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M. E. Meza-Aviña et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1827–1830
OH
O
HO
HO
OH
OH
O
N
N
S
N
O
N
N
N
O
O
P
N
N
O
O
HN
S
O
HO
O
N
O
O
O
N
O
O
O
N
Scheme 1. Synthesis of acylated piperidones.
n
H
LPI
ML191
ML192
ML193
CID 23612552
CID 1434953
CID 1261822
analogues since the bond angle for the C
a
will be similar to that
IC50 = 1.1 µM
IC50 = 0.70 µM
IC50 = 0.22 µM
of the cyclopropane analogues, however, this structure is much
flatter.
Figure 1. LPI and lead antagonists of GPR55.12
With a handful of acylated piperidones prepared, the final two
steps first involved a reductive coupling of aryl hydrazides (3t–z)
with the previously synthesized piperidones (2a–f) to yield hydra-
zides 4 (Scheme 2).14 These compounds were then cyclocarbony-
lated using triphosgene to yield oxadiazolones 5.15 The reductive
coupling reactions proceeded smoothly but the products of that
step were often unstable to silica gel chromatography. Therefore,
the unpurified products were treated with triphosgene without
further purification. This modification of the synthesis typically
improved the yields of the final compounds (see Supplementary
data for individual yields).
Similar to the cyclopropane starting materials (1a–f), the hydra-
zides (3t–z) were selected to probe the electronic and steric oppor-
tunities in the binding site. Based on the current model (Fig. 2), the
aromatic ring adjacent to the oxadiazolone is involved in an inter-
action with M3.36(105) and F6.48(239). Additionally, the oxadia-
zolone contributes as the key interaction between the basic
carbonyl oxygen with the ammonium of K2.60(80). Thus, electron
rich aromatic rings adjacent to the oxadiazolone should make the
carbonyl oxygen more basic and strengthen this interaction.
A targeted exploration of the SAR of all six acids (1a–f) with
hydrazide 3t and all seven hydrazides (3t–z) with acid 1a (Fig. 3
and Table 1) was performed instead of synthesizing and exploring
the biological activity of all 42 permutations of the six acids and
seven hydrazides. Acid 1a and hydrazide 3t were chosen as the
constants since these were the most simplified pieces consisting
of an unsubstituted phenyl ring. Unfortunately, there were solubil-
ity issues with some of the compounds (e.g., 5bt and 5bv), so addi-
tional combinations were required to elucidate the effect of the
different areas of the scaffold.
compounds were desired that modified the electronics and sterics
of these areas. Hence, the ML191 synthetic studies reported herein
were undertaken to explore the SAR of this oxadiazolone class of
compounds. ML191 was also chosen as the lead antagonist since
there are very few structurally related compounds that could be
purchased and screened compared to the available compounds
for ML192 and ML193.
Our synthetic approach to GPR55 antagonists was designed so
that many different structures could be accessed to rapidly explore
initial SAR, along with validating or modifying our current model
(Fig. 2).11 The synthesis begins with the coupling of a carboxylic
acid to 4-piperidone by first forming the acid chloride (Scheme 1).
The different acids chosen, based on the initial hit, modify the elec-
tronics and sterics of this section of the molecule. Relative to
ML191, compound 2a reduces the steric impact, 2b increases the
electron-density in the aromatic ring, and compounds 2c and 2d
decrease the electron-density. Compounds 2e and 2f were selected
to examine the influence of steric bulk at the position of the cyclo-
propane ring. The largest change in overall structure relates to the
1-naphthoic acid derivative (2f). Although the naphthalene ring is
structurally different, this analogue can position the distal
aromatic ring in a similar position as the phenyl rings of the other
Compounds were initially screened via an image-based cell
assay to identify antagonist activity. The rationale for using the
b-arrestin recruitment assay was to provide a fair comparison of
IC50 values since our initial report employed this assay.11,12 Briefly,
U2OS cells overexpressing GPR55 and barr2-GFP were exposed to
LPI (6 lM; EC80) resulting in the recruitment of b-arrestin. Antago-
nist activity was evaluated by ligand-mediated inhibition of LPI-
induced receptor activation. This strategy quickly identified the
compounds that had IC50 values higher than 15
excluded from further analysis (Fig. 3).
lM which were
Concentration response curves were generated for compounds
that were active at concentrations below 15 M employing both
l
the image-based b-arrestin recruitment assay and the DiscoveRx
PathHunterÒ chemiluminescent b-arrestin complementation
assay. In the DiscoveRx PathHunterÒ system, CHO-K1 cells stably
expressing GPR55 (fused with a b-galactosidase enzyme fragment),
and b-arrestin (fused to an N-terminal deletion mutant of b-galac-
tosidase) were used to quantitate the inhibition of LPI-induced
b-arrestin activity. (Fig. 4, Table 1). Hence, antagonist activity
was evaluated through the use of two differential means of
b-arrestin quantitation, in two different cellular backgrounds
Figure 2. (A) Docking and key interactions between ML191 and GPR55. ML191
(green) has a key H-bond interaction with K2.60 (pink). ML191 also has p-stacking
or other van der Waals interactions with F169, F3.33, F6.55, M7.39, and Y3.32 (all
mustard). The interactions with M7.39 and F6.55 appear to hinder the rotation of
M3.36 and F6.48 (both purple) which are considered the toggle switch for GPR55.
(B) Electrostatic potential map of ML191. This figure is adapted from previously
published work, see Ref. 12.
Meza-Avi?a, Maria Elena
