Lactam and oxazolidinone derived potent 5-hydroxytryptamine 6 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2014.03.049

Lactam and oxazolidinone derived potent 5-hydroxytryptamine 6 (5-HT ₆) receptor antagonists have been disclosed. One potent member from the lactam series, racemic compound 14 (K_i of 2.6 nM in binding assay, IC₅₀ of 15 nM in functional cAMP antagonism assay) was separated into corresponding enantiomers that displayed the effect of chirality on binding potency (K_i of 1.6 nM and 3000 nM, respectively). The potent enantiomer displayed an IC₅₀ of 8 nM in cAMP antagonism assay, selectivity against a number of family members as well as brain permeability in rats after 6 h post oral administration.

Full text of DOI:10.1016/j.bmcl.2014.03.049

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2094–2097
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Lactam and oxazolidinone derived potent 5-hydroxytryptamine
6 receptor antagonists
⇑
Greg Hostetler, Derek Dunn, Beth Ann McKenna, Karla Kopec, Sankar Chatterjee
Cephalon, Inc., 145 Brandywine Parkway, West Chester, PA 19380-4245, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Lactam and oxazolidinone derived potent 5-hydroxytryptamine 6 (5-HT6) receptor antagonists have been
disclosed. One potent member from the lactam series, racemic compound 14 (Ki of 2.6 nM in binding
assay, IC50 of 15 nM in functional cAMP antagonism assay) was separated into corresponding enantio-
mers that displayed the effect of chirality on binding potency (Ki of 1.6 nM and 3000 nM, respectively).
The potent enantiomer displayed an IC50 of 8 nM in cAMP antagonism assay, selectivity against a number
of family members as well as brain permeability in rats after 6 h post oral administration.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 22 December 2013
Revised 15 March 2014
Accepted 17 March 2014
Available online 25 March 2014
Keywords:
5-Hydroxytryptamine
Antagonist
Lactam
Oxazolidinone
CNS
O
O
The 5-hydroxytryptamine 6 (5-HT6) receptor is one of the
prominent players of the serotonin receptor family.1 Due to its
exclusive distribution in the central nervous system (CNS), this
receptor has emerged as a promising target for the pharmacologi-
cal intervention for the treatment of several CNS-related disorders,
for example, cognitive function in Alzheimer’s disease and schizo-
phrenia, anxiety, obesity, depression and sleep–wake activity.2–5
Thus, the discovery of novel and potent inverse agonists/antago-
nists of this receptor has become a recent area of research for the
pharmaceutical industries.6,7 It had been revealed that SB-
S
N
NH
N
I
Figure 1. Structures of compound I.
query whether the motif itself was needed for the potency of this
class of compounds.
742457 (compound I, Fig. 1),
a potent 5-HT6 antagonist,
Accordingly, the spiro bicyclic system in compound 1a was sim-
plified to generate a pair of series around the central cyclic moiety
(ring B). They are represented by generic structures E (lactam ser-
ies) and F (oxazolidinone series), respectively, (Fig. 2) making them
unique motifs for the receptor’s antagonism. In addition, they were
also notable for the absence of any sulfonamide or sulfone moiety
in the framework, a frequent feature of literature reported potent
inverse agonists/antagonists from various laboratories.7 In this Let-
ter, we disclose some preliminary results from our ongoing explo-
ration from both series.
demonstrated significant improvement in global function in the
treatment of dementia in Alzheimer’s disease in a phase IIb pla-
cebo-controlled study.8
In search of novel and potent 5-HT6 receptor inverse agonists/
antagonists, our team profiled our corporate chemical library on
a high throughput screening (HTS) platform. From this exercise,
the team encountered the 1-thia-4,7-diaza-spiro[4.4]nonane-3,6-
dione-derived ‘hit’ compound 1a [Ki of 5.70 lM against human
5-HT6 (h5-HT6) receptor, Fig. 2].9 Subsequently, compound 1a
acted as a launching pad for additional exploration of the series.
Scheme
1 depicts the representative synthesis of various
While
a research program was aimed at developing the
N-substituted phenyl lactams. Commercially available appropriate
aryl bromide 2 and 4-phenyllactam 3 were coupled under Buch-
wald condition to generate racemic lactams 4 and 6–17 (Table 1).10
Scheme 2 illustrates the route that was utilized to synthesize
compound 5 (Table 1). Commercially available amine 18 and acid
chloride 19 were coupled under basic condition to generate an
intermediate amide (not shown) that was subsequently treated
structure-activity relationship (SAR) around the central [5,5]-spiro
motif (rings B/C),9 a parallel program was initiated to answer to the
⇑
Corresponding author.
E-mail address: Sankar.Chatterjee24@gmail.com (S. Chatterjee).
http://dx.doi.org/10.1016/j.bmcl.2014.03.049
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

G. Hostetler et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2094–2097
2095
O
Table 1 (continued)
Br
c
Compound
P
X
Y, Y
O
h5-HT6 (Ki nM)
HN
+
X
z
N
16
H
4900
2
3
a
HN
N
O
N
X
17
Ph
O
10
N
N
HN
z
a
Binding assay against recombinant h5-HT6; internal ligand [3H]LSD (binding of
4 and 6 - 17
this ligand to the h5-HT6 membranes was saturable with Bmax = 6.2 pmol/mg
protein and Kd = 2.3 nM). Ki value of a test compound was then calculated according
to the Cheng-Prusoff method.
Scheme 1. Reagents and conditions: (a) CuI, K2CO3, K3PO4, trans-N,N-dimethyl-1,2-
cyclohexanediamine, 1,4-dioxane, 110 °C, 50–60%.
b
All compounds were tested as racemates.
Average of three experiments.
c
d
SEM: 0.3 (n = 3).
Table 1
Biological data for compounds 1a–b and 4–17a,b
R
3
4
5
2
1
N
Y
Y
O
B
O
B
A
A
P
N
N
Y
R
X
S
D
X
C
O
N
z
D
c
H
Compound
P
X
Y, Y
—
h5-HT6 (Ki nM)
1a
1b
—
—
5700
88
1a R = H
1b R = OMe
E Y = CH2
F Y = O
HN
HN
HN
F
4
5
6
Ph
O
54
Figure 2. Structures of HTS-hit compound 1a, 1b and current series of lactams E
and oxazolidinones F.
2-Cl-Ph
2-Cl-Ph
O
100
650
Cl
O
H, H
Cl
tBoc
N
+
H
N
NH2
7
Ph
O
1200
18
19
a, b
8
9
Ph
Ph
O
O
140
70
HN
HN
O
R
N
N
N
N
HN
HN
10
11
Ph
Ph
O
O
84
48
20 R = tBoc
5 R = H.HCl
Cl
c
N
Scheme 2. Reagents and conditions: (a) iPrNEt2, CH2Cl2, MeOH, 0 °C to room temp,
quantitative; (b) Me3S+–OÀ, 60% NaH in oil, DMSO 0 °C to room temp, overnight,
40–50%; (c) 4 N HCl in dioxane, room temp; 2 h, quantitative.
N
HN
N
12
13
Ph
Ph
O
O
4100
N
with the carbanion generated from trimethylsulfoxide to generate
lactam 20.11 Deprotection of the t-boc group in compound 20 gen-
erated compound 5.
N
N
>30,000
S
Scheme 3 displays a representative synthesis of oxazolidinone
derivatives containing a benzazepine moiety. Commercially avail-
able bromoketone of general structure 21 was reduced to corre-
sponding bromohydroxy derivative of general structure 22 that
was subsequently coupled with compound 18 (Scheme 2) to gen-
erate the carbamate 23. Cyclization of the carbamate 23 generated
the tBoc-protected oxazolidinone 24 that on subsequent deprotec-
tion generated the oxazolidinone derivatives 25–29 (Table 2).
Binding properties of the target compounds were assessed
against recombinant human 5-HT6 (h5-HT6) receptors by
N
14
15
Ph
Ph
O
O
2.6d
HN
HN
N
55
N
N

2096
G. Hostetler et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2094–2097
O
OH
O
O
Br
tBoc
N
Br
R
N
a
c
N
H
O
b
N
O
X
X
Br
21
X
22
X
24 R = tBoc
25 - 29 R = H.HCl
d
23
Scheme 3. Reagents and conditions: (a) NaBH4, MeOH, 0 °C to room temp, quantitative; (b) triphosgene, Et3N, CH2Cl2, À20 °C to room temp, compound 18, overnight, 40–
50%; (c) 60% NaH in oil; THF, 0 °C to room temp, overnight, 60–65%; (d) 4 N HCl in dioxane, room temp; 2 h, quantitative.
displacement of [3H]LSD and reported as Ki values from an average
of three experiments.12 The biological data for the lactam series
has been displayed in Table 1, while the similar data for the oxazo-
lidinone series has been depicted in Table 2, respectively.
Identification of compound 1a as a modestly active ‘‘hit’’ mole-
cule encouraged us to profile additional available derivatives from
the same class. This offered a more potent analog 1b (Fig. 1 and
Table 1, Ki of 88 nM). In our previous disclosure, the group de-
scribed how combination of these two results led us to postulate
and introduce an azepine moiety (ring F compound 4, Table 1) in
the western hemisphere of the designed molecules in the first
generation of targets9 as well as in the current series.
From the lactam series, as shown in Table 1, the first member of
this series, compound 4 (Ki 54 nM) displayed potency, being ca.
100-fold more potent than HTS-hit compound 1a. This indicated
that combination of a central lactam moiety in conjunction with
an azepine system in the western region of the molecule could in-
deed replace the architecturally rigid [5,5]-spiro bicyclic system of
compound 1a. The enhancement of the potency also revealed that
flexibility in the entire architecture of the system would be bene-
ficial. Introduction of an ortho-chloro group in the phenyl ring
(eastern region) generated compound 5 reducing the potency by
two-fold indicating additional lipophilicity/steric bulk might not
be beneficial to the potency from this site. The reduction of the car-
bonyl moiety of the lactam nucleus in compound 5 generated cor-
responding pyrrolidinyl derivative 6 with a reduction in potency
ca. six-fold. Thus, the carbonyl group of the lactam moiety was
necessary in a productive binding. Next, the symmetrical azepine
ring system of compound 4 was replaced with an unsymmetrical
azepine system to generate compound 7 resulting in ca. 20-fold
reduction in potency indicating the spatial importance of the ba-
sic-N atom at that region for potency. To expand the scope of the
SAR-exploration, the benzazepine moiety of parent compound 4
was next replaced with a series of structurally variant heterocycles
to generate compounds 8–17. Introduction of a tetrahydroisoquin-
oline moiety produced compound 8 with ca. 2.5-fold drop in
potency. On the other hand, the replacement of the entire bicyclic
framework (benzazepine or tetrahydroisoquinoline) in the western
region with a phenyl ring tethered either to a piperidinyl ring
(compound 9) or a piperizinyl ring (compound 10) was proven to
be less detrimental. In a pair of pyridine derivatives 11–12, the rel-
ative position of the pyridyl-N atom with respect to the lactam
moiety and the basic piperizinyl moiety affected potency
significantly (cf. compound 11 being ca. 85-fold more potent than
compound 12). In a similar fashion, the replacement of the
six-membered pyridyl nucleus with a five-membered 1,3-thiazolyl
nucleus was detrimental to the potency (cf. compound 13 vs com-
pound 11). Returning to the theme of the necessity of a central
bicyclic scaffold for potency, a series of quinoline based analogs,
compounds 14–17 were generated. In this series also, the relative
position of the quinoline-N atom with respect to the lactam moiety
and the basic piperizinyl moiety influenced the activity of the ana-
logs, cf. compound 14 vs compound 15, the former being ca., 20-
fold more potent than the latter. The deletion of the phenyl ring at-
tached to the lactam moiety in compound 14 generated compound
16 with ca. 1900-fold loss in potency revealing the importance of
the presence of a lipophilic aromatic moiety on activity in the east-
ern region. On the other hand, the replacement of the piperizinyl
ring in compound 14 with a homopiperizine ring generated com-
pound 17 resulting in a loss of only ca. four-fold potency.
Table 2
Biological data for compounds 1a and 25–34a,b
O
X
N
O
6
5
1
R
2
4
3
c
Compound
X
R
h5-HT6 (Ki nM)
1a
5,700
HN
HN
HN
HN
HN
25
26
27
28
29
30
H
16
2-F
15
47
87
10
2-Cl
2-OMe
3-Cl
2-Cl
O
240
HN
31
2-Cl
140
HN
HN
N
N
32
33
2-Cl
2-Cl
1900
150
HN
HN
34
2-Cl
330
In parallel, in the oxazolidinone series also (Table 2), first few
analogs were developed incorporating an azepine moiety in the
western region (compounds 25–29). As shown, initial analog 25
was ca. 360-fold more potent than the HTS-hit compound 1a. This
indicated that similar to a lactam moiety, a central oxazolidinone
moiety could also replace architecturally rigid [5,5]-spiro bicyclic
a
Binding assay against recombinant h5-HT6; internal ligand [3H]LSD (binding of
this ligand to the h5-HT6 membranes was saturable with Bmax = 6.2 pmol/mg
protein and Kd = 2.3 nM). Ki value of a test compound was then calculated according
to the Cheng-Prusoff method.
b
All compounds were tested as racemates.
Average of three experiments.
c

G. Hostetler et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2094–2097
2097
3. Shimizu, S.; Mizuguchi, Y.; Ohno, Y. CNS Neurol. Disord. Drug Targets 2013, 12,
861.
4. Wesołowska, A. Pharmacol. Rep. 2010, 62, 564.
5. Ly, S.; Pishadari, B.; Lok, L. L.; Hajos, M.; Kocsis, B. ACS Chem. Neurosci. 2013, 4,
191.
6. Marazziti, D.; Baroni, S.; Borsini, F.; Picchetti, M.; Falaschi, V.; Catena-Dell, M. O.
Curr. Med. Chem. 2013, 20, 371.
7. (a) Rossé, G.; Schaffhauser, H. Curr. Top. Med. Chem. 2010, 10, 207; (b) Heal, D. J.;
Smith, S. L.; Fisas, A.; Codony, X.; Buschmann, H. Pharmacol Ther. 2008, 117,
207.
8. Maher-Edwards, G.; Zvartau-Hind, J.; Hunter, A. J.; Gold, M.; Hopton, G.; Jacobs,
G.; Davy, M.; Williams, P. Curr. Alzheimer Res. 2010, 7, 374.
9. Hostetler, G.; Dunn, D.; McKenna, B. A.; Kopec, K.; Chatterjee, S. Chem. Bio. Drug
Design 2014, 83, 149.
10. Klapars, A.; Huang, X.; Buchwald, S. L. J. Am. Chem. Soc. 2002, 124, 7421.
11. Metzger, H.; Seelert, K. Angew. Chem., Int. Ed. Engl. 1963, 2, 624.
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.
13. Sebben, M.; Ansanay, H.; Bockaert, J.; Dumuis, A. Neuro. Rep. 1994, 5, 2553.
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
1. Vitalis, T.; Ansorge, M. S.; Dayer, A. G. Front. Cell Neurosci. 2013, 7, 93.
2. Ramirez, M. J. Alzheimers Res. Ther. 2013, 5, 15.