Biochemical evaluation of a series of synthetic chalcone and hydrazide derivatives as novel inhibitors of cruzain from trypanosoma cruzi

Article abstract of DOI:10.1590/S0103-50532010000100021

Chagas' disease, a parasitic infection widely distributed throughout Latin America, is a major public health problem with devastating consequences in terms of human morbidity and mortality. The enzyme cruzain is the major cysteine protease from Trypanosoma cruzi, the etiologic agent of American trypanosomiasis or Chagas' disease, and has been selected as an attractive target for the development of novel trypanocidal drugs. In the present work, we describe the synthesis and inhibitory effects of a series of thirty-three chalcone and seven hydrazide derivatives against the enzyme cruzain from T. cruzi. Most of the compounds showed promising in vitro inhibition (IC50 values in the range of 20-60 μM), which suggest the potential of these compounds as lead candidates for further development. Twelve compounds have not been reported before, and four of them (7, 13, 16 e 18) are among the most potent inhibitors of the series.

Full text of DOI:10.1590/S0103-50532010000100021

J. Braz. Chem. Soc., Vol. 21, No. 1, 142-150, 2010.
Printed in Brazil - ©2010 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00
Biochemical Evaluation of a Series of Synthetic Chalcone and Hydrazide
Derivatives as Novel Inhibitors of Cruzain from Trypanosoma cruzi
Deise M. Borchhardt,a Alessandra Mascarello,b Louise Domeneghini Chiaradia,b
Ricardo J. Nunes,b Glaucius Oliva,a Rosendo A. Yunes*,b and Adriano D. Andricopulo*,a
aLaboratório de Química Medicinal e Computacional, Centro de Biotecnologia Molecular Estrutural,
Instituto de Física de São Carlos, Universidade de São Paulo, 13566-970 São Carlos-SP, Brazil
bLaboratório Estrutura e Atividade, Centro de Ciências Físicas e Matemáticas, Departamento de
Química, Universidade Federal de Santa Catarina, 88040-900 Florianópolis-SC, Brazil
A doença de Chagas, uma infecção parasitária amplamente distribuída na América Latina, é
um problema grave de saúde pública com conseqüências devastadoras em termos de morbidade
e mortalidade humana. A enzima cruzaína é a principal cisteíno protease do Trypanosoma cruzi,
agente etiológico da tripanossomíaseAmericana ou doença de Chagas, e foi selecionada como alvo
atrativo para o desenvolvimento de novos fármacos tripanocidas. No presente trabalho, a síntese e
os efeitos inibitórios de uma série de trinta e três chalconas e sete hidrazidas são descritos contra
a enzima cruzaína de T.cruzi. A maioria dos compostos mostraram inibição promissora in vitro
(valores de IC50 na faixa de 20-60 μM), o que sugere o potencial desses compostos como candidatos
a líderes para contínuo desenvolvimento. Doze compostos são inéditos, sendo que quatro destes
(7, 13, 16 e 18) estão entre os inibidores mais potentes da série.
Chagas’disease, a parasitic infection widely distributed throughout Latin America, is a major
public health problem with devastating consequences in terms of human morbidity and mortality.
The enzyme cruzain is the major cysteine protease from Trypanosoma cruzi, the etiologic agent
of American trypanosomiasis or Chagas’ disease, and has been selected as an attractive target
for the development of novel trypanocidal drugs. In the present work, we describe the synthesis
and inhibitory effects of a series of thirty-three chalcone and seven hydrazide derivatives against
the enzyme cruzain from T. cruzi. Most of the compounds showed promising in vitro inhibition
(IC50 values in the range of 20-60 μM), which suggest the potential of these compounds as lead
candidates for further development. Twelve compounds have not been reported before, and four
of them (7, 13, 16 e 18) are among the most potent inhibitors of the series.
Keywords: chalcones, Trypanosoma cruzi, cruzain, Chagas’ disease, inhibition
Introduction
including poor efficacy, and serious side effects. Therefore,
there is an urgent need for new chemotherapeutic agents
with novel mechanisms of action.1-3
Chagas’ disease, caused by the protozoan parasite
Trypanosoma cruzi, is a major cause of illness, morbidity,
long-term disability, and death in Latin America. It is
estimated that about 16-18 million people are infected
and other 100 million are at risk of infection, with more
than 50,000 deaths each year. In spite of the alarming
health, economic, and social consequences of this parasitic
infection, the limited existing drug therapy (nifurtimox and
benznidazole) suffers from a combination of drawbacks
Enzymes are extremely attractive targets for small
molecule drug intervention in human diseases.1,2 One
important drug target is the major cysteine protease from
T. cruzi, cruzain (EC 3.4.22.51). This enzyme is implicated
in several vital processes of the parasite, playing a pivotal
role during the infection of host cells, replication, and
metabolism. The identification and design of potent and
selective cruzain inhibitors is, therefore, of great importance.
About one decade ago, in a review of inhibitors of cysteine
proteases, Otto and Schirmeister4 cited a series of chalcone
*e-mail: aandrico@if.sc.usp.br; ryunes@qmc.ufsc.br

Vol. 21, No. 1, 2010
Borchhardt et al.
143
derivatives as inhibitors of malarial cysteine proteases.
However, the potential of this class of inhibitors has not
been fully explored, in spite of its synthetic versatility
and potential low toxicity.5 To the best of our knowledge,
only two chalcones were reported as inhibitors of T. cruzi
cruzain.6 In that respect, there is a justifiable interest in
developing structure-activity relationships (SAR) for this
class of compounds. On the other hand, acylhydrazides (a
class of compounds structurally related to chalcones) have
been successfully investigated against T. cruzi cruzain.7 In
addition, both chalcone and acylhydrazide derivatives were
active in vitro against T. brucei and T. cruzi.8,9
dimethoxyacetophenone (prepared as previously described,
with yield of 95%);13 chalcones 7-20 are derived from
3,4-methylenedioxyacetophenone; chalcones 21-27 are
derived from 2-naphthylacetophenone; chalcones 28-31 are
derived from 2,4,5-trimethoxyacetophenone (prepared as
previously described, with yield of 81%);14 and chalcones
32 and 33 are derived from 2,4-dimethoxyacetophenone.
The benzylated vanillin (3-methoxy-4-(phenylmethoxy)-
benzaldehyde) used to prepare chalcone 33 was obtained
according to the procedure of Tsai and Klinman,15 with
yield of 88%.
The hydrazides (34-40) were prepared by condensation
of the obtained 3,4,5-trimethoxybenzohydrazide (41)
(previously prepared as described, with yield of 80%)16
and corresponding aromatic aldehydes, with yields ranging
from 61 to 72% (Scheme 2).17
As part of our research program aimed at discovering
novel T. cruzi cruzain inhibitors, we have synthesized and
evaluated a series of chalcones and hydrazides.10-12
Results and Discussion
All synthesized compounds, including those that had
been previously reported (chalcones 1, 2, 4, 5, 8-12, 14,
17, 19, 21, 23-26, 28, 31-33; and hydrazides 34, 35, 38 and
40)18,19 were well characterized by 1H NMR, 13C NMR, IR
and elemental analysis. Detailed spectral characterization
(1H NMR, 13C NMR, IR and elemental analysis) for all
novel compounds (3, 6, 7, 13, 15, 16, 18, 20, 22, 27, 29,
30, 36, 37 and 39) is presented in the experimental part. 1H
NMR spectra revealed that all the structures are configured
E (JHα-Hβ 15-16 Hz).
Synthesis
The chalcone derivatives (Table 1) were prepared
by aldolic condensation of aromatic aldehydes and
corresponding acetophenone (Scheme 1), with yields
ranging from 18 to 92%.12 Chalcones 1 and 2 are
derived from 3,4-methylenedioxybenzaldehyde;
chalcones 3-6 are derived from 2-hydroxy-3-bromo-4,6-
O
O
O
a
+
R
CH3
R
R'
H
R'
1-33
Compd.
1
R
R’
Compd.
2
R
R’
3,4-methylenedioxy-Ph
3-NO2-Ph
4-Br-Ph
3,4-methylenedioxy-Ph
1-naphthyl
2-OH-Ph
3
2-OH-3-Br-4,6-OCH3-Ph
2-OH-3-Br-4,6-OCH3-Ph
3,4-methylenedioxy-Ph
3,4-methylenedioxy-Ph
3,4-methylenedioxy-Ph
3,4-methylenedioxy-Ph
3,4-methylenedioxy-Ph
3,4-methylenedioxy-Ph
3,4-methylenedioxy-Ph
2-naphthyl
4
2-OH-3-Br-4,6-OCH3-Ph
2-OH-3-Br-4,6-OCH3-Ph
3,4-methylenedioxy-Ph
3,4-methylenedioxy-Ph
3,4-methylenedioxy-Ph
3,4-methylenedioxy-Ph
3,4-methylenedioxy-Ph
3,4-methylenedioxy-Ph
3,4-methylenedioxy-Ph
2-naphthyl
5
4-OCH3-Ph
6
4-O(CH2)3CH3-Ph
4-Br-Ph
7
2,6-Cl-Ph
8
9
4-OCH3-Ph
10
12
14
16
18
20
22
24
26
28
30
32
2,4,5-OCH3-Ph
4-CH3-Ph
11
13
15
17
19
21
23
25
27
29
31
33
3,4,5-OCH3-Ph
1-naphthyl
Ph
2-Cl-Ph
2-CH3-furan
tiophene
3-OCH3-Ph
3-OCH3-4-OBn-Ph
4-OCH3-Ph
4-O(CH2)3CH3-Ph
2,6-OCH3-Ph
4-F-Ph
2-naphthyl
2,4,5-OCH3-Ph
3,4-methylenedioxy-Ph
2-CH3-furan
2-naphthyl
2-naphthyl
2-naphthyl
2-naphthyl
2-naphthyl
2,4,5-OCH3-Ph
2,4,5-OCH3-Ph
tiophene
2,4,5-OCH3-Ph
1-naphthyl
2,4,5-OCH3-Ph
2,4,5-OCH3-Ph
3-OCH3-Ph
2,4-OCH3-Ph
2,4,5-OCH3-Ph
2,4-OCH3-Ph
3-OCH3-4-OBn-Ph
Scheme 1. Synthesis of chalcones 1-33. a: KOH 50% m/v, methanol, rt, 24 h.

144
Biochemical Evaluation of a Series of Synthetic Chalcone and Hydrazide Derivatives
J. Braz. Chem. Soc.
Scheme 2. Synthesis of 3,4,5-trimethoxybenzohydrazide and hydrazide derivatives 34-40. a: (CH3)2SO4, K2CO3, acetone, TBAI, reflux, 12 h. b: H2NNH2.
H2O, MeOH, reflux, 5 h. c: respective aldehyde, MeOH, reflux, 2 h.
Biochemical evaluation of the synthetic compounds
potential of these compounds as a new class of small
molecule inhibitors of T. cruzi cruzain. Future efforts
to optimize these compounds will involve the design,
synthesis and SAR studies of a variety of more water-
soluble derivatives, as well as investigation about the
importance of methylenedioxyphenyl moiety side in the
structure of chalcones through the determination of the
possible mechanism of enzyme inhibition and inhibitor
binding mode. Efforts in this direction are in progress, and
will be reported in due course.
As can be seen (Table 1), among the 40 derivatives
evaluated against T. cruzi cruzain, compounds 1, 3-10,
12-14, 16, 18, 20, 21, 25, 28-30, 33-36, and 38-40 have
IC50 values in the range of 20-100 μM.
The IC50 values of compounds 15, 22, 26, 27 (IC50s
>
25 μM), 11, 17, 19, 23, 24, and 37 (IC50s > 50 μM), as well
as of compounds 2, 31 and 32 (the weakest inhibitors of
the series with IC50s > 100 μM), could not be determined
accurately due to solubility limitations that appeared to
decrease the sensitivity of the assay. The most potent
inhibitors of the series, 1, 5, 7, 10, 13, 16, and 18 (IC50
values between 20 and 25 μM) represent a new class of
lead candidates for further development. Furthermore, with
a single exception of compound 5, all chalcone derivatives
possess the methylenedioxyphenyl (1,3-benzodioxole)
moiety, which suggests a significant contribution of this
group to the observed activity. In addition, it is worth noting
that chalcones 7, 13, 16 and 18 are newly synthesized
compounds and are among the most promising compounds
for further SAR studies. On the other hand, the hydrazide
derivatives (34-40) investigated in this work showed only
moderate inhibitory potency, with IC50 values in the range
of 40-60 μM.
Experimental
Physicochemical data of the synthesized compounds
The purified compounds were obtained in yields ranging
from 18 to 92%. Melting points were determined with a
Microquímica MGAPF-301 apparatus and are uncorrected.
IR spectra were recorded with an Abb Bomen FTLA 2000
spectrometer on KBr disks. NMR (1H and 13C NMR)
were recorded on a Varian Oxford AS-400 (400 MHz)
spectrometer, using tetramethylsilane as an internal
standard. Elemental analysis was carried out with a CHNS
EA 1110. Percentages of C and H were in agreement with
the product formula (within + 0.4% of theoretical values
of C).
Conclusions
General procedure for the synthesis and purification of
A series of chalcones and hydrazides have been
prepared and evaluated in vitro against cruzain from
T. cruzi. Most compounds showed promising inhibitory
activity against the parasite enzyme, with IC50 values in
the low micromolar range. Our results demonstrate the
chalcones (1-33)
The chalcones were synthesized as shown in Scheme 1.
All reagents used in this work were analytical grade and
were purchased from Merck and Sigma-Aldrich. Exceptions

Vol. 21, No. 1, 2010
Borchhardt et al.
145
Table 1. Structures and IC50 values of a series of synthetic chalcone and hydrazide derivatives as inhibitors of T. cruzi cruzain
Compd.
Structure
IC50a (mM) Compd.
Structure
IC50a (mM) Compd.
Structure
IC50a (mM)
O
OH
O
O
O
O
O
1
20
45
2
>100
20
3
37
82
Br
4
5
6
7
20
8
30
9
35
10
13
16
19
22
25
25
11
14
17
20
23
26
>50
50
12
15
18
21
24
27
30
25
>25
22
23
>50
46
O
O
O
>50
>25
50
60
O
CH3
>50
>25
>50
>25
28
31
100
29
32
48
30
33
100
37
>100
>100
34
55
35
38
60
40
36
39
50
47
37
40
>50
50
a IC50 values are representative of three independent experiments performed in triplicate and the standard deviations were within 10% of the mean values
reported for each compound.

146
Biochemical Evaluation of a Series of Synthetic Chalcone and Hydrazide Derivatives
J. Braz. Chem. Soc.
are 2-hydroxy-3-bromo-4,6-dimethoxyacetophenone,
2,4,5-trimethoxyacetophenone and 3-methoxy-4-
(phenylmethoxy)-benzaldehyde, which were prepared
as previously described.13-15 The chalcones (1-33) were
prepared by magnetic stirrer of the acetophenone derivative
(1.2 mmol), methanol (20 mL), KOH 50% m/v (5 mL),
and the corresponding aldehyde (1.2 mmol), at room
temperature for 24 h. Distilled water and hydrochloric acid
(10%) were added to the reaction for total precipitation of
the products.The compounds were then obtained by vacuum
filtration and later recrystallized from dichloromethane and
hexane. Chalcones 1, 2, 4, 5, 8-12, 14, 17, 19, 21, 23-26, 28,
31-33 were previously described in the literature,18 while
compounds 3, 6, 7, 13, 15, 16, 18, 20, 22, 27, 29 and 30
are novel compounds which have not been reported. The
chalcones (3-6) showed yields in the range of 40-50% due
the presence of the bromine atom in the starting material
acetophenone, which stabilizes the enolate intermediate
of the reaction making it less reactive. This effect was not
observed for the chalcones (7-20), which showed yields in
the range of 70-98%.
115.19 (C3, C5), 124.46 (Cα), 128.04 (C1), 130.71 (C2,
C6), 143.72 (Cβ), 160.02 (C4), 168.46 (C2’), 168.82 (C6’),
176.12 (C4’), 198.07 (C=O); IR (KBr) νmax/cm-1 3450,
2945, 1615, 1554, 1221, 963; Anal. Calc. for C21H23BrO5:
C 57.94; H 5.33. Found: C 57.27; H 5.27.
(2E)-1-(1,3-Benzodioxol-5-yl)-3-(2,6-dichlorophenyl)-2-
propen-1-one (7)
Light yellow solid; mp 118-119 ºC; yield: 90%;
1H NMR (CDCl3) d 6.08 (s, 2H, -OCH2O-), 6.90 (d, 1H,
J 8.0 Hz, H5’), 7.21 (t, 1H, J 7.6 Hz, H4), 7.38 (d, 2H, J
8.4 Hz, H3, H5), 7.54 (s, 1H, H2’), 7.62 (d, 1H, J 16.0
Hz, Hα), 7.64 (d, 1H, J 8.0 Hz, H6’), 7.83 (d, 1H, J 16.0
Hz, Hβ); 13C NMR (CDCl3) d 102.16 (-OCH2O-), 108.23
(C5’), 108.76 (C2’), 125.40 (C6’), 128.00 (Cα), 129.07
(C3, C5), 129.99 (C1’), 130.49 (C4), 132.75 (C6), 132.98
(C2), 135.40 (C1), 137.50 (Cβ), 148.62 (C3’), 152.23 (C4’),
188.22 (C=O); IR (KBr) νmax/cm-1 1655, 1242 (C=O), 1595
(C=C), 1310, 1033 (C-O), 2905, 1503, 1439, 1107, 979,
927, 786, 743 (Ar); Anal. calc. for C16H10Cl2O3: C 59.84;
H 3.14. Found: C 59.69; H 3.26.
(2E)-1-(2’-Hydroxy,3’-bromo,4’,6’-dimethoxyphenyl)-3-
(2E)-1-(1,3-Benzodioxol-5-yl)-3-(1-naphthyl)-2-propen-
(1-naphthyl)-2-propen-1-one (3)
1-one (13)
1
1
Yellow solid; mp 218-219ºC; yield: 42%; H NMR
Yellow solid; mp 137-139 ºC; yield: 91%; H NMR
(acetone-d6) d 4.06 (s, 3H, o-OCH3), 4.14 (s, 3H, p-OCH3),
6.51 (s, 1H, H5’), 7.60 (d, 1H, J 7.6 Hz, H10), 7.61 (m, H5),
7.62 (d, J 7.6 Hz, H8), 7.70 (t, 1H, J 7.6 Hz, H9), 8.03 (m,
3H, H4, H6), 8.09 (d, 1H, J 15.4 Hz, Hα), 8.38 (d, 1H, J 8.4
Hz, H3), 8.66 (d, 1H, J 15.4 Hz, Hβ); 13C NMR (CDCl3)
d 56.39 (p-OCH3), 56.62 (o-OCH3), 87.41 (C3’), 92.24
(C5’), 107.20 (C1’), 123.90 (Cα), 125.46 (C10), 125.68
(C3), 126.52 (C9), 127.15 (C5), 128.96 (C4), 129.79 (C8),
130.86 (C6), 132.00 (C2), 133.00 (C7), 133.97 (C1), 140.42
(Cβ), 162.27 (C6’), 162.55 (C2’), 163.46 (C4’), 192.91
(C=O); IR (KBr) νmax/cm-1: 3438 (OH), 1624, 1218 (C=O),
1552 (C=C), 1347, 1040 (C-O), 2952, 1415, 1314, 1250,
1129, 973, 800, 767, 659 (Ar);Anal. calc. for C21H17BrO4:
C 61.03; H 4.15. Found: C 59.99; H 4.01.
(CDCl3) d 6.09 (s, 2H, -OCH2O-), 6.93 (d, 1H, J 8.0 Hz,
H5’), 7.53 (d, 1H, J 16.0 Hz, Hα), 7.54 (d, 1H, J 8.0 Hz,
H10), 7.58 (m, 1H, H4), 7.59 (d, 1H, J 8.0 Hz, H6’), 7.61
(s, 1H, H2’), 7.61 (m, 1H, H5), 7.72 (dd, 1H, J 8.0/1.0 Hz,
H9), 7.90 (m, 1H, H8), 7.93 (d, 1H, J 8.0 Hz, H6), 8.28 (d,
1H, J 8.0 Hz, H3), 8.66 (d, 1H, J 16.0 Hz, Hβ); 13C NMR
(CDCl3) d 102.13 (-OCH2O-), 108.19 (C2’), 108.73 (C5’),
109.99 (C10), 123.80 (C6’), 124.63 (Cα), 125.05 (C3),
125.23 (C9), 125.66 (C5), 126.52 (C4), 127.17 (C8), 128.96
(C6), 130.90 (C2), 131.99 (C1’), 133.77 (C7), 133.96 (C1),
141.46 (Cβ), 148.60 (C3’), 153.81 (C4’), 188.29 (C=O);
IR (KBr) νmax/cm-1 1646, 1253 (C=O), 1584 (C=C), 1343,
1037 (C-O), 2919, 1498, 1444, 1307, 1115, 978, 930, 806,
773, 732, 709 (Ar); Anal. calc. for C20H14O3: C 79.46; H
4.67. Found: C 78.53; H 4.80.
(2E)-1-(2’-Hydroxy-3’-bromo-4’,6’-dimethoxyphenyl)-3-
(4-buthoxyphenyl)-2-propen-1- one (6)
(2E)-1-(1,3-Benzodioxol-5-yl)-3-(2-chlorophenyl)-2-
1
Yellow solid; mp 172-173 ºC; yield: 47%; H NMR
propen-1-one (15)
1
(CDCl3) d 0.98 (t, 3H, -CH3), 1.47-1.60 (m, 2H, -CH2CH3),
1.75-1.81 (m, 2H, -CH2CH2CH3), 3.98 (s, 3H, OCH3), 3.99
(s, 3H, OCH3), 4.01 (t, 2H, -OCH2-), 6.06 (s, 1H, H5’),
6.91 (d, 2H, J 8.4 Hz, H3, H5), 7.54 (d, 2H, J 8.4 Hz, H2,
H6), 7.75 (d, 1H, J 15.6 Hz, Hα), 7.83 (d, 1H, J 15.6 Hz,
Hβ), 14.96 (OH); 13C NMR (acetone-d6) d 13.42 (CH3),
19.18 (-CH2CH3), 31.28 (- CH2CH2CH3), 56.25 (OCH3),
67.86 (-OCH2-), 88.37 (C5’), 94.61(C3’), 104.43 (C1’),
Cream solid; mp 112-113 ºC; yield: 87%; H NMR
(CDCl3) d 6.08 (s, 2H, -OCH2O-), 6.90 (d, 1H, J 8.0 Hz,
H5’), 7.31 (m, 1H, H4), 7.34 (m, 1H, H5), 7.44 (m, 1H,
H3), 7.45 (d, 1H, J 16.0 Hz, Hα), 7.54 (s, 1H, H2’), 7.65
(dd, 1H, J 8.0/1.0 Hz, H6’), 7.74 (m, 1H, H6), 8.16 (d, 1H,
J 16.0 Hz, Hβ); 13C NMR (CDCl3) d 101.91 (-OCH2O-),
107.94 (C2’), 124.52 (C6’), 124.89 (Cα), 127.04 (C5),
127.75 (C6), 130.30 (C3), 131.03 (C4), 132.72 (C1’),

Vol. 21, No. 1, 2010
Borchhardt et al.
147
133.38 (C1), 135.43 (C2), 140.06 (Cβ), 148.36 (C3’),
151.85 (C4’), 188.15 (C=O); IR (KBr) νmax/cm-1 1653,
1241 (C=O), 1598 (C=C), 1319, 1037 (C-O), 2900, 1489,
1435, 1106, 975, 924, 813, 751, 569 (Ar); Anal. calc. for
C16H11ClO3: C 67.03; H 3.87. Found: C 67.14; H 4.05.
(Cα), 127.46 (C1), 130.14 (C1’, C2, C6), 144.25 (Cβ),
148.22 (C3’), 152.11 (C4’), 161.22 (C4), 181.79 (C=O);
IR (KBr) νmax/cm-1 1654, 1240 (C=O), 1596 (C=C), 1330,
1030 (C-O), 2953, 2863, 1504, 1438, 1291, 1173, 1110,
973, 924, 807, 571 (Ar);Anal. calc. for C20H20O4: C 74.06;
H 6.21. Found: C 74.28; H 6.46.
(2E)-1-(1,3-Benzodioxol-5-yl)-3-(5-methylfuran-2-yl)-2-
propen-1-one (16)
(2E)-1-(2-Naphthyl)-3-(2,6-dimethoxyphenyl)-2-propen-
Light orange solid; mp 98-99 ºC; yield: 87%; 1H NMR
(CDCl3) d 2.39 (CH3), 6.06 (s, 2H, -OCH2O-), 6.13 (d,
1H, J 4.0 Hz, H4), 6.61 (d, 1H, J 4.0 Hz, H5), 6.89 (d, 1H,
J 8.0 Hz, H5’), 7.33 (d, 1H, J 16.0 Hz, Hα), 7.52 (d, 1H,
J 16.0 Hz, Hβ), 7.67 (d, 1H, J 8.0 Hz, H6’), 7.54 (s, 1H,
H2’); 13C NMR (CDCl3) d 14.25 (CH3), 102.02 (-OCH2O-),
108.10 (C2’), 108.60 (C5’), 109.52 (C4), 117.46 (C5),
118.21 (C6’), 124.73 (Cα), 130.71 (Cβ, C1), 133.34 (C1’),
148.44 (C3’), 150.64 (C1), 151.73 (C4’), 155.95 (C3),
187.98 (C=O); IR (KBr) νmax/cm-1 1651, 1251 (C=O),
1601 (C=C), 1360, 1036 (C-O), 2908, 1567, 1492, 1438,
1291, 1192, 1107, 1010, 994, 936, 906, 846, 792, 612, 539
(Ar); Anal. calc. For C15H12O4: C 70.31; H 4.72. Found: C
70.09; H 4.79.
1-one (22)
1
Cream solid; mp 109- 111 ºC; yield: 79%; H NMR
(CDCl3) d 3.94 (s, 6H, OCH3), 6.61 (d, 1H, J 8.0 Hz, H3,
H5), 7.31 (t, 1H, J 8.0 Hz, H4), 7.55 (dd, 1H, J 8.0/1.0 Hz,
H5’), 7.60 (dd, 1H, J 8.0/1.0 Hz, H6’), 7.90 (d, 1H, J 8.0
Hz, H4’), 7.93 (d, 1H, J 8.0 Hz, H10’), 8.00 (d, 1H, J 8.0
Hz, H9’), 8.11 (d, 1H, J 8.0 Hz, H7’), 8.13 (d, 1H, J 16.0
Hz, Hα), 8.33 (d, 1H, J 16.0 Hz, Hβ), 8.53 (s, 1H, H2’); 13C
NMR (CDCl3) d 56.15 (OCH3), 104.00 (C3, C5), 113.19
(C1), 125.08 (Cα), 125.40 (C10’), 126.76 (C5’), 128.01
(C7’), 128.25 (C9’), 128.51 (C6’), 129.76 (C2’), 130.06
(C4), 131.76 (C4’), 132.84 (C8’), 135.52 (C1’), 136.07
(Cβ), 136.53 (C3’), 160.62 (C2, C6), 192.40 (C=O). IR
(KBr) νmax/cm-1 1645, 1256 (C=O), 1581 (C=C), 1320, 1011
(C-O), 2934, 2833, 1470, 1439, 1177, 1110, 851, 757, 618,
535, 459 (Ar); Anal. calc. for C21H18O3: C 79.23; H 5.70.
Found: C 79.14; H 5.69.
(2E)-1-(1,3-Benzodioxol-5-yl)-3-(thiophen-2-yl)-2-propen-
1-one (18)
1
Beige solid; mp 101-103 ºC; yield: 91%; H NMR
(CDCl3) d 6.07 (s, 2H, -OCH2O-), 6.90 (d, 1H, J 4.0 Hz,
H5’), 7.09 (d, 1H, J 8.0 Hz, H4), 7.29 (d, 1H, J 16.0 Hz,
Hα), 7.35 (d, 1H, J 4.0 Hz, H5), 7.41 (d, 1H, J 4.0 Hz, H3),
7.52 s (H2’), 7.63 (d, 1H, J 8.0 Hz, H6’), 7.93 (d, 1H, J 16.0
Hz, Hβ); 13C NMR (CDCl3) d 102.09 (-OCH2O-), 108.16
(C2’), 108.59 (C5’), 120.68 ($C6’), 124.81 (Cα), 128.54
(C4), 128.79 (C5), 132.13 (Cβ), 133.12 (C1’), 136.94 (C3),
140.72 (C1), 148.52 (C3’), 151.93 (C4’), 187.87 (C=O);
IR (KBr) νmax/cm-1 1644, 1264 (C=O), 1581 (C=C), 1361,
1040 (C-O), 1500, 1443, 1325, 1301, 1202, 1109, 960, 910,
799, 697, 567 (Ar); Anal. calc. for C14H10O3S: C 65.10; H
3.90. Found: C 64.73; H 3.63.
(2E)-1-(2-Naphthyl)-3-(5-methylfuran-2-yl)-2-propen-1-
one (27)
Gold yellow solid; mp 85-87 ºC; yield: 92%; 1H NMR
(CDCl3) d 2.43 (s, 3H, CH3), 6.15 (d, 1H, J 4.0 Hz, H4),
6.66 (d, 1H, J 4.0 Hz, H5), 7.54 (d, 1H, J 16.0 Hz, Hα),
7.58 (m, 1H, H5’), 7.61 (m, 1H, H6’), 7.62 (d, 1H, J 16.0
Hz, Hβ), 7.90 (d, 1H, J 8.0 Hz, H4’), 7.94 (d, 1H, J 8.0 Hz,
H10’), 8.01 (d, 1H, J 8.0 Hz, H9’), 8.12 (d, 1H, J 8.0 Hz,
H7’), 8.57 (s, 1H, H2’); 13C NMR (CDCl3) d 14.09 (CH3),
109.43 (C4), 117.52 (C5), 118.36 (C10’), 124.51 (Cα),
126.67 (C5’), 127.80 (C7’), 128.25 (C9’), 128.46 (C6’),
129.56 (C2’), 129.76 (C4’), 130.80 (Cβ), 132.63 (C8’),
135.43 (C1’), 135.75 (C3’), 150.45 (C1), 155.96 (C3),
189.69 (C=O); IR (KBr) νmax/cm-1 1648, 1284 (C=O), 1557
(C=C), 1362, 1012 (C-O), 3056, 1595, 1182, 1114, 819,
780, 710, 614, 535, 473 (Ar); Anal. calc. for C18H14O2: C
82.42; H 5.38. Found: C 81.68; H 5.30.
(2E)-1-(1,3-Benzodioxol-5-yl)-3-(4-buthoxyphenyl)-2-
propen-1-one (20)
1
Cream solid; mp 102-104 ºC; yield: 92%; H NMR
(CDCl3) d 0.99 (t, 3H, J 8.0 Hz, CH3), 1.51 (m, -CH2- CH3),
1.79 (m, -CH2-CH2-CH3), 4.01 (t, 2H, J 8.0 Hz, -O-CH2-),
6.06 (s, 2H, -OCH2O-), 6.89 (d, 1H, J 8.0 Hz, H5’), 6.92
(d, 1H, J 8.0 Hz, H3, H5), 7.37 (d, 1H, J 16.0 Hz, Hα),
7.53 (s, 1H, H2’), 7.58 (d, 1H, J 8.0 Hz, H2, H6), 7.65 (d,
1H, J 8.0 Hz, H6’), 7.77 (d, 1H, J 16.0 Hz, Hβ); 13C NMR
(CDCl3) d 13.84 (CH3), 19.22 (-CH2-CH3), 31.21 (-CH2-
CH2-CH3), 67.87 (-O-CH2-), 101.81 (-OCH2O-), 107.87
(C2’), 108.44 (C5’), 114.88 (C3, C5), 119.18 (C6’), 124.46
(2E)-1-(2’,4’,5’-Trimethoxyphenyl)-3-(1-naphthyl)-2-
propen-1-one (29)
1
Yellow solid; mp 162-164 ºC; yield: 87%; H NMR
(CDCl3) d 3.90 (s, 3H, o-OCH3), 3.92 (s, 3H, m-OCH3),
3.98 (s, 3H, p-OCH3), 6.56 (s, 1H, H3’), 7.45 (s, 1H, H6’),
7.51 (t, 1H, J 8.0 Hz, H9), 7.53 (m, 1H, H5), 7.59 (d, 1H,
H4), 7.72 (d, 1H, J 16.0 Hz, Hα), 7.85 (d, 1H, J 8.0 Hz,

148
Biochemical Evaluation of a Series of Synthetic Chalcone and Hydrazide Derivatives
J. Braz. Chem. Soc.
H8), 7.88 (m, 2H, H6, H10), 8.31 (d, 1H, J 8.0 Hz, H3),
8.55 (d, 1H, J 16.0 Hz, Hβ); 13C NMR (CDCl3) d 56.37
(o-OCH3), 56.49 (m-OCH3), 57.01 (p-OCH3), 97.26 (C3’),
113.50 (C6’), 120.67 (C1’), 123.99 (Cα), 125.21 (C10),
125.67 (C3), 125.95 (C9), 126.23 (C5), 128.87 (C4), 130.11
(C8), 130.36 (C6), 132.03 (C2), 133.96 (C7), 138.96 (Cβ),
141.46 (C5’), 143.67 (C1), 153.94 (C2’), 155.18 (C4’),
191.12 (C=O); IR (KBr) νmax/cm-1 3456, 774 (C-S), 1660,
1214 (C=O), 1610 (C=C), 1321, 1027 (C-O), 2834, 1516,
846 (Ar).
ethanol to give white solids. Hydrazides 34, 35, 38 and 40
were previously described in the literature,19 while 36, 37
and 39 are novel compounds which have not been reported.
(2E)-N’-(3’-Bromo-4’-hydroxy-5’-methoxybenzilidene)-
3,4,5-trimethoxybenzohydrazide (36)
1
White solid, mp 244-250 ºC; yield: 72%; H NMR
(DMSO-d6) d 3.73 (s, 3H, m-OCH3), 3.87 (s, 6H, m-OCH3),
3.90 (s, 3H, p-OCH3), 7.22 (s, 2H, H2, H6), 7.33 (s, 1H,
H2’), 7.43 (s, 1H, H6’), 8.33 (s, 1H, -N=CH), 10.03 (br,
1H, OH), 11.68 (s, 1H, -CONH); 13C NMR (DMSO-d6)
d 56.78 (m-OCH3), 56.72 (p-OCH3), 60.81 (m- OCH3),
105.85 (C2, C6), 108.88 (C6’), 110.03 (C3’), 125.05
(C1), 127.30 (C2’), 129.29 (C1’), 142.10 (C4), 146.49
(-N=CH), 147.46 (C4’), 149.32 (C5’), 153.37 (C3, C5),
163.16 (-CONH); IR (KBr) νmax/cm-1 3364 (N-H), 1646,
1251 (C=O), 1569 (C=N), 1309, 1027 (C-O), 2985, 1505,
1426, 1296, 1143, 1002 (Ar).
(2E)-1-(2’,4’,5’-Trimethoxyphenyl)-3-(thiophen-2-yl)-2-
propen-1-one (30)
1
Yellow solid; mp 117-119 ºC; yield: 86%; H NMR
(CDCl3) d 3.90 (s, 3H, o-OCH3), 3.94 (s, 3H, m- OCH3),
3.96 (s, 3H, p-OCH3), 6.54 (s, 1H, H3’), 7.07 (t, 1H, J 4.0
Hz, H4), 7.31 (d, 1H, J 4.0 Hz, H5), 7.37 (d, 1H, J 4.0 Hz,
H3), 7.39 (s, 1H, H6’), 7.47 (d, 1H, J 16.0 Hz, Hα), 7.84 (d,
1H, J 16.0 Hz, Hβ); 13C NMR (CDCl3) d 56.37 (o-OCH3),
56.45 (m-OCH3), 57.01 (p-OCH3), 97.33 (C3’), 113.38
(C6’), 120.56 (C1’), 126.51 (Cα), 128.09 (C5), 128.41
(C4), 131.31 (C3), 134.63 (C1’, Cβ), 141.38 (C5’), 153.88
(C2’), 155.13 (C4’), 189.21 (C=O); IR (KBr) νmax/cm-1:
3099, 729 (C-S), 1651, 1267 (C=O), 1606 (C=C), 1307,
1023 (C-O), 2997, 2940, 2829, 1582, 1504, 1406, 1206,
1137, 847, 510 (Ar).
(2E)-N’-(4’-Hydroxy-5’-methoxybenzilidene)-3,4,5-
trimethoxybenzohydrazide (37)
1
White solid; mp 198ºC; yield: 72%; H NMR
(DMSO-d6) d 3.71 (s, 3H, m-OCH3), 3.82 (s, 6H,
m-OCH3), 3.85 (s, 3H, p-OCH3), 6.84 (d, 1H, J 8.0
Hz, H3’), 7.08 (d, 1H, J 8.0 Hz, H2’), 7.20(s, 2H,
H2, H6), 7.30 (s, 1H, H6’), 8.34 (s, 1H, -N=CH),
9.55 (br, 1H, OH), 11.52 (s, 1H, -CONH); 13C NMR
(DMSO-d6) d 56.25 (p-OCH3), 56.77 (m-OCH3), 60.81
(m-OCH3), 105.78 (C2, C6), 109.64 (C6’), 116.15
(C3’), 122.83 (C2’), 126.36 (C1’), 129.45 (C1), 140.96
(C4), 148.74 (-N=CH), 149.08 (C4’), 149.73 (C5’),
153.37 (C3, C5), 162.99 (-CONH); IR (KBr) νmax/cm-1
3418 (N-H), 1664, 1230 (C=O), 1584 (C=N), 1338, 1030
(C-O), 3265, 2885, 1612, 1495, 1411, 1097 (Ar).
General procedure for the synthesis and purification of
3,4,5-trimethoxybenzohydrazide (41)16
3,4,5-trimethoxybenzohydrazide was obtained from
a mixture of gallic acid (6 mmol), dimethyl sulphate
(28 mmol), K2CO3 (26 mmol) and tetrabutylammonium
iodide (TBAI) (0.1 g) in acetone (30 mL). The mixture was
refluxed for 12 h, after which the precipitate was filtered
and washed with acetone. The ester obtained (6 mmol)
was treated with a solution of 99% N2H4 (4.4 mmol) in
methanol (20 mL) and refluxed for 5 h and then kept
at room temperature overnight. The solid obtained was
filtered and recrystallized from hot methanol to afford the
3,4,5-trimethoxybenzohydrazide (80%).
(2E)-N’-(4’-buthoxybenzilidene)-3,4,5-trimethoxy-
benzohydrazide (39)
White solid; mp 119ºC; yield: 61%; 1H NMR (CDCl3)
d0.96(t,CH3),1.47(m,-CH2-CH3),1.71(m,-CH2-CH2-CH3),
4.04 (t, -O-CH2-), 3.39 (s, 9H, m-OCH3), 3.45 (s, 3H,
p-OCH3), 3.85 (s, 3H, OCH3), 7.04 (d, 2H, J 8.0 Hz,
H3’, H5’), 7.24 (s, 2H, H2, H6), 7.68 (d, 2H, J 8.0 Hz,
H2’, H6’), 8.41 (s, 1H, -N=CH), 11.61 (s, 1H, -CONH);
13C NMR (CDCl3) d 14.38 (CH3), 19.41 (-CH2-CH3), 31.36
(-CH2-CH2-CH3), 56.78 (m-OCH3), 60.83 (p-OCH3),
68.02 (-O-CH2-), 105.80 (C2, C6), 115.50 (C3’, C5’),
127.37 (C1’), 129.35 (C1), 129.40 (C2’, C6’), 148.41
(-N=CH), 153.37 (C3, C5), 161.03 (C4’), 163.07 (C=O);
IR (KBr) νmax/cm-1 3236 (N-H), 1652, 1237 (C=O), 1577
(C=N), 1330, 1027 (C-O), 2961, 2875, 1509, 1413, 1237,
1123, 998, 848 (Ar).
General procedure for the synthesis and purification of N´-
substituted benzylidene-3,4,5-trimethoxybenzohydrazide
(34-40)17
The hydrazides were synthesized as shown in Scheme 2.
3,4,5-trimethoxybenzohydrazide (2 mmol) was mixed with
the appropriate aldehyde (2 mmol) in methanol (15 mL)
and refluxed for 2 h. After cooling, the crude product was
collected by filtration, washed and recrystallized from hot

Vol. 21, No. 1, 2010
Borchhardt et al.
149
Biochemical evaluation of the synthetic compounds
References
Materials
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The activity of the enzyme was measured and quantified
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Acknowledgments
We gratefully acknowledge financial support from
CNPq, FAPESP and CAPES, Brazil. We thank Prof. Ana
Paula C. A. Lima from the Federal University of Rio de
Janeiro for the T. cruzi cruzain clone and the Department
of Chemistry, UFSC, for the chemical analysis.
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Received: April 16, 2009
Web Release Date: October 29, 2009
FAPESP helped in meeting the publication costs of this article.