Synthesis and biological evaluation of benzimidazole-5-carbohydrazide derivatives as antimalarial, cytotoxic and antitubercular agents

Article abstract of DOI:10.1016/j.bmc.2011.01.050

A series of N′-substituted-2-(5-nitrofuran or 5-nitrothiophen-2-yl)- 3H-benzo[d]imidazole-5-carbohydrazide derivatives were synthesized and investigated for their abilities to inhibit β-hematin formation, hemoglobin hydrolysis and in vivo for their antima

Full text of DOI:10.1016/j.bmc.2011.01.050

Bioorganic & Medicinal Chemistry 19 (2011) 2023–2029
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of benzimidazole-5-carbohydrazide
derivatives as antimalarial, cytotoxic and antitubercular agents
José Camacho a, Arthur Barazarte a, Neira Gamboa b, Juan Rodrigues b, Rosario Rojas c, Abraham Vaisberg c,
Robert Gilman c, Jaime Charris a,
⇑
a Laboratorio de Síntesis Orgánica, Universidad Central de Venezuela, Aptdo. 47206, Los Chaguaramos, 1041-A Caracas, Venezuela
b Unidad de Bioquímica, Facultad de Farmacia, Universidad Central de Venezuela, Aptdo. 47206, Los Chaguaramos, 1041-A Caracas, Venezuela
c Laboratorios de Investigacion y Desarrollo, Facultad de Ciencias y Filosofia, Universidad Peruana Cayetano Heredia, Lima, Perú
a r t i c l e i n f o
a b s t r a c t
A series of N0-substituted-2-(5-nitrofuran or 5-nitrothiophen-2-yl)-3H-benzo[d]imidazole-5-carbohyd-
razide derivatives were synthesized and investigated for their abilities to inhibit b-hematin formation,
hemoglobin hydrolysis and in vivo for their antimalarial efficacy in rodent Plasmodium berghei. Selected
analogues were screened for their antitubercular activity against sensitive MTB H37Rv and multidrug-
resistant MDR-MTB strains, and cytotoxic activity against a panel of human tumor cell lines and two non-
tumourogenic cell lines. Compounds 3a, 5a, f, 6g were the most promising as inhibitors of b-hematin
formation, however, their effect as inhibitors of hemoglobin hydrolysis were marginal. The most active
compounds to emerge from the in vitro and in vivo murine studies were 3a and 6i, suggesting an anti-
malarial activity via inhibition of b-hematin formation and are as efficient as chloroquine. The cytotoxic
and antitubercular activities of the present compounds were not comparable with those of the standard
drugs employed. But, however, compound 5b showed better antitubercular activity compared to rifam-
pin against multidrug-resistant MDR-MTB strains. Compounds 3a, 6i and 5b showed a good safety index.
Ó 2011 Elsevier Ltd. All rights reserved.
Article history:
Received 9 December 2010
Revised 20 January 2011
Accepted 24 January 2011
Available online 1 February 2011
Keywords:
Antimalarial
Benzimidazole
Plasmodium berghei
b-Hematin
Tuberculosis
Cancer
1. Introduction
process is also thought to be the molecular target of other quino-
line antimalarials.4 Hemozoin was originally considered to be
Malaria caused 350–500 million clinical episodes annually and
result in over one million deaths, most of which affect children un-
der 5 years old in sub-Saharan Africa. Malaria is the fifth cause of
death from infectious diseases worldwide (after respiratory infec-
tions, HIV/AIDS, diarrhoeal diseases and tuberculosis). Recent esti-
mates show that as many as 3.3 billion people live in areas at risk
of malaria in 109 countries. In addition to its health toll, malaria
puts a heavy economic burden on endemic countries and contrib-
utes to the cycle of poverty people face in many countries.1 Malaria
mortality and morbidity began to increase in the 1980s due to a
combination of factors such as the increase in parasite and vector
resistance to the current antimalarial drugs and insecticides, the
weakening of traditional malaria control programs, rapid decen-
tralization and integration into deteriorating primary health ser-
vice, and the development of humanitarian crisis situations in
many malaria-endemic areas. This dramatic increase led to a com-
pelling and urgent necessity for new antimalarials, with mecha-
nisms of action different from the existing ones, and to identify
new drug targets.2 Chloroquine has recently been shown to inhibit
hemozoin formation within the parasite food vacuole.3 This
formed by the polymerization of heme, but it has now been dem-
onstrated to be a crystalline cyclic dimmer of ferriprotoporphyrin
IX.5–8 Thus, hemozoin synthesis, a process unique to the malaria
parasite, offers a logical and valuable potential target for new anti-
malarial drug development. New drugs that attack the same vital
target of chloroquine but that are not subject to the same resis-
tance mechanism would be highly desirable.
Despite the availability of highly potential antitubercular
agents, tuberculosis (TB) remains primary cause of comparatively
high mortality worldwide. The statistics shows that around 32%
of the world’s population is infected by Mycobacterium tuberculosis,
the main causal agent of TB and today more people die from tuber-
culosis than ever before.9–11 Problems in the chemotherapy of TB
arise when patients develop bacterial resistance to the first-line
drugs. Therefore, the development of new drugs with activity
against multi drug-resistant TB, extensively drugs-resistant TB
and latent TB is a priority task.
Benzimidazole scaffolds is one of the privileged structures in
medicinal chemistry. Indeed, various examples featuring these
particular scaffolds have been prepared, many exhibiting remark-
able biological activities.12–18
On the other hand, nitroaromatic compounds are very impor-
tant group, which have been used extensively in the treatment of
⇑
Corresponding author. Fax: +58 2126052707.
E-mail addresses: jaimecharris11@cantv.net, jaime.charris@ucv.ve (J. Charris).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.01.050

2024
J. Camacho et al. / Bioorg. Med. Chem. 19 (2011) 2023–2029
anaerobic infections, and are under continuum investigation. There
is a direct proof that free-radical metabolites are involved in many
applications including important antitumor, antiparasitic and
antibacterial agents.19–23 After extensive literature search, it was
observed that, till date enough effort has not been made to com-
bine these moieties as a single molecular scaffold and identify
new candidates that may be value in designing new, potent, selec-
tive and less toxic antimalarial and antitubercular agents. The
choice of the carbohydrazide moiety was motive to the relation
with the structure of the isoniazid and pyrazinamide agents used
for tuberculosis chemotherapy. In view of this data, we reported
the synthesis of nitrofuran and nitrothiophene incorporated with
bezimidazole and carbohydrazide which possessed wide variety
of biological activity encouraging antimalarial activity against
Plasmodium berghei in vivo and antitubercular activity against
M. tuberculosis H37Rv and MDR. Additionally, we also report the
cytotoxic activity against several cancer cell lines.
(NO2) with 1H NMR a singlet around 10.5 and 10.6 ppm accounted
for NH vanished on D2O exchange, doublets around 7.5 and
0
0
7.9 ppm J: 3–4 Hz assigned to protons H3 and H4 , respectively
and benzimidazole moiety protons as doublets around 7.7 ppm J:
8.5 Hz assigned to proton H7, doublet of doublets around 7.9 ppm
J: 8.5 and 2.0 Hz assigned to proton H6, and doublets around
8.3 ppm J: 2.0 Hz assigned to proton H4. The synthetic route lead-
ing to the title compounds is summarized in Scheme 1.
2.2. Biological
All analogs of those derivatives were tested in vitro for their
effects as inhibitors of b-hematin formation, inhibition of hemo-
globin hydrolysis, and in vivo for their efficacy in a murine model
(Table 1). The first mentioned in vitro assay was used to assess
the abilities of the derivatives N0-substituted-2-(5-nitrofuran
or 5-nitrothiophen-2-yl)-3H-benzo[d]imidazole-5-carbohydrazide
derivatives 5a–i, 6a–i to inhibit b-hematin formation. To evaluate
the potential antimalarial activity of compounds, we tested the
ability of these compounds to inhibit heme crystallization, con-
sidering that heme can crystallize spontaneously under acid and
low oxygen condition found in the vacuole of the parasite.25 Re-
sults that showed more than 90% of inhibition of heme crystalli-
zation were considered significant (compounds 3a, 5a, f, 6g Table
1), results reveal that the compounds were as active as chloro-
quine (94.19 0.36%) inducing the inhibition of heme crystalliza-
tion. The 5-nitrofuranyl moiety appeared to be favourable for the
potential antimalarial activity, since most of the compounds pos-
sessing showed measurable levels of inhibition of b-hematin for-
mation, except compound 6g having 5-nitrothiophene-2-yl
showed good activity. All compounds were tested for their capac-
ity of inhibiting haemoglobin proteolysis, in an in vitro assay
which uses trophozoyte-rich extract to digest the native haemo-
globin of mice. The electrophoretic analysis indicated that only
compound 6c was partially effective inhibiting the proteolysis
of haemoglobin 44.93 1.12% compared with leupeptin (LEU)
and pepstatin (PEP) 91.62 0.69 and 95.45 0.66, respectively
Table 1.
2. Results and discussion
2.1. Synthesis
Our strategy for synthesis of 5a–i and 6a–i, is a very simple one
as illustrated in Scheme 1. In the procedure here described 3a,b
were obtained by the reaction of 3,4-diaminobenzoic acid 1 with
5-nitro-2-furaldehyde 2a and 5-nitro-2-thiophenecarbaldehyde
2b, to obtain the title compounds in good yields (Scheme 1). In this
case nitrobenzene was used as an oxidant following an analogous
procedure.24 Compounds 3a–b were allowed to react with thionyl
chloride to obtain the corresponding chloro derivatives, com-
pounds 4a–b were not isolated from the reaction medium, so were
condensed with the appropriately substituted aryl acid hydrazides
in DCM and DMAP to afford the target molecules. The final com-
pounds were purified by recrystallization from ethanol, and the
structure of the compounds was confirmed by IR, 1H NMR and ele-
mental analysis. The IR spectra of target compounds 5a–i and 6a–i
showed the broad stretching band around 3400 and 3200 cmꢀ1
due to (NH) and around 1530–1510 and 1350–1340 cmꢀ1 due
H
NH2
N
i
+
OHC
X
NO2
N
X
2a,b
NO2
HO2C
NH2
HO2C
3a,b
1
ii
H
N
X
NO2
N
ClOC
4a,b
iii
7
4
H
N 1
O
H
N
X
NO2
3
N
R1
N
H
O
5a-i, 6a-i
Scheme 1. Synthesis of N0-substituted-2-(5-nitrofuran or 5-nitrothiophe-2-yl)-3H-benzo[d]imidazole-5-carbohydrazide derivatives 5a–i, 6a–i. (i) nitrobenzene,
D
. (ii) SOCl2,
benzene, DMF, . (iii) CH2Cl2, DMAP, 0 °C? rt, X: O, S. R1: H, phenyl, naphthyl.
D

J. Camacho et al. / Bioorg. Med. Chem. 19 (2011) 2023–2029
2025
Table 1
Inhibition of b-hematin synthesis (IHS), globin proteolysis (IGP) (%), effect of benzimidazole derivatives (20 mg/kg) on parasitemia at fourth day post-infection (%P) and survival
days (SD) of P. berghei infected-mice by benzimidazole derivatives
No.
R1
IbHS
IC50
(
lM)
IGP
%P
4.02 0.45
SD
3a
3b
5a
5b
5c
5d
5e
5f
5g
5h
5i
6a
6b
6c
6d
6e
6f
6g
6h
6i
Leu
Pep
CQ
SS
95.43 0.58*
57.63 1.96
94.05 1.9*
79.21 0.63
73.43 5.88
59.62 3.61
72.44 1.38
94.76 0.49*
69.38 2.45
84.63 1.75
31.74 4.89
87.39 0.97
86.18 0.73
78.59 0.74
25.48 1.91
61.64 4.48
70.77 6.95
92.36 0.48*
76.49 2.7
75.76 0.99
—
8.43
0
0
17 1.26
21.09
8.63
10.92
10.30
23.35
13.13
8.71
14.40
14.06
ND
9.88
10.78
15.96
ND
24.50
21.36
9.56
19.12
11.10
—
11.5 2.06
25.8 0.86
10.14 3.35
3.38 1.17
27.4 1.08
5.98 1.56
4.8 2.22
9.4 0.6
12.2 1.15
8.2 0.86
13.6 1.28
12.4 2.29
9.8 1.07
11.8 1.98
12.2 1.65
12.2 0.58
10.6 0.81
ND
10.04 1.53
12 1.41
7.4 0.4
10.8 1.59
10.6 1.57
13.8 2.35
10 1.3
H
Ph
3-ClPh
4-ClPh
3-MeOPh
3,4-MeOPh
3,4,5-MeOPh
3-PhOPh
3-OH-2-Naphtyl
H
Ph
3-ClPh
4-ClPh
3-MeOPh
3,4-MeOPh
3,4,5-MeOPh
3-PhOPh
3-OH-2-naphtyl
—
24.24 1.14
0
38.98 0.92
33.75 1.74
2.82 0.85
34.53 1.16
0
ND
12.95 3.8
ND
12.87 0.43
36.16 1.12
39.19 0.45
44.93 1.10**
12.72 1.21
13.36 0.98
38.18 0.87
ND
18.72
3
10.12 2.13
27.2 0.91
13.52 3.71
24.8 1.32
16.06 2.64
9.36 3.59
7.28 1.67
1.8 0.49
—
0
11 2.4
14.08 0.88
91.62 0.69
95.45 0.66
24.12 1.16
—
18.8 2.05
—
—
—
—
—
—
—
—
—
—
94.19 0.36
—
1.3 0.3
21.8 2.31
>30
11.66 1.66
X 3a, (5a–i) = O; X 3b (6a–i) = S; Leu = leupeptin; Pep = pepstatin; CQ = chloroquine; SS = saline solution.
The results are expressed by the mean standard error of the mean.
*
p >0.05 compared to chloroquine.
p >0.05 compared to leupeptin (LEP) and pepstatin (PEP).
**
p <0.05.
p <0.001 compared to saline solution and chloroquine. n = 6.
Compounds 5a–i and 6a–i were tested in infected mice with
P. berghei ANKA, a chloroquine-susceptible strain of murine malar-
ia. Compounds were given to mice (chloroquine or 5a–i, 6a–i in
20 mg kgꢀ1, ip once daily) for four consecutive days (days 1–4
post-infection). At day fourth post-infection, the parasitemia was
determined; the survival days were monitored and compared with
control mice receiving a saline solution (untreated mice). Control
mice died within 8.2 0.37 days post-infection, compounds 3a,
5f and 6i increased the survival time for 17 1.26, 12.2 1.65
and 18.8 2.05 days, respectively, while chloroquine prolonged
the survival time of the infected mice to 30 days. Compounds 3a,
5f and 6i were able to reduce and delay the progression of malaria
(4.02 0.45, 4.8 2.22 and 1.8 0.49%), respectively but did
not eradicate the infection compared with chloroquine 1.3 0.3
(Table 1).
On the other hand, due to the urgent need to discover new
drugs for the treatment of TB, several colorimetric methods for
rapid screening of pure compounds or crude natural product ex-
tracts have been developed.26–28 The microplate Alamar blue assay
(MABA) is a rapid, nonradioactive, and inexpensive method that is
based on the color change of the redox dye Alamar blue for the
detection of viable M. tuberculosis.29,30 Members of our group have
developed a variant of this assay that uses tetrazolium bromide in-
stead of the MABA.31 This tetrazolium microplate assay (TEMA)
determines MIC values as quickly (6–7 days) and accurately as
the MABA procedure. Compounds 3a, 5a,b,e–h and 6e,g,i were
tested in vitro for their antimycobacterial activity against sensitive
M. tuberculosis H37Rv strain and a multidrug-resistant (MDR) clin-
ical isolate. Results revealed that compounds 5b exhibited a mod-
erated antimycobacterial activity, with a MIC value of 12.5 lg/mL
Table 2
Cytotoxicity and antimycobacterial activity of benzimidazole derivatives against 3T3, BALB/3T3 clone A31 embryonic mouse fibroblast cells; Vero, normal African green monkey
kidney epithelial cells; H460, human large cell lung cancer; DU145, human prostate carcinoma; MCF-7, human breast adenocarcinoma; M-14, human melanoma; HT-29, human
colon adenocarcinoma; K562, human chronic myelogenous leukemia cells; sensitive MTB H37Rv strain; multidrug-resistant MDR strain of MTB
No.
GI50
DU145
125
15.63
15.63
15.63
15.63
15.63
>7.8
(
l
g/mL)
MCF-7
>250
62.5
MIC (
l
g/mL)
MDR
3T3
VERO
>250
H460
M-14
HT-29
K562
H37Rv
3a
5a
5b
5e
5f
5g
5h
6e
6g
6i
62.5
62.5
125
250
250
250
>25
>25
12.5
25
>25
>25
>25
>25
>25
>25
—
>25
>25
6.25
25
25
25
62.5
31.25
62.5
62.5
15.63
31.25
>7.8
62.5
62.5
62.5
0.3
31.25
15.63
7.81
15.63
15.63
>7.8
15.63
62.5
62.5
3.3
62.5
31.25
31.25
15.63
31.25
>7.8
31.25
62.5
62.5
5.3
31.25
15.63
15.63
15.63
31.25
>7.8
15.63
31.25
31.25
0.3
31.25
15.63
31.25
31.25
>7.8
15.63
31.25
31.25
0.7
31.25
31.25
62.5
31.25
>7.8
62.5
15.63
31.25
31.25
>7.8
15.63
31.25
62.5
25
25
>250
62.5
62.5
62.5
0.6
31.25
31.25
0.8
>25
>25
—
4?32
2?16
C-P
INH
RIF
>5
0.125
0.063
C-P, Cisplatin; INH, isoniazid; RIF, rifampin.

2026
J. Camacho et al. / Bioorg. Med. Chem. 19 (2011) 2023–2029
against sensitive M. tuberculosis H37Rv strain and 6.25 lg/mL
against the multidrug-resistant (MDR) clinical isolates, compared
with reference drugs isoniazid (INH) and rifampin (RIF) MIC
4. Experimental protocols
4.1. Chemistry
0.063 and 32 lg/mL (Table 2).
Cytotoxic activity of compounds 3a, 5a,b,e–h and 6e,g,i have
been assayed using a murine fibroblast nontransformed cell line
as control, one normal African green monkey kidney epithelial
cells and seven human cancerous cell lines. The results of cyto-
toxic activity are summarized in Table 2. The results showed that
compounds 3a, 5a,b,e–h and 6e,g,i displayed moderate to weak
Melting points were determined on a Thomas micro hot
stage apparatus and are uncorrected. Infrared spectra were deter-
mined as KBr pellets on a Shimadzu model 470 spectrophotometer.
The 1H NMR spectra were recorded using a Jeol Eclipse 270
(270 MHz) spectrometer using DMSO-d6, and are reported in
ppm downfield from the residual DMSO. Elemental analyses were
performed on a Perkin–Elmer 2400 CHN analyser, results were
within 0.4% of the predicted values for all compounds. Chemical
reagents were obtained from Aldrich Chemical Co., USA. All sol-
vents were distilled and dried in the usual manner.
activity (7.81–250
lines.
lg/mL) against control and cancerous cell
2.3. Structure–activity relationship study
Having confirmed the activity of compound 3a, we embarked
a hit-to-lead exploration program focusing on CO2H of
4.1.1. General procedure for the synthesis of 2-(5-nitrofuran or
5-nitrothiophen-2-yl)-3H-benzo[d]imidazole-5-carboxylic acid
3a–b
on
benzimidazole nucleus. The first step toward lead optimization
was incorporation of formylhydrazido group. The inhibition of
heme polymerization study data of this compound showed the
same activity that 3a, but the activity in vivo against P. berghei
infected mice was marginal, the cytotoxic and antimicobacterial
activities also were marginal. One significant problem with
accurate measurements of kinetic constant and other activities
for this compound has been solubility. Hence we planned to intro-
duce the aromatic group with electron withdrawing and donating
groups at different position to study its influence on activity.
Different analogs with H, chlorine, hydroxyl, aryloxy, and methoxy
groups were synthesized. In majority of the cases, compounds
having H, chlorine, phenoxy and monomethoxy groups exhibited
marginal activity as antimalarial, antimicobacterial and cytotoxic.
However, compounds with di- or tri-methoxy substituents and a
5-nitrofuranyl or 5-nitrothiophene-2-yl moiety in position 2 of
benzimidazole exhibited good activity in vitro as inhibitors against
heme polymerization study. In exceptional case, N0-(3-hydroxy-2-
naphthyl)-2-(5-nitrothiophen-2-yl)-3H-benzo[d]-imidazole-5-car-
bohydrazide 6i was found better in vivo as antimalarial.
A mixture of the 5-nitro-2-furaldehyde/5-nitro-2-thiophen-
carbaldehyde 2a–b (5 mmol), 3,4-diaminobenzoic acid 1 (5 mmol),
in nitrobenzene (25 mL) was refluxed for 8 h. The solvent was
evaporated to dryness under reduced pressure, n-heptane was
added (10 mL) and the solid thus obtained was collected by filtra-
tion, washed with methanol and ethyl ether. Further purification
was accomplished by recrystallization from ethanol.
4.1.1.1. 2-(5-Nitrofuran-2-yl)-3H-benzo[d]imidazole-5-carbox-
ylic acid 3a32
. Green solid. Yield 85%. mp 294–296 °C; IR (KBr)
cmꢀ1: 3632, 3152, 1692, 1626 (CO2H, CH@CH), 1546 (NO2). 1H
0
RMN, DMSO-d6, d ppm, 80 °C: 7.53 (d, 1H, H4 , J = 3.9 Hz); 7.70 (d,
0
1H, H7, J = 8.6 Hz); 7.83 (d, 1H, H3 , J = 3.9 Hz); 7.92 (dd, 1H, H6,
J = 1.3, 8.6 Hz); 8.24 (s, 1H, H4); 13.25 (br s, 1H, OH). Anal. Calcd
for C12H7N3O5: C, 52.76; H, 2.58; N, 15.38. Found: C, 52.80; H,
2.56; N, 15.41.
4.1.1.2. 2-(5-Nitrothiophen-2-yl)-3H-benzo[d]imidazole-5-car-
boxylic acid 3b. Brown solid. Yield 89%. mp > 300 °C; IR (KBr)
cmꢀ1: 3280, 1696, 1619 (CO2H, CH@CH), 1516 (NO2). 1H RMN,
DMSO-d6, d ppm, 80 °C: 7.68 (d, 1H, H7, J = 8.7 Hz); 7.87 (d, 1H,
3. Conclusion
0
0
H4 , J = 4.3 Hz); 7.90 (dd, 1H, H6, J = 1.2, 8.7 Hz); 8.19 (d, 1H, H3 ,
J = 4.3 Hz); 8.21 (s, 1H, H4); 13.40 (br s, 1H, OH). Anal. Calcd for
The present study describes the synthesis and the in vitro, and
in vivo antimalarial, antimycobacterial and tumor cells activity of
tripartite hybrids from pharmacophores benzimidazole, 5-nitrofu-
ran or 5-nitrothiophene-2-yl and substituted aryl acid hydrazides.
In summary, some of them showed good selectivity index between
the parasite, antimycobacterial and tumor cells. Compounds 3a,
5a,f, 6g exhibited potential effects as inhibitors of b-hematin
formation, 5b against sensitive M. tuberculosis strain and multi-
drug-resistant (MDR) clinical isolate. The study confirms that anti-
malarial mechanism of action could be similar to that of
chloroquine, as most of the compounds form an association com-
plex with b-hematin and thereby inhibit hemozoin formation.
The results provide basic information to establish that a 5-nitrothi-
ophene-2-yl moiety on position two of the bezimidazole is not very
essential for an inhibitory activity of heme dimerization, however,
a 5-nitrofuranyl moiety is essential and opens new vistas for de-
sign of new antimalarial agents. Rationally, such a combination
of antitropozoal pharmacophores and other functionalities offers
many attractive features for accelerating antimalarial, antimyco-
bacterial. Due to the better activity as inhibitors of b-hematin for-
mation, compounds 3a, 5a,f, 6g, 4-day suppressive test compound
6i, and against tested MT-MDR clinical isolates, compound 5b have
been selected for further development and studies to acquire more
information about structure–activity relationships and pharmaco-
kinetic are in progress in our laboratories.
C12H7N3O4S: C, 49.83; H, 2.44; N, 14.53. Found: C, 49.79; H, 2.46;
N, 14.54.
4.1.2. General procedure for the synthesis of N0-substituted-2-
(5-nitrofuran or 5-nitrothiophen-2-yl)-3H-benzo[d]imidazole-
5-carbohydrazide derivatives 5a–i, 6a–i
A mixture of 3a,b (0.3 mmol), thionyl chloride (1.5 mmol), and
benzene 25 mL was gradually heated to boiling, whereupon the
acid chloride dissolved. After refluxing for 12 h, excess of thionyl
chloride and solvent were removed in vacuo. The acid chloride
was not characterized, then to a solution of acid chloride, DMAP
(1.5 mmol) in dry CH2Cl2 (10 mL) was slowly added a dry CH2Cl2
(10 mL) solution of carbohydrazide respective (0.4 mmol)
(30 min) at 0 °C, and the mixture was stirred for 36 h at rt. The sol-
vent was removed in vacuo and the residue was slowly added
water 5% KOH at 0 °C and the mixture stirred for 30 min. The solid
was washed with water, methanol and ethyl ether, recrystallized
from a mixture of ethanol and water (1:1) to give 5a–i, 6a–i.
4.1.2.1. N0-Formyl-2-(5-nitrofuran-2-yl)-3H-benzo[d]imidazole-
5-carbohydrazide 5a. Yield 66%. mp 250 °C (dec). IR (KBr), cmꢀ1
:
3132 (NH), 1650, 1604 (Ar) 1344 (NO2). 1H RMN, DMSO-d6, d ppm:
0
7.57 (t, 1H, H3 , J = 4.0 Hz); 7.77 (d, 1H, H7, J = 8.8 Hz); 7.90 (d, 1H,
0
H4 , J = 4.0 Hz); 7.92 (dd, 1H, H6, J = 1.5, 8.8 Hz); 8.27 (s, 1H, H4);

J. Camacho et al. / Bioorg. Med. Chem. 19 (2011) 2023–2029
2027
9.03 (s, 1H, CHO); 10.68 (s, 1H, NH); 10.80 (s, 1H, NH). Anal. Calcd
for C13H9N5O5: C, 49.53; H, 2.88; N, 22.21. Found: C, 49.50; H, 2.92;
N, 22.30.
RMN, DMSO-d6, d ppm: 7.08 (d, 1H, Ar, J = 7.68 Hz); 7.20 (t, 1H,
00
H5 , J = 7.32 Hz); 7.25 (dd, 1H, Ar, J = 2.20, 8.08 Hz); 7.44 (t, 2H,
000,5000
H3
, J = 7.3 Hz); 7.54–7.57 (m, 3H, Ar); 7.60(d, 1H, H3´ ,
J = 3.5 Hz); 7.73–7.76 (m, 2H, Ar); 7.89 (d, 1H, H4´ , J = 3.5 Hz);
7.92 (dd, 1H, H6, J = 1.2, 8.6 Hz); 8.29 (s, 1H, H4); 10.56 (s, 1H,
NH); 10.61 (s, 1H, NH). Anal. Calcd for C25H17N5O6: C, 63.11; H,
3.54; N, 14.49. Found: C, 63.19; H, 3.60; N, 14.40.
4.1.2.2.
N0-Benzoyl-2-(5-nitrofuran-2-yl)-3H-benzo[d]imidaz-
ole-5-carbohydrazide 5b. Yield 55%. mp 220–222 °C. IR (KBr),
cmꢀ1: 3133 (NH), 1602 (Ar) 1342 (NO2). 1H RMN, DMSO-d6, d
00 00
0
ppm: 7.54 (t, 2H, H3 ,5 , J = 7.7 Hz); 7.57 (d, 1H, H3 , J = 3.6 Hz);
N0-(3-Hydroxy-2-naphthyl)-2-(5-nitrofuran-2-yl)-3H-
5i. Yield 60%.
00
7.61 (t, 1H, H4 , J = 7.3 Hz); 7.75 (d, 1H, H7, J = 8.4 Hz); 7.88 (d,
4.1.2.9.
benzo[d]imidazole-5-carbohydrazide
1H, H4´ , J = 3.6 Hz); 7.92 (dd, 1H, H6, J = 1.6, 8.6 Hz); 7.94 (d, 2H,
H2 ,6 , J = 7.4 Hz); 8.28 (d, 1H, H4, J = 1.6 Hz); 10.64 (s, 1H, NH);
10.74 (s, 1H, NH). Anal. Calcd for C19H13N5O5: C, 58.32; H, 3.45;
N, 17.90. Found: C, 58.37; H, 3.48; N, 18.01.
mp >260 °C. IR (KBr), cmꢀ1: 3424 (NH), 1651, 1625 (Ar), 1507,
1344 (NO2). 1H RMN, DMSO-d6, d ppm: 5.6 (br s, 1H, OH); 7.36
(s, 1H, Ar); 7.52 (t, 1H, Ar, J = 7.4 Hz); 7.60 (d, 1H, H3´ , J = 3.3 Hz);
7.66 (t, 1H, Ar J = 6.3 Hz); 7.75–7.78 (m, 2H, Ar); 7.89 (d, 1H, H4´ ,
J = 3.5 Hz); 7.91–7.93 (m, 2H, Ar); 8.29 (s, 1H, H4); 8.57 (s, 1H,
Ar); 10.74 (s, 1H, NH); 10.79 (s, 1H, NH). Anal. Calcd for
00 00
4.1.2.3. N0-(3-Chlorobenzoyl)-2-(5-nitrofuran-2-yl)-3H-benzo[d]-
imidazole-5-carbohydrazide 5c. Yield 73%. mp 190 °C. IR (KBr),
cmꢀ1: 3408, 3232 (NH), 1683, 1645 (Ar), 1520, 1344 (NO2). 1H
RMN, DMSO-d6, d ppm: 7.58 (t, 1H, H5 , J = 7.5 Hz); 7.61–7.65 (m,
2H, Ar); 7.65 (d, 1H, H3 , J = 3.0 Hz); 7.76 (d, 1H, H7, J = 9.0 Hz);
C23H15N5O6: C, 60.40; H, 3.31; N, 15.31. Found: C, 60.42; H, 3.37;
00
N, 15.49.
0
00
7.89–7.92 (m, 2H, Ar); 7.97 (s, 1H, H2 ); 8.27 (s, 1H, H4); 10.66 (s,
4.1.2.10. N’-Formyl-2-(5-nitrothiophen-2-yl)-3H-benzo[d]imid-
azole-5-carbohydrazide 6a. Yield 77%. mp 220 °C (dec). IR (KBr)
cmꢀ1: 3424, 3248 (NH), 1686, 1651 (Ar), 1523, 1334 (NO2). 1H
RMN, DMSO-d6, d ppm: 7.57 (d, 1H, H3´ J = 4.2 Hz); 7.90–8.08 (m,
2H, Ar); 8.14–8.24 (m, 3H, Ar); 9.16 (s, 1H, CHO); 10.46 (s, 1H,
NH); 10.68 (s, 1H, NH). Anal. Calcd for C13H9N5O4S: C, 47.13; H,
2.74; N, 21.14. Found: C, 47.10; H, 2.85; N, 21.32.
1H, NH); 10.68 (s, 1H, NH). Anal. Calcd for C21H19N5O5Cl: C,
59.24; H, 2.84; N, 16.45. Found: C, 58.97; H, 3.03; N, 16.70.
4.1.2.4. N0-(4-Chlorobenzoyl)-2-(5-nitrofuran-2-yl)-3H-benzo[d]-
imidazole-5-carbohydrazide 5d. Yield 68%. mp 178–180 °C. IR
(KBr), cmꢀ1: 3269 (NH), 1699, 1683, 1594 (Ar), 1542, 1347 (NO2).
1H RMN, DMSO-d6, d ppm: 7.60–7.62 (m, 3H, Ar); 7.75 (d, 1H, H7,
0
J = 8.4 Hz); 7.88 (dd, 1H, H6, J = 2.0, 8.4 Hz); 7.92 (d, 1H, H4 ,
4.1.2.11. N’-Benzoyl-2-(5-nitrothiophen-2-yl)-3H-benzo[d]imid-
azole-5-carbohydrazide 6b. Yield 51%. mp >300 °C. IR (KBr),
cmꢀ1: 3312 (NH), 1683 (Ar), 1520, 1342 (NO2). 1H RMN, DMSO-
00 00
00 00
J = 4.0 Hz); 7.96 (d, 2H, H2 ,6
, J = 8.4 Hz); 8.25 (d, 1H, H4,
J = 2.0 Hz); 10.53 (s, 1H, NH); 10.57 (s, 1H, NH). Anal. Calcd for
21H19N5O5Cl: C, 59.24; H, 2.84; N, 16.45. Found: C, 59.26; H,
C
d6,
d
ppm: 7.54 (t, 2H, H3 ,5 , J = 7.7 Hz); 7.58 (d, 1H, H3´ ,
2.89; N, 16.37.
00
J = 4.3 Hz); 7.61 (t, 1H, H4 , J = 7.3 Hz); 7.74 (d, 1H, H7, J = 8.3 Hz);
7.88 (dd, 1H, H6, J = 1.3, 8.3 Hz); 7.92 (d, 1H, H4´ , J = 4.3 Hz); 7.94
4.1.2.5. N0-(3-Methoxybenzoyl)-2-(5-nitrofuran-2-yl)-3H-benzo-
[d]imidazole-5-carbohydrazide 5e. Yield 73%. mp 182–184 °C.
IR (KBr), cmꢀ1: 3296 (NH), 1686, 1603 (Ar), 1350 (NO2). 1H RMN,
DMSO-d6, d ppm: 3.83 (s, 3H, OCH3); 7.18 (dd, 1H, Ar, J = 2.7,
00 00
(d, 2H, H2 ,6 , J = 7.4 Hz); 8.27 (d, 1H, H4, J = 1.3 Hz); 10.63 (s, 1H,
NH); 10.74 (s, 1H, NH). Anal. Calcd for C19H13N5O4S: C, 56.02; H,
3.22; N, 17.19. Found: C, 55.79; H, 3.23; N, 17.46.
0
8.3 Hz); 7.38–7.54 (m, 3H, Ar); 7.65 (d, 1H, H3 , J = 3.5 Hz); 7.77
(d, 1H, H7, J = 9.0 Hz); 7.91 (dd, 1H, H6, J = 1.8, 9.0 Hz); 7.94 (d,
4.1.2.12.
N’-(3-Chlorobenzoyl)-2-(5-nitrothiophen-2-yl)-3H-
benzo[d]imidazole-5-carbohydrazide 6c. Yield 54%. mp 190 °C
0
1H, H4 , J = 3.5 Hz); 8.28 (d, 1H, H4, J = 1.8 Hz); 10.54 (s, 1H, NH);
(dec). IR (KBr), cmꢀ1: 3472 (Ar), 1700 (Ar), 1521, 1336 (NO2). 1H
10.61 (s, 1H, NH). Anal. Calcd for C20H15N5O6: C, 57.01; H, 3.59;
N, 16.62. Found: C, 57.15; H, 3.58; N, 16.78.
00
RMN, DMSO-d6, d ppm: 7.58 (t, 1H, H5 , J = 7.8 Hz); 7.68 (d, 1H,
Ar, J = 8.1 Hz); 7.74 (d, 1H, H7, J = 8.4 Hz); 7.88 (dd, 1H, H6, J = 1.7,
00
0
8.4 Hz); 7.91 (d, 1H, H6 J = 8.1 Hz); 7.95 (d, 1H, H3 , J = 4.8 Hz);
4.1.2.6.
N0-(3,4-Dimethoxybenzoyl)-2-(5-nitrofuran-2-yl)-3H-
00
0
7.97 (s, 1H, H2 ); 7.98 (d, 1H, H4 , J = 4.8 Hz); 8.25 (d, 1H, H4,
J = 1.7 Hz); 10.65 (s, 1H, NH); 10.68 (s, 1H, NH). Anal. Calcd for
19H12N5O4ClS: C, 51.65; H, 2.74; N, 15.85. Found: C, 51.82; H,
benzo[d]imidazole-5-carbohydrazide 5f. Yield 87%. mp 208 °C
(dec). IR (KBr), cmꢀ1: 3392, 3232 (NH), 1683, 1651 (Ar), 1507,
1341 (NO2). 1H RMN, DMSO-d6, d ppm: 3.83 (s, 3H, OCH3); 3.84 (s,
3H, OCH3); 7.08 (d, 1H, Ar, J = 6.3 Hz); 7.55–7.60 (m, 2H, Ar); 7.57
C
2.83; N, 15.97.
0
0
(d, 1H, H3 , J = 3.7 Hz); 7.76 (d, 1H, H7, J = 8.3 Hz); 7.88 (d, 1H, H4 ,
J = 3.6 Hz); 7.90 (dd, 1H, H6, J = 1.2, 8.3 Hz); 8.27 (s, 1H, H4); 10.29
(br s, 1H, NH); 10.42 (bs, 1H, NH). Anal. Calcd for C21H17N5O7: C,
55.88; H, 3.79; N, 15.51. Found: C, 56.02; H, 3.68; N, 15.70.
4.1.2.13.
N0-(4-Chlorobenzoyl)-2-(5-nitrothiophen-2-yl)-3H-
benzo[d]imidazole-5-carbohydrazide 6d. Yield 48%. mp 185 °C
(dec). IR (KBr) cmꢀ1: 3471 (NH), 1699, 1635 (Ar), 1518, 1336
1
0
(NO2). H RMN, DMSO-d6, d ppm: 7.57 (d, 1H, H3 , J = 3.7 Hz);
00 00
7.61 (d, 2H, H3 ,5 , J = 8.4 Hz); 7.76 (d, 1H, H7, J = 8.4 Hz); 7.90
4.1.2.7. N0-(3,4,5-Trimethoxybenzoyl)-2-(5-nitrofuran-2-yl)-3H-
benzo[d]imidazole-5-carbohydrazide 5g. Yield 81%. mp 160 °C
(dec). IR (KBr), cmꢀ1: 3472 (NH), 1694, 1649 (Ar), 1580, 1341
(NO2). 1H RMN, DMSO-d6, d ppm: 3.86 (s, 3H, OCH3); 3.91 (s, 6H,
0
(dd, 1H, H6, J = 1.5, 8.4 Hz); 7.91 (d, 1H, H4 , J = 3.7 Hz); 7.96 (d,
2H, H2 ,6 , J = 8.4 Hz); 8.27 (d, 1H, H4, J = 1.5 Hz); 10.55 (s, 1H,
NH); 10.57 (s, 1H, NH). Anal. Calcd for C19H12N5O4ClS: C, 51.65;
H, 2.74; N, 15.85. Found: C, 51.63; H, 2.77; N, 16.13.
00 00
00 00
0
OCH3); 7.30 (s, 2H, H2 ,6 ); 7.58 (d, 1H, H3 , J = 3.8 Hz); 7.76 (d,
0
1H, H7, J = 8.6 Hz); 7.90 (d, 1H, H4 , J = 3.8 Hz); 7.91 (dd, 1H, H6,
4.1.2.14. N0-(3-Methoxybenzoyl)-2-(5-nitrothiophen-2-yl)-3H-
benzo[d]imidazole-5-carbohydrazide 6e. Yield 68%. mp 147–
149 °C. IR (KBr) cmꢀ1: 3472 (NH), 1683 (Ar), 1578, 1337 (NO2).
1H RMN, DMSO-d6, d ppm: 3.84 (s, 3H, OCH3); 7.16 (dd, 1H, Ar,
J = 1.5, 8.6 Hz); 8.28 (s, 1H, H4); 10.38 (br s, 1H, NH); 10.47 (br s,
1H, NH). Anal. Calcd for C22H19N5O8: C, 54.89; H, 3.98; N, 14.55.
Found: C, 55.09; H, 3.83; N, 14.68.
4.1.2.8. N0-(3-Phenoxybenzoyl)-2-(5-nitrofuran-2-yl)-3H-benzo-
[d]imidazole-5-carbohydrazide 5h. Yield 52%. mp 210–212 °C.
IR (KBr), cmꢀ1: 3472 (NH), 1654, 1649 (Ar), 1580, 1341 (NO2). 1H
00
00
J = 2.2, 8.0 Hz); 7.45 (t, 1H, H5 , J = 7.7 Hz); 7.49 (s, 1H, H2 ); 7.53
(d, 1H, Ar, J = 7.7 Hz); 7.75 (d, 1H, H7, J = 8.4 Hz); 7.88–7.92 (m,
2H, Ar); 8.24–8.25 (m, 2H, Ar); 10.46 (s, 1H, NH); 10.50 (s, 1H,

2028
J. Camacho et al. / Bioorg. Med. Chem. 19 (2011) 2023–2029
NH). Anal. Calcd for C20H15N5O5S: C, 54.92; H, 3.46; N, 16.01.
Found: C, 55.03; H, 3.52; N, 16.25.
4.2.2. Parasite, experimental host and strain maintenance
Male Balb-C mice, weighing 18–22 g were maintained on a
commercial pellet diet and housed under conditions approved
by Ethics Committee. P. berghei (ANKA strain), a rodent malaria
parasite, was used for infection. Mice were infected by ip injection
with 1 ꢁ 106 infected erythrocytes diluted in phosphate buffered
saline solution (PBS, 10 mM, pH 7.4, 0.1 mL). Parasitemia was
monitored by microscopic examination of Giemsa stained
smears.33
4.1.2.15. N0-(3,4-Dimethoxybenzoyl)-2-(5-nitrothiophen-2-yl)-
3H-benzo[d]imidazole-5-carbohydrazide 6f. Yield 48%. mp
198 °C (dec). IR (KBr), cmꢀ1: 3568, 3296 (NH), 1670, 1598 (Ar),
1518, 1337 (NO2). 1H RMN, DMSO-d6, d ppm: 3.75 (s, 3H, OCH3);
00
0
3.80 (s, 3H, OCH3); 7.08 (d, 1H, H5 , J = 8.4 Hz); 7.53 (d, 1H, H3 ,
00
J = 3.3 Hz); 7.58 (dd, 1H, H6 , J = 1.5, 8.4 Hz); 7.76 (d, 1H, H7,
0
J = 8.6 Hz); 7.88 (dd, 1H, H6, J = 2.3, 8.6 Hz); 7.92 (d, 1H, H4 ,
4.2.3. Parasite extracts
J = 3.1 Hz); 8.24–8.26 (m, 2H, Ar); 10.39 (s, 1H, NH); 10.51 (s, 1H,
NH). Anal. Calcd for C21H17N5O6S: C, 53.96; H, 3.67; N, 14.98.
Found: C, 54.12; H, 3.69; N, 14.90.
Blood of infected animals, at a high level of parasitemia
(30–50%), was collected by cardiac puncture with an heparinized
syringe and the blood pool was centrifuged (500g ꢁ 10 min, 4 °C).
Plasma and buffy coat were removed and the red blood cells
(RBC) pellet was washed twice with chilled PBS-Glucose (5.4 %).
The washed RBC pellet was centrifuged on a discontinuous percoll
gradient (80–70% percoll in PBS-Glucose, 20,000g ꢁ 30 min ꢁ
4 °C).34 The upper band (mature forms) was removed by aspiration,
collected in eppendorf tubes and washed twice with chilled PBS-
Glucose and the infected erythrocytes were lysed with the non-
ionic detergent saponin (0,1% in PBS ꢁ 10 min). 1 mL of cold PBS
was added and the samples were centrifuged (13,000g ꢁ 5 min,
4 °C) to remove erythrocyte cytoplasm content (including erythro-
cyte haemoglobin). The free parasites were mixed PBS-Glucose (5.4
%), and subjected to three freeze–thaw cycles (ꢀ70 °C/+37 °C). The
final homogenate was used in the haemoglobin hydrolysis inhibi-
tion assay.35
4.1.2.16.
N0-(3,4,5-Trimethoxybenzoyl)-2-(5-nitrothiophen-2-
yl)-3H-benzo[d]imidazole-5-carbohydrazide 6g. Yield 76%. mp
285 °C (dec). IR (KBr), cmꢀ1: 3348 (NH), 1644 (Ar), 1584, 1520,
1334 (NO2). 1H RMN, DMSO-d6, d ppm: 3.73 (s, 3H, OCH3); 3.85
(s, 6H, OCH3); 7.29 (s, 2H, H2 ,6 ); 7.50 (d, 1H, H3 , J = 4.5 Hz);
7.74 (d, 1H, H7, J = 8.4 Hz); 7.88 (dd, 1H, H6, J = 2.1, 8.4 Hz); 7.94
(d, 1H, H4 , J = 4.5 Hz); 8.22 (d, 1H, H4, J = 2.1 Hz); 10.48 (s, 1H,
NH); 10.56 (s, 1H, NH). Anal. Calcd for C22H19N5O7S: C, 53.12; H,
3.85; N, 14.08. Found: C, 53.16; H, 3.79; N, 14.09.
00 00
0
0
4.1.2.17. N0-(3-Phenoxybenzoyl)-2-(5-nitrothiophen-2-yl)-3H-
benzo[d]imidazole-5-carbohydrazide
6h. Yield
82%.
mp
200 °C(dec). IR (KBr), cmꢀ1: 3472 (NH), 1683 (Ar), 1578, 1337
(NO2). 1H RMN, DMSO-d6, d ppm: 7.08 (d, 2H, Ar, J = 7.7 Hz); 7.23
(t, 1H, Ar, J = 7.3 Hz); 7.25 (dd, 1H, Ar, J = 2.2, 8.1 Hz); 7.44 (t, 2H,
4.2.4. Mice native hemoglobin
000,5000
H3 , J = 7.3 Hz); 7.54–7.57 (m, 3H, Ar); 7.74–7.76 (m, 2H, Ar);
Native hemoglobin from non-infected mice was obtained by
treating one volume of pellet erythrocytes with two volumes of
water. The resulting solution was used as the substrate in the inhi-
bition of the hemoglobin hydrolysis assay.
7.88 (dd, 1H, H6, J = 1.7, 8.6 Hz); 7.93 (d, 1H, H4´, J = 4.6 Hz); 8.26
(d, 1H, H4, J = 1.7); 10.56 (s, 1H, NH); 10.61 (s, 1H, NH). Anal. Calcd
for C25H17N5O5S: C, 60.12; H, 3.43, N, 14.02. Found: C, 60.13; H,
3.47; N, 14.17.
4.2.5. Inhibition of hemoglobin hydrolysis
4.1.2.18. N0-(3-Hydroxy-2-naphthyl)-2-(5-nitrothiophen-2-yl)-
3H-benzo[d]imidazole-5-carbohydrazide 6i. Yield 89%. mp
230 °C (dec). IR (KBr), cmꢀ1: 3472 (NH), 1651, 1650 (Ar), 1521,
1336 (NO2). 1H RMN, DMSO-d6, d ppm: 5.4 (br s, 1H, OH); 7.35
(s, 1H, Ar); 7.52 (t, 1H, Ar, J = 6.9 Hz); 7.57 (d, 1H, H3´ , J = 4.5 Hz);
7.66 (t, 1H, Ar, J = 6.3 Hz); 7.76–7.77 (m, 1H, Ar); 7.88 (dd, 1H,
H6, J = 1.6, 8.4 Hz); 7.91 (d, 1H, Ar, J = 8.4 Hz); 7.94 (d, 1H, H4´ ,
J = 4.5 Hz); 8.26 (d, 1H, H4, J = 1.6 Hz); 8.58 (s, 1H, Ar); 10.73 (s,
1H, NH); 10.79 (s, 1H, NH). Anal. Calcd for C23H15N5O5S: C,
58.35; H, 3.19; N, 14.79. Found: C, 58.37; H, 3.23; N, 14.67.
The proteolytic effect of the parasite extract on the native mice
hemoglobin was assayed using 96-wells tissue culture plate
(Greiner Bio-One). The assay mixture contained: mice native
hemoglobin (10
and acetate buffer (0.2 M, pH 5.4) to a final volume of 100
The compounds (10 M) were incorporated in the incubation mix-
lL), parasite extract (50
lL), GSH (10
lL, 10
l
M),
lL.
l
ture dissolved in DMSO. The incubations were carried out at 37 °C
for 18 h and the reactions were stopped by addition of reduced
sample buffer. The degree of digestion was evaluated electropho-
retically by SDS–PAGE by visual comparison of the globin bands
(14 kDa). A DMSO control was electrophoresed at the same time.
Once the bands were obtained, the densitometer registered the
band densities reported as intensity/mm2 SD, so we proceeded
to check the densities in order to have a percentage of inhibition
of hemoglobin hydrolysis.
4.2. Biological assays
4.2.1. Inhibition of heme polymerization
The heme polymerization assay was performed according to an
already published protocol,25 briefly, a solution of hemin chloride
(50 lL, 4 mM), dissolved in DMSO (5.2 mg/mL), was distributed
in 96-well micro plates. Different concentrations (100–5 mM) of
4.2.6. 4-Day suppressive test
Balb-C mice (18–23 g) were infected iv (using caudal vein) with
106 infected red blood cells with P. berghei (n = 6). Two hours after
infection, treatment began with the best compounds tested in the
in vitro assays. These were dissolved in DMSO (0.1 M), diluted with
Saline-Tween 20 solution (2%). Each compound (20 mg/kg) was
administered once by ip for 4 days. At day four, the parasitemia
was counted by examination of Giemsa stained smears. Chloro-
quine (25 mg/kg) was used as a positive control. The survival time
beyond the control group (without drug treatment) was recorded.
The results were expressed as percentage of parasitemia (% of par-
asitemia) and survival days of each compound-treated group over
the control (non-treated group).36
the compounds dissolved in DMSO, were added in triplicate in test
wells (50
(50 L). b-Hematin formation was initiated by the addition of ace-
tate buffer (100 L 0.2 M, pH 4.4). The plates were incubated at
lL). Controls contained either water (50 lL) or DMSO
l
l
37 °C for 48 h to allow for completion of the reaction and centri-
fuged (4000 rpm ꢁ 15 min, IEC-CENTRA, MP4R). After discarding
the supernatant, the pellet was washed twice with DMSO
(200 lL) and finally, dissolved in NaOH (200 lL, 0.2 N). The
solubilized aggregates were further diluted 1:2 with NaOH
(0.1 N) and absorbance recorded at 405 nm (Microplate Reader,
BIORAD-550). The results were expressed as a percentage of
inhibition of flavoprotein (FP) polymerization.

J. Camacho et al. / Bioorg. Med. Chem. 19 (2011) 2023–2029
2029
4.2.7. Anti-Mycobacterium tuberculosis assay
A suspension of MTB was made by mixing growth from slants
(20–30 days old) with 100 L of Tween 80 into 0.2% bovine serum
References and notes
1. WHO World Malaria Report 2010 (<http://rbm.who.int/wmr/>, 2010).
2. Miller, L. H.; Baruch, D. I.; Marsh, K.; Doumbo, O. K. Nature (London) 2002, 415,
673.
3. (a) Egan, T. J.; Ncokazi, K. K. J. Inorg. Biochem. 2005, 99, 1532; (b) Joshi, A. A.;
Viswanathan, C. L. Anti-Infec. Agents Med. Chem. 2006, 5, 105.
4. Tilley, L.; Loria, P.; Foley, M. Chloroquine and Other Quinoline Antimalarials. In
Antimalarial Chemotherapy; Rosenthal, P. J., Ed.; Humana Press: Totowa, N.J.,
2001; p 87.
5. Sullivan, D. J.; Gluzman, I. Y.; Russell, D. G.; Goldberg, D. E. Proc. Natl. Acad. Sci.
U.S.A. 1996, 93, 11865.
6. Slater, A.; Swiggard, W.; Orton, B.; Flitter, W.; Goldberg, D.; Cerami, A.;
Henderson, G. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 325.
7. Pagola, S.; Stephens, P.; Bohle, D.; Kosar, A.; Madesen, S. Nature (London) 2000,
404, 307.
8. Egan, T. J. J. Inorg. Biochem. 2006, 100, 916.
l
albumin. Turbidity of the suspension was then adjusted to a
McFarland standard No. 1 (3 ꢁ 107 CFU/mL) by adding Tween 80
and bovine serum albumin. The bacterial suspension (300 lL)
was further transferred to 7.2 mL of 7H9GC broth (4.7 g of
Middlebrook 7H9 broth base, 20 mL of 10% glycerol, 1 g of Bacto
Casitone, 880 mL of distilled water, 100 mL of OADC (oleic acid,
albumin, dextrose, catalase). For the bioassay, the compounds
were resuspended in DMSO at a concentration of 4 mg/mL (stock
solution). These stock solutions were further diluted with
appropriate volumes of 7H9GC broth to yield final concentration
of 0.4–25
antibiotics used as positive controls were 0.125–32
isoniazid and 0.063–16 g/mL for rifampin. The drug (100
was mixed in the wells with 100 L of bacterial inoculums,
resulting in final bacterial concentration of approximately
l
g/mL. Final drug concentrations ranges of standard
g/mL for
L)
9. Fidock, D. A. Nature 2010, 465.
l
10. WHO Global tuberculosis control: a short update to the 2009 report http://
www.who.int/tb/publications/global_report/2009/update/en/index.html.
11. Balganesh, T. S.; Alzari, P. M.; Cole, S. T. Trends Pharmacol. Sci. 2008, 29, 576.
12. Boiani, M.; González, M. Mini-Rev. Med. Chem 2005, 5, 409.
13. Friedman, M. D.; Stotter, P. L.; Porter, T. H.; Folkers, K. J. Med. Chem. 1973, 16,
1314.
14. Tong, Y.; Bouska, J. J.; Ellis, P. A.; Johnson, E. F.; Leverson, J.; Liu, X.; Marcotte, P.
A.; Olson, A. M.; Osterling, D. J.; Przytulinska, M.; Rodríguez, L. E.; Shi, Y.; Soni,
N.; Stavropoulos, J.; Thomas, S.; Donawho, C. K.; Frost, D. J.; Luo, Y.; Girand, V.
L.; Penning, T. D. J. Med. Chem. 2009, 52, 6803.
15. Pérez-Villanueva, J.; Santos, R.; Hernández-Campo, A.; Giulianotti, M. A.;
Castillo, R.; Medina-Franco, J. L. Med. Chem. Commun. 2011, 2, 44.
16. Serdons, K.; Verduyckt, T.; Vanderghinste, D.; Borghgraef, P.; Cleynhens, J.; Van
Leuven, F.; Kung, H.; Bormans, G.; Verbruggen, A. Eur. J. Med. Chem. 2009, 44,
1415.
17. Kobayashi, K.; Uchiyama, M.; Takahashi, H.; Kawamoto, H.; Ito, S.; Yoshizumi,
T.; Nakashima, H.; Kato, T.; Shimizu, A.; Yamamoto, I.; Asai, M.; Miyazoe, H.;
Ohno, A.; Hirayama, M.; Ozaki, S.; Tani, T.; Ishii, Y.; Tanaka, T.; Mochidome, T.;
Tadano, K.; Fukuroda, T.; Ohta, H.; Okamoto, O. Bioorg. Med. Chem. Lett. 2009,
19, 3096.
l
l
l
a
1.2 ꢁ 106 CFU/mL. The wells in column 11 served as inoculum-
only controls. Solvent (DMSO) was included in every experiment
as a negative control. Plants were sealed in plastic bags and then
incubated at 37 °C for 5 days. On day 5, 50 lL of tetrazolium-
Tween 80 mixture {1.5 mL of tetrazolium[3-(4,5-dimethylthia-
zol-2-yl)2,5-diphenyltetrazolium bromide] at a dilution of 1 mg/
mL, in absolute ethanol and 1.5 mL of 10% Tween 80} was added
to the wells, and the plate was incubated at 37 °C for 24 h. After
this period, the growth of the microorganism was visualized by
the change in color of the dye from yellow to purple. The tests
were carried out in triplicate. MIC is defined as the lowest drug
concentration that prevents the aforementioned change in
color.37
18. Welsch, M. E.; Snyder, S. A.; Stockwell, B. R. Curr. Opin. Chem. Biol. 2010, 14, 1.
19. Cho, Y.; Ioerger, T. R.; Sacchettini, J. C. J. Med. Chem. 2008, 51, 5984.
20. Kim, P.; Kang, S.; Boshoff, H. I.; Jiricek, J.; Collins, M.; Singh, R.; Manjunatha, U.
H.; Niyomrattanakit, P.; Zhang, L.; Goodwin, M.; Dick, T.; Keller, T. H.; Dowd, C.
S.; Barry, C. E. J. Med. Chem. 2009, 52, 1329.
4.2.8. Cell growth inhibition bioassay
The compounds were screened for cytotoxicity against
a
21. Brain-Isasi, S.; Quezada, C.; Pessoa, H.; Morello, A.; Kogan, M. J.; Alvarez-Lueje,
A. Bioorg. Med. Chem. 2008, 16, 7622.
22. Siriram, D.; Yogeeswari, P.; Dhakla, P.; Senthilkumar, P.; Banerjee, D.;
Manjashetty, T. H. Bioorg. Med. Chem. Lett. 2009, 19, 1152.
23. Sriram, D.; Yogeeswari, P.; Kumar, D. R.; Senthilkumar, P.; Bhat, P.; Srividya, M.
Bioorg. Med. Chem. Lett. 2010, 20, 4313.
24. Charris, J.; Camacho, J.; Ferrer, R.; Lobo, G.; Barazarte, A.; Gamboa, N.;
Rodrigues, J.; López, S. J. Chem. Res. 2006, 769.
25. Baelmans, R.; Deharo, E.; Muñoz, V.; Sauvain, M.; Ginsburg, H. J. Esp. Parasitol.
2000, 4, 243.
26. Yajko, D. M.; Madej, J. J.; Lancaster, M. V.; Sanders, C. A.; Cawthon, V. L.; Gee, B.;
Babst, A.; Hadlev, W. K. J. Clin. Microbiol. 1995, 33, 2324.
27. Gómez-Flores, R.; Gupta, S.; Tamez-Guerra, R.; Mehta, R. T. J. Clin. Microbiol.
1995, 33, 1842.
28. Palomino, J. C.; Martin, A.; Camacho, M.; Guerra, H.; Swings, J.; Portales, F.
Antimicrob. Agents Chemother. 2002, 46, 2720.
29. Collins, L. A.; Franzblau, C. Antimicrob. Agents Chemother. 1997, 41, 1004.
30. Franzblau, S. G.; Witzig, R. S.; Mclaughlin, J. C.; Torres, P.; Madico, G.;
Hernandez, A.; Degnan, M. T.; Cook, M. B.; Quenzer, V. K.; Ferguson, R. M.;
Gilman, R. H. J. Clin. Microbiol. 1988, 36, 362.
panel of human tumor cell lines and two nontumorogenic cell
lines, using previously reported methodologies38 at a maximum
concentration of 250 lg/mL. Briefly, growth inhibition was
evaluated by preparing serial dilutions of each compound and
incubating the cells in 96-well plates for 48 h at 37 °C. Appropri-
ate solvents controls were tested for comparison. The percent
inhibition of cell growth relative to the control was evaluated
colorimetrically using a sulforhodamine B dye according to a
published procedure39 by comparison to the control. The GI50
value was defined as the concentration of test sample resulting
in a 50% reduction of absorbance as compared with untreated
controls that received a serial dilution of the solvent in which
the test samples were dissolved, and was determined by linear
regression analysis.
31. Caviedes, L.; Delgado, J.; Gilman, R. H. J. Clin. Microbiol. 2002, 40, 1873.
32. Bavin, P. M. G. J. Med. Chem. 1966, 9, 788.
33. Dorn, A.; Stoffel, R.; Matile, H.; Bubendorf, A.; Ridley, R. Nature (London) 1995,
374, 269.
34. Deharo, E.; Gautret, P.; Ginsburg, H.; Chabaud, A.; Landau, I. Parasitol. Res. 1994,
80, 159.
35. Rosenthal, P. Exp. Parasitol. 1995, 80, 272.
36. Peters, W.; Robinson, B. L. Parasitic Infection Models. In Handbook of
Antimalarial Models of Infection; Zak, O., Sande, M., Eds.; Academic Press:
London, 1999; p 757.
37. Rojas, R.; Caviedes, L.; Aponte, J.; Vaisberg, A.; Lewis, W.; Lamas, G.; Sarasara,
C.; Gilman, R.; Hammond, G. J. Nat. Prod. 2006, 69, 845.
4.3. Data analysis
Data were statistically analyzed using one-way ANOVA and
t-tests for specific group comparisons; assuming 95% of confidence
according GRAPHPAD Prism 3.02.40
Acknowledgments
38. Wood, C. A.; Lee, K.; Vaisberg, A.; Kingston, D.; Neto, C. Chem. Pharm. Bull. 2001,
49, 1477.
39. Skehan, P.; Storeng, R.; Scudiero, D. J. Nat. Cancer Inst. 1990, 4, 1107.
40. GRAPHPAD Prism Software 4.02 for windows. May 17th 1992–2004.
We thank the IIF-FF and CDCH-UCV (Grants IIF: 01.2009,
PG. 06-7548-2009/1), and CYTED-RIDIMEDCHAG programmes for
financial support.