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