Lipid-like sulfoxides and amine oxides as inhibitors of mast cell activation

Article abstract of DOI:10.1016/j.ejmech.2011.02.068

The drug miltefosine is a prototypic lipid-like compound thought to modulate membrane environments and thereby indirectly prevent receptor-mediated signaling events. In addition to its primary therapeutic indications in cancer and leishmaniasis, miltefosi

Full text of DOI:10.1016/j.ejmech.2011.02.068

European Journal of Medicinal Chemistry 46 (2011) 2147e2151
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Lipid-like sulfoxides and amine oxides as inhibitors of mast cell activation
,
b
,
,
,
*
José Batista a 1, Georg Schlechtingen b 1, Tim Friedrichson b, Tobias Braxmeier b, Jürgen Bajorath a
a Department of Life Science Informatics, B-IT, LIMES, Program Unit Medicinal Chemistry and Chemical Biology, Rheinische Friedrich-Wilhelms-Universität Bonn, Dahlmannstr. 2,
D-53113 Bonn, Germany
b JADO Technologies GmbH, Tatzberg 47-51, D-01307 Dresden, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The drug miltefosine is a prototypic lipid-like compound thought to modulate membrane environments
and thereby indirectly prevent receptor-mediated signaling events. In addition to its primary therapeutic
indications in cancer and leishmaniasis, miltefosine has also been shown to block immunoglobulin E
receptor-dependent mast cell activation. Miltefosine and other alkylphospholipids that are active in mast
cell degranulation assays contain a positively charged nitrogen and a phosphate group that are important
for activity. In addition to alkylphospholipids, ceramides are also known to act on membrane environ-
ments and inhibit mast cell activation. We have systematically searched a very large compound collec-
tion for other lipid-like inhibitors of mast cell activation. Analogs of an initially identified screening hit
were synthesized and preliminary SAR information was collected, leading to the identification of sulf-
oxide and amine oxide containing lipid-like compounds as new inhibitors of mast cell activation. Sulf-
oxide and amine oxide derivatives were found to be only slightly less active than miltefosine.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Received 11 November 2010
Received in revised form
21 February 2011
Accepted 26 February 2011
Available online 5 March 2011
Keywords:
Mast cell activation and degranulation
Immunoglobulin E receptor
Membrane microdomains
Lipid rafts
Lipid-like compounds
Membrane-directed inhibitors
Computational filtering
Molecular similarity
1. Introduction
still is an unconventional approach to therapy. For this purpose,
compounds would be required to enter rafts and perturb the regular
Allergic diseases are associated with deregulated mast cell acti-
vation [1]. Among other effects, mast cell activation and degranu-
lation depend on signal transduction through the immunoglobulin E
receptor [2]. This receptor is known tobe concentrated in membrane
microdomains having a characteristic lipid composition and a high
degree of lipid ordering [2]. These microdomains are often called
lipid rafts [3]. Mast cell activation can be prevented by blocking the
immunoglobulin E receptor, for example, through the use of
monoclonal antibodies. Signaling through the immunoglobulin E
receptor depends on specific receptoreligand interactions and also
on the structural integrity of its lipid raft environment [2]. Hence, in
addition to blocking the receptoreligand interactions, an alternative
but as of yet only little explored route to the treatment of allergic
diseases might be to selectively alter the structure and organization
of immunoglobulin E receptor containing membrane microdomains
and thereby prevent effective immunoglobulin E receptor-depen-
dent signaling. Targeting membrane structures instead of receptors
packing of their characteristic phospho- and glycolipids, thus indi-
rectly affecting receptor function. However, there is some prior
evidence for this potential mechanism of action. The drug miltefo-
sine (hexadecylphosphocholine), an alkylphospholipid, is currently
approved for the treatment of breast cancer metastasis and leish-
maniasis [4,5]. Miltefosine (compound 1, Fig. 1) consists of a polar
head group that is similar to plasmalogen phospholipids. Plasmal-
ogen phospholipids are known to be enriched in immunoglobulin E
receptor containing lipid rafts [6]. It is thus likely that miltefosine
mightalsoenterthesemembrane domains and therebycompromise
their structural integrity. If so, then miltefosine would also be
expected to affect immunoglobulin E receptor-dependent signaling,
although it had originally not been implicated in such effects.
However, this hypothesis has been tested and it has indeed been
shown that miltefosine blocked immunoglobulin
E receptor-
dependent mast cell activation both in vivo and in vitro [7]. There-
fore, miltefosine is thought to perturb the structure of immuno-
globulin E receptor containing lipid rafts, although there currently is
no detailed mechanistic information available.
Following up on these findings, we have previously reported
other alkylphospholipid structures that inhibit the activation of
Rat Basophilic Leukemia clone 2H3 (RBL-2H3) cells, a commonly
* Corresponding author. Tel.: þ49 228 2699 306; fax: þ49 228 2699 341.
E-mail address: bajorath@bit.uni-bonn.de (J. Bajorath).
The contributions of these authors should be considered equal.
1
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.02.068

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J. Batista et al. / European Journal of Medicinal Chemistry 46 (2011) 2147e2151
fingerprints, as provided by the MOE Software Toolkit, and
compared to compounds representing currently known inhibitory
chemotypes including betaines and ceramides. Only compounds
with less than 70% 2D Tanimoto similarity to any known inhibitor
were further considered in order to focus the search on new che-
motypes. A total of 179 compounds with 90 unique head groups
met this dissimilarity criterion. These candidate molecules were
subjected to visual inspection and only four compounds were
ultimately selected for experimental evaluation, as rationalized in
the results and discussion section. Fig. 2 shows these four selected
chemotypes. Compound 4 was acquired from Timtec, compound 5
and 6 from SigmaeAldrich, and compound 7 from Chemdiv.
2.2. Degranulation assay
Inhibition of degranulationwas determined as percent reduction
of
b-hexosaminidase release compared to antigen-stimulated
Fig. 1. Known lipid-like inhibitors of mast cell activation. Compound 1 is miltefosine.
Its head group on the left contains a quaternary nitrogen and a phosphate group.
Compound 2 has a head group that is chemically distinct from miltefosine, but also
contains a charged nitrogen. Compound 3 represents the ceramide (sphingolipid)
chemotype.
release used as a positive control after subtraction of unspecific
release as a negative control. Relative inhibition was calculated as: %
Inhibition ¼ 100 ꢀ (1 ꢁ (test compound ꢁ negative control)/(posi-
tive control ꢁ negative control)). The assay was performed on RBL-
2H3 cells. Cells were seeded in 24 well plates in a volume of 1 ml
medium (70% Minimum Essential Medium with Earle’s Salts, 20%
used in vitro cell culture model for mast cells [8]. RBL-2H3 cells
were also used as a cell culture model in our current study. Milte-
fosine and other alkylphospholipids that are active in degranula-
tion assays contain a positively charged quaternary amine and
a negatively charged phosphate group (compound 1, Fig. 1), i.e. they
are betaines. Through a miltefosine-based pharmacophore search,
we have also identified another lipid-like inhibitor of mast cell
activation with a head group that is chemically distinct from
alkylphospholipids [8] (compound 2, Fig. 1). This inhibitor also
contains a quaternary nitrogen and, in this case, a delocalized
positive charge, but no negatively charged group (that might mimic
a phosphate group). There is currently only one other chemotype
known that is not a betaine, but active in RBL-2H3 cell degranula-
tion assays, i.e. ceramide, a sphingolipid [9,10] (compound 3, Fig. 1).
In this study, we have searched for other lipid-like inhibitors of
mast cell activation that are chemically distinct from alkylphos-
pholipids and ceramides as potential starting points for the
generation of membrane-directed anti-allergic agents.
RPMI 1640 Medium, 10% FBS (fetal bovine serum) and 2 mM
glutamine). After incubation the medium was replaced with 400
L-
ml
fresh medium containing 0.4
were washed with 800 l pre-warmed Tyrode’s buffer (TyB). After
washing 380 l TyB containing a test compound or no compound
were added and the cells were incubated for 1 h at 37 ꢂC. Cells were
activated by addition of 20 l DNP-HSA (dinitrophenol-human
serum albumin; 0.1 g/ml inTyB) for 15 min. Plates were centrifuged
at 4 ꢂC and transferred to an ice bath. Duplicate aliquots (25
l) of
supernatants were transferred to a black 96-well plate. 100 l MUG
substrate solution (2.5 mM 4-methylumbelliferyl-N-acetyl-
mg/ml anti-DNP IgE. The next day cells
m
m
m
m
m
m
b-D-
glucosaminide (Calbiochem, no. 474502) in 0.05 M citrate) was
added and plates were incubated for 30 min at 37 ꢂC. The reaction
was terminated by addition of 150 ml stop solution (0.1 M NaHCO3/
0.1 M Na2CO3, pH 10). Fluorescence was measured at 365 nm exci-
tation and 440 nm emission wavelengths. As a negative control,
supernatant of non-stimulated cells without anti-DNP IgE was
measured to detect unspecific
b-hexosaminidase release. As
2. Experimental
2.1. Computational analysis
Approximately 4.2 million in silico-formatted compounds
collected from medicinal chemistry vendor sources were subjected
to a computational filtering protocol. Compound filtering was
carried out using the Molecular Operation Environment (MOE) [11].
To ensure the desired lipid-like character of candidate structures
the following rules were initially applied: (1) molecular size:
between 18 and 50 non-hydrogen atoms, (2) lipid-likeness: pres-
ence of a C11 or longer saturated alkyl chain, (3) charge state: no
charged atoms, (4) head group: hydrophobic substituent with
a maximum of three atoms permitted. The last criterion ensured
that the alkyl chain was the only major hydrophobic substructure.
Only 1868 compounds passed these filters. These lipid-like
compounds were further filtered for the following undesired
chemical moieties: (a) polyether, (b) polyfluorine, (c) adamantane-
like rings (d) nitrile, (e) nitrite, (f) azo-coupling groups, (g) alkyl
chain with 22 or more carbon atoms. A total of 956 compounds did
not contain any of these moieties and were further analyzed. The
956 selected database compounds were encoded as MACCS
Fig. 2. Test compounds. Shown are four different lipid-like compounds that were
selected as candidate inhibitors. Assay results are reported for each compound.

J. Batista et al. / European Journal of Medicinal Chemistry 46 (2011) 2147e2151
2149
a control for total
were treated with 400
150 mM NaCl, 5 mM EDTA and 1% (w/v) Triton X-100) at room
temperature. Antigen-stimulated -hexosaminidase release served
as positive control. IC50 values were determined from concentra-
tion-response data (concentration vs. inhibition of degranulation)
with a non-linear five point parameter curve fitting procedure using
SigmaPlot software [10].
b
-hexosaminidase content, the remaining cells
3. Results
ml lysis buffer (25 mM Tris$HCl, pH 7.5,
In order to search for lipid-like inhibitors that depart from the
alkylphospholipid and ceramide structures, we have carried out
a rule-based computational filtering procedure of a very large
compound collection that was augmented by molecular similarity
analysis. Through filtering and exclusion of compounds with
obvious 2D structural similarity to alkylphospholipids and ceram-
ides, we ultimately reduced w4.2 million database compounds to
179 lipid-like structures with 90 unique head groups (the lipid-
likeness constraint eliminated the majority of database
compounds). We then subjected this relatively small set of candi-
date head groups to visual inspection in order to eliminate
compounds that had no H-bond acceptor functions, which are
likely to be important for activity in light of currently available
active chemotypes, or that had only limited accessibility to H-bond
acceptors (e.g. because of proximal ring systems). We also only
considered compounds that were commercially available. These
additional criteria further reduced the number of candidates, and
we finally selected only four compounds for experimental evalua-
tion in RBL-2H3 cell degranulation assays (shown in Fig. 2).
b
2.3. Toxicity assay
The maximum tolerated concentration (Mtc) was determined as
the highest dose of a test compound that did not cause toxic cellular
toxicity determined by lactate dehydrogenase release using
a commercially available cytotoxicity test (Promega Cytotox-One
cat. #67891).
2.4. Synthesis
Dodecyl methyl sulfoxide (compound 8, Fig. 3) was purchased
from SigmaeAldrich (no. 641588) and N,N-dimethyltetradecyl-
amine-N-oxide (compound 14, Fig. 3) from Anatrace, Ohio (no.
T360). Sulfoxides were prepared from the corresponding thioethers
by oxidation with H2O2 in hexafluoroisopropanol (HFIP) [12],
occasionally with addition of CH2Cl2 to improve solubility. This
method yielded clean monooxygenation without any further
oxidation to sulfones. Except for dithiane (compound 16, Fig. 3),
thioethers were prepared by alkylation of commercial thiols
following known procedures (Fig. 3). Experimental details and
analytical data are provided as Supplementary information.
Disulfoxides were obtained as mixtures of stereoisomers. In the
case of compound 13 (Fig. 3), oxidation of racemic trans-thioether
(compound 19, Fig. 3) yielded two equipotent stereoisomers (S]O
epimers), which were separated by flash chromatography. The
available spectroscopic data did not allow for unequivocal assign-
ment of configuration at the S-atom. The bioassay results presented
for compound 13 (Fig. 3) refer to the faster-eluting, more soluble
stereoisomer, which was the main product of this synthesis.
While compound 4 was found to be inactive, compounds 5 and 6
displayed weak inhibition of degranulation at 25
By contrast, compound 7 showed more than 70% inhibition under
these conditions and only very little toxicity (Mtc 100 M). Hence,
mM concentration.
m
this compound was considered an attractive hit. Its head group
contained a cyclohexyl acetate and a sulfoxide group, representing
a previously unobserved inhibitory chemotype lacking charged
atoms. Compound 7 also had the shortest (C12) saturated alkyl tail
among the four test compounds.
In order to better understand key features of this head group,
several analogs were synthesized and tested, as shown in Fig. 4.
Because the oxygen-rich test compounds 5 and 6 in Fig. 2 were only
very weakly active, we first investigated the role of the sulfoxide
group by generating compound 8 where the head group was
reduced to only a sulfoxide (Fig. 4). Interestingly, this compound
already showed 66% inhibition at 25 mM. Thus, it was only slightly
less active than compound 7, which confirmed the importance of
the sulfoxide group. For sulfoxides, we varied the length of the
saturated alkyl “lipid tail” from C10 to C16 and found that C12
analogs were most active. Additional C12 analogs were generated
in order to investigate the presentation of sulfoxide group(s) in
different chemical environments. For example, adding a second
sulfoxide group in a six-atom alkyl chain head group reduced
inhibition (compound 9, Fig. 4). However, presenting a second
sulfoxide group in an aliphatic six-membered ring (compound 10,
Fig. 4) further increased the inhibitory activity observed for
compound 7 to 80% at 25 mM. By contrast, presenting single or dual
sulfoxide groups in head groups containing an aromatic ring again
reduced inhibition (compounds 11 and 12, Fig. 4). The comparison
of compounds 12 and 13 in Fig. 4 demonstrates that the presence of
an aromatic instead on an aliphatic ring was unfavorable. Taken
together, the analogs of our initial hit shown in Fig. 4 revealed some
preliminary SAR information. With 80% inhibition at 25
mM, cor-
responding to an IC50 value of 10.7 M, compound 10 was the most
m
active sulfoxide derivative we identified. Similar to compound 7,
compound 10 also displayed only very little cellular toxicity in our
assays.
Considering the previously observed relevance of nitrogen
containing betaine structures for inhibition, we also reasoned that
it should be interesting to replace the active sulfoxide head group
by an amine oxide. These head groups differ only in a single sulfur/
nitrogen atom and have comparable polarity. We initially tested
a C12 amine oxide, which displayed weak but detectable activity in
Fig. 3. Synthesis of sulfoxides 9e13. i: dodecyl iodide, NaOH/EtOH (75%), ii: EtBr,
NaOH/EtOH (88%), iii: H2O2/HFIP, iv: BuLi; dodecyl iodide, ꢁ78 ꢂC; 0 ꢂC (47%), v: MeI,
K2CO3 (98%), vi: dodecane thiol, NEt3. Yields of oxidation vii: 9 (33%), 10 (54%), 11 (55%),
12 (86%), 13 (64%).
RBL-2H3 cell degranulation assays, with 40% inhibition at 25 mM. In

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J. Batista et al. / European Journal of Medicinal Chemistry 46 (2011) 2147e2151
currently known to be active in RBL-2H3 cell degranulation assays,
used as a model system for mast cell activation, include alkyl-
phospholipids and ceramides. The characteristic feature of active
alkylphospholipids is the presence of a charged nitrogen and
a phosphate group. From the comparison of different alkylphos-
pholipids, it was possible to derive a simple pharmacophore [8]. In
order to increase the probability of identifying other active che-
motypes, we deliberately did not require the presence of a nega-
tively charged acceptor function when defining the
pharmacophore. This simple query was then utilized to discover
a new active chemotype containing a charged nitrogen (compound
2, Fig. 1) [8]. In order to further expand the search for other che-
motypes as potential inhibitors of mast cell activation, we imple-
mented a computational filtering protocol in this study that was
augmented by molecular similarity calculations to eliminate
candidate compounds that were structurally similar to known
inhibitory chemotypes. Furthermore, we analyzed candidate
compounds based on the hypothesis that sterically well-accessible
hydrogen bond acceptors should generally be important for activity
in lipid raft environments. A compound with a sulfoxide containing
head group was found to display considerable activity in RBL-2H3
cell degranulation assays. Given the activity of betaines and the
sulfoxide head group, both of which are strong dipoles, we
reasoned that amine oxides might also be interesting candidates for
evaluation as mast cell degranulation inhibitors. We then indeed
found that amine oxides were active in RBL-2H3 cell degranulation
assays. However, in this case, most active analogs were obtained for
C14 saturated alkyl chains, whereas sulfoxides were most active
with C12 alkyl chains. Analyzing the origins of different alkyl chain
length preferences for chemically simple sulfoxide and amine oxide
head groups should provide interesting opportunities for further
studies.
Fig. 4. Analogs of compound 7. Assay results are reported. “Mtc” gives the maximum
tolerated compound concentration in toxicity assays. Compound 10 was the most
active sulfoxide derivative.
this case, we also varied the length of the saturated alkyl chain from
C10 to C18 and found that the C14 amine oxide analog was highly
5. Conclusions
active (compound 14, Fig. 5), with 95% inhibition at 25
IC50 value of 10.6 M. Both compounds 10 and 14 were only slightly
less active in RBL-2H3 cell degranulation assays than compound 2
M) that were tested as
mM and an
m
In this study, we have identified sulfoxide and amine oxide
containing lipid-like molecules as new membrane-directed inhib-
itors of mast cell activation. By preparing analogs of an initially
identified hit (compound 7) we have established that a sulfoxide
head group was sufficient for inhibition. However, sulfoxide groups
presented in an aliphatic chain showed reduced inhibition and the
addition of aromatic rings to sulfoxide containing head groups was
also unfavorable. By contrast, two sulfoxide groups present in an
aliphatic ring yielded the currently most active analog (compound
10). Similar to compound 7, this analog caused only very little
cellular toxicity and was only slightly less active than miltefosine in
degranulation assays. Furthermore, we found that compound 14
containing an amine oxide head group was also comparably active.
(IC50 ¼ 5.5
m
M) and miltefosine (IC50 ¼ 4.6
m
controls. Thus, these findings suggest that lipid-like compounds
with sulfoxide and amine oxide containing head groups should
merit further exploration as membrane-directed inhibitors of mast
cell activation.
4. Discussion
Evidence is accumulating that well-structured membrane
microdomains of specific composition, termed lipid rafts, provide
an environment for certain receptors that contributes to the regu-
lation of their signaling functions [3]. However, the approach of
targeting membrane microdomains that are enriched with a ther-
apeutically relevant receptor using lipid-like synthetic compounds
is still in its infancy. Although miltefosine is a marketed drug, the
molecular mechanisms by which so-called raftophilic compounds
might act are just beginning to be understood [3]. We have set out
to target lipid rafts containing the immunoglobulin E receptor in
order to control signaling events leading to mast cell activation that
are implicated in allergic diseases. Compound classes that are
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ejmech.2011.02.068.
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