In vivo and in vitro anti-leishmanial activities of 4-nitro-N-pyrimidinand N-pyrazin-2-ylbenzenesulfonamides, and N2-(4-nitrophenyl)-N 1propylglycinamide

Article abstract of DOI:10.1016/j.bmc.2009.09.030

A series of compounds containing the nitrobenzene and sulfonamido moieties were synthesized and their leishmanicidal effect was assessed in vitro against Leishmania infantum promastigotes. Among the compounds evaluated, the p-nitrobenzenesulfonamides 4Aa

Full text of DOI:10.1016/j.bmc.2009.09.030

Bioorganic & Medicinal Chemistry 17 (2009) 7449–7456
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
In vivo and in vitro anti-leishmanial activities of 4-nitro-N-pyrimidin-
and N-pyrazin-2-ylbenzenesulfonamides, and N2-(4-nitrophenyl)-N1-
propylglycinamide
M. Auxiliadora Dea-Ayuela a,c, Encarna Castillo b, Marta Gonzalez-Alvarez a,f, Celeste Vega d,e
,
Miriam Rolón d,e, Francisco Bolás-Fernández c, Joaquín Borrás f, M. Eugenia González-Rosende a,
*
a Department of Chemistry, Biochemistry and Molecular Biology, University CEU Cardenal Herrera, Edificio Seminario s/n, 46113-Moncada, Valencia, Spain
b Department of Physiology, Pharmacology and Toxicology, University CEU Cardenal Herrera, Edificio Seminario s/n, 46113-Moncada, Valencia, Spain
c Department of Parasitology, Faculty of Pharmacy, Complutense University of Madrid, Plaza Ramón y Cajal s/n, 28040-Madrid, Spain
d Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Asunción, Campus Universitario San Lorenzo, San Lorenzo, Paraguay
e Centro para el Desarrollo de la Investigación Científica (Fundación Moisés Bertoni/Laboratorio Díaz Gill), Eligio Ayala 1384, Asunción, Paraguay
f Department of Inorganic Chemistry, University of Valencia, Vicent Andrés Estellés s/n, 46180-Burjassot, Valencia, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 May 2009
Revised 15 August 2009
Accepted 17 September 2009
Available online 20 September 2009
A series of compounds containing the nitrobenzene and sulfonamido moieties were synthesized and their
leishmanicidal effect was assessed in vitro against Leishmania infantum promastigotes. Among the com-
pounds evaluated, the p-nitrobenzenesulfonamides 4Aa and 4Ba, and the p-nitroaniline 5 showed signif-
icant activity with a good selectivity index. In a Balb/c mice model of L. Infantum, administration of
compounds 4Aa, 4Ba or 5 (5 mg/kg/day for 10 days, injected ip route) led to a clear-cut parasite burden
reduction (ca. 99%). In an attempt to elucidate their mechanism of action, the DNA interaction of 4Aa and
5 was investigated by means of viscosity studies, thermal denaturation and nuclease activity assay. Both
compounds showed nuclease activity in the presence of copper salt. The results suggest that compounds
4Aa, 4Ba and 5 represent possible candidates for drug development in the therapeutic control of
leishmaniasis.
Keywords:
Leishmania infantum
Nitrobenzene
Nuclease activity
Sulfonamides
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
the classic treatment of leishmaniasis. Pentamidine, amphotericin
B and miltefosine have been introduced as second-line therapy.
Protozoan parasitic diseases have plagued mankind for centu-
ries and continue to cause significant public health problems.
Leishmaniasis comprises a group of diseases with distinct clinical
manifestations caused by different species of the protozoan para-
site Leishmania. This type of infection is considered within the most
relevant group of neglected tropical diseases and targeted by the
WHO and other health organizations for prevention, control and
eradication.1,2 It is estimated that human leishmaniasis is distrib-
uted worldwide with a prevalence of 12 million individuals in 88
countries and 350 million individuals at risk of infection.3,4 In addi-
tion, over the last decade Leishmania-HIV co-infections are becom-
ing a major health problem in affected areas.5,6
Current treatment of leishmaniasis is based on chemotherapy
with some attempts at immunotherapy. Unfortunately, there is
no vaccine against Leishmania infection and vector control is diffi-
cult. Therefore chemotherapy is the main approach to control this
worldwide spread disease.7–11 Pentavalent antimonials have been
However, the resistance to antimonials has become a severe prob-
lem and treatment with second-line drugs is generally expensive
and frequently complicated by the occurrence of toxic side ef-
fects.12–14 Consequently, the development of novel, efficient and
safe chemotherapeutic agents with leishmanicidal activity is
imperative.
In this context, sulfonamides have long been the subject of
pharmaceutical interest as a result of their potent biological activ-
ities.15,16 Recently, it has been established that sulfonamides in the
presence of Cu(II) and reducing agents have strong nuclease
activity.17 Moreover, sulfonamide derivatives exhibiting anti-leish-
manial activity have been described.18 Thus, a series of N-quinolin-
8-yl-arylsulfonamides have been reported as potent in vitro
leishmanicidal compounds, being the quinolinsulfonamide
1
(Fig. 1) the most active compound.19 On the other hand it has been
shown that nitro aromatic derivatives possess substantial activity
against Leishmania. In those studies compounds 2 and 3 were
identified as new anti-leishmanial compounds.20–22
Based on the anti-leishmanial activity of nitrobenzene deriva-
tives and aryl sulfonamides, we synthesized a series of com-
* Corresponding author. Tel.: +34 961 369 000; fax: +34 961 395 272.
E-mail address: eugenia@uch.ceu.es (M.E. González-Rosende).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.09.030

7450
M. A. Dea-Ayuela et al. / Bioorg. Med. Chem. 17 (2009) 7449–7456
F
F
Pr
2. Results and discussion
Pr
N
O2N
S
H3CO
CN
O2N
NO2
2.1. Leishmanicidal activity
SO2NH
NO2
The compounds synthesized to investigate the anti-leishmanial
activity were sulfonamides 4A–F, sulfadiazine, p-nitroaniline 5,
guanidines 6a–f and imidazolidinones 7a,b (Fig. 2).
Sulfonamides 4 were easily obtained by reaction of the amine
with the corresponding sulfonyl chloride according to the litera-
ture23 (Scheme 1). The synthesis of p-nitroaniline 5, guanidines 6
and imidazolidinones 7 was carried out according to a procedure
previously described.24
The leishmanicidal activity of sulfadiazine and compounds 4, 5,
6 and 7 was evaluated in vitro against cultured promastigotes of L.
infantum (MCAN/ES/92/BCN 83), according to a modification of a
previously reported procedure.25 Whereas guanidines 6 and imi-
dazolidinones 7 had no effect on the growth of L infantum prom-
SO2NH
N
2
3
1
Figure 1. Sulfonamides and nitrobenzene derivatives with leishmanicidal activity.
pounds that have both nitrobenzene and sulfonamide entities and
we evaluated their leishmanicidal effect against Leishmania infan-
tum promastigotes (Fig. 2). Compounds exhibiting a good selectiv-
ity index were also tested in vivo in an acute murine model of
leishmaniasis. Studies to elucidate the mechanism of action were
also carried out and the interaction with DNA was investigated by
viscosity studies, thermal denaturation and nuclease activity
assays.
astigotes at concentrations as high as 100 lg/mL, pyrimidines
Herein we report on the evaluation of compounds 4–7 as poten-
tial antiparasitics against L. infantum. The results demonstrate that
the benzenesulfonamides 4Aa and 4Ba, and the p-nitroaniline
derivative 5 are promising candidates to be considered as lead
compounds for new leishmanicidal drugs, since they are effective
against promastigotes of L. infantum at concentrations in the
micromolar range and display excellent in vivo efficacies in a
mouse model of leishmaniosis, with no toxicity against mamma-
lian cells. Compounds 4Aa and 5 showed nuclease activity in the
presence of copper salt, therefore it is not possible to discard
DNA cleavage for their mechanism of action.
4Aa,b,k, pyrazine 4Ba, pyridine 4Bd, thiazoles 4Ca,b,d–f, isoxazole
4Da, pyrazole 4Dd, indazole 4E, indanes 4Fa–c and aniline 5 were
found to be active compounds. The p-nitrobenzenesulfonamides
4Aa (92.7% growth inhibition, GI) 4Ba (90.8% GI), 4Da (85.6% GI),
4E (88% GI) and 4Fa (91.9% GI), and the p-nitroaniline 5 (85% GI)
were the most active compounds. In the case of compounds 4Aa,
4Ba, 4Da, 4E and 5, the leishmanicidal activity was associated with
a lack of toxicity to murine macrophages (IC50 >256 lM) (Table 1).
As shown in Table 1, the anti-leishmanial activity of sulfona-
mides 4A follows the order 4Aa > 4Ab > 4Ae ꢀ 4Ai, being the p-ni-
tro derivative 4Aa the most active compound. The removal of the
R1
X
R2
N
N
NHSO2R
R1
NHSO2R
N
S
R1
N
NHSO2R
4A
4B
4C
4Aa: R = 4-NO2Ph R1 = H
4Ab: R = 2-NO2Ph R1 = H
4Ac: R = 3-NO2Ph R1 = H
4Ba: X = N
4Bb: X = N
4Bc: X = N
4Bd: X = CH2 4-NHSO2(4-NO2Ph)
4Be: X = CH2 3-NHSO2(4-NO2Ph)
4Bf: X = CH2 2-NHSO2(4-NO2Ph)
2-NHSO2(4-NO2Ph)
2-NHSO2(4-ClPh)
2-NHSO2Ph
4Ca: R = 4-NO2Ph R1 = H
4Cb: R = 4-ClPh 1 = H
4Cc: R = 4-NO2Ph R1 = CH3 R2 = H
R2 = H
R2 = H
R
4Ad: R = CH3
4Ae: R = Ph
4Af: R = 4-FPh
4Ag: R = 4-ClPh
4Ah: R = 4-BrPh
R1 = H
4Cd: R = 4-ClPh
4Ce: R = Ph
4Cf: R = 4-NO2Ph R1 = H
R
R
1 = CH3 R2 = H
1 = CH3 R2 = H
R2 = CH3
R
1 = H
R
R
1 = H
1 = H
R1 = H
4Ai: R = 4-CH3OPh R1 = H
4Aj: R = 4-NO2Ph R1 = CH3
4Ak: R = 4-ClPh
R1 = CH3
NHSO2R
NHSO2R
NHSO2
NO2
N
N
H3C
X
N
H
4D
4E
4F
4Da: X = O 4-NO2Ph
4Db: X = O 4-ClPh
4Dc: X = O Ph
4Fa: R = 4-NO2Ph
4Fb: R = 4-ClPh
4Fc: R = Ph
4Dd: X = N 4-NO2Ph
NO2
SO2 R
N
H2N
HN
N
SO2 R
N
HN
O
N
NHSO2
NH2
CH2CONHR1
HN
NHCH2CONHPr
5
7
6
Sulfadiazine
7a: R = 4-NO2Ph
7b: R = 4-CH3OPh
6a: R = 4-NO2Ph R1 = H
6b: R = 4-CH3Ph R1 = H
6c: R = 4-BrPh
R1 = H
6d: R = 4-CH3OPh R1 = H
6e: R = 3-NO2Ph R1 = Et
6f: R = 3-NO2Ph R1 = Pr
Figure 2. Structures of the series of compounds evaluated against L. infantum.

M. A. Dea-Ayuela et al. / Bioorg. Med. Chem. 17 (2009) 7449–7456
7451
N
N
4Aa (NO2?NH2) and the mode of action of 4Aa on L. infantum by
the classical route of inhibiting the novo folate synthesis could
be discarded. Probably, sulfonamides 4 and p-nitroaniline 5 enter
the parasite and undergo redox cycling producing reactive oxygen
species (ROS) that can peroxidatively damage membrane lipids,
proteins or DNA.12 In fact, several nitroaromatic compounds have
been shown that increase ROS generation in Leishmania parasites.21
Sulfonamides 4Aa, 4Ba, and p-nitroaniline 5, the most active
compounds in the in vitro anti-promastigote assay with a good
therapeutic index, were then tested in vivo in an acute murine
model of visceral leishmaniosis. Compounds 4Aa, 4Ba and 5 were
assayed at concentrations of 5 mg/kg/dose administered by the
intraperitoneal route daily up to a total of 10 doses using a modi-
fication of a method previously described.26 The assayed com-
pounds displayed high levels of activity, with significant
suppression of parasite burden in spleen and liver compared to
the parasite burden in the tissues of the controls (Table 2). No
apparent signs of toxicity, such as gross weight loss or hair loss,
were observed in any of the animals at the end of the assay,
strongly suggesting that 4Aa, 4Ba and 5 are well tolerated by the
infected mice.
N
N
R
SO2Cl
N
H
SO2R
NH2
py
0 ºC
4a-i
Scheme 1. Synthesis of sulfonamides 4.
Table 1
In vitro activities of compounds 4, 5 and sulfadiazine against L. infantum promastig-
otes and J774 macrophagesa,b
Compound
Growth inhibition (%)
IC50
L. infantum
(lM)
J774
4Aa
4Ab
4Ac
4Ad
4Ae
4Af
4Ag
4Ah
4Ai
92.7
40.6
—
100 2.6
>256
—
—
>256
—
>256
ND
—
—
ND
—
—
20.3
—
—
—
—
—
—
—
29.6
33.3
71.5
90.8
25.3
37.0
46.3
35.7
39.3
58.6
65.0
36.7
74.5
69.0
55.6
85.6
15.7
17.9
71.5
88.2
91.9
49.9
45.3
—
>256
>256
224 32.2
66.2 3.4
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
126 0.7
>256
>256
164 10.7
14.5 0.2
<1
ND
ND
>256
>256
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
>256
ND
ND
>256
>256
61 5.6
ND
ND
—
4Aj
4Ak
4Ba
4Bb
4Bc
4Bd
4Be
4Bf
4Ca
4Cb
4Cc
4Cd
4Ce
4Cf
4Da
4Db
4Dc
4Dd
4E
4Fa
4Fb
4Fc
2.2. DNA binding properties
In an attempt to gain insight into the anti-leishmanial mecha-
nism of action of compounds 4Aa and 5, the mode and propensity
for binding to calf thymus DNA (CT-DNA) was investigated by vis-
cosity studies, thermal denaturation and nuclease activity assay.
Viscosity measurements were carried out on calf thymus DNA
by varying the concentration of sulfonamide 4Aa or p-nitroaniline
5. Hydrodynamic measurements that are sensitive to length
changes are regarded as the least ambiguous and the most critical
tests of binding in solution. A classical intercalation model results
in the lengthening of the DNA helix as the base pairs are separated
to accommodate the binding ligand, leading to an increase of DNA
viscosity. By contrast, compounds that bind exclusively in DNA
grooves by partial and/or non-classical intercalation under the
same conditions typically cause a less pronounced (positive or neg-
ative) or no change in DNA solution viscosity.27 The results ob-
tained with compounds 4Aa or 5 reveal that both of them have
no effect on the relative viscosity of CT-DNA at 25 °C or at 37 °C.
These findings indicate that the interaction does not involve a clas-
sic intercalation.
>256
>256
—
Sulfadiazine
5
85.0
156 26
>256
a
ND, not determined.
—, No activity detected.
b
Thermal behaviour of DNA in the presence of some compounds
can give insight into their conformational changes when tempera-
ture is raised and information about the interaction strength of the
complexes with DNA. The double-stranded DNA tends to dissociate
gradually to single strands when increasing the solution tempera-
ture thus generating a hyperchromic effect on the absorption spec-
tra of DNA bases (kmax = 260 nm). In order to identify this
transition process, the melting temperature (Tm), which is defined
as the temperature where the half of the double strand plasmid is
electron withdrawing nitro group, as in sulfonamide 4Ae, de-
creases the potency against the parasites and the replacement of
the nitro group on the benzene ring of compound 4Aa by an elec-
tron-donating methoxy group as in compound 4Ai, causes a dimi-
nution in the antiparasitic activity. In addition, sulfonamides 4Af
and 4Ah with electron-withdrawing substituents in para position
were also ineffective. The same pattern is observed in the pyrida-
zine (4Ba–c), isoxazole (4Da–c) and indane (4Fa–c) series. There-
fore, the electronic effect of the substituents on the benzene ring
seems to be not too relevant for the leishmanicidal activity and
our findings indicate that the p-nitrobenzene moiety is essential
for the antiparasitic activity.
In an attempt to explain the lack of activity of the other two p-
nitrophenyl derivatives 6a and 7a, we were suspect of a higher
hydrophilic character of 6a and 7 to enter the parasite in compar-
ison to 4Aa, 4Ba and 5. We used the software ACD/Labs to estimate
their log P. The log P values of 6a (0.10) and 7a (À0.34) were signif-
icantly lower than 4Aa (1.04), 4Ba (1.20) and 5 (1.54).
Table 2
In vivo anti-leishmanial effect of compounds 4Aa, 4Ba and 5
Compound
Mean SDb% suppressiona
Spleen
Liver
4Aa
5
4Ba
99.82 0.11c
99.89 0.10c
99.44 0.93c
97.58 4.03c
97.95 3.88c
99.28 0.92c
a
Reductions of parasite burdens in spleens and livers of treated mice in relation
It is noteworthy that the substitution of the NO2 group of com-
pound 4Aa by NH2 (antibacterial sulfadiazine) resulted in a signif-
icant loss of activity. This finding suggests that the observed
activity is not probably due to the reduced form of compound
to those in the control (untreated) groups.
b
The standard deviation (SD) was calculated by comparing individual data for
the treated animals with the average value for the control group.
c
P <0.0005 compared with controls.

7452
M. A. Dea-Ayuela et al. / Bioorg. Med. Chem. 17 (2009) 7449–7456
transformed into a single strand one, is a valuable parameter.28
Intercalation of natural or synthesized organics and metallointer-
calators generally results in a considerable increase in the melting
point temperature (Tm).29 The results indicate that both com-
pounds 4Aa and 5 do not produce significant changes in the Tm va-
lue (Tm control = 53.8 °C, Tm 4Aa = 54.2 °C, Tm 5 = 54.0 °C) and
therefore no stabilization of DNA takes place in the presence of
compounds 4Aa and 5.
The activity of compounds 4Aa and 5 as chemical nucleases was
studied using supercoiled pUC18 DNA in cacodylate buffer (pH
6.0). Figure 3 shows that sulfonamide 4Aa and p-nitroaniline 5
did not present ability to damage DNA plasmid at the assayed con-
centrations. Further assays were carried out in order to evaluate
the ability to cleave pUC18 plasmid DNA by copper(II)–ligand
(1:1) mixtures. The nuclease activity of the mixtures ranging from
3. Conclusion
In summary, sulfonamides 4Aa, 4Ba and the p-nitroaniline 5
constitute new antiparasitic agents with high selectivity indices
versus mammalian cells and emerge as candidates for further eval-
uation against kinetoplastid parasites. Since the synthesis of these
compounds is straightforward, a wide variety of analogues can be
prepared to optimize in vitro and in vivo activities. In addition, gi-
ven the economic realities of chemotherapy for leishmaniasis, the
inexpensive large-scale synthesis of appropriate candidates would
allow their future development if in vivo activity can be achieved.
Synthesis and pharmacological evaluation of related derivatives to
determine structural features for the anti-leishmanial activity are
currently in progress.
15 to 45
chloride in the presence of ascorbate (50-fold excess relative cop-
per(II) concentration) (Fig. 4). At concentrations of 15 M or
30 M, the Cu(II)–ligand (1:1) mixtures were not able to induce
DNA cleavage, whereas at the concentration of 45 M a complete
lM was examined and compared with that of the cupric
4. Experimental section
4.1. Chemistry
l
l
l
4.1.1. General
degradation of supercoiled form to yield circular and linear forms
was induced (lanes 10 and 14). This activity was higher than that
of copper(II) salt at the same conditions (compare lanes 10 and
14 with lane 6). Taking into consideration that the ligands do not
exhibit nuclease activity alone, it is evident that Cu(II) do contrib-
ute to the DNA cleavage process. The results indicate that 4Aa and
5 do not interact nor cleave the DNA by themselves but they do so
in the presence of copper salt. This behaviour is probably due to a
redox process in the presence of the metal transition. The activa-
tion of a Fenton-type or a Haber–Weiss reactions produces the
generation of oxygen active species (hydroxyl, superoxide, singlet
oxygen-like species and hydrogen peroxide) which then cleave
the DNA.30 Further studies will make possible to identify the reac-
tive oxygen species that mediate the DNA strand scission by mon-
itoring the quenching of DNA cleavage in the presence of ROS
scavengers in solution.
All reagents were purchased from Aldrich and used without
purification. All experiments were made under nitrogen atmo-
sphere. Melting points were determined with a Kofler hot-stage
apparatus and are uncorrected. Column chromatography was per-
formed using silica gel (Merck 60, 70–230 mesh). 1H and 13C NMR
spectra were recorded on a Bruker AC-300 instrument. Chemical
shifts (d values) and coupling constants (J values) are given in
ppm and hertz, respectively. HRMS were obtained using a VG Auto-
spec TRIO 1000 instrument. The ionization mode used in mass
spectra was electron impact (EI) or fast atom bombardment
(FAB). Sulfadiazine was obtained from Sigma Chemical Co. Com-
pounds 4Aa, 4Ad, 4Ah, 4Ai, 5, 6a–d and 7a,b were synthesized as
previously described.24
4.1.2. General procedure for the synthesis of sulfonamides 423
To an ice-cooled solution of 2-aminopyrimidine (1.9 g,
20 mmol) in pyridine (8 mL) was slowly added the corresponding
sulfonyl chloride (30 mmol) in pyridine (6 mL). The mixture was
stirred at 0 °C for 2 h and allowed to reach room temperature.
Water was added (100 mL) and the solid was collected and recrys-
tallized from MeOH/CH2Cl2.
4.1.2.1. 2-Nitro-N-pyrimidin-2-ylbenzenesulfonamide (4Ab).
Yield: 93%; mp: 137–139 °C; 1H NMR (300 MHz, DMSO-d6):
d = 6.98 (t, J = 4.8 Hz, 1H), 7.24 (td, J = 8.4, J = 1.3, 1H), 7.71 (td,
J = 8.4, J = 1.5, 1H), 8.05 (dd, J = 8.4, J = 1.5, 1H), 8.17 (dd, J = 8.4,
J = 1.3, 1H), 8.51 (d, J = 4.8, 2H), 10.11 (s, 1H, NH); 13C NMR
(75 MHz, DMSO-d6): d = 114.6 (CH), 123.0 (CH), 123.6 (CH), 125.8
(CH), 130.9 (C), 135.0 (CH), 139.7 (C), 158.5 (CH), 159.3 (C);
HRMS-FAB+: m/z [M+H] calcd for C10H9N4O4S: 281.0344, found:
281.0339.
Figure 3. Agarose gel electrophoresis of pUC18 plasmid DNA treated with
compounds 4Aa and 5. Incubation time 60 min (37 °C). (1) kDNA/EcoR1 + HindIII
Marker; (2) control; (3) 4Aa 6
4Aa 30 M; (8) 4Aa 45 M; (9) 4Aa 60
(13) 5 15 M; (14) 5 30 M; (15) 5 45
l
M; (4) 4Aa 9
M; (10) 5 6
M; (16) 5 60 l
l
M; (5) 4Aa 12
M; (11) 5 9
M.
l
M; (6) 4Aa 15
l
M; (7)
l
l
l
l
l
M; (12) 5 12 l
M;
l
l
l
4.1.2.2. 3-Nitro-N-pyrimidin-2-ylbenzenesulfonamide (4Ac)
Yield: 50%; mp: 217–220 °C (lit31 217–219 °C); 1H NMR
(300 MHz, DMSO-d6): d = 7.06 (t, J = 4.9, 1H), 7.89 (t, J = 8.1, 1H),
8.40 (d, J = 8.1, 1H), 8.46 (d, J = 8.1, 1H), 8.52 (d, J = 4.9, 2H), 8.70
(s, 1H); 13C NMR (75 MHz, DMSO-d6): d = 114.9 (CH), 122.7 (CH),
127.3 (CH), 131.1 (CH), 133.8 (CH), 143.4 (C), 147.9 (C), 157.4 (C),
158.5 (CH); HRMS (FAB+): m/z [M+H] calcd for C10H9N4O4S:
281.0344, found: 281.0356.
Figure 4. Agarose gel electrophoresis of pUC18 plasmid DNA treated with the
mixture of copper chloride and the ligands and 50-fold-excess of ascorbate.
Incubation time 60 min (37 °C). (1) kDNA/EcoR1 + HindIII Marker; (2) control; (3)
4.1.2.3. N-Pyrimidin-2-ylbenzenesulfonamide (4Ae). Yield: 97%;
mp: 227–229 °C (lit32 229–230 °C); 1H NMR (300 MHz, DMSO-d6):
d = 7.03 (t, J = 4.9, 1H), 7.56 (m, 3H), 7.96 (dd, J = 7.9, J = 1.3, 2H),
8.47 (d, J = 4.9, 2H); 13C NMR (75 MHz, DMSO-d6): d = 116.0 (CH),
127.8 (CH), 129.2 (CH), 133.2 (CH), 140.0 (C), 157.4 (C), 158.6 (CH).
pUC18 + ascorbate; (4) CuCl2 15
45 M; (8) CuCl2 + 4Aa 15 M; (9) CuCl2 + 4Aa 30
5 45 M; (12) CuCl2 + 5 15 M; (13) CuCl2 + 5 30
l
M; (5) CuCl2 30
M; (10) CuCl2 + 4Aa 45
M; (14) CuCl2 + 5 45 l
lM; (6) CuCl2 45
l
M; (7) 4Aa
M; (11)
M.
l
l
l
l
l
l
l

M. A. Dea-Ayuela et al. / Bioorg. Med. Chem. 17 (2009) 7449–7456
7453
4.1.2.4. 4-Fluoro-N-pyrimidin-2-yl-benzenesulfonamide (4Af)
Yield: 80%; mp: 183–186 °C (lit33 184.5–185 °C); 1H NMR
(300 MHz, DMSO-d6): d = 7.05 (t, J = 4.9, 1H), 7.41 (t, J = 9.0, 2H),
8.03 (dd, J = 9.0, J = 5.0, 2H), 8.50 (d, J = 4.9, 2H), 11.0 (s, 1H, NH);
13C NMR (75 MHz, DMSO-d6): d = 116.1 (CH), 116.5 (CH), 131.0
(CH), 136.9 (C), 157.4 (C), 158.7 (CH), 162.5 (C).
d = 6.86 (td, J = 7.1, J = 0.94, 1H), 7.27 (d, J = 8.8, 1H), 7.81 (td J = 9,
J = 1.8, 1H), 7.94 (dd, J = 7.1, J = 1.8, 1H), 8.09 (d, J = 8.8, 2H), 8.34
(d, J = 8.8, 2H); 13C NMR (75 MHz, DMSO-d6): d = 114.4 (CH),
115.5 (CH), 124.6 (CH), 128.6 (CH), 128.0 (CH), 140.3 (C), 142.8
(CH), 149.0 (C), 149.4 (CH), 154.6 (C); HRMS-EI+: m/z calcd for
C11H9N3O4S: 279.0313, found: 279.0314.
4.1.2.5. 4-Chloro-N-pyrimidin-2-yl-benzenesulfonamide (4Ag)
Yield: 80%; mp: 199–202 °C (lit34 203–204 °C); 1H NMR
(300 MHz, DMSO-d6): d = 7.06 (t, J = 4.9, 1H), 7.63 (d, J = 8.1, 2H),
7.98 (d, J = 8.1, 2H), 8.51 (d, J = 4.9, 2H), 12.03 (s, 1H, NH); 13C
NMR (75 MHz, DMSO-d6): d = 116.1 (CH), 129.4 (CH), 129.8 (CH),
138.1 (C), 139.7 (C), 157.0 (C), 158.7 (CH).
4.1.2.14. 4-Nitro-N-thiazol-2-yl-benzenesulfonamide (4Ca)
Yield: 90%; mp: 263–265 °C (lit40 260–261 °C); 1H NMR
(300 MHz, DMSO-d6): d = 6.95 (d, J = 3.3, 1H), 7.30 (d, J = 3.3, 1H),
8.11 (d, J = 8.5, 2H), 8.40 (d, J = 8.5, 2H); 13C NMR (75 MHz,
DMSO-d6) d: 109.4 (CH), 124.8 (CH), 125.2 (CH), 127.6 (CH),
148.1 (C), 149.6 (C), 169.7 (C).
4.1.2.6. N-(4,6-Dimethyl-pyrimidin-2-yl)-4-nitro-benzenesul-
fonamide (4Aj). Yield: 65%; mp: 218–220 °C (lit.37 215 °C); 1H
NMR (300 MHz, DMSO-d6): d = 2.25 (s, 6H), 6.72 (s, 1H), 8.18 (d,
J = 8.8, 2H), 8.35 (d, J = 8.8, 2H).
4.1.2.15. 4-Chloro-N-thiazol-2-yl-benzenesulfonamide (4Cb)
Yield: 90%; mp: 198–200 °C (lit.34 201–202 °C); 1H NMR
(300 MHz, DMSO-d6): d = 6.80 (d, J = 3.3, 1H); 7.25 (d, J = 3.3, 1H);
7.6 (d, J = 8.5, 2H); 8.80 (d, J = 8.5, 2H); 13C NMR (75 MHz, DMSO-
d6): d = 109.0 (CH); 125.0 (CH); 128.0 (CH); 129.4 (CH); 137.1
(C); 141.6 (C); 169.4 (C).
4.1.2.7. N-(4,6-Dimethyl-pyrimidin-2-yl)-4-chloro-benzenesul-
fonamide (4Ak). Yield: 54%; mp: 185–187 °C (lit.38 181–182 °C);
1H NMR (300 MHz, DMSO-d6): d = 2.25 (s, 6H), 6.75 (s, 1H), 7.62
(d, J = 8.5, 2H), 7.96 (d, J = 8.5, 2H), 12.14 (s, 1H, NH); 13C NMR
(75 MHz, DMSO-d6): d = 22.9 (CH3), 113.1 (CH), 128.8 (CH), 130.2
(C), 130.4 (CH), 137.4 (C), 140.5 (C), 156.2 (C).
4.1.2.16. 4-Nitro-N-5-methyl-thiazol-2-yl-benzenesulfonamide
(4Cc). Yield: 88%; mp: 235–240 °C (lit.41 255 °C); 1H NMR
(300 MHz, DMSO-d6): d = 2.17 (s, 3H), 7.05 (s, 1H), 8.03 (d, J = 8.5,
2H), 8.35 (d, J = 8.5, 2H).
4.1.2.8. 4-Nitro-N-pyrazin-2-yl-benzenesulfonamide (4Ba)
Yield: 86%; mp: 233–237 °C; 1H NMR (300 MHz, DMSO-d6):
d = 8.2 (m, 4H), 8.40 (m, 3H); 13C NMR (75 MHz, DMSO-d6):
d = 124.9 (CH), 129.2 (CH), 133.7 (CH), 139.5 (CH), 142.1 (CH),
145.8 (C), 148.1 (C), 150.3 (C); HRMS-EI+: m/z calcd for
C10H9N4O4S: 281.0344, found: 281.0351.
4.1.2.17. 4-Chloro-N-5-methyl-thiazol-2-yl-benzenesulfon-
amide (4Cd). Yield: 50%; mp: 102–104 °C; 1H NMR (300 MHz,
DMSO-d6): d = 2.06 (s, 3H); 6.33 (s, 1H); 7.58 (d, J = 8.5, 2H); 7.75
(d, J = 8.5, 2H); HRMS-EI+: m/z calcd for C10H9ClN2O2S2: 287.9793,
found: 287.9794.
4.1.2.18. N-5-Methyl-thiazol-2-yl-benzenesulfonamide (4Ce)
Yield: 40%; mp: 149.1–152 °C; 1H NMR (300 MHz, DMSO-d6):
d = 2.15 (s, 3H), 6.46 (s, 1H), 7.61 (m, 3H), 7.85 (d, J = 8.3, 2H);
13C NMR (75 MHz, DMSO-d6): d = 13.7 (CH3); 102.6 (CH); 116.4
(C); 126.1 (CH); 129.2 (CH); 132.2 (CH); 135.0 (C); 165.8 (C).
4.1.2.9. 4-Chloro-N-pyrazin-2-yl-benzenesulfonamide (4Bb)
Yield: 40%; mp: 190–193 °C; 1H NMR (300 MHz, DMSO-d6):
d = 7.67 (d, J = 8.5, 2H), 7.95 (d, J = 8.5, 2H); 8.22 (dd, J = 2.8,
J = 1.5, 1H), 8.22 (d, J = 1.5, 1H); 13C NMR (75 MHz, DMSO-d6):
d = 129.5 (CH), 129.7 (CH), 135.5 (CH), 138.4 (C), 139.2 (CH),
139.3 (C), 142.3 (CH), 148.4 (C), HRMS-EI+: m/z calcd for
C10H9N3O2SCl: 270.0104, found: 270.0107.
4.1.2.19. 4-Nitro-N-4-methyl-thiazol-2-yl-benzenesulfonamide
(4Cf). Yield: 70%; mp: 189.5–193.2 °C (lit.42 197–9 °C). 1H NMR
(300 MHz, DMSO-d6): d = 2.10 (s, 3H), 6.45 (s, 1H), 8.05 (d, J = 8.5,
2H); 8.37 (d, J = 8.5, 2H).
4.1.2.10. N-Pyrazin-2-yl-benzenesulfonamide (4Bc). Yield: 70%;
mp: 204–208 °C; 1H NMR (300 MHz, DMSO-d6): d = 7.60 (m, 3H),
7.93 (d, J = 8, 2H); 8.22 (m, 2H), 8.35 (d, J = 1.3, 1H); 13C NMR
(75 MHz, DMSO-d6): d = 127.4 (CH), 129.6 (CH), 133.6 (CH), 135.2
(CH), 139.2 (CH), 140.2 (C), 142.4 (CH), 148.4 (C); HRMS-FAB+: m/
z calcd for C10H10N3O2S: 236.0493, found: 236.0495.
4.1.2.20. N-4-Methyl-thiazol-2-yl-benzenesulfonamide (4Cg)
Yield: 57%; mp: 164–166 °C (lit.43 161–162 °C). 1H NMR
(300 MHz, DMSO-d6): d = 2.17 (s, 3H); 6.97 (s, 1H); 7.55 (m, 3H);
7.77 (d, J = 8.1, 2H).
4.1.2.11. 4-Nitro-N-pyridin-4-yl-benzenesulfonamide (4Bd)
Yield: 40%; mp: 290–294 °C (lit.39 293–295 °C); 1H NMR
(300 MHz, DMSO-d6): d = 6.97 (d, J = 7.53, 2H), 8.02 (d, J = 7.53,
2H), 8.04 (d, J = 8.86, 2H), 8.31 (d, J = 8.86, 2H); 13C NMR
(75 MHz, DMSO-d6): d = 115.4 (CH), 124.5 (CH), 127.8 (CH), 139.4
(C), 139.5 (CH), 149.1 (C), 150.0 (C).
4.1.2.21. N-(5-Methyl-isoxazol-3-yl)-4-nitro-benzenesulfon-
amide (4Da). Yield: 90%; mp: 190–193 °C (lit.44 194–195 °C); 1H
NMR (300 MHz, DMSO-d6): d = 2.29 (s, 3H), 6.16 (s, 1H), 8.10 (d,
J = 9.2, 2H), 8.42 (d, J = 9.2, 2H); 13C NMR (75 MHz, DMSO-d6) d:
12.4 (CH3), 95.8 (CH), 125.2 (CH), 128.8 (CH), 144.9 (C), 149.5 (C),
157.4 (C), 171.0 (C).
4.1.2.12. 4-Nitro-N-pyridin-3-yl-benzenesulfonamide (4Be)
Yield: 88%; mp: 240–243 °C; 1H NMR (300 MHz, DMSO-d6):
d = 7.33 (dd, J = 8.2, J = 4.8, 1H), 7.51 (ddd, J = 8.2, J = 2.6, J = 1.5,
1H), 8.02 (d, J = 8.85, 2H), 8.31 (m, 2H), 8.37 (d, J = 8.85, 2H), 10.9
(s, 1H, NH); 13C NMR (75 MHz, DMSO-d6): d = 124.5 (CH), 125.2
(CH), 128.6 (CH), 128.7 (CH), 134.0 (C), 142.6 (CH), 144.7 (C),
146.3 (CH), 150.3 (C); HRMS-EI+: m/z calcd for C11H9N3O4S:
279.0313, found: 279.0299.
4.1.2.22. N-(5-Methyl-isoxazol-3-yl)-4-chlorobenzenesulfona-
mide (4Db). Yield: 60%; mp: 158.5–159.9 °C; 1H NMR (300 MHz,
DMSO-d6): d = 2.29 (s, 3H); 6.14 (s, 1H); 7.70 (d, J = 8.8, 2H); 7.85
(d, J = 8.8, 2H), 11.5 (br s, 1H, NH); 13C NMR (75 MHz, DMSO-d6)
d: 12.4 (CH3), 95.8 (CH), 129.0 (CH), 129.9 (CH), 138.6 (C), 138.7
(C), 149.5 (C), 157.6 (C), 171.0 (C); HRMS-EI+: m/z calcd for
C10H9N2O3SCl: 272.0022, found: 282.0019.
4.1.2.23. N-(5-Methyl-isoxazol-3-yl)-benzenesulfonamide (4Dc)
Yield: 90%; mp: 105–108 °C (lit.45 112.5 °C); 1H NMR (300 MHz,
DMSO-d6): d = 2.29 (s, 3H); 6.14 (s, 1H); 7.62 (m, 3H); 7.87 (d,
4.1.2.13. 4-Nitro-N-pyridin-2-yl-benzenesulfonamide (4Bf)
Yield: 63%; mp: 185–186 °C; 1H NMR (300 MHz, DMSO-d6):

7454
M. A. Dea-Ayuela et al. / Bioorg. Med. Chem. 17 (2009) 7449–7456
J = 8.2, 2H); 13C NMR (75 MHz, DMSO-d6) d: 12.4 (CH3), 95.7 (CH),
127.0 (CH), 129.8 (CH), 133.8 (CH), 139.7 (C), 157.6 (C), 171.0 (C).
NH), 8.16 (dd, J = 7.7, J = 1.3, 1H), 8.37 (ddd, J = 7.7, J = 2.2, J = 1.3,
1H), 8.45 (t, J = 2.2, 1H); 13C NMR (75 MHz, DMSO-d6): d = 14.4
(CH3), 33.2 (CH2), 43.2 (CH2), 120.2 (CH), 125.8 (CH), 130.6 (CH),
131.7 (CH), 145.6 (C), 147.1 (C), 156.6 (C@N), 167.6 (C@O);
HRMS-EI+: m/z calcd for C11H15N5O5S: 329.0793, found: 329.0788.
4.1.2.24. N-(5-Methyl-1H-pyrazol-3-yl)-4-nitro-benzenesulfon-
amide (4Dd). Yield: 95%; mp: 167–171 °C. 1H NMR (300 MHz,
DMSO-d6): d = 1.94 (s, 3H), 5.15 (s, 1H), 8.13 (d, J = 8.8, 2H), 8.40
(d, J = 8.8, 2H); 13C NMR (75 MHz, DMSO-d6) d: 14.2 (CH3), 89.4
(CH), 125.3 (CH), 129.1 (CH), 142.3 (C), 146.0 (C), 152.6 (C), 152.8
(C). HRMS-EI+: m/z calcd for C10H10N4O4S: 282.0422, found:
282.0423.
4.1.3.3. 2-[2-(3-Nitrobenzenesulfonyl)guanidino]-N-propyl-
acetamide (6f). A solution of 8 (0.54 g, 1.63 mmol) in propyl-
amine (5 mL) was heated at reflux for 6 h. The excess of amine
was removed under reduced pressure and the residue was purified
by column chromatography. Eluent AcOEt. Yield: 0.34 g (61%); mp:
108–110 °C; 1H NMR (300 MHz, DMSO-d6): d = 0.80 (t, J = 7.3, 3H),
1.35 (sextet, J = 7.3, 2H), 2.96 (q, J = 6.5, 2H), 3.72 (d, J = 5.2, 2H),
6.96 (s, 1H, NH), 7.07 (s, 1H, NH), 7.80 (t, J = 8.10, 1H), 7.95 (t,
J = 5.2, 1H, NH), 8.16 (d, J = 8.1, 1H), 8.37 (d, J = 8.1, 1H), 8.45 (s,
1H); 13C NMR (75 MHz, DMSO-d6): d = 11.6 (CH3), 22.6 (CH2),
40.6 (CH2), 43.6 (CH2), 120.7 (CH), 126.3 (CH), 131.1 (CH), 132.2
(CH), 146.0 (C), 147.9 (C), 157.1 (C@N), 168.2 (C@O); HRMS-EI+:
m/z calcd for C12H17N5O5S: 343.0950, found: 343.0952.
4.1.2.25. N-(1H-Indazol-5-yl)-4-nitro-benzenesulfonamide (4E)
Yield: 73%; mp: 234–236 °C; 1H NMR (300 MHz, DMSO-d6): d = 7.08
(d, J = 8.8, 1H), 7.44 (m, 2H), 7.93 (d, J = 8.7, 2H), 8.00 (s, 1H), 8.31 (d,
J = 8.7, 2H), 10.5 (br s, 1H, NH); 13C NMR (75 MHz, DMSO-d6) d:
111.2 (CH), 114.1 (CH), 122.9 (CH), 123.2 (C), 124.9 (CH), 128.7
(CH), 129.6 (C), 133.9 (CH), 138.2 (C), 145.1 (C), 150.0 (C); HRMS-
EI+: m/z calcd for C13H10N4O4S: 318.0422, found: 318.0429.
4.1.2.26. N-Indan-5-yl-4-chloro-benzenesulfonamide (4Fb)
Yield: 84%; mp: 121–123 °C; 1H NMR (300 MHz, DMSO-d6): d = 1.80
(m, 2H), 2.69 (m, 4H), 6.78 (dd, J = 7.9, J = 1.3, 1H), 6.90 (s, 1H), 7.00
(d, J = 7.9, 1H), 7.54 (d, J = 8.2, 2H), 7.68 (d, J = 8.2, 2H), 10.1 (br s, 1H,
NH); 13C NMR (75 MHz, DMSO-d6) d: 25.4 (CH2), 32.0 (CH2), 32.6
(CH2), 117.4 (CH), 119.5 (CH), 125.0 (CH), 129.0 (CH), 129.7 (CH),
135.7 (C), 137.9 (C), 138.8 (C), 140.4 (C), 145.1 (C); HRMS-EI+: m/z
calcd for C15H14ClNO2S: 307.0433, found: 307.0439.
4.2. Leishmanicidal assays
4.2.1. In vitro leishmanicidal assay
4.2.1.1. Parasite. An autochthonous isolate of L. infantum (MCAN/
ES/92/BCN 83) obtained from an asymptomatic dog from the Prio-
rat region (Catalunya, Spain) and given by Professor Montserrat
Portús was used. Promastigotes were cultured in Schneider’s Insect
Medium (Sigma, St. Louis, MO) at 26 °C supplemented with 20%
heat-inactivated foetal bovine serum (FBS) (Sigma, St. Louis, MO)
4.1.2.27. N-Indan-5-yl-benzenesulfonamide (4Fc). Yield: 91%;
mp: 150–154 °C; 1H NMR (300 MHz, DMSO-d6): d = 1.92 (m, 2H),
2.72 (m, 4H), 6.84 (dd, J = 8.1, J = 2.1, 1H), 6.94 (s, 1H), 7.02 (d,
J = 8.1, 1H), 7.53 (m, 3H), 7.72 (d, J = 7.9, 2H), 10.1 (br s, 1H, NH);
13C NMR (75 MHz, DMSO-d6) d: 25.4 (CH2), 31.9 (CH2), 32.6
(CH2), 117.1 (CH), 118.9 (CH), 124.9 (CH), 127.0 (CH), 129.5 (CH),
134.0 (CH), 136.0 (C), 139.9 (C), 140.0 (C), 145.4 (C); HRMS-EI+:
m/z calcd for C15H15NO2S: 273.0823, found: 273.0826.
and 100 U/mL of penicillin plus 100
St. Louis, MO) in 25 mL culture flasks.
lg/mL of streptomycin (Sigma,
4.2.1.2. Promastigote assay. The assay was performed using a
modification of a previous method.25 Promastigotes (2.5 Â 105par-
asites/well) were cultured in 96-well plastic plates. Compounds
were dissolved in dimethylsulfoxide (DMSO). Different dilutions
of the compounds (a first screening using 1, 10 and 100
and then 252, 126, 63, 31.5, 15.7, 7.8, 3.9, 1.9 M for active com-
pounds) up to 200 L final volume were added. After 48 h at
26 °C, 20 L of 2.5 mM resazurin solution was added and the fluo-
lg/mL,
4.1.3. Synthesis of guanidines (6e) and (6f)
Prepared as described for analogues 6a–d,24 starting from com-
pound 8.
l
l
l
rescence intensity (535 nm-excitation wavelength- and 590 nm-
emission wavelength-) was determined with a fluorometer Infinite
200 (Tecan i-Control) to calculate growth inhibition (%). The solu-
tion of resazurin was prepared at 2.5 mM in phosphate buffered
4.1.3.1. 2-[(2-{[(3-Nitrophenyl)sulfonyl]imino}pyrimidin-1(2H)-
yl]acetamide (8). To a stirred suspension of sulfonamide 4c (1 g,
3.6 mmol) in dimethylformamide, DMF (12 mL), diisopropylethyl-
amine, DIPEA (0.65 mL, 3.6 mmol) was added dropwise under nitro-
gen. After 40 min iodoacetamide (0.66 g, 3.6 mmol) was added. The
reaction mixture was stirred at room temperature for 16 h and then
poured onto water (100 mL). The resulting solid was collected and
dried to give compound 8. Yield: 1.1 g (90%); mp: 240 °C (decomp);
1H NMR (300 MHz, DMSO-d6): d = 4.79 (s, 2H, CH2), 6.94 (dd, J = 6.6,
J = 4.5, 1H), 7.46 (s, 1H, NH), 7.78 (t, J = 8.1, 1H), 7.86 (s, 1H, NH), 8.20
(ddd, J = 8.1, J = 1.7, J = 0.9, 1H), 8.35 (ddd, J = 8.1, J = 1.7, J = 0.9, 1H),
8.42 (dd, J = 6.6, J = 2.2, 1H), 8.56 (t, J = 1.7, 1H), 8.64 (dd, J = 4.5,
J = 2.2, 1H); 13C NMR (75 MHz, DMSO-d6) d: 55.1 (CH2), 109.1 (CH),
122.0 (CH), 126.2 (CH), 130.6 (CH), 133.4 (CH), 145.5 (C), 147.6 (C),
152.4 (CH), 154.9 (C), 164.9 (CH), 167.1 (C@O).
solution (PBS), pH 7.4, and filtered through 0.22 lm prior use. All
tests were carried out in triplicate. Resazurin sodium salt was ob-
tained from Sigma–Aldrich (St. Louis, MO) and stored at 4 °C pro-
tected from light.
4.2.2. Cytotoxicity assay
4.2.2.1. Cells. J774 murine macrophages were grown in RPMI
1640 medium (Sigma) supplemented with 10% heat-inactivated
FBS (30 min at 56 °C), penicillin G (100 U/mL) and streptomycin
(100 lg/mL). For the experiments, cells in the pre-confluence
phase were harvested with trypsin. Cell cultures were maintained
at 37 °C in a humidified environment with 5% CO2.
4.1.3.2. 2-[2-(3-Nitrobenzenesulfonyl)guanidino]-N-ethylaceta-
mide (6e). To a solution of 8 (0.54 g, 1.63 mmol) in methanol
(20 mL), ethylamine 2 M in methanol (5 mL) was added. The reac-
tion mixture was heated at reflux for 6 h and then concentrated
under reduced pressure. The remaining oil was purified by column
chromatography. Eluent AcOEt/MeOH (9:1). Yield: 0.19 g (38%);
mp: 170–172 °C; 1H NMR (300 MHz, DMSO-d6): d = 0.97 (t,
J = 7.3, 3H), 3.04 (quintet, J = 7.3, 2H), 3.72 (d, J = 5.2, 2H), 6.96 (s,
1H, NH), 7.08 (s, 1H, NH), 7.81 (t, J = 8.1, 1H), 7.95 (t, J = 5.2, 1H,
4.2.2.2. Cytotoxicity assay on macrophages. Cell viability was
evaluated using a modification of a method previously described.35
J774 macrophages were seeded (5 Â 104 cells/well) in 96-well flat-
bottom microplates with 200 lL of RPMI 1640 medium. The cells
were allowed to adhere for 24 h at 37 °C in 5% CO2, then the medium
was replaced by different concentrations of the test compounds in
200
were also included. To evaluate cell viability, 20
resazurin solution were added and the plates were returned to the
lL of medium and exposed to for another 24 h. Growth controls
l
L of a 2.5 mM

M. A. Dea-Ayuela et al. / Bioorg. Med. Chem. 17 (2009) 7449–7456
7455
incubator for another 3 h. Reduction of resazurin was determined by
measurement of the fluorescence intensity (535 nm-excitation
wavelength- and 590 nm-emission wavelength-). Background was
subtracted. Each concentration was assayed three times. Medium
and controls were used in each test as blanks.
ate buffer. Final concentrations ranged from 1 to 30
times were measured with a digital stopwatch. Time readings were
l
M. The flow
1/3
recorded by triplicate. Data were analyzed as (
ratio of the concentration of complex and DNA, where
cosity of DNA in the presence of complex and
g
/g0)
versus the
is the vis-
g
g0 is the viscosity of
DNA alone. Viscosity values were calculated from the observed
flow time of a DNA-containing solution corrected from the flow
4.2.3. In vivo leishmanicidal assay
4.2.3.1. Mice. Balb/c mice of 6–8 weeks of age were purchased
from Harlan Ibérica S.A. (Barcelona, Spain) and allocated in the Ani-
mal House Unit of the Complutense University under controlled
food, light/darkness cycles and temperature conditions. All proce-
dures for animal manipulations were approved by the Complu-
tense University Institutional Animal Care and Use Committee
following Spanish law.
time of buffer alone (t0),
g
= t À t0.
4.3.2. Thermal denaturation experiments
DNA-melting experiments were carried out by monitoring the
absorbance (260 nm) of CT-DNA (100 lM NP) at different tempera-
tures in the absence or presence of the compounds at 4:1 DNA/com-
plex ratio. Measurements were performed with an Agilent 8453 UV–
vis spectrophotometer equipped with a Peltier temperature con-
trolled sample cell and driver (Agilent 89090A). The solution con-
taining the compound and CT-DNA in phosphate buffer (1 mM
phosphate, 2 mM NaCl, pH 7.2) was continuously stirred and heated
at 1 °C minÀ1 rate of temperature increase. The investigated interval
of temperature ranged from 20 to 90 °C. The melting point was ob-
tained with the first derivative using the Savitsky-Golay algorithm.
4.2.3.2. Preparation of Leishmania infantum parasites for
experimental infection. L. infantum amastigotes (MCAN/ES/92/
BCN 83) harvested from spleens of infected hamsters were cul-
tured in NNN medium added with penicillin (200 IU) and strepto-
mycin (2 mg/mL) for 2 days up to transformation into
promastigotes. Thereafter they were transferred to C-199 medium
supplemented with 1% 10 mM adenin in 50 mM Hepes, 0.25%
hemin in 50% triethanolamine, 0.348 g/L sodium bicarbonate,
25 mM Hepes, 20% heat-inactivated foetal bovine serum (FBS, Sig-
4.3.3. Nuclease activity: pUC18 DNA cleavage
Reactions of DNA cleavage produced by the compounds were
ma), 100 lg/mL penicillin and 100 lg/mL streptomycin, at pH 7.2.
undertaken by mixing 13
L of pUC18 (0.25 g/ l), 6
pound at increasing concentrations to obtain final concentrations
between 6 M and 60 M. To assay nuclease activity in the pres-
ence of a copper salt, the samples contained 7 L of 0.1 M cacodyl-
ate buffer (pH 6.0), 1 L of pUC18 (0.25 g/ L), 6 L of a mixture
CuCl2 + compound solution at increasing concentrations to obtain
final concentrations between 15 M and 45 M and 6 L of reduc-
ing agent (ascorbate) in cacodylate buffer. The mixtures were al-
lowed to stand for 1 h at 37 °C. After that, 3 L of a loading
buffer solution consisting of 0.25% bromophenol blue, 0.25% xylene
cyanol and 30% glycerol were added. Then the solution was sub-
jected to electrophoresis on a 0.8% agarose gel in 0.5 Â TBE buffer
(0.045 M tris, 0.045 M boric acid and 1 mM EDTA) containing
2 lL/100 mL of a solution of ethidium bromide (10 mg/mL) at
80 V for about 2 h. The gel was photographed on a capturing Gel
l
L of 0.1 M cacodylate buffer (pH 6.0),
Under these conditions the maximum number of metacyclic forms
as determined by its resistance to complement lysis is achieved by
day 7th. After 7 days the primary culture promastigotes were har-
vested by centrifugation at 2000g for 15 min, washed with phos-
phate buffer saline (PBS) at pH 7.2 and finally resuspended in
PBS. Promastigotes were counted using the Neubauer haemocy-
tometer and the final suspension adjusted to provide 107 prom-
astigotes per inoculum. Groups of 6 animals each were infected
with 107 promastigotes per animal, given by the intracardiac (IC)
route following anaesthesia with sodium pentobarbital.
1
l
l
l
l
L of a solution of the tested com-
l
l
l
l
l
l
l
l
l
l
l
4.2.3.3. Protocol. Mice were randomly assigned to three groups.
One group was kept as untreated control. Treatment started on
the 55 day post-infection and lasted for 10 continuous days. Ani-
mals were dosed once daily and the compounds were administered
at 5 mg/kg given by the intraperitoneal route in a 0.1 mL final vol-
ume of physiological saline solution. Five days later, the mice were
sacrificed and the parasitic burden was evaluated.
Printer plus TDI.
4.4. Statistics
4.2.3.4. Estimation of parasite burden. The liver and spleen
from each animal were aseptically removed, weighted and after-
wards samples of each were homogenised in cold PBS-glucose-
EDTA solution using a steel stainless tissue grinder. Cell debris
was eliminated by passage trough a glass wool column. The sus-
pension obtained was centrifuged at 2000g for 15 min at 4 °C.
Thereafter the supernatants were discarded and the pellets resus-
pended in C-199 medium supplemented with 1% adenin, 0.2%
hemin, sodium bicarbonate, 25 mM Hepes, antibiotics and 20%
FBS. 200 mL of the suspension were transferred to each of the
96-well microtiter plate containing NNN medium added with anti-
biotics. Parasite burden were estimated by the limit dilution assay
according to Hill et al. and Titus et al.36
The concentration of the test compounds that produced a 50%
reduction in infected cells (IC50) was determined from least-
squares linear regression of growth rate. Mann–Whitney test was
used to determine the statistical significance of the values ob-
tained. Parasite suppression was determined by comparing each
experimental value of the parasite burden with the mean value
for the controls. Parasite burdens were analyzed by Student’s t-
test.
Acknowledgements
The authors thank to the Generalitat Valenciana (Project
GVPRE2008/77) and the Universidad CEU Cardenal Herrerra (Pro-
ject PRCEU-UCH08–07) for financial support. M. González-Alvarez
and J. Borrás acknowledge CYCIT (Project CTQ2007–63690/BQU).
4.3. DNA interactions
4.3.1. Viscosity measurements
References and notes
Viscosity experiments were carried out with a semimicro Ubbe-
lodhe viscosimeter immersed in a Julabo ME16G thermostatted
water-bath maintained to 25.0 0.1 °C or to 37.0 0.1 °C. Solutions
of the compounds in [DMF/cacodylate buffer (pH 6) 2:10] were
1. WHO. Tropical disease research. Progress 2003–2004. Special Programme for
Research & Training in Tropical Diseases. Geneva, 2005.
2. Hotez, P. J.; Molyneux, D. H.; Fenwick, A.; Kumaresan, J.; Sachs, S. E.; Sachs, J. D.;
Savioli, L. N. Eng. J. Med. 2007, 357, 1018.
added to a calf thymus DNA, CT-DNA (50 lM) solution in cacodyl-

7456
M. A. Dea-Ayuela et al. / Bioorg. Med. Chem. 17 (2009) 7449–7456
3. Leishmaniasis http://www.who.int/tdr/diseases/leish/default.htm (accessed in
2008).
4. Desjeux, P. Comp. Immunol. Microbiol. Infect. Dis. 2004, 27, 305.
5. World Health Organization. 2008. Burden of disease at http://www.who.int/
leishmaniasis/burden/en/ (accessed in 2008).
25. Ordóñez-Gutiérrez, L.; Espada-Fernández, R.; Dea-Ayuela, M. A.; Torrado,
J. J.; Bolás Fernández, F.; Alunda, J. M. Int. J. Antimicrob. Agents 2007, 30,
325.
26. Yardley, V.; Khan, A. A.; Martin, M. B.; Slifer, T. R.; Araujo, F. G.; Moreno, S. N.;
Docampo, R.; Croft, S. L.; Oldfield, E. Antimicrob. Agents Chemother. 2002, 46,
929.
27. Liu, J.; Zhang, T.; Lu, T.; Qu, L.; Zhou, H.; Zhang, Q.; Ji, L. J. Inorg. Biochem. 2002,
91, 269.
28. Tselepi-Kalouli, E. T.; Katsaros, N. J. Inorg. Biochem. 1989, 37, 271.
29. Kelly, J. M.; Tossi, A. B.; McConnell, D. J.; OhUigin, C. Nucleic Acid Res. 1985, 13,
6017.
6. Cruz, I.; Nieto, J.; Moreno, J.; Cañavate, C.; Desjeux, P.; Alvar, J. Indian J. Med. Res.
2006, 123, 357.
7. Mishra, J.; Saxena, A.; Singh, S. Curr. Med. Chem. 2007, 14, 1153.
8. Croft, S. L.; Barret, M. P.; Urbina, J. A. Trends Parasitol. 2005, 21, 508.
9. Davis, A. J.; Kedzierski, L. Curr. Opin. Invest. Drugs 2005, 6, 163.
10. Sundar, S.; Chatterjee, M. Indian J. Med. Res. 2006, 123, 345.
11. Croft, S. L.; Seifert, K.; Yardley, V. Indian J. Med. Res. 2006, 123, 399.
12. Croft, S. L.; Sundar, S.; Fairlamb, A. H. Clin. Microbiol. Rev. 2006, 19, 111.
13. Ouellette, M.; Drummelsmith, J.; Papadopoulou, B. Drug Resist. Update 2004, 7,
257.
30. Drevensenk, P.; Zupancic, T.; Pihlar, B.; Jerala, R.; Kolitsch, U.; Plaper, A.; Turel,
I. J. Inorg. Biochem. 2005, 99, 432.
31. English, J. P.; Clark, J. H.; Shepherd, R. G.; Marson, H. W.; Krapcho, J.; Roblin, R.
O. J. Am. Chem. Soc. 1946, 68, 1039.
14. Piñero, J. E.; Jiménez, I. A.; Valladares, B.; Ravelo, A. G. Expert Opin. Ther. Patents
2004, 14, 1113.
32. English, J. P.; Chappell, D.; Bell, P. H.; Roblin, R. O. J. Am. Chem. Soc. 1942, 64,
2516.
15. Reddy, N. S.; Mallireddigari, M. R.; Consenza, S.; Gumireddy, K.; Bell, S. C.;
Reddy, E. P.; Reddy, M. V. Bioorg. Med. Chem. Lett. 2004, 14, 4093.
16. Stranix, B. R.; Lavallée, J. F.; Sevigny, G.; Yelle, J.; Perron, V.; LeBerre, N.;
Herbart, D.; Wu, J. J. Bioorg. Med. Chem. Lett. 2006, 16, 3459.
17. González-Alvarez, M.; Alzuet, G.; Borrás, J.; Castillo-Agudo, L.; Montejo-
Bernardo, J. M.; Gutiérrez-Rodríguez, A. J. Biol. Inorg. Chem. 2008, 13, 1249.
18. Peixoto, M. P.; Beverly, S. M. Antimicrob. Agents Chemother. 1987, 31, 1575.
19. Everson da Silva, L.; Joussef, A. C.; Kramer Pacheco, L.; Gaspar da Silva, D.;
Steindel, M.; Andrade Rebelo, R. Bioorg. Med. Chem. 2007, 15, 7553.
20. George, T. G.; Johnsamuel, J.; Delfín, D. A.; Yakovich, A.; Mukherjee, M.; Phelps,
M. A.; Dalton, J. T.; Sackett, D. L.; Kaiser, M.; Brun, R.; Werbovetz, K. A. Bioorg.
Med. Chem. 2006, 14, 5699.
33. Sveinbjornsson, A.; Vanderwerf, C. A. J. Am. Chem. Soc. 1951, 73, 869.
34. Grigorovskii, A. M.; Dykhanov, N. N.; Kimen, Z. M. Zhurnal Obshchei Khimii
1957, 27, 531.
35. Rolón, M.; Seco, E.; Vega, C.; Nogal, J.; Escario, J.; Gómez-Barrio, A.; Malpartida,
F. Int. J. Antimicrob. Agents 2006, 28, 104.
36. (a) Hill, J. O.; North, R. J.; Collins, F. M. Infect. Immun. 1983, 39, 1087;
(b) Titus, R. G.; Marchand, M.; Boon, T.; Louis, J. A. Parasite Immunol.
1985, 7, 545.
37. Rose, F. L.; Swain, G. J. Chem. Soc. 1945, 689.
38. Hultquist, M. E.; Germann, R. P.; Webb, J. S.; Wright, W. B.; Roth, B.; Smith, J.
M.; SubbaRow, Y. J. Am. Chem. Soc. 1951, 73, 2558.
39. Kravtsov, D. N.; Nesmeyanov, A. N. Izvestiya Akademii Nauk SSSR, Seriya
Khimicheskaya 1967, 5, 1024.
40. Ganellin, C. R.; Triggle, D. J. Dictionary of Pharmacological Agents; Chapman and
Hall: London, 1997.
21. (a) Delfín, D. A.; Morgan, R. E.; Zhu, X.; Werbovetz, K. A. Bioorg. Med. Chem.
2009, 17, 820; (b) Delfín, D. A.; Bhattacharjee, A. K.; Yakovich, A. J.; Werbovetz,
K. A. J. Med. Chem. 2006, 49, 4196.
22. Bhattacharya, G.; Herman, J.; Delfín, D. A.; Salem, M. M.; Barszcz, T.; Mollet, M.;
Riccio, G.; Brun, R.; Werbovetz, K. A. J. Med. Chem. 2004, 47, 1823.
23. Acero-Alarcón, A.; Armero-Alarte, T.; Jordá-Gregori, J. M.; Rojas-Argudo, C.;
Zaballos-García, E.; Server-Carrio, J.; Ahjyaje, F. Z.; Sepúlveda-Arques, J.
Synthesis 1999, 12, 2124.
41. Newbery, G. Patent US 243338, 1947.
42. Fosbinder, R. J.; Walter, L. A. J. Am. Chem. Soc. 1939, 61, 2032.
43. Sprague, J. M.; Kissinger, L. W. J. Am. Chem. Soc. 1941, 63, 578.
44. Xu, X.; Weitzberg, M.; Keyes, R. F.; Li, Q.; Wang, R.; Wang, X.; Zhang, X.; Frevert,
E. U.; Camp, H. S.; Beutel, B. A.; Sham, H. L.; Gu, Y. G. Bioorg. Med. Chem. 2007,
17, 1803.
24. González-Rosende, M. E.; Castillo, E.; Asins, B.; Mamouni, R.; Sepúlveda-
Arques, J. Tetrahedron 2007, 63, 8709.
45. Uno, T.; Machida, K.; Hanai, K. Chem. Pharm. Bull. 1966, 14, 756.