Discovery of novel and selective tertiary alcohol containing inhibitors of the norepinephrine transporter

Article abstract of DOI:10.1016/j.bmcl.2005.12.061

A novel series of tertiary alcohol containing 2-substituted benzyl morpholines have been discovered as potent and selective inhibitors of the norepinephrine transporter. Efficient synthetic routes were developed featuring a highly diastereoselective nucleophilic addition of benzyl Grignard reagents to enantiopure (4-benzylmorpholin-2-yl)phenylmethanone (11) as the key synthetic step. In vitro binding affinity for the norepinephrine, dopamine and serotonin transporters and in vivo examination of a select compound (16) in a pharmacodynamic animal model for norepinephrine reuptake inhibition are presented.

Full text of DOI:10.1016/j.bmcl.2005.12.061

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2022–2025
Discovery of novel and selective tertiary alcohol
containing inhibitors of the norepinephrine transporter
Manuel J. Cases-Thomas,a,* John J. Masters,b Magnus W. Walter,a Gordon Campbell,a
Louise Haughton,a Peter T. Gallagher,a David R. Dobson,a Vincent Mancuso,c
Benjamin Bonnier,c Thierry Giard,c Thierry Defrance,c Michel Vanmarsenille,c
Andrew Ledgard,a Craig White,a Sivi Ouwerkerk-Mahadevan,c Francoise J. Brunelle,c
Nancy A. Dezutter,c Camy A. Herbots,c Joel Y. Lienard,c Jeremy Findlay,a
Lorna Hayhurst,a John Boot,a Linda K. Thompsonb and Susan Hemrick-Lueckeb
aEli Lilly and Company Ltd, Lilly Research Centre, Erl Wood Manor, Windlesham, Surrey GU20 6PH, UK
bEli Lilly and Company, Lilly Research Laboratories, Indianapolis, IN 46285, USA
cEli Lilly and Company, Lilly Development Centre, S.A. Mont-Saint-Guibert 1348, Belgium
Received 26 October 2005; revised 16 December 2005; accepted 19 December 2005
Available online 18 January 2006
Abstract—A novel series of tertiary alcohol containing 2-substituted benzyl morpholines have been discovered as potent and selec-
tive inhibitors of the norepinephrine transporter. Efficient synthetic routes were developed featuring a highly diastereoselective
nucleophilic addition of benzyl Grignard reagents to enantiopure (4-benzylmorpholin-2-yl)phenylmethanone (11) as the key syn-
thetic step. In vitro binding affinity for the norepinephrine, dopamine and serotonin transporters and in vivo examination of a select
compound (16) in a pharmacodynamic animal model for norepinephrine reuptake inhibition are presented.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Monoamine reuptake inhibitors are widely used for the
treatment of major psychiatric disorders1 (Fig. 1).
taTM).4 Given the high degree of validation of mono-
amine reuptake inhibition as a clinically proven mode
of action, novel compounds with improved metabolic
Selective serotonin reuptake inhibitors (SSRIs), such as
fluoxetine (ProzacTM), have been widely used to treat pa-
tients with depression. Selective norepinephrine reup-
take inhibitors (SNRIs) are also used for the
treatment of depression (reboxetine 3, EdronaxTM)2
and the treatment of attention deficit hyperactivity dis-
order (atomoxetine 2, StratteraTM).3 More recently, a
compound with a dual activity profile, i.e., exhibiting
reuptake inhibition of both serotonin and norepineph-
rine, has been approved by the FDA for treatment of
depression and the management of pain associated with
diabetic peripheral neuropathy (duloxetine 4, Cymbal-
O
R
O
O
O
O
S
N
H
N
N
H
H
(+/-)-3 Reboxetine
4 Duloxetine
(+/-)-1 R = 4-CF3 (Fluoxetine)
2 R = 2-Me (Atomoxetine)
O
N
R
H
S
N
O
HO
O
Keywords: Depression; Atomoxetine; Attention deficit hyperactivity
disorder; ADHD; Norepinephrine; Serotonin; Dopamine; Reuptake;
Inhibitor; Selective norepinephrine reuptake inhibitor; a-MMT; Met-
ariminol; Morpholine; Tertiary alcohol.
O
N
H
N
H
7
5
6
*
Corresponding author. Tel.: +44
cases_manuel@lilly.com
0
1276 483108; e-mail:
Figure 1. Mono and dual amine reuptake inhibitors.
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.12.061

M. J. Cases-Thomas et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2022–2025
2023
and/or pharmacological properties are of great interest
to industrial and academic researchers alike.
sium chloride to ketone 9 produced a mixture of all pos-
sible isomers (de ꢀ80%). After silica gel
chromatographic separation of the diasteromers and
removal of the N-benzyl protecting group by catalytic
phase transfer hydrogenolysis, each of the single enanti-
omers (13–16) was obtained by chiral HPLC.12
We have recently disclosed novel monoamine reuptake
inhibitors containing an arylthiomorpholine motif as
in 5 or based on a quinolone containing scaffold as in
6.5,6 Herein, we would like to report the discovery, syn-
thesis and biological activity of a series of tertiary alco-
hol containing benzyl morpholines as exemplified by
generic structure 7.7
A key synthetic step in the preparation of 16–25 consist-
ed of a highly diastereoselective addition of an appropri-
ately substituted benzyl Grignard reagent to ketone 11
(de >95%). Debenzylation of the resultant adduct was
achieved by hydrogenolysis, or carbamate exchange
with a-chloro ethyl chloroformate (ACE-Cl) followed
by methanolysis, to provide the target molecules 16–25
as single enantiomers in good overall yields.
The synthesis of the target molecules 13–25 is detailed in
Scheme 1.8 Reaction of 2-chloro acrylonitrile with
N-benzyl ethanolamine, followed by treatment with
potassium tert-butoxide, afforded the N-benzyl protect-
ed 2-cyanomorpholine 8 reliably in a one-pot proce-
dure.9 Treatment of this nitrile with 2-methoxybenzyl
magnesium chloride afforded the ketone 9 in good over-
all yield. Similarly, addition of phenyl magnesium chlo-
ride to the nitrile 8 provided the racemic ketone 10; this
was readily resolved by chiral HPLC or chiral SFC
affording the ketones 11 and 12 in quantities of up to
several hundred grams.10,11 Addition of phenyl magne-
X-ray crystallographic analysis of the 2-bromo-ana-
logue 21 allowed determination of absolute stereochem-
istry (2S,20R) (Fig. 2).13 Assignment of the (2S,20R)
stereochemistry for products 16–20 and 22–25 was by
analogy to this result. The high degree of diastereoselec-
tivity in the addition step of benzyl Grignard reagents to
(4-benzylmorpholin-2-yl)phenylmethanone (11) can be
rationalised on the basis of a chelation-controlled nucle-
ophilic attack of the organometallic reagent.
O
H
H
N
N
O
N
OH
NH
O
N
Cl
i, ii
iii
All compounds presented in Table 1 were tested as single
enantiomers for their binding affinity to the norepineph-
rine (NET), serotonin (SERT) and dopamine (DAT)
transporters.14 All the 2-methoxy-substituted isomers
13–16 displayed binding affinity to the NET and poor
affinity to the SERT and DAT. The most active isomer,
16, was also the single enantiomer resulting from the
synthetic route described from ketone 11; this therefore
suggests that 16 also contained the (2S,20R) stereochem-
istry. Compounds 17 and 18, which contain methoxy
substitution in the 3- and 4-position, respectively, dis-
played reduced binding affinity to the transporters.
These results along with the binding affinity for the
NET of the unsubstituted construct 19 indicated a pref-
erence for substitution in the 2-position for the NET
(within this select set of compounds).
+
O
Ph
Ph
Ph
iv
9
8
O
O
O
H
O
N
O
N
O
v
+
N
Ph
11
Ph
Ph
10
12
O
HO
H
13-major
14-major
15-minor
16-minor
vi, vii, v
O
9
N
H
ClH
R
viii, vii
HO
O
16-25
Br
11
HO
O
N
2'
2
ClH
H
N
ClH
H
Scheme 1. Reagents and conditions: (i) Et2O or neat 15 ꢁC to rt, 24 h.
(ii) THF, t-BuOK 0 ꢁC 3 h; 70–80%. (iii) 1-Methyl magnesium
chloride-2-methoxy benzene 0 ꢁC to rt, 1.5 h, then 5 N HCl, 0 ꢁC,
58%. (iv) Phenyl magnesium chloride 0 ꢁC to rt, 1.5 h, then 5 N HCl,
0 ꢁC, 70%. (v) Chiral preparative HPLC resolution or chiral
preparative SFC resolution.10 (vi) Phenyl magnesium chloride
ꢁ10 ꢁC, 0.5 h; then SiO2 chromatographic separation of diasteromers
(vii) Pd/C, HCO2NH4, EtOAc; or ACE-Cl, THF followed by MeOH;
40–75%. (viii) Mg turnings, 1,2-dibromoethane, 1-halomethyl-substi-
tuted benzene, Et2O 0 ꢁC to rt, 0.5 h; or commercially available
benzylic Grignard reagents, add compound 11 in Et2O, ꢁ20 ꢁC to rt,
2 h; 40–100%.
21
Mg
O
Mg
HO Nu
O
O
O
O
Nu
H
H
Nu-
N
Ph
Ph
Ph
Figure 2. Structure derived from the X-ray diffraction13 of 21 and
rationalised stereochemical outcome of the addition reaction of benzyl
Grignard reagents to (4-benzylmorpholin-2-yl)phenylmethanone, 11.

2024
M. J. Cases-Thomas et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2022–2025
Table 1. NET, SERT and DAT binding affinity for compounds 13–25
R
HO
O
13-25
N
ClH
H
Compound
R
Kia (nM)
SERT
NET
DAT
13
14
15
16
17
18
19
20
21
22
23
24
25
2-OMe isomer 1
2-OMe isomer 2
2-OMe isomer 3
2-OMe isomer 4
3-OMe
160.8 28.4
156 25
(5 5.7%)b
(0.3 0.7%)b
(1.4 0.7)b
(4.7 2%)b
(4.6 1.6%)b
(3.7 2.5)b
56
6
3.2 0.4
(5.5 0.7%)b
(2.8 1.4%)b
(4.3 4.7%)b
(4.2 1.5%)b
(4.6 0.7%)b
(13 3.6%)b
(2.4 2.2%)b
(2.7 4%)b
(3.8 3.1%)b
(4.3 2.2%)b
(6.2 0.1%)b
(5.5 3.5%)b
(20.8 4.8%)b
(24.1 1.1%)b
(3.8 3.2%)b
(5.1 3.5%)b
(6.2 2.1%)b
(2.9 4%)b
(19.8 0.8%)b
(25.2 9.5%)b
148 12.3
30 9.7
4-OMe
H
2-Cl
2-Br
2-OEt
11.9 2.4
5.2 1
7.7 3.7
2-Oi-Pr
2-SCF3
2-Ph
54.4 12.7
3.7
(14.8 4.9%)b
(3 0.6%)b
1
a Ki values are means of at least three experiments.
b Values in parentheses represent the %-displacement of radioligand at 100 nM for NET and SERT, and 1000 nM for DAT. For experimental details
of assay conditions, see reference section.14 Minimum significant ratio (MSR) for binding assays: NET, 2.5; SERT, 3.0; DAT, 1.6.
A similar preference for 2-substitution has previously
been observed and reported for a series of arylthiometh-
yl morpholine-based inhibitors of the NET (e.g., 5).5
Additional compounds with substitution in the 2-posi-
tion of the benzylic functionality (20–25) also displayed
measurable binding affinity to the NET and good selec-
tivity versus the SERT and DAT. Introduction of 2-
chloro or 2-bromo functionality (20 and 21, respective-
ly) led to a significant increase in NET binding affinity
compared to 19. Alkoxy substitution in the 2-position
was found to be particularly effective in yielding com-
pounds with high binding affinity to the NET. The
NET binding affinity of 2-methoxy-substituted analogue
16 was comparable to those of atomoxetine 2 (Ki
(nM) = 5)15 and reboxetine 3 (Ki (nM) = 1.1 0.2).1
Larger alkoxy substitution, as in 22 and 23, also retained
high binding affinity to the NET. The 2-trifluoromethyl-
thio-substituted 24 had somewhat reduced binding affin-
ity to the NET, while 2-phenyl substitution (25) afforded
comparable binding to the most potent compound 16.
induced cortical norepinephrine depletion model in rats.
In this animal model, a subcutaneous dose of a-MMT
produces through metabolic conversion the neurotoxin
metaraminol which is subsequently transported via the
NET into neurons resulting in a depletion of cortical
norepinephrine.18,19 Blockade of the NET by inhibitors
prevents transport of metaraminol into neurons, thereby
maintaining cortical NE levels. After a 3 mg/kg subcuta-
neous dose of compound 16, a 70 6% blockade of a-
MMT-induced depletion of cortical norepinephrine
was observed. Further, despite extensive in vitro metab-
olism in rat liver microsomes, compound 16 also showed
significant dose-dependent activity after oral administra-
tion with a measured ED50 = 18 mg/kg (Fig. 3).20
In summary, we have discovered a novel series of highly
selective and potent norepinephrine reuptake inhibitors
containing a tertiary alcohol motif. An efficient synthetic
1.2
Further in vitro characterization of 16 included evalua-
tion of the extent of its metabolism in various species liv-
er microsomes. Compound 16 was found to be
extensively metabolised in rat liver microsomes, but sig-
nificantly less in dog and human liver microsomes.16 The
benzylic tertiary alcohol functionality in 16 was found to
be both chemically and configurationally stable under
aqueous acidic conditions. After treatment of 16 with
0.1 N HCl at 40 ꢁC, less than 5% loss of parent was ob-
served after 72 h.17
1.0
*
Control
#
0.8
0.6
*
#
α-MMT alone*
*
0.4
0.2
3
10
30
Dose of 16 (mg/kg, p.o.)
The in vivo pharmacodynamic activity of compound 16
was investigated in an a-methyl-m-tyrosine (a-MMT)-
Figure 3. Blockade of a-MMT-induced depletion of cortical norepi-
nephrine concentrations after oral administration of 16.

M. J. Cases-Thomas et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2022–2025
2025
with 100% ethanol/0.3% DMEA (dimethylethylamine) as
eluent using a flow rate of 150 ml/min and UV-detection at
300 nm. (2) SFC Multigram II supplied by Mettler Toledo
Autochem. Column: Chiralpak AD-H 25 cm · 21.0 mm
i.d. 5 l (micron); flow rate: 70 ml/min; modifier: 30%
MeOH, 70% CO2 with 0.2% DEMA (diethylmethyl-
amine); temperature: 35 ꢁC; outlet pressure: 100 bar;
injection: 1 ml (500 mg); detection: UV at 260 nm.
route providing access to the targets in enantiomerically
pure form has been developed. Compound 16 had the
greatest binding affinity to the NET within the group of
compounds presented and this inhibitor was found to be
stable under aqueous acid conditions. Despite extensive
metabolism in rat liver microsomes, 16 was found to have
potent oral in vivo activity in the a-MMT pharmacody-
namic model of NET inhibition. Further investigations
and results with compound 16, and other select com-
pounds within this structural series of NET inhibitors,
will be reported in future communications.
11. Analytical data for (4-benzylmorpholin-2-yl)phenylmeth-
anone, enantiomer 1: pale yellow solid, mp 80.1–82.2 ꢁC,
20
½aꢂD ꢁ29.4ꢁ (MeOH, 12.40 g/l). 1H NMR (400 MHz,
CDCl3) d 2.3 (m, 2H), 2.7 (dd, J = 11.5, 2 Hz, 1H), 3.1 (dt,
J = 11.5, 2 Hz, 1H), 3.5 (d, J = 13 Hz, 1H), 3.6 (d,
J = 13 Hz, 1H), 3.8 (td, J = 11, 2.5 Hz, 1H, 6), 4.0 (dt,
J = 11, 2 Hz, 1H), 4.9 (dd, J = 10, 2.5 Hz, 1H), 7.2–7.3 (m,
5H), 7.4–7.5 (m, 2H), 7.6–7.7 (m, 1H), 7.9–8.0 (m, 2H).
ESI-MS C18H19NO2 MS (ES+) m/z = 282 [M+H]+. Ana-
lytical data for 4-(benzylmorpholin-2-yl)phenylmetha-
none, enantiomer 2: pale yellow solid, mp 82.9–83.6 ꢁC,
Acknowledgments
We thank Greg Stephenson for the X-ray structure,
David C. Evans, Ann-Helene Klugery and John Reilly
for helpful contributions. Our special thanks to Jyll
Brown and Paul Greenstock for their invaluable
collaboration.
20
½aꢂD +29.7ꢁ (MeOH, 11.78 g/l). Other data identical to
enantiomer 1.
12. See Ref. 8 for detailed experimental procedures for
Grignard addition and deprotection. Enantiomer separa-
tion was achieved using the following methods: isomers 1
and 2 chiral HPLC on a Chiralpak AD 10 lm column
(supplied from Chiral technologies, Illkirch, France) with
85% heptane: 15% ethanol (0.2% DMEA) as eluent using a
flow rate of 12 ml/min and UV detection at 260 nm.
Isomers 3 and 4 chiral HPLC on a Chiralpak AD 10 lm
column (supplied from Chiral technologies, Illkirch,
France) with 85% heptane: 15% ethanol (0.2% DMEA)
as eluent using a flow rate of 12 ml/min and UV detection
at 260 nm.
13. CCDC 291659 contains the supplementary crystallograph-
ic data for this paper. These data can be obtained free of
charge via www.ccdc.ac.uk/data_request/cif, or by email-
ing data_request@ccdc.cam.ac.uk, or by contacting The
Cambridge Crystallographic Data Centre, 12, Union
Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
14. NET assay: Gobel, J.; Saussy, D. L.; Goetz, A. S. J.
Pharmacol. Toxicol. Methods 1999, 42, 237; SERT/DAT
assay: Ramamoorthy, S.; Giovanetti, E.; Qian, Y.; Blak-
ely, R. D. J. Biol. Chem. 1998, 273, 2458.
15. Bymaster, F. P.; Katner, J. S.; Nelson, D. L.; Hemrick-
Luecke, S. K.; Threlkeld, P. G.; Heiligenstein, J. H.;
Morin, S. M.; Gehlert, D. R.; Perry, K. W. Neuropsycho-
pharmacology 2002, 27, 699.
16. Remaining parent compound 16 after 15 min incubation
in liver microsomes (1 lmol/l, 0.5 mg/ml protein) of
different species: Human 98%, Dog 60% and Rat 0%.
17. Compound 15 was shown to be configurationally stable in
solution for at least 3 days at 40 ꢁC, in water, 0.1 N HCl
and in buffer solutions over a pH range of 2–8.
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10. Separation was achieved using one of two methods: (1)
Chiral HPLC on a Diacel chiralpak AD 20 lM column