Oximes: Metabolic activation and structure-allergenic activity relationships

Article abstract of DOI:10.1021/jm701092n

Metabolic activation of chemicals (prohaptens) in the skin can cause allergic contact dermatitis. We have explored structure-allergenic activity relationships for seven potential oxime prohaptens using the local lymph node assay and a GSH trapping screen

Full text of DOI:10.1021/jm701092n

J. Med. Chem. 2008, 51, 2541–2550
2541
Oximes: Metabolic Activation and Structure-Allergenic Activity Relationships
†
†
†
‡
,†
Moa Andresen Bergström, Sofia I Andersson, Kerstin Broo, Kristina Luthman, and Ann-Therese Karlberg*
Department of Chemistry, Dermatochemistry and Skin Allergy, and Department of Chemistry, Medicinal Chemistry, Göteborg UniVersity,
SE-412 96 Göteborg, Sweden
ReceiVed September 4, 2007
Metabolic activation of chemicals (prohaptens) in the skin can cause allergic contact dermatitis. We have
explored structure-allergenic activity relationships for seven potential oxime prohaptens using the local
lymph node assay and a GSH trapping screen with liver microsomes. The general structure-allergenic
activity relationships found were that an R,ꢀ-unsaturation is necessary for an oxime to be a prohapten and
that increased steric hindrance around this double bond leads to reduction in sensitizing capacity. We also
found that sensitizing oximes can be distinguished in vitro from nonsensitizers by monitoring of mono-
oxidized (+16 Da) GSH conjugates in the GSH trapping screen. However, care should be taken when
interpreting data from GSH trapping screens, as nonsensitizers may also form GSH conjugates via alternative
mechanisms. This investigation emphasizes the importance of considering cutaneous bioactivation in toxicity
assessment of chemicals used in contact with the skin.
Introduction
ment (OECD) as a stand-alone test method for determination
of sensitizing capacity. However, there is an urgent need for
alternative nonanimal-based methods due to European Union
legislation such as REACH (Registration, Evaluation, Autho-
risation and Restriction of Chemicals) and the seventh Amend-
Biotransformation of xenobiotic chemicals, such as drugs,
food additives, and cosmetics, can give rise to reactive and toxic
intermediates. This phenomenon is known as bioactivation or
1
metabolic activation, and the processes involved are often
oxidative, catalyzed by members of the cytochrome P450
9
,10
ment to the Cosmetics Directive.
One of the challenges in
a
2
the development of alternative assays is the identification and
potency assessment of prohaptens, which become transformed
(
P450 ) enzyme superfamily. The reactive metabolites formed
can interact with cells in numerous ways, including covalent
modification of cellular components (e.g., proteins and DNA),
which in turn may lead to tissue necrosis, carcinogenicity, and
1
1–14
to skin sensitizers via bioactivation in the skin.
By
definition, prohaptens may also be activated before skin contact
1
5–17
1
(e.g., by air oxidation
), though, in the following sections,
immunotoxicity. Bioactivation in the skin can lead to develop-
the term prohapten will refer only to allergens that are activated
via skin metabolism. Alternative methods for the prediction of
contact allergy that do not include metabolic activation will not
identify prohaptens as sensitizers. For the development and
evaluation of robust alternative methods, it is important that
the mechanisms involved in prohapten activation are investigated
and that structure–activity relationships (SARs) for prohaptens
are explored. The information obtained from SAR studies can
also be used in in silico predictive approaches and in the design
of non- or only weakly-sensitizing compounds with minimal
loss in desired activity (e.g., biological, technical, or cosmetic).
ment of allergic contact dermatitis (ACD), the clinical symp-
3
,4
tom of contact allergy. ACD is the most frequent manifesta-
5
tion of immunotoxicity in humans, and it is estimated that
1
0–15% of the adult European population is sensitized to one
6
,7
or more chemicals. ACD is classified as a T-lymphocyte-
mediated delayed-type or type-IV hypersensitivity reaction and
is caused by the binding of reactive chemicals (haptens) to
4
carrier macromolecules, such as proteins in the skin. To prevent
ACD in a sensitized individual, a life-long avoidance of the
allergenic compound is necessary. Thus, it is important to
identify contact allergens and to evaluate their sensitizing
potency to make proper risk assessments.
The development of assays for prediction of skin sensitizing
potential is a task of great importance. Currently, the only
reliable methods to determine allergenic activity are based on
Currently, the glutathione (GSH) trapping screen with hepatic
subcellular fractions is one of the most commonly used in vitro
techniques for investigation of potential bioactivation and
subsequent formation of reactive metabolites/intermediates. It
1
8,19
is used widely in drug discovery
studies of prohaptens.
and has also been used in
in vivo tests, one of which is the local lymph node assay
1
3,14
8
This assay is based on the ability of
(
LLNA). The LLNA has been evaluated and accepted by the
the GSH thiol group to efficiently react with and trap electro-
philic compounds formed primarily by phase I metabolic
conversions. The conjugates formed can be identified using LC-
MS techniques, where detection of the m/z 272 fragment
U.S. Food and Drug Administration (FDA) and is recommended
by the Organisation for Economic Co-operation and Develop-
*
To whom correspondence should be addressed. Telephone: +46-31-
(corresponding to deprotonated γ-glutamyl-dehydroalanyl-gly-
7
724726. Fax: +46-31-7723840. E-mail: karlberg@chem.gu.se.
†
Department of Chemistry, Dermatochemistry and Skin Allergy, Göte-
cine) in the negative ionization mode has been shown to be a
highly efficient tool to detect GSH conjugates of different
borg University.
‡
Department of Chemistry, Medicinal Chemistry, Göteborg University.
Abbreviations: ACD, allergic contact dermatitis; P450, cytochrome
20
a
structural classes. Additional information regarding the nature
P450; ESI, electrospray ionization; FDA, Food and Drug Administration;
FCAT, Freund’s complete adjuvant test; GSH, glutathione; HLM, human
liver microsome; LLNA, local lymph node assay; MLM, mouse liver
microsome; MW, microwave irradiation; OECD, organisation for economic
co-operation and development; SAR, structure-activity relationship; SIM,
selected ion monitoring.
and chemical structure of the reactive metabolites trapped can
then be gained from subsequent tandem MS analysis. Incuba-
tions of contact allergens with GSH alone has also been utilized
in assays for the correlation of the electrophilic character with
2
1–23
sensitizing capacity.
1
0.1021/jm701092n CCC: $40.75
 2008 American Chemical Society
Published on Web 03/28/2008

2
542 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8
Bergström et al.
a
Scheme 1. Proposed Mechanism of Cutaneous Metabolic
Activation of R-Carvoxime
Scheme 2. Synthesis of oximes 3–6 and 7
Chart 1. Structures of Oximes Studied for Structure-Allergenic
Activity Relationships
a
Reagents and conditions: NH2OH·HCl, NaOAc, MeOH/water 9:1,
MW, 150 °C, 5 min.
The present study concerns oximes as prohaptens. Oximes
have numerous industrial applications, for example, as anti-
skinning agents in paint, blocking agents in the polymer industry,
and chelators in the metals industry. Certain oximes have also
2
4
25
Figure 1. Dose-response curves for oximes 2 (O), 3 (+), 4 (9), 5
been shown to have anti-inflammatory, antiallergic, anti-
(
2), 6 (∆), 7 (×), and 8 (0) tested in the local lymph node assay
2
6
27
dotal, and antiepileptic properties. In addition, oximes are
common metabolic intermediates formed from benzyl- and
(
LLNA). The horizontal, dotted line marks a stimulation index (SI) of
3, the cutoff limit for a compound to be considered a sensitizer.
2
8
alkylamines via, for example, P450-mediated oxidation.
However, despite the wide use of oximes, their contact allergenic
activity has been poorly studied. We have previously shown
that the R,ꢀ-unsaturated R-carvoxime (1, Scheme 1), is a
phobicity was considered important to, as accurately as possible,
allow comparison of allergenic activity in relation to structure
and to avoid variations in skin penetration capacities. Oximes
2
9
prohapten of strong sensitizing potency. Studies performed
2
–6 contain the same cyclic structure but have a varying
in guinea pigs revealed that a ꢀ-methyl substituted analogue of
substitution pattern at the R- and ꢀ-positions, and 2 is a saturated
analogue of 3. To study the influence of conformational
flexibility on the sensitizing capacity, compound 7, an acyclic
analogue of 3, was included in the series. To investigate if an
oxime conjugated with an aromatic ring could induce skin
sensitization, benzaldoxime (8) was also included. R,ꢀ-Unsatur-
ated oximes have previously been shown to be poor electro-
1
had retained allergenic activity, whereas a saturated analogue
2
9
of 1 was inactive. In subsequent mechanistic studies, it was
found that 1 is metabolically activated by epoxidation of the
R,ꢀ-unsaturation (Scheme 1) and that the R,ꢀ-epoxy oxime
metabolites formed were sensitizers of extreme potency and
highly chemically reactive toward a nucleophilic model pep-
14
2
9
tide. The reason for the high reactivity and sensitizing capacity
of the R,ꢀ-epoxy oxime metabolites was proposed to be due to
the formation of allylic nitroso intermediates by tautomerisation
philes. Therefore, any sensitizing effect observed is expected
to be caused by conversion of 2–8 into reactive intermediates
in the skin.
1
4
(
Scheme 1). In the present study, we have explored SARs for
Synthesis. Oximes 3–7 were synthesized using microwave-
assisted heating in moderate to good yields (51–71%) from their
corresponding ketones and hydroxylamine hydrochloride (Scheme
oximes and investigated the possibility of correlating in vitro
studies of metabolic activation of oximes with their sensitizing
potency. For this reason, a series of seven oximes (2–8, Chart
2
). The E-isomer was preferentially formed in all cases and
1
) were screened for sensitizing capacity using the LLNA and
exclusively for 4 and 6, as this minimizes steric interactions
between the hydroxyl group of the oxime and the proximal
hydrogen or methyl substituent in the R-position. The microwave-
assisted heating allowed the preparation of 3–7 with reduced
reactive metabolite-trapping studies were carried out using the
GSH trapping screen together with human and mouse liver
microsomes (HLMs or MLMs).
3
0–33
reaction times compared to previously reported procedures.
Results and Discussion
34
Skin Sensitizing Potency. Currently, the murine LLNA is
the method of choice for identification and potency assessment
of contact allergens and was used to determine the sensitizing
potency of oximes 2–8 (Figure 1, Table S1, Supporting
Information). Oxime 2 was inactive at the concentrations tested
Design of Oximes Used in SAR Investigation. To investi-
gate structure-allergenic activity relationships for oximes, a
series of seven compounds (2–8) was designed. Similarity within
the series in chemical structure, molecular weight, and hydro-

Metabolic ActiVation of Oximes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8 2543
(
0.10–20% w/v) and was classified as a weak sensitizer/
Scheme 3. Formation of Glutathione (GSH) Conjugates from the
Isomers of the R,ꢀ-Epoxy Oxime 9
a
nonsensitizing compound. This result is in agreement with a
previous study of a saturated analogue of 1, which was shown
29
to be a nonsensitizer in guinea pigs. The nonsubstituted oxime
3
was found to be a strong sensitizer (EC3: 0.055 M, 0.61%
w/v), thus verifying that an R,ꢀ-unsaturation is necessary for
an oxime to be a sensitizer. The R- and ꢀ-methyl substituted
oximes 4 and 5, respectively, were weaker sensitizers compared
to 3 (4: 0.16 M, 2.0% w/v; 5: 0.18 M, 2.2% w/v) and were
classified as moderate sensitizers. In comparison, the R-methyl
substituted oxime 1 has previously been tested in the LLNA
and was found to be a strong sensitizer (36 mM, 0.60%
2
9
w/v). The difference observed in sensitizing capacity between 1
and 4 might be due to a difference in skin penetration, as the only
structural disparity between these compounds is the pendant
isopropenyl group in the 5-position of 1. The R,ꢀ-dimethyl
substituted oxime 6 was found to be a weak sensitizer/nonsensi-
tizing compound as it was inactive at the concentrations tested
(0.10–20% w/v). Previous sensitization experiments in guinea pigs
showed that a similar R,ꢀ-dimethyl substituted R,ꢀ-unsaturated
oxime, the ꢀ-methyl analogue of 1 is a sensitizer, although the
number of responding animals were lower compared to a corre-
29
sponding experiment with 1. The differences between the LLNA
and the FCAT test procedures could account for this discrepancy.
35
In the FCAT, the test compound is administered intradermally
together with an immunostimulatory adjuvant (which increases the
8
sensitivity at the point of sensitization), whereas, in the LLNA,
the test compound is applied topically. The LLNA results for 3–6
show that increased alkyl substitution around the double bond of
R,ꢀ-unsaturated oximes leads to a reduction in their sensitizing
capacity. This phenomenon is probably due to the methyl groups
inducing steric hindrance at the structural moiety that is important
for the metabolic activation, thus partly or completely preventing
the metabolic activation reaction.
a
Enantiomeric pairs of GSH conjugates are indicated as a, b, c, and d.
The conformationally more flexible and nonsubstituted R,ꢀ-
unsaturated oxime 7 was notably less sensitizing (EC3: 0.79
M, 9.0% w/v; classification: moderate sensitizer) compared to
its cyclic analogue 3. The importance of considering confor-
mational flexibility together with metabolic activation has been
a cell proliferation not involving the formation of memory
T-cells. This can result in difficulties in distinguishing between
weak sensitizers and nonsensitizing compounds.
38
Preparation of GSH Conjugates of r,ꢀ-Epoxy Oxime 9.
Compound 9 is the proposed R,ꢀ-epoxy oxime metabolite of 3
36,37
acknowledged for mutagenic chemicals,
however, it has not
(
Scheme 3). GSH conjugates of 9 were prepared to aid the
yet been suggested for contact allergenic prohaptens. However,
a similar reactivity pattern has previously been observed in our
laboratory when investigating SARs for potentially prohaptenic
development of an LC-MS method for the analysis of incubation
mixtures for GSH conjugates formed from putative R,ꢀ-epoxy
oxime metabolites of 2–7. Using LC-MS, four major products
were found in the reaction between 9 and GSH (Figure 2A).
According to mass accuracy analysis and tandem MS, each of
these products were found to be GSH conjugates of 9, as they
exhibited a molecular ion at m/z 435.1550 ((10 ppm) and a
fragmentation pattern that support a conjugate formed by
addition of GSH to 9 (Figure 3, Scheme 4). A full interpretation
of the MS fragmentation pattern is provided in the Supporting
Information (Scheme S1). The presence of ions at m/z 306 and
1
3
alkenes. We found that the monoterpene ꢀ-phellandrene is a
moderate sensitizer in the LLNA (EC3: 0.41 M), whereas its
acyclic 3,4-seco analogue was found to be inactive (EC3 > 1.8
1
3
M). The chemical structures of the compounds tested were,
in both this and the present study, carefully chosen to avoid
variations in skin penetration capacities. Hence, the differences
observed in sensitizing capacity for cyclic versus acyclic
prohaptens could be due to an inherent dissimilarity in the
metabolic activation processes of such compounds. However,
the hypothesis that noncyclic prohaptens are less sensitizing
compared to their cyclic analogues needs further verification
before general SARs can be formulated.
3
60 corresponding to loss of glycine and pyroglutamate from
the GSH moiety, respectively (Scheme 4), showed that 9 had
been trapped by the thiol function of GSH. We have previously
shown that the conjugation reaction between GSH and the
putative R,ꢀ-epoxy oxime metabolites formed from 1 proceeds
exclusively via addition of the GSH thiol function to the
Benzaldoxime (8) failed to induce an EC3 value at the
concentrations tested (0.10–20% w/v), thus showing that oximes
conjugated with an unsubstituted phenyl ring are weak or
nonsensitizing compounds. It was not considered necessary to
retest 2, 6, and 8 in higher concentrations, as the chosen
concentration interval allows a distinction between strong,
moderate, and weak contact allergens. In addition, false positive
results can be obtained for compounds (e.g., irritants) that
typically when tested in high concentrations are able to induce
1
4
R-position of the R,ꢀ-epoxy oxime. This addition can take
place at both faces of the proposed nitroso intermediate formed
from the R,ꢀ-epoxy oxime (Scheme 1) and may also lead to
14
inversion of the oxime stereochemistry. When these reactivity
patterns are applied to the reaction between GSH and 9 (which
is a mixture of four diastereomers), a total of eight distinct
conjugates may be formed (Scheme 3). As these eight conjugates

2
544 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8
Bergström et al.
oxime 9 in the microsomal incubations, the incubation mixtures
were spiked with synthetically prepared conjugates, and the mass
spectra of the metabolically formed GSH conjugates were
compared with those of the synthetically prepared. Both the
spiking and the mass spectra could confirm that 3 is metaboli-
cally converted to 9 by both HLMs and MLMs (data not shown).
The mass spectra of all four compounds formed in the incubation
mixtures contained the same fragments (e.g., m/z 417, 288, and
2
55; Scheme 4) as were found in the mass spectra of the
synthetically formed GSH conjugates. As 3 was shown to be a
strong sensitizer in the LLNA and R,ꢀ-epoxy oximes have
1
4
previously been shown to be sensitizers of extreme potency,
these results indicate that the allergenic activity observed for 3
is a result of its metabolic conversion to 9 in the skin.
Substantial amounts of ions matching the +16 Da criterion
were also found in the MLM and HLM incubations containing
the R,ꢀ-unsaturated oximes 4, 5, and 7 (Table 1, Figure 4C,D,F).
The MLM incubation mixtures of 4, 5, and 7 were therefore
reanalyzed using tandem MS for fragmentation pattern com-
parison with the +16 Da conjugates formed from 3. All +16
Da conjugates exclusively formed in the NADPH-containing
incubations of 4, 5 and 7 were found to exhibit fragments of
the same origin as found for oxime 3, thus confirming that the
compounds detected were GSH-trapped monooxidized metabo-
lites, most likely R,ꢀ-epoxy oximes. In addition, compounds 4,
Figure 2. Chromatograms of glutathione (GSH) conjugates formed
in reaction mixtures of R,ꢀ-epoxy oxime 9 and GSH (A) and in mouse
(
B) and human (C) liver microsomal incubations of oxime 3. Analysis
was performed in the negative ionization mode and the m/z 433 ion
was monitored.
consist of four enantiomeric pairs, only four peaks will appear
using standard nonchiral chromatographic techniques (Figure
5
, and 7 were all moderate sensitizers in the LLNA. As oximes
, 5, and 7 are structurally related to oximes 1 and 3, it is likely
2
A). Therefore, GSH conjugates formed from 9 most likely
4
correspond to the GSH conjugates shown in Scheme 3. The
involvement of a nitroso intermediate in the reaction between
that these oximes exert their allergenic activity via metabolic
activation to R,ꢀ-epoxy oximes. Oxime 6 was inactive in the
LLNA, which was proposed to be due to a partial or complete
prevention of metabolic epoxidation of the R,ꢀ-unsaturated
double bond. Hence no, or only small amounts of, +16 Da GSH
conjugates were expected to be formed in the liver microsomal
incubations of 6. Indeed, only small amounts of +16 Da
conjugates could be detected in the incubations with 6 (Figure
9
and GSH was indicated by the change in E/Z ratio of the
1
oxime moiety, from 4:1 for 9 (as determined by H NMR, Figure
S1, Supporting Information) to approximately 1:1 (Figure 2A,
LC-MS data).
GSH Trapping Experiments. Oximes 2–8 were incubated
with MLMs or HLMs in the presence of GSH to identify
reactive metabolites and to investigate the possibility of predict-
ing skin sensitization capacity of oximes using the GSH trapping
screen. Even though the metabolic activity of the skin differs
from that of the liver, the same overall metabolic processes are
4
E), thus confirming this hypothesis. Compounds 2 and 8 were
also inactive in the LLNA. As 2 is a saturated oxime, no +16
Da GSH conjugates were expected to be identified in the
incubations, unless 2 was activated via an alternative pathway.
It is possible that oxime 8 could form a monooxidized reactive
metabolite via epoxidation of the aromatic ring, however this
was not expected to occur as 8 was inactive in the LLNA. No
3
9
considered to occur. The molecular weights of the GSH
conjugates formed in the MLM and HLM incubations of
oximes 2–8 are shown in Table 1. Chromatograms of the
conjugates formed in MLM incubations are shown in Figure
+
16 Da GSH conjugates were identified in the incubations with
and 8 (Figure 4A and G), thus confirming that these
4
. The results from the MLM and HLM incubations are
2
qualitatively similar; however, greater amounts of metabolites
were overall formed from the MLMs as compared to the
HLMs (data not shown).
compounds cannot be bioactivated to form reactive +16
metabolites.
1
. Detection of GSH Conjugates Formed from r,ꢀ-Epoxy
2. Detection of Additional GSH Conjugates Formed in
Liver Microsomal Incubations. Despite the fact that a good
correlation between sensitizing oximes (3, 4, 5, and 7) and the
presence of +16 Da conjugates in the GSH trapping screen had
been observed, we found it of interest to study the formation
of additional conjugates. Monitoring of the m/z 272 ion in the
negative ion mode has previously been demonstrated as a useful
tool for detection of unknown GSH conjugates of different
structural classes. Even though the +16 Da conjugates were
the predominant GSH conjugates found in the MLM and HLM
incubations of oximes 2–8, various additional GSH conjugates
could be identified when using SIM detection of the m/z 272
ion (Figure 4H-N). The molecular weights of the GSH
conjugates identified were obtained from the total ion chro-
matogram run in scan mode and are indicated in Table 1 and
Figure 4H-N. Conjugates corresponding to an addition of GSH
to the corresponding ketone (-15 Da) of the oximes were
identified in the MLM incubations of 3–7 and HLM incubations
Oxime Metabolites. Analogously to the reaction between GSH
and 9, it was assumed that conjugates formed by trapping of
R,ꢀ-epoxy oxime metabolites with GSH would have a molecular
weight of +16 Da (one oxygen atom) in addition to the
combined masses of the parent oxime and GSH (Scheme 5).
P450-mediated hydroxylation, which also results in monooxi-
dized metabolites was not considered to be an important
bioactivation pathway for the compounds studied. As aliphatic
alcohols are nonelectrophilic, the C-hydroxylated metabolites
are likely equally poorly reactive toward GSH as the parent
2
0
4
0
oxime itself. In addition, aliphatic alcohols are nonallergenic.
Four peaks containing ions matching this criterion were identi-
fied in the MLM and HLM incubations of 3 (Figure 2B,C).
These peaks were all absent in the NADPH-free control. The
retention times of the compounds formed matched the retention
times of the four GSH conjugates formed in the reaction between
9
and GSH. To examine the presence of GSH-trapped R,ꢀ-epoxy

Metabolic ActiVation of Oximes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8 2545
Figure 3. Tandem MS spectra of glutathione conjugates formed from R,ꢀ-epoxy oxime 9: A, retention time 9.2 min; B, 9.9 min; C, 10.3 min; and
D, 10.7 min.
of 3–5 and 7. These conjugates were also observed in the
NADPH-free controls. Oxime hydrolysis to the corresponding
ketone analogues may occur both enzymatically and nonenzy-
formed after dehydration (loss of water, 18 Da) of a +16 Da
conjugate. This would explain the appearance of -2 Da
conjugates in incubations with oximes 3–7, but not the observa-
tion of -2 Da conjugates in incubations with oximes 2 and 8
as these oximes cannot be metabolically converted to R,ꢀ-epoxy
oximes. However, the formation of -2 Da conjugates from
oximes does not seem important for the development of contact
allergy to oximes, since 2, 6, and 8 were inactive in the LLNA.
4
1
matically and has previously been observed for 2. R,ꢀ-
Unsaturated ketones, which can be formed from 3–7, are soft
electrophiles and react rapidly and efficiently with soft nucleo-
philes, such as GSH. Hence, the absence of –15 Da conjugates
in the incubations with 2 and 8 may be due to the corresponding
ketones of these oximes not being sufficiently reactive toward
GSH. R,ꢀ-Unsaturated ketones, but not nonconjugated ketones,
3
. Inherent Reactivity of Oximes 2–8 Toward GSH.
29,42
Conjugates corresponding to a direct addition of GSH to the
oxime ((0 Da) were identified in the incubations of oximes 3,
are also potential skin sensitizers.
Carvone, the correspond-
ing ketone of 1, is a considerably weaker sensitizer (EC3: 0.86
M) than both 1 (EC3: 0.036 M) and the R,ꢀ-epoxy oxime
5
, and 7. Similarly to the -15 Da and some of the +16 Da
14,29
GSH conjugates, these conjugates were also present in the
NADPH-free controls (marked with an asterisk in Figure 4).
To investigate their origin, reactions of oximes 2–8 and GSH
in buffer without liver microsomes were carried out. In general,
only minor amounts of GSH conjugates were identified. Oximes
are poor electrophiles, but do react with good nucleophiles, such
metabolites formed from 1 (EC3: 0.00088–0.0011 M).
Thus,
it is likely that the main contribution to the activation of R,ꢀ-
unsaturated oximes occurs via the epoxide rather than the ketone
pathway. The impact of the ketone metabolites on the sensitizing
potency of oximes is most likely minimal as ketones are usually
not sufficiently potent. However, previous studies in guinea pigs
sensitized to 1 showed that about 40% (6/15 animals) responded
when tested with carvone, indicating that the alternative route
2
9
as the thiol function of GSH. Conjugates resulting from a
direct reaction between the oxime and GSH were identified in
incubations with 3, 4, 5, and 7. GSH conjugates formed from
the corresponding ketone of the oximes were found in incuba-
tions with 3 and 7. Trace amounts of conjugates with a mass
corresponding to a mass gain of one oxygen atom (+16 Da) in
2
9
exists.
Conjugates corresponding to a loss of 2 Da in molecular
weight relative to the weight of the parent oximes and GSH
were identified in all incubations. Such conjugates may be

2
546 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8
Bergström et al.
Scheme 4. Proposed Structures for Major Tandem MS Fragments of Glutathione Conjugates of R,ꢀ-Epoxyoxime 9
Table 1. LC-MS Analyses of the Liver Microsomal Incubations of
Oximes 2–8 with Glutathione (GSH); Molecular Weights (Da) of
Identified GSH Conjugates
above, in comparison to 1, the R,ꢀ-unsaturated ketone carvone
is considerably more reactive toward sulfur nucleophiles, but
29,42
is a >20-fold weaker sensitizer in the LLNA compared to 1.
a
a
experiments
MLM
experiments
MLM
Furthermore, the formation of +16 Da conjugates via S-
oxidation of (0 Da conjugates does not appear to be important
in the assessment of skin sensitizing oximes using the GSH
trapping screen, as all the sensitizing oximes studied (3, 4, 5,
and 7) afforded significant amounts of unique +16 Da conju-
gates in the NADPH-containing incubations only.
substrate
HLM
substrate
HLM
2
434 (+14) 434 (+14)
18 (-2) 418 (-2)
6
462 (+16) 462 (+16)
444 (-2) 444 (-2)
32 (-15)
4
4
3
434 (+16) 434 (+16)
4
4
4
18 ((0)
418 ((0)
416 (-2)
7
8
436 (+16) 436 (+16)
16 (-2)
420 ((0)
418 (-2)
420 ((0)
418 (-2)
In summary, we have explored structure-allergenic activity
relationships for oximes and investigated the possibility of
correlating in vitro studies of metabolic activation of oximes
with their sensitizing potency. The general structure-allergenic
activity relationships were that an R,ꢀ-unsaturation is necessary
for an oxime to be a prohapten and that increased steric
hindrance around this double bond leads to reduction in
sensitizing capacity. In addition, we have shown that an aromatic
aldoxime was a weak/nonsensitizing compound and that an
acyclic R,ꢀ-unsaturated oxime was significantly less sensitizing
compared to its cyclic analogue. We recommend that these
SARs are applied in the design of novel commercially used
chemicals and that they should be incorporated in in silico
predictive databases. We also found that sensitizing oximes can
be distinguished in vitro from nonsensitizers by monitoring of
the formation of monooxidized (+16 Da) GSH conjugates in
the GSH trapping screen. However, care should be taken when
interpreting data from the GSH trapping screen as nonsensitizers
may also form significant amounts of GSH conjugates via
alternative mechanisms. Hence, this investigation highlights the
significance of a solid mechanistic understanding of prohapten
activation in the development of novel nonanimal-based assays
for prediction of contact allergenic activity.
03 (-15) 403 (-15)
4
05 (-15) 405 (-15)
4
5
448 (+16) 448 (+16)
4
30 (-2)
430 (-2)
442 (+14) 426 (-2)
426 (-2)
4
17 (-15) 417 (-15)
448 (+16) 448 (+16)
4
4
4
32 ((0)
30 (-2)
432 ((0)
430 (-2)
17 (-15) 417 (-15)
a
The numbers given within parentheses denote the change in molecular
weight relative to the weight of the parent oximes and GSH. Conjugates
formed in the reaction between an R,ꢀ-epoxy oxime metabolite and GSH
are assumed to have gained 16 Da. Conjugates formed from a ketone
metabolite (formed via hydrolysis of the corresponding oxime) are assumed
to have lost 15 Da. MLM ) mouse liver microsomes, HLM ) human liver
microsomes.
addition to the combined masses of the oxime and GSH were
detected in incubations with 4 and 7. These conjugates probably
result from autoxidation of the sulfide moiety to form the
corresponding sulfoxide of the adducts formed in a direct
reaction between the oxime and GSH ((0 Da), as previously
29
has been shown for 1. Taken together, these experiments show
that R,ꢀ-unsaturated oximes have an inherent capacity to form
conjugates with GSH without metabolic activation. However,
the importance of the inherent electrophilicity of R,ꢀ-unsaturated
oximes for their allergenic activity is likely to be minimal as
the chemical reactivity of these oximes toward biological
nucleophiles is very low in comparison with that of contact
allergens of equal or lower sensitizing capacity. As mentioned
Experimental Section
Caution: Skin contact with 3–5, 7, and 9 must be aVoided. These
compounds are skin-sensitizers (3–5 and 7) or probable skin-
sensitizers (9) and must therefore be handled with care.

Metabolic ActiVation of Oximes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8 2547
Figure 4. Chromatograms of glutathione (GSH) conjugates formed in mouse liver microsomal incubations of oximes 2 (A and H), 3 (B and I), 4
C and J), 5 (D and K), 6 (E and L), 7 (F and M), and 8 (G and N). Analysis was performed in the negative ionization mode, and the following
(
ions were monitored: m/z 435.1 (A and F), 433.1 (B), 447.1 (C and D), 461.2 (E), 443.1 (G), and 272 (H-N). In chromatograms H-N, the mass
of identified molecular ions corresponding to the GSH conjugates detected are indicated. Peaks marked with an asterisk (*) were detected also in
the NADPH-free controls. Peaks marked with a hash (#) correspond to an impurity in the GSH buffer.
Scheme 5. Formation of +16 Da Glutathione (GSH) Conjugates
aluminum sheets (Silica gel 60 F254) developed using anisaldehyde
dip (2.1 mL of acetic acid, 5.1 mL of anisaldehyde, and 7 mL of
a
via Metabolic Conversion of R,ꢀ-Unsaturated Oximes
H
2
SO
4
in 186 mL of ethanol) or permanganate dip (1.0 g KMnO
4
,
6
.7 g K
2
CO , and 1.7 mL of 5% aqueous NaOH in 100 mL of
3
water) followed by heating. Column chromatography was performed
on wet packed silica (Silica gel 60, 230–400 mesh). The purity of
synthesized and purchased test compounds was determined to be
g98% (GC/MS) before testing. Pooled HLMs from male and
female donors, female CD-1 MLMs, and an NADPH-regenerating
system were purchased from BD Biosciences (Woburn, MA).
Instrumentation. The microwave-assisted reactions were carried
out using a Biotage Initiator instrument. Purity analyses were
performed on a Hewlett-Packard gas chromatograph (model 6890)
connected to a mass spectrometer (Hewlett-Packard model 5973).
The GC was equipped with a HP-1MS fused silica capillary column
(30 m × 0.25 mm, 0.25 µm, Agilent Technologies, Palo Alto, CA).
Helium was used as carrier gas and the flow rate was 1.2 mL/min.
The temperature program started at 35 °C for 1 min, increased by
a
R1, R2, and R3 ) alkyl or H.
Chemicals and Biochemicals. Unless otherwise indicated,
reagents were obtained from commercial suppliers and used without
further purification. Cyclohexanone oxime (2) and syn-benzal-
doxime (8) were purchased from Fluka. 2-Methylcyclohex-2-enone,
1
0 °C/min, and ended at 250 °C for 5 min. For mass spectral
analysis, the mass spectrometer was used in the scan mode detecting
ions with m/z values ranging from 50 to 500.
2
,3-dimethylcyclohex-2-enone and 2,3-epoxycyclohexanone oxime
LC-MS analyses were performed using atmospheric pressure
electrospray ionization (ESI) on a Hewlett-Packard 1100 HPLC-
MS. The system included a vacuum degasser, a gradient pump, an
4
3,44
(
9) were synthesized as previously described.
were monitored using thin layer chromatography on silica-plated
The reactions

2
548 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8
Bergström et al.
autoinjector, a column thermostat, a diode array detector, and a
single quadropole mass spectrometer. The electrospray interface
was used with the following spray chamber settings: nebulizer
pressure, 40 psig; capillary voltage, 3.5 kV; drying gas temperature,
product was purified by column chromatography on silica gel (25%
ethyl acetate in hexanes) affording a 3:1 E/Z mixture of 3 as a
colorless solid (1.27 g, 71%). NMR spectral data were consistent
3
0
with literature data for 3.
3
50 °C; and drying gas flow rate, 10 L/min.
(E)-2-Methylcyclohex-2-enone Oxime (4). 2-Methylcyclohex-
2-enone (1.76 g, 16 mmol) was reacted as described in the general
procedure. The crude product was purified by recrystallization from
hexanes affording 4 as colorless needles (1.01 g, 51%). NMR
Determination of accurate mass was performed using ESI on a
Micromass Q-Tof 2 instrument (a hybrid quadropole-time-of-flight
mass spectrometer). The mass spectrometer was connected to a
Hewlett-Packard 1100 HPLC system, which included a vacuum
degasser, a gradient pump, a column thermostat, and a HTS PAL
autoinjector. The following mass spectrometric source conditions
were used: capillary voltage, 3.1 kV; cone voltage, 35 V; MCP,
3
1
spectral data were consistent with literature data for 4.
3-Methylcyclohex-2-enone Oxime (5). 3-Methylcyclohex-2-
enone (1.76 g, 16 mmol) was reacted as described in the general
procedure. The crude product was purified by column chromatog-
raphy on silica gel (40% ethyl acetate in hexanes) affording a 6:4
E/Z mixture of 5 as a colorless solid (1.75 g, 70%). NMR spectral
2
350 V; TOF, 9.10 kV; source temperature, 120 °C; and desolvation
temperature, 320 °C. Leucine enkephalin at m/z 556.2771 was used
as lock mass and the instrument was calibrated with sodium formate.
A measured mass with a e10 ppm deviation from the theoretical
mass was considered accurate.
3
2
data were consistent with literature data for 5.
(E)-2,3-Dimethylcyclohex-2-enone Oxime (6). 2,3-Dimethyl-
cyclohex-2-enone (1.50 g, 12 mmol) was reacted as described in
the general procedure. The crude product was purified by recrys-
tallization from hexanes affording 6 as a pale yellow solid (0.86 g,
51%). NMR spectral data were consistent with literature data
LC-MS Methods for Analysis of GSH Conjugates. The
microsomal incubations, the reactivity experiments, and the mixture
of 9 and GSH were analyzed for GSH conjugates in the negative
ionization mode. For each sample, 20 µL (reactivity experiments),
3
1
for 6.
1
0 µL (liver microsomal incubations), or 0.5 µL (GSH conjugates
Hex-4-en-3-one Oxime (7). Hex-4-en-3-one (1.50 g, 15.3 mmol)
was reacted as described in the general procedure. The crude product
was purified by column chromatography on silica gel (15% ethyl
acetate in hexanes), affording a 11:9 E/Z mixture of 7 as a colorless
oil (1.01 g, 58%). NMR spectral data were consistent with literature
of 9) were injected onto a HyPURITY 3 µm C18 150 mm × 3.0
mm column (Thermo Electron Corp., Bellafonte, PA), and the
analytes were eluted with a gradient flow at 0.4 mL/min. The flow
was diverted to waste for the first 5 min of each run. Mobile phase
A consisted of 0.005% pentafluoropropanoic acid, 0.1% formic acid,
and 3% acetonitrile in water and mobile phase B of 0.005%
pentafluoropropanoic acid, 0.1% formic acid, and 50% acetonitrile
in water. A linear gradient of 0–50% B in A in 20 min was
followed by a second gradient of 50–100% B in 1 min and ended
with 4 min of isocratic elution at 100% B. The column was
equilibrated with 100% A for 10 min between each run. The
detector was used in the scan mode (% cycle time 40, fragmentor
voltage 70 eV), detecting ions at m/z 100–500, and in the SIM
mode, detecting the deprotonated γ-glutamyl-dehydroalanyl-
glycine ion at m/z 272 (% cycle time, 30; fragmentor voltage,
3
3
data for 7.
Incubation of Oximes 2–8 with GSH and Liver Micro-
somes. Each microsomal incubation contained HLMs or MLMs
(1 mg/mL of protein), substrate (200 µM), GSH (5.0 mM), 100
mM potassium phosphate buffer (pH 7.4), and an NADPH-
+
regenerating system (1.3 mM NADP , 3.3 mM glucose-6-
phosphate, 0.4 U/mL glucose-6-phosphate dehydrogenase, and 3.3
mM magnesium chloride) in a total volume of 250 µL. The
incubations were performed in duplicate and initialized by addition
of the NADPH-regenerating system after 3 min of preincubation
at 37 °C. Control experiments were run in the absence of the
NADPH-regenerating system. After 2 h, the incubation mixtures
were terminated by addition of acetonitrile (250 µL) and centrifuged
at 3000 rpm and 4 °C for 10 min. The supernatants (450 µL) were
concentrated to dryness with a stream of nitrogen. The samples
were reconstituted with 1% acetonitrile in water (100 µL), mixed
for 10 min at 300 rpm and analyzed using the LC-MS analysis
method described above. Incubations run with MLMs, which
contained ions with a mass corresponding to one or more +16 Da
GSH conjugates, were reanalyzed for accurate mass.
1
20 eV) and the deprotonated molecular ion of a GSH-trapped R,ꢀ-
epoxy oxime metabolite (% cycle time 30, fragmentor voltage 70
-
eV, m/z of [MGSH conjugate of epoxy oxime - H] of oximes 2–8: 435.1
(
2 and 7), 433.1 (3), 447.1 (4 and 5), 461.2 (6), and 443.1 (8)).
Accurate mass analysis in the positive ionization mode was
performed on the mixture of 9 and GSH (ca. 5 µM) and selected
samples from the liver microsomal incubations (diluted 1:100). For
each sample, 20 µL were injected onto an ACE 3 µm C18 100
mm × 2.1 mm column (Advanced Chromatograpy Technologies,
Aberdeen, U.K.). Mobile phase A consisted of 0.1% formic acid
in water and mobile phase B of 100% acetonitrile. Each run was
Reaction of r,ꢀ-Epoxy Oxime 9 with GSH. Compound 9 (2.5
mg, 0.02 mmol) and GSH (6 mg, 0.02 mM) were added to
potassium phosphate buffer (1 mL, 100 mM, pH 7.4). The mixture
was shaken for 5 min and was subsequently diluted with buffer (3
equiv) and analyzed using LC-MS as described above. A chro-
matogram of the conjugates formed is shown in Figure 2A. The
exact masses of the conjugates formed (singly protonated molecular
2
5 min using a gradient flow of 0.3 mL/min. The analytes were
initially eluted isocratically with 5% B in A for 5 min, followed
by a gradient of 5–30% B in 15 min and a second gradient of
3
0–95% B in 1 min. After this, the flow was held at 95% B in A
for 2 min, whereafter it returned to 5% B in 0.1 min, where it stayed
for 1.9 min. The flow was split post-column to lead 0.12 mL/min
into the mass detector. MS spectra were acquired in the positive
ionization mode using a scan range between m/z 100–700 Da or
using the following tandem MS settings: set mass, m/z 435; scan
range, m/z 100–600; collision energy, 15 eV.
General Procedure for Preparation of Oximes Using Mi-
crowave-Assisted Heating. To a solution of ketone (ca. 1 M) in
methanol/water 9:1 in a 10–20 mL microwave reaction vial were
added hydroxylamine hydrochloride (1.1 equiv) and sodium acetate
+
ions, MH ) as determined by Q-Tof analysis were 435.1568 (ret.
time 9.2 min), 435.1565 (9.9 min), 435.1561 (10.3 min), and
435.1574 (10.7 min), which corresponds to mass deviations of <10
ppm from the calculated mass (435.1550 Da). To confirm the
presence of these conjugates in liver microsomal incubations of 3,
the major GSH conjugates formed from 9 were separated and
fractions containing the individual conjugates were collected. The
same LC method as described above was used, but with the
following modification: a linear gradient of 0–25% eluent B in A
in 20 min was followed by a second gradient of 25–100% of B in
A for 1 min and ended with 4 min of isocratic elution at 100% B.
For each run, 10 µL of a 20 mM solution of GSH conjugates of 9
was injected. The combined fractions were lyophilized to dryness.
Reaction of Oximes 2–8 with GSH. A mixture of oxime (200
µM) and GSH (5 mM) in potassium phosphate buffer (100 mM,
pH 7.4) was gently shaken in a water bath at 37 °C for 2 h. These
mixtures were analyzed using the LC-MS analysis method described
above.
(
1.2 equiv). The vial was sealed and the mixture was heated in a
microwave cavity at 150 °C for 5 min and was then allowed to
cool to room temperature. The mixture was filtered, diluted with
water (80 mL), and extracted with chloroform (3 × 50 mL). The
organic phases were washed with saturated aqueous sodium
bicarbonate (2 × 50 mL), water (50 mL), and brine (50 mL), dried
over magnesium sulfate, and concentrated under reduced pressure.
Cyclohex-2-enone Oxime (3). Cyclohex-2-enone (1.54 g, 16
mmol) was reacted as described in the general procedure. The crude

Metabolic ActiVation of Oximes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8 2549
Sensitization Experiments using the Local Lymph Node
76/769/EEC and Commission Directives 91/155/EEC, 93/67/EEC, 93/
34
1
05/EC and 2000/21/EC. Off. J. Eur. Union 2006, L 396, 1-849.
Assay (LLNA). All animal procedures were approved by the local
ethics committee. The procedures were performed according to the
OECD guidelines (Guideline No. 429; Skin Sensitization: Local
Lymph Node Assay). Female CBA/Ca mice, 8 weeks of age, were
purchased from Harlan (Horst, Netherlands) or Scanbur (Sollentuna,
Sweden). The assay was performed using the test concentrations
shown in Table 1. Briefly, groups of female CBA/Ca mice (n ) 3
or 4) received 25 µL of the test compound dissolved in vehicle
(
10) EU. Directive 2003/15/EC of the European Parliament and of the
Council of 27 February 2003 amending Council Directive 76/768/
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(
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(
(
acetone/olive oil 4:1) on the dorsum of the ears daily for three
consecutive days. Control animals were treated in the same way
with vehicle alone. All mice were injected intravenously five days
after the first treatment, with 250 µL of phosphate-buffered saline
(
(
3
containing 20 µCi of [ H]thymidine. Five hours later, draining
auricular lymph nodes were excised and pooled for each group,
and a single cell suspension of lymph node cells was prepared.
The thymidine incorporation was measured by ꢀ-scintillation
counting. Results are expressed as mean dpm/lymph node for each
experimental group and as stimulation index (SI), that is, test group/
control group ratio. Test materials that at one or more concentrations
caused an SI greater than 3 were considered to be positive in the
LLNA. EC3 values (the estimated concentration required to induce
9
27–936.
(
15) Karlberg, A. T. Contact allergy to colophony. Chemical identifications
of allergens, sensitization experiments and clinical experiences. Acta
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(
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45
an SI of 3) were calculated by linear interpolation. The sensitizing
potency of the test compounds was classified according to the
following: <0.1, extreme; g0.1–<1, strong; g1–<10, moderate;
8
14.
4
6,47
g10–<100, weak.
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Acknowledgment. We thank Petri Karhunen for skillful
technical assistance, Dr. J. Lars G. Nilsson and Dr. Lars Weidolf
for valuable discussions, and the Biotransformation group in
Clinical Pharmacology & DMPK, AstraZeneca R&D Mölndal,
for kindly letting us use their Q-Tof instrument. This work was
financially supported by The Swedish Animal Welfare Agency.
(
18) Doss, G. A.; Baillie, T. A. Addressing metabolic activation as an
integral component of drug design. Drug Metab. ReV. 2006, 38, 641–
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49.
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Supporting Information Available: Full data of the LLNA
(
1
responses of compounds 2–8, a H NMR spectrum of oxime 9,
and a full interpretation of the MS fragmentation pattern of GSH
conjugates of 9. This material is available free of charge via the
Internet at http://pubs.acs.org.
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