6-[2-(4-Aryl-1-piperazinyl)ethyl]-2H-1,4-benzoxazin-3(4H)-ones: Dual-acting 5-HT1 receptor antagonists and serotonin reuptake inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.08.084

Investigation of a series 6-[2-(4-aryl-1-piperazinyl)ethyl]-2H-1,4-benzoxazin-3(4H)-ones has led to the discovery of potent 5-HT_1A/1B/1D receptor antagonists with and without additional SerT affinity. Modulation of the different target activities gave compounds with a range of profiles suitable for further in vivo characterization.

Full text of DOI:10.1016/j.bmcl.2008.08.084

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5653–5656
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
6-[2-(4-Aryl-1-piperazinyl)ethyl]-2H-1,4-benzoxazin-3(4H)-ones: Dual-acting
5-HT1 receptor antagonists and serotonin reuptake inhibitors
b
b
b
a
Steven M. Bromidge a, , Barbara Bertani , Manuela Borriello , Stefania Faedo , Laurie J. Gordon ,
Enrica Granci b, Matthew Hill a, Howard R. Marshall a, Luigi P. Stasi b, Valeria Zucchelli b, Giancarlo Merlo b,
Alessia Vesentini b, Jeannette M. Watson a, Laura Zonzini b
*
a Neurosciences CEDD, GlaxoSmithKline, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK
b Neurosciences CEDD, GlaxoSmithKline, Medicines Research Centre, Via A. Fleming 4, Verona 37135, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Investigation of a series 6-[2-(4-aryl-1-piperazinyl)ethyl]-2H-1,4-benzoxazin-3(4H)-ones has led to the
discovery of potent 5-HT1A/1B/1D receptor antagonists with and without additional SerT affinity.
Modulation of the different target activities gave compounds with a range of profiles suitable for further
in vivo characterization.
Received 28 July 2008
Revised 22 August 2008
Accepted 23 August 2008
Available online 28 August 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
5-HT1A/1B/1D receptor antagonists
Serotonin reuptake inhibitors
5-HT1 autoreceptors
Antidepressant
Over the past decades, a large body of pre-clinical and clinical
evidence has confirmed a link between extracellular levels of sero-
tonin (5-HT) and a number of psychiatric indications, including
anxiety and depression.1 In fact, the most effective antidepressant
agents in current clinical use are the selective serotonin reuptake
inhibitors (SSRIs) which inhibit serotonin transporters (SerT) and
consequently elevate synaptic 5-HT levels.2 Despite the success
of SSRIs, one undesirable characteristic is a long latency to thera-
peutic onset which is hypothesized to be due to the requirement
for desensitisation of 5-HT1 autoreceptors to maintain increased
5-HT levels.3
5-HT1 autoreceptors are located on both cell bodies (5-HT1A and
5-HT1D receptor subtypes) and nerve terminals (5-HT1B and 5-HT1D
receptor subtypes). They are widely distributed in the brain and in
addition to SerT are known to have a major role in the control of
synaptic 5-HT levels.4 For instance neurochemical studies have
demonstrated that acute co-administration of SSRIs with 5-HT1A
and/or 5-HT1B/1D receptor antagonists gave a greater increase in
extracellular brain 5-HT levels with respect to SSRIs alone.4,5 Inter-
estingly, microdialysis studies also revealed that concomitant
blockade of 5-HT1 autoreceptors in the absence of a SerT compo-
nent resulted in an immediate increase in central 5-HT levels.5
From the above, it follows that blockade of 5-HT1A/1B/1D autore-
ceptors, with or without concomitant SerT inhibition, should rap-
idly increase brain 5-HT to therapeutically effective levels and
provide a fast onset of antidepressant/anxiolytic action relative to
current therapies. This hypothesis is supported by a meta-analysis
of clinical trials data which suggested that the 5-HT1A autoreceptor
antagonist ( )-pindolol hastens the therapeutic response to SSRIs.6
Previous publications from this group have described the
development of novel compounds containing the 5-(piperidinyl-
ethyloxy)quinoline and benzoxazinone units as both mixed SerT/
5-HT1A/1B/1D receptor antagonists or ‘selective’ 5-HT1A/1B/1D recep-
tor antagonists.7 We also recently disclosed a new class of quinoli-
nyl-piperazinyl ethylbenzoxazinones, in particular SB-744185 (1),
as a highly potent, orally bioavailable and brain penetrant mixed
SSRI-5-HT1A/1B/1D receptor antagonists (Fig. 1).8
This letter reports further exploration of the structure–activity
relationships (SAR) in this novel series. In one aspect, we sought
N
N
H
N
O
N
(1) SB-744185
O
* Corresponding author. Tel.: +44 01279627031; fax: +44 01279622790.
Figure 1. Structure of SB-744185 (1) a dual 5-HT1A/1B/1D receptor antagonist and
E-mail address: steve.bromidge@gsk.com (S.M. Bromidge).
SerT inhibitor.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.084

5654
S. M. Bromidge et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5653–5656
to modulate the different target activities in order to identify 5-
HT1A/1B/1D receptor antagonists with varying levels of selectivity
between the different 5-HT1 autoreceptors and the serotonin
transporter for detailed comparison of their in vivo profiles in
terms of efficacy versus side-effects.
was prepared by alkylation of 2-fluoro-6-nitrophenol 22 with
methyl bromoacetate to give 23 which was then subject to nitro
reduction and concomitant cyclization. The 8- or 7-fluorobenzox-
azinone 24 was converted to 25 by Friedel–Crafts acylation with
chloroacetyl chloride followed by carbonyl reduction to give the
key chloroethyl derivative 26 which was alkylated with
2-methyl-5-(1-piperazinyl)quinoline.
The 5-fluoro analogue 6 was prepared as shown in Scheme 2.
Thus, treatment of chloro-4-fluoro-2-methoxybenzene 27 with
chloroacetyl chloride and aluminum chloride gave acylation and
concomitant O-demethylation to afford the phenol ethanone 28.
Carbonyl reduction to 29, followed by nitration to 30 and subse-
quent hydrogenation gave the amino phenol 31 which was acyl-
ated with chloroacetyl chloride and cyclized to the key
chloroethyl 5-F-benzoxazinone 32 which was finally alkylated
with 2-methyl-5-(1-piperazinyl)-quinoline to afford 6.
The novel compounds9 (Table 1) were prepared as described be-
low. The N-methylated analogue 2 was prepared by alkylation of 1
with MeI using sodium hydride in DMF in 55% yield. The ethyl ana-
logue 3 was prepared by N-alkylation of 6-(2-chloroethyl)-2H-1,4-
benzoxazin-3(4H)-one with ethyl iodide followed by coupling with
2-methyl-5-(1-piperazinyl)quinoline.
The 8- and 7-fluoro analogues 4 and 5 were prepared according
to Scheme 1 from the key appropriately substituted fluorobenzox-
azinone 24. The 7-fluorobenzoxazinone 24b was commercially
available, while the corresponding 8-fluorobenzoxazinone 24a
The analogues 7–10 (Table 2) containing piperazine modifica-
tion were prepared by alkylation of the appropriately substituted
2-methyl-5-piperazinyl-quinolines with 6-(2-chloroethyl)-2H-
1,4-benzoxazin-3(4H)-one. The 2-methyl-5-piperazinyl-quinolines
were themselves prepared by Buchwald amination of 2-methyl-
quinolin-5-yl trifluoromethanesulfonate with the appropriate
unsubstituted or mono BOC protected piperazine. Substituted
quinolines 11–16 (Table 3) were similarly prepared via triflation
of the appropriately substituted 2-methyl-5-hydroxyquinoline
and Buchwald amination with BOC-piperazine.10 Compounds 17–
21 (Table 4) were prepared as above by alkylation of the appropri-
ate commercially available or known arylpiperazines with 6-(2-
chloroethyl)-2H-1,4-benzoxazin-3(4H)-one.
The affinities of the compounds for h5-HT1A, h5-HT1B, and h5-
HT1D receptors, stably expressed in CHO cells, were evaluated by
displacement binding using [3H]-WAY100635 (5-HT1A) and [3H]-
5-HT (5-HT1B and 5-HT1D), respectively, as radioligands. The affin-
ities of the compounds for the reuptake site of the hSerT, stably
expressed in epithelial pig kidney (LLCPK) cells, were assessed by
displacement of [3H]-citalopram binding.9 The intrinsic activities
Table 1
Binding affinities (pKia) for human 5-HT1A/1B/1D and SerT and intrinsic activity (IA):
substitution of the benzoxazinoneb
N
N
R1
N
5
8
N
O
7
R2
O
Compound
R1
R2
pKi
5-HT1A (IA)
5-HT1B (IA)
5-HT1D (IA)
SerT
1
2
3
4
5
6
H
H
H
H
8-F
7-F
5-F
9.6 (0.2)
9.4 (0.3)
9.3 (0.0)
8.9 (0.0)
9.6 (0.2)
9.6 (0.3)
9.3 (0.1)
9.1 (0.2)
9.0 (0.0)
8.8 (0.3)
9.4 (0.1)
8.9 (0.2)
9.8 (0.0)
9.5 (0.0)
9.4 (0.0)
9.7 (0.0)
9.6 (0.0)
9.4 (0.0)
8.8
7.7
7.1
8.9
9.1
8.3
Me
Et
H
H
H
a
Each pKi determination lies within 0.3 log units of the mean with a minimum of
3 replicates.
b
Compounds were characterized and purity assessed using 1H NMR and LC–MS.
O
H
N
H
N
NO2
NO2
O
Cl
O
(i)
(ii)
(iii)
N
OMe
7
7
OH
22
O
O
O
8
8
F
24 a) = 8-F
b) = 7-F
F
F
F
O
25 a) = 8-F
23
b) = 7-F
H
N
Cl
O
N
(v)
(iv)
H
N
O
N
7
O
8
F
7
4 = 8-F
5 = 7-F
O
26 a) = 8-F
b) = 7-F
8
F
Scheme 1. Reagents and conditions: (i) BrCH2CO2Me, K2CO3, acetone, reflux, 5 h (93%); (ii) iron powder, acetic acid, rt, 14 h (71%); (iii) ClCH2COCl, AlCl3, 80 °C, 5 h (50–65%);
(iv) triethylsilane, TFA, 0 °C to rt, overnight (72–91%); (v) 2-methyl-5-(1-piperazinyl)quinoline, NaI/Na2CO3, NMP, 120 °C, 12 h (24–43%).
O
Cl
Cl
Cl
Cl
Cl
Cl
Cl
(i)
(iii)
(ii)
OH
OMe
F
F
OH
F
OH
F
29
30
27
28
NO2
F
H
(vii)
Cl
N
N
F
Cl
N
O
(v)
(vi)
H
N
(iv)
N
O
OH
F
O
6
32
31
NH2
O
Scheme 2. Reagents and conditions: (i) ClCH2COCl, AlCl3, 70 °C, 3 h (38%); (ii) triethylsilane, TFA, 0 °C to rt, 2 h (42%); (iii) cHNO3, glacial acetic acid, 0 °C, 45 min (70%); (iv)
H2, 10% Pd/C, EtOH, atmosphere/rt, 24 h (100%); (v) ClCH2COCl, NaHCO3, THF, 0 °C, 30 min; (vi) K2CO3, butan-2-one, reflux, 1 h (71%); (vii) 2-methyl-5-(1-
piperazinyl)quinoline, NaI/Na2CO3, NMP, 120 °C, 3 h (46%).
1

S. M. Bromidge et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5653–5656
5655
Table 2
Table 4
Binding affinities (pKia) for human 5-HT1A/1B/1D and SerT and intrinsic activity (IA):
Binding affinities (pKia) for human 5-HT1A/1B/1D and SerT and intrinsic activity (IA):
substitution of the piperazine ringa
replacement of the quinoline ringa
Ar
N
H
H
N
N
O
N
N
linker
O
O
O
Compound
Ar
pKi
Compound
Linker
pKi
5-HT1A (IA)e
9.6 (0.2)
5-HT1B (IA)
5-HT1D (IA)
9.8 (0.0)
SerT
8.8
d
5-HT1A (IA)e
5-HT1B (IA)
5-HT1D (IA)
9.8 (0.0)
SerT
8.8
d
1
7
8
9
9.6 (0.2)
8.5 (0.0)
8.8 (0.0)
9.1 (0.2)
6.9 (0.8)
9.3 (0.1)
N
N
N
1
9.3 (0.1)
N
N
N
N
8.1 (0.0)
8.7 (0.0)
9.1 (0.1)
6.9 (0.2)
8.8 (0.0)
9.3 (0.0)
9.5 (0.0)
7.6 (0.0)
9.4
9.1
8.0
8.4
17
18
N
8.8 (1.0)
9.4 (0.8)
9.2 (0.5)
9.4 (0.7)
9.6 (0.4)
9.6 (0.7)
9.1
8.0
N
N
N
N
19
20
8.5 (0.6)
9.3 (0.3)
9.7 (1.2)
9.2 (0.2)
9.4 (0.4)
9.2 (0.6)
9.5 (0.0)
9.7 (0.4)
9.8 (0.5)
8.7
8.2
8.6
N
10
N
N
a
See Table 1.
N
O
21
Table 3
Binding affinities (pKia) for human 5-HT1A/1B/1D and SerT and intrinsic activity (IA):
a
substitution of the quinoline ringa
See Table 1.
R2
N
N
Table 5
Rat pharmacokinetic profile for compounds 1, 2, and 19
H
N
N
O
R1
Compound
Cli rat; humana
ml/min/g liver
CLbb
ml/min/kg
Vss
(L/kg)
t1/2
(h)
Fpo
(%)
Br:Bl
O
Compound
R1
R2
pKi
1
2
19
1.3; <0.5
0.8; 1.1
ND
5
8
4
2.0
2.7
1.1
5.1
4.4
2.8
68
59
67
1.6
2.2
0.3
5-HT1A (IA)e
5-HT1B (IA)
5-HT1D (IA)
SerT
d
1
11
12
13
14
15
16
Me
Me
Me
Me
Me
Me
Ph
H
8-F
9.6 (0.2)
8.4 (0.2)
8.6 (0.7)
9.5 (0.0)
9.9 (0.4)
9.2 (1.2)
7.0 (0.4)
9.3 (0.1)
8.6 (0.0)
9.3 (0.3)
9.2 (0.0)
9.5 (0.0)
9.1 (0.3)
7.5 (0.2)
9.8 (0.0)
9.5 (0.0)
9.7 (0.0)
9.7 (0.0)
9.8 (0.0)
9.4 (0.3)
7.9 (0.0)
8.8
8.4
8.8
9.1
9.1
7.8
9.8
a
Intrinsic clearance in liver microsomes.
In vivo data determined by 0.5 mg/kg iv and 1 mg/kg po administration in rat
b
8-Cl
7-F
7-Cl
7-Me
H
reduced 5-HT1A/1B/1D affinities by about an order of magnitude
while SerT affinity was maintained or even increased. The data
suggest the (S)-enantiomer 7 has somewhat higher affinity for SerT
than 5-HT1A/1B/1D receptors compared to the (R)-enantiomer 8. In
contrast, the racemic 2-methyl analogue 9 showed reduced SerT
affinity relative to 1 but maintained sub-nanomolar 5-HT1A/1B/1D
target affinities combined with low intrinsic activities. The bridged
piperazine 10 suffered a drop in all target activities in particular for
a
See Table 1.
of the compounds at h5-HT1A, h5-HT1B and h5-HT1D receptors were
determined using a [35S]-GTP
cS binding assay, with data reported
relative to the maximum response elicited by 5-HT.9
The initial focus of this work centered on exploration of the ben-
zoxazinone moiety (Table 1). Our previous studies7 suggested the
presence of the secondary lactam N–H is crucial for SerT activity
and so the alkylated analogues 2 and 3 were prepared. N-substitu-
tion with methyl or ethyl maintained the impressive sub-nanomo-
lar 5-HT1A/1B/1D receptor affinities and low intrinsic activities with
the anticipated reduction in SerT affinities relative to 1 resulting
in at least 25- and 80-fold selectivity for 5-HT1A/1B/1D receptors over
SerT for 2 and 3, respectively. The effects of introducing fluoro sub-
stituents into available positions on the benzoxazinone aromatic
ring was also explored (Table 1). While the 8-fluoro 4 and 7-fluoro
5 analogues had profiles almost identical to 1, the 5-fluoro analogue
6 maintained high 5-HT1A/1B/1D receptor affinities with a modest
reduction in SerT.
5-HT1A/1B/1D
.
Introduction of substituents into the quinoline ring also re-
vealed a range of interesting profiles and subtle effects on intrinsic
activity (Table 3). 8-Fluoro substitution 11 afforded ꢀ10-fold drop
in 5-HT1A/1B affinities but maintained 5-HT1D and SerT affinities.
The corresponding 8-chloro analogue 12 had a similar profile but
with increased intrinsic activity at 5-HT1A indicative of partial
agonism in this particular recombinant receptor system. In con-
trast, the 7-fluoro substitution 13 maintained high and balanced
affinities across all four targets with zero intrinsic activities. The
7-chloro analogue 14 gave the highest 5-HT1A affinity yet observed,
but again accompanied by increased 5-HT1A intrinsic activity.
Interestingly, the corresponding 7-methyl analogue 15 showed a
similar 5-HT1A/1B/1D affinity profile but even higher 5-HT1A intrinsic
A limited exploration of the effects of substitution of the pipera-
zine was also undertaken (Table 2). 3-Methyl substitution (7 and 8)

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S. M. Bromidge et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5653–5656
activity (IA = 1.2, i.e., full agonist) and a 10-fold drop in SerT affin-
ity. Replacing the 2-methyl quinoline substituent with phenyl 16
gave the highest SerT activity yet achieved in conjunction with a
dramatic drop in 5-HT1A/1B/1D affinities (ꢀ100-fold selectivity for
SerT).
A selection of aryl replacements for the 5-quinoline were also
introduced (Table 4). In general 5-HT1A/1B/1D sub-nanomolar affin-
ities were maintained with good SerT affinity. Notably, significant
increases in intrinsic activity at all three receptors were observed,
particularly for 5-HT1A, highlighting once again strict structural
requirements for maintaining low intrinsic activity.
References and notes
1. (a) Hirschfeld, R. M. A. J. Clin. Psychiatry 2000, 61(Suppl. 6), 4; (b) Lucki, I. Biol.
Psychiatry 1998, 44, 151; (c) Naughton, M.; Mulrooney, J. B.; Leonard, B. E.
Human Psychopharmacol. 2000, 15, 397.
2. Vaswani, M.; Linda, F. K.; Ramesh, S. Prog. Neuro-Psychopharm. Biol. Psychiatry
2003, 27, 85.
3. (a) Wood, M. D.; Thomas, D. R.; Watson, J. M. Exp. Opin. Investig. Drugs 2002, 11,
457; (b) Blier, P.; De Montigny, C. Neuropsychopharmacology 1999, 21, 91S.
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Pharmacol. 2004, 8, 251.
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7. (a) Atkinson, P. J.; Bromidge, S. M.; Duxon, M. S.; Gaster, L. M.; Hadley, M. S.;
Hammond, B.; Johnson, C. N.; Middlemiss, D. N.; North, S. E.; Price, G. W.; Rami,
H. K.; Riley, G. J.; Scott, C. M.; Shaw, T. E.; Starr, K. R.; Stemp, G.; Thewlis, K. M.;
Thomas, D. R.; Thompson, M.; Vong, A. K. K.; Watson, J. M. Bioorg. Med. Chem.
Lett. 2005, 15, 737; (b) Lovell, P. J.; Blaney, F. E.; Goodacre, C. J.; Scott, C. M.;
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Bioorg. Med. Chem. Lett. 2007, 17, 1033; (c) Ward, S. E.; Johnson, C. N.; Lovell, P.
J.; Scott, C. M.; Smith, P. W.; Stemp, G.; Thewlis, K. M.; Vong, A. K.; Watson, J. M.
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W.; Starr, K. R.; Watson, J. M. Bioorg. Med. Chem. Lett. 2008, submitted (BMCL-
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9. Bertani, B.; Borriello, M.; Bozzoli, A.; Bromidge, S. M.; Granci, E.; Leslie, C.;
Serafinowska, H.; Stasi, L.; Vong, A.; Zucchelli, V. WO Patent 2004046124, 2004.
10. 5-Hydroxy-2,7-dimethylquinoline was prepared by bromination of 2,7-
dimethyl-5,6,7,8-tetrahydroquinoline-5-one followed by heating under basic
conditions. 5-Hydroxy-2-phenylquinoline was prepared by demethylation of
5-methoxy-2-phenylquinoline (Ubeda, J. I.; Villacampa, M.; Avendano, C.
Synthesis 1998, 8, 1176) using HBr.
Having successfully identified compounds with a range of
pharmacological profiles across 5-HT1 autoreceptors and SerT,
their suitability for progression to in vivo studies was inves-
tigated in rat pharmacokinetic studies. Several compounds
such as
2 and 19 demonstrated good oral bioavailability
combined with reasonable half-lives and high brain exposure
(Table 5).
In conclusion, exploration of SAR of a series 6-[2-(4-aryl-1-pip-
erazinyl)ethyl]-2H-1,4-benzoxazin-3(4H)-ones led to the discovery
of highly potent 5-HT1A/1B/1D receptor antagonists both with and
without additional SerT activity. Modulation of the balance be-
tween the different target affinities allowed us to identify tool
compounds for in vivo comparison of efficacy versus side-effect
profiles with the ultimate aim to identify faster acting antidepres-
sants with reduced side-effect burden.