Phenylsulfonylfuroxans as modulators of multidrug-resistance-associated protein-1 and P-glycoprotein

Article abstract of DOI:10.1021/jm100066y

A series of furoxan derivatives were studied for their ability to interact with P-gp and MRP1 transporters in MDCK cells overexpressing these proteins. 3-Phenylsulfonyl substituted furoxans emerged as the most interesting compounds. All of them were capable of inhibiting P-gp, and a few also were capable of inhibiting MRP1. Substituents at the 4-position of 3-phenylsulfonylfuroxan scaffold were able to modulate the selectivity and the intensity of inhibition. In some cases, they reverted MRP1 inhibitor activity, namely, they were capable of potentiating MRP1 dependent efflux. When compounds 16 and 17 were coadministered with doxorubicin, they restored a high degree of the activity of the antibiotic. Preliminary immunoblotting studies carried out on these two compounds indicate that they are capable of nitrating P-gp, which in this form is likely unable to efflux the antibiotic.

Full text of DOI:10.1021/jm100066y

J. Med. Chem. 2010, 53, 5467–5475 5467
DOI: 10.1021/jm100066y
Phenylsulfonylfuroxans as Modulators of Multidrug-Resistance-Associated Protein-1
and P-Glycoprotein
Roberta Fruttero,† Marco Crosetti,† Konstantin Chegaev,† Stefano Guglielmo,† Alberto Gasco,*,† Francesco Berardi,‡
Mauro Niso,‡ Roberto Perrone,‡ Maria Antonietta Panaro,§ and Nicola Antonio Colabufo*,‡
†Dipartimento di Scienza e Tecnologia del Farmaco, Universitaꢀ degli Studi di Torino, Via Pietro Giuria 9, 10125 Torino, Italy,
‡Dipartimento Farmacochimico, Facoltaꢁ di Farmacia, Universitaꢀ degli Studi di Bari “A. Moro”, Via Orabona, 4, 70125 Bari, Italy, and
§CIMAOL, Universitaꢀ degli Studi di Bari “A. Moro”, Piazza Giulio Cesare, 70124 Bari, Italy
Received January 16, 2010
A series of furoxan derivatives were studied for their ability to interact with P-gp and MRP1
transporters in MDCK cells overexpressing these proteins. 3-Phenylsulfonyl substituted furoxans
emerged as the most interesting compounds. All of them were capable of inhibiting P-gp, and a few
also were capable of inhibiting MRP1. Substituents at the 4-position of 3-phenylsulfonylfuroxan
scaffold were able to modulate the selectivity and the intensity of inhibition. In some cases, they reverted
MRP1 inhibitor activity, namely, they were capable of potentiating MRP1 dependent efflux. When
compounds 16 and 17 were coadministered with doxorubicin, they restored a high degree of the activity
of the antibiotic. Preliminary immunoblotting studies carried out on these two compounds indicate that
they are capable of nitrating P-gp, which in this form is likely unable to efflux the antibiotic.
Introduction
in MDR. P-gp contains 12 transmembrane helices organized in
two membrane spanning domains (MSDs), each containing six
transmembrane helices and two nucleotide binding domains
(NBDs) responsible for ATP binding. MRP1-6 transporters
differ from P-gp because they present three MSDs and the
additional domain contains five transmembrane domains.11
Several strategies5,10 have been employed to reverse MDR
including coadministration of the antineoplastic agents with
a MDR inhibitor such as elacridar, tariquidar and lani-
quidar (Chart 1). These complex molecules, belonging to third
generation P-gp inhibitors, have been studied in clinical
trials.12-17 Preliminary results show that they have signi-
ficant pharmacokinetic and pharmacodynamic limitations.
In particular, they inhibited the CYP3A4 enzyme affecting
chemotherapeutic detoxification and they showed poor selec-
tivity toward other ABC transporters not involved in MDR.17
Recently, it has been reported that reduced endogenous
NO production could be a possible mechanism responsible
for MDR in HT29-dx doxorubicin-resistant human colon
cancer cells and consequently that its restoration reverses
resistance.18 Moreover, exogenous NO influences the devel-
opment of MDR. Indeed, it was found that S-nitroso-
N-acetyl-D,L-penicillamine, S-nitrosoglutathione, and sodium
nitroprusside, three well-known NO-donors, when used at
100 μM, markedly reduced the efflux of doxorubicin in HT29-
dx and induced tyrosine nitration in MRPs transporters.18 In
addition, other studies reported that an inadequate supply of
oxygen could induce MDR in solid cancers because several
conventional anticancer drugs require oxygen for their
maximal activity.19 It has been demonstrated that hypoxia-
induced MDR could be reverted by low concentrations (from
0.1 nM to 1 μM) of NO mimetics.20,21
Multidrug resistance (MDRa) generates cancer cells un-
responsive to antineoplastic drugs treatment through several
mechanisms.1 Among them, the most extensively studied is
the increased efflux of chemotherapeutic agents from cells due
to a number of ATP binding cassette (ABC) transporters.2
Human ABC transporters belong to a family of 49 genes
classified into seven subfamilies: ABC-A, ABC-B, ABC-C,
ABC-D, ABC-E, ABC-F, ABC-G.3,4 They use the energy of
ATP hydrolysis to extrude compounds by a complex translo-
cation process.5 Some of these transporters play physiological
functions in several barriers and are also involved in CNS
disorders such as Alzheimer’s disease, Parkinson’s disease, and
epilepsy.6 They are localized in the luminal membrane of the
endothelial cells constituting barriers such as the blood-brain
barrier, blood-cerebrospinal fluid barrier, and blood-testis
barrier.7 This strategic localization permits modulation of the
absorption and excretion of xenobiotics across these barriers.8
Moreover, because they efflux drugs,9,10 reducing their con-
centration in tumor cells, overexpression of transporters in
several tumor cell lines and tumor tissues results in MDR.
Among these transporters, ABC-B1, better known as P-glyco-
protein (P-gp), and ABC-C1-6, multidrug resistance associated
proteins (MRP1-6) are the most representative pumps involved
*To whom correspondence should be addressed. For A.G.: phone,
þ39-011-670-7670; fax, þ39-011-670-7286; e-mail, alberto.gasco@
unito.it. For N.A.C.: phone, þ39-080-544-2727; fax, þ39-080-544-
2231; e-mail, colabufo@farmchim.uniba.it.
aAbbreviations: P-gp, P-glycoprotein; MDCK, Madin-Darby canine
kidney; MRP, multidrug resistance associated proteins; MDR, multidrug
resistance; ABC, ATP binding cassette; MSDs, membrane spanning
domains; NBDs, nucleotide binding domains; NO, nitric oxide; DBU,
1,8-diazabicyclo[5.4.0]undec-7-ene; MTT, 3-[4,5-dimethylthiazol-2-yl]-
2,5-diphenyltetrazolium bromide.
Furoxan (1,2,5-oxadiazole 2-oxide) (1) (Figure 1) is an old
heterocyclic system well-known to chemists because of its
r
2010 American Chemical Society
Published on Web 07/16/2010
pubs.acs.org/jmc

5468 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Chart 1. P-gp Inhibitors
Fruttero et al.
these products with free intracellular thiols or thiol groups
of proteins.26-29
The present work represents a first attempt to develop
MDR modulating agents bearing a furoxan ring as the
structural determinant. For this purpose, an extended series
of furoxan derivatives have been tested for their ability to
interact with P-gp and MRP1 transporters in MDCK cells
overexpressing these proteins. The structures of the com-
pounds studied are collected in Figure 1.
First, we examined diphenylfuroxan derivative (2) and
a series of 3-/4-phenylfuroxan isomer pairs (a,b) bearing at
4-/3-positions R groups with different stereoelectronic and
lipophilic properties (3a,b-10a,b). From the screening of these
products, the isomer pair 10a and 10b, bearing as an R
substituent the electron-withdrawing and highly lipophilic phe-
nylsulfonyl group, emerged as the most interesting. Conse-
quently, differently substituted phenylsulfonylfuroxan isomer
pairs (11a,b-13a,b) and bis(phenylsulfonyl)furoxan derivative
14 were considered. In addition, a number of 3-phenylsulfonyl
substituted furoxans bearing alkoxy groups at the 4-position
(15-21), characterized by having different sizes, shapes, and
lipophilicity, or a phenoxy moiety (22) as well as the two
furazans (1,2,5-oxadiazoles) 26 and 27 were taken into account.
Results
Chemistry. Most of compounds listed in Figure 1 (2, 3a,
b-11a,b, 14, and 15) were prepared according to published
methods (see Experimental Section). The synthetic pathways
used to prepare the remaining products are reported in
Scheme 1. The action of thiophenol on the already described
4-nitrofuroxan-3-carboxamide (23) in acetonitrile solution
afforded the expected phenylthiofuroxan derivative 24b.
This product was partly transformed into the isomer 24a
by irradiation with the full mercury arc of an immersion
medium pressure lamp. The isomer mixture was enriched in
24a by grinding in cold methanol and then resolved by flash
chromatography. The two isomers treated with pertrifluoro-
acetic acid afforded the expected phenylsulfonylfuroxan-
carboxamides 12a and 12b, respectively. The two furox-
ancarboxamide isomers, dissolved in THF, were treated
under nitrogen with pyridine and then with trifluoroacetic
Figure 1. Phenylfuroxan, phenylsulfonylfuroxan, and phenylsul-
fonylfurazan derivatives.
intriguing chemistry and a dispute regarding its structure.22
In the recent past, there has been renewed interest in furoxan
derivatives because it was found that they can release nitric
oxide under the action of thiols.23-25 The mechanism of this
release is complex and not yet fully understood. The first
step most likely involves interaction of the electrophilic
3-position of furoxan ring with the nucleophilic -SH group,
followed by ring opening and then by NO release. A number
of biological actions of furoxans are associated, or likely
associated, with NO release, following the interaction of

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5469
Scheme 1. Synthesis of Phenylsulfonylfuroxans 12a,b-13a,b,
15-22, and Phenylsulfonylfurazans 26 and 27a
using calcein-AM fluorescent probes in MDCK-MDR1 and
MDCK-MRP1 cell lines.33 These cells overexpressed only P-gp
or MRP1 transporters, respectively, so that the observed bio-
logical effects can be ascribed to the inhibition of these pumps.
Calcein-AM is a lipophilic P-gp and MRP1 substrate able
to cross the cell membrane. Inside the cell compartment it is
hydrolyzed by endogenous cytoplasmic esterases, yielding
highly fluorescent calcein. This hydrolyzed compound is not
a P-gp or MRP1 substrate, and it cannot cross the cell
membrane via passive diffusion because it is hydrophilic.
Thus, a rapid increase in the fluorescence of cytoplasmic
calcein can be monitored. P-gp and MRP1 transporters,
present in the cell membrane, rapidly efflux the calcein-AM
before its entrance into the cytosol, resulting in a reduction in
the fluorescent signal due to a decrease in the accumulation of
calcein. Evaluation of P-gp or MRP1 activity in the presence
of pumps inhibitors can be performed in a competitive
manner. Compounds that block P-gp and MRP1 pumps
inhibit calcein-AM efflux, increasing fluorescent calcein accu-
mulation. Calcein measurement into the cells was plotted
versus log[drug], and for each compound an EC50 value was
obtained from nonlinear iterative curve fitting by Prism,
version 3.0, GraphPad software.34
All compounds were tested for their inhibiting activity
against P-gp and MRP1, and the results are reported in Table 1.
All 3-phenylfuroxans derivatives 2 and 3a-10a were found
to be inactive (EC50 > 100 μM) toward P-gp and MRP1, with
the sole exception of compound 10a bearing a 4-phenyl-
sulfonyl substituent. This product displayed a moderate
effect toward each transporter (EC50=46 and 26.5 μM,
respectively). In the corresponding 4-phenylfuroxan series,
compound 10b displayed the best P-gp inhibiting acti-
vity (EC50 = 10 μM) and a weak MRP1 blocking effect
(EC50 = 64 μM). Also in this series, 3-nitrofuroxan deriva-
tive 7b displayed moderate inhibition toward P-gp (EC50
=
53 μM), while it was a moderate inducer of the MRP1 pump
(EC50=45 μM). Moreover, the 3-CN substituted compound
8b moderately inhibited the MRP1 pump (EC50 =42 μM),
while it was inactive toward P-gp (EC50>100 μM). All the
other isomers belonging to 4-phenylfuroxan series were found
to be inactive vs both P-gp and MRP1 (EC50>100 μM).
Among 3-phenylsulfonyl and 4-phenylsulfonyl derivatives
11a,b-13a,b and 14, compounds 11a-13a, belonging to the
3-phenylsulfonyl series, and 3,4-diphenylsulphonyl deriva-
tive 14 (EC50 from 3.0 to 50 μM) were found to be more
potent with respect to compound 10a in inhibiting P-gp.
In particular, the best results were obtained for compound
14 (EC50 = 3.0 μM), while 4-carboxamide derivative 12a
a Reaction conditions: (a) ROH, DBU, CH2Cl2, room temp; (b)
PhSH, Et3N, MeCN, -10 °C; (c) hν, CH2Cl2, room temp; (d) H2O2,
CF3COOH; (e) (CF3CO)2O, Py, THF, 0 °C.
anhydride to yield the corresponding furoxancarbonitriles
13a and 13b. The structures assigned to these compounds
were confirmed by 13C NMR spectroscopy, on the basis of the
knowledge that in a furoxan isomer pair the Nþ-O- moiety
exerts a shielding influence on the resonance of the 13C linked
to the 3-position of the ring with respect to the corresponding
13C-linked to the 4-position.30,31 Consequently, the structure
of 4-CN (-13CN, 105.9) and of 3-CN (-13CN, 103.4 ppm)
was assigned to 13a, and 13b, respectively.
The preparation of 4-alkoxy-3-phenylsulfonylfuroxans
16-21, phenoxy substituted furoxan 22, and 3-alkoxy-
4-phenylsulfonylfurazans 26 and 27 was carried out in
CH2Cl2 solution by treating the appropriate alcohols with
14 or with 25 in the case of furazans in the presence of 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU). The 3-phenylsulfo-
nyl structure was assigned to furoxan products on the basis
of the knowledge that 14 undergoes selective nucleophilic
displacement of the 4-phenylsulfonyl group by alcohols in
THF under basic conditions.32
displayed comparable activity with respect to 10a (EC50
=
50 μM vs 46 μM). Moreover, compounds 12a, 13a, and 14
displayed unexpected MRP1 inducing activity (EC50 from
15.1 to 85.8 μM), whereas 4-methylsubstituted furoxan 11a
was the best MRP1 inhibiting agent (EC50 = 5.1 μM). In the
corresponding 4-phenylsulfonyl series compounds 11b-13b
were found to be less potent than the corresponding isomers
(EC50 > 52.6 μM) in inhibiting the P-gp pump, while only
compound 12b inhibited MRP1 (EC50 = 13 μM).
In Table 1 the results obtained working with phenoxy or
alkoxy substituted furoxans 15-22 are reported. With the
exception of 4-ethoxy (15) and 4-phenoxy (22) derivatives,
which displayed moderate P-gp inhibitory activity (EC50
=
12 and 20.5 μM, respectively), all the other alkoxy derivatives
16-21 showed high P-gp inhibitory activity (EC50 from 2.15
to 4.60 μM). This effect is not linearly dependent on their
Biochemical Studies. P-gp and MRP1 inhibiting activities of
tested compounds were performed by fluorescence measurement

5470 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Fruttero et al.
Table 1. P-gp and MRP1 Modulating Activities of Phenylfuroxans (2-10) and Phenylsulfonylfuroxans (11-22)
EC50 (μM) ( SEMa
MRP1
P-gp
compd
R
R0
inhibiting activity
inducing activity
2
3a
Ph
Ph
Ph
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
98 ( 5.0
CH3
Ph
3b
4a
CH3
Ph
>100
>100
NH2
Ph
4b
5a
NH2
Ph
>100
>100
OCH3
Ph
5b
6a
OCH3
Ph
>100
>100
CONH2
Ph
NO2
6b
7a
CONH2
Ph
NO2
>100
>100
7b
8a
Ph
CN
53 ( 2.5
>100
>100
45 ( 9.0
Ph
CN
85 ( 4.0
42 ( 1.5
>100
8b
9a
Ph
SO2NH2
Ph
Ph
SO2NH2
Ph
>100
>100
9b
>100
10a
10b
11a
11b
12a
12b
13a
13b
14
SO2Ph
Ph
CH3
46 ( 2.5
10 ( 0.4
28.3 ( 5.7
>100
26.5 ( 1.2
64 ( 2.5
5.1 ( 0.8
>100
SO2Ph
SO2Ph
CH3
SO2Ph
CONH2
SO2Ph
CN
SO2Ph
CONH2
SO2Ph
CN
50 ( 3.0
52.6 ( 2.0
7.4 ( 0.5
55.4 ( 2.0
3.0 ( 0.2
12 ( 0.4
2.26 ( 0.2
3.35 ( 0.2
4.60 ( 0.1
3.31 ( 0.2
2.15 ( 0.5
2.23 ( 0.2
20.5 ( 3.0
1.50b
85.8 ( 9.0
13 ( 1.2
29.5 ( 3.0
72 ( 8.0
15.1 ( 2.0
49.1 ( 5.0
7.61 ( 0.8
12 ( 1.5
SO2Ph
SO2Ph
OC2H5
OC4H9
OC6H13
OC8H17
OC10H21
O-iso-C3H7
O-iso-C4H9
OPh
SO2Ph
SO2Ph
SO2Ph
SO2Ph
SO2Ph
SO2Ph
SO2Ph
SO2Ph
SO2Ph
15
16
17
18
8.7 ( 1.0
8.0 ( 0.9
19
20
63 ( 8.0
46 ( 5.0
13.6 ( 2.0
21
22
MC18
MK571
2.80b
2.85b
a The values are the mean ( SEM from two independent experiments with samples in duplicate. b See ref 35.
lipophilicity. The most active products were n-butoxy 16 and
the two branched alkoxy derivatives 20 and 21. The behavior
of this class of products against MRP1 protein is surprising.
Indeed, the highly lipophilic substances 18 and 19, bearing
long aliphatic chains, display high inhibitor activity (EC50
=
8.7 and 8.0 μM, respectively), while the remaining products
15-17 and 20-22 induced MRP1 activation, similar to
compounds 12a, 13a, and 14. This effect, shown in Figure 2
for compounds 14, 16, 17, was seen as lower intracellular
calcein accumulation in treated cells than basal levels in
untreated cells. The best activation was observed with com-
pounds 16 and 17 (EC50 = 7.61 and 12 μM, respectively).
Compounds 14, 16, 17 and 19-21, which displayed the
best P-gp inhibitory activity (EC50 from 2.15 to 3.35 μM),
were tested at 10 μM in antiproliferative assay (MTT)
employing MDCK-MDR1 cells, which are insensitive to
doxorubicin antineoplastic treatment. Products 14, 16, 17,
and 19 showed very low antiproliferative action, while 20 and
21 decreased cell viability of 30% and 45%, respectively. For
this reason, only 14, 16, 17, and 19 were tested in MTT
coadministrated with doxorubicin to check their ability to
Figure 2. Calcein efflux P-gp- and MRP1-mediated. Gray bars
indicate the inducer activity of compounds at 50 μM in MDCK-
MRP1 cells, whereas black bars indicate inhibition activity at 50 μM
tested compounds in MCDK-MDR1 cells.
restore the toxicity of the antineoplastic agent. The results
are reported in Figure 3
To have a preliminary indication of whether NO could be
involved in the mechanism of doxorubicin accumulation in-
duced by 3-phenylsulfonylfuroxan derivatives, MDCK-MDR1

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5471
Figure 3. Antiproliferative effect of 0.1 μΜ doxorubicin (white bar) and of 10 μM compounds 14, 16, 17, 19 (black bars) at 48 h in MDCK-
MDR1 cells. In comparison, each compound (10 μM) was administered for 24 h. After washout, each compound was coadministrated with
doxorubicin (0.1 μM) at the same concentration for 24 h (gray bars).
Table 2. P-gp and MRP1 Inhibiting Activities of Phenylsulfonyl-
furazans (26, 27)
EC50 ( SEMa ( μM)
compd
R
P-gp
MRP1
Figure 4. Immunoprecipitation and immunoblotting experiments
in MCDK-MDR1 cells: detection of P-gp (A) and nitro-Pgp bands
in no treated cells (B) and in cells treated with 10 μM compounds
16 (C) and 17 (D).
26
27
OC4H9
OC6H13
49.7 ( 2.5
32.2 ( 1.6
1.50b
>100
>100
2.80b
2.85b
MC18
MK571
a The values are the mean ( SEM from two independent experiments
with samples in duplicate. b See ref 35.
cells were incubated for 2 days with either 16 or 17. The whole
cellular lysate was then immunoprecipitated with a specific
anti-nitrotyrosine antibody, and immunoprecipitated pro-
teins were subjected to Western blotting, using anti P-gp
antibody (Figure 4). In these experiments β-actin levels were
used as protein loading control. Figure 4 indicates a high level
of nitrotyrosine residues in the cells treated with the two
furoxans.
nucleophilic -SH group. A similar mechanism of NO release
was recently proposed to explain the capacity of furoxan deri-
vatives to inhibit thioredoxin glutathione reductase (TGR), a
selenocysteine-containing flavoenzyme required by trematode
flatwarms of the genus Schistosoma to maintain proper
cellular redox balance.29 Analogously, the moderate activity
of the two furazans 26 and 27 listed in Table 2 (EC50 = 49.7
and 32.2 μM, respectively), which were synthesized for com-
parison since theyweredeprived of the capacityto release NO,
could arise from the interaction of the -SH group of trans-
porter with the carbon of the heteroring bearing the phenyl-
sulfonyl substituent. This interaction should lead to the
formation of tetrahedral intermediate followed by the simple
displacement of phenylsulfonyl, a good leaving group under
the action of thiols in furazan system, without any ring
opening and NO release.
The results reported in Figure 3 show that combination
treatment of 0.1 μM doxorubicin with either 10 μM 14 or 19
partly decreased cell viability (15% and 20%, respectively),
while compounds 16 and 17 at the same concentration induce a
comparable high viability decrease (65%). These results, com-
bined with those of Western blotting, suggest that NO inhibits
the efflux of doxorubicin from MDCK-MDR1 cells and
provide proof that furoxan derivatives can reverse drug toler-
ance through NO-dependent pump inactivation. Likely, NO
reduces the number of functionally active transporters modify-
ing the conformation of a site crucial for the drug transport.
Discussion
The activity profile of furoxans as modulators of MRP1
and P-gp(Table1)isnot simple, anditsinterpretationrequires
attentive study. The preliminary results obtained in the im-
munoblotting experiments working with 16 and 17 support
the possibility that NO is involved in P-gp inhibitory activity
of 3-phenylsulfonylfuroxan derivatives. The NO responsible
for nitration could be derived from their interaction with free
cellular thiols. Another interesting possibility is that NO
mightbeproducedfroma nucleophilicattackatthe 3-position
of the furoxan system by a cysteine residue of the protein
transporter. This attack should induce the formation of a
tetrahedral intermediate, followed by sequential ring opening
to quaternary nitroso derivative, NO production, and the
probable release of heterocycle scaffold from protein. Then
NO so formed could nitrate the transporter, inducing mod-
ulation of its activity. In this case, the whole process should
depend not only upon the electrophilic reactivity of the
3-furoxan position but also upon both the docking of the
product to the protein and its appropriate alignment with the

5472 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Fruttero et al.
Finally, the finding that some furoxans, in particular
compounds 16 and 17 (Figure 2), are able to potentiate
MRP1 dependent efflux is interesting. Indeed, while P-gp
inducers have been already reported and studied,36 MRPs
inducing agents were not as yet described in literature. These
products are potential tools to investigate MRP1 induction
mechanism and to characterize specific binding sites for
substrates, inhibitors, and inducers of this transporter.37
(m, 3H), 7.67-7.74 (m, 2H) (C6H5), 7.78 (s, 1H), 8.56 (s, 1H)
(CONH2). 13C NMR (75 MHz, DMSO-d6) δ ppm 111.0, 125.6,
129.8, 130.3, 135.0, 155.7, 157.0. MS (EI) m/z 237 [M]þ. Anal.
(C9H7N3O3S) C, H, N.
3-Phenylthiofuroxan-4-carboxamide (24a). A solution of 24b
(5.5 g, 23.0 mmol) in CH2Cl2 (100 mL) in a quartz reactor
was irradiated with the full mercury arc of an immersion
medium pressure lamp (125W, Photochemical Reactors Ltd.,
Buckinghamshire, HP16 ODR, U.K.) for 1 h at room tempera-
ture. The organic solvent was removed under reduced pressure,
and the obtained solid was treated with cold MeOH (50 mL).
The resulting mixture was filtered and the filtrate was evapo-
rated to give a mixture of two isomers in ∼1:1 molar ratio. The
title product was partially purified by flash chromatography
(eluent 7/3 PE/AcOEt) and finally crystallized from CCl4 to give
pale-yellow crystals. Yield 10%, mp = 115.5-116.5 °C (CCl 4).
1H NMR (300 MHz, DMSO-d6) δ ppm 7.37-7.45 (m, 5H,
C6H5), 8.28 (s, 1H), 8.55 (s, 1H) (CONH2). 13C NMR (75 MHz,
DMSO-d6) δ ppm 109.9, 127.8, 128.5, 129.5, 130.7, 153.3, 157.6.
MS (EI) m/z 237 [M]þ. Anal. (C9H7N3O3S) C, H, N.
Conclusions
The results obtained in the present work show that the
furoxan system can be used to design both P-gp and MRP1
ligands. In particular, 3-phenylsulfonyl substituted furoxan
appears to be the most interesting and flexible scaffold for
designing inhibitors of both transporters. Substituents at the
4-position of this substructure can modulate the selectivity and
the intensity of inhibition. In some cases, they can display
interesting MRP1 inducing activity; namely, they can potenti-
ate MRP1 dependent efflux. Results obtained working with
selected members of the 3-phenylsulfonylfuroxan series show
that the compounds 16 and 17 are able to restore in high degree
the antiproliferative activity of doxorubicin when coadminis-
tered with this antineoplastic agent. Preliminary immunoblot-
ting studies carried out on these two same compounds indicate
that they are capable of nitrating P-gp, which in this form is
likely unable to efflux the chemotherapeutic agent.
4-Phenylsulfonylfuroxan-3-carboxamide (12b). To a solution
of 24b (0.50 g, 2.1 mmol) in a CH2Cl2/CF3COOH mixture
(5 mL/5 mL) a solution of 88% H2O2 (0.5 mL, 18 mmol) in
CF3COOH (5 mL) was added dropwise at 0 °C. The cooling
bath was removed, and the mixture was stirred at room tem-
perature for 2 h. Then it was poured into H2O (50 mL) and
extracted with EtOAc (2 ꢀ 30 mL). The organic extract was
washed with H2O, brine, dried, and evaporated. The obtained
solid was crystallized from MeOH to give the title compound as
a white crystalline solid. Yield 68%, mp = 188-188.5 °C
Experimental Section
1
(MeOH). H NMR (300 MHz, DMSO-d6) δ ppm 7.74-7.88
Chemistry. 1H and 13C NMR spectra were recorded on a
Bruker Avance 300 at 300 and 75 MHz, respectively, using
SiMe4 as the internal standard. Low resolution mass spectra
were recorded with a Finnigan-Mat TSQ-700. Melting points
(m, 2H), 7.88-7.93 (m, 1H), 8.10-8.13 (m, 3H), 8.57 ppm (s,
1H) (C6H5 þ CONH2). 13C NMR (75 MHz, DMSO- d6) δ ppm
109.1, 129.3, 129.8, 135.9, 136.6, 153.7, 157.4. MS (CI) m/z 270
[M þ H]þ. Anal. (C9H7N3O5S) C, H, N.
€
were determined with a capillary apparatus (Buchi 540). Flash
3-Phenylsulfonylfuroxan-4-carboxamide (12a). The product
was obtained by the same procedure used to synthesized 12b,
starting from 24a. Yield 24%, mp = 177.5-178 °C (i-PrOH/
column chromatography was performed on silica gel (Merck
Kieselgel 60, 230-400 mesh ASTM). PE stands for 40-60
petroleum ether. The progress of the reactions was followed
by thin layer chromatography (TLC) on 5 cm ꢀ 20 cm plates
with a layer thickness of 0.2 mm. Anhydrous magnesium sulfate
was used as the drying agent for the organic phases. Organic
solvents were removed under vacuum at 30 °C. Purities of all
new compounds were g95% and were determined by elemental
analysis and HPLC. Elemental analyses (C, H, N) of the new
compounds dried at 20 °C at a pressure of <10 mmHg for 24 h
were performed at the University of Geneva, Switzerland, and
the results are within (0.4% of the theoretical values. HPLC
analyses were performed with a HP 1100 chromatograph sys-
tem (Agilent Technologies, Palo Alto, CA) equipped with a
quaternary pump (model G1311A), a membrane degasser
(G1379A), a diode array detector (DAD) (model G1315B)
integrated in the HP1100 system, and a Zorbax Extend C18
column (150 mm ꢀ 4.6 mm, 5 μm particle size). Data analysis
was done using a HP ChemStation system (Agilent Techno-
logies). Compounds 9a,b38 and 2339 were synthesized according
to the literature. The preparation of products 2, 3a,b-11a,b, 14,
25, and 15 is reported in a number of references that are collected
in the bibliography of reviews 22 and 40.
4-Phenylthiofuroxan-3-carboxamide (24b). To a solution of 23
(2.1 g, 12.0 mmol) in CH3CN (25 mL) a mixture of Et3N (1.7
mL, 12.0 mmol) and thiophenol (1.3 mL, 13 mmol) in CH3CN
(15 mL) was added dropwise at -10 °C. The reaction mixture
was stirred at -10 °C for 1 h, then poured into H2O (75 mL) and
extracted with CH2Cl2 (2 ꢀ 50 mL). The organic solvent was
washed with H2O (30 mL), brine, dried, and evaporated. The
obtained solid was washed with cold MeOH, filtered, and used
without further purification. Yield 79%. An analytically pure
sample was obtained by crystallization, mp = 146-149 °C (dec
MeOH). 1H NMR (300 MHz, DMSO-d6) δ ppm 7.25-7.56
1
H2O). H NMR (300 MHz, DMSO-d6) δ ppm 7.75-7.81 (m,
2H), 7.89-7.94 (m, 1H), 8.08-8.11 (m, 2H) (C6H5), 8.49 (s, 1H),
8.68 (s, 1H) (CONH2). 13C NMR (75 MHz, DMSO-d6) δ ppm
115.6, 128.6, 129.6, 136.2, 136.6, 150.5, 156.6. MS (CI) m/z 270
[M þ H]þ. Anal. (C9H7N3O5S) C, H, N.
4-Phenylsulfonylfuroxan-3-carbonitrile (13b). To a solution of
12b (0.30 g, 1.1 mmol) in dry THF (15 mL) kept under positive
N2 pressure, pyridine (0.18 mL, 2.2 mmol) was added at 0 °C
followed by (CF3CO)2O (0.30 mL, 2.2 mmol). The cooling bath
was removed, and the mixture was stirred at room temperature
for 1 h. Then it was poured into H2O (50 mL) and extracted with
EtOAc (2 ꢀ 25 mL). The organic extract was washed with H2O,
brine, dried, and evaporated. The obtained oil was solidified by
treating with cold PE and crystallized from hexane to give the
title compound as a white crystalline solid. Yield 81%, mp =
89.5-90 °C (C6H14). 1H NMR (300 MHz, CDCl3) δ ppm
7.70-7.75 (m, 2H), 7.83-7.90 (m, 1H), 8.13-8.16 (m, 2H)
(C6H5). 13C NMR (75 MHz, CDCl3): δ ppm 93.8, 103.4,
129.4, 130.4, 136.0, 136.6, 157.1. MS (EI) m/z 251 [M]þ. Anal.
(C9H5N3O4S) C, H, N.
3-Phenylsulfonylfuroxan-4-carbonitrile (13a). The product
was obtained by the same procedure used to synthesize 13b,
starting from 12a. Yield 64%, mp = 103-103.5 °C (C6H14). 1H
NMR (300 MHz, CDCl3) δ ppm 7.68-7.73 (m, 2H), 7.83-7.88
(m, 1H), 8.09-8.16 (m, 2H) (C6H5). 13C NMR (75 MHz,
CDCl3) δ ppm 105.9, 115.0, 129.0, 129.9, 130.3, 136.4, 136.8.
MS (EI) m/z 251 [M]þ. Anal. (C9H5N3O4S) C, H, N.
General Procedure for the Synthesis of 4-Alkyl(aryl)oxy-3-
phenylsulfonylfuroxans (16-22). To a mixture of the corre-
sponding alcohol or phenol (1.5 mmol) and DBU (3.0 mmol)
in CH2Cl2 (15 mL), 3,4-bisphenylsulfonylfuroxan (1.0 mmol)
was added in one portion. The reaction mixture was stirred at

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5473
room temperature for 2 h. Then the organic solvent was washed
with H2O (20 mL), 1 M HCl (2 ꢀ 10 mL), brine, dried, and
evaporated. The obtained solid was purified by crystallization.
4-Butoxy-3-phenylsulfonylfuroxan (16). Yield 75%, mp =
137.9, 148.8, 161.4. MS (CI) m/z 283 [M þ H]þ. Anal. (C12H14-
N2O4S) C, H, N.
3-Hexyloxy-4-phenylsulfonylfurazan (27). Eluent 6/4 PE/
CH2Cl2 v/v, colorless oil. Yield 80%. 1H NMR (300 MHz,
CDCl3) δ ppm 0.91 (t, 3H, CH3), 1.30-1.42 (m, 6H, 3CH2),
1.80-1.87 (m, 2H, OCH2CH2), 4.36 (t, 2H, OCH2), 7.60-7.65
(m, 2H), 7.73-7.78 (m, 1H), 8.08-8.11 (m, 2H) (C6H5). 13C
NMR (75 MHz, CDCl3) δ ppm 14.0, 22.5, 25.2, 28.5, 31.3, 74.0,
129.0, 129.6, 135.4, 137.9, 148.8, 161.4. MS (CI) m/z 311 [M þ
H]þ. Anal. (C14H18N2O4S) C, H, N.
1
96-97 °C (i-PrOH). H NMR (300 MHz, DMSO-d6) δ ppm
0.93 (t, 3H, CH3), 1.33-1.45 (m, 2H, CH3CH2), 1.69-1.79 (m,
2H, OCH2CH2), 4.40 (t, 2H, OCH2), 7.74-7.79 (m, 2H),
7.89-7.94 (m, 1H), 8.01-8.04 (m, 2H) (C6H5). 13C NMR (75
MHz, DMSO-d6) δ ppm 13.4, 18.3, 29.9, 71.2, 110.4, 128.3,
130.0, 136.1, 137.2, 158.9. MS (CI) m/z 299 [M þ H]þ. Anal.
(C12H14N2O5S) C, H, N.
Biology. Materials. Cell culture reagents were purchased from
Celbio s.r.l. (Milan, Italy). CulturePlate 96-well plates were
purchased from PerkinElmer Life Science; calcein-AM, MTT
(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide),
doxorubicin, and all reagents for immunoprecipitation and im-
munoblotting assays were obtained from Sigma-Aldrich (Milan,
Italy). MK571 was purchased fromCalbiochem (San Diego, CA).
Cell Cultures. MDCK-MDR1 and MDCK-MRP1 are a gift of
Prof. P. Borst, NKI-AVL Institute, Amsterdam, The Netherlands.
MDCK-MDR1 and MDCK-MRP1 were grown in DMEM high
glucose supplemented with 10% fetal bovine serum, 2 mM
glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin in a
humidified incubator at 37 °C with a 5% CO2 atmosphere.
Calcein-AM Experiment. These experiments were carried out
as described by Feng et al. with minor modifications.33 Each
cell line (50 000 cells per well) was seeded into a black Culture-
Plate 96-well plate with 100 μL of medium and allowed to
become confluent overnight. An amount of 100 μL of test
compounds was solubilized in culture medium and added to
monolayers followed by incubation at 37 °C for 30 min.
Calcein-AM was added in 100 μL of phosphate buffered saline
(PBS) to yield a final concentration of 2.5 μM, and the plate was
incubated for 30 min. Each well was washed three times with ice
cold PBS. Saline buffer was added to each well, and the plate
was read in a Victor3 microplate reader (PerkinElmer) at
excitation and emission wavelengths of 485 and 535 nm,
respectively. In these experimental conditions calcein cell accu-
mulation in the absence and in the presence of tested com-
pounds was evaluated and fluorescence basal level was
estimated in untreated cells. In treated wells the increase of
fluorescence with respect to basal level was measured. EC50
values were determined by fitting the fluorescence increase
percentage versus log [dose].34
Antiproliferative Assay. Determination of cell growth was
performed using the MTT assay at 24 and 48 h.41,42 On day 1,
20 000 cells/well were seeded into 96-well plates in a volume of
100 μL. On day 2, the various drugs alone or in combination
with doxorubicin were added. In all the experiments, the various
drug solvents (ethanol, DMSO) were added to each control to
evaluate possible solvent cytotoxicity. After the established
incubation time with drugs, 0.5 mg/mL MTT was added to each
well, and after 3 h of incubation at 37 °C the supernatant was
removed. The formazan crystals were solubilized using 100 μL
of DMSO, and the absorbance values at 570 and 630 nm were
determined on a microplate reader Victor 3.
Immunoprecipitation and Immunoblotting Analysis. In an im-
munoprecipitation assay, treated and untreated cells (2 days)
were harvested and lysed in ice-cold lysis buffer (0.5 M Tris-HCl,
1.86 g/mL EDTA, 1 M NaCl, 0.001 g/mL digitonin, 4 U/mL
aprotinin, 2 μM leupeptin, and 100 μM PMSF). Lysates were
centrifuged at 13800g for 20 min at 4 °C. The protein concen-
tration was determined by the Bradford method. Then 1 mL of
clear lysates was incubated with 5 μL of mouse monoclonal anti-
human P-glycoprotein (MDR) antibody overnight with contin-
uous rotation at 4 °C. Protein A-sepharose beads (30 μL) were
then added, and the samples were gently rocked at 4 °C for 3 h.
After five washes with lysis buffer, the beads were recovered and
resuspended in 40 μL of 2ꢀ SDS sample buffer (4% SDS, 0.125
mol/L Tris-HCl, 20% glycerol, and 0.04% bromophenol blue,
pH 6.8) and then boiled for 5 min. The coimmunoprecipitation
4-Hexyloxy-3-phenylsulfonylfuroxan (17). Yield 55%, mp =
72-73 °C (i-PrOH/H2O). 1H NMR (300 MHz, CDCl3) δ ppm
0.87 (t, 3H, CH3), 1.25-1.43 (m, 6H, 3CH2), 1.75-1.84 (m, 2H,
OCH2CH2), 4.34 (t, 2H, OCH2), 7.52-7.57 (m, 2H), 7.66-7.71
(m, 1H), 7.86-8.00 (m, 2H) (C6H5). 13C NMR (75 MHz,
CDCl3) δ ppm 14.1, 22.6, 25.4, 28.5, 31.4, 71.8, 110.6, 128.7,
129.8, 135.7, 138.3, 159.2. MS (CI) m/z 327 [M þ H]þ. Anal.
(C14H18N2O5S 0.25H2O) C, H, N.
3
4-Octyloxy-3-phenylsulfonylfuroxan (18). Yield 90%, mp =
1
78-80 °C (MeOH/H2O). H NMR (300 MHz, DMSO-d6) δ
ppm 0.87 (t, 3H, CH3), 1.24-1.38 (m, 10H, 5CH2), 1.70-1.77
(m, 2H, CH2), 4.39 (t, 2H, OCH2), 7.73-7.80 (m, 2H),
7.88-7.93 (m, 1H), 8.00-8.03 (m, 2H) (C6H5). 13C NMR (75
MHz, DMSO-d6) δ ppm 13.7, 21.8, 24.8, 27.6, 28.2, 28.3, 30.9,
71.2, 110.1, 128.0, 129.7, 135.9, 137.0, 158.6. MS (CI) m/z 355
[M þ H]þ. Anal. (C16H22N2O5S) C, H, N.
4-Decyloxy-3-phenylsulfonylfuroxan (19). Yield 88%, mp =
1
65-67 °C (MeOH/H2O). H NMR (300 MHz, DMSO-d6) δ
ppm 0.84 (t, 3H, CH3), 1.12-1.35 (m, 14H, 7CH2), 1.72-1.74
(m, 2H, CH2), 4.38 (t, 2H, OCH 2), 7.70-7.75 (m, 2H),
7.88-7.93 (m, 1H), 8.00-8.03 (m, 2H) (C6H5). 13C NMR (75
MHz, DMSO-d6) δ ppm 14.2, 22.4, 25.3, 28.1, 28.8, 29.0, 29.2,
31.6, 71.7, 110.7, 128.6, 130.3, 136.4, 137.6, 159.2. MS (CI) m/z
383 [M þ H]þ. Anal. (C18H26N2O5S) C, H, N.
4-Isopropoxy-3-phenylsulfonylfuroxan (20). Yield 37%,
1
mp = 101-103 °C (i-PrOH). H NMR (300 MHz, CDCl3) δ
ppm 1.47 (d, 6H, 2CH3), 5.10 (qi, 1H, OCH), 7.60-7.64 (m, 2H),
7.73-7.78 (m, 1H), 8.04-8.07 (m, 2H) (C6H5). 13C NMR (75
MHz, CDCl3) δ ppm 21.1, 76.4, 110.4, 128.3, 129.9, 136.0, 137.2,
158.0. MS (CI) m/z 285 [M þ H]þ. Anal. (C11H12N2O5S)
C, H, N.
4-Isobutoxy-3-phenylsulfonylfuroxan (21). Yield 94%, mp
100-102 °C (i-PrOH). 1H NMR (300 MHz, CDCl3) δ ppm
1.05 (d, 6H, 2CH3), 2.21 (qi, 1H, CH2CH), 4.18 (d, 2H, OCH2),
7.60-7.65 (m, 2H), 7.73-7.79 (m, 1H), 8.05-8.08 (m, 2H)
(C6H5). 13C NMR (75 MHz, CDCl3) δ ppm 18.8, 27.8, 77.2,
110.4, 128.5, 129.7, 135.6, 138.3, 159.2. MS (CI) m/z 299 [M þ
H]þ. Anal. (C12H14N2O5S) C, H, N.
4-Phenyloxy-3-phenylsulfonylfuroxan (22). Yield 60%, mp
102-104 °C (i-PrOH). 1H NMR (300 MHz, CDCl3) δ ppm
7.29-7.34 (m, 3H), 7.42-7.47 (m, 2H), 7.61-7.71 (m, 2H),
7.76-7.81 (m, 1H), 8.08-8.11 (m, 2H) (2C6H5). 13C NMR (75
MHz, CDCl3) δ ppm 110.8, 119.8, 126.8, 128.6, 129.8, 130.0,
135.8, 138.0, 152.6, 158.5. MS (CI) m/z 319 [M þ H]þ. Anal.
(C14H10N2O5S) C, H, N.
General Procedure for the Synthesis of 3-Alkyloxy-4-phenyl-
sulfonylfurazans (26, 27). To a mixture of the appropriate
alcohol (1.5 mmol) and DBU (3.0 mmol) in CH2Cl2 (15 mL),
3,4-bisphenylsulfonylfurazan (1.0 mmol) was added in one
portion, and the reaction mixture was stirred at room tempera-
ture for 24 h. Then the organic solvent was washed with H2O (20
mL), 1 M HCl (2 ꢀ 10 mL), brine, dried, and evaporated. The
obtained oil was purified by flash chromatography.
3-Butoxy-4-phenylsulfonylfurazan (26). Eluent 1/1 PE/CH2Cl2
v/v, colorless oil. Yield 81%. 1H NMR (300 MHz, DMSO-d6) δ
ppm 0.97 (t, 3H, CH3), 1.37-1.50 (m, 2H, CH3CH2), 1.77-1.86
(m, 2H, OCH2CH2), 4.37 (t, 2H, OCH2), 7.60-7.65 (m, 2H),
7.73-7.79 (m, 1H), 8.08-8.12 (m, 2H) (C6H5). 13C NMR (75
MHz, DMSO-d6) δ ppm 13.6, 18.8, 30.5, 73.7, 129.0, 129.6, 135.4,

5474 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Fruttero et al.
(IP) proteins dissociated from the beads were used for immuno-
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Immunoblotting was done as described below. Protein
samples (25 μg/lane) and prestained standards were loaded
onto 10% sodium dodecyl sulfate (SDS) precast polyacryla-
mide gels. After electrophoresis, the resolved proteins were
transferred from the gel to nitrocellulose membranes. A blot-
ting buffer (20 mM Tris/150 mM glycine, pH 8, 20% (v/v)
methanol) was used for membrane saturation and blotting.
Primary antibodies (mouse monoclonal anti-human P-glyco-
protein (MDR), rabbit anti-nitrotyrosine, and β-actin mono-
clonal antibody, 1:2000) were used, followed by horseradish
peroxidase-linked secondary antibody (goat anti-mouse IgG,
goat anti-rabbit, 1:1000) and visualized by chemiluminescence
detection.
(16) Pusztai, L.; Wagner, P.; Ibrahim, N.; Rivera, E.; Theriault, R.;
Booser, D.; Symmans, F. W.; Wong, F.; Blumenschein, G.;
Fleming, D. R.; Rouzier, R.; Boniface, G.; Hortobagyi, G. N.
Phase II study of tariquidar, a selective P-glycoprotein inhibitor, in
patients with chemotherapy-resistant, advanced breast carcinoma.
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