3,4-Dihydro-2H-benzoxazinones as dual-acting 5-HT1A receptor antagonists and serotonin reuptake inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.11.031

Investigation of halogen substitution in lead compound 1 has led to the identification of analogues which combine high affinity for 5-HT_1A receptors and potent serotonin reuptake inhibitory activity. Several compounds show an improved selectivity over 5-HT_1B and 5-HT_1D receptors and a superior pharmacokinetic profile in the rat.

Full text of DOI:10.1016/j.bmcl.2006.11.031

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1033–1036
3,4-Dihydro-2H-benzoxazinones as dual-acting 5-HT1A receptor
antagonists and serotonin reuptake inhibitors
Peter J. Lovell,a,* Frank E. Blaney,b Caroline J. Goodacre,a Claire M. Scott,a
Paul W. Smith,a Kathryn R. Starr,a Kevin M. Thewlis,a Antonio K. K. Vong,a
Simon E. Warda and Jeannette M. Watsona
aPsychiatry Centre of Excellence for Drug Discovery, GlaxoSmithKline, New Frontiers Science Park, Third Avenue,
Harlow, Essex CM19 5AW, UK
bDepartment of Computational and Structural Sciences, GlaxoSmithKline, New Frontiers Science Park, Third Avenue,
Harlow, Essex CM19 5AW, UK
Received 27 October 2006; revised 8 November 2006; accepted 12 November 2006
Available online 15 November 2006
Abstract—Investigation of halogen substitution in lead compound 1 has led to the identification of analogues which combine high
affinity for 5-HT1A receptors and potent serotonin reuptake inhibitory activity. Several compounds show an improved selectivity
over 5-HT1B and 5-HT1D receptors and a superior pharmacokinetic profile in the rat.
Ó 2006 Elsevier Ltd. All rights reserved.
H
Drugs which selectively inhibit the reuptake of serotonin
N
O
(SSRIs) and therefore elevate 5-HT levels in the brain
are the most effective antidepressant agents in use.
Although they offer several advantages over the older
tricyclic antidepressants, they still suffer several limita-
tions. They are only effective in approximately 70% of
the depressed population and induce side effects such
as nausea and sexual dysfunction.1 In addition, several
weeks of treatment with SSRIs are required before the
onset of antidepressant activity.2 The latency to thera-
peutic onset is thought to be due to the time taken to
desensitise 5-HT1A autoreceptors and SSRIs only acute-
ly elevate brain 5-HT levels after this desensitisation
process has occurred.3 Therefore, a combined SSRI-5-
HT1A receptor antagonist should have a faster onset
of action than an SSRI alone. Indeed, in preclinical
studies, co-administration of a 5-HT1A antagonist with
an SSRI results in an immediate increase in CNS 5-
HT levels4 and also shortens the onset of anxiolytic
activity in a rat model of anxiety.5
N
O
O
N
1
A series of 3,4-dihydro-2H-benzoxazinones 1 have al-
ready been described (inter alia) as 5-HT1A receptor li-
gands with potent 5-HT reuptake inhibition.7
However, further profiling highlighted significant affini-
ty for 5-HT1B and 5-HT1D receptors and only moderate
in vivo metabolic stability (CLb 50 mL/min/kg) in rat.
This Letter now describes the further optimization of
the pharmacological and pharmacokinetic profiles of
this series of compounds.
In an attempt to rationalise the in vivo clearance data,
in vitro metabolic studies were carried out. Metabolite
identification using rat microsomes suggested that both
the quinoline and benzoxazinone ring systems in 1 were
potentially vulnerable to oxidation and prompted the
preparation of halogenated analogues. Halogenated
quinoline and benzoxazinone analogues were prepared
according to Schemes 1 and 2.8 Reaction of di- and
Furthermore, pindolol (a 5-HT1A receptor antagonist)
has been reported to accelerate the antidepressant action
of SSRIs in several clinical trials.6
Keywords: 5-HT1A; SSRI; Benzoxazinone.
*
Corresponding author.
Peter.J.Lovell@gsk.com
Tel.:
+44
1279
627679; e-mail:
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.11.031

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P. J. Lovell et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1033–1036
Scheme 1. Reagents and condition: (i) crotonaldehyde, 5 N HCl, reflux (22–86%); (ii) NaOMe, MeOH (38–79%), then BBr3, CH2Cl2 (51–82%); (iii)
H2, Pd/C, EtOH then BBr3, CH2Cl2 (48–78%); (iv) BBr3, CH2Cl2 (51–82%); (v) BrCH2CH2Br, K2CO3, MEK, 85 °C (71–81%).
Scheme 2. Reagents and condition: (i) HNO3, AcOH (58–89%); (ii) NaBH4, MeOH (79–99%); (iii) NBS, PPh3 (48–76%); (iv) P(OEt)3; (v) N-Boc-4-
piperidone, t-BuOK, THF (74–88% over two steps); (vi) LiCl, DMF (34–57%); (vii) H2, Pd/C, EtOH (78–99%); (viii) chloroacetylchloride (63–74%);
(ix) trifluoroacetic acid, CH2Cl2 (76–99%); (x) diisopropylethylamine, isopropanol, reflux (39–74%).
tri-substituted anilines with crotonaldehyde gave the
corresponding quinolines 2–6. The 5-halo substituent
in both 2 and 3 could be selectively displaced with sodi-
um methoxide and then reacted with BBr3 to afford 7-
halo quinolinols 7 and 8. Similarly, the 8-halo quinoli-
nols 10 and 11 were derived from methoxy precursors
5 and 6, respectively. Regioselective synthesis of the 6-
fluoroquinoline analogue 9 however required the use
of a blocking group; an ortho-bromo substituent was
used to control the Skraup reaction as this could be
readily removed by hydrogenation. Alkylation of phe-
nols 7–11 was achieved using 1,2 dibromoethane to af-
ford the coupling partners 12–16. The synthesis of
halogenated benzoxazinones (Scheme 2) required con-
struction of the parent ring system. Nitration of substi-
tuted anisoles gave compounds 17–19. Reduction of the
aldehyde to the benzyl alcohol allowed formation of the
benzyl bromide using NBS and subsequent transforma-
tion into the benzyl phosphonate. Wadsworth–Emmons
coupling with N-Boc-4-piperidone ensued in high yield
and was followed by a straightforward three-step se-
quence for the construction of the oxazinone ring.
Removal of the Boc-protecting group gave piperidines
20–22 which were coupled with 12–16 to afford target
compounds 23–37.
All compounds were evaluated using the displacement
of tritiated WAY100635 from human cloned 5-HT1A
receptors expressed in CHO cells.9 The potency at the
5-HT reuptake site (serotonin transporter, SerT) was as-
sessed by measuring the inhibition of reuptake of tritiat-
ed 5-HT into rat cortical synaptosomes.10
Halogenation at either the 6- or 8-position of the
quinoline ring gave a 10-fold reduction in potency at
5-HT1A receptors (Table 1) when compared to 1. In con-
trast, 5-HT1A affinity could be maintained with a small
substituent at the quinoline 7-position, but more inter-
estingly this modification introduced high selectivity
over 5-HT1B and 5-HT1D receptors. These selectivity
data could be rationalised by docking 1 into our
5-HT1 receptor homology models (Fig. 1).11 These sug-
gest that a 7-F or 7-Cl substituent could be accommo-
dated by the 5-HT1A receptor and would reside
Table 1. 5-HT1 receptor binding affinities, SerT potencya,b,c
Compound
R4
5-HT1A pKia
5-HT1B pKia
5-HT1D pKia
SerT pKi
1
23
24
25
26
27
28
H
8.6
7.7
7.4
9.1
8.8
7.7
7.3
8.0
6.1
8.8
7.1
7.2
6.4
6.1
6.6
8.6
8.1
8.1
7.9
7.8
8.0
7.0
8.0
8-F
8-Cl
7-F
7-Cl
7-CN
6-F
6.8
<6.0
5.9
6.0
7.5
a All pKi values represent the mean of at least three experiments, each within 0.3 of the mean.
b Receptors and radioligands used in binding assays: 5-HT1B human cloned receptors in CHO cells, [3H]5-HT; 5-HT1D human cloned receptors on
CHO cells, [3H]5-HT.
c All new compounds gave satisfactory NMR, MS and analytical data.

P. J. Lovell et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1033–1036
1035
between residues Ile 310 and Phe 309. However, these
residues are leucine and tryptamine, respectively, in 5-
HT1B and 5-HT1D receptors which implies that the pri-
mary origins of selectivity are due to a steric interaction.
Encouragingly, PK profiling of both 25 and 26 in the rat
revealed an increase in metabolic stability (Table 2),
which substantiated our in vitro metabolism data.
CNS penetration however remained poor and modifica-
tion of the benzoxazinone now became the focus for fur-
ther investigation.
In general, halogen substitution on the benzoxazinone
ring (compounds 29–31) was well tolerated with the 7-
fluoro isomer 30 showing some enhancement in 5-
HT1A binding (Table 3). As expected, the electron-rich
8-position of the benzoxazinone ring showed the great-
est susceptibility towards oxidation. Indeed, pharmaco-
kinetic profiling of all three fluoro benzoxazinone
isomers (Table 2) revealed that 31 achieved the greatest
increase in metabolic stability. More importantly the 5-
fluoro isomer 29 showed a dramatic 10-fold increase in
brain to blood ratio compared to 1, presumably due to
a modification of polarity by a neighbouring group
effect.
Figure 1. Compound 1 docked into a 5-HT1A homology model.11
Table 2. Rat PK profile for halogenated analoguesa
Compound Clb Fpo Brain:
(mL/min/kg) blood ratio
50 0.4
1
45
Further investigation now focused on the effects of com-
bining halogenation in both of the quinoline and ben-
zoxazinone rings. This strategy led to the identification
of several highly potent and selective 5-HT1A receptor
antagonists 32–37 with potent 5-HT reuptake inhibition
(Table 3). No significant improvements to metabolic sta-
bility were made with these analogues (Table 2). Howev-
er, CNS penetration studies again revealed the beneficial
effects of benzoxazinone substitution, in particular the 5-
fluoro analogue 32 showed a dramatic increase in brain
to blood ratio. Oral bioavailability in the rat was good
for analogues 32–37 and would not present a limiting
factor for subsequent in vivo experiments. The dihalo-
genated analogues were further profiled against a range
Halogenated
quinolines
25
26
25
25
41
56
—
0.2
Halogenated 29
benzoxazinones 30
31
41
36
22
4.0
—
—
33
Dihalogenated
analogues
32
33
34
35
36
37
27
28
19
18
25
16
39
43
54
60
88
51
6.4
1.2
0.4
a 3 mg/kg dose administered orally.
Table 3. Receptor binding affinities in radioligand binding assaysa,b
Compound
R5
R4
pKi 5-HT
pKi
pKi
dopamine
1A
1B
1D
SerT
2A
2B
2C
6
7
a1b
D2
D3
1
29
30
31
32
33
34
35
36
37
H
H
8.6
8.6
9.3
8.3
8.8
8.6
9.4
8.6
8.4
8.0
8.0
8.6
7.7
9.1
6.4
5.8
6.0
5.7
6.5
6.2
8.8
9.1
8.5
9.4
7.0
6.2
6.6
6.1
7.1
6.2
8.1
7.8
8.1
8.2
7.6
7.2
8.1
7.8
7.2
7.2
5.5
—
5.8
—
<5.1
—
<5
—
—
—
<5
<5
<6
6.7
—
5.6
—
5.9
—
6.2
—
5-F
7-F
8-F
5-F
5-F
7-F
7-F
8-F
8-F
H
H
—
—
—
—
—
—
—
—
—
—
—
—
—
—
H
7-F
7-Cl
7-F
7-Cl
7-F
7-Cl
5.7
5.4
5.7
<5
<5
5.5
—
5.6
5.4
5.9
<5.3
<5
6.9
6.9
7.4
7.4
7.1
7.1
5.9
6.4
6.1
5.6
<6
5.8
5.9
5.9
5.7
5.5
<5
5.6
6.1
6.0
6.3
5.7
5.4
5.9
5.8
6.0
<6
<5.1
5.5
5.1
<5.2
<5
5.6
a All pKi values represent the mean of at least three experiments, each within 0.3 of the mean.
b Receptors and radioligands used in binding assays: 5-HT1A/1B/1D receptors as above; 5-HT2A (human cloned receptors in HEK 293 cells; [3H]-
ketanserin); 5-HT2B (human cloned receptors in HEK 293 cells; [3H]5-HT); 5-HT2C (human cloned receptors in HEK 293 cells; [3H]-mesulergine);
5-HT6 (human cloned receptors in HeLa cells; [3H]-LSD); 5-HT7(a) (human cloned receptors in HEK 293 cells; [3H]-5-CT); D2 (human cloned
receptors in CHO cells; [125I]-iodosulpiride); D3 (human cloned receptors in CHO cells; [125I]-iodosulpiride).

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P. J. Lovell et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1033–1036
of monoamine receptors (Table 3) and generally were
found to be highly selective, though modest affinity
was seen for 5-HT7 receptors.
HT1B and 5-HT1D subtypes, which can be attributed to
halogen substitution at the quinoline 7-position. Halo-
gen substitution also has a marked effect on the pharma-
cokinetic profile in the rat and has afforded compounds
with improved metabolic stability and CNS penetration
which were suitable for in vivo evaluation.
A decision to further profile compounds 34 and 35 was
based on their excellent 5-HT1A and SerT activities, 100-
fold selectivity over 5-HT1B and 5-HT1D receptors and
promising PK profiles. The functional activity of com-
pounds 34 and 35 was measured using GTPcS binding
in HEK293 cells expressing 5-HT1A receptors, with
intrinsic activity (IA) being expressed relative to the 5-
HT response (5-HT = 1).9 Neither compound displayed
5-HT1A receptor agonist activity, giving IA values of
zero.
References and notes
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De Montigny, C.; Chaput, I.; Blier, P. Int. Acad. Biomed.
Drug Res. 1993, 5, 8.
In vitro radioligand binding studies in native tissue from
several species (rat, guinea pig, marmoset) with 34 and
35 showed nanomolar affinity (pKi > 9) for the high ago-
nist affinity state of 5-HT1A receptors. A minimal differ-
ence was observed for the low agonist binding affinity
state which is consistent with both compounds showing
low intrinsic activity.
3. De Montigny, C.; Chaput, I.; Blier, P. J. Clin. Psycho-
pharmacol. 1987, 7(6 Suppl.), 24S.
4. Romero, L.; Hervas, I.; Artigas, F. Neurosci. Lett. 1996,
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5. Duxon, M. S.; Starr, K. R.; Upton, N. Br. J. Pharmacol.
2000, 130, 1713.
6. (a) Artigas, F.; Perez, V.; Alvarez, E. Arch. Gen. Psychi-
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Watson, J. P.; Isaac, M. T. J. Affect. Disord. 1997, 44, 101;
(c) Perez, V.; Gilaberte, I.; Faries, D.; Alvarez, E.; Artigas,
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Franchini, L.; Sforzini, L.; Gasperini, M.; Smeraldi, E.;
Perez, J. J. Clin. Psychopharmacol. 1997, 17, 446; (e)
Puzantian, T.; Kawase, K. Pharmacotherapy 1999, 19,
205; (f) Perez, V.; Soler, J.; Puigdemont, D.; Alvarez, E.;
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7. 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(3), 737.
Ex vivo binding studies with both 34 and 35 showed a
dose dependent displacement of both [3H]WAY100635
and [3H]8-OH DPAT binding in rat cortex. Approxi-
mately 50% inhibition of binding of both radioligands
was observed with 34 after a 1 mg/kg oral dose, suggest-
ing good in vivo occupancy of 5-HT1A receptors. This
was further supported in a 5-HT1A receptor pharmaco-
dynamic model12 in which 35 attenuated 8-OH DPAT-
induced locomotor activity (LMA) in rats with an
ED50 of 3 mg/kg following oral dosing. There were no
effects on LMA per se up to 10 mg/kg.
Ex vivo [3H]5-HT uptake studies show that both com-
pounds also inhibited uptake in rat cortical synapto-
somes with an ED50 of 3–5 mg/kg, demonstrating
similar in vivo occupancy of SerT.
8. Johnson, C. N.; Rami, H. K.; Stemp, G.; Thewlis, K.;
Thompson, M.; Vong, A. K. K.; WO patent 2002034754,
2002.
9. Gaster, L. M.; Wyman, P. A.; Flynn, S. T.; WO patent 99/
07700, 1999.
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pharmacology 1987, 93, 193.
11. Construction of receptor models was based on the X-ray
crystal structure of bovine rhodopsin and the sequence
homology with 5-HT1 receptors; Palczewski, K.; Kuma-
saka, T.; Hori, T.; Behnke, C. A.; Motoshima, H.; Fox, B.
A.; Le Trong, I.; Teller, D. C.; Okada, T.; Stenkamp, R.
E.; Yamamoto, M.; Miyano, M. Science 2000, 289, 739.
12. Forster, E. A.; Cliffe, I. A.; Bill, D. J.; Dover, G. M.;
Jones, D.; Reilly, Y.; Fletcher, A. Eur. J. Pharmacol. 1995,
281(1), 81.
Similarly, 34 potently inhibited 8-OH DPAT-induced
LMA with an ED50 < 1 mg/kg, which is consistent with
34 showing a 10-fold greater affinity for 5-HT1A
receptors.
In conclusion, halogenation of both the quinoline and
benzoxazinone rings present in 1 has led to the discovery
of highly potent and dual-acting 5-HT1A receptor antag-
onists and serotonin reuptake inhibitors. The dihalogen-
ated analogues display an excellent selectivity profile
over a range of monoamine receptors, in particular 5-