Synthesis and antimalarial activities of some furoxan sulfones and related furazans

Article abstract of DOI:10.1016/j.ejmech.2005.05.001

Furoxan derivatives bearing a sulfone moiety at position 3 or 4 were synthesized and tested for their antimalarial action on the chloroquine- sensitive D10 and the chloroquine-resistent W2 strains of Plasmodium falciparum. The furazan analogues were consi

Full text of DOI:10.1016/j.ejmech.2005.05.001

European Journal of Medicinal Chemistry 40 (2005) 1335–1340
www.elsevier.com/locate/ejmech
Short communication
Synthesis and antimalarial activities of some furoxan
sulfones and related furazans
Ubaldina Galli a, Loretta Lazzarato b, Massimo Bertinaria b, Giovanni Sorba a,
Alberto Gasco b,*, Silvia Parapini c, Donatella Taramelli c
a Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, Università degli Studi del Piemonte Orientale,
Via Bovio 6, 28100 Novara, Italy
b Dipartimento di Scienza e Tecnologia del Farmaco, Università degli Studi di Torino, Via Pietro Giuria 9, 10125 Torino, Italy
c Istituto di Microbiologia, Università degli Studi di Milano, Via Pascal 36, 20133 Milano, Italy
Received 15 February 2005; accepted 27 May 2005
Available online 24 June 2005
Abstract
Furoxan derivatives bearing a sulfone moiety at position 3 or 4 were synthesized and tested for their antimalarial action on the chloroquine-
sensitive D10 and the chloroquine-resistent W2 strains of Plasmodium falciparum. The furazan analogues were considered for comparison.
The most active compounds were the products in which the –SO2R groups are at the 3-position of the furoxan system. These latter substances
displayed an antimalarial activity in the µM range, possibly related in part to their ability to release NO.
© 2005 Elsevier SAS. All rights reserved.
Keywords: Plasmodium falciparum; Nitric oxide; Antimalarial; Furoxanyl sulfones
1. Introduction
Furoxan ring (1,2,5-oxadiazole 2-oxide) I (Chart 1), is an
heterocyclic system known to chemists thanks to its intrigu-
ing chemistry and an argument on its structure [1]. Furoxan
derivatives have recently received particular attention in view
of their ability to release nitric oxide (NO) [2]. In pH 7.4 buff-
ered water solution at 37 °C, the release of NO requires the
Chart 1
.
presence of thiol cofactors. Several of these products were
studied as cardiovascular agents principally for their vasodi-
lating properties [3,4].
proved to inhibit the catalytic activity of cysteine proteases
[6]. There are scattered reports in literature that exogenous
NO also displays cytotoxic and cytostatic effects against
viruses and microbial agents [7] including protozoa [6]. The
NO-donors S-nitroso glutathione (GSNO) and S-nitroso-L-
cysteine (SNOCys) kill Plasmodium falciparum [8]. Their
50% inhibitory concentrations (IC50) were found to be very
close, approximately 40 µM. A number of furoxan deriva-
tives proved to display antimicrobial activity [4]. Some of
them inhibited in vitro the growth of a number of protozoa
including T. vaginalis, E. histolytica and T. cruzi [4]. In this
last case electrochemical studies suggested that trypanosoma
cell damage could be caused by oxidative stress associated
Endogenous NO is a potent antimicrobial agent. Together
with reactive oxygen intermediates (ROI), NO is one of the
toxic mediators released by activated macrophages against
pathogens. NO-mediated cellular toxicity is due to the gen-
eration of reactive species and/or inhibition of essential
enzymes [5]. In particular S-nitrosilation of cysteine contain-
ing proteins seems to be a widespread mechanism for the anti-
parasitic effect of NO [6]. A number of NO-donors, namely
molecules able to release NO in physiological conditions,
* Corresponding author. Tel.: +39 011 670 7670; fax: +39 011 670 7286.
E-mail address: alberto.gasco@unito.it (A. Gasco).
0223-5234/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2005.05.001

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U. Galli et al. / European Journal of Medicinal Chemistry 40 (2005) 1335–1340
with the presence of the N+–O– moiety [9]. A benzofuroxa-
nyl thiourea derivative was evaluated as inhibitor of tripano-
some cysteine proteinases displaying interesting activity.
In addition, 4-(phenylsulfonyl)-3-(((2-dimethylamino)-
ethyl)thio)furoxan oxalate proved to be able to inhibit the
activity of the HIV-1 reverse transcriptase, an enzyme con-
taining cysteine residue(s) [4], and Leishmania infantum cys-
teine proteinase [10]. Until now, the potential antimalarial
activity of the furoxans has not been evaluated in spite of an
urgent need for new antimalarials because of the widespread
resistance of malaria parasites to conventional drugs, such as
chloroquine (CQ) II (Chart 1) [11].
connected not only with their potential ability to release NO
[13,14] and to induce oxidant stress owing to the presence of
the N+–O– moiety [9] but also to their capacity of undergoing
nucleophilic attack by a number of nucleophiles, which
includes the thiol group present in cysteine proteinases, with
consequent displacement of the SO2R groups (Scheme 1) [15]
or a prevalent addition on the vinyl moiety in the case of the
derivatives 4a, 4b (Scheme 2). The furazan analogues (series
c), deprived of the N+–O– moiety, but able to react with thiol
groups (see Schemes) [15], were also included for compari-
son.
In the context of our research on the furoxan pharmaco-
chemistry, we decided to study selected derivatives of this
heterocyclic system as potential antimalarial compounds. In
this paper, we report the synthesis of new furoxan sulfones
derivatives (4a, 4b–7a, 7b) and preliminary results of their in
vitro activity against P. falciparum, the aetiological agent of
the most deadly form of human malaria. The antimalarial
activity of the sulfones used as intermediates for the prepara-
tion of the new compounds was also evaluated (derivatives
1a, 1b, 3a, 3b) [12]. The choice of this kind of products is
2. Results and discussion
For the preparation of the final vinyl sulfones 4a, 4b, 4c
we started from the corresponding thioethers 2a, 2b, 2c
(Scheme 1). The intermediates 2a, 2b were prepared through
nucleophilic substitution of the phenylsulfonyl group in 1a,
1b by 2-mercaptoethanol, using a procedure we have already
described [12]. The intermediate 2c was synthesized in a simi-
lar manner from the parent phenylsulfonylfurazan derivative
Scheme 1
TEA, CH2Cl2, 0 °C.
.
a) 2-Mercaptoethanol, 50% aq. NaOH, THF for 2a, 2c; 2-mercaptoethanol, MeO–Na+, –10 °C for 2b; b) MCPBA, CH2Cl2, 0 °C to r.t.; c) MsCl,
Scheme 2.
a) EtOH, EtO–Na+, THF, –45 °C; b) EtSH, TEA, THF, 0 °C; c) p-anisidine, THF, r.t.

U. Galli et al. / European Journal of Medicinal Chemistry 40 (2005) 1335–1340
1337
Table 1
1c. The thioethers were transformed into the corresponding
sulfones 3a, 3b, 3c by action of m-chloroperbenzoic acid
(MCPBA) in CH2Cl2. These last products were dehydrated
in CH2Cl2 to the final vinyl sulfones, by action of mesyl chlo-
ride (MsCl) in the presence of triethylamine (TEA). In order
to obtain differently substituted alkylsulfonyl furoxan and
furazan derivatives, these Michael-type systems were let to
react with three different models of nucleofiles, namely
p-anisidine, ethanol and ethanethiol (Scheme 2). These
reagents contain as nucleophilic center a N, O, S atom, respec-
tively, and are characterized by having different nucleophilic
power. p-Anisidine was chosen as nitrogen nucleophile also
in view of the facility to isolate the final amines 7a, 7b, 7c.
These products display low basicity (e.g. 7c pKa =
2.19 0.03) and consequently they exist at physiological pH
prevalently in the uncharged form.
The reactions with ethanol were run in THF at –45 °C, in
the presence of catalytic amount of sodium ethoxide to give
in fairly good yields the corresponding ethers 5a, 5b, 5c. Simi-
larly both the reactions in THF with ethanethiol at 0 °C, in
the presence of TEA, and the reactions in THF with
p-anisidine at room temperature, gave in very good yields the
expected sulfur 6a, 6b, 6c, and nitrogen 7a, 7b, 7c adducts.
The high reactivity of the furoxanyl and furazanyl vinyl sul-
fones towards the nucleophilic agents makes these products
useful scaffolds for the synthesis of a variety of sulfones con-
taining either the furoxan or the furazan system. The ability
of the furoxan sulfones to produce nitrite, which is the prin-
cipal oxidative product of nitric oxide in aerobic water solu-
tion, under the action of an excess of cysteine (5:1) was evalu-
ated by the Griess reaction according to a procedure
previously described [12]. The results are collected in Table 1.
Analysis of the data shows that the 3-RSO2-furoxan deriva-
tives are more potent NO-donors than the corresponding
4-RSO2-isomers.
To ascertain their antimalarial activity, all of the sulfones
were screened in vitro against the chloroquine-sensitive (CQ-
S), D10, and the chloroquine-resistant (CQ-R), W2 strain of
P. falciparum. The antiplasmodial activities are expressed as
50% inhibitory concentrations (IC50) in the two P. falci-
parum strains and are compared to that of CQ (Table 1). The
products in which the R–SO2-group is at the 3-position of the
furoxan system (series b) displayed antimalarial activity in
the low micromolar range, without significant differences
between the CQ-R or CQ-S strain. These compounds proved
to be more potent than the corresponding 3-phenyl isomers
(series a), which, in turn, were more potent than the furazan
analogues (series c). They also appear to be more active than
the NO-donors GSNO and SNOcys. Within each series, the
vinyl derivatives, 4a, 4b, 4c were the least active products.
The furoxan derivatives belonging to series b also had sig-
nificant NO releasing properties with respect to the furoxans
of series a. This statement, in connection with the minor activ-
ity of the furazan analogues deprived of the ability to release
NO, could suggest nitric oxide played a role in the mecha-
nism of parasite death. Additional studies are necessary to
In vitro antimalarial activity of furoxanyl and furazanyl sulfones and nitrite
production (% NO2– mol/mol) by furoxan derivatives
–
Compound
1a
1b
1c
3a
3b
3c
4a
4b
4c
5a
5b
5c
6a
6b
6c
% NO2 S.E. D10 IC50 (µM)a
W2 IC50 (µM)a
7.04 2.11
3.07 0.89
32.86 9.74
40.36 14.83
3.03 0.85
> 78.65
< 1
6.67 2.53
3.76 0.33
35.87 16.59
41.54 15.12
4.41 1.61
> 78.65
10.3 0.1
///
< 1
9.64 0.21
///
< 1
56.13 12.29
22.04 2.97
76.66 11.30
22.79 4.46
5.36 1.94
> 70.84
50.74 10.98
18.31 4.12
70.85 12.91
17.22 4.19
5.33 0.20
> 70.84
10.5 0.8
///
< 1
10.0 0.5
///
< 1
9.67 4.01
4.23 1.68
59.79 10.22
14.08 3.37
4.73 0.92
42.76 13.13
0.025 0.011
7.79 4.38
3.59 0.89
55.93 15.72
12.82 2.59
4.47 0.41
37.98 11.71
0.35 0.17
10.0 0.4
///
7a HCl
7b HCl
7c
< 1
20.8 0.7
///
Chloroquine ///
a Compounds were tested against CQ-S, D10, and CQ-R, W2 strains of
P. falciparum using the pLDH method. All values are mean S.D. of three
different experiments conducted in duplicate.
confirm this possibility since higher NO-release of these prod-
ucts might parallel their higher ability to induce oxidative
stress and/or to undergo nucleophilic substitutions.
3. Experimental protocols
3.1. Chemistry
Melting points were measured with a capillary apparatus
(Stuart Smp 3) and are uncorrected. All the compounds were
routinely checked by IR (FT-IR Thermo-Nicolet Avatar),
1H-and 13C-NMR (BrukerAvance 300 and Jeol ECP300) and
mass spectrometry (Finnigan-Mat TSQ-700). The following
abbreviations were used to indicate the peak multiplicity:
s = singlet; d = doublet; t = triplet; m = multiplet. Flash col-
umn chromatography was performed on silica gel (Merck Kie-
selgel 60, 230–400 meshASTM); thin layer chromatography
(TLC) was carried out on 5 × 20 cm plates with a layer thick-
ness of 0.25 mm using the indicated eluents. Petroleum ether
40–60 °C (PE) was used as a co-eluent. Anhydrous magne-
sium sulfate was used as drying agent for the organic phases.
Analysis (C, H, N) of the new compounds was performed by
REDOX (Monza) and the results are within 0.4%. Struc-
tures 1a [16], 1b [16], 1c [16], 2a [12], 2b [12], were synthe-
sized according to methods described in the literature. Tet-
rahydrofuran (THF) was distilled immediately before use
from Na and benzophenone under a positive atmosphere of
N2. All reactions were carried out three times without any
attempt to optimize the yields.
3.1.1. 2-[(4-Phenylfurazan-3-yl)thio]ethanol (2c)
The title compound was prepared in the same manner as
2a. Eluent (PE/EtOAc 80:20 v/v). Yield 98%. H-NMR
1

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U. Galli et al. / European Journal of Medicinal Chemistry 40 (2005) 1335–1340
(CDCl3) d 3.42 (t, 2H, –OCH2CH2S–), 3.97 (t, 2H,
–OCH2CH2S–), 7.45–7.81 (m, 5H, Ph); 13C-NMR (CDCl3)
d 35.5 (–OCH2CH2S–), 60.5 (–OCH2CH2S–), 125.1 (Ci Ph),
128.1, 129.1 (Co, Cm Ph), 130.9 (Cp Ph), 151.1 (C4 Furaz.),
152.6 (C3 Furaz.).
–SO2CH=CHaHb), 7.50–7.75 (m, 5H, Ph); 13C-NMR
(CDCl3) d 116.6 (C3 Furox.), 124.6 (Ci Ph), 128.7, 129.5
(Co, Cm Ph), 131.7 (Cp Ph), 133.5 (–SO2CH=CH2), 135.6
(–SO2CH=CH2), 154.7 (C4 Furox.); MS (EI) m/z 252 (M)+;
drying conditions: r.t.; 24 h, pressure < 10 mmHg. Anal.
(C10H8N2O4S) C, H, N.
3.1.2. General procedure for the preparation of 3a, 3b, 3c
To a solution of 2-[(3-phenylfuroxan-4-yl)thio]ethanol (2a)
(6.10 g, 25.63 mmol) in CH2Cl2 (60 ml), kept at 0 °C, MCPBA
(14.31 g, 64.08 mmol) was added. The reaction was allowed
to reach room temperature (r.t.) and was completed in 20 h.
The mixture was filtered under vacuum and the precipitate
was washed with CH2Cl2. To the filtrate were added ice and
NaOH 2 M until pH 8–9, after separation the aqueous layer
was extracted with CH2Cl2 (60 ml). The organic layers were
dried and evaporated. The crude product was purified by flash
chromatography.
3.1.3.3. 3-Phenyl-4-(vinylsulfonyl)furazan (4c). Eluent
(PE/EtOAc 90:10 v/v).Yield 79%. M.p. 56.4–57.2 °C (from
1
diisopropylether). H-NMR (CDCl3) d 6.43–6.46 (m, 1H,
3
–SO2CH=CHaHb, JHH = 9.9 Hz), 6.65–6.71 (m, 1H,
3
–SO2CH=CHaHb, JHH = 17.3 Hz), 7.00–7.09 (m, 1H,
–SO2CH=CHaHb), 7.55–7.99 (m, 5H, Ph); 13C-NMR
(CDCl3) d, 121.7 (Ci Ph), 127.8, 128.0 (Co, Cm Ph), 130.4
(Cp Ph), 132.6 (–SO2CH=CH2), 134.2 (–SO2CH=CH2), 150.9
(C3 Furaz.), 154.0 (C4 Furaz.); MS (EI) m/z 236 (M)+; dry-
ing conditions: r.t.; 24 h, pressure < 10 mmHg. Anal.
(C10H8N2O3S) C, H, N.
3.1.2.1. 2-[(3-Phenylfuroxan-4-yl)sulfonyl]ethanol (3a). Elu-
ent (PE/EtOAc 70:30 v/v). Yield 69%. M.p. 62–63 °C (from
EtOAc/PE, Ref. [12] 62–63 °C).
3.1.4. General procedure for the preparation of 5a, 5b, 5c
To a solution of 3-phenyl-4-(vinylsulfonyl)furoxan (4a)
(200 mg; 0.79 mmol) in dry THF (5 ml), kept under inert
atmosphere at –45 °C, a solution of sodium ethoxide 0.636 M
(440 µl; 0.4 eq) was slowly added. At the end of the addition
the reaction was completed. The mixture was washed with a
saturated solution of NH4Cl (5 ml), dried and evaporated. The
crude product was purified by flash chromatography.
3.1.2.2. 2-[(4-Phenylfuroxan-3-yl)sulfonyl]ethanol (3b). Elu-
ent (CH2Cl2).Yield 80%. M.p. 79–81 °C (from Et2O/PE, Ref.
[12] 80–81 °C).
3.1.2.3. 2-[(4-Phenylfurazan-3-yl)sulfonyl]ethanol (3c). Elu-
ent (PE/EtOAc 70:30 v/v).Yield 78%. M.p. 60.5–62 °C (from
EtOAc/PE, Ref. [12] 60.5–62 °C).
3.1.4.1. 4-[(2-Ethoxyethyl)sulfonyl]-3-phenylfuroxan
(5a). Eluent (PE/EtOAc 80:20 v/v).Yield 63%. M.p. 66–67 °C
1
3.1.3. General procedure for the preparation of 4a, 4b, 4c
To a stirred solution of 2-[(3-phenylfuroxan-4-yl)sulfo-
nyl]ethanol (3a) (0.84 g, 3.11 mmol) in CH2Cl2 (10 ml), kept
at 0 °C, TEA (1.08 ml, 7.76 mmol) and MsCl (0.29 ml,
3.72 mmol) were added. After 10 min the reaction was com-
pleted. The mixture was washed with a saturated solution of
NH4Cl (5 ml), dried and evaporated. The crude product was
purified by flash chromatography.
(from diisopropylether). H-NMR (CDCl3) d 1.06 (t, 3H,
–OCH2CH3), 3.38 (q, 2H, –OCH2CH3), 3.50 (t, 2H,
–SO2CH2CH2–), 3.84 (t, 2H, –SO2CH2CH2–), 7.54–7.86 (m,
5H, Ph); 13C-NMR (CDCl3) d 14.8 (–OCH2CH3), 55.1
(–OCH2CH3), 63.3 (–SO2CH2CH2–), 66.9 (–SO2CH2CH2–),
112.4 (C3 Furox.), 120.6 (Ci Ph), 129.2 (Co, Cm Ph, over-
lapped signals), 131.6 (Cp Ph), 157.7 (C4 Furox.); MS (EI)
m/z 298 (M)+; drying conditions: r.t.; 24 h, pres-
sure < 10 mmHg. Anal. (C12H14N2O5S) C, H, N.
3.1.3.1. 3-Phenyl-4-(vinylsulfonyl)furoxan (4a). Eluent
(PE/EtOAc 90:10 v/v). Yield 94%. M.p. 98.5–99.5 °C (from
diisopropylether). 1H-NMR (CDCl3) d 6.45 (m, 1H,
3.1.4.2. 3-[(2-Ethoxyethyl)sulfonyl]-4-phenylfuroxan
(5b). Eluent (PE/EtOAc 90:10 v/v). Yield 51%. M.p. 58.6–
59.6 °C (from diisopropylether). 1H-NMR (CDCl3) d 1.01 (t,
3H, –OCH2CH3), 3.34 (q, 2H, –OCH2CH3), 3.58 (t, 2H,
–SO2CH2CH2–), 3.78 (t, 2H, –SO2CH2CH2–), 7.49–7.74 (m,
5H, Ph); 13C-NMR (CDCl3) d 14.8 (–OCH2CH3), 53.4
(–OCH2CH3), 63.7 (–SO2CH2CH2–), 67.1 (–SO2CH2CH2–),
118.0 (C3 Furox.), 125.0 (Ci Ph), 128.7, 129.6 (Co, Cm Ph),
131.6 (Cp Ph), 155.1 (C4 Furox.); MS (EI) m/z 298 (M)+;
drying conditions: r.t.; 24 h, pressure < 10 mmHg. Anal.
(C12H14N2O5S) C, H, N.
3
–SO2CH=CHaHb, JHH = 9.8 Hz), 6.65 (m, 1H,
3
–SO2CH=CHaHb, JHH = 16.5 Hz), 6.90–6.98 (m, 1H,
–SO2CH=CHaHb), 7.54–7.92 (m, 5H, Ph); 13C-NMR
(CDCl3) d 112.0 (C3 Furox.), 120.2 (Ci Ph), 128.8, 129.1
(Co, Cm Ph), 131.6 (Cp Ph), 134.4 (–SO2CH=CH2), 135.3
(–SO2CH=CH2), 157.9 (C4 Furox.); MS (EI) m/z 252 (M)+;
drying conditions: r.t.; 24 h, pressure < 10 mmHg. Anal.
(C10H8N2O4S) C, H, N.
3.1.3.2. 4-Phenyl-3-(vinylsulfonyl)furoxan (4b). Eluent
(PE/EtOAc 95:5 v/v).Yield 88%. M.p. 94–95 °C (from diiso-
3.1.4.3. 3-[(2-Ethoxyethyl)sulfonyl]-4-phenylfurazan
propylether). 1H-NMR (CDCl3)
d
6.41 (m, 1H,
(5c). Eluent (PE/EtOAc 80:20 v/v).Yield 63%. M.p. 35–36 °C
3
1
–SO2CH=CHaHb, JHH = 9.4 Hz), 6.65 (m, 1H,
(from diisopropylether). H-NMR (CDCl3) d 0.98 (t, 3H,
3
–SO2CH=CHaHb, JHH = 16.3 Hz), 6.74–6.80 (m, 1H,
–OCH2CH3), 3.33 (q, 2H, –OCH2CH3), 3.64 (t, 2H,

U. Galli et al. / European Journal of Medicinal Chemistry 40 (2005) 1335–1340
1339
–SO2CH2CH2–), 3.86 (t, 2H, –SO2CH2CH2–), 7.54–7.92 (m,
5H, Ph); 13C-NMR (CDCl3) d 14.9 (–OCH2CH3), 56.6
(–OCH2CH3), 63.5 (–SO2CH2CH2–), 67.1 (–SO2CH2CH2–),
123.7 (Ci Ph), 129.4, 129.9 (Co, Cm Ph), 131.9 (Cp Ph), 152.9
(C4 Furaz.), 155.6 (C3 Furaz.); MS (EI) m/z 282 (M)+; dry-
ing conditions: r.t.; 24 h, pressure < 10 mmHg. Anal.
(C12H14N2O4S) C, H, N.
0.79 mmol) in dry THF (3 ml). The reaction was completed
after 1.45 h. The mixture was concentrated under reduced
pressure and the residue was treated with EtOAc (15 ml). To
the resulting solution oxalic acid 0.5 M in EtOAc (4 ml) and
then water (10 ml) were added. After separation the organic
layer was dried and evaporated. The crude product was puri-
fied by flash chromatography.
3.1.5. General procedure for the preparation of 6a, 6b, 6c
To a solution of 3-phenyl-4-(vinylsulfonyl)furoxan (4a)
(200 mg; 0.79 mmol) in dry THF (3 ml), kept under inert
atmosphere at 0 °C, ethanethiol (117 µl; 1.58 mmol) and TEA
(220 µl; 1.58 mmol) were added. At the end of the addition
the reaction was completed. The mixture was washed with a
saturated solution of NH4Cl (5 ml) and then extracted with
EtOAc (2 × 10 ml). The combined organic layers were dried
and evaporated. The crude product was purified by flash chro-
matography.
3.1.6.1. 4-Methoxy-N-{2-[(3-phenylfuroxan-4-yl)sulfonyl]-
ethyl}aniline (7a). Eluent (PE/EtOAc 90:10 v/v).Yield 88%.
1H-NMR (CDCl3) d 3.62–3.75 (m, 7H, –SO2CH2CH2–,
–OCH3, overlapped signals), 6.47 (2H, A2′B2′ system), 6.77
(2H, A2′B2′ system), 7.48–7.73 (m, 5H, Ph); 13C-NMR
(CDCl3) d 38.5 (–SO2CH2CH2–), 53.0 (–SO2CH2CH2–), 55.9
(–OCH3), 114.6, 115.3 (Co, Cm PhOCH3), 112.2 (C3 Furox.),
120.1 (Ci Ph), 128.9, 129.2 (Co, Cm Ph), 131.8 (Cp Ph), 139.6
(Cp PhOCH3), 153.2 (Ci PhOCH3), 157.6 (C4 Furox.); MS
(EI) m/z 375 (M)+; drying conditions: r.t.; 24 h, pres-
sure < 10 mmHg.Anal. (C17H17N3O5S) C, H, N. For the phar-
macological tests the product was treated with Et2O/HCl to
give the corresponding hydrochloride.Yield 81%. M.p. 148 °C
dec (from dry EtOAc).
3.1.5.1. 4-{[2-(Ethylthio)ethyl]sulfonyl}-3-phenylfuroxan
(6a). Eluent (PE/EtOAc 95:5 v/v).Yield 95%. M.p. 70–71 °C
(from EtOAc/PE). 1H-NMR (CDCl3) d 1.27 (t, 3H,
–SCH2CH3), 2.58 (q, 2H, –SCH2CH3), 3.02 (t, 2H,
–SO2CH2CH2–), 3.77 (t, 2H, –SO2CH2CH2–), 7.54-7.91 (m,
5H, Ph); 13C-NMR (CDCl3) d 14.8 (–SCH2CH3), 24.0
(–SCH2CH3), 26.5 (–SO2CH2CH2–), 54.9 (–SO2CH2CH2–),
112.0 (C3 Furox.), 120.5 (Ci Ph), 129.2, 129.6 (Co, Cm Ph),
132.1 (Cp Ph), 157.6 (C4 Furox.); MS (CI) m/z 315 (M + 1)+;
drying conditions: r.t.; 24 h, pressure < 10 mmHg. Anal.
(C12H14N2O4S2) C, H, N.
3.1.6.2. 4-Methoxy-N-{2-[(4-phenylfuroxan-3-yl)sulfonyl]-
ethyl}aniline (7b). Eluent (PE/EtOAc 90:10 v/v).Yield 98%.
1H-NMR (CDCl3) d 3.54 (m, 4H, –SO2CH2CH2–), 3.74 (s,
3H, –OCH3), 6.41 (2H,A2′B2′ system), 6.72 (2H,A2′B2′ sys-
tem), 7.40–7.57 (m, 5H, Ph); 13C-NMR (CDCl3) d 38.8
(–SO2CH2CH2–), 52.7 (–SO2CH2CH2–), 55.8 (–OCH3),
114.8, 115.2 (Co, Cm PhOCH3), 117.3 (C3 Furox.), 124.6
(Ci Ph), 128.7, 129.6 (Co, Cm Ph), 131.7 (Cp Ph), 139.6 (Cp
PhOCH3), 153.4 (Ci PhOCH3), 154.8 (C4 Furox.); MS (EI)
m/z 375 (M)+; drying conditions: r.t.; 24 h, pres-
sure < 10 mmHg.Anal. (C17H17N3O5S) C, H, N. For the phar-
macological tests the product was treated with Et2O/HCl to
give the corresponding hydrochloride.Yield 80%. M.p. 166–
168 °C dec (from dry MeOH).
3.1.5.2. 3-{[2-(Ethylthio)ethyl]sulfonyl}-4-phenylfuroxan
(6b). Eluent (PE/EtOAc 95:5 v/v). Yield 95%. M.p. 73.5–
74.5 °C (from EtOAc/PE). 1H-NMR (CDCl3) d 1.22 (t, 3H,
–SCH2CH3), 2.52 (q, 2H, –SCH2CH3), 2.88 (t, 2H,
–SO2CH2CH2–), 3.66 (t, 2H, –SO2CH2CH2–), 7.50-7.75 (m,
5H, Ph); 13C-NMR (CDCl3) d 14.5 (–SCH2CH3), 23.7
(–SCH2CH3), 26.4 (–SO2CH2CH2–), 53.3 (–SO2CH2CH2–),
117.1 (C3 Furox.), 124.6 (Ci Ph), 128.8, 129.7 (Co, Cm Ph),
131.9 (Cp Ph), 155.2 (C4 Furox.); MS (CI) m/z 315 (M + 1)+;
drying conditions: r.t.; 24 h, pressure < 10 mmHg. Anal.
(C12H14N2O4S2) C, H, N.
3.1.6.3. 4-Methoxy-N-{2-[(4-phenylfurazan-3-yl)sulfonyl]-
ethyl}aniline (7c). Eluent (PE/EtOAc 90:10 v/v). Yield 93%.
M.p. 79–80 °C (from diisopropylether). 1H-NMR (CDCl3) d
3.66–3.79 (m, 7H, –SO2CH2CH2–, –OCH3, overlapped sig-
nals), 6.53 (2H, A2′B2′ system), 6.78 (2H, A2′B2′ system),
7.49–7.90 (m, 5H, Ph); 13C-NMR (CDCl3) d 38.6
(–SO2CH2CH2–), 54.6 (–SO2CH2CH2–), 55.7 (–OCH3),
114.8, 115.1 (Co, Cm PhOCH3), 122.9 (Ci Ph), 129.1, 129.4
(Co, Cm Ph), 131.8 (Cp Ph), 139.5 (Cp PhOCH3), 153.2 (Ci
PhOCH3), 152.4 (C4 Furaz.), 154.8 (C3 Furaz.); MS (EI) m/z
359 (M)+; drying conditions: r.t.; 24 h, pressure < 10 mmHg.
Anal. (C12H14N2O3S2) C, H, N.
3.1.5.3. 3-{[2-(Ethylthio)ethyl]sulfonyl}-4-phenylfurazan
(6c). Eluent (PE/EtOAc 95:5 v/v). Yield 95%. M.p. 33.5–
34.5 °C (from diisopropylether). 1H-NMR (CDCl3) d 1.25 (t,
3H, –SCH2CH3), 2.57 (q, 2H, –SCH2CH3), 3.01 (t, 2H,
–SO2CH2CH2–), 3.83 (t, 2H, –SO2CH2CH2–), 7.50–7.96 (m,
5H, Ph); 13C-NMR (CDCl3) d 14.5 (–SCH2CH3), 23.6
(–SCH2CH3), 26.1 (–SO2CH2CH2–), 56.3 (–SO2CH2CH2–),
122.9 (Ci Ph), 129.2, 129.4 (Co, Cm Ph), 131.8 (Cp Ph), 152.5
(C4 Furaz.), 154.4 (C3 Furaz.); MS (EI) m/z 298 (M)+; dry-
ing conditions: r.t.; 24 h, pressure < 10 mmHg. Anal.
(C12H14N2O3S2) C, H, N.
3.1.7. NO-release: detection of nitrite
A solution of the appropriate compound (20 µL, 10−2 M)
in dimethyl sulfoxide (DMSO) was added to 1980 µl of
50 mM phosphate buffer (pH 7.4) in the presence of 0.5 mM
L-cysteine. The final concentration of drug was 10−4 M.After
3.1.6. General procedure for the preparation of 7a, 7b, 7c
p-Anisidine (195 mg; 1.58 mmol) was added to a solution
of 3-phenyl-4-(vinylsulfonyl)furoxan (4a) (200 mg;

1340
U. Galli et al. / European Journal of Medicinal Chemistry 40 (2005) 1335–1340
1 h at 37 °C, 1 ml of the reaction mixture was treated with
250 µl of Griess reagent [sulfanilamide (4 g), N-naphthyl-
ethylenediamine dihydrochloride (0.2 g), 85% phosphoric
acid (10 ml) in distilled water (final volume: 100 ml)]. After
10 min at room temperature, the absorbance was measured at
540 nm (Shimadzu UV-2501PC spectrophotometer). 10–
70 nmol ml–1 sodium nitrite standard solutions were used for
the calibration curve [14]. The yields in nitrite was expressed
as % NO2– (mol/mol) S.E.
Acknowledgments
This work was supported by the grants from Turin Univer-
sity to A.G. and the Italian Minister of the University and
Research, PRIN 2003-062554004 to D.T.
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3.2.1. Parasite cultures
P. falciparum cultures were carried out according to Trager
and Jensen’s with slight modifications [17]. The CQ-sensitive,
strain D10 and the CQ-resistant, strain W2 were maintained
at 5% hematocrit (human type A-positive red blood cells) in
RPMI 1640 (Gibco BRL, NaHCO3 24 mM) medium with
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plasma, 20 mM Hepes (Biological Industries, Kibbutz, Israel),
2 mM Glutammine (Biological Industries, Kibbutz, Israel).
All the cultures were maintained at 37 °C in a standard gas
mixture consisting of 1% O2, 5% CO2, 94% N2.
3.2.2. Parasite growth and drug susceptibility assay
Compounds were dissolved in either water (chloroquine)
or DMSO and then diluted with a medium to achieve the
required concentrations (final DMSO concentration < 1%,
which is non-toxic to the parasite). Drugs were placed in
96 wells flat-bottom microplates (COSTAR) and serial dilu-
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and incubated for 72 h at 37 °C. Parasite growth was deter-
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