Metabolic and chemical origins of cross-reactive immunological reactions to arylamine benzenesulfonamides: T-cell responses to hydroxylamine and nitroso derivatives

Article abstract of DOI:10.1021/tx900329b

Exposure to sulfamethoxazole (SMX) is associated with T-cell-mediated hypersensitivity reactions in human patients. T-cells can be stimulated by the putative metabolite nitroso SMX, which binds irreversibly to protein. The hydroxylamine and nitroso deriva

Full text of DOI:10.1021/tx900329b

184
Chem. Res. Toxicol. 2010, 23, 184–192
Metabolic and Chemical Origins of Cross-Reactive Immunological
Reactions to Arylamine Benzenesulfonamides: T-Cell Responses to
Hydroxylamine and Nitroso Derivatives
J. Luis Castrejon,† Sidonie N. Lavergne,† Ayman El-Sheikh,† John Farrell,†
James L. Maggs,† Sunil Sabbani,‡ Paul M. O’Neill,‡ B. Kevin Park,† and Dean J. Naisbitt*,†
MRC Centre for Drug Safety Science, Department of Pharmacology and Therapeutics, School of Biomedical
Sciences, The UniVersity of LiVerpool, LiVerpool L69 3GE, United Kingdom, and Department of Chemistry, The
UniVersity of LiVerpool, LiVerpool L69 7ZD, United Kingdom
ReceiVed September 11, 2009
Exposure to sulfamethoxazole (SMX) is associated with T-cell-mediated hypersensitivity reactions in
human patients. T-cells can be stimulated by the putative metabolite nitroso SMX, which binds irreversibly
to protein. The hydroxylamine and nitroso derivatives of three arylamine benzenesulfonamides, namely,
sulfamethozaxole, sulfadiazine, and sulfapyridine, were synthesized, and their T-cell stimulatory capacity
in the mouse was explored. Nitroso derivatives were synthesized by a three-step procedure involving the
formation of nitro and hydroxylamine sulfonamide intermediates. For immune activation, female Balb-c
strain mice were administered nitroso sulfonamides four times weekly for 2 weeks. After 14 days, isolated
splenocytes were incubated with the parent compounds, hydroxylamine metabolites, and nitroso derivatives
to measure antigen-specific proliferation. To explore the requirement of irreversible protein binding for
spleen cell activation, splenocytes were incubated with nitroso derivatives in the presence or absence of
glutathione. Splenocytes from nitroso sulfonamide-sensitized mice proliferated and secreted interleukin
(IL)-2, IL-4, IL-5, and granulocyte monocyte colony-stimulating factor following stimulation with nitroso
derivatives but not the parent compounds. Splenocytes from sensitized mice were also stimulated to
proliferate with hydroxylamine and nitroso derivatives of the structurally related sulfonamides. The addition
of glutathione inhibited the nitroso-specific T-cell response. Hydroxylamine metabolites were unstable
in aqueous solution: Spontaneous transformation yielded appreciable amounts of nitroso and azoxy
compounds as well as the parent compounds within 0.1 h. T-cell cross-reactivity with nitroso sulfonamides
provides a mechanistic explanation as to why structurally related arylamine benzenesulfonamides are
contraindicated in hypersensitive patients.
availability of synthetic stable and reactive metabolites for
functional studies, and the accessibility of clinical samples and
animal models of immunogenicity.
Introduction
It is estimated that up to 5% of hospital admissions are caused
by adverse drug reactions (1). Most of these are dose-dependent
(type A), on-target toxicities that represent an exaggeration of
the known primary and/or secondary pharmacology of the drug.
They are normally avoidable through a reduction in dose or
improved therapeutic drug monitoring (2–4). Off-target toxicity
is unpredictable and, although less common, has a tendency to
be more severe due to the immune system and its ability to
disseminate the initial chemical signal (5, 6). A proportion of
these off-target toxicitiessso-called hypersensitivity reactionss
are provoked by drug-specific T-cells (7–9). Of the drugs
associated with a high incidence of hypersensitivity reactions,
sulfamethoxazole (SMX;1 1a) is the model selected by many
research groups to explore the phenotype and function of
antigen-specific T-cells and mechanisms of antigen presentation.
This is because of the drug’s well-defined metabolism, the
SMX (1a) is an aromatic amine and is metabolized to an
N-acetylated form, which is rapidly excreted in urine (10).
Moreover, in humans, approximately 2% of the drug is
metabolized by CYP2C8/9 to a hydroxylamine (sulfamethox-
azole hydroxylamine, SMX-NHOH; 3a), an oxidative metabolite
that circulates in the blood and is eliminated in urine (11–14).
Spontaneous oxidation of SMX-NHOH in aqueous solution
generates nitroso sulfamethoxazole (SMX-NO; 4a), an unstable
product prone to further oxidation generating nitro SMX (2a)
(15), thiol-mediated reduction (16–18), and conjugation with
SMX-NHOH (3a), forming azo and azoxy dimers (19). In spite
of these competing reactions, SMX-NO (4a) has been shown
to bind covalently to serum proteins (20–22) at cysteine residues
(20, 23), to liver tissue (24), and to the surface of immune cells
(14, 25, 26). A complex and partly metastable series of haptenic
structures, including cysteine-linked N-hydroxysulfenamide
(semimercaptal), sulfinamide, N-hydroxysulfinamide, and N-
hydroxysulfonamide adducts, have recently been characterized
on model proteins and peptides exposed to SMX-NO (4a) (23).
* To whom correspondence should be addressed. Tel: 0044 151 7945346.
Fax: 0044 151 7945540. E-mail: dnes@liv.ac.uk.
† Department of Pharmacology and Therapeutics.
‡ Department of Chemistry.
1 Abbreviations: GM-CSF, granulocyte monocyte colony-stimulating
factor; IL, interleukin; SD, sulfadiazine; SD-NHOH, sulfadiazine hydroxy-
lamine; SD-NO, nitroso sulfadiazine; SMX, sulfamethoxazole; SMX-
NHOH, sulfamethoxazole hydroxylamine; SMX-NO, nitroso sulfamethox-
azole; SP, sulfapyridine; SP-NHOH, sulfapyridine hydroxylamine; SP-NO,
nitroso sulfapyridine; THF, tetrahydrofuran.
SMX-NO (4a) is a potent immunogen in experimental
animals (20, 25, 27, 28). T-cells are stimulated by SMX-NO
(4a) via a classical hapten mechanism involving protein-complex
formation, processing, and the presentation of derived peptides
10.1021/tx900329b  2010 American Chemical Society
Published on Web 12/02/2009

T-Cell Responses to N-Oxygenated Sulfonamides
Chem. Res. Toxicol., Vol. 23, No. 1, 2010 185
dimethyl sulfoxide, Hank’s balanced salt solution, RPMI-1640,
streptomycin, L-glutamine, HEPES, and penicillin were purchased
from Sigma Chemical Co. (Poole, Dorset, United Kingdom).
Lymphoprep was obtained from Nycomed (Birmingham, United
Kingdom), and [3H]-thymidine was obtained from Moravek (CA).
The medium consisted of RPMI, supplemented with 10% fetal
bovine serum, HEPES buffer (25 mM), L-glutamine (2 mM),
penicillin (100 U/mL), and streptomycin (100 µg/mL).
NMR Spectroscopy and HPLC. The sulfonamide metabolites
were characterized using NMR spectroscopy, and the purity was
obtained by HPLC. NMR data were obtained with a Bruker, Inc.,
spectrometer (Coventry, United Kingdom). 1H NMR spectra were
obtained at 400 MHz. All spectra were recorded in 120 µL of
DMSO-d6. Chemical shifts (δ) are expressed in ppm relative to
residual peak 2.50 (ppm). For HPLC analysis, samples were eluted
at room temperature on a Prodigy 5 µm ODS-2 column (150 mm
× 4.6 mm i.d.; Phenomenex, Macclesfield, Cheshire, United
Kingdom) using linear gradients of acetonitrile in 0.1% (v/v) formic
acid: 20-50% over 20 min for SMX derivatives and 5-50% over
20 min for SD and SP derivatives. The eluent flow rate was 0.9
mL/min.
Synthesis of Nitroso SMX. SMX-NO (4a) was synthesized by
a three-step procedure described previously (15). 4-Nitrobenzyl-
sulfonylchloride (10 g) was added slowly to a cooled solution of
3-amino-5-methylisoxazole (20 mL of pyridine, 0 °C). After 24 h,
the reaction mixture was filtered, and the resultant brown residue
was washed with an excess of distilled water. The crude product
was recrystallized from ethyl acetate:toluene (1:3 v/v) to give 2a
as a white powder (nitro SMX; 2a). To synthesize SMX-NHOH
(3a), nitro SMX (1 g; 2a) was suspended in THF (100 mL) and
distilled water (10 mL). Sodium hypophosphite (1.12 g; 10 mL of
distilled water) and palladium/carbon catalyst (0.1 g) were then
added to the stirred solution to reduce the nitro moiety. The reaction
was followed by TLC using dichloromethane-ethyl acetate (65:
35, v/v), until all of the starting material had reacted (approximately
20 min). The product was extracted with diethyl ether (200 mL),
and the combined organic extracts were dried with magnesium
sulfate and filtered, and then, the solvent was evaporated under
reduced pressure. The resulting crude yellow oil was recrystallized
using trichloromethane to give a white powder [SMX-NHOH (3a)].
To synthesize SMX-NO (4a), SMX-NHOH (1 g; 3a) was dissolved
in ethanol (70 mL) and added to an iron(III) chloride solution (5.99
g in 50 mL of water) at room temperature. After 5 min, the reaction
mixture was filtered. A bright yellow powder (SMX-NO; yield,
30-40%) was collected as a final product. Analytical data have
been described previously (9).
Figure 1. Proliferative response of splenocytes isolated from (A)
C57BL/6 and (B) Balb-c strain mice. The mice were immunized with
SMX-NO (4a) (5 mg/kg), and the cells were cultured in the presence
of SMX (1a), SMX-NHOH (3a), and SMX-NO (4a) for 72 h.
Proliferation was measured by incorporation of [3H]-thymidine. Results
represent the mean ( SD for three animals, with incubations carried
out in triplicate. Statistical analysis compares drug-treated splenocytes
with cell incubations containing DMSO alone (*p < 0.05). Splenocytes
from na¨ıve mice were not stimulated with SMX (1a), SMX-NHOH
(3a), or SMX-NO (4a).
in the context of MHC molecules (19). SMX-NO also stimulates
T-cells isolated from blood and blister fluid of hypersensitive
patients. In contrast to animal models, incubation of SMX (1a)
with peripheral blood mononuclear cells and T-cell clones from
allergic patients stimulates a proliferative response (9, 27, 29, 30).
The fine specificity of the interaction of drugs with MHC
and specific T-cell receptors and their ability to stimulate a T-cell
response is an important consideration with respect to patient
management and administration of structurally related drugs.
It is well-established that nonantimicrobial (nonarylamine)
sulfonamides, even if they possess a free amino group on the
sulfonamide moiety and are associated with hypersensitivity
reactions (31), are safe to use in SMX (1a) hypersensitive
patients (33–35) in the absence of cross-reactivity (35). How-
ever, antimicrobial (arylamine) benzenesulfonamides, often
related closely to SMX in structure, should be avoided. Indeed,
the interaction of T-cells with a panel of antimicrobial sulfona-
mides containing the same core structure has been explored in
detail; T-cells obtained from blood and blister fluid in patients with
toxic epidermal necrolysis respond to as many as six antimicrobial
sulfonamides and display clear differences in the minimum
concentration needed for T-cell stimulation (9, 32, 34–38). The
lack of a panel of synthetic nitroso sulfonamide derivatives
has thus far precluded structure-activity analysis of T-cell
responses to protein-reactive drug antigens. Therefore, the
aim of this study was to synthesize hydroxylamine and nitroso
derivatives of three antimicrobial sulfonamides, namely, SMX
(1a), sulfapyridine (SP; 1b), and sulfadiazine (SD; 1c) (Figure
1; Scheme 1), and to study their T-cell stimulatory capacity
in a mouse model.
Synthesis of Nitroso SP. Similar to the synthesis of SMX-NO
(4a), three-step procedures were used for the synthesis of nitroso
sulfapyridine (SP-NO; 4b) and nitroso sulfadiazine (SD-NO; 4c).
However, palladium/carbon was replaced as the catalyst for nitro
reduction because it was associated with excessive reduction and
liberation of the parent amine. Several catalysts (palladium, zinc,
and Raney nickel) and hydrogen donors (sodium phosphinite,
ammonium chloride, and hydrazine) were compared in different
solvent systems (THF/water, DMF/ethanol/water, and dichloroet-
hane) to define the most appropriate experimental conditions.
A Raney nickel catalyst and hydrazine in dichloroethane/ethanol
were used for the synthesis of SP hydroxylamine (3b). Nitro SP
(2b) [2.5 g, 100 mL of ethanol/dichloroethane (1:1, v/v)] was cooled
(0 °C) and mixed with Raney nickel catalyst (≈0.1 g), and hydrazine
(0.9 g) was added dropwise. The temperature was maintained
between 0 and 10 °C for the duration of the experiment. The
reaction was followed by TLC (1:9; methanol/dichloromethane).
After 3 h, the solution was filtered through a Celite bed, and the
crude solid was washed with acetone to extract the hydroxylamine,
and then, the solvent was evaporated. The product was dried under
vacuum for 2 h and recrystallized using methanol/dichloromethane
(1:9, v/v). The solution was left overnight in an ice bath, and the
resultant white precipitate was filtered and dried under vacuum to
give 3b as a final product. SP-NO (4b) was synthesized by
following the procedure described above for SMX-NO (4a), with
Experimental Procedures
Caution: Sulfonamides and their oxidation deriVatiVes are
sensitizing compounds. Therefore, they must be handled carefully,
and skin contact should be aVoided.
Materials. 4-Nitrobenzylsulfonylchloride, 2-aminopyrimidine,
anhydrous pyridine, 2-aminopyridine, 3-amino-5-methylisoxazole,
hydrazine, Raney nickel, dichloroethane, sodium hypophosphite,
palladium, tetrahydrofuran (THF), methanol, ethanol, chloroform,
magnesium sulfate, iron chloride, dichloromethane, ethyl acetate,

186 Chem. Res. Toxicol., Vol. 23, No. 1, 2010
Castrejon et al.
the following modifications. First, the reaction was stirred for 30
min. Second, on completion of the reaction, the volume was reduced
by half to promote precipitation of the product.
(3a), SP-NHOH (3b), SD-NHOH (3c) (all 5-50 µM), SMX (1a),
SP (1b), and SD (1c) (all 40-800 µM) for 72 h (37 °C, 5% CO2).
To measure proliferation, [3H]-thymidine (0.5 µCi) was added to
each well for the final 16 h. Cells were harvested, and [3H]-
thymidine incorporation was measured as counts per min on a
ꢀ-counter (PerkinElmer Life Sciences, Cambridge, United King-
dom).
1
Nitro SP (2b). White solid (7.70 g), mp ) 160 °C. H NMR
(400 MHz, d-DMSO): δ 8.37 (dt, J ) 9.3 and 2.5 Hz, 2H), 8.13
(dt, J ) 9.3 and 2.5 Hz, 2H), 7.98 (dd, J ) 5.9 and 1.1 Hz, 1H),
7.85 (ddd, J ) 8.9, 7.0, and 1.9 Hz, 1H), 7.31 (d, J ) 8.9 Hz, 1H),
6.9 (ddd, J ) 7.0, 6.0, and 0.94 Hz), 3.47 (br s, 1H). IR (KBr,
cm-1): 2970, 1631, 1350, 1142, 964, 733. HRMS: Found [M +
Na]+, 302.0206; C11H9N3O4SNa required, 302.0211.
Assessment of the ex Vivo Proliferative Response of Spleno-
cytes to Sulfonamide Derivatives in the Presence of Glu-
tathione. Splenocytes from sensitized mice were incubated with
SMX-NO (4a), SP-NO (4b), SD-NO (4c) [25 µM; except for SD-
NO (4c), 10 µM], SMX-NHOH (3a), SD-NHOH (3c), or SP-NHOH
(3b) [25 µM; except for SMX-NHOH (3a), 50 µM] for analysis of
proliferation using the conditions described above. Glutathione (1
mM) was added to certain incubations. After 72 h (37 °C, 5% CO2),
[3H]-thymidine (0.5 µCi) was added for the last 16 h of the
experiment to measure antigen-specific spleen cell proliferation.
Assessment of the Stability of Sulfonamide Hydroxylamines
by Liquid Chromatography-Mass Spectrometry. Aliquots (75
µL) of solutions derived from cell incubations treated with SMX-
NHOH (3a), SP-NHOH (3b), or SD-NHOH (3c) (0.1, 4, 24, and
72 h) were chromatographed at room temperature on a Prodigy 5
µm ODS-2 column (150 mm × 4.6 mm i.d.; Phenomenex) using
linear gradients of acetonitrile in 0.1% (v/v) formic acid: 20-50%
over 20 min for SMX and 5-50% over 20 min for SD and SP.
The eluent flow rate was 0.9 mL/min. The LC system consisted of
two Jasco PU980 pumps (Jasco UK, Great Dunmow, Essex, United
Kingdom) and a Jasco HG-980-30 mixing module. Eluted com-
pounds were monitored at 254 nm with a Jasco UV-975 spectro-
photometer. Eluate split-flow to the LC-MS interface was ca. 40
µL/min. A Quattro II mass spectrometer (Waters Corp., Manchester,
United Kingdom) fitted with the standard coaxial electrospray
source was operated using nitrogen as the nebulizing and drying
gas. The interface temperature was 80 °C, the electrospray capillary
voltage was 3.8 kV, and the cone voltage was 40 V. The instrument
was set up for selected ion monitoring in the positive-ion mode as
follows: channel dwell time, 200 ms; cycle time, 230 ms; and span,
0.5 amu.
Assessment of Cytokine Secretion. Supernatant from incuba-
tions of immunized splenocytes stimulated with SMX-NO (4a), SP-
NO (4b), or SD-NO (4c) (all 25 µM) was collected after 72 h and
stored at -70 °C for analysis of cytokine secretion. Concentrations
of interleukin (IL)-2, IL-4, IL-5, TNF-R, granulocyte monocyte
colony-stimulating factor (GM-CSF), and IFN-γ were measured
in the supernatant using a mouse cytokine/chemokine LINCOplex
multiplex assay kit (LINCO Research Inc., Hampshire, United
Kingdom). Cytokine secretion (pg/mL) was measured using a Bio-
Plex Suspension Array System (model Luminex100) operating with
BioRad Bio-Plex Manager 3.0 Software (BioRad, Hertfordshire,
United Kingdom).
Statistical Analysis. Values to be compared were initially
analyzed for non-normality by the Shapiro-Wilks test. Because the
data were found to be non-normally distributed, we used the
Mann-Whitney test for comparison of the two groups. A p value
<0.05 was considered statistically significant.
Sulfapyridine Hydroxylamine (SP-NHOH, 3b). White solid
1
(0.473 g), mp ) 160 °C. H NMR (400 MHz, d-DMSO): δ 8.93
(br s, 1H), 8.63 (br s, 1H), 8.09 (dd, J ) 5.1 and 1.1 Hz, 1H),
7.66-7.71 (m, 3H), 7.12 (d, J ) 8.5 Hz, 1H), 6.92 (ddd, J ) 7.0,
5.3, and 0.7 Hz, 1H), 6.86 (d, J ) 8.7 Hz, 2H). IR (KBr, cm-1):
2670, 1631, 1389, 1242, 1130, 1080, 879, 775. HRMS: Found [M
+ Na]+, 288.0418; C11H11N3O3SNa required, 288.0419.
SP-NO (4b). Green compound (0.074 g), mp ) 150 °C. 1H NMR
(400 MHz, d-DMSO): δ 8.21 (d, J ) 8.5 Hz, 2H), 8.09 (d, J ) 8.5
Hz, 2H), 7.98 (d, J ) 5.3 Hz, 1H), 7.84 (ddd, J ) 8.8, 7.2, and 1.6
Hz, 1H), 7.32 (d, J ) 8.7 Hz, 1H), 6.89 (t, J ) 6.5 Hz, 1H). IR
(KBr, cm-1): 3352, 2974, 1631, 1450, 1385, 1354, 1296, 1146,
999, 883, 775. HRMS: Found [M + H]+, 264.0438; C11H10N3O3S
required, 264.0443.
Synthesis of Nitroso SDZ. As described above for SP, a Raney
nickel catalyst and hydrazine in dichloroethane/ethanol were used
for the synthesis of SD hydroxylamine (3c). The reaction was
followed by TLC (1:9; methanol/dichloromethane), and after 1.5 h,
the solution was filtered through a Celite bed, the solvent was
evaporated, and the crude solid was washed with acetone to extract
the hydroxylamine. The product was then dried under vacuum for
2 h and recrystallized using 2-propanol. The solution was left
overnight in an ice bath, and the resultant white precipitate was
filtered and dried under vacuum to give 3c as a final product. SD-
NO (4c) was synthesized by following the procedure described
above for SP-NO (4b).
1
Nitro SD (2c). Yellow solid (8.37 g), mp ) 270 °C. H NMR
(400 MHz, d-DMSO): δ 8.53 (d, J ) 4.9 Hz, 2H), 8.4 (dt, J ) 9.4
and 2.5 Hz, 2H), 8.22 (dt, J ) 9.4 and 2.5 Hz, 2H), 7.07 (t, J )
4.9 Hz, 1H), 3.42 (br s, 1H). IR (KBr, cm-1): 2970, 1581, 1520,
1442, 1346, 1165, 1088, 957, 798, 737. HRMS: Found [M + H]+,
281.0332; C10H9N4O4S required, 281.0345.
Sulfadiazine Hydroxylamine (SD-NHOH, 3c). Orange solid
(0.701), mp ) 220 °C. 1H NMR (400 MHz, d-DMSO): δ 8.97 (br
s, 1H), 8.65 (br s, 1H), 8.48 (d, J ) 4.7 Hz, 2H), 7.76 (dt, J ) 9.5
and 2.0 Hz, 2H), 7.01 (t, J ) 4.9 Hz, 1H), 6.85 (dt, J ) 9.5 and
2.3 Hz, 2H). IR (KBr, cm-1): 3321, 2974, 1577, 1493, 1442, 1315,
1146, 1088, 945, 795. HRMS: Found [M + Na]+, 289.0359;
C10H10N4O3SNa required, 289.0371.
SD-NO (4c). Brown solid (0.05 g), mp ) 230 °C. 1H NMR (400
MHz, d-DMSO): δ 8.44-8.60 (m, 2H), 8.25-8.36 (m, 2H),
8.06-8.17 (m, 2H), 6.99-7.12 (m, 1H). IR (KBr, cm-1): 2970,
1581, 1500, 1442, 1412, 1346, 1169, 1088, 953, 795. HRMS: Found
[M + Na]+, 287.0203; C10H8N4O3SNa required, 287.0215.
Immunizing Protocol. Female Balb-c (16-20 g) and C57/Bl6
(20-30 g) mice were purchased from Charles River U.K. Ltd.
(Kent, United Kingdom). Mice were administered SMX-NO (4a)
(5 mg/kg; n ) 4), SP-NO (4b) (5 mg/kg; n ) 3), or SD-NO (4c)
(5 mg/kg; n ) 3) in DMSO (100 µL) via i.p. injection four times
weekly for 2 weeks according to a protocol described previously
(25). On completion of this dosing regimen, animals were killed,
and the spleen was removed for analysis of splenocyte proliferation.
Results
Synthesis of Sulfonamide Hydroxylamine and Nitroso
Derivatives. Nitroso sulfonamides can be synthesized via a
three-step procedure involving: (1) nucleophilic coupling of
4-nitrobenzenesulfonyl chloride to the relevant side chain, (2)
catalytic hydrogenation of nitro intermediates, and (3) oxidation
of the hydroxylamine intermediates with iron(III) chloride
(Schemes 1 and 2).
Nitro-SMX (2a), SP (2b), and SD (2c) were synthesized in
high yields and purity using an established literature method
(15). Nitro-SMX (2a) reduction and the liberation of SMX-
NHOH (3a) were performed using an established procedure with
palladium-carbon as the catalyst and sodium phosphinite as
Assessment of the ex Vivo Proliferative Response of Spleno-
cytes to Sulfonamides and Sulfonamide Derivatives. Spleens
were homogenized in Hank’s balanced salt solution (10 mL) and
filtered to liberate a single cell suspension. Red cells were removed
by centrifugation with Lymphoprep. The splenocytes were washed,
and viability was assessed using Trypan Blue. Cells were suspended
in culture medium at a concentration of 1.5 × 106 cells/mL and
placed in a round-bottom 96-well plate (1.5 × 105/well) with SMX-
NO (4a), SP-NO (4b), SD-NO (4c) (all 1-25 µM), SMX-NHOH

T-Cell Responses to N-Oxygenated Sulfonamides
Chem. Res. Toxicol., Vol. 23, No. 1, 2010 187
Scheme 1. Structures of SMX (1a), SP (1b), and SD (1c)
SP-NO (4b) and SD-NO (4c). For SP-NHOH (3b), the reaction
mixture was filtered after 30 min, yielding a bright green product
that was found to be SP-NO (4b) on analysis. For SD-NHOH
(3c), a pale yellow product, SD-NO (4c) was detected after
15-20 min.
N-Oxygenated Derivatives of SMX, SD, and SP
Stimulate a T-Cell Response in Na¨ıve Mice. Preliminary
experiments were designed to explore the immunogenicity of
SMX-NO (4a) (5 mg/kg; 4 × weekly for 2 weeks) in BALB/c
and C57BL/6 strain mice. Higher SMX-NO (4a) concentra-
tions were not used due to overt toxicity following repeated
dosing (data not shown). Splenocytes isolated from BALB/c
and C57BL/6 strain mice administered SMX-NO (4a) were
stimulated to proliferate with SMX-NO (4a) (Figure 1). The
response to SMX-NO (4a) was significantly stronger with
splenocytes from Balb/c strain mice; thus, this strain of mouse
was selected for all subsequent investigations.
Scheme 2. Three-Step Synthesis of the Hydroxylamine and
Nitroso Derivatives of SMX (1a), SP (1b), and SD (1c)
As described previously (27), splenocytes from mice exposed
to SMX-NO (4a) (5 mg/kg; 4 × weekly for 2 weeks)
proliferated following stimulation with SMX-NO (4a) and SMX-
NHOH (3a) but not SMX (1a) (Figure 2A). Similar results were
obtained with SP-NO (4b)- and SD-NO (4c)-immunized mice.
Splenocytes were stimulated to proliferate with nitroso and
hydroxylamine derivatives but not the parent compounds (Figure
2B,C). Hydroxylamine- and nitroso-specific proliferation was
concentration-dependent up to 25-50 µM. Concentrations above
50 µM inhibited splenocyte proliferation (data not shown).
The strength of the maximum proliferative response of
splenocytes from SD-NO- (4c) immunized mice to SD-NO (4c)
was significantly lower than that observed with SMX-NO (4a)-
stimulated and SP-NO (4b)-stimulated splenocytes from SMX-
NO (4a)- and SP-NO (4b)-immunized mice, respectively [SMX-
NO (4a)-immunized: 0 µM, 2967 ( 1073.14 cpm; SMX-NO
(4a): 25 µM, 26120.41 ( 8891.24 cpm; p < 0.05. SP-NO (4b)-
immunized: 0 µM, 3225.11 ( 2386.57 cpm; SP-NO (4b): 25
µM, 35592.11 ( 11310.10 cpm; p < 0.05. SD-NO (4c)-
immunized: 0 µM, 193.55 ( 127.77 cpm; SD-NO (4c): 5 µM,
4120.55 ( 3529.75 cpm; p < 0.05]. Splenocytes from na¨ıve
mice, that is, mice not exposed to any of the sulfonamides or
their derivatives, were not stimulated specifically with SMX
(1a), SP (1b), SD (1c), or their respective hydroxylamine or
nitroso derivatives.
N-Oxygenated Derivatives of SMX, SD, and SP Stimulate
a T-Cell Response in Mice Immunized with Other Nitroso
Derivatives. Splenocytes from mice immunized with SMX-NO
(4a) were additionally stimulated to proliferate in the presence
of SD-NHOH (3c) and SD-NO (4c). Hydroxylamine and nitroso
derivatives of SP (3b or 4b) stimulated a weak response and
only at the highest concentration tested (Figure 3A). A prolifera-
tive response of splenocytes from SD-NO (4c)- and SP-NO (4b)-
immunized mice was detected with hydroxylamine and nitroso
derivatives of all three sulfonamides (Figure 3B,C). The
proliferative response was stronger when splenocytes from
SMX-NO (4a)-, SP-NO (4b)-, and SD-NO (4c)-treated mice
were stimulated with the compound against which the animals
had been immunized.
the hydrogen donor (15). The syntheses of SP-NHOH (3b) and
SD-NHOH (3c) were more challenging as the initial experiments
using palladium-carbon and sodium phosphinite in THF-water
were unsuccessful (data not shown). Nitro-SP (2b) and nitro-
SD (2c) were, indeed, largely insoluble in THF. Furthermore,
as hydrogen transfer occurred almost immediately, it was not
possible to prevent excessive reduction. To resolve these issues,
several experimental approaches used previously in the reduction
of nitrobenzene derivatives were tested (39–43). Choices of
solvent, catalyst, hydrogen donor, and temperature were all
found to influence the rate of hydrogen transfer (Table 1). The
selective reduction of nitro-SP (2b) and nitro-SD (2c) was
eventually performed using Raney nickel catalysis with hydra-
zine as the hydrogen donor in a dichloroethane/ethanol solvent
system maintained at 0 °C. The reactions were stopped as soon
as the starting material was consumed. Following recrystalli-
zation using methanol/chloroform or 2-propanol, both SP-
NHOH (3b) and SD-NHOH (3c) were obtained in high purity.
As reported previously, SMX-NHOH (3a) oxidizes rapidly
in the presence of iron(III) chloride (44, 45) SMX-NO (4a), a
bright yellow insoluble product, was detected in high yields and
purity after 5 min. A similar procedure was used to synthesize
Table 1. Reagents and Conditions Used To Optimize the Synthesis of SP and SD Hydroxylamines (3b and 3c)
catalyst
hydrogen donor
solvent, conditions
duration
result summary
A
B
C
D
E
F
palladium
zinc
palladium
zinc
Raney nickel
Raney nickel
sodium phosphinite
ammonium chloride
hydrazine
ammonium chloride
hydrazine
THF, water, room temperature
7 min
30 min
24 h
20 min
3 h
hydroxylamine, amine and nitro
hydroxylamine, amine and nitro
no reaction
hydroxylamine and nitro
SP hydroxylamine (95% pure)
SD hydroxylamine (95% pure)
DMF, water, room temperature
DMF, water, ethanol, THF, room temperature
DMF, water, ethanol, room temperature
dichloroethane, ethanol, 0 °C
hydrazine
dichloroethane, ethanol, 0 °C
1.5 h

188 Chem. Res. Toxicol., Vol. 23, No. 1, 2010
Castrejon et al.
Figure 4. Proliferative response of splenocytes from Balb-c strain mice
immunized with (A) SMX-NO (4a), (B) SP-NO (4b), or (C) SD-NO
(4c) (all 5 mg/kg) and cultured with hydroxylamine or nitroso
derivatives in the presence or absence of glutathione. Proliferation was
measured by incorporation of [3H]-thymidine. Results represent the
mean ( SD for three animals, with incubations carried out in triplicate.
Statistical analysis compares drug-treated splenocytes with cell incuba-
tions containing DMSO alone (p < 0.05).
of lymphocytes from allergic patients and animal models of
SMX (1a) immunogenicity (25, 29). In this study, glutathione
pretreatment significantly reduced the proliferation of spleno-
cytes from SMX-NO (4a)-, SP-NO (4b)-, and SD-NO (4c)-
immunized mice stimulated with the corresponding hydroxy-
lamine and nitroso derivatives. [3H]-Thymidine incorporation
was decreased almost to control levels (Figure 4).
Rapid Degradation of SMX, SD, and SP Hydroxy-
lamines in Solution. To investigate the fate of SMX-NHOH
(3a), SP-NHOH (3b), and SD-NHOH (3c) in solution, spleno-
cytes were incubated in complete medium for 0.1-72 h (the
duration of the proliferation assay), and products of oxidation,
reduction, and self-conjugation were analyzed by LC-MS.
SMX-NHOH (3a) (Rt, 8.2 min; m/z [270]+) was detectable
at 0.1 h. Weak signals for SMX (1a) (Rt, 9.5 min; m/z 254 [M
+ 1]+) and azoxy SMX (Rt, 24.0 min; m/z 519 [M + 1]+) were
also detected. After 1 h, the signal for SMX-NHOH (3a) had
all but disappeared. Degradation of SMX-NHOH (3a) coincided
with the appearance of SMX (1a) and azoxy SMX in a time-
dependent fashion. Low levels of SMX-NO (4a) were detected
between 1 and 4 h (Rt, 18.3 min; m/z 268 [M + 1]+).
SP-NHOH (3b) degraded rapidly in culture. A weak signal
(Rt, 9.6 min; m/z 266 [M + 1]+) was detectable at the earliest
time point analyzed (0.1 h). Interestingly, although low levels
of SP (1b) (Rt, 9.5 min; m/z 250 [M + 1]+) and azoxy SP (Rt,
17.1 min; m/z 511 [M + 1]+) were detectable at each time point
tested, the intensity of the respective signals did not increase
with time.
Figure 2. Proliferative response of splenocytes from Balb-c strain mice
immunized with (A) SMX-NO (4a), (B) SD-NO (4c), or (C) SP-NO
(4b) (all 5 mg/kg) and cultured with their respective hydroxylamine
and nitroso derivatives for 72 h. Proliferation was measured by
incorporation of [3H]-thymidine. Results represent the mean ( SD for
three animals, with incubations carried out in triplicate. Statistical
analysis compares drug-treated splenocytes with cell incubations
containing DMSO alone (*p < 0.05). Splenocytes from na¨ıve mice were
not stimulated specifically with SMX (1a), SP (1b), SD (1c), or their
respective hydroxylamine or nitroso derivatives.
Incubation of SD-NHOH (3c) with splenocytes resulted in a
similar degradation profile. SD-NHOH (3c) (Rt, 7.9 min; m/z
267 [M + 1]+) was detected between 0 and 1 h. A weak signal
for azoxy SD (Rt, 17.5 min; m/z 513 [M + 1]+) was detected at
0.1 h, and the intensity of the signal increased with time. SD
(1c) and SD-NO (4c) were not detected throughout the incuba-
tion period.
Cytokine Secretion from Splenocytes Stimulated by
SMX-NO, SD-NO, and SP-NO. Cytokines secreted by antigen-
stimulated splenocytes from mice immunized with nitroso
sulfonamides were analyzed using a mouse cytokine multiplex
assay kit. SMX-NO (4a) stimulated splenocytes from SMX-
NO (4a)-immunized mice and secreted the Th2 cytokines IL-4
and IL-5 and IL-2 and GM-CSF (Table 2). Similar results were
obtained following antigen stimulation of splenocytes from SP-
NO (4b)-immunized mice (Table 2). In contrast, splenocytes
Figure 3. Proliferative response of splenocytes from Balb-c strain mice
immunized with (A) SMX-NO (4a), (B) SP-NO (4b), or (C) SD-NO
(4c) (all 5 mg/kg) and cultured with different hydroxylamine or nitroso
derivatives. Proliferation was measured by incorporation of [3H]-
thymidine. Results represent the mean ( SD for three animals, with
incubations carried out in triplicate. Statistical analysis compares drug-
treated splenocytes with cell incubations containing DMSO alone (*p
< 0.05).
Glutathione Prevents the Selective Stimulation of Sple-
nocytes by Hydroxylamine and Nitroso Derivatives of
SMX, SD, and SP. Glutathione prevents the conversion of
hydroxylamine metabolites to nitroso compounds and rapidly
reduces synthetic nitroso compounds (15, 16, 18, 25). Further-
more, glutathione inhibits SMX-NO (4a)-specific stimulation

T-Cell Responses to N-Oxygenated Sulfonamides
Chem. Res. Toxicol., Vol. 23, No. 1, 2010 189
Table 2. Cytokine Secretion by Antigen-Stimulated Splenocytes from Nitroso Sulfonamide-Immunized Mice
cytokine (pg/m)
immunogen
antigen
IL-2
IL-4
IL-5
GM-CSF
IFN-γ
TNF-R
SMX-NO (4a)
0
<10
NDb
ND
ND
25.4 ( 4.8
39.4 ( 8.5
16.9 ( 4.7
16.6 ( 4.7
<10
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
SMX-NO
SP-NO
SD-NO
0
SMX-NO
SP-NO
SD-NO
0
75.6 ( 1.6a,c
44.3 ( 1.2c
67.3 ( 1.3c
27.9 ( 3.9
60.4 ( 9.3c
122.4 ( 15.4c
104.7 ( 12.7c
10.0 ( 0.8
68.8 ( 1.3c
<10
18.5 ( 0.9c
<10
39.1 ( 3.5c
<10
82.6 ( 6.5c
32.2 ( 8.7c
31.2 ( 18.6c
ND
10.2 ( 0.9c
10.0 ( 1.2
23.7 ( 1.8c
94.2 ( 7.9c
21.0 ( 2.9c
<10
<10
ND
SP-NO (4b)
SD-NO (4c)
18.1 ( 2.7c
48.2 ( 5.7c
11.7 ( 2.1c
ND
105.5 ( 32.1c
202.9 ( 28.7c
45.0 ( 7.9c
ND
ND
ND
<10
25.8 ( 8.9
13.0 ( 1.4
12.3 ( 0.2
<10
16.0 ( 2.0
21.0 ( 9.5
SMX-NO
SP-NO
SD-NO
16.7 ( 0.2c
<10
<10
ND
<10
54.3 ( 3.4c
12.3 ( 0.8c
ND
a Mean ( SEM. b ND, not detected. c P < 0.05.
from SD-NO (4c)-immunized mice secreted IL-2 and IL-4 alone
and only when stimulated with SD-NO (4c) or SMX-NO (4a)
(Table 2). Splenocytes from na¨ıve mice did not secrete detect-
able cytokine when stimulated with SMX (1a), SP (1b), SD
(1c), or their respective hydroxylamine or nitroso derivatives.
activated with the pyridine derivative SP (1b). SMX (1a)
derivatives were synthesized in high yields and purity using a
previously described three-step procedure (15) (Scheme 2). first,
coupling of 4-nitrobenzenesulfonyl chloride to 3-amino-5-
methylisoxazole, liberating nitro-SMX (2a); second, reduction
of nitro-SMX (2a) to SMX-NHOH (3a); and finally, oxidation
of SMX-NHOH (3a) to SMX-NO (4a). Essentially, the same
approach was used to synthesize SP (1b) and SD (1c) derivatives
with the exception that alternative conditions had to be sought
for the nitro reduction. Use of a palladium-carbon catalyst with
sodium phosphinite as the hydrogen donor in THF/water to
reduce nitro SP (2b) and SD (2c) gave a mixture of products in
low yield. By optimizing the reaction conditions (catalyst,
hydrogen donor, and solvent; Table 1), it was eventually possible
to synthesize both SP-NHOH (3b) and SD-NHOH (3c) in
acceptable yields and high purity (Table 2). The use of Raney
nickel as the catalyst and hydrazine as the hydrogen donor
prevented excessive reduction, and the reaction was easily
monitored over 1.5-3 h.
SMX-NO (4a), SP-NO (4b), and SD-NO (4c) (5 mg/kg, i.p.)
induced an immune response in mice at the same dose. Spleen
cells isolated from sensitized mice proliferated following in vitro
stimulation with the nitroso compound against which the animals
were immunized. The proliferative response of splenocytes was
concentration-dependent, a significant response being detected
at inducer concentrations between 5 and 100 µM (Figure 2).
Inhibition of splenocyte proliferation, at concentrations above
100 µM (results not shown), conformed with the previously
described in vitro cytotoxicity of sulfonamide hydroxylamines
and nitroso derivatives (56–58).
Discussion
Drug-specific T-cells have been isolated from the blood and
skin of allergic patients but not control subjects exposed to the
same drug without any adverse event (9, 46–48). The mecha-
nism by which drugs interact with immunological receptors and
stimulate T-cells remains an intriguing area of research since
the majority of drugs associated with a high incidence of
hypersensitivity are low molecular weight chemicals and
therefore are not classical antigens, that is, proteins requiring
processing prior to the presentation of derived, MHC-restricted
peptides to specific T-cell receptors.
Selective covalent modification of nucleophilic residues on
protein is thought to be a prerequisite for the activation of an
immune response directed against a drug or chemical allergen
(49, 50). Using SMX (1a) as a model compound, several
research groups have shown that the protein-reactive derivative
SMX-NO (4a) (25, 26, 51) is at least 3 orders of magnitude
more immunogenic than the parent compound (20, 25, 27, 28),
stimulates blood- and skin-derived T-cells from allergic patients
(9, 29, 30), and activates na¨ıve T-cells from healthy volunteers
(52). Furthermore, we have established a causal relationship
between oxidative metabolism of SMX (1a) and costimulatory
signaling in dendritic cells by showing that 1-aminobenzotria-
zole, a nonspecific P450 and myeloperoxidase inhibitor, attenu-
ates SMX (1a)-mediated upregulation of CD40 (14). The
presentation of a distinct antigenic determinant to T-cells from
allergic patients is similarly dependent on metabolism of SMX
(1a) (unpublished observation). The aim of the current inves-
tigation was to evaluate whether nitroso sulfonamide-specific
T-cells can be stimulated with structurally related nitroso
sulfonamides through T-cell receptor cross-reactivity.
Nitroso sulfonamides are known to degrade readily in solution
and are not detectable after 5 min in cell cultures (19). Their
disappearance from aqueous solutions has been associated with
the formation of an azoxy dimer (19), adducts of cysteine and
cysteinyl sulfoxy acids residues on protein that presumably
representimportantantigenicdeterminantsforT-cells(17,23,26,51),
and nitro and hydroxylamine intermediates, products of autoxi-
dation and reduction by glutathione and antioxidants, respec-
tively (15, 16, 19, 59). Thus, the exposure of splenocytes to
nitroso derivatives in the proliferation assay is probably low
for most of the incubation period and likely several orders of
magnitude lower than the starting concentration.
To accomplish this objective, hydroxylamine and nitroso
derivatives of SMX (1a), SP (1b), and SD (1c) were synthesized.
SP (1b) and SD (1c) are also antimicrobial, that is, arylamine,
benzenesulfonamides that have been associated with hypersen-
sitivity reactions (53) and are metabolized to a hydroxylamine
by human hepatic microsomes (54, 55). However, SP (1b) and
SD (1c) were selected particularly because they are for the most
part ineffective at stimulating SMX (1a)-specific T-cell clones
from allergic patients (32, 36–38). The pyrimidine derivative
SD (1c) stimulated a weak response in 10-15% of SMX (1a)-
specific T-cell clones, while only one out of 21 clones were
The hydroxylamine derivative of SMX (3a) is a well-
characterized human metabolite; approximately 2% of an oral
dose of SMX (1a) is excreted as the hydroxylamine in urine
(60, 61). However, hydroxylamines are susceptible to oxidation,
reduction, and self-conjugation reactions (15, 16, 27, 57). Thus,
when exploring the T-cell stimulatory capacity of hydroxylamine

190 Chem. Res. Toxicol., Vol. 23, No. 1, 2010
Castrejon et al.
metabolites, it is important to relate proliferation of splenocyte
cultures to analysis of compound stability. Each hydroxylamine
[SMX-NHOH (3a), SP-NHOH (3b), and SD-NHOH (3c)] was
found to degrade rapidly in the culture medium, and no more
than trace amounts were detected after 1 h. Despite this,
exposing spleen cells isolated from immunized mice to hy-
droxylamine stimulated a proliferative response (Figure 2). With
SMX-NHOH (3a), low levels of SMX-NO (4a) were detected
even after 4 h. For SP-NHOH (3b) and SD-NHOH (3c), nitroso
formation was confirmed indirectly through the characterization
of azoxy dimers, compounds originating from the conjugation
of hydroxylamine and nitroso species (19). Interestingly, SP-
NHOH (3b) appeared to be particularly unstable in culture
medium. Only a weak signal corresponding to SP-NHOH (3b)
was detected by mass spectrometry at the earliest time point
tested (0.1 h), which is in agreement with the previously
described short half-life of SP-NHOH (3b) (58). Furthermore,
unlike SMX-NHOH (3a) and SD-NHOH (3c), products of
reduction and dimerization were only detected at low levels
throughout the incubation period. These results indicate that the
formation of SP-NO (4b) protein adducts may be favored over
other reactions. To further investigate this possibility, specific
antidrug antibodies are being generated in ongoing experiments.
NO (4b)-immunized mice, but not those from SD-NO (4c)-
immunized mice, secreted GM-CSF, a cytokine involved in
phagocyte production and the differentiation of Langerhans cells
into mature dendritic cells.
In conclusion, our data show that splenocytes from mice
immunized against the synthetic nitroso derivative of a given
antimicrobial (arylamine) benzenesulfonamide can recognize in
vitro the nitroso derivative of another antimicrobial sulfonamide.
In addition, the inhibitory effect of glutathione on this in vitro
proliferation supports the potential role of drug-protein adduct
formation in sulfonamide-specific T cell proliferation. These data
support the hapten hypothesis of immune recognition of drugs
by immune cells and suggest that the response of T-cells from
allergic human donors against noncovalently associated parent
drugs may be a consequence of T-cell receptor cross-reactivity.
They provide additional justification for the contraindication of
structurally related antimicrobial sulfonamides in sulfonamide
allergic patients.
Acknowledgment. J.L.C. is a Ph.D. student funded by the
The Mexican Council for Science and Technology (CONACyT).
This work also received funding from the Wellcome Trust
(Project Grant No. 078598/Z/05/Z) as part of the Centre for
Drug Safety Science supported by the Medical Research Council
(Grant No. G0700654).
In agreement with previous studies focusing on the immu-
nogenicity of SMX (1a) metabolites (25, 28), neither SMX (1a),
SP (1b), nor SD (1c) stimulated splenocytes from nitroso-
sulfonamide-immunized mice. Splenocytes from SMX-NO
(4a)-, SP-NO (4b)-, and SD-NO (4c)-immunized mice prolifer-
ated in the presence of all three nitroso sulfonamides (Figure
3A-C). The concentrations of nitroso metabolite associated with
splenocyte proliferation were lower than those needed to deplete
intracellular glutathione and/or decrease cell viability (17, 19).
Glutathione, which is one of a panel of low molecular weight
thiols that prevent the spontaneous oxidation of sulfonamide
hydroxylamines (16), the reaction of nitroso sulfonamides with
protein (16, 17, 59), and the direct toxicity of sulfonamide
metabolites (58), effectively inhibited the splenocyte prolifera-
tion, indicating that protein adduct formation is needed to
stimulate specific T-cells. Collectively, these results imply that
although the drug structure per se contributes toward the
selectivity of the antigen-T-cell receptor binding interaction, the
protein (or the derived peptide) covalently modified at specific
cysteine residues provides the principal signal that determines
whether or not a T-cell responds.
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