G. Hostetler et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2094–2097
2097
12. Receptor binding assay:
system of compound 1a to impart activity. In addition, compound
25 displayed ca. three-fold more potency than the corresponding
lactam derived compound 4 (Table 1). Due to the lack of a detailed
knowledge of the three-dimensional structure of the ligand–recep-
tor complex, it had been postulated that extra oxygen in compound
25 with its available lone pairs of electrons might be involved in
some additional beneficial binding. Incorporation of an electron-
withdrawing but sterically small ortho-fluoro group in the phenyl
ring (eastern region) to generate compound 26 was not detrimen-
tal to the potency. However, the incorporation of sterically bulkier
ortho-chlorine atom generating compound 27 reduced the potency
ca. three-fold compared to the compound 25, reminiscent of simi-
lar effect from the lactam series. Introduction of an ortho-methoxy
group also reduced the potency by ca. five-fold indicating steric
bulk in the ortho position of the phenyl ring in the western region
might be detrimental to potency. Interestingly, relocating the chlo-
rine atom from the ortho to the meta position in the same phenyl
ring brought back the potency of the parent compound (cf. com-
pound 29 vs compound 25). The effect of the replacement of the
azepine system in the western region with linear or fused bicyclic
systems on the potency of the compound 25 was next investigated
generating compounds 30–34. This limited maneuver was not pro-
ductive to offer a more potent compound than compound 25.
At this stage, the emergence of potent compound 14 (Ki of
2.6 nM, Table 1) dictated us to focus on this compound for further
exploration. 5-HT6 receptor is positively coupled to adenylyl cy-
clase, and the activation of the receptor leads to increased produc-
tion of cyclic adenosine monophosphate (cAMP).13 Thus,
compound 14 was evaluated in a cAMP antagonism assay.14 In this
functional assay, this compound displayed an IC50 of 15 nM indi-
cating it to be an antagonist of the receptor. In order to probe
the effect of the chirality on the binding activity, compound 14
was separated into respective enantiomers via chiral HPLC. The
enantiomers (>98% ee) displayed ca. 1700-fold difference in bind-
ing potency (Ki of 1.6 nM vs 3000 nM); in addition, the more potent
isomer displayed an IC50 of 8 nM in the cAMP antagonism assay.
The determination of the absolute configuration of either isomer
remains an active area of research. However, the more potent
enantiomer was profiled against several members of the serotonin
Membrane preparation. Membranes were prepared from CHO-K1 cells stably
transfected with the human 5-HT6 receptor (Euroscreen; ES-316-C). The cells
were grown in Gibco Advanced DMEM-F12 (Cat # 12634010) containing 2%
dialyzed FBS (Hyclone Cat
# SH30079.03). The cells were harvested in
phosphate buffered saline (PBS) containing 0.1 mM EDTA and pelleted by
centrifugation (1000g), the supernatant was discarded and the pellets were
stored at À80 °C prior to membrane preparation. Membranes were then
prepared as follows. Briefly, frozen cell pellet was resuspended in a lysis buffer
containing 5 mM Tris–HCl (pH 7.5), 5 mM EDTA and 1 complete EDTA-free
protease inhibition tablet (Roche Applied Science, Indianapolis, IN) per 50 mL
buffer and homogenized with a tissue homogenizer. The cell lysate was then
centrifuged at 40,000g for 30 min at 4 °C to collect the membranes. The
membrane pellets were washed in membrane buffer (50 mM Tris–HCl (pH
7.5), 0.6 mM EDTA, 5 mM MgCl2, 1 complete EDTA-free protease inhibitor
tablet per 50 mL buffer) using a tissue homogenizer. The membranes were
centrifuged at 40,000g for 30 min at 4 °C and the pellets were resuspended in
membrane buffer containing 250 mM sucrose, and protein concentration was
determined using the Comassie Plus kit (Pierce Biotechnology, Rockford, IL).
Assay protocol. Membranes prepared from cells expressing recombinant
human 5-HT6 receptor (h5-HT6) were resuspended in assay buffer containing
50 mM Tris HCl (pH 7.4), 4 mM CaCl2, 10
lg/mL saponin and 0.1% (w/v)
ascorbic acid. Membranes were preincubated using 1.75
l
g membrane protein
and 0.25 mg FlashBlue scintillation beads (Perkin Elmer catalogue # FB001) per
well at 4 °C for 30 min. Vehicle or test compound and 4 nM [3H]LSD
(PerkinElmer catalogue
room temperature in
#
a
NET638) were added and incubated for 3 h at
final volume of 80 in 96-well plate. Test
lL
a
compounds or assay controls for total and non-specific binding were diluted
in DMSO as 100Â solutions and serially diluted by half log concentrations on a
Perkin Elmer JANUS Automated Workstation. Serotonin (10 lM final
concentration) was used to determine non-specific binding in the assay.
Plates were read using the Microbeta Trilux 1450 LSC and luminescence
counter. Data were analyzed by nonlinear regression using the dose-response
equation (variable slope) to calculate IC50 in XLfit4 (ID Business Solutions Inc.):
y = (Bottom + ((Top À Bottom)/(1 + ((IC50/x)^Hill slope))))
Binding of [3H]LSD to the h5-HT6 membranes was saturable with
Bmax = 6.2 pmol/mg protein and Kd = 2.3 nM. Ki value was then calculated
family displaying Ki of 3.1 lM against 5-HT2A, >30 lM against 5-
HT2B and 5-HT2C, respectively. In PK studies (rats), this enantiomer
was detected in the brain even after 6 h post oral administration
(dose: 5 mg/kg) indicating that the compound was crossing the
blood–brain barrier. Additional profiling of both the isomers as
well as the racemate is ongoing.15
In conclusion, in this Letter, we disclosed lactam and oxazolid-
inone-derived potent h5-HT6 receptor antagonists. Based on its
superior potency, a lactam derivative, racemate compound 14
(Ki of 2.6 nM) was profiled further. Separated potent enantiomer
(Ki of 1.6 nM) displayed selectivity against a number of family
members as well as brain permeability in rats after 6 h post oral
administration.
according to Cheng-Prussof method using the equation: Ki app = IC50
/
(1+([radioligand]Kd)). The method was an adaptation from patent
a
application, as described in Bacon, E. R.; Bailey, T. R.; Dunn, D. D.; Hostetler,
G. A.; McHugh, R. J.; Morton, G. C.; Rossé, G. C.; Salvino, J. M.; Sundar, B. G.;
Tripathy, R. Patent application, 2011, WO 2011/087712, 1.
14. cAMP antagonism assay. cAMP level in the cells were determined using a
homogeneous time-resolvedfluroscent (HTRF(R)
)
assay, (Cat
Cisbio, Bedford, MA) in cell line stably expressing human 5HT6 receptor.
Briefly, 18 L of cells in the cell density of 5000 cells per well were plated to
# 62AM4PEC,
l
white 384-well OptPlates (Cat # 600727, PerkinElmer Life Sciences, Boston,
MA). 100 nL of test compounds were added to cells and the plate was pre-
incubated at room temperature for 10 min (final DMSO concentration was
0.5%). The cells were then stimulated with
concentration of 10 nM for 30 min at room temperature. The reaction was
stopped by the addition of 12 L of cAMP-d2 in lysis buffer, followed by
addition of 12 L of anti cAMP-cryptate in lysis buffer. The plate was incubated
6 lL of 5-HT at its EC50
l
l
Acknowledgments
at room temperature for 1 h and read on multi-labeled plate reader Envision
2102 or 2104 (PerkinElmer Life Sciences, Boston, MA). The fluorescent ratio
(665 nM/590 nM Â 104) was calculated, which was inversely proportional to
the level of cAMP in the sample. The method was an adaptation from Gabriel
et al. Assay Drug Dev. Technol. 2003, 1, 291.
Authors wish to acknowledge Dr. Edward R. Bacon for his sup-
port and encouragement during the course of this research.
Authors thank one of the reviewers for constructive criticism to
improve the quality of the manuscript.
15. In absence of any experimental data on the structures of the complexes of the
receptor bound to the potent compound disclosed in Ref. 9 and the compound
14 from this manuscript, the authors refrained from comparing the SAR
between two series. When such data becomes available, it would be interesting
to see whether the both compound classes bind in the same manner to the
receptor or in a different way.
References and notes