Communications
doi.org/10.1002/cmdc.202100301
ChemMedChem
antagonists[6a] (Figure 1A). Prior to the deorphanization of
GPR183, a small molecule inverse agonist of GPR183 named
GSK682753A (2) was identified; this compound was later shown
to also inhibit 7α,25-OHC (1)-mediated GPR183 activity.[2b,3] In
2014, screening of a library containing around 100 K com-
pounds resulted in the discovery of the GPR183 agonist NIBR51
(3).[8] This agonist was then used to rescreen the same
compound library for GPR183 antagonists, leading to the
identification of NIBR127 (4), which in turn was chemically
optimized to give the more potent GPR183 antagonist NIBR189
(5).[8] Using the molecular scaffold 6 (Figure 1A) of the known
GPR183 antagonist 5 as starting point, we here report the
discovery of a novel series of small molecule GPR183 agonists.
To identify commercially available compounds that were
structurally related to the known GPR183 antagonist 5[8] we first
conducted a substructure screen of the Enamine Screening
Collection (~2.7 M compounds) using the Markush formula 7
(Figure 1B) as search query. The results were further filtered for
“drug-likeness” based on chemical criteria and subsequently
clustered to ensure chemical diversity. A total of 79 compounds
(9–87, supplementary Figure S1 and Table S1) were manually
selected for experimental screening; these were all piperazine
diamides of the general structure 8 (Figure 1B).
G protein activation induced by the 79 compounds was
detected using CHO-K1 cells that were transiently transfected
with GPR183 and a chimeric Gα subunit Gqi4myr that is
recognized as Gαi by Gαi-coupled receptors, but activates Gαq
pathways,[9] consequently enabling Ca2+ release. Calcium
release was measured for 100 seconds after ligand addition by
utilizing a fluorescent indicator, and data was extracted as the
change in fluorescence over time. Based on the G protein
signaling efficacy induced by 10 μM of each compound (Fig-
ure 2, top), we selected 10 of the compounds (9–18) for further
dose-response experiments (Figure 2, middle). Here, com-
pounds 15 and 16 displayed the most favorable agonist
properties, combining acceptable efficacy and potency (EC50 of
209 nM and 179 nM, respectively). As previously shown, the
antagonist 5 did not display any intrinsic activity, while the
endogenous agonist 1 activated the receptor with a potency
(EC50) of 17 nM (Figure 2, shown as reference curves).
As all the screened compounds were built on a central
piperazine diamide core, the structural variation was in the two
distal ring systems and the spacers (Figure 1B). Of the 10 active
compounds (Figure 3), four contained the same (E)-alkene
spacer as the antagonist 5, two contained an ethylene spacer,
and four contained the oxy-methylene spacer found in the
known antagonist 4. However, the non-systematic structural
variation in the distal ring systems made it difficult to identify
clear structure-activity relationship (SAR) trends in this com-
pound series.
Figure 3. Structures of the 10 compounds (9–18) that showed agonistic
activity in the initial screening, grouped by type of two-atom spacer (C=C,
CÀ C, CÀ O).
methanone (93) via coupling with the corresponding acyl
chlorides (Scheme 1). The same starting material (93) was
reacted with the corresponding carboxylic acids in the presence
of EDC to afford 90 and 91 in excellent yields. Coupling of Boc-
piperazine (94) and chloroacetyl chloride yielded intermediate
95. This was further reacted with 1-naphthol and Boc-depro-
tected to give 96. Subsequent amide coupling employing 1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) led to com-
pound 92 in excellent yield.
As Gαi is the direct G protein-signaling pathway elicited by
GPR183,[2a] we switched to this pathway for the functional tests
of the synthesized compounds 88–92, and included 15 and 16
for comparison with the calcium release experiment in the
initial screen. Hence, the GPR183 agonist activity of the five in-
house compounds was experimentally tested using a BRET
assay to determine Gαi coupling at various concentrations of
the compounds. CHO-K1 cells were transiently transfected with
GPR183 and the CAMYEL (cAMP sensor using YFP-Epac-Rluc)
BRET biosensor, which changes conformation in response to
cAMP levels; consequently, activation of Gαi leads to a rise in
BRET signal.
While the reference compounds 88–90 were devoid of
agonist activity (Figure 4A–C), the crossover compounds 91 and
92 displayed agonist profiles similar to the initial agonist hits 16
and 15, respectively, i.e. similar potency and partial agonist
properties, meaning that the efficacy did not reach that of the
full agonist 1 (Figure 4D–E). The potency of the reference
endogenous agonist 1 obtained here (19 nM) (Figure 4H) was
comparable to the value in the calcium assay, as were the
We therefore designed and synthesized five additional
compounds (Scheme 1): three reference compounds (88–90)
that contained distal unsubstituted phenyl rings, as well as two
crossover compounds (91 and 92) that combined the Western
and Eastern ring systems of the top agonist hits 15 and 16.
The substituted piperazines 88 and 89 were synthesized in
one step from commercially available phenyl(piperazin-1-yl)
ChemMedChem 2021, 16, 1–6
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