ACS Medicinal Chemistry Letters
Letter
S2R in a radioligand displacement assay using [3H]-(+)-
pentazocine to selectively mark S1R and [3H]-ditolylguanidine,
in the presence of cold (+)-pentazocine, to mark S2R. The
binding affinities, expressed as inhibition constant (Ki), are
reported in Table 1. The compounds showed nanomolar
affinity at both SR subtypes with Ki values ranging from 195 to
0.5 nM for S1R, and from 182 to 0.59 nM for S2R.
Compounds 9, 11, and 13 exhibited a moderate selectivity
for S1R over S2R with a S1R/S2R ratio of 24, 43, and 34,
respectively. The binding results, supported by docking
calculation, allowed us to disclose some structure−activity
relationships. The narrowing and expansion of the carbocyclic
ring (1 and 2, respectively) preserved the high affinity of the
lead compound III at both subtypes (KiS1R = 4.2 and 1.7 nM
and KiS2R = 3.0 and 8.1 nM, respectively). Compounds 1 and
2 presented a binding mode analogue to that of compound III
(Figure SI-1B). In contrast, scaffold rigidification obtained by
condensing one (for compounds 3−5) or two (for 6) benzene
rings caused a significant drop in affinity for both SR subtypes.
Similarly, opening of the spirocyclic portion into the more
flexible derivative 7 led to a reduced affinity at both subtypes
(KiS1R = 68 and 76 nM and KiS2R = 85 and 63 nM for E and
Z isomers, respectively). These results highlighted that a bulky
aromatic ring, condensed or appended to the 1,3-dithiolane
core, is detrimental for affinity. As suggested by docking
calculations, although the secondary binding site of S1R might
tolerate bulky groups, the rigid and constrained conformation
assumed by these molecules prevented an easy fit within the
pocket. Thus, compounds 3−7 assumed the reverse binding
mode, as previously observed for I and II.
The benzylpiperidine ring was located in the secondary site,
while the bulkier spirocyclic moieties were accommodated in
the wider and longer primary hydrophobic pocket. However,
this led to an unsuitable alignment between the protonated
piperidine nitrogen atom and the Glu172, justifying the
reduced affinity of compounds 3−7 compared to III (Figure
SI-1C). No diastereoselectivity between the E/Z isomers was
observed. The structural simplification of the 1,4-dithiaspiro-
decane moiety of III with a less sterically hindered and more
flexible structure (8 and 9) resulted in a subnanomolar affinity
(KiS1R = 2.0 and 0.59 nM and KiS2R = 0.5 and 12 nM, for 8
and 9, respectively). Compounds 8 and 9 showed a binding
mode comparable to that described for III, 1−3 (Figure 2A).
However, the opening of the cycloalkylspiro portion induced a
slight shift of these compounds toward the secondary pocket,
(i) straightening the salt bridge between the protonated amine
and Glu172 and (ii) allowing the benzyl-tail to be engaged in
an additional π−π face-to-edge stacking with Tyr103.
Figure 2. Docking of compound 9 (A, stick, violet carbon) and 11 (B,
stick green carbon) into S1R binding site. The reference crystal
structure used for the docking calculation (PDB ID: 6DK1) of S1R is
shown in teal cartoon. Important interacting residues are in stick
representation. Model atoms except for carbons are color-coded with
protein carbons (teal), oxygen (red), nitrogen (blue), and sulfur
(yellow). Bridge salt and π−π interactions are represented as yellow
and magenta dotted lines, respectively. Part of the β-barrel has been
hidden for a clearer visualization of the binding site.
Recent studies suggested that both S1R and S2R modulators
might have a positive effect in neuroprotection.5,10 Therefore,
we evaluated the in vitro neuroprotective capacity of selected
high affinity ligands. According to the binding affinities,
compounds I, II, and 11 were chosen for their S1R selectivity,
whereas III and 9 were selected as S1R/S2R mixed
compounds. ROS are normally produced in neurons and the
resulting oxidative stress is one of the major contributors to
cell death in neurodegenerative disorders. Herein, we assessed
the capability of compounds I−III, 9, and 11 to protect SH-
SY5Y cells from oxidative damage induced by two potent ROS
generating insults, namely rotenone and oligomycin.21 At first,
the selected compounds were investigated, as single agents, for
cytotoxicity (Table SI-2) in human SH-SY5Y cell line to define
the experimental doses for the neuroprotective studies.22 The
neuroprotective capacity was reported as percentage of cell
viability after treatment with the compound in the presence of
the toxic stimuli.
At 1 μM concentration, compounds I−III and 11 were able
to significantly prevent cell damage induced by rotenone
(Figure 3). Interestingly, I and II were effective also at the
higher dose of 5 μM. To investigate the role of S1R in this
process, the neuroprotective activity of our molecules was
tested in the presence of PB212, a S1R antagonist.23 All
compounds displayed a significant decrease of their neuro-
protective capacity in combination with PB212 (P < 0,001 at 1
μM). Altogether these results suggested the involvement of
sigma receptors in the neuroprotective effect of the proposed
compounds.
The high S1R affinity was maintained by replacing the
piperidine with the piperazine (Ki = 0.6 and 2.1 nM for 10 and
11, respectively). As expected, the presence of the
benzylpiperazine reverted the binding poses of 10 and 11 in
order to efficiently align the protonated nitrogen with Glu172
(Figure 2B). This binding mode fulfilled the Glennon’s
pharmacophore, preserving the distances of the two hydro-
phobic groups of the molecule to the central protonated site.
Finally, no significant differences in terms of affinity and
selectivity were observed by replacing the dithiolane (10 and
11) with the dioxolane bioisoster (12 and 13, Figure SI-1D).
To sum up, the SAR investigation pointed out that (i)
structural simplification of the spiro portion allowed achieving
sub/low nanomolar affinity for SRs, while (ii) the introduction
of bulky groups led to a drop in affinity for both SR subtypes.
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ACS Med. Chem. Lett. 2020, 11, 1028−1034