Design and synthesis of new (E)-cinnamic N-acylhydrazones as potent antitrypanosomal agents

Article abstract of DOI:10.1016/j.ejmech.2012.05.041

We report herein the synthesis and trypanocidal profile of new (E)-cinnamic N-acylhydrazones (NAHs) designed by exploiting molecular hybridization between the potent cruzain inhibitors (E)-1-(benzo[d][1,3]dioxol-5-yl)-3-(4-bromophenyl) prop-2-en-1-one and (E)-3-hydroxy-N'-((2-hydroxynaphthalen-1-yl)methylene)-7- methoxy-2-naphthohydrazide. These derivatives were evaluated against both amastigote and trypomastigote forms of Trypanosoma cruzi and lead us to identify two compounds that were approximately two times more active than the reference drug, benznidazole, and with good cytotoxic index. Although designed as cruzain inhibitors, the weak potency displayed by the best cinnamyl NAH derivatives indicated that another mechanism of action was likely responsible for their trypanocide action.

Full text of DOI:10.1016/j.ejmech.2012.05.041

European Journal of Medicinal Chemistry 54 (2012) 512e521
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design and synthesis of new (E)-cinnamic N-acylhydrazones as potent
antitrypanosomal agents
,
Samir A. Carvalho a b, Larisse O. Feitosa a, Márcio Soares a, Thadeu E.M.M. Costa a, Maria G. Henriques a,
Kelly Salomão c, Solange L. de Castro c, Marcel Kaiser d, Reto Brun d, James L. Wardell e,
Solange M.S.V. Wardell f, Gustavo H.G. Trossini g, Adriano D. Andricopulo h, Edson F. da Silva a,
,i,
*
Carlos A.M. Fraga b
a Instituto de Tecnologia em Fármacos e Farmanguinhos, Fundação Oswaldo Cruz, 21041-250 Rio de Janeiro, RJ, Brazil
b Programa de Pós-Graduação em Química, Instituto de Química, Universidade Federal do Rio de Janeiro, 21949-900 Rio de Janeiro, RJ, Brazil
c Laboratório de Biologia Celular, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, 21040-900, Rio de Janeiro, RJ, Brazil
d Medical Parasitology and Infection Biology, Swiss Tropical Institute, CH-4002 Basel, Switzerland
e Centro de Desenvolvimento Tecnológico em Saúde (CDTS), Fundação Oswaldo Cruz, 21040-900 Rio de Janeiro, RJ, Brazil
f CHEMSOL, 1 Harcourt Road, Aberdeen AB15 5NY, Scotland, UK
g Departamento de Farmácia, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, 05508-000 São Paulo, Brazil
h Laboratório de Química Medicinal e Computacional, Centro de Biotecnologia Molecular Estrutural, Instituto de Física de São Carlos, Universidade de São Paulo,
13560-590 São Carlos, Brazil
i Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, PO Box 68023, 21941-902 Rio de Janeiro, RJ, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
We report herein the synthesis and trypanocidal profile of new (E)-cinnamic N-acylhydrazones (NAHs)
designed by exploiting molecular hybridization between the potent cruzain inhibitors (E)-1-(benzo[d]
[1,3]dioxol-5-yl)-3-(4-bromophenyl)prop-2-en-1-one and (E)-3-hydroxy-N0-((2-hydroxynaphthalen-1-
yl)methylene)-7-methoxy-2-naphthohydrazide. These derivatives were evaluated against both amasti-
gote and trypomastigote forms of Trypanosoma cruzi and lead us to identify two compounds that were
approximately two times more active than the reference drug, benznidazole, and with good cytotoxic
index. Although designed as cruzain inhibitors, the weak potency displayed by the best cinnamyl NAH
derivatives indicated that another mechanism of action was likely responsible for their trypanocide
action.
Received 23 February 2012
Received in revised form
18 May 2012
Accepted 30 May 2012
Available online 7 June 2012
Keywords:
N-Acylhydrazone
Cinnamic acid
Chagas’ disease
Trypanosoma cruzi
Cruzain inhibitors
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
million people in Latin America, of whom 30e40% either have
or will develop cardiomyopathy, digestive megasyndromes, or
Chagas’ disease (CD) is caused by the intracellular obligatory
parasite Trypanosoma cruzi. CD is the major cause of infectious
cardiopathy and represents an important public health
problem. It is broadly dispersed in 18 developing countries in
South and Central America [1] and affects approximately eight
both [2]. Recently, CD has become a major concern due to
globalization, which results in immigration of infected indi-
viduals to non-endemic regions, thus spreading the disease [3].
Nifurtimox and benznidazole (Bzn) were empirically introduced
into the clinical therapy regime for CD over four decades ago.
Abbreviations: AMC, 7-amino-4-methylcoumarin; Bzn, benznidazole; CD, Chagas’ disease; DMES, Dulbecco’s modified Eagle medium; DMSO, dimethylsulfoxide; EDC, 1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide; ESI, electrospray ionization; FBS, fetal bovine serum; HOBt, N-hydroxybenzotriazole; HPLC, high pressure liquid chroma-
tography; IR, infrared; MS, mass spectra; MTT(4,5-dimethylthiazol-2yl)-2,5-dimethyl tetrazolium bromide, 3-; NAH, N-acylhydrazone; NMR, nuclear magnetic resonance;
ppm, parts per million; SI, selectivity index; TLC, thin-layer chromatography.
* Corresponding author. Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, PO Box 68023, 21941-902 Rio de Janeiro, RJ, Brazil. Tel.: þ55 21 2562
6503; fax: þ55 21 2562 6478.
E-mail address: cmfraga@ccsdecania.ufrj.br (C.A.M. Fraga).
URL: http://www.farmacia.ufrj.br/lassbio
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.05.041

S.A. Carvalho et al. / European Journal of Medicinal Chemistry 54 (2012) 512e521
513
Neither drug is ideal because they present variable results
depending on the phase of the disease (they are only effective
in the acute and recent chronic phases of the infection), the
dose and duration of the treatment, and patient age and
endemic region, as well as showing undesirable secondary side
effects [4].
T. cruzi contains a cathepsin L-like cysteine protease termed
cruzain (cruzipain), responsible for the major proteolytic activity of
all life-cycle stages of this parasite [5]. Selective inhibitors of this
protease cure T. cruzi infection in animal models using both mice [6]
and dogs [7], making this enzyme a prime target for potential
trypanocidal drugs [8].
The privileged status of the N-acylhydrazone framework [9]
has been widely exploited in the design of new bioactive
compounds with different pharmacological profiles [10], and the
pharmacophoric character of this group has been identified
during the development of some cysteinyl protease inhibitors
[11e13]. Moreover, chalcones [(2E)-1,3-diphenylprop-2-en-1-
one] share some similarity with the four-atom acylhydrazone
linker and modeling studies suggest that they could occupy
similar positions and adopt similar binding modes in the active
site of the malarial falcipain-2 [14], a related cysteine protease
from Plasmodium falciparum [15] that has more than 90%
homology with cruzain at the binding site residues [16,17]. In
addition, the unsaturated aryl ketone subunit of chalcones can
act as good Michael acceptors, which would result in the irre-
versible inhibition of cruzain through the formation of a covalent
adduct with the nucleophilic amino acid cysteine in the active
site. Although the anti-T. cruzi activity of chalcones is well
described [18], there are few studies associated with cruzain
inhibition assays [11,18].
Fig. 1. Design of cinnamic N-acylhydrazone derivatives (3e26) and their
conformationally-modified analogs (27e30). See Table 1 for the nature of substituents
R1eR7.
2. Results and discussion
2.1. Chemistry
The planned synthetic route exploited to achieve the new NAH
derivatives (3e30) are depicted in Schemes 1 and 2. (E)-Cinnamic
acids (31aed) were employed as starting materials. Non-
commercially available (E)-cinnamic acids (31e and 31f) were
obtained in 75% and 70% yield, respectively, through the
KnoevenageleDoebner reaction of the corresponding aromatic
aldehyde (32a and 32b) with malonic acid in basic media [20,21].
The preparation of the cinnamic N-acylhydrazide derivatives
involved the use of general methodology developed by Zhang and
coworkers [22]. This methodology involved the pre-formation of
esters and/or activated amides, followed by nucleophilic substitu-
tion of these intermediates with hydrazine hydrate. Through the
use of HOBt/EDC as coupling reagent, N-acylhydrazides (33ae33f)
were obtained in yields ranging from 83 to 92% (Scheme 1). Acyl-
hydrazide derivatives 33g and 33h were respectively prepared from
3-phenylpropionic acid (34) and 2-naphtoic acid (35) by Fischer
esterification and hydrazinolysis of the respective ester intermedi-
ates (Scheme 2) [23]. The target NAH derivatives (3e30) were
obtained in good yields (Table 1) by acid catalyzed condensation of
the hydrazines (33ae33h) with the corresponding aromatic alde-
hydes (ArCHO) in ethanol [23], as described in Schemes 1 and 2.
1H NMR spectra and HPLC chromatograms of the synthesized
NAH derivatives (3e30) were consistent with the presence of only
one geometric isomeratthe iminebondlevel, which X-ray diffraction
data indicated as having the relative configuration (E). The molecular
structure of (2E)-N-[(E)-4-chlorobenzylidene]-3-phenylprop-2-
enohydrazide (5) in its mono-hydrate [24] was very similar to that
of the bromo analog (6) reported here (Fig. 2). As described previ-
ously for other NAH derivatives [25], cinnamyl N-acylhydrazones
(3e30) exist in equilibrium between the two stable conformers sin-
periplanar (sp) and antiperiplanar (ap) in solution. These conformers
are able to interconvert through rotation of the amide bond.
Regarding the chemical shifts of these conformers, it is believed that
the signal from the imine hydrogen being more downshield matches
the ap conformer, as described by Palla and colleagues [26].
In our effort to develop potent trypanocidal compounds, we
constructed a new class of cinnamic N-acylhydrazone (NAH)
derivatives designed by molecular hybridization between two
classes of cruzain inhibitors represented by the chalcone derivative
(1) (IC50 ¼ 20
mM) [19] and the naphthyl NAH derivative (2)
(IC50 ¼ 0.6 M) [11]. This class aimed to enhance the inhibitory
m
profile against the targeted enzyme by offering two electrophilic
sites (A and B) capable of binding with the nucleophilic cysteine
residue of cruzain (Fig. 1).
Both intracellular amastigotes and circulating trypomasti-
gotes forms of T. cruzi are relevant in drug development assays,
since they are present in the vertebrate host during the acute
and chronic phases of Chagas’ disease. For this reason, we first
synthesized and evaluated the activity of the cinnamyl N-acyl-
hydrazones (3e17), presenting substituents with different ster-
eoelectronic properties only in the phenyl subunit D (Fig. 1),
against amastigote and trypomastigote forms of T. cruzi. Next,
aiming to enhance the trypanocidal activity observed in the
screening of NAH derivatives 3e17, we proposed structural
changes in the phenyl ring C of two potent compounds (Fig. 1).
For each new substituent on the phenyl subunit C, two new
cinnamic NAH derivatives were synthesized (18e25) (Table 1). In
addition, NAH derivative 26, the retroisostere of 17, was planned
to investigate how the exchange of substituents between the
two phenyl subunits (C) and (D) influenced the trypanocidal
activity (Table 1). Unsaturated derivatives (27e28) and the
naphthyl analogs (29e30) were structurally designed to inves-
tigate how the degree of conformational freedom and the loss of
the pharmacophoric vinyl amide subunit would influence the
trypanocidal activity (Table 1). Then, new cinnamic NAH deriv-
atives (18e30) were evaluated in vitro against the bloodstream
trypomastigote form of the parasite. The six most active
compounds and Bzn were evaluated for toxicity to mammalian
cells.

514
S.A. Carvalho et al. / European Journal of Medicinal Chemistry 54 (2012) 512e521
Table 1
Physical and spectral properties, in vitro trypanocidal activity, cytotoxicity and inhibition of cruzain of the cinnamic and structurally-related N-acylhydrazone derivatives
(3e30).
SIc
b
b
b
b
R1
R2
R3
R4
R5
R6
R7
Yield (%) Mp (ꢀC) Conformational IC50
IC50
IC50
IC50
ratio (ap/sp0)a
amastigote tripomastigote cytotoxicity
cruzain
3
4
5
6
7
8
9
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
H
H
H
H
H
H
H
OH
H
H
H
H
H
H
H
H
H
OH
H
F
Cl
Br
NO2
OCH3
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
93
83
81
80
94
76
80
85
80
90
89
87
88
229
190
211
220
233
219
290
240
229
180
187
193
219
185
240
221
255
222
228
206
253
193
265
216
151
197
209
197
e
1:0.9
1:0.6
1:0.9
1:0.9
1:1
>100
>100
>100
ND
>1100
>1100
>1100
>1100
>1100
>1100
>1100
5.9 ꢁ 1.2
107.0 ꢁ 21.0
>1100
>1100
>1100
127.0 ꢁ 10.0
71.0 ꢁ 14.0
>1100
ND
ND
ND
ND
ND
ND
ND
81
1:0.8
1:0.9
1:0.4
1:0.3
1:0.9
1:0.6
1:0.4
1:0.4
1:0.6
1:0.6
1:0.4
1:0.7
ND
1:0.8
1:0.3
1:0.8
1:0.3
1:0.8
1:0.8
1:0.6
1:1
>100
>100
6.6
>100
61
>100
>100
73
70
40
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Bzn
420.2 ꢁ 20.1
52.2 ꢁ 4.0
71
OH
ND
OeCH2eO
OCH3 OCH3
OH
OCH3 OH
OCH3 OCH3 OCH3 91
OCH3 OH
H
OCH3 OH
H
OCH3 OH
H
OCH3 OH
H
OCH3 OH
H
H
ND
ND
ND
ND
ND
ND
OCH3
NO2
H
NO2
H
NO2
H
NO2
H
NO2
H
H
NO2
H
NO2
e
90
85
83
82
83
87
87
90
85
81
85
88
89
88
e
OeCH2eO
OeCH2eO
OCH3 OCH3 OCH3 OH
OCH3 OCH3 OCH3
NO2
NO2
OCH3 OCH3
OCH3 OCH3
NO2
H
H
H
H
H
5.6 ꢁ 1.4
281.3 ꢁ 14.8
74.8 ꢁ 11.0
50
>1100
ND
H
78.8 ꢁ 13.0
18.0 ꢁ 4.0
17.0 ꢁ 0.4
16.0 ꢁ 2.0
65.0 ꢁ 15.0
>1100
79.0 ꢁ 13.0
18.0 ꢁ 1.4
>1100
ND
H
2413.0 ꢁ 100
45.9 ꢁ 6.0
83.9 ꢁ 6.0
44.7 ꢁ 3.0
134
32
116
OCH3 OCH3 OH
OCH3 OCH3
H
550.2 ꢁ 17.8
H
OH
H
H
OH
H
1865.0 ꢁ 80.1
H
H
OCH3
H
H
H
ND
ND
ND
OH
H
H
H
H
H
H
1630 ꢁ 77.0
>100
90
OCH3 OH
H
OCH3 OH
e
ND
ND
ND
0.122
ND
H
H
OH
H
H
1:1
1:0.9
e
93.0 ꢁ 12.0
93.0 ꢁ 20.0
10.8 ꢁ 0.4
ND
ND
e
e
e
e
e
3840.7 ꢁ 110.2
356
a
Relative ratio of amide bond conformers identified from the relative integration of signals related to the NeNH subunit of the NAH group.
b
c
IC50
(mM).
SI ¼ Cytotoxicity IC50/Trypanocide IC50 (trypomastigote forms of T. cruzi); ND ¼ Not determined.
Scheme 1. Reagents and conditions: a) malonic acid, pyridine, piperidinecat., reflux, 1 h; b) (1) HOBt, EDC, CH3CN, rt, 2 h; (3) inverse addition to hydrazine, CH3CN, cyclohexane,
0e10 ꢀC; c) ArCHO, EtOH, HClcat., reflux.

S.A. Carvalho et al. / European Journal of Medicinal Chemistry 54 (2012) 512e521
515
Comparing the trypanocidal activity of the o-hydroxyphenyl
and 5-NO2evanillinyl containing derivatives, it was observed that
three of the five most active (10, 18 and 22) contained an o-hydroxy
substituted phenyl group. Moreover, when comparing the activity
of each pair alone (10 and 17, 18 and 19, 20 and 21, 22 and 23 and 24
and 25), only for compound 20 did the presence of
a 5-
NO2evanillinyl unit result in superior trypanocidal activity. For
the pair of NAH derivatives 22 and 23, the trypanocidal activity was
equal (Table 1). Furthermore, the retro-isosteric replacement
between the substituents attached to the phenyl subunits C and D
improved activity against trypomastigotes for 26 (IC50 ¼ 78.8
more than the corresponding analog 17 (IC50 > 150 M) (Table 1).
The reduction of the cinnamic double bond of compound 10
(IC50 ¼ 5.9 M) resulted in a remarkable decrease in the trypano-
cidal activity, as evidenced by the corresponding saturated analog
27 (IC50 ¼ 18.0 M). This profile could be related to the augmented
mM)
m
Scheme 2. Reagents and conditions: a) PdCl2, HCO2H, H2O, NaOH, 65 ꢀC, 90%; b) EtOH,
H2SO4, reflux, 2 h; c) NH2NH2 (aq. 80%), reflux, 3 h (33g, 90% yield, 2 steps)/(33h, 85%
yield, 2 steps); d) ArCHO, EtOH, HClcat., reflux.
m
m
conformational freedom acquired by 27, allowing it to adopt
different conformations from those of the most active NAH deriv-
ative 10 and decreasing the antiprotozoal activity. On the other
hand, a more pronounced decline was observed in comparison of
the trypanocidal activity of 10 with a more restricted conforma-
tional analog 29. It is possible that reducing the degrees of confor-
mational freedom of the double bond linked to the aromatic ring (C)
prevented 29 from adopting the bioactive conformation of 10.
Next, we evaluated the inhibitory effects of the six most active
compounds against the enzyme cruzain from T. cruzi (Table 1)
by monitoring the liberation of the fluorescent leaving
group 7-amino-4-methylcoumarin from the peptide substrate
ZePheeArgeAMC [14,19]. The results demonstrated that these
compounds (Table 1) did not substantially inhibit this enzyme,
indicating that another mechanism of action must be involved in
their trypanocidal activity. Derivatives 10, 21 and 23 had the best
2.2. Trypanocide activity
The in vitro screening of the cinnamyl NAH derivatives (3e17)
against intracellular amastigote forms of T. cruzi showed that
compound 10, with an o-hydroxyphenyl group attached to the
imine unit, was the most active (IC50 ¼ 6.6
mM). This superior try-
panocidal profile in comparison to the other derivatives could be
explained by the ability of the o-hydroxybenzylidene N-acylhy-
drazone framework to form an electrophilic quinone methide
intermediate that could interact with nucleophilic sites in target
enzymes of T. cruzi, e.g. cysteine proteases [13].
The trypanocidal activity of the other derivatives varied in ring C
was not as pronounced as for compound 10. The comparison
between the potencies of 5-NO2evanillinyl (17) and vanillinyl (15)
NAH derivatives (IC50 ¼ 40 and 73
mM, respectively) (Table 1)
revealed that the presence of the NO2 group significantly increased
the trypanocidal activity, suggesting that this group may interfere
with the redox system of the parasite. Piperonyl (12) and 3,4,5-
trimethoxyphenyl (16) derivatives had only modest trypanocidal
inhibitory profile with IC50 values ranging from 44.7 to 52.2 mM. On
the other hand, the cruzain inhibitory activity of unsaturated
derivative 27 could not be determined accurately due to solubility
limitations that decreased the sensitivity of the assay.
activity, with IC50 values of 61 mM and 70 mM, respectively (Table 1).
The six most active derivatives (IC50 ꢂ 20
mM) and Bzn had their
From these initial results, we proposed novel cinnamyl NAH
derivatives (18e25) with structural changes in the phenyl subunit
(C) (Fig. 1). For all of the new substituents introduced on the
aromatic ring (C), two novel derivatives led to the highest trypa-
nocidal activity in the first series: 2-hydroxyphenyl and 5-
NO2evanillinyl.
activity against mammalian cells measured using the macrophage
lineage J774 and the selectivity index (SI) (LC50 for cytotoxicity
divided by IC50 for trypanocidal activity) was calculated. In this
context, the five compounds with SI ꢃ50 (i.e., 10, 18, 21, 23 and 27)
were considered as good candidates for subsequent studies on
trypanocidal activity in a murine model [27,28].
Next, all NAH derivatives, including 3e17, had their trypanocidal
activity evaluated on bloodstream trypomastigotes (Table 1). In
general, the presence of o-hydroxyphenyl provided better activity,
corroborating the results obtained in the first series of assays on
amastigotes. The derivatives that presented the most pronounced
trypanocidal activity were 10 and 18, with IC50 values of 5.6 and
3. Conclusions
We have discovered five new potent trans-cinnamic N-acylhy-
drazones compounds with low cytotoxicity and excellent trypa-
nocidal activity. Two of them (10 and 18) were two-fold more
potent than the reference drug Bzn against trypomastigotes of
T. cruzi. Although designed as cruzain inhibitors, none of the most
active NAH derivatives were potent inhibitors of this enzyme,
suggesting that a more complex mechanism of action is related to
the displayed trypanocidal activity.
5.9 mM, respectively (Table 1). The structural modifications intro-
duced in the phenyl subunit C confirmed the pharmacophoric
sketch of the introduced substituents [13,19], since most of the
derivatives 18e25 were equally or more active than their unsub-
stituted counterparts (Table 1).
4. Experimental section
4.1. General procedures
Melting points were determined on a Buchi apparatus and are
uncorrected. Infrared spectra were recorded on a Thermo Nicolet
Nexus 670 spectrometer in potassium bromide pellets and
frequencies are expressed in cmꢄ1 1H NMR and 13C NMR spectra
.
were recorded, at room temperature, on a Bruker Avance 500,
Bruker Avance 400, Bruker DRX-300 and Bruker DRX-200
Fig. 2. Atom arrangements for (E)-N0-(4-bromobenzylidene)cinnamohydrazide (6).

516
S.A. Carvalho et al. / European Journal of Medicinal Chemistry 54 (2012) 512e521
spectrometers operating at 500, 400, 300 and 200 MHz (1H)/125,
100, 75, 50 MHz (13C), respectively. Chemical shifts are reported in
ppm (d) downfield from tetramethylsilane used as internal stan-
added. The reaction medium was extracted with ethyl acetate
(3 ꢅ 30 mL) and aqueous sodium bicarbonate (1 ꢅ 50 mL). The
organic phases were joined and the solvent was evaporated under
reduced pressure to furnish the respective N-acylhydrazines
(33ae33f), as described next.
dard. Low resolution mass spectra (MS) were obtained by electron-
spray ionization in an LC/MS micromass ZQ 4000. Microanalysis
data were obtained using a PerkineElmer 240 analyzer, using
a PerkineElmer AD-4 balance.
The progress of all reactions was monitored by TLC, which was
performed on 2.0 ꢅ 6.0 cm aluminum sheets precoated with
silica gel 60 (HF-254, Merck) to a thickness of 0.25 mm. The
developed chromatograms were viewed under ultraviolet light
(254e265 nm).
4.3.1. (2E)-3-Phenylacrylohydrazide (33a)
90% yield; beige solid, mp 116e117 ꢀC (Lit. [30] mp 116e117 ꢀC);
1H NMR (400 MHz, DMSO-d6)
d 4.48 (s, 2H, eCONHeNH2), 6.55 (d,
J ¼ 16.0 Hz,1H, CH]CHeCOOH), 7.33e7.46 (m, 4H, CH]CHeCOOH,
Ar(C3)H, Ar(C4)H and Ar(C5)H), 7.53e7.55 (d, 2H, Ar(C2)H and Ar(C6)
H), 9.37 (s, 1H, eCONHeNH2) ppm: 13C NMR (100 MHz, DMSO-d6)
d
120.28 (CH]CHeCOOH), 127.43 (Ar(C2)H and Ar(C6)H), 128.91
4.2. General procedure for the synthesis of (E)-cinnamic acids (31e
and 31f)
(Ar(C3)H and Ar(C5)H), 129.38 (Ar(C4)H), 134.92 (Ar(C1)), 138.16
(CH]CHeCOOH) and 164.47 (CH]CHeCOOH) ppm; IR (KBr) nmax
cmꢄ1: 3292 and 3026 (
n NeH); 1664 (n C]O); MS (ESI) m/z: 185.2
The corresponding benzaldehyde derivative 32a or 32b
(33 mmol) and malonic acid (72 mmol) were dissolved in a mixture
of 15 mL of pyridine and 0.25 mL of piperidine and heated under
reflux until TLC analysis indicated the end of the reaction. The
reaction medium was cooled, neutralized with HCl (10% aq.),
filtered in Buchner funnel, washed with water and dried, providing
the appropriate (E)-cinnamic acids 31e and 31f as described next.
(Mþ.[þNa]) (100%). Anal. Calcd for C9H10N2O: C: 66.65; H: 6.21; N:
17.27. Found: C: 66.58; H: 6.20; N: 17.24.
4.3.2. (2E)-3-(1,3-Benzodioxol-5-yl)acrylohydrazide (33b)
92% yield; beige solid, mp 151e152 ꢀC (Lit. [31] mp 151e152 ꢀC);
1H NMR (400 MHz, DMSO-d6)
d 6.05 (s, 2H, OeCH2eO), 6.39 (d,
J ¼ 16.0 Hz, 1H, CH]CHeCOOH), 6.93 (d, J ¼ 8.0 Hz, 1H, Ar(C5)H),
7.12 (dd, J ¼ 8.0 Hz, J ¼ 1.4 Hz, 1H, Ar(C6)H), 7.33 (d, J ¼ 1.4 Hz, 1H,
Ar(C2)H), 7.45 (d, J ¼ 16.0 Hz, 1H, CH]CHeCOOH) ppm: 13C NMR
4.2.1. (2E)-3-(3,4-Dimethoxy-5-nitrophenyl)acrylic acid (31e)
87% yield; orange solid, mp 235 ꢀC; 1H NMR (400 MHz, DMSO-
(100 MHz, CDCl3) d 101.24 (OeCH2eO), 106.03 (Ar(C2)H), 108.29
d6)
d
3.88 (s, 3H, Ar(C3)OCH3), 3.94 (s, 3H, Ar(C4)OCH3), 6.61 (d, 1H,
(Ar(C5)H), 115.36 (Ar(C6)H), 123.85 (CH]CHeCOOH), 128.63
(Ar(C1)), 141.37 (CH]CHeCOOH), 148.00 (Ar(C4)), 149.03 (Ar(C3))
and 167.00 (CH]CHeCOOH) ppm: IR (KBr) nmax cmꢄ1: 3050 and
J ¼ 16.0 Hz, CH]CHeCOOH), 7.45 (d, 1H, J ¼ 16.0 Hz, CH]
CHeCOOH), 7.57 (d, 1H, J ¼ 2.0 Hz, Ar(C2)H) and 7.62 (d, 1H,
J ¼ 2.0 Hz, Ar(C6)H) ppm; 13C NMR (100 MHz, DMSO-d6)
d
56.83
3100 (
n
NeH), 1664 (
n
C]O); MS (ESI) m/z: 229.0 (Mþ.[þNa])
(Ar(C3)OCH3), 61.71 (Ar(C4)OCH3), 115.37 (Ar(C2)H), 115.56 (Ar(C6)
H), 121.21 (CH]CHeCOOH), 130.76 (Ar(C1)), 141.57 (CH]
CHeCOOH), 142.35 (Ar(C5)NO2), 144.74 (Ar(C4)OCH3), 153.60
(Ar(C3)OCH3) and 167.32 (CH]CHeCOOH) ppm; IR (KBr)
(100%). Anal. Calcd for C10H10N2O3: C: 58.25; H: 4.89; N: 13.59.
Found: C: 58.14; H: 4.88; N: 13.54.
4.3.3. (2E)-3-(3,4,5-Trimethoxyphenyl)acrylohydrazide (33c)
85% yield; white solid, mp 153e154 ꢀC (Lit. [30] mp 153e154 ꢀC);
nmax cmꢄ1: 3383e3049 (
n
OeH), 1654 (n C]O), 1608 (n N]O), 1276
(n
CeOeC); MS (ESI) m/z: 253.1, 252.1 (100%). Anal. Calcd
1H NMR (400 MHz, DMSO-d6)
d 3.67 (s, 3H, Ar(C4)OCH3), 3.80 (s, 6H,
for C11H11NO6: C: 52.18; H: 4.79; N: 5.53. Found: C: 51.99; H: 4.78;
N: 5.54.
Ar(C3)OCH3 and Ar(C5)OCH3), 4.41 (s, 2H, eCONHeNH2), 6.48 (d,
J ¼ 16.0 Hz, 1H, CH]CHeCOOH), 6.87 (s, 2H, Ar(C2)H and Ar(C6)H),
7.38 (d, J ¼ 16.0 Hz, 1H, CH]CHeCOOH), 9.22 (s, 1H, eCONHeNH2)
4.2.2. (2E)-3-(4-Hydroxy-3-methoxy-5-nitrophenyl)acrylic acid
(31f)
ppm: 13C NMR (100 MHz, DMSO-d6)
d 55.87 (Ar(C3)OCH3 and Ar(C5)
OCH3), 60.07 (Ar(C4)OCH3), 104.88 (Ar(C2)H and Ar(C6)H), 119.53
(CH]CHeCOOH), 130.53 (Ar(C1)), 138.34 (CH]CHeCOOH), 138.60
(Ar(C4)), 153.06 (Ar(C3) and Ar(C5)), 164.58 (CH]CHeCOOH) ppm:
90% yield; yellow solid, mp 249 ꢀC (Lit. [29] mp 249 ꢀC); 1H NMR
(500 MHz, DMSO-d6)
d 3.95 (s, 3H, Ar(C3)OCH3), 6.61 (d, 1H,
J ¼ 16.0 Hz, CH]CHeCOOH), 7.56 (d, 1H, J ¼ 16.0 Hz, CH]
IR (KBr) nmax cmꢄ1: 3317 and 3240 (
n NeH), 1579 (n C]O), 1247 (n
CHeCOOH), 7.64 (s, 1H, Ar(C2)H), 7.77 (s, 1H, Ar(C6)H) ppm; 13C
CeOeC): MS (ESI) m/z: 251.2 (100%). Anal. Calcd for C12H16N2O4: C:
NMR (125 MHz, DMSO-d6)
d
56.86 (Ar(C3)OCH3), 113.88 (Ar(C2)H),
57.13; H: 6.39; N: 11.10. Found: C: 56.99; H: 6.40; N: 11.13.
117.19 (Ar(C6)H), 119.07 (CH]CHeCOOH), 125.15 (Ar(C1)), 137.33
(Ar(C5)NO2), 142.32 (CH]CHeCOOH), 143.83 (Ar(C4)OeH), 149.69
4.3.4. (2E)-3-(3,4-Dimethoxyphenyl)acrylohydrazide (33d)
(Ar(C3)OCH3), 167.58 (CH]CHeCOOH) ppm; IR (KBr) nmax cmꢄ1
:
90% yield; white solid, mp 217e218 ꢀC (Lit. [31] mp
3050 and 3100 (
n
OeH), 1664 (
n
C]O); MS (ESI) m/z: 241.9 (100%).
217e217.5 ꢀC); 1H NMR (400 MHz, DMSO-d6)
d 3.76 and 3.78 (s, 6H,
Anal. Calcd for C10H9NO6: C: 50.22; H: 3.79; N: 5.86. Found: C:
50.29; H: 3.78; N: 5.84.
Ar(C3)OCH3 and Ar(C4)OCH3), 6.42 (d, J ¼ 16.0 Hz, 1H, CH]
CHeCOOH), 6.96 (d, J ¼ 8.00 Hz, 1H, Ar(C5)H), 7.10 (dd, J ¼ 8.00,
1H, Ar(C6)H), 7.13 (d, 1H, Ar(C2)H), 7.37 (d, J ¼ 8.0 Hz, 1H, CH]
CHeCOOH), 9.23 (s, 1H, eCONHeNH2) ppm: 13C NMR (100 MHz,
4.3. General procedure for the synthesis of cinnamic acid
hydrazides (33ae33f)
DMSO-d6) d 55.43 (Ar(C4)OCH3), 55.55 (Ar(C3)OCH3), 110.11 (Ar(C5)
H), 111.76 (Ar(C2)H), 117.88 (Ar(C1)), 121.17 (CH]CHeCOOH), 127.70
(Ar(C6)H), 138.34 (CH]CHeCOOH), 148.88 (Ar(C3)OCH3), 150.06
To a suspension of the corresponding cinnamic acid (31ae31f)
(6.67 mmol, 1.0 equiv) in 15 mL CH3CN at room temperature, was
added HOBt (8 mmol,1.2 equiv) in a single portion, followed by EDC
(8 mmol, 1.2 equiv). The reaction was stirred at room temperature
for 2 h, the time required for the complete consumption of cin-
namic acid. The resulting mixture was then slowly added to
a solution of hydrazine hydrate (13.34 mmol, 2.0 equiv) in 15 mL of
CH3CN kept between 0 and 10 ꢀC. The reaction was usually
completed upon the end of addition. Then, 15 mL of water was
(Ar(C4)OCH3), 164.92 (CH]CHeCOOH) ppm: IR (KBr) nmax cmꢄ1
:
3321 and 3236 (n NeH), 1510 (n C]O) and 1263 (n CeOeC): MS
(ESI) m/z: 223.2 (100%). Anal. Calcd for C11H14N2O3: C: 59.45; H:
6.35; N: 12.60. Found: C: 59.41; H: 6.36; N: 12.57.
4.3.5. (2E)-3-(3,4-Dimethoxy-5-nitrophenyl)acrylohydrazide (33e)
83% yield; orange solid, mp 164e165 ꢀC; 1H NMR (400 MHz,
DMSO-d6) d 3.87 (s, 3H, Ar(C3)OCH3), 3.93 (s, 3H, Ar(C4)OCH3), 6.60

S.A. Carvalho et al. / European Journal of Medicinal Chemistry 54 (2012) 512e521
517
(d, J ¼ 16.0 Hz, 1H, CH]CHeCOOH), 7.45 (d, J ¼ 16.0 Hz, 1H, CH]
4.5. General procedure for the synthesis of NAH derivatives (3e30)
To solution of the corresponding hydrazide derivative
CHeCOOH), 7.57 (d, J ¼ 2.0 Hz, 1H, Ar(C2)H), 7.62 (d, J ¼ 2.0 Hz,
1H, Ar(C6)H), 9.34 (eCONHeNH2) ppm: 13C NMR (100 MHz, DMSO-
a
d6)
d
56.83 (Ar(C3)OCH3), 61.71 (Ar(C4)OCH3), 115.37 (Ar(C2)H),
(33ae33h) (0.829 mmol) in absolute ethanol (90 mL) containing
a catalytic amount of 37% aq. hydrochloric acid was added
0.87 mmol of desired aromatic aldehyde derivative. The mixture
was stirred at reflux for 60 min, when extensive precipitation was
observed. The reaction mixture was then poured into cold water,
neutralized with 10% aqueous sodium bicarbonate solution. The
precipitate formed was filtered out and dried, furnishing the target
compounds in the yields as described next.
115.56 (Ar(C6)H), 121.21 (CH]CHeCOOH), 130.76 (Ar(C1)), 141.57
(CH]CHeCOOH), 142.35 (Ar(C5)NO2), 144.74 (Ar(C4)OCH3), 153.60
(Ar(C3)OCH3) and 167.32 (CH]CHeCOOH) ppm: IR (KBr) nmax
cmꢄ1: 3383e3049 (
n
NeH), 1654 (n C]O), 1608 (n N]O) and 1276
(n CeOeC): MS (ESI) m/z: 267.2 (100%). Anal. Calcd for C11H13N3O5:
C: 49.44; H: 4.90; N: 15.72. Found: C: 49.35; H: 4.89; N: 15.68.
4.3.6. (2E)-3-(4-Hydroxy-3-methoxy-5-nitrophenyl)acrylohydrazide (33f)
85% yield; yellow solid, mp 160e161 ꢀC; 1H NMR (500 MHz,
4.5.1. (E)-N0-Benzylidenecinnamohydrazide (3)
DMSO-d6)
d
3.92 (s, 3H, Ar(C3)OCH3), 6.58 (d, 1H, J ¼ 16.0 Hz, CH]
93% yield; white solid, mp 229 ꢀC (Lit. [34] mp 225.5e227.5); 1H
CHeCOOH), 7.54 (d, 1H, J ¼ 16.0 Hz, CH]CHeCOOH), 7.62 (s, 1H,
NMR (400 MHz, DMSO-d6)
d
6.71 (d, J ¼ 16.0 Hz, 1H, CH]
Ar(C2)H), 7.74 (s, 1H, Ar(C6)H) ppm; 13C NMR (125 MHz, DMSO-d6)
CHeCOOH), 7.40e7.47 (m, 6H, Ar(C3)H, Ar(C4)H, Ar(C5)H, Ar(C3 )
0
0
0
0
0
d
56.86 (Ar(C3)OCH3), 113.88 (Ar(C2)H), 117.19 (Ar(C6)H), 119.07
H, Ar(C4 )H and Ar(C5 )H), 7.57e7.66 (m, 2H, Ar(C2 )H and Ar(C6 )H),
7.70e7.76 (m, 3H, CH]CHeCOOH, Ar(C2)H and Ar(C6)H), 8.06 and
8.24 (s, 1H, eN]CHe), 11.52 and 11.68 (s, 1H, eCONHe): 13C NMR
(CH]CHeCOOH), 125.15 (Ar(C1)), 137.33 (Ar(C5)NO2), 142.32 (CH]
CHeCOOH),143.83 (Ar(C4)OeH),149.69 (Ar(C3)OCH3),167.58 (CH]
CHeCOOH) ppm: IR (KBr) nmax cmꢄ1: 3383e3049 (
n
NeH), 1654 (
n
(100 MHz, DMSO-d6) d 117.08 and 120.21 (CH]CHeCOOH), 126.89
0
0
0
C]O), 1608 (
n
N]O) and 1276 (
n
CeOeC): MS (ESI) m/z: 252.1
and 127.09 (Ar(C3 )H) and (Ar(C5 )H), 127.73 and 128.20 (Ar(C2 )H
0
(100%). Anal. Calcd for C10H11N3O5: C: 47.43; H: 4.38; N: 16.59.
Found: C: 47.28; H: 4.38; N: 16.66.
and Ar(C6 )H), 128.79, 128.95 and 129.00 (Ar(C2)H, Ar(C3)H, Ar(C5)H
0
and Ar(C6)H), 129.78 and 129.86 (Ar(C4 )H or Ar(C4)H), 130.02
0
0
(Ar(C4)H or Ar(C4 )H),134.15 and 134.26 (Ar(C1 )),134.63 and 134.77
(Ar(C1)), 140.56 and 142.11 (CH]CHeCOOH), 143.17 and 146.69
(eN]CHe), 161.41 and 165.99 (CH]CHeCOOH) ppm: IR (KBr) nmax
4.4. General procedure for the synthesis of 2-naphthoic and 3-
phenylpropanoic acid hydrazides (33g and 33h)
cmꢄ1: 2989 and 2893 (
n NeH), 1647 (n C]O), 1570 (n C]C): MS
(ESI) m/z: 273.1 (Mþ.[þNa]) (100%). Anal. Calcd for C16H14N2O: C:
76.78; H: 5.64; N: 11.19. Found: C: 76.65; H: 5.63; N: 11.21.
To a solution of the corresponding carboxylic acid derivative
(34) or (35) (3 mmol) in 10 mL of ethanol was added a catalytic
amount of concentrated H2SO4 and the mixture was refluxed for
2 h, when analysis by TLC indicated the end of the reaction. Then,
3 mL of 80% hydrazine monohydrate was added. The reaction
mixture was maintained under reflux for 3 h, when TLC indicated
the total consumption of the ester intermediate. The media was
poured onto ice and the resulting precipitate was filtered out,
affording the corresponding acylhydrazide derivatives (33g) and
(33h) as described next.
4.5.2. (E)-N0-(4-Fluorobenzylidene)cinnamohydrazide (4)
83% yield; white solid, mp 190 ꢀC; Spectroscopic data are
described in Supplementary Material. Anal. Calcd for C16H13N2FO:
C: 71.63; H: 4.88; N: 10.44. Found: C: 71.76; H: 4.89; N: 10.41.
4.5.3. (E)-N0-(4-Chlorobenzylidene)cinnamohydrazide (5)
81% yield; white solid, mp 211 ꢀC (Lit. [35] mp 211.3 ꢀC); Spec-
troscopic data are described in Supplementary Material. Anal. Calcd
for C16H13ClN2O: C: 67.49; H: 4.60; N: 12.45. Found: C: 67.64; H:
4.61; N: 12.50.
4.4.1. 3-Phenylpropanohydrazide (33g)
90% yield; beige solid, mp 160e161 ꢀC (Lit. [32] mp 160e161 ꢀC);
4.5.4. (E)-N0-(4-Bromobenzylidene)cinnamohydrazide (6)
1H NMR (400 MHz, DMSO-d6)
d
2.30 (t,
J
¼
8.0 Hz, 2H,
80% yield; white solid, mp 220 ꢀC; Spectroscopic data are
described in Supplementary Material. Anal. Calcd for C16H13BrN2O:
C: 58.38; H: 3.98; N: 8.51. Found: C: 58.22; H: 3.97; N: 8.50.
CH2eCH2eCOOH), 2.79 (t, J ¼ 8.0 Hz, 2H, CH2eCH2eCOOH), 4.19 (s,
2H, eCONHeNH2), 7.21 (m, 5H, Ar(C2)H, Ar(C3)H, Ar(C4)H, Ar(C5)H
and Ar(C6)H), 8.95 (s, 1H, eCONHeNH2): 13C NMR (100 MHz,
DMSO-d6)
d
30.36 (CH2eCH2eCOOH), 35.24 (CH2eCH2eCOOH),
4.5.5. (E)-N0-(4-Nitrobenzylidene)cinnamohydrazide (7)
125.96 (Ar(C4)H), 128.18 (Ar(C3)H and Ar(C5)H), 128.27 (Ar(C2)H
94% yield; yellow solid, mp 233 ꢀC; Spectroscopic data are
described in Supplementary Material. Anal. Calcd for C16H13N3O3:
C: 65.08; H: 4.44; N: 14.23. Found: C: 65.23; H: 4.43; N: 14.19.
and Ar(C6)H), 140.90 (Ar(C1)) and 173.76 (CH2eCH2eCOOH) ppm:
IR (KBr) nmax cmꢄ1: 3311 and 3180 (
n NeH), 1618 (n C]O): MS (ESI)
m/z: 187.1 (Mþ.[þNa]) (100%). Anal. Calcd for C9H12N2O: C: 65.83;
H: 7.37; N: 17.06. Found: C: 65.77; H: 7.35; N: 16.98.
4.5.6. (E)-N0-(4-Methoxybenzylidene)cinnamohydrazide (8)
76% yield; beige solid, mp 219 ꢀC (Lit. [35] mp 218e219 ꢀC);
Spectroscopic data are described in Supplementary Material.
Anal. Calcd for C17H16N2O2: C: 72.84; H: 5.75; N: 9.99. Found: C:
72.99; H: 5.76; N: 9.95.
4.4.2. 2-Naphthohydrazide (33h)
85% yield; white solid, mp 153 ꢀC (Lit. [33] mp 152 ꢀC); 1H NMR
(400 MHz, DMSO-d6)
d 4.56 (s, 2H, eCONHeNH2), 7.58 (m, 2H,
Ar(C5)H and Ar(C7)H), 7.95 (m, 4H, Ar(C6)H, Ar(C8)H, Ar(C3)H and
Ar(C4)H), 8.42 (s, 1H, Ar(C1)H), 9.91 (s, 1H, eCONHeNH2): 13C NMR
4.5.7. (E)-N0-(4-Hydroxybenzylidene)cinnamohydrazide (9)
(100 MHz, DMSO-d6)
d
123.82 (Ar(C3)H), 126.64 (Ar(C7)H), 127.24
80% yield; yellow solid, mp 290 ꢀC (Lit. [35] mp 290e291 ꢀC); Spec-
troscopic data are described in Supplementary Material. Anal. Calcd for
C16H14N2O2:C:72.16;H:5.30;N:10.52.Found:C:72.08;H:5.31;N:10.49.
(Ar(C5)H), 127.47 (Ar(C2)CONHeNH2), 127.56 (Ar(C4)H), 127.83
(Ar(C6)H), 128.78 (Ar(C8)H), 130.63 (Ar(C1)H), 132.14 (Ar(C9)),
134.03 (Ar(C10)) and 165.88 (Ar(C2)CONHeNH2) ppm: IR (KBr) nmax
cmꢄ1: 3311 and 3180 (
n
NeH), 1618 (
n
C]O): MS (ESI) m/z: 209.0
4.5.8. (E)-N0-(2-Hydroxybenzylidene)cinnamohydrazide (10)
(Mþ.[þNa]) (100%). Anal. Calcd for C11H10N2O: C: 70.95; H: 5.41; N:
85% yield; yellow solid, mp 240 ꢀC (Lit. [35] mp 240 ꢀC); 1H NMR
15.04. Found: C: 70.74; H: 5.40; N: 15.00.
(500 MHz, DMSO-d6)
d
6.71 (d, J ¼ 16.0 Hz, 1H, CH]CHeCOOH),

518
S.A. Carvalho et al. / European Journal of Medicinal Chemistry 54 (2012) 512e521
0
0
0
0
0
6.88e6.95 (m, 2H, Ar(C3 )H and Ar(C5 )H), 7.31 (m, 1H, Ar(C4 )H),
7.41e7.48 (m, 3H, Ar(C3)H, Ar(C4)H and Ar(C5)H), 7.55e7.58 (m, 1H,
116.36 (Ar(C3 )H), 118.75 and 120.31 (Ar(C1 )), 119.33 and 119.46
0
(Ar(C5 )H), 123.91 and 124.27 (Ar(C6)H), 126.50 and 128.97 (Ar(C1)),
0
0
0
Ar(C6 )H), 7.65e7.70 (m, 3H, CH]CHeCOOH, Ar(C2)H and Ar(C6)H),
129.30 and 129.37 (Ar(C6 )H), 130.95 and 131.31 (Ar(C4 )H), 140.83
0
8.39 and 8.46 (s, 1H, eN]CHe), 10.10 and 11.18 (s, 1H, Ar(C2 )OeH),
and 141.91 (CH]CHeCOOH), 146.82 and 148.03 (Ar(C4)), 146.85
11.50 and 11.95 (s, 1H, eCONHe): 13C NMR (125 MHz, DMSO-d6)
116.10 and 116.39 (Ar(C3 )H), 117.22 and 118.77 (CH]CHeCOOH),
and 148.95 (Ar(C3)), 156.31 and 157.34 (eN]CHe), 161.47 (Ar(C2 )),
0
d
166.02 (CH]CHeCOOH) ppm: IR (KBr) nmax cmꢄ1: 3045 (
n
OeH),
0
0
0
119.37, 119.49, 119.70 and 120.30 (Ar(C1 ) and Ar(C5 )H), 127.86 and
128.17 (Ar(C2)H and Ar(C6)H), 129.01 and 129.07 (Ar(C3)H and Ar(C5)
2900 (n NeH), 1675 (n C]O), 1500 (n C]C), 1261 (n CeO): MS
(ESI) m/z: 309.1 (100%). Anal. Calcd for C17H14N2O4: C: 65.80; H:
0
0
H), 129.33 (Ar(C4)H or Ar(C4 )H), 130.03 (Ar(C4)H or Ar(C4 )H), 131.06
4.55; N: 9.03. Found: C: 65.64; H: 4.56; N: 9.06.
0
and 131.42 (Ar(C6 )H), 134.56 and 134.83 (Ar(C1)), 140.56 and 140.96
(CH]CHeCOOH), 142.00 and 146.99 (eN]CHe), 156.38 and 157.36
4.5.17. (2E)-3-(1,3-Benzodioxol-5-yl)-N0-[(1E)-4-hydroxy-3-
methoxy-5-nitrobenzylidene] acrylohydrazide (19)
83% yield; yellow solid, mp 255 ꢀC; Spectroscopic data
are described in Supplementary Material. Anal. Calcd for
C18H15N3O7: C: 56.11; H: 3.92; N: 10.91. Found: C: 55.98; H: 3.93;
N: 10.87.
(Ar(C2 )),161.23 and 165.80 (CH]CHeCOOH). IR (KBr) nmax cmꢄ1: 3251
0
(n OeH), 3059 (n NeH), 1651 (n C]O), 1546 (n C]C),1268 (n CeO): MS
(ESI) m/z: 289.2 (Mþ.[þNa]) (100%). Anal. Calcd for C16H14N2O2: C:
72.16; H: 5.30; N: 10.52. Found: C: 72.01; H: 5.31; N: 10.49.
4.5.9. (E)-N0-(3,4-Dihydroxybenzylidene)cinnamohydrazide (11)
80% yield; yellow solid, mp 229 ꢀC; Spectroscopic data are
described in Supplementary Material. Anal. Calcd for C16H14N2O3:
C: 68.07; H: 5.00; N: 9.92. Found: C: 68.28; H: 4.99; N: 9.89.
4.5.18. (2E)-N0-[(1E)-2-Hydroxybenzylidene]-3-(3,4,5-trimethoxyphenyl)
acrylohydrazide (20)
82% yield; white solid, mp 222 ꢀC; Spectroscopic data are
described in Supplementary Material. Anal. Calcd for C19H20N2O5:
C: 64.04; H: 5.66; N: 7.86. Found: C: 64.19; H: 5.67; N: 7.89.
4.5.10. (E)-N0-(3,4-Methylenedioxybenzylidene)cinnamohydrazide (12)
90% yield; yellow solid, mp 180 ꢀC; Spectroscopic data are
described in Supplementary Material. Anal. Calcd for C17H14N2O3:
C: 69.38; H: 4.79; N: 9.52. Found: C: 69.50; H: 4.78; N: 9.54.
4.5.19. (2E)-N0-[(1E)-4-Hydroxy-3-methoxy-5-nitrobenzylidene]-
3-(3,4,5-trimethoxyphenyl) acrylohydrazide (21)
83% yield; orange solid, mp 228 ꢀC; 1H NMR (400 MHz, DMSO-
4.5.11. (E)-N0-(3,4-Dimethoxybenzylidene)cinnamohydrazide (13)
89% yield; yellow solid, mp 187 ꢀC; Spectroscopic data are
described in Supplementary Material. Anal. Calcd for C18H18N2O3:
C: 69.66; H: 5.85; N: 9.03. Found: C: 69.43; H: 5.86; N: 9.07.
d6)
d
3.70 (s, 3H, Ar(C4)OCH3), 3.84 and 3.85 (s, 6H, Ar(C3)OCH3 and
0
Ar(C5)OCH3), 3.96 and 3.99 (s, 3H, Ar(C3 )OCH3), 6.66 (d, J ¼ 16.0 Hz,
1H, CH]CHeCOOH), 6.98 and 7.11 (s, 2H, Ar(C2)H and Ar(C6)H),
0
7.56e7.64 (m, 2H, CH]CHeCOOH and Ar(C2 )H), 7.75e7.81 (s, 1H,
0
0
Ar(C6 )H), 8.01 and 8.18 (s, 1H, eCONHe), 10.88 (s, 1H, Ar(C4 )OeH),
4.5.12. (E)-N0-(3-Hydroxy-4-methoxybenzylidene)cinnamohydrazide (14)
87% yield; yellow solid, mp 193 ꢀC; Spectroscopic data are
described in Supplementary Material. Anal. Calcd for C17H16N2O3:
C: 68.91; H: 5.44; N: 9.45. Found: C: 69.13; H: 5.45; N: 9.42.
11.57 and 11.73 (s, 1H, eCONHe): 13C NMR (100 MHz, DMSO-d6)
d
55.82 and 55.92 (Ar(C3)OCH3 and Ar(C5)OCH3), 56.31 and 56.69
0
(Ar(C4)OCH3), 60.13 (Ar(C3 )OCH3), 105.23 and 105.62 (Ar(C2)H and
Ar(C6)H), 111.90 and 112.22 (Ar(C2 )H), 115.91 and 116.07 (Ar(C6 )H),
116.70 and 119.41 (CH]CHeCOOH), 125.11 (Ar(C1 )), 130.23 and
130.42 (Ar(C1)), 137.12 (Ar(C5 )), 139.04 (Ar(C4)), 140.87 (CH]
0
0
0
4.5.13. (E)-N0-(4-Hydroxy-3-methoxybenzylidene)
cinnamohydrazide (15)
0
CHeCOOH), 142.16 and 144.82 (eN]CHe), 143.84 and 144.10
88% yield; yellow solid, mp 219 ꢀC (Lit. [35] mp 218e219 ꢀC);
Spectroscopic data are described in Supplementary Material.
Anal. Calcd for C17H16N2O3: C: 68.91; H: 5.44; N: 9.45. Found: C:
69.11; H: 5.43; N: 9.47.
(Ar(C4 )), 149.81 (Ar(C3 )), 153.11 (Ar(C3) and Ar(C5)), 161.54 and
166.16 (CH]CHeCOOH) ppm: IR (KBr) nmax cmꢄ1: 3333 (
OeH),
2966 ( NeH), 1695 ( C]O), 1535 ( C]C), 1260 ( CeO): MS
0
0
n
n
n
n
n
(ESI) m/z: 430.2 (100%). Anal. Calcd for C20H21N3O8: C: 55.68; H:
4.91; N: 9.74. Found: C: 55.82; H: 4.90; N: 9.70.
4.5.14. (E)-N0-(3,4,5-Trimethoxybenzylidene)cinnamohydrazide (16)
91% yield; white solid, mp 185 ꢀC; Spectroscopic data are
described in Supplementary Material. Anal. Calcd for C19H20N2O4:
C: 67.05; H: 5.92; N: 8.23. Found: C: 66.93; H: 5.91; N: 8.19.
4.5.20. (2E)-3-(3,4-Dimethoxy-5-nitrophenyl)-N0-[(1E)-(2-
hydroxybenzylidene)]acrylohydrazide (22)
87% yield; yellow solid, mp 206 ꢀC; 1H NMR (500 MHz, DMSO-
d6)
d
3.88 (s, 3H, Ar(C4)OCH3), 3.95 and 3.98 (s, 3H, Ar(C3)OCH3),
4.5.15. (E)-N0-(4-Hydroxy-3-methoxy-5-nitrobenzylidene)
cinnamohydrazide (17)
6.74 (d, J ¼ 16.0 Hz, 1H, CH]CHeCOOH), 7.84e7.91 (m, 2H, Ar(C3 )H
0
0
0
and Ar(C5 )H), 7.21e7.29 (t, 1H, Ar(C4 )H), 7.53e7.70 (m, 4H, CH]
90% yield; yellow solid, mp 240 ꢀC; Spectroscopic data are
described in Supplementary Material. Anal. Calcd for C17H15N3O5:
C: 59.82; H: 4.43; N: 12.31. Found: C: 60.02; H: 4.44; N: 12.27.
CHeCOOH, Ar(C2)H, Ar(C6)H and Ar(C6 )H); 8.38 and 8.43 (s, 1H,
0
0
eN]CHe), 10.07 and 11.14 (s, H, Ar(C2 )OeH), 11.51 and 11.96 (s, 1H,
eCONHe); 13C NMR (125 MHz, DMSO-d6)
d
56.66 (Ar(C3)OCH3),
61.71 (Ar(C4)OCH3), 114.56 (Ar(C6)H), 115.67 and 116.08 (Ar(C2)H),
4.5.16. (2E)-3-(1,3-Benzodioxol-5-yl)-N0-[(1E)-2-hydroxybenzylidene]
acrylohydrazide (18)
116.27 and 116.37 (Ar(C3 )H), 118.70 and 120.23 (Ar(C1 )), 119.35 and
0
0
0
119.43 (Ar(C5 )H), 121.66 (CH]CHeCOOH), 126.53 and 129.37
95% yield; beige solid, mp 221 ꢀC; 1H NMR (400 MHz, DMSO-d6)
(Ar(C6 )H), 131.01 and 131.09 (Ar(C1)), 131.40 and 131.46 (Ar(C4 )H),
138.61 and 139.82 (CH]CHeCOOH), 140.92 and 147.35 (eN]
CHe), 142.01 and 142.29 (Ar(C5)), 144.68 and 144.93 (Ar(C4)),
0
0
d
6.08 (s, 2H, OeCH2eO), 6.51 (d, J ¼ 16.0 Hz, 1H, CH]CHeCOOH),
0
0
6.88e6.92 (m, 2H, Ar(C3 )H and Ar(C5 )H), 6.97 (d, J ¼ 8.0 Hz, 1H,
Ar(C5)H), 7.15 (d, J ¼ 8.0 Hz, 1H, Ar(C6)H), 7.22 (s, 1H, Ar(C2)H), 7.28
0
153.53 and 153.66 (Ar(C3)), 156.40 and 157.39 (Ar(C2 )), 160.86 and
0
0
(t, 1H, Ar(C4 )H), 7.52e7.59 (m, 2H, CH]CHeCOOH and Ar(C6 )H),
8.34 and 8.42 (s, 1H, eN]CHe), 11.20, 11.36 and 11.80 (s, 1H,
165.55 (CH]CHeCOOH); 13C NMR DEPT (100 MHz, DMSO-d6)
d
56.66 (Ar(C3)OCH3), 61.71 (Ar(C4)OCH3), 114.56 (Ar(C6)H), 115.67
eCONHe and s, 1H, Ar(C2 )OeH): 13C NMR (100 MHz, DMSO-d6)
and 116.08 (Ar(C2)H), 116.27 and 116.37 (Ar(C3 )H), 119.35 and
0
0
0
d
101.56 (OeCH2eO), 106.42 and 106.82 (Ar(C2)H), 108.57 and
119.43 (Ar(C5 )H), 121.66 (CH]CHeCOOH), 126.53 and 129.37
0
0
108.66 (Ar(C5)H), 115.22 and 117.61 (CH]CHeCOOH), 116.05 and
(Ar(C6 )H), 131.40 and 131.46 (Ar(C4 )H), 138.61 and 139.82 (Ar(C3)),

S.A. Carvalho et al. / European Journal of Medicinal Chemistry 54 (2012) 512e521
519
140.92 and 147.35 (eN]CHe) ppm; IR (KBr) nmax cmꢄ1: 3284 (
OeH), 1676 ( C]O), 1537 ( C]C), 1355 ( (N]O)2), 1274 ( CeO):
n
117.12 and 117.29 (Ar(C3 )H), 120.69 and 120.82 (Ar(C5 )H), 127.09
0
0
0
n
n
n
n
and 127.16 (Ar(C6 )H), 128.86 and 129.18 (Ar(C2)H and Ar(C6)H),
MS (ESI) m/z: 370.3 (100%). Anal. Calcd for C18H17N3O8: C: 58.22; H:
4.61; N: 11.32. Found: C: C: 58.41; H: 4.60; N: 11.34.
129.40 (Ar(C3)H and Ar(C5)H), 130.67 (Ar(C4)H), 132.46 and 132.73
0
(Ar(C4 )H), 144.17 and 148.55 (eN]CHe), 157.20 and 157.93
(Ar(C2 )) ppm: IR (KBr) nmax cmꢄ1: 3275 (
n
OeH), 3082 (
n
NeH),
0
4.5.21. (2E)-3-(3,4-Dimethoxy-5-nitrophenyl)-N0-[(1E)-(4-
hydroxy-3-methoxy-5-nitro benzylidene)]acrylohydrazide (23)
87% yield; yellow solid, mp 253 ꢀC; 1H NMR (500 MHz, DMSO-
w1670 (
n
C]O), w1530 (
n
C]C): MS (ESI) m/z: 291.1 (Mþ.[þNa])
(100%). Anal. Calcd for C16H16N2O2: C: 71.62; H: 6.01; N: 10.44.
Found: C: 71.55; H: 5.99; N: 10.48.
d6)
d
3.89 (s, 3H, Ar(C4)OCH3), 3.95 and 3.98 (s, 6H, Ar(C3)OCH3 and
0
Ar(C3 )OCH3), 6.77 (d, 1H, J ¼ 16.0 Hz, CH]CHeCOOH), 7.59e7.83
4.5.26. N0-[(1E)-4-Hydroxy-3-methoxy-5-nitrobenzylidene]-3-
phenylpropanohydrazide (28)
0
0
(m, 5H, CH]CHeCOOH, Ar(C2)H, Ar(C6)H, Ar(C2 )H and Ar(C6 )H),
8.00 and 8.18 (s, 1H, eN]CHe); 11.66 and 11.78 (s, 1H, eCONHe);
88% yield; orange solid, mp 197 ꢀC; Spectroscopic data are
described in Supplementary Material. Anal. Calcd for C17H17N3O5:
C: 59.47; H: 4.99; N: 12.24. Found: C: 59.60; H: 4.98; N: 12.21.
13C NMR (125 MHz, DMSO-d6)
d
56.45 and 56.60 (Ar(C3 )OCH3),
0
0
56.70 (Ar(C3)OCH3), 61.77 (Ar(C4)OCH3), 112.07 and 112.19 (Ar(C2 )
0
H), 114.43 and 115.77 (Ar(C6 )H), 115.40 and 115.57 (Ar(C2)H), 116.02
and 116.26 (Ar(C6)H), 119.46 and 122.14 (CH]CHeCOOH), 124.87
4.5.27. N0-[(1E)-2-Hydroxybenzylidene]-2-naphthohydrazide (29)
89% yield; beige solid, mp 209 ꢀC; Spectroscopic data are
described in Supplementary Material. Anal. Calcd for C18H14N2O2:
C: 74.47; H: 4.86; N: 9.65. Found: C: 74.61; H: 4.85; N: 9.62.
0
and 124.97 (Ar(C1 )), 131.12 and 131.31 (Ar(C1)), 137.13 and 137.19
0
(Ar(C5 )), 138.37 and 139.62 (CH]CHeCOOH), 141.41 and 145.41
0
(eN]CHe), 142.20 (Ar(C5)), 143.91 and 144.33 (Ar(C4 )), 144.72
0
(Ar(C4)), 149.81 and 149.88 (Ar(C3 )), 153.60 and 153.67 (Ar(C3)),
161.06 and 165.77 (CH]CHeCOOH); 13C NMR DEPT (125 MHz,
4.5.28. N0-[(1E)-4-Hydroxy-3-methoxy-5-nitrobenzylidene]-2-
naphthohydrazide (30)
0
DMSO-d6)
d
56.45 and 56.60 (Ar(C3 )OCH3), 56.70 (Ar(C3)OCH3),
61.77 (Ar(C4)OCH3), 112.07 and 112.19 (Ar(C2 )H), 114.43 and 115.77
88% yield; yellow solid, mp 197 ꢀC; Spectroscopic data are
described in Supplementary Material. Anal. Calcd for C19H15N3O5:
C: 62.46; H: 4.14; N: 11.50. Found: C: 62.33; H: 4.13; N: 11.52.
0
0
(C Ar(C6 )H), 115.40 and 115.57 (Ar(C2)H), 116.02 and 116.26 (Ar(C6)
H), 119.46 and 122.14 (CH]CHeCOOH), 138.37 and 139.62 (CH]
CHeCOOH), 141.41 and 145.41 (eN]CHe).ppm: IR (KBr) nmax
cmꢄ1: 3284 (
w1320 (
n
OeH), 3012 (
n
NeH), 1666 (
n
C]O), w1550 (
n
C]C),
4.6. Activity against intracellular amastigote forms [36]
n
(N]O)2), 1274 ( CeO): MS (ESI) m/z: 445.3 (100%). Anal.
n
Calcd for C19H18N4O9: C: 51.12; H: 4.06; N: 12.55. Found: C: 51.25;
H: 4.05; N: 12.50.
Rat skeletal muscle myoblasts (L6 cells) were seeded in 96-well
microtiter plates at 2000 (cells/well)/100
with 10% fetal bovine serum (FBS) and 2 mM
24 h, 5000 trypomastigotes of T. cruzi (Tulahuen strain C2C4 con-
taining the -galactosidase (Lac Z) gene) was added. After 48 h, the
medium was removed from the wells and replaced by 100 L of
fresh medium with or without a serial drug dilution. Seven 3-fold
dilutions were used covering a range from 0.123 to 90 g/mL. The
mL RPMI 1640 medium
L-glutamine. After
4.5.22. (2E)-3-(3,4-Dimethoxyphenyl)-N0-[(1E)-(2-hydroxybenzylidene)]
acrylohydrazide (24)
b
90% yield; beige solid, mp 193 ꢀC; Spectroscopic data are
described in Supplementary Material. Anal. Calcd for C18H18N2O4:
C: 66.25; H: 5.56; N: 8.58. Found: C: 66.19; H: 5.55; N: 8.60.
m
m
plates were incubated at 37 ꢀC in 5% CO2 for 4 days. Then the
substrate CPRG/Nonidet (50 mL) was added to all wells. The color
reaction that developed during the following 2e6 h was read
photometrically at 540 nm. From the sigmoidal inhibition curve,
IC50 values were calculated. Bzn was used as the standard drug.
4.5.23. (2E)-3-(3,4-Dimethoxyphenyl)-N0-[(1E)-(4-hydroxy-3-
methoxy-5-nitrobenzylidene)] acrylohydrazide (25)
85% yield; yellow solid, mp 265 ꢀC; Spectroscopic data are
described in Supplementary Material. Anal. Calcd for C19H19N3O7:
C: 56.86; H: 4.77; N: 10.47. Found: C: 57.04; H: 4.76; N: 10.44.
4.7. Activity against bloodstream trypomastigote forms [37]
4.5.24. (2E)-3-(4-Hydroxy-3-methoxy-5-nitrophenyl)-N0-[(1E)-
benzylidene]acrylohydrazide (26)
Stock solutions of NAH derivatives were prepared in DMSO.
Bloodstream trypomastigote forms of T. cruzi (Y strain) were isolated
from infected Swiss mice and re-suspended with Dulbecco’s modi-
fied Eagle medium plus 10% fetal calf serum (DMES) to a parasite
81% yield; yellow solid, mp 216 ꢀC; Spectroscopic data are
described in Supplementary Material. Anal. Calcd for C17H15N3O5:
C: 59.82; H: 4.43; N: 12.31. Found: C: 59.67; H: 4.44; N: 12.35.
concentration of 10 ꢅ 106 cells/mL. This suspension (100
mL) was
4.5.25. N0-[(1E)-2-Hydroxybenzylidene]-3-phenylpropanohydrazide (27)
85% yield; white solid, mp 151 ꢀC; 1H NMR (400 MHz, DMSO-d6)
added to the same volume of each NAH derivative, previously
prepared at twice the desired concentrations in DMES. Stock solu-
tions of the NAH derivatives were prepared in dimethylsulfoxide
d
2.53 (m, 2H, CH2eCH2eCOOH), 2.90 (m, 2H, CH]CHeCOOH),
(DMSO) and they were assayed in the range of 0.5e2000 m
g/mL1,
0
0
0
6.82e6.92 (m, 2H, Ar(C3 )H and Ar(C5 )H), 7.19 (m, 1H, Ar(C4 )H),
7.24e7.31 (m, 5H, Ar(C2)H, Ar(C3)H, Ar(C4)H, Ar(C5)H and Ar(C6)H),
with the final concentration of DMSO never exceeding 0.5%, which
0
7.49 (d, J ¼ 8.0 Hz, 1H, Ar(C6 )H), 8.25 and 8.32 (s, 1H, eN]CHe),
has no deleterious effect on the parasites [38].
10.10 and 11.14 (s, 1H, Ar(C4)OeH), 11.24 and 11.59 (s, 1H,
eCONHe). 13C NMR (100 MHz, DMSO-d6)
d
30.86 and 31.41
4.8. Cytotoxic effect on murine macrophages
(CH2eCH2eCOOH), 34.72 and 36.26 (CH2eCH2eCOOH), 117.12 and
0
0
117.29 (Ar(C3 )H), 119.11 and 120.13 (Ar(C1 )), 120.69 and 120.82
Cellular viability in the presence and absence of the NAH deriv-
atives (3e30) was determined by Mosmans assay using MTT (3-(4,5-
dimethylthiazol-2yl)-2,5-dimethyl tetrazolium bromide; Merck) as
previously described [39]. The macrophage cell line J774 was plated
in flat-bottomed 96-well plates (2.5 ꢅ 106 cells/mL) for 1 h in
controlled atmosphere (5% CO2, 37 ꢀC), and non-adherent cells were
washed by gentle flushing with RPMI 1640. Adherent cells were
0
0
(Ar(C5 )H), 127.09 and 127.16 (Ar(C6 )H), 128.86 and 129.18 (Ar(C2)H
and Ar(C6)H), 129.40 (Ar(C3)H and Ar(C5)H), 130.67 (Ar(C4)H),
132.46 and 132.73 (Ar(C4 )H), 141.43 and 141.79 (Ar(C1)), 144.17 and
0
0
148.55 (eN]CHe), 157.20 and 157.93 (Ar(C2 )), 169.84 and 174.95
(CH]CHeCOOH) ppm; RMN-13C DEPT (100 MHz, DMSO-d6): 30.86
and 31.41 (CH2eCH2eCOOH), 34.72 and 36.26 (CH2eCH2eCOOH),

520
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cultured in the presence of medium alone, 3% Tween 20 and the
compounds (0.1e100 g/mL) in a triplicate assay. After 18 h, 20 L of
5 mg/mL MTT solution was added to each well and after 4 h later the
supernatant was discharged and dimethylsulfoxide (DMSO) (100
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m
mL/well) was added for solubilization of the formazan crystals and
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Cruzain truncated in the C-terminal extension was obtained
from Escherichia coli (strain M15 or DH5a containing the expression
plasmid) [40]. The substrate ZePheeArgeAMC and all reagents for
buffer preparation were purchased from SigmaeAldrich. Stock
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The highly purified enzyme (0.64 nM), in 50 mM sodium phos-
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5 mM DTT, was incubated with the N-acylhydrazone derivatives for
5 min at room temperature followed by the addition of the fluoro-
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measurements were done using 355 nm as the excitation wave-
length and 460 nm as the emissionwavelength. Cruzain activity was
measured as an increase of fluorescence intensity by cleavage of the
substrate ZePheeArgeAMC and release of aminocoumarin.
The IC50 values were calculated using nonlinear regression
analysis employing the Sigma-Plot enzyme kinetics module and it
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inhibitor, relatively to the activity in the absence of inhibitor. At
least seven inhibitor concentrations with inhibition range between
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Acknowledgments
This work was supported by grants from CNPq (BR.), FAPERJ
(BR.), and FIOCRUZ (BR.). The in vitro screening of trypanocidal
activity of the amastigotes of T. cruzi was supported by DNDi (SW.).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
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