6, 131.9, 129.8, 128.3, 123.9, 120.1, 114.3, 111.7, 55.4,
40.2, 14.0; HRMS ESI(+): calcd for C21H24NO2 (M+H)+ 322.1802,
found 322.1812; UV–vis kmax (nm) in CH2Cl2: 401.
C
5.1.13. (1E,4E)-2-Methyl-1-(4-nitrophenyl)-5-phenylpenta-1,4-
dien-3-one (2i)
Percent yield: 92%; mp: 100–101 °C; 1H NMR (400 MHz, CDCl3)
dH 8.28 (d, J = 8 Hz, 2H, ArH), 7.73 (d, J = 16 Hz, 2H, ArH), 7.62 (m,
2H, ArH), 7.58 (d, J = 12 Hz, 2H, @CH), 7.55 (s, 1H, CH3C@CH),
7.42 (m, 3H, ArH), 7.36 (d, J = 16 Hz, 2H, @CH), 2.19 (d, J = 4 Hz,
3H, @CCH3); 13C NMR (100 MHz, CDCl3) dC 192.2, 147.3, 144.6,
142.6, 141.6, 135.3, 134.8, 130.6, 130.3, 129.0, 128.4, 123.7,
121.4, 14.1; HRMS ESI(+): calcd for C18H16NO3 (M+H)+ 294.1125,
found 294.1134; UV–vis kmax (nm) in CH2Cl2: 308.
5.1.19. (1E,4E)-1-(4-Dimethylamino)phenyl-2-methyl-5-(4-
nitrophenyl)penta-1,4-dien-3-one (2o)
Percent yield: 57.6%; mp: 212–215 °C; 1H NMR (400 MHz,
CDCl3) dH 1H NMR (400 MHz, CDCl3) d 8.25 (d, J = 8 Hz, 2H, @CH),
7.74 (d, J = 8 Hz, 2H, ArH), 7.62 (d, J = 8 Hz, 2H, ArH), 7.58 (s, 1H,
CH3-C@CH), 7.48 (d, J = 8 Hz, 2H, ArH), 6.73 (d, J = 12 Hz, 2H,
ArH), 3.05 (s, 6H, N(CH3)2), 2.25 (d, J = 1.6 Hz, 3H, @CCH3); 13C
NMR (100 MHz, CDCl3) dC 191.3, 150.6, 148.1, 141.9, 141.4, 139.0,
133.8, 132.3, 128.6, 126.2, 124.1, 123.4, 111.7, 40.1, 13.8; HRMS
ESI(+): calcd for C20H21N2O3 (M+H)+ 337.1547, found 337.1562;
UV–vis kmax (nm) in CH2Cl2: 418.
5.1.14. (1E,4E)-5-(4-Methoxyphenyl)-2-methyl-1-(4-
nitrophenyl)penta-1,4-dien-3-one (2j)
Percent yield: 63%; mp: 74–76 °C; 1H NMR (400 MHz, CDCl3) dH
8.27 (d, J = 8 Hz, 2H, ArH), 7.71 (d, J = 16 Hz, 1H, @CH), 7.58 (d, 4H,
ArH), 7.52 (s, 1H, @CH), 7.26 (s, 1H, CH3C@CH), 6.93 (d, J = 8 Hz, 2H,
ArH), 3.87 (s, 3H, OCH3), 2.19 (d, J = 1.6 Hz, 3H, @CCH3); 13C NMR
(100 MHz, CDCl3) dC 192.2, 161.7, 147.2, 144.5, 142.8, 141.7,
134.6, 130.2, 130.2, 127.5, 123.3, 119.1, 114.5, 55.5, 14.2; HRMS
ESI(+): calcd for C19H18NO4 (M+H)+ 324.1230, found 322.1250;
UV–vis kmax (nm) in CH2Cl2: 316.
5.1.20. (1E,4E)-1,5-Bis(4-dimethylamino)phenyl-2-
methylepenta-1,4-dien-3-one (2p)
Percent yield: 60%; mp: 146–147 °C; 1H NMR (400 MHz, CDCl3)
dH 7.63 (d, J = 16 Hz, 2H, @CH), 7.63 (d, J = 8 Hz, 2H, ArH), 7.44 (d,
J = 8 Hz, 2H, ArH), 7.30 (s, 1H, CH3–C@CH), 6.73 (d, J = 8 Hz, 2H,
ArH), 6.68 (d, J = 8 Hz, 2H, ArH), 3.03 (d, J = 0 Hz, 12H, N(CH3)2),
2.23 (d, J = 1.6 Hz, 3H, @CCH3); 13C NMR (100 MHz, CDCl3) dC
192.8, 151.6, 150.3, 143.3, 138.5, 134.7, 131.8, 129.90, 124.2,
123.4, 117.4, 111.9, 111.8, 40.2, 14.1; HRMS ESI(+): calcd for
5.1.15. (1E,4E)-2-Methyl-1,5-bis(4-nitrophenyl)penta-1,4-dien-
3-one (2k)
Percent yield: 65%; mp: 188–189 °C; 1H NMR (400 MHz, CDCl3)
dH 8.31 (t, J = 0 Hz, J = 4 Hz, 1H, ArH), 8.29 (t, J = 0 Hz, J = 4 Hz, 2H,
ArH), 8.23 (t, J = 0 Hz, J = 4 Hz, 1H, ArH), 7.74 (m, 3H, ArH and
CH3-C@CH), 7.60 (d, J = 8 Hz, 3H, @CHand ArH), 7.49 (d, J = 16 Hz,
1H, @CH), 2.21 (d, J = 1.6 Hz, 3H, @CCH3); 13C NMR (100 MHz,
CDCl3) dC 191.2, 148.6, 147.4, 142.1, 141.3, 141.3, 141.0, 136.4,
130.3, 128.9, 125.0, 124.3, 123.8, 14.0; HRMS ESI(+): calcd for
C
22H27N2O (M+H)+ 335.2118, found 335.2124. UV–vis kmax (nm) in
CH2Cl2: 422.
5.2. Biological activity
5.2.1. Parasites and cells
Leishmania amazonensis promastigote forms (MHOM/BR/Josefa)
were maintained at 25 °C in Warren’s medium (brain heart infusion
plus haemin and folic acid) pH 7.0, supplemented with 10% Fetal Bo-
vine Serum (FBS, Gibco Invitrogen, Grand Island, NY, USA). Epimas-
tigote 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 (epithelialcells fromthe kid-
ney of the monkey Macaca mulatta) in DMEM supplemented with
10% FBS at 37 °C in a humidified 5% CO2 atmosphere. J774A1 murine
macrophages were maintained in tissue flasks with RPMI 1640 med-
ium (Gibco Invitrogen Corporation, New York, USA) pH 7.6, with so-
C
18H15N2O5 (M+H)+ 339.0910, found 339.0910; UV–vis kmax (nm)
in CH2Cl2: 304.
5.1.16. (1E,4E)-5-(4-Dimethylamino)phenyl-2-methyl-1-(4-
nitrophenyl)penta-1,4-dien-3-one (2l)
Percent yield: 61%; mp: 129–131 °C; 1H NMR (400 MHz, CDCl3)
dH 8.26 (d, J = 8 Hz, 2H, ArH), 7.71 (d, J = 16 Hz, 1H, @CH), 7.56 (d,
J = 12 Hz, 2H, ArH), 7.52 (d, J = 8 Hz, 2H, ArH), 7.48 (s, 1H, @CH),
7.12 (d, J = 16 Hz, 1H, @CH), 3.68 (d, J = 8 Hz, 2H, ArH), 3.05 (s,
6H, N(CH3)2), 2.19 (d, J = 1.6 Hz, 3H, @CCH3); 13C NMR (100 MHz,
CDCl3) dC 192.4, 152.1, 147.0, 145.8, 143.1, 142.1, 133.6, 130.4,
130.2, 123.7, 122.5, 116.2, 111.8, 40.1, 14.4; HRMS ESI(+): calcd
for C20H21N2O3 (M+H)+ 337.1547, found 337.1568; UV–vis kmax
(nm) in CH2Cl2: 415.
dium bicarbonate and L-glutamine added, and supplemented with
10% FBS at 37 °C in a 5% CO2–air mixture.
5.2.2. Antiproliferative assay
5.1.17. (1E,4E)-1-(4-Dimethylamino)phenyl)-2-methyl-5-
phenylpenta-1,4-dien-3-one (2m)
The effects of synthesized compounds were evaluated in prom-
astigotes of L. amazonensis and epimastigotes of T. cruzi. The inoc-
ulum (1 ꢁ 106 cells/mL) was introduced into 24-well plate
containing the compounds dissolved in dimethylsulfoxide (DMSO)
and Warren’s medium or LIT in several concentrations
Percent yield: 67%; mp: 60–62 °C; 1H NMR (400 MHz, CDCl3) dH
7.68 (s, 1H, @CH), 7.61 (m, 2H, ArH), 7.56 (s, 1H, CH3–C@CH), 7.47
(d, J = 12Hz, 2H, @CH), 7.46 (d, J = 0Hz, 2H, ArH), 7.39 (m, 2H, ArH),
6.73 (d, J = 8 Hz, 2H, ArH), 3.04 (s, 6H, N(CH3)2), 2.24 (d, J = 1.6 Hz,
3H, @CCH3); 13C NMR (100 MHz, CDCl3) dC 150.6, 142.2, 142.1,
140.1, 135.6, 134.2, 132.0, 129.8, 128.8, 128.1, 123.7, 122.4,
111.7, 40.12, 13.9; HRMS ESI(+): calcd for C20H22NO (M+H)+
292.1610, found 292.1705; UV–vis kmax (nm) in CH2Cl2: 401.
(1.0–100.0 lM). The final concentration of DMSO did not exceed
1%. Cell grown was determined by counting the parasites with a
Neubauer hemocytometer after incubation for 72 h at 25 °C for
L. amazonensis or for 96 h at 28 °C for 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.
5.1.18. (1E,4E)-1-(4-Dimethylamino)phenyl-5-(4-
methoxyphenyl)-2-methylepenta-1,4-dien-3-one (2n)
Percent yield: 79%; mp: 110–112 °C; 1H NMR (400 MHz, CDCl3)
dH 7.62 (d, J = 16 Hz, 1H, @CH), 7.56 (d, J = 8 Hz, 2H, ArH), 7.54 (brs,
1H, CH3–C@CH), 7.45 (d, J = 8 Hz, 2H, ArH), 7.34 (s, J = 16 Hz, 1H,
5.2.3. Viability of trypomastigote forms of T. cruzi
The tissue-culture-derived trypomastigote forms (1 ꢁ 107 cells/mL)
were resuspended in DMEM and added in duplicate to 96-well

Z. Ud Din et al. / Bioorg. Med. Chem. 22 (2014) 1121–1127
1127
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pounds (1.0–100.0 M). Parasites were incubated for 24 h at
l
37 °C in a 5% CO2 atmosphere. The results were obtained by
observing motility, allowing the determination of the viability of
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5.2.4. Cytotoxicity assay
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a
suspension of 2.5 ꢁ 105 cells/mL was cultured in DMEM.
After 24 h, the different compounds were added to each well
(10.0–1000.0 lM) and the plates were incubated for 48 h for mac-
rophages or 96 h for LLCMK2 cells in a 5% CO2–air mixture at 37 °C.
Following incubation, MTT assay was performed (2 mg/mL stock
solution, 50
was stopped, and the formazan crystals were solubilized by adding
DMSO (150
L/well).45 The relative amount of formazan/well pro-
lL/well). After 4 h of incubation, the MTT processing
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l
duced by viable cells was determined spectrophotometrically at
570 nm by blanking against an appropriate control. The CC50 val-
ues (50% cytotoxic concentration) were estimated and the selectiv-
ity index (SI) was used to compare cytotoxicity between cells and
protozoa (ratio: CC50 of cells divided by CC50 of the compound in
the protozoa).
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Acknowledgments
The authors are grateful to Fundação de Amparo à Pesquisa do
Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvi-
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support and Third World Academy of Science (TWAS) for Ph.D. fel-
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nior (Espírito Santo Federal University, UFES, Brazil) for Gaussian
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