Antileishmanial activity screening of 5-nitro-2-heterocyclic benzylidene hydrazides

Article abstract of DOI:10.1016/j.bmc.2008.05.076

A series of 53 nitro derivatives rationally designed were obtained by parallel synthesis and screened against Leishmania donovani. Six compounds exhibited IC₅₀ values lower than standard drugs. Brief SAR analysis revealed that substitution is important to the activity. Nitrothiophene analogues were more potent than the nitrofuran ones. This was attributed to the ability of sulfur atoms in accommodating electrons from nitro group, which facilitate its reduction and therefore the formation of free radicals lethal to parasites.

Full text of DOI:10.1016/j.bmc.2008.05.076

Bioorganic & Medicinal Chemistry 16 (2008) 6724–6731
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antileishmanial activity screening of 5-nitro-2-heterocyclic
benzylidene hydrazides
Daniela G. Rando a, , Mitchell A. Avery b,c, Babu L. Tekwani c, Shabana I. Khan c, Elizabeth I. Ferreira a
*
a Faculdade de Ciências Farmacêuticas, Universidade de São Paulo—Av. Prof. Lineu Prestes, 580 CEP 05508-900, R. Afonso Celso, 718 apto 144 Vila Mariana,
04119-060 São Paulo, SP, Brazil
b Department of Medicinal Chemistry, University of Mississippi, 417 Faser Hall, University, MS 38677, USA
c National Center for Natural Products Research (NCNPR), University of Mississippi, Thad Cochran Research Center, University, MS 38677-1848, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 53 nitro derivatives rationally designed were obtained by parallel synthesis and screened
against Leishmania donovani. Six compounds exhibited IC50 values lower than standard drugs. Brief
SAR analysis revealed that substitution is important to the activity. Nitrothiophene analogues were more
potent than the nitrofuran ones. This was attributed to the ability of sulfur atoms in accommodating elec-
trons from nitro group, which facilitate its reduction and therefore the formation of free radicals lethal to
parasites.
Received 22 November 2007
Revised 26 May 2008
Accepted 29 May 2008
Available online 5 June 2008
Keywords:
Ó 2008 Elsevier Ltd. All rights reserved.
Nitroderivatives
Antileishmanial agents
Parallel synthesis
SAR
1. Introduction
recent years, widespread resistance to pentavalent antimonial
agents has been observed, especially in cases of Leishmania/HIV
Leishmaniasis is defined as a cluster of vector-borne diseases
with diverse clinical manifestations, caused by the obligate intra-
cellular protozoan parasite of the genus Leishmania.1 Its manifes-
tations include three broad groups of disorders: visceral
leishmaniasis, cutaneous leishmaniasis, and mucocutaneous leish-
maniasis. These manifestations are caused by a total of about 21
leishmanial species, which are transmitted by around 30 species
of phlebotomine sandflies.2–4 Visceral leishmaniasis, the only
lethal form of this parasitosis, is mainly caused byLeishmania dono-
vani.5 Mucocutaneous and cutaneous manifestations, on the other
hand, can be caused by a variety of species. There are over 14 rec-
ognized species of Leishmania within the subgenera Leishmania and
Viannia, which may produce lesions in man.6,7
co-infection.9
These agents have been improved with the advent of new formu-
lations or dosage regimens but the need for new compounds with
better potency and toxicity profiles is still urgent.10,11 Substantial ef-
forts have been made towards this goal and several manuscripts on
antileishmanial therapy can be found on the literature.12–16
This paper reports the synthesis of 53 nifuroxazide derivatives
(Fig. 1 and Table 1), rationally designed and obtained by parallel
synthesis methodology, and their biological screening against
L. donovani. A brief structure–activity analysis is included as well.
2. Design and chemical discussion
Leishmaniasis is endemic in areas of the tropics, subtropics, and
Southern Europe, in settings ranging from rain forests in the Ameri-
castothedesertsinWesternAsia, andfromruraltoperiurbanareas.6
The treatment of leishmaniasis is far from satisfactory. Since the
1940s, the pentavalent antimony compounds sodium stibogluco-
nate (PentostamÒ, Glaxo Wellcome, UK) and meglumine antimoni-
ate (GlucantimeÒ, Rhône-Poulenc Rorer, France) have been the
mainstays of antileishmanial therapy.6,8 These drugs present high
toxicity besides requiring parenteral administration for extended
periods, especially in cases of visceral leishmaniasis. Moreover, in
Fifty-three nitro compounds were designed based on nifuroxaz-
ide structure backbone (Fig. 1), an antibacterial agent for the treat-
ment or prevention of a wide range of infections of genitourinary
tract as well as an adjunct therapy for patients with second- and
third-degree burns.17
The core structure allows different patterns of substitution in
the aromatic ring generating several analogues with different
physical–chemical properties but with almost similar steric
characteristics. The R groups were chosen based on two parame-
ters—r and p constants. These constants represent, respectively,
the relative electronic and lipophilic contributions of a proper
substituent (R) to the global structure. Both kinds of contributions
could interfere with the nitro group reduction by unspecific nitro-
* Corresponding author. Tel./fax: +55 11 3726 4429.
E-mail address: dgrando@usp.br (D.G. Rando).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.05.076

D. G. Rando et al. / Bioorg. Med. Chem. 16 (2008) 6724–6731
6725
Figure 1. Nifuroxazide: lead compound and structural substitutions in the synthetic analogues.
reductases and consequent free radical formation, the most accept-
able mechanism of action to nitro derivatives.18 This criterion also
allows rational development of diversity and 2D QSAR studies as
well. Mono- and di-substituted analogues were synthesized and
considered in this work.
Structure diversity was also obtained by using bioisosterism be-
tween nitrofuran and nitrothiophene, that is, between oxygen and
sulfur since they present the same number of electrons in the va-
lence shell but differ in several physical–chemical properties, such
as van der Waals radii, electronegativity, and lipophilicity.
The compounds were prepared by solution phase parallel syn-
thesis in three steps according to classical synthetic procedures,
as indicated in Scheme 1.19
The compounds were obtained in moderate to high yield and
their purity was determined by HPLC (Table 1).
It was observed that the main variable at parallel synthetic ap-
proach applied to obtain these compounds was the time to accom-
plish the kinetic equilibrium, which varied from 15 to 60 min to
the considered derivatives.
nitrofuran analogues (14/O, 25/O, 26/O, and 32/O) presented lower
activity than the non-substituted analogue (1/O) and only three
substituted nitrothiophene analogues (3/S, 11/S, and 32/S) pre-
sented lower activity than the non-substituted analogue (1/S).
Apparently, substitution is important to improve the antileishma-
nial activity of the compounds. However, it was not possible to ob-
serve any standard behaviour or quantitative relationship between
the physical–chemical parameters chosen and the biological activ-
ity studied.
Compounds 4/O, 4/S, 12/S, and 27/O have not shown any activ-
ity at tested concentrations probably because of a solubility prob-
lem. Analogues 4/O, 4/S, and 12/S are all dimethylamine
substituted derivatives, and could not have exhibited activity as a
result of protonation at pH culture medium, lowering their avail-
ability. It is noteworthy that compound 12/O, although also a
dimethylamine substituted derivative, showed to be active. Other
factors might have contributed to its activity and deserves further
studies.
Biological activity can also be displayed as histogram plots as
shown in Figure 2.
3. Biological evaluation and discussion
Those histogram plots show that nitrothiophene analogues
exhibited much higher activities than the nitrofuran analogues.
A hypothesis, which has been advanced to explain this effect,
is that sulfur free d atomic orbitals from nitrothiophene ring
can accommodate more electrons than permitted by the octet
theory. Being adjacent to a nitro-linked carbon, the sulfur free
d orbitals could accept the nitro group electrons thus increasing
the susceptibility of the latter to reduction by unspecific
nitroreductases.
The 53 obtained analogues were tested, in vitro, against L. dono-
vani promastigote forms at concentrations ranging from 10 to
0.01 lg/mL, as described in Section 5. The obtained IC50 and IC90
values are summarized in Table 2.
The nitroderivatives exhibited very promising activities when
compared to the standard drugs pentamidine and amphotericin
B. The nitrothiophene analogues 2/S (4-Cl), 6/S (4-OH), 8/S (4-
CF3), and 18/S (3,4-Cl), and the nitrofuran analogues 18/O (3,4-Cl)
and 28/O (3-NH2; 4-CH3) exhibited activity higher than or close
to the standard drugs.
4. Conclusions
These analogues can be tested in Leishmania amastigote forms
and are good candidates to antileishmanial in vivo assay as well.
Hansch analysis (2D-QSAR) studies were performed by applying
BuildQSAR and BilinProgramà softwares. Linear, parabolic, and
bilinear models were taken into account but no statistically accept-
able model could be established when considering classical elec-
In conclusion, a series of novel 5-nitroheterocyclic benzohyd-
razide derivatives were designed and synthesized. The antileish-
manial screening revealed the following six promising analogues,
which exhibited IC50 values lower than standard drugs pentami-
dine and amphotericin B: 2/S (4-Cl), 6/S (4-OH), 8/S (4-CF3), 18/S
(3,4-Cl), 18/O(3,4-Cl), and 28/O (3-NH2; 4-CH3). They are good can-
didates for further in vivo antileishmanial assays. Nitrothiophene
derivatives showed better activity profile than the nitrofuran ana-
logues. Further studies applying CoMFA and CoMSIA are being per-
formed by our group to explore the possibility of existing
quantitative structure–activity relationships when considering ste-
ric contributions.
tronic and lipophilic parameters (e.g., LogP,
r, p, and MR). This
lack of correlation may indicate that the chosen parameters were
not the most appropriate or that parameters related with steric com-
ponents could be of greater importance.
Nonetheless, qualitative analyses revealed interesting informa-
tion about the biological behaviour of the derivatives, as will be de-
scribed below.
The non-substituted derivatives showed low activity profiles
when compared to the substituted ones. Only four substituted
5. Experimental
5.1. Parallel synthetic procedures and analytical details19
´
Developed by Oliveira, D.B and Gáudio, A.C. from Universidade Federal do Espırito
Mono- and di-substituted benzoic methyl esters were classi-
cally obtained from the corresponding benzoic acid in the presence
of excess methanol and sulfuric acid as catalyst, after 4–6 h reflux.
Santo—Brazil freeware (http://tau.cce.ufes.br/dfis/anderson/htmL/
buildqsar_ing.htm).
à
Developed by Kubinyi (http://home.t-online.de/home/kubinyi).

6726
D. G. Rando et al. / Bioorg. Med. Chem. 16 (2008) 6724–6731
Table 1
Synthetic library obtained by parallel synthesis
Nitrofuran derivatives
R groups
Nitrothiophene derivatives
R groups Yield (%)
Yield (%)
Puritya (%)
Puritya (%)
Rpara
Rmeta
Rpara
Rmeta
1/O
2/O
3/O
4/O
5/O
6/O
7/O
8/O
H
Cl
H
H
H
H
H
H
H
H
85
95
52
44
70
80
88
83
81
95
73
60
87
88
83
86
71
81
90
72
56
30
75
59
49
53
59
100
100
98
98
86
1/S
2/S
3/S
4/S
5/S
6/S
7/S
8/S
H
Cl
H
H
H
H
H
H
H
H
91
87
85
61
93
86
93
87
84
93
85
88
86
83
87
93
73
80
85
60
53
85
50
23
39
63
99
100
99
99
97
99
99
100
98
99
99
99
98
99
99
91
97
99
99
99b
99
99b
98
99b
99
98
OMe
N(Me)2
NH2
OH
Me
CF3
NO2
H
H
H
H
H
OMe
N(Me)2
NH2
OH
Me
CF3
NO2
H
H
H
H
H
99
100
100
97
99
98
99
99
98
99
100
99
99
100
99
9/O
H
Cl
9/S
H
Cl
10/O
11/O
12/O
14/O
15/O
16/O
17/O
18/O
19/O
22/O
23/O
24/O
25/O
26/O
27/O
28/O
30/O
32/O
10/S
11/S
12/S
14/S
15/S
16/S
17/S
18/S
19/S
22/S
23/S
24/S
25/S
28/S
29/S
30/S
32/S
OMe
N(Me)2
OH
Me
CF3
NO2
Cl
OMe
N(Me)2
OH
Me
CF3
NO2
Cl
H
H
Cl
F
H
H
Cl
F
F
Cl
F
Cl
F
F
NO2
NO2
OMe
OH
Cl
Me
Me
Cl
OMe
OH
NH2
NH2
NO2
NH2
OMe
SO2NH2
NO2
NO2
OMe
Me
Me
Me
Cl
OMe
OH
NH2
NH2
OH
OMe
SO2NH2
99
98b
99
98b
100b
99
99
a
Purity determined by HPLC.
b
Purity determined by HPLC after liquid chromatography purification. Code number/O = furan derivatives and code number/S = thiophene derivatives.
Scheme 1. Synthetic approach to obtain the library of compounds.
The hydrazide intermediates were obtained from reaction of the
(Argonaut Technology), which allowed up to 20 simultaneous reac-
tions but with smaller volumes.
Since all reactions were produced in parallel, an initial screen-
ing was performed to determine the best conditions to a batch of
reactions.
benzoic methyl ester with excess of hydrazine 90% in water and
under heating (60 °C) and stirring. Crystals were obtained by cool-
ing the reaction vessels with cold water followed by filtering the
precipitate.
The final products were synthesized by coupling the hydrazides
with the nitro-heterocyclic aldehyde (nitrofuran or nitrothiophene
aldehydes) at equimolar proportions (1:1). The reactants, dissolved
in a mixture of water/ethanol (2:5), were kept under reflux by
times ranging from 15 min to 1 h.
All steps above were carried out in parallel using two different
automatized apparatus. For the first two steps a First Mate Bench-
top Synthesizer (Argonaut Technology) was used. It allows 16
simultaneous reactions in large volumes. The last step was accom-
plished using an Argonaut Quest 210 Parallel Organic Synthesizer
1H NMR spectra were recorded on a Bruker 400 MHz Advance
Systems spectrometer in DMSO-d6 using tetramethylsilane (TMS)
as an internal standard. Chemical shifts were expressed in part
per million (d) and the first order values reported for coupling con-
stants (J) in Hertz. Splitting patterns were described as follows: s
(singlet), d (doublet), t (triplet), m (multiplet), and br s (broad
singlet).
Elemental analyses were performed by a Perkin-Elmer CHN/SO
Series II Analyzer and were within 0.4% of the theoretical values.
Some compounds presented one ( ) or two ( ) water molecules
*
**

D. G. Rando et al. / Bioorg. Med. Chem. 16 (2008) 6724–6731
6727
Table 2
adsorbed, which were considered on elemental analysis results.
Molecules with high hygroscopicity could not be submitted to ele-
IC50 and IC90 values of the nitroderivative analogues
mental analysis ( ).
Mass spectra were recorded using Waters Alliance LC/MS
Systems.
Flash chromatography, when necessary, was performed using
300 mesh silica gel and a mixture of ethyl acetate/hexane (2:1)
as eluent. The yields were calculated after purification. HPLC puri-
ties were determined on an HPLC Alliance, Waters, and processed
with Water Empower II software.
Samples
MW
IC50
M)
IC90
M)
Samples MW
IC50
M)
IC90
M)
***
(
l
(
l
(
l
(
l
1/O
2/O
3/O
4/O
5/O
6/O
7/O
8/O
259.22 11.19
293.66 7.83
289.24 8.99
302.29 NAa
29.70
23.84
25.93
NAa
1/S
2/S
3/S
4/S
5/S
6/S
7/S
8/S
275.28 6.54
309.73 1.26
305.31 7.21
318.35 NAa
290.3 2.24
291.28 0.89
289.31 4.49
343.28 0.82
320.28 2.81
309.73 2.74
305.31 8.52
318.35 NAa
291.28 2.75
289.31 4.84
343.28 3.79
320.28 2.78
25.43
4.84
26.20
NAa
274.23 2.66
275.22 2.40
273.24 10.98
327.22 8.86
304.22 6.57
293.66 7.83
289.24 10.37
302.29 7.94
275.22 10.17
273.24 19.76
327.22 10.39
304.22 9.86
328.11 1.68
14.59
17.80
28.55
24.45
22.35
24.18
26.62
24.15
27.98
>36.60
24.14
25.97
26.91
25.51
19.25
20.25
48.94
131.47
97.51
NAa
>34.45
4.46
7.95
1.89
5.31
9/O
9/S
5.1.1. N0-[(5-Nitrofuran-2-yl)methyl]benzohydrazide (1/O)
1H NMR (300 MHz, d ppm, DMSO-d6): 7.26 (s, 1H, H4), 7.54–7.57
(d, J = 7.05, 2H, H12 and H14), 7.60–7.62 (d, J = 6.55, 1H, H13), 7.78–
7.79 (d, J = 3.27, 1H, H3), 7.90–7.92 (d, J = 6.30, 2H, H11 and H15),
8.40 (s, 1H, H6), 12.23 (s, 1H, H8); MS (m/z): 258.08 [Mꢀ1]. Anal.
Calcd for C12H9N3O4: *C, 51.94; H, 3.25; N, 15.15. Found: C,
51.64; H, 3.87; N, 15.08.
10/O
11/O
12/O
14/O
15/O
16/O
17/O
18/O
19/O
22/O
23/O
24/O
25/O
26/O
27/O
28/O
30/O
32/O
Pentamidine
Amphotericin
B
10/S
11/S
12/S
14/S
15/S
16/S
17/S
18/S
19/S
22/S
23/S
24/S
25/S
28/S
29/S
30/S
32/S
6.78
32.75
NAa
7.55
19.01
6.41
14.27
0.87
4.92
4.67
3.91
18.36
18.34
4.70
344
311
0.41
2.15
295.2
9.49
5.1.2. 4-Chloro-N 0-[(5-nitrofuran-2-yl)methylidene]-
benzohydrazide (2/O)
311.65 6.42
334.24 7.39
320.22 9.90
304.26 16.43
290.23 26.98
338.66 NAa
288.26 0.62
303.27 8.90
372.74 38.45
0.63 1.06
327.5 1.92
350
336
320
304
305
319
2.00
5.45
4.69
2.24
2.30
2.19
1H NMR (300 MHz, d ppm, DMSO-d6): 7.27 (d, 1H, H4), 7.59–
7.61 (d, J = 7.55, 2H, H12 and H14), 7.76–7.78 (d, J = 3.58, 1H, H3),
7.91–7.94 (d, J = 6.76, 2H, H11 and H15), 8.37 (s, 1H, H6), 12.28 (s,
1H, H8); MS (m/z): 292.01 [Mꢀ1]. Anal. Calcd for C12H8ClN3O4:
*C, 46.20; H, 2.57; N, 13.48. Found: C, 45.08, H, 2.88; N, 12.93.
4.59
4.39
17.17
2.88
52.76
88.53
388.5 8.06
5.1.3. 4-Methoxy-N0-[(5-nitrofuran-2-yl)methylidene]-
benzohydrazide (3/O)
1.1
1.19
1H NMR (300 MHz, d ppm, DMSO-d6): 3.83 (s, 3H, OCH3), 7.06–7.08
(d, J = 7.58, 2H, H12 and H14), 7.25 (br s, 1H, H4), 7.79–7.81 (d, J = 3.42,
a
NA = non-active at tested concentrations. Code number/O = furan derivatives
and code number/S = thiophene derivatives.
Figure 2. Histogram plot of the biological activity for nitrofuran and nitrothiophene derivatives.

6728
D. G. Rando et al. / Bioorg. Med. Chem. 16 (2008) 6724–6731
1H, H3), 7.90–7.92(d, J = 8.31, 2H, H11 and H15), 8.38(s, 1H, H6), 12.12(s,
1H, H8); MS (m/z): 288.14 [Mꢀ1]. Anal. Calcd for C13H11N3O5: C, 53.98;
H, 3.83; N, 14.53. Found: C, 53.84; H, 3.58; N, 14.41.
H15), 7.79 (br s, 1H, H11), 8.40 (S, 1H, H6), 12.19 (s, 1H, H8); MS
(m/z): 287.90 [Mꢀ1]. Anal. Calcd for C13H11N3O5: C, 53.98; H,
3.83; N, 14.53. Found: C, 53.96; H, 3.48; N, 14.39.
5.1.4. 4-(Dimethylamino)-N0-[(5-nitrofuran-2-yl)methylidene]-
benzohydrazide (4/O)
5.1.12. 3-(Dimethylamino)-N0-[(5-nitrofuran-2-yl)methylidene]-
benzohydrazide(12/O)
1H NMR (300 MHz, d ppm, DMSO-d6): 2.98 (s, 6H, N(CH3)2),
6.73–6.75 (d, J = 8.8, 2H, H12 and H14), 7.19–7.20 (d, J = 3.91, 1H,
H4), 7.78–7.80 (m, 3H, H3, H11 and H15), 8.36 (s, 1H, H6), 11.93 (s,
1H NMR (300 MHz, d ppm, DMSO-d6): 2.95 (s, 6H, N(CH3)2), 6.94–
6.95 (d, J = 7.83, 1H, H13), 7.17 (br s, 2H, H11 and H15), 7.25 (br s, 1H,
H4), 7.31–7.33 (m, 1H, H12), 7.79 (br s, 1H, H3), 8.41 (s, 1H, H6), 12.10
(s, 1H, H8); MS (m/z): 300.90 [Mꢀ1]. Anal. Calcd for C14H14N4O4: *C,
52.45; H, 4.37; N, 17.48. Found: C, 52.78; H, 4.82; N, 17.25.
*
1H, H8); MS (m/z): 301.16 [Mꢀ1]. Anal. Calcd for C14H14N4O4: C,
52.50; H, 4.38; N, 17.50. Found: C, 52.66; H, 5.08; N, 17.68.
5.1.5. 4-Amino-N0-[(5-nitrofuran-2-yl)methylidene]-
benzohydrazide (5/O)
5.1.13. 3-Hydroxy-N0-[(5-nitrofuran-2-yl)methylidene]-
benzohydrazide (14/O)
1H NMR (300 MHz, d ppm, DMSO-d6): 5.89 (s, 2H, NH2), 6.58–
6.60 (d, J = 8.31, 2H, H12 and H14), 7.19–7.21 (d, J = 3.67, 1H, H4),
7.65–7.69 (d, J = 8.07, 2H, H11 and H15), 7.77–7.79 (d, J = 3.42, 1H,
H3), 8.35 (s, 1H, H6), 11.85 (s, 1H, H8); MS (m/z): 273.04 [Mꢀ1].
Anal. Calcd for C12H10N4O4: C, 52.55; H, 3.65; N, 20.44. Found: C,
52.06; H, 3.52; N, 19.70.
1H NMR (300 MHz, d ppm, DMSO-d6): 6.96–7.04 (d, J = 3.78, 1H,
H4), 7.24–7.34 (m, 4H, H3, H12, H13 and H15), 7.79–7.82 (m, 1H,
H11), 8.40 (s, 1H, H6), 9.84 (br s, 1H, OH), 12.17 (s, 1H, H8); MS
(m/z): 274.08 [Mꢀ1]. Anal. Calcd for C14H14N4O4: C, 52.37; H,
3.30; N, 15.27. Found: C, 52.11; H, 3.19; N, 15.18.
5.1.14. 3-Methyl-N0-[(5-nitrofuran-2-yl)methylidene]-
benzohydrazide (15/O)
5.1.6. 4-Hydroxy-N0-[(5-nitrofuran-2-yl)methylidene]-
benzohydrazide (6/O)
1H NMR (300 MHz, d ppm, DMSO-d6): 2.40 (s, 3H, CH3), 7.26–7.27
(d, J = 2.5, 1H, H4), 7.44 (br s, 2H, H12 and H13), 7.70–7.73 (m, 2H, H11
and H15), 7.79–7.80 (d, J = 3.5, 1H, H3), 8.40 (s, 1H, H6), 12.20 (s, 1H,
H8); MS (m/z): 272.17 [Mꢀ1]. Anal. Calcd for C13H11N3O4: C, 57.14;
H, 4.03; N, 15.38. Found: C, 56.36; H, 3.94; N, 15.40.
1H NMR (300 MHz, d ppm, DMSO-d6): 6.84–6.86 (d, J = 8.31, 2H,
H12 and H14), 7.21 (br s, 1H, H4), 7.77–7.80 (m, 3H, H3, H11 and H15),
8.35 (s, 1H, H6), 10.21 (br s, 1H, OH), 12.01 (s, 1H, H8); MS (m/z):
274.17 [Mꢀ1]. Anal. Calcd for C12H9N3O5: C, 52.36; H, 3.27; N,
15.27. Found: C, 52.28; H, 3.13; N, 15.09.
5.1.15. N0-[(5-Nitrofuran-2-yl)methylidene]-3-(trifluoromethyl)-
benzohydrazide (16/O)
5.1.7. 4-Methyl-N0-[(5-nitrofuran-2-yl)methylidene]-
benzohydrazide (7/O)
1H NMR (300 MHz, d ppm, DMSO-d6): 7.30 (br s, 1H, H4), 7.79
(br s, 2H, H12 and H3), 7.98–7.99 (d, J = 6.8, 1H, H13), 8.21–8.23
(d, J = 7.6, 2H, H11 and H15), 8.39 (s, 1H, H6), 12.42 (s, 1H, H8); MS
(m/z): 326.01 [Mꢀ1]. Anal. Calcd for C13H8F3N3O4: C, 47.72; H,
2.46; N, 12.84. Found: C, 47.49; H, 2.23; N, 12.80.
1H NMR (300 MHz, d ppm, DMSO-d6): 2.36 (s, 3H, CH3), 7.25 (br
s, 1H, H4), 7.33–7.35 (d, J = 6.16, 2H, H12 and H14), 7.79–7.82 (m,
3H, H3, H11 and H15), 8.39 (s, 1H, H6), 12.17 (s, 1H, H8); MS (m/z):
*
271.10 [Mꢀ1]. Anal. Calcd for C13H11N3O4: C, 53.56; H, 3.78; N,
14.42. Found: C, 53.42; H, 4.27; N, 14.33.
5.1.16. 3-Nitro-N0-[(5-nitrofuran-2-yl)methylidene]-
benzohydrazide (17/O)
5.1.8. N0-(5-Nitrofuran-2-yl)methylidene]-4-(trifluoromethyl)-
benzohydrazide (8/O)
1H NMR (300 MHz, d ppm, DMSO-d6): 7.32 (br s, 1H, H4), 7.80
(br s, 1H, H3), 7.84–7.87 (t, J = 7.70, 1H, H12), 8.35–8.37 (d,
J = 7.09, 1H, H11), 8.41 (s, 1H, H15), 8.45–8.47 (d, J = 7.82, 1H,
1H NMR (300 MHz, d ppm, DMSO-d6): 7.30 (br s, 1H, H4), 7.79
(br s, 1H, H3), 7.91–7.93 (d, J = 7.55, 2H, H12 and H14), 8.10–8.13
(d, J = 7.35, 2H, H11 and H15), 8.40 (s, 1H, H6), 12.40 (s, 1H, H8);
MS (m/z): 326.00 [Mꢀ1]. Anal. Calcd for C13H8F3N3O4: C, 47.72;
H, 2.46; N, 12.84. Found: C, 47.50; H, 2.16; N, 12.68.
H
13), 8.74 (s, 1H, H6), 12.51 (s, 1H, H8); MS (m/z): 303.11 [Mꢀ1].
*
Anal. Calcd for C12H8N4O6: C, 44.69; H, 2.48; N, 17.38. Found: C,
45.50; H, 2.75; N, 17.42.
5.1.9. 4-Nitro-N0-[(5-nitrofuran-2-yl)methylidene]-
benzohydrazide (9/O)
5.1.17. 3,4-Dichloro-N0-[(5-nitrofuran-2-yl)methylidene]-
benzohydrazide (18/O)
1H NMR (300 MHz, d ppm, DMSO-d6): 7.31 (br s, 1H, H4), 7.79
(br s, 1H, H3), 8.14–8.15 (d, J = 4.97, 2H, H11 and H15), 8.37–8.39
(d, J = 8.74, 3H, H6, H12 and H14), 12.49 (s, 1H, H8); MS (m/z):
303.03 [Mꢀ1]. Anal. Calcd for C12H8N4O6: *C, 44.69; H, 2.48; N,
17.38. Found: C, 45.30; H, 2.88; N, 17.48.
1H NMR (300 MHz, d ppm, DMSO-d6): 7.30 (br s, 1H, H4), 7.79–
7.80 (d, J = 3.9, 1H, H12), 7.84–7.85 (d, J = 4.8, 1H, H11), 7.89 (br s,
1H, H15), 8.15 (br s, 1H, H3), 8.37 (br s, 1H, H6), 12.34 (s, 1H, H8);
*
MS (m/z): 327.85 [Mꢀ1]. Anal. Calcd for C12H7Cl2N3O4: C, 41.62;
H, 2.02; N, 12.14. Found: C, 42.04; H, 2.51; N, 11.91.
5.1.10. 3-Chloro-N0-[(5-nitrofuran-2-yl)methylidene]-
benzohydrazide (10/O)
5.1.18. 3,4-Difluoro-N0-[(5-nitrofuran-2-yl)methylidene]-
benzohydrazide (19/O)
1H NMR (300 MHz, d ppm, DMSO-d6): 7.26–7.29 (d, J = 9.74, 1H,
1H NMR (300 MHz, d ppm, DMSO-d6): 7.29 (br s, 1H, H4), 7.62–
7.64 (d, J = 8.8, 1H, H12), 7.79 (m, 2H, H3 and H15), 7.94–7.98 (m, 1H,
H12), 7.57–7.58 (d, J = 2.78, 1H, H4), 7.68 (br s, 1H, H3), 7.76–7.79 (d,
J = 10.13, 1H, H13), 7.86 (br s, 1H, H6), 7.91–7.94 (d, J = 9.54, 1H,
H
11), 8.36 (s, 1H, H6), 12.27 (s, 1H, H8); MS (m/z): 293.94 [Mꢀ1].
*
H
11), 8.35– 8.38 (d, J = 9.34, 1H, H15), 12.30 (s, 1H, H8); MS (m/z):
Anal. Calcd for C12H7F2N4O4: C, 46.00; H, 2.24; N, 13.42. Found:
C, 46.18 H, 2.74; N, 13.46.
*
292.02 [Mꢀ1]. Anal. Calcd for C12H8ClN3O4: C, 46.20; H, 2.57; N,
13.48. Found: C, 44.34; H, 3.29; N, 12.73.
5.1.19. 3-Chloro-4-fluoro-N0-[(5-nitrofuran-2-yl)methylidene]-
benzohydrazide (22/O)
5.1.11. 3-Methoxy-N0-[(5-nitrofuran-2-yl)methylidene]-
benzohydrazide (11/O)
1H NMR (300 MHz, d ppm, DMSO-d6): 7.29 (br s, 1H, H4), 7.58–
7.63 (m, 1H, H12), 7.78–7.79 (d, J = 3.42, 1H, H3), 7.89–7.99 (m, 1H,
1H NMR (300 MHz, d ppm, DMSO-d6): 3.82 (s, 3H, OCH3), 7.19
(br s, 1H, H13), 7.27 (br s, 1H, H12), 7.42–7.48 (m, 3H, H3, H4 and
H11), 8.12–8.14 (d, J = 6.34, 1H, H15), 8.34 (s, 1H, H6), 12.27 (s, 1H,

D. G. Rando et al. / Bioorg. Med. Chem. 16 (2008) 6724–6731
6729
H8); MS (m/z): 309.92 [Mꢀ1]. Anal. Calcd for C12H7ClFN3O4: *C,
5.1.28. N0-[(5-Nitrothiophen-2-yl)methylidene]benzohydrazide
43.70; H, 2.12; N, 12.75. Found: C, 43.89; H, 2.66; N, 13.51.
(1/S)
1H NMR (300 MHz, d ppm, DMSO-d6): 7.51–7.60 (m, 4H, H4, H12
H13 and H14), 7.88–7.90 (d, J = 6.9, 2H, H11 and H15), 8.12 (br s, 1H,
,
5.1.20. 3-Methoxy-4-nitro-N0-[(5-nitrofuran-2-yl)methylidene]-
benzohydrazide (23/O)
H3), 8.67 (s, 1H, H6), 12.22 (s, 1H, H8); MS (m/z): 274.00 [Mꢀ1].
Anal. Calcd for C12H9N3O3S: C, 52.31; H, 3.27; N, 15.26. Found: C,
52.21; H, 3.24; N, 15.34.
1H NMR (300 MHz, d ppm, DMSO-d6): 4.01 (s, 3H, OCH3), 7.32 (br
s, 1H, H4), 7.61–7.63 (d, J = 7.34, 1H, H11), 7.78 (s, 1H, H15), 7.80 (br s,
1H, H3), 8.03–8.05 (d, J = 7.80, 1H, H12), 8.41 (s, 1H, H6), 12.38 (s, 1H,
H8); MS (m/z): 332.94 [Mꢀ1]. Anal. Calcd for C12H7ClFN3O4: C, 46.70;
H, 2.99; N. 16.78. Found: C, 46.67; H, 2.88; N, 16.68.
5.1.29. 4-Chloro-N0-[(5-Nitrothiophen-2-yl)methylidene]-
benzohydrazide (2/S)
1H NMR (300 MHz, d ppm, DMSO-d6): 7.60–7.62 (m, 3H, H4, H12
and H14), 7.91–7.93 (d, J = 7.8, 2H, H11 and H15), 8.12 (s, 1H, H3),
8.65 (s, 1H, H6), 12.28 (s, 1H, H8); MS (m/z): 307.95 [Mꢀ1]. Anal.
Calcd for C12H8ClN3O3S: C, 46.49; H, 2.58; N, 13.56. Found: C,
46.22; H, 2.39; N, 13.70.
5.1.21. 3-Hydroxy-4-nitro-N0-[(5-nitrofuran-2-yl)methylidene]-
benzohydrazide (24/O)
1H NMR (300 MHz, d ppm, DMSO-d6): 7.29 (br s, 1H, H4), 7.42–
7.44 (d, J = 7.8, 1H, H11), 7.59 (br s, 1H, H15), 7.78 (br s, 1H, H3),
8.00–8.02 (d, J = 8.1, 1H, H12), 8.38 (s, 1H, H6), 12.37 (s, 1H, H8);
MS (m/z): 318.95 [Mꢀ1]. Anal. Calcd for C12H8N4O7: C, 42.60; H,
5.1.30. 4-Methoxy-N0-[(5-nitrothiophen-2-yl)methylidene]-
benzohydrazide (3/S)
*
2.37; N, 16.57. Found: C, 42.59; H, 2.93; N, 16.37.
1H NMR (300 MHz, d ppm, DMSO-d6): 3.83 (s, 3H, OCH3), 7.05–
7.07 (d, J = 8.60, 2H, H12 and H14), 7.55–7.56 (d, J = 3.18, 1H, H4),
7.88–7.90 (d, J = 7.80, 2H, H11 and H15), 8.11-8.12 (d, J = 3.9, 1H,
H3), 8.66 (s, 1H, H6), 12.10 (s, 1H, H8); MS (m/z): 304.07 [Mꢀ1].
Anal. Calcd for C13H11N3O4S: C, 51.15; H, 3.61; N, 13.77. Found:
C, 50.48; H, 3.42, N, 13.72.
5.1.22. 3-Amino-4-methoxy-N0-[(5-nitrofuran-2-yl)methylidene]-
benzohydrazide(25/O)
1H NMR (300 MHz, d ppm, DMSO-d6): 3.83 (s, 3H, OCH3), 4.97
(s, 2H, NH2), 6.88–6.90 (d, J = 8.3, 1H, H12), 7.17 (s, 1H, H4), 7.19–
7.22 (d, J = 9.0, 2H, H11 and H15), 7.79–7.80 (d, J = 2.9, 1H, H3),
8.36 (s, 1H, H6), 12.06 (s, 1H, H8); MS: 305.05 [M+1]. Anal. Calcd
for C13H12N4O5: not determined.
5.1.31. 4-(Dimethylamino)-N0-[(5-nitrothiophen-2-yl)methylidene]-
benzohydrazide (4/S)
5.1.23. 3-Amino-4-hydroxy-N0-[(5-nitrofuran-2-yl)methylidene]-
benzohydrazide (26/O)
1H NMR (300 MHz, d ppm, DMSO-d6): 2.97 (s, 6H, N(CH3)2),
6.72–6.74 (d, J = 8.8, 2H, H12 and H14), 7.49–7.50 (d, J = 3.9, 1H,
H4), 7.77–7.79 (d, J = 8.8, 2H, H11 and H15), 8.09–8.10 (d, J = 4.2,
1H, H3), 8.63 (s, 1H, H6), 11.91 (s, 1H, H8); MS (m/z): 317.16
1H NMR (300 MHz, d ppm, DMSO-d6): 4.75 (s, 2H, NH2), 6.71–
6.73 (m,1H, H12), 7.02–7.06 (m, 1H, H15), 7.16–7.21 (m, 2H, H4
and H11), 7.76–7.79 (m, 1H, H3), 8.34 (s, 1H, H6), 11.93 (s, 1H,
H8); MS (m/z): 289.01 [Mꢀ1]. Anal. Calcd for C12H10N4O5: C.
49.66; H, 3.45; N, 19.31. Found: C, 48.80; H, 3.56; N, 18.07.
*
[Mꢀ1]. Anal. Calcd for C14H14N4O3S: C, 50.00; H, 4.17; N, 16.67.
Found: C, 49.91; H, 4.51; N, 16.80.
5.1.32. 4-Amino-N0-[(5-nitrothiophen-2-yl)methylidene]-
benzohydrazide (5/S)
5.1.24. 4-Chloro-3-nitro-N0-[(5-nitrofuran-2-yl)methylidene]-
benzohydrazide (27/O)
1H NMR (300 MHz, d ppm, DMSO-d6): 5.86 (s, 2H, NH2), 6.56–
6.58 (d, J = 8.3, 2H, H12 and H14), 7.48–7.49 (d, J = 3.7, 1H, H4),
7.62–7.64 (d, J = 8.3, 2H, H11 and H15), 8.08–8.09 (d, J = 3.9, 1H,
H3), 8.59 (S, 1H, H6), 11.82 (s, 1H, H8); MS (m/z): 289.01 [Mꢀ1].
1H NMR (300 MHz, d ppm, DMSO-d6): 7.29 (br s, 1H, H4), 7.80
(br s, 1H, H3), 7.86–7.88 (d, J = 8.1, 1H, H12), 8.05–8.07 (d, J = 8.8,
1H, H11), 8.29 (s, 1H, H15), 8.40 (s, 1H, H6), 12.42 (s, 1H, H8); MS
(m/z): 336.90 [Mꢀ1]. Anal. Calcd for C12H7ClN4O6: C, 42.54; H,
2.07; N, 16.54. Found: C, 42.78; H, 2.81; N, 14.42.
*
Anal. Calcd for C12H10N4O3S: C, 49.60; H, 3.44; N, 19.29. Found:
C, 49.07; H, 3.37; N, 19.26.
5.1.25. 3-Amino-4-methyl-N0-[(5-nitrofuran-2-yl)methylidene]-
benzohydrazide (28/O)
5.1.33. 4-Hydroxy-N0-[(5-nitrothiophen-2-yl)methylidene]-
benzohydrazide (6/S)
1H NMR (300 MHz, d ppm, DMSO-d6): 2.07 (s, 3H, CH3), 5.08 (s,
2H, NH2), 7.01 (br s, 2H, H12 and H11), 7.14 (br s, 1H. H15), 7.20 (d,
1H, H4), 7.75–7.76 (d, J = 2.9, 1H, H3), 8.38 (s, 1H, H6), 12.11 (s, 1H,
H8); MS (m/z): 286.90 [Mꢀ1]. Anal. Calcd for C13H12N4O4: C, 49.66;
H, 3.45; N, 19.31. Found: C, 49.61; H, 3.24; N, 19.56.
1H NMR (300 MHz, d ppm, DMSO-d6): 6.85–6.87 (d, J = 8.3, 2H,
H12 and H14), 7.53–7.54 (d, J = 3.9, 1H, H4), 7.78–7.80 (d, J = 8.1,
2H, H11 and H15), 8.11–8.12 (d, J = 4.2, 1H, H3), 8.64 (s, 1H, H6),
10.2 (s, 1H, OH), 12.01 (s, 1H, H8); MS (m/z): 290.01 [Mꢀ1]. Anal.
Calcd for C12H9N3O4S: *C, 49.48; H, 3.11; N, 14.43. Found: C,
49.52; H, 2.79; N, 14.35.
5.1.26.3-Methoxy-4-methyl-N0-[(5-nitrofuran-2-yl)methylidene]-
benzohydrazide(30/O)
5.1.34. 4-Methyl-N0-[(5-nitrothiophen-2-yl)methylidene]-
benzohydrazide (7/S)
1H NMR (300 MHz, d ppm, DMSO-d6):2.21(s, 3H, CH3), 3.87 (s, 3H,
OCH3), 7.27(br s, 1H, H4), 7.29–7.31 (d, J = 7.58, 1H, H12), 7.43–7.45 (d,
J = 7.80, 2H, H11 and H15), 7.79–7.80 (d, J = 2.9, 1H, H3), 8.41 (s, 1H, H6),
12.14 (s, 1H, H8);MS(m/z):302.01[Mꢀ1]. Anal. CalcdforC14H13N3O5:
C, 55.40; H, 4.29; N, 13.90. Found: C, 53.95; H, 4.30; N, 13.38.
1H NMR (300 MHz, d ppm, DMSO-d6): 2.37 (s, 3H, CH3), 7.33–
7.35 (d, J = 7.6, 2H, H12 and H14), 7.55–7.56 (d, J = 2.9, 1H, H4),
7.80–7.82 (d, J = 6.6, 2H, H11 and H15), 8.11–8.12 (d, J = 3.9, 1H,
H3), 8.67 (s, 1H, H6), 12.15 (s, 1H, H8); MS (m/z): 288.07 [Mꢀ1].
*
Anal. Calcd for C13H11N3O3S: C, 50.76; H, 3.58; N, 13.67. Found:
5.1.27. 2-Chloro-5-({2-[(5-nitrofuran-2-yl)methylidene]-
hydrazino}carbonyl)benzene sulfonamide (32/O)
C, 51.07; H, 3.99; N, 13.64.
1H NMR (300 MHz, d ppm, DMSO-d6): 7.31 (br s, 1H, H4), 7.77–
7.80 (m, 3H, H3 and SO2NH2), 7.85–7.87 (d, J = 7.83, 1H, H12), 8.12–
8.14 (d, J = 7.30, 1H, H11), 8.41 (br s, 1H, H15), 8.52 (s, 1H, H6), 12.48
(s, 1H, H8); MS (m/z): 370.88 [Mꢀ1]. Anal. Calcd for C12H9ClN4O6S:
C, 38.66; H, 2.42; N, 15.03. Found: C, 39.20; H, 2.49; N, 15.33.
5.1.35. N0-[(5-Nitrothiophen-2-yl)methylidene]-4-(trifluoro-
methyl)benzohydrazide (8/S)
1H NMR (300 MHz, d ppm, DMSO-d6): 7.58–7.59 (d, J = 2.77, 1H,
H4), 7.90–7.92 (d, J = 7.81, 2H, H12 and H14), 8.08–8.11 (d, J = 8.81,
3H, H3, H11 and H15), 8.66 (s, 1H, H6), 12.40 (s, 1H, H8); MS (m/z):

6730
D. G. Rando et al. / Bioorg. Med. Chem. 16 (2008) 6724–6731
341.96 [Mꢀ1]. Anal. Calcd for C13H8F3N3O3S: C, 45.44; H, 2.33; N,
8.32–8.34 (d, J = 7.6, 1H, H11), 8.43–8.45 (d, J = 7.8, 1H, H13), 8.66
(s, 1H, H15), 8.72 (s, 1H, H6), 12.50 (s, 1H, H8); MS (m/z): 319.03
[Mꢀ1]. Anal. Calcd for C12H8N4O5S: **C, 44.17; H, 2.45; N, 17.18.
Found: C, 44.76; H, 2.44; N, 16.75.
12.23. Found: C, 45.21; H, 2.10; N, 12.42.
5.1.36. 4-Nitro-N0-[(5-Nitrothiophen-2-yl)methylidene]-
benzohydrazide (9/S)
1H NMR (300 MHz, d ppm, DMSO-d6): 7.60 (br s, 1H, H4), 8.12–
8.13 (d, J = 7.1, 3H, H3, H11 and H15), 8.36–8.38 (d, J = 8.3, 2H, H12
and H14), 8.66 (s, 1H, H6), 12.49 (s, 1H, H8); MS (m/z): 318.98
[Mꢀ1]. Anal. Calcd for C12H8N4O5S: C, 45.00; H, 2.52; N, 17.49.
Found: C, 44.62; H, 2.27; N, 17.26.
5.1.44. 3,4-Dichloro-N0-[(5-nitrothiophen-2-yl)methylidene]-
benzohydrazide (18/S)
1H NMR (300 MHz, d ppm, DMSO-d6): 7.63–7.64 (d, J = 4.4, 1H,
H4), 7.85–7.87 (d, J = 8.3, 1H, H12), 7.89–7.91 (d, J = 8.8, 1H, H11),
8.17 (br s, 2H, H3 and H15), 8.66 (s, 1H, H6), 12.37 (s, 1H, H8); MS
(m/z): 343.87 [Mꢀ1]. Anal. Calcd for C12H7Cl2N3O3S: C, 41.86; H,
2.03; N, 12.21. Found: C, 41.16; H, 2.06; N, 11.81.
5.1.37. 3-Hydroxy-N0-[(5-nitrothiophen-2-yl)methylidene]-
benzohydrazide (10/S)
1H NMR (300 MHz, d ppm, DMSO-d6): 7.55–7.59 (m, 2H, H4 and
5.1.45. 3,4-Difluoro-N0-[(5-nitrothiophen-2-yl)methylidene]-
benzohydrazide (19/S)
H12), 7.67–7.69 (d, J = 7.3, 1H, H13), 7.85–7.87 (d, J = 7.3, 1H, H11),
7.94 (s, 1H, H15), 8.12–8.13 (d, J = 3.4, 1H, H3), 8.65 (s, 1H, H6),
12.29 (s, 1H, H8);MS (m/z): 290.02 [Mꢀ1]. Anal. Calcd for
1H NMR (300 MHz, d ppm, DMSO-d6): 7.61–7.67 (m, 2H, H4 and
H12), 7.80 (br s, 1H, H15), 7.94–7.98 (t, J = 9.8, 1H, H11), 8.12–8.13
(d, J = 2.0, 1H, H3), 8.65 (s, 1H, H6), 12.27 (s, 1H, H8); MS (m/z):
309.93 [Mꢀ1]. Anal. Calcd for C12H7F2N3O3S: C, 46.30; H, 2.25; N,
13.50. Found: C, 47.43; H, 2.18; N, 13.67.
C12H9N3O4S: C, 46.49; H, 2.58; N, 13.56. Found: C, 45.26; H, 2.54;
N, 13.27.
5.1.38. 3-Methoxy-N0-[(5-nitrothiophen-2-yl)methylidene]-
benzohydrazide (11/S)
5.1.46.3-Chloro-4-fluoro-N0-[(5-nitrothiophen-2-yl)methylidene]-
benzohydrazide(22/S)
1H NMR (300 MHz, d ppm, DMSO-d6): 3.82 (s, 3H, OCH3), 7.18
(br s, 1H, H13), 7.44–7.46 (m, 3H, H3, H4 and H12), 7.57 (br s, 1H,
1H NMR (300 MHz, d ppm, DMSO-d6): 7.62–7.64 (d, J = 8.3, 2H,
H4 and H12), 7.95 (br s, 1H, H3), 8.13–8.15 (br s, 2H, H11 and H15),
8,65 (s, 1H, H6), 12,31 (s, 1H, H8); MS (m/z): 324.90 [Mꢀ1]. Anal.
Calcd for C12H7ClFN3O3S: *C, 43.97; H, 2.14; N, 12.82. Found: C,
41.95; H, 2.66; N, 12.33.
H
15), 8.12 (br s, 1H, H11), 8.68 (s, 1H, H6), 12.18 (s, 1H, H8); MS
(m/z): 304.10 [Mꢀ1]. Anal. Calcd for C13H11N3O4S: *C, 48.29; H,
3.41; N, 13.00. Found: C, 48.04; H, 3.84; N, 13.04.
5.1.39. 3-(Dimethylamino)-N0-[(5-nitrothiophen-2-yl)methyl-
idene]benzohydrazide (12/S)
5.1.47. 3-Methoxy-4-nitro-N0-[(5-nitrothiophen-2-yl)methyl-
idene]benzohydrazide (23/S)
1H NMR (300 MHz, d ppm, DMSO-d6): 2.95 (s, 6H, N(CH3)2),
6.93–6.95 (d, J = 8.3, 1H, H13), 7.16 (br s, 2H, H11 and H15), 7.30–
7.34 (t, J = 8.3, 1H, H12), 7.55–7.56 (d, J = 3.9, 1H, H4), 8.11–8.13
(d, J = 4.4, 1H, H3), 8.70 (s, 1H, H6), 12.10 (s, 1H, H8); MS (m/z):
1H NMR (300 MHz, d ppm, DMSO-d6): 3.99 (s, 3H, H16), 7.57–
7.62 (m, 2H, H4 and H11), 7.75 (br s, 1H, H15), 8.01–8.03 (d,
J = 7.8, 1H, H12), 8.14 (br s, 1H, H3), 8.68 (s, 1H, H6), 12.37 (s, 1H,
H8); MS (m/z): 348.96 [Mꢀ1]. Anal. Calcd for C13H10N4O6S:
C = 44.60; H, 2.86; N, 16.00. Found: C, 44.32; H, 2.93; N, 15.76.
*
317.12 [Mꢀ1]. Anal. Calcd for C14H14N4O3S: C, 50.00; H, 4.17; N,
16.67. Found: C, 49.29; H, 4.63; N, 16.60.
5.1.40. 3-Hydroxy-N0-[(5-nitrothiophen-2-yl)methylidene]-
benzohydrazide (14/S)
5.1.48. 3-Hydroxy-4-nitro-N0-[(5-nitrothiophen-2-yl)-
methylidene]benzohydrazide (24/S)
1H NMR (300 MHz, d ppm, DMSO-d6): 7.00 (br s, 1H, H4), 7.28–
7.33 (m, 3H, H12, H13 and H15), 7.57 (br s, 1H, H3), 8.13 (s, 1H, H11),
8.66 (s, 1H, H6), 12.15 (s, 1H, H8); MS (m/z): 290.04 [Mꢀ1]. Anal.
Calcd for C12H9N3O4S: C, 49.44; H, 3.09; N, 14.42. Found: C,
49.28; H, 2.85; N, 14.49.
1H NMR (300 MHz, d ppm, DMSO-d6): 7.42–7.44 (d, J = 8.3, 1H,
H15), 7.59–7.61 (m, 2H, H4 and H11), 8.00–8.02 (d, J = 8.3, 1H,
H
12), 7.13–8.14 (d, J = 3.7, 1H, H3), 8.67 (s, 1H, H6), 12.37 (s, 1H,
H8); MS (m/z): 334.93 [Mꢀ1]. Anal. Calcd for C12H8N4O6S: *C,
40.68; H, 2.26; N, 15.82. Found: C, 41.68; H, 2.73; N, 15.74.
5.1.41. 3-Methyl-N0-[(5-nitrothiophen-2-yl)methylidene]-
benzohydrazide (15/S)
5.1.49. 3-Amino-4-methoxy-N0-[(5-nitrothiophen-2-yl)-
methylidene]benzohydrazide (25/S)
1H NMR (300 MHz, d ppm, DMSO-d6): 2.38 (s, 3H, H16), 7.41 (br
s, 2H, H12 and H13), 7.56 (br s, 1H, H4), 7.69 (br s, 2H, H11 and H15),
8.11 (br s, 1H, H3), 8.66 (s, 1H, H6), 12.18 (s, 1H, H8); MS (m/z):
1H NMR (300 MHz, d ppm, DMSO-d6): 3.83 (s, 3H, OCH3), 4.99
(S, 2H, NH2), 6.89–6.91 (d, J = 8.1, 1H, H12), 7.15–7.19 (m, 2H, H11
and H15), 7.53–7.54 (d, J = 3.9, 1H, H4), 8.12-8.13 (d, J = 3.9, 1H,
H3), 8.64 (s, 1H, H6), 11.99 (s, 1H, H8); MS (m/z): 318.99 [Mꢀ1].
Anal. Calcd for C13H12N4O4S: not determined.
*
288.12 [Mꢀ1]. Anal. Calcd for C13H11N3O3S: C, 50.81; H, 3.58; N,
13.68. Found: C, 50.78, H, 4.13; N, 13.85.
5.1.42. N0-[(5-Nitrothiophen-2-yl)methylidene]-3-(trifluoro-
methyl)benzohydrazide (16/S)
5.1.50. 3-Amino-4-hydroxy-N0-[(5-nitrothiophen-2-yl)-
methylidene]benzohydrazide (26/S)
1H NMR (300 MHz, d ppm, DMSO-d6): 7.61-7.62 (d, J = 3.4, 1H,
H4), 7.78–7.82 (t, J = 7.6, 1H, H12), 7.98–8.00 (d, J = 7.3, 1H, H13),
8.13–8.14 (d, J = 3.4, 1H, H 3), 8.20–8.23 (m, 2H, H11 and H15),
8.67 (s, 1H, H6), 12.40 (s, 1H, H8); MS (m/z): 341.96 [Mꢀ1]. Anal.
Calcd for C13H8F3N3O3S: *C, 43.18; H, 2.21; N, 11.63. Found: C,
43.38; H, 2.57; N, 11.53.
1H NMR (300 MHz, d ppm, DMSO-d6): 4.75 (br s, 2H, NH2), 6.71-
6.74 (d, J = 8.3, 1H, H12), 7.06–7.08 (d, J = 8.1, 1H, H15), 7.17 (br s,
1H, H11), 7.50–7.51 (d, J = 4.3, 1H, H4), 8.10–8.11 (d, J = 4.3, 1H,
H3), 8.64 (s, 1H, H6), 11.96 (s, 1H, H8); MS (m/z): 304.90 [Mꢀ1].
Anal. Calcd for C12H10N4O4S: not determined.
5.1.51. 3-Amino-4-methyl-N0-[(5-nitrothiophen-2-yl)-
methylidene]benzohydrazide (28/S)
5.1.43. 3-Nitro-N0-[(5-nitrothiophen-2-yl)methylidene]-
benzohydrazide (17/S)
1H NMR (300 MHz, d ppm, DMSO-d6): 2.10 (s, 3H, CH3), 5.11 (s,
2H, NH2), 7.00–7.06 (m, 2H, H12 and H11), 7.13 (br s, 1H, H15), 7.53–
7.54 (d, J = 3.7, 1H, H4), 7.75–7.76 (d, J = 3.9, 1H, H3), 8.98 (s, 1H,
1H NMR (300 MHz, d ppm, DMSO-d6): 7.59–7.60 (d, J = 3.3, 1H,
H4), 7.82–7.86 (t, J = 8.1, 1H, H12), 8.11–8.12 (d, J = 4.0, 1H, H3),

D. G. Rando et al. / Bioorg. Med. Chem. 16 (2008) 6724–6731
6731
H6), 12.00 (s, 1H, H8); MS: 305.05 [M+1]. Anal. Calcd for
13H12N4O3S: C, 45.90; H, 3.53; N, 16.47. Found: C, 45.18, H, 3.33,
N, 17.45.
tions from 50 to 3.1
l
g/mL. Drug dilutions were prepared directly
C
in cell suspension in 96-well plates and were incubated at 26 °C for
48 h and growth of Leishmania promastigotes was determined by
Alamar Blue assay.20 Standard fluorescence was measured on a
Fluostar Galaxy plate reader (BMG Lab Technologies) at excitation
wavelength of 544 nm and emission wavelength of 590 nm.
Pentamidine and amphotericin B were used as the standard
antileishmanial agents. Percentual growth was calculated and plot-
ted versus test concentration for computing the IC50 values and
5.1.52. 3-Hydroxy-4-methyl-N0-[(5-nitrothiophen-2-yl)-
methylidene]benzohydrazide (29/S)
1H NMR (300 MHz, d ppm, DMSO-d6): 2.16 (s, 3H, CH3), 7.17–
7.91 (d, J = 7.8, 1H, H12), 7.26–7.28 (d, J = 7.8, 1H, H11), 7.32 (s,
1H, H15), 7.52–7.53 (d, J = 3.9, 1H, H4), 8.10–8.12 (d, J = 4.2, 1H,
H3), 8.64 (s, 1H, H6), 9.72 (s, 1H, OH), 12.13 (s, 1H, H8); MS (m/z):
303.98 [Mꢀ1]. Anal. Calcd for C13H11N3O4S: not determined.
IC90
.
References and notes
5.1.53. 3-Methoxy-4-methyl-N0-[(5-nitrothiophen-2-yl)-
methylidene]benzohydrazide (30/S)
1. Sundar, S.; Rai, M. Curr. Opin. Infect. Dis. 2002, 15, 593.
2. Desjeux, P. Clin. Dermatol. 1996, 14, 417.
3. Bates, P. A. Int. J. Parasitol. 2007, 37, 1097.
4. Ashford, R. W. Ann. Trop. Med. Parasitol. 1997, 91, 693.
5. Chappuis, F.; Sundar, S.; Hailu, A.; Ghalib, H.; Rijal, S.; Peeling, R. W.; Alvar, J.;
Boelaert, M. Nat. Rev. Microbiol. 2007, 5, 873.
6. Reithinger, R.; Dujardin, J. C.; Louzir, H.; Pirme, C.; Alexander, B.; Brooker, S.
Lancet Infect. Dis. 2007, 7, 581.
1H NMR (300 MHz, d ppm, DMSO-d6): 2.21 (s, 3H, CH3), 3.86 (s,
3H, OCH3), 7.27–7.30 (d, J = 7.34, 1H, H12), 7.42 (br s, 2H, H11 and
H15), 7.56–7.57 (d, J = 3.7, 1H, H4), 8.14–8.16 (d, J = 3.9, 1H, H3),
8.70 (s, 1H, H6), 12.13 (s, 1H, H8); MS (m/z): 317.97 [Mꢀ1]. Anal.
Calcd for C14H13N3O4S: C, 52.70; H, 4.08; N, 13.17. Found: C,
52.72; H, 3.97; N, 12.94.
7. Amato, V. S.; Tuon, F. F.; Bacha, H. A.; Neo, V. A.; Nicodemo, A. C. Acta Trop.
2008, 105, 1.
8. Mishra, J.; Saxena, A.; Singh, S. Curr. Med. Chem. 2007, 14, 1153.
9. Alvar, J.; Cañavate, C.; Gutiérrez-Solar, B.; Jimenez, M.; Laguna, F.; Lopez-Velez,
R.; Molina, R. J. Moreno. Clin. Microbiol. Rev. 1997, 10, 298.
10. Berman, J. D. Clin. Infect. Dis. 1999, 28, 49.
5.1.54. 2-Chloro-5-({2-[(5-nitrothiophen-2-yl)methylidene]-
hydrazino}carbonyl) benzene sulfonamide (32/S)
1H NMR (300 MHz, d ppm, DMSO-d6): 7.60–7.61 (d, J = 2.7, 1H,
H4), 7.76 (br s, 2H, SO2NH2), 7.84–7.86 (d, J = 8.1, 1H, H12), 8.11–
8.14 (m, 2H, H3 and H11), 8.50 (s, 1H, H15), 8.68 (s, 1H, H6), 12.50
(s, 1H, H8); MS (m/z): 386.85 [Mꢀ1]. Anal. Calcd for C12H9ClN4O5S2:
C, 37.07; H, 2.32; N, 14.41. Found: C, 37.35; H, 2.24; N, 14.62.
11. Herwaldt, B. L. Lancet 1999, 354, 1191.
12. Salem, M. M.; Werbovetz, K. A. Curr. Med. Chem. 2006, 13, 2571.
13. Aveniente, M.; Pinto, E. F.; Santos, L. S.; Rossi-Bergmann, B.; Barata, L. E. S.
Bioorg. Med. Chem. 2007, 15, 7337.
14. Fan, X.; Zhang, X.; Bories, C.; Loiseau, P. M.; Torrence, P. F. Bioorg. Chem. 2007,
35, 121.
15. Fuertes, M. A.; Nguewa, P. A.; Castilla, L.; Alonso, C.; Pérez, J. M. Curr. Med.
Chem. 2008, 15, 433.
16. Gross, L.; Lorente, S. O.; Jimenez, C.; Yardley, V.; Rattray, L.; Wharton, H.; Little,
S.; Croft, S. L.; Ruiz-Perez, L. M.; Gonzalez-Pacanowska, D.; Gilbert, I. H. J. Med.
Chem. 2006, 49, 6049.
17. Raether, W.; Hänel, H. Parasitol. Res. 2003, 90, S19.
18. Orna, M. V.; Mason, R. P. J. Biol. Chem. 1989, 264, 12379.
19. Rando, D. G.; Sato, D. N.; Siqueira, L. J. A.; Malvezzi, A.; Leit, C. Q. F.; do_Amaral,
A. T.; Ferreira, E. I.; Tavares, L. C. Bioorg. Med. Chem. 2002, 10, 557.
20. Mikus, J.; Steverding, D. Parasitol. Int. 2000, 48, 265.
5.2. Antileishmanial assays
Antileishmanial activity of the compounds was tested in vitro
against a culture of L. donovani promastigotes. The parasites were
grown in RPMI 1640 medium supplemented with 10% fetal calf
serum (Gibco Chem Co.) at 26 °C. A 3-day-old culture was diluted
to 5 ꢁ 105 promastigotes/mL. Samples were tested at concentra-