Epiminocyclohepta[b]indole analogs as 5-HT6 antagonists

Article abstract of DOI:10.1016/j.bmcl.2012.01.022

A new series of epiminocyclohepta[b]indoles with potent 5-HT₆ antagonist activity were discovered and optimized using in vitro protocols. One compound from this series was progressed to advanced pharmacokinetic (PK) studies followed by 5-HT6 receptor occupancy studies. The compound was found to have excellent oral absorption, a highly favorable PK profile and demonstrated pharmacodynamic interaction with the 5-HT₆ receptor as shown by ex vivo autoradiography.

Full text of DOI:10.1016/j.bmcl.2012.01.022

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1494–1498
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Epiminocyclohepta[b]indole analogs as 5-HT6 antagonists
Alan J. Henderson a, , Peter R. Guzzo a, Animesh Ghosh b, Jagjit Kaur b, Jia-Man Koo b,
⇑
Kassoum Nacro b, Shailaja Panduga b, Rashmi Pathak b, Bharat Shimpukade b, Valentina Tan b,
Kai Xiang b, Jonathan D. Wierschke c, Matthew L. Isherwood b
a AMRI, 26 Corporate Circle, PO Box 15098, Albany, NY 12212-5098, USA
b AMRI Singapore Research Centre, Singapore Science Park II, Singapore 117525, Singapore
c AMRI Bothell, 22215 26th Avenue, SE Bothell, WA 98021, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of epiminocyclohepta[b]indoles with potent 5-HT6 antagonist activity were discovered and
optimized using in vitro protocols. One compound from this series was progressed to advanced pharma-
cokinetic (PK) studies followed by 5-HT6 receptor occupancy studies. The compound was found to have
excellent oral absorption, a highly favorable PK profile and demonstrated pharmacodynamic interaction
with the 5-HT6 receptor as shown by ex vivo autoradiography.
Received 16 December 2011
Revised 3 January 2012
Accepted 9 January 2012
Available online 14 January 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Serotonin
5-HT6
SAR
Cognition
Weight loss
5-HT6 is a serotonin receptor that is expressed in various re-
gions of the brain, including the hypothalamus and pre-frontal cor-
tex.1 Due to the fact that these receptors are almost exclusively
located within the CNS,1 5-HT6 represents an attractive target as
it is unlikely that modulation of the receptor will be associated
with any of the peripheral side effects that often plague CNS tar-
geted programs. Modulation of the 5-HT6 receptor in pre-clinical
animal models has been shown to induce promising efficacy in
conditions associated with sleep,2 anxiety and depression,3 epi-
lepsy4 and pain.5 However, most of the focus of 5-HT6 research
(particularly antagonists) has centered on weight loss and cogni-
tion. Modulation of the 5-HT6 receptor has been shown to produce
significant weight loss in rodent models of obesity,6 defining the
receptor as a promising new target for weight management. Fur-
thermore, antagonism of the 5-HT6 receptor has demonstrated
beneficial effects in treating cognitive deficits associated with con-
ditions such as Alzheimer’s disease and schizophrenia.7 A number
of 5-HT6 antagonists are currently undergoing clinical evaluation,
with the most advanced compound SB-742457 in Phase II trials
for treating cognitive deficits associated with Alzheimer’s disease.8
Through a process of rational drug design, and the use of
ligand–receptor pharmacophore models, we initiated a 5-HT6 pro-
gram to identify potent antagonists of the receptor which had an
excellent in vitro profile coupled with favorable pharmacokinetic
properties consistent with oral dosing. The aryl-substituted epimi-
nocyclohepta[b]indole motif 1 was identified as a scaffold with the
potential to interact with the 5-HT6 receptor.
NH
O
O
S
Ar
N
1
In order to test this concept, three phenyl-substituted arylsulfones
were synthesized with the arylsulfone located on various positions
of the indole ring. All of the compounds were prepared as race-
mates. 5-HT6 binding affinities were determined via a scintillation
proximity assay using membranes expressing the human 5-HT6
receptor.9 A clear SAR trend developed, with the R8 substituted
phenylsulfone 4 having very good affinity for 5-HT6, whilst the R6
and R7 substituted analogs (2 and 3, respectively) showed a much
weaker interaction with the target. In order to fully characterize
compound 4, it was resolved into its constituent (À) and (+) enan-
tiomers (4a and 4b, respectively) via chiral chromatography. Both
enantiomers had very similar 5-HT6 binding affinities. Determina-
tion of functional activity through measurement of cAMP levels in
a cell-based assay10 showed that both enantiomers were 5-HT6
antagonists, with the (+) enantiomer having slightly improved affin-
ity for the receptor in this instance (Table 1).
Based on these data, SAR development around the 8-substituted
aryl sulfones was undertaken. A general synthesis was developed
⇑
Corresponding author.
E-mail address: alan.henderson@amriglobal.com (A.J. Henderson).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.022

A. J. Henderson et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1494–1498
1495
Table 1
Table 2
SAR of phenylsulfonyl-substituted epiminocyclohepta[b]indoles
SAR of 8-arylsulfonyl epiminocyclohepta[b]indoles
R1
NH
R8
N
O
Ar
O
S
R7
N
R6
N
R2
a,b
b,c,d
Compound
R6
R7
H
R8
5-HT6 Ki
(nM)
5-HT6 Kb
(nM)
Compound
Ar
R1
R2
5-HT6 Kia,b (nM)
4
9
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
2-Fluorophenyl
3-Fluorophenyl
4-Fluorophenyl
2-Chlorophenyl
3-Chlorophenyl
4-Chlorophenyl
3-Trifluoromethylphenyl
3-Trifluoromethoxyphenyl
3-Aminophenyl
3-Hydroxyphenyl
3-Cyanophenyl
H
Me
Et
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
Me
H
8.2
7.8
91
54
52
350
4.6
4.1
60
4.8
1.8
37
2.4
7.2
6.3
11
2
3
4
–SO2Ph
H
H
814
131
8.2
15
H
H
H
H
–SO2Ph
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
H
H
H
–SO2Ph
–SO2Ph
–SO2Ph
4a (À)
11
3.9
Et
4b (+)
9.6
i-Pr
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Displacement of [3H]LSD from human 5-HT6 receptor membranes.9
Values are means of at least two determinations where each determination is
a
b
within 50% of the mean value shown.
Production of cAMP from CHO-1 cells expressing the human 5-HT6 receptor.10
c
d
All compounds were antagonists (<15% agonist activity).
for the rapid production of racemic 8-arylsulfone epiminocyclo-
hepta[b]indoles (Scheme 1). Commercially available 4-bro-
mophenylhydrazine and nortropinone were reacted together under
Fisher-indole11 conditions to generate the epiminocyclohep-
ta[b]indole intermediate. This was isolated and Boc-protected with-
out purification, providing the N-Boc intermediate 5. The yield for
this two-step transformation was low, due to a significant propor-
tion of the hydrazone intermediate apparently failing to cyclize
during the Fisher-Indole cyclization. Various attempts to optimize
this step were unsuccessful. N-methylation of the indole was
achieved under standard conditions to produce 6, which was sub-
jected to a palladium-catalyzed aryl sulfonylation12 using arylsulfi-
nic acid sodium salts (which could be prepared in a single step from
the sulfonyl chloride13) generating 7. The yields for this particular
conversion were substrate-dependent, and although the reaction
generally appeared to progress cleanly using standard monitoring
techniques, high yields were never achieved. Removal of the Boc
group under acidic conditions delivered the target racemates 8 as
hydrochloride salts. These were further purified by chiral chroma-
tography to give the constituent enantiomers if deemed necessary.
Initial SAR development around racemic 8-arylsulfonyl epimi-
nocyclohepta[b]indoles is shown in Table 2. Early efforts were fo-
cused on substitution of the basic nitrogen (R1) where it was
found, through limited analoging, that methyl was tolerated
whereas extending to ethyl had a detrimental effect on 5-HT6 bind-
ing affinity. It was decided to halt exploration of basic nitrogen
substituents at this stage, as it was apparent that no potency gains
were likely, coupled with the fact that the N-alkyl analogs were
predicted to suffer from poor metabolic stability. Next, our atten-
tion turned to the N-substituent of the indole (R2). Once again,
35
a
Displacement of [3H]LSD from human 5-HT6 receptor membranes.9
Values are means of at least two determinations where each determination is
b
within 50% of the mean value shown.
limited SAR exploration was undertaken here, as it was quickly
determined that the methyl substituent was likely optimal based
on the drop in potency for the unsubstituted (11), ethyl (12) and
isopropyl (13) analogs. The main focus of our attention was on
modifications of the pendant arylsulfone (Ar). Initial efforts cen-
tered on substituted phenyl rings. Monosubstitution with either
fluoro or chloro in the 4-position led to a decrease in potency com-
pared to the parent phenyl compound (4 vs 16 and 19, respec-
tively). Substitution at the 2- and 3-position was well tolerated
and a slight increase in potency was observed (14, 15, 17 and
18). In the case of the chloro-substituted analogs, there was a def-
inite preference for meta substitution, with the 3-chlorophenyl
analog 18 being approximately threefold more potent than the 2-
substituted regioisomer 17. Compound 18 was also approximately
fivefold more potent than the parent compound 4 suggesting there
was significant potential to improve the 5-HT6 binding affinity
through arylsulfone substitutions. Taking the slightly improved
potency of 18 into account, as well as the added synthetic chal-
lenge associated with preparing 2-substituted analogs, the SAR
focus was concentrated on 3-substituted arylsulfones. Introduction
of lipophilic substituents such as trifluoromethyl (20, clogP = 4.0)
and trifluoromethoxy (21, clogP = 4.1) were well tolerated, with
20 having similar potency to the 3-chlorophenyl analog 18.
NBoc
NBoc
Br
Br
NH
a,b
c
Br
+
NHNH2
O
N
H
5
N
25-30%
90-95%
over 2 steps
6
NBoc
N
O
O
NH
N
O
O
d
S
e
S
Ar
Ar
•HCl
quant.
24-53%
8
7
Scheme 1. Reagents and conditions: (a) EtOH, HCl, reflux; (b) Boc2O, K2CO3, i-PrOH, H2O, 0 °C to room temperature; (c) NaH, MeI, DMF, 0 °C to room temperature; (d)
ArSO2Na, Pd2(dba)3, xantphos, Cs2CO3, toluene, reflux; (e) HCl, Et2O, MeOH, CH2Cl2, room temperature.

1496
A. J. Henderson et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1494–1498
Somewhat surprisingly, polar substituents were also tolerated,
with the 3-amino compound 22 (clogP = 2.1) and 3-hydroxy ana-
log 23 (clogP = 2.7) having similar affinity to the parent phenyl 4.
This was encouraging, as it offered an opportunity to significantly
alter the physicochemical properties of the scaffold whilst still
retaining 5-HT6 affinity. However, not all substituents were toler-
ated; the 3-cyano analog 24 (clogP = 2.6) was approximately four-
fold less potent for 5-HT6 than the parent compound.
each other. A similar trend was noticed upon determination of
Kb’s, where it was also shown that all of the analogs were 5-HT6
antagonists. In vitro determination of potential metabolic liability
was assessed through percentage of compound remaining after
one hour upon incubation with both rat and human liver micro-
somes. The majority of compounds tested were found to have good
metabolic stability (>50% remaining after 1 h in both species).
Higher significance was placed on compounds that exhibited
>75% of parent remaining after 1 h with trifluoromethylphenyl
analog 20b, both enantiomers of the 1-indolyl (31a and 31b) and
5-indolyl (33a and 33b) and the benzthiophenyl analog 34b having
a particularly good profile. Further profiling in the bidirectional
Caco2 flux assay highlighted potential permeability and efflux is-
sues within the NH indole substituted compounds, with both enan-
Attention was then switched to modifications of the phenyl ring
itself by introducing heteroatoms into the structure (Table 3). All
attempts at replacing the phenyl ring with an unsubstituted pyri-
dine were unsuccessful, with significant drops in potency regis-
tered for all three regioisomers 25, 26 and 27. Similarly,
introduction of a thiophene (28), C-linked pyrazole (29) or N-
linked pyrrole (30) resulted in reduced 5-HT6 affinity, suggesting
that heteroaromatic rings were not well tolerated as phenylsulfo-
nyl replacements. However, this problem was somewhat circum-
vented by introduction of the N-linked indole 31 where a marked
improvement in potency was achieved and sub-nM affinity was
apparent for the first time. Interestingly, the indole ring was found
to be a highly flexible substituent; switching the point of connec-
tivity to the 3-position (32) or 5-position (33) was not detrimental
to 5-HT6 affinity highlighting that both C-linked and N-linked
heteroaryl sulfones could be tolerated. Indeed, the 5-substituted
indole 33 turned out to be extremely potent, having greater affinity
for 5-HT6 than all other compounds. Furthermore, replacement of
the NH of the indole with a sulfur atom (benzthiophene 34) im-
parted a significant increase in potency over the comparable indole
32 (approximately fourfold) suggesting the enhanced 5-HT6 affin-
ity was not due to any H-bonding interactions of the indole NH.
This was confirmed by preparing the N-methyl indole 35, which
was extremely potent.
Table 3
SAR of 8-heteroarylsulfonyl epiminocyclohepta[b]indoles
R1
N
O
Ar
O
S
N
R2
Compound
Ar
R1
R2
5-HT6 Kia,b (nM)
25
26
27
28
29
30
31
32
33
34
35
2-Pyridyl
3-Pyridyl
4-Pyridyl
3-Thiophenyl
3-(1-Methyl)pyrazolyl
1-Pyrrolo
1-Indolyl
3-Indolyl
5-Indolyl
3-Benzthiophenyl
3-(1-Methyl)indolyl
H
H
H
H
H
H
H
H
H
H
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
192
156
>10000
51
1010
32
0.85
1.3
0.32
0.33
0.81
Selected compounds from Tables 2 and 3 were chirally resolved
to deliver their constituent enantiomers, which were subjected to
more intensive in vitro profiling (Table 4). It was found that the
sets of enantiomers had similar profiles with the (À) and (+)
enantiomers having 5-HT6 binding affinities within 1–3-fold of
a
Displacement of [3H]LSD from human 5-HT6 receptor membranes.9
Values are means of at least two determinations where each determination is
b
within 50% of the mean value shown.
Table 4
SAR of chirally pure 8-arylsulfonyl epiminocyclohepta[b]indoles
NH
N
O
Ar
O
S
Compound Ar
Optical rotationa 5-HT6 Ki (nM)b,c 5-HT6 Kb (nM)c,d,e RLM% remaining HLM% Remaining Caco2 Permeability Efflux ratioi
at 1 hf
at 1 hg
A-Bh
18a
18b
20a
20b
31a
31b
32a
32b
33a
33b
34a
34b
3-Chlorophenyl
3-Chlorophenyl
(À)
2.7
1.9
3.7
1.5
1.2
0.68
1.2
0.74
0.37
0.28
0.36
0.28
3.5
4.0
15
12
10
9.7
2.8
2.6
1.6
1.5
3.4
2.5
63
72
65
79
88
86
34
57
77
91
67
80
76
93
78
100
82
96
72
86
89
99
52
78
7.49
8.55
16.5
24.8
15
6
(+)
4.4
2.8
1.8
2.2
1.9
2.5
8.5
40
3-Trifluoromethylphenyl (À)
3-Trifluoromethylphenyl (+)
1-Indolyl
1-Indolyl
3-Indolyl
3-Indolyl
5-Indolyl
5-Indolyl
3-Benzthiophenyl
3-Benzthiophenyl
(À)
(+)
(À)
(+)
(À)
(+)
(À)
(+)
12
0.41
1.05
0.55
ND
0.24
15.7
ND
3.7
2.8
ND, not determined.
a
Optical rotation determined via rotation of polarized light using a polarimeter.
b
c
d
e
f
Displacement of [3H]LSD from human 5-HT6 receptor membranes.9
Values are means of at least two determinations where each determination is within 50% of the mean value shown.
Production of cAMP from CHO-1 cells expressing the human 5-HT6 receptor.10
All compounds were antagonists (<15% agonist activity).
Rat liver microsomes.
g
h
i
Human liver microsomes.
Apical to basolateral flux (Â10À6 cm/s)
Ratio of basolateral to apical flux/apical to basolateral flux.

A. J. Henderson et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1494–1498
1497
Table 5
important as it indicated preferential partitioning of the drug in
the CNS over the periphery (Table 6).
Oral exposure data in Sprague Dawley rats.
Compound 20b was then subjected to pharmacodynamic
assessment through determination of 5-HT6 receptor occupancy.
Occupancy was measured using ex vivo methodology, with autora-
diography techniques employed to determine the level of occu-
pancy14 at three different doses (Fig. 1). Two timepoints were
chosen (6 and 24 h) to obtain an indication of the duration of ac-
tion of compound 20b. It was found that 20b caused a dose-related
increase in 5-HT6 receptor occupancy after 6 h, although there was
a plateau apparent between the 30 and 90 mg/kg doses, where the
occupancy was found to be in the 75–80% range. Interestingly,
5-HT6 receptor occupancy was sustained over 24 h at all doses,
with very little change between the 6 and 24 h timepoints. This
was fully anticipated, as the previously determined half life of
the compound suggested it should have a long duration of action.
In summary, SAR development around the parent 8-arylsulfonyl
epiminocyclohepta[b]indole identified a series of potent 5-HT6
antagonists. In vitro assays were used to triage compounds for pro-
gression into in vivo studies. Through this process, compound 20b
was identified as a 5-HT6 antagonist with excellent oral exposure, a
favorable PK profile and high levels of sustained 5-HT6 receptor
occupancy upon acute dosing. This profile suggests 20b would be
suitable for once-daily dosing.
Compound
Dose (mg/kg)
Cmax (0–6 h) (ng/mL)
Tmax (0–6 h)
20b
31b
30 (po)
30 (po)
732 176
41 3.7
6
6
tiomers of the 3-indolyl (32a and 32b) and the (À) enantiomer of
the 5-indolyl (33a) substituents having poor permeability coupled
with significant efflux. The (À) enantiomer of the 5-indolyl substi-
tuent 33b was not tested in this assay, but was anticipated to have
a similar profile to 33a. Trifluoromethylphenyl analog 20b and 1-
indolyl analog 31b were classified as being highly permeable with
non-significant efflux. These two compounds were selected for
in vivo assessment based on their attractive profile.
Compounds 20b and 31b were orally dosed to Sprague Dawley
rats at 30 mg/kg, and plasma samples were taken over the follow-
ing 6 h to obtain a preliminary indication of oral exposure
(Table 5).
Compound 20b was found to have an excellent PK profile; plas-
ma levels increased steadily up to the terminal point (6 h). 31b had
significantly reduced oral exposure compared to 20b, although,
once again, the Cmax over the 6 h timecourse occurred upon termi-
nation. Based on these findings, 20b was selected for detailed phar-
macokinetic and pharmacodynamic assessment.
Acknowledgments
Upon oral and iv dosing to Sprague Dawley rats, 20b had a
robust PK profile. At a 10 mg/kg oral dose, 20b had a long half life
and excellent bioavailability. This was driven in part by its low
clearance and moderate volume of distribution. The compound
also exhibited a favorable brain to plasma ratio which was
We would like to thank Sharon Cheetham and Jean Viggers of
RenaSci Consultancy for generating receptor occupancy data. We
would also like to thank AMRI’s DMPK group for provision of bio-
analysis data.
Table 6
Detailed PK data for 20b
CLb (mL/min/kg)
5.7
Vss (L/kg)
Fa,b
Brain/plasma ratioa (6 h)
1.3
a
a
a
a
b
Compound
Cmax (ng/mL)
Tmax (h)
t1/2 (h)
AUC0-inf (h ng/mL)
20b
916
4
10
26687
4.7
91%
a
Obtained from 10 mg/kg po dose.
Obtained from 2 mg/kg iv dose.
b
120.0
100.0
80.0
60.0
40.0
20.0
0.0
***
***
***
***
***
***
10 mg/kg
6 H
Vehicle
24 H
Vehicle
6 H
30 mg/kg 90 mg/kg
10 mg/kg 30 mg/kg 90 mg/kg
24 H 24 H 24 H
6 H
6 H
Fig. 1. Ex vivo 5-HT6 receptor occupancy data determined in Sprague Dawley rats.14 All values had p < 0.0001. Receptor occupancy values compared to vehicle (0%) at 6 h
were 60%, 76% and 78% at the 10, 30 and 90 mg/kg doses respectively. Receptor occupancy values compared to vehicle (0%) at 24 h were 48%, 74% and 84% at the 10, 30 and
90 mg/kg doses respectively.

1498
A. J. Henderson et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1494–1498
was calculated using the Cheng–Prusoff equation: (Ki = IC50/(1+(L/KD)), where
References and notes
L = concentration of radioligand in the assay, and KD = affinity of the
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9. Human 5-HT6 binding affinities were measured using a scintillation proximity
assay (SPA) format. Samples were incubated in 50 mM Tris-HCl, pH 7.5, 5 mM
MgCl2, 1 mM EDTA (4% DMSO final) with 10 nM [N-methyl-3H]-LSD (Perkin–
activity was determined using 10 lM 5-HT as the Emax control and was
expressed as the ratio of cAMP produced in a test well to the average cAMP
produced in the Emax control wells. All cell-based experiments were performed
in duplicate using 10 concentrations of test compound. IC50/EC50 values were
determined using XLfit4.1 curve fitting software from IDBS Ltd. For
antagonists, the apparent dissociation constant (Kb) was calculated using a
modified
Cheng–Prusoff
equation:
Kb = IC50/(1 + (A/EC50A)),
where
A = concentration of reference agonist in the assay and EC50A = EC50 value of
the reference agonist. An antagonist was defined as having an Emax <15% in the
agonist mode.
11. Humphrey, G. R.; Kuethe, J. T. Chem. Rev. 2006, 106, 2875.
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beads (PVT-PEI-WGA, GE Healthcare) per well in a final volume of 0.2 mL. Total
binding control contained compound dilution buffer only; nonspecific binding
14. 5-HT6 receptor occupancy was determined in the caudate putamen. Brain
sections were incubated using
[
[
125I]SB258585 (total binding) or
125I]SB258585 and methiothepin (non-specific binding). Radioactivity was
was determined in the presence of 100 lM 5-hydroxytryptamine. Specific
measured in a b-imager. Two determinations were made per animal, and 5
animals were used per dose group. Data was presented as mean specific
binding and as a percentage of the vehicle-treated control which was taken as
100%.
binding was determined by subtracting nonspecific binding from total binding.
All experiments were performed in duplicate using ten concentrations of
competing ligand. IC50 values were determined from specific binding data
using XLfit4.1 curve fitting software from IDBS Ltd. The inhibition constant (Ki)