Design and synthesis of morpholine derivatives. SAR for dual serotonin & noradrenaline reuptake inhibition

Article abstract of DOI:10.1016/j.bmcl.2008.03.050

Single enantiomer (SS) and (RR) 2-[(phenoxy)(phenyl)methyl]morpholine derivatives 5, 8-23 are inhibitors of monoamine reuptake. Target compounds were prepared using an enantioselective synthesis employing a highly specific enzyme-catalysed resolution of racemic n-butyl 4-benzylmorpholine-2-carboxylate (26) as the key step. Structure-activity relationships established that serotonin and noradrenaline reuptake inhibition are functions of stereochemistry and aryl/aryloxy ring substitution. Consequently, selective SRI, selective NRI and dual SNRIs were all identified. One of these compounds, a potent and selective dual SNRI, (SS)-5a was selected as a candidate for further pre-clinical evaluation.

Full text of DOI:10.1016/j.bmcl.2008.03.050

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2562–2566
Design and synthesis of morpholine derivatives.
SAR for dual serotonin & noradrenaline reuptake inhibition
Paul V. Fish,a,* Christopher Deur,c Xinmin Gan,c Keri Greene,c David Hoople,b
Malcolm Mackenny,a Kimberly S. Para,c Keith Reeves,b Thomas Ryckmans,a Cory Stiff,c
Alan Stobie,a Florian Wakenhuta and Gavin A. Whitlocka
aDepartment of Genitourinary Chemistry, Pfizer Global Research and Development, Sandwich Laboratories,
Ramsgate Road, Sandwich, Kent CT13 9NJ, UK
bDepartment of Synthetic Services, Pfizer Global Research and Development, Sandwich Laboratories, Ramsgate Road,
Sandwich, Kent CT13 9NJ, UK
cDepartment of Chemistry, Pfizer Global Research & Development, Ann Arbor Laboratories, Ann Arbor, MI 48105, USA
Received 6 March 2008; revised 14 March 2008; accepted 16 March 2008
Available online 20 March 2008
Abstract—Single enantiomer (SS) and (RR) 2-[(phenoxy)(phenyl)methyl]morpholine derivatives 5, 8–23 are inhibitors of mono-
amine reuptake. Target compounds were prepared using an enantioselective synthesis employing a highly specific enzyme-catalysed
resolution of racemic n-butyl 4-benzylmorpholine-2-carboxylate (26) as the key step. Structure–activity relationships established
that serotonin and noradrenaline reuptake inhibition are functions of stereochemistry and aryl/aryloxy ring substitution. Conse-
quently, selective SRI, selective NRI and dual SNRIs were all identified. One of these compounds, a potent and selective dual SNRI,
(SS)-5a was selected as a candidate for further pre-clinical evaluation.
ꢀ 2008 Elsevier Ltd. All rights reserved.
Selective inhibition of serotonin (5-HT) and noradrena-
line (NA) reuptake (SNRI) has been shown to be an
attractive dual pharmacology approach for the treat-
ment of a number of diseases.1,2 For example, dual 5-
HT/NA reuptake inhibitor duloxetine (1) has shown
clinical efficacy in the treatment of depression, pain,
and urinary incontinence.3,4
amine7 templates. In this letter, we disclose some factors
that modulate 5-HT and NA reuptake inhibition in a
morpholine scaffold as a surrogate for the biogenic
amine.
Reboxetine (2), a selective NA reuptake inhibitor used
clinically in the treatment of depression,8 was selected
as a suitable chemical starting point. As an initial ven-
ture, we elected to introduce a 3-Cl and a 4-Cl substitu-
ent onto the aryloxy ring of reboxetine to explore the
effect upon SRI and NRI activity.6,7 In addition, the
influence of the two vicinal chiral centres on monoamine
reuptake inhibition was explored by the preparation of
these targets as racemic, single diastereoisomers 3–5.9
O
NHMe
NH
S
O
O
EtO
1: duloxetine
2: ( )-reboxetine
The combination of the 2-OEt-4-Cl with the syn diaste-
reoisomer 4ab furnished a promising lead as SRI activity
had been introduced and NRI activity retained giving a
dual SNRI with 25-fold selectivity over dopamine (DA)
reuptake inhibition (DRI) (Table 1). The 2-OEt-3-Cl,
syn diastereoisomer 3ab was also a balanced SNRI but
with weaker affinity at both transporters. The SRI and
NRI profiles of the corresponding 2-OMe-4-Cl ana-
logues 5ab and 5cd were similar to those of 4ab and
As part of our research efforts to identify potential drug
candidates, we have recently reported SNRIs derived
from piperazine,5 3-amino-pyrrolidine,6 and benzyl-
Keywords: Serotonin and noradrenaline reuptake inhibitor; SNRI;
Morpholine; Enzyme-catalysed resolution.
*
Corresponding author. Tel.: +44 (0) 1304 644589; fax.: +44 (0) 1304
651987; e-mail: paul.fish@pfizer.com
0960-894X/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.03.050

P. V. Fish et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2562–2566
2563
Table 1. In vitro inhibition of monoamine reuptake for racemic diasteroisomers (3–5) and single enantiomers (5, 8–23)a,b,c
O
O
O
O
R
R
R
R
NH
NH
NH
NH
O
O
O
O
R2
R4
R2
R4
R2
R4
R2
R4
R3
R3
R3
R3
3-5, 8-23
(SS)-syn-a
3-5, 8-23
(RR)-syn-b
3-5, 8-23
(RS)-anti-c
3-5, 8-23
(SR)-anti-d
Compound
R
R2, R3, R4
Stereochemistry
5-HT Ki (nM)
NA Ki (nM)
DA Ki (nM)
clogP
duloxetine
reboxetine
3ab
3cd
S
5
45
5
435
4.26
3.26
3.82
3.82
4.05
4.05
3.52
3.52
3.52
3.52
2.95
2.95
3.81
3.81
3.78
3.78
3.20
3.20
3.22
3.22
4.26
2.48
3.67
3.82
3.64
3.66
3.66
3.66
3.97
4.02
4.02
H
2-OEt
SS/RR
SS/RR
RS/SR
SS/RR
RS/SR
SS/RR
RS/SR
SS
1400
240
340
60
>10,000
>10,000
>10,000
1540
1670
5050
2200
1570
1790
3080
NT
H
2-OEt, 3-Cl
2-OEt, 3-Cl
2-OEt, 4-Cl
2-OEt, 4-Cl
2-OMe, 4-Cl
2-OMe, 4-Cl
2-OMe, 4-Cl
2-OMe, 4-Cl
2-OMe, 4-F
2-OMe, 4-F
2-Cl, 4-F
275
1490
15
H
4ab
4cd
H
H
220
28
390
36
5ab
5cd
H
H
85
320
8
5a
5b
H
110
11
H
RR
SS
420
6.0
610
12
8a
8b
H
740
22
H
RR
9a
9b
H
SS
260
20
1170
760
H
2-Cl, 4-F
2-Me, 4-F
RR
SS
830
18
10a
10b
11a
11b
12a
12b
13a
14a
15a
16a
17a
18a
19a
20a
21a
22a
23a
H
185
34
1250
950
H
2-Me, 4-F
2-F, 3-F
2-F, 3-F
RR
860
10
H
SS
3390
12
2600
380
H
RR
SS
130
12
H
2,3-OCH2CH2-
2,3-OCH2CH2-
2-Cl, 3-Cl
1800
20
NT
H
RR
350
17
5750
420
H
SS
SS
100
110
30
H
2-OMe, 4-CN
2-OMe, 4-Br
2-OMe, 4-CF3
2-Cl, 4-OMe
2-OMe, 4-Cl
2-OMe, 4-Cl
2-OMe, 4-Cl
2-OMe, 4-Cl
2-OMe, 4-Cl
2-OMe, 4-Cl
210
12
NT
NT
H
SS
H
SS
41
96
160
28
NT
H
SS
SS
1090
NT
2-F
3-F
4-F
2-Me
3-Me
4-Me
70
58
22
SS
9
29
10,800
NT
NT
SS
62
70
SS
SS
84
16
69
88
NT
1650
SS
180
a Nomenclature used throughout: a = SS; b = RR; c = RS; d = SR; ab is the racemic SS/RR syn-diastereoisomer.
b See Ref. 25 for details of assay conditions. Monoamine reuptake Ki values are geometric means of at least three experiments. Differences of <2-fold
should not be considered significant.
c NT, not tested.
4cd, with again a preference for the syn diastereoisomer
5ab. This modification of the aryloxy ring (i.e.,
4ab ! 5ab) brought a 2-fold increase in SRI activity
resulting in a potent, balanced SNRI with >100-fold
selectivity over DRI. The SAR within these compounds
3–5 showed the anti diastereoisomers to be inferior in
both SRI and NRI activity compared to the syn and
were discontinued.
pounds as the contribution to the observed effects
may not be equal for each enantiomer; this is
further complicated when seeking dual activities
at two biological targets.12 Hence, compound 5ab
was then prepared as the two single enantiomers
(SS)-5a and (RR)-5b so as to determine the influ-
ence of absolute stereochemistry on the SRI and
NRI activity.
Compound 5ab has physicochemical properties consis-
tent with CNS target space10 (m. wt. 333; clogP 3.52;
Target compounds 5, 8–23 (Table 1) were prepared
using a new enantioselective synthesis starting from
the racemic morpholine ester 7 (Fig. 1).13 This route
was attractive to us as it allowed for the late stage instal-
lation of both the aryl and aryloxy rings. The key (R)
and (S) morpholine acids 6 were efficiently prepared
on large scale by an enzyme-catalysed resolution of race-
mic ester 7.
2
logD7.4 1.9; pKa 7.7; HBD 1; HBA 4; PSA 40 A )
11
˚
and was selected as a lead for our drug discovery
programme.
Caution must be exercised when evaluating SAR
from the biological activities of racemic com-

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P. V. Fish et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2562–2566
tent NRI with SRI activity, whereas (RR)-5b was a
O
*
R
O
*
selective SRI. This split in SRI versus NRI activity
was also observed with five additional pairs of enantio-
mers 8–12. In some cases, selective SRI (e.g., 9b) and
selective NRI (e.g., 8a, 11a) were achieved. Overall,
there was a broad range in both SRI and NRI activities
and the ratios of activity (SRI:NRI, 350:1 to 1:40).
NH
*
X
N
PG
O
O
R4
R2
R3
PG = BOC or Bn
5, 8-23: Proposed targets
6: (R) & (S) acids (X = OH)
7: (RS) ester (X = O-n-Bu)
Both 5a and 5b were screened for off-target pharmacol-
ogy against a panel of 70 receptors, enzymes and ion
channels. Compound 5b was found to have significant
binding affinity for the histamine H1 receptor18
(Ki = 26 nM), whereas 5a was inactive at H1 (<20% at
10 lM). It is of note that neither 5a or 5b had confirmed
functional activity against any other target in this panel
at 63 lM. It was subsequently determined that 8b–12b
also had affinity for the H1 receptor (Ki = 10–200 nM),
whereas 8a–12a were inactive. The modest NRI activity
combined with H1 receptor affinity resulted in the dis-
continuation of the (RR)-series.
Figure 1. Target compounds, retrosynthesis and homochiral synthons.
Racemic ester 26, prepared in two steps from N-benzy-
lethanolamine (24) and chloroacrylonitrile,14 was
screened against a panel of lipases (Scheme 1). Lipase
Candida rugosa15 was completely stereospecific, catalyz-
ing the hydrolysis of the (S)-ester to give (S)-acid 28
whilst leaving the (R)-ester 27 untouched.16,17 The N-
benzyl protecting group of 27 was then exchanged for
the N-BOC amine 29 as it had a beneficial effect on
the yields of subsequent reactions and simplified depro-
tection at the final step. Base hydrolysis of the n-butyl
ester 29 gave the acid as the Li salt 30, and activation
of 30 with 1-propanephosphonic anhydride (T3P)
followed by the reaction with HN(Me)OMe gave Wein-
reb amide 31. Treatment of 31 with Grignard reagents
ArMgX gave the aryl ketones 32 with no detectable loss
in e.e. (Scheme 2). Reduction of 32 with Zn(BH4)2 cre-
ated the second stereocentre giving alcohol 33 with good
diastereoselectivity (RS:RR 16:1), and then the reaction
of 33 with MeSO2Cl gave mesylate 34. The displacement
of the MsO group of 34 with phenols ArOH gave the
corresponding (RR)-aryloxy ethers 35 in good yield
and with complete inversion of the benzylic stereocentre,
and finally deprotection of the N-BOC amine with HCl
afforded the (RR)-target compounds 5,8–23. The (SS)-
enantiomers were prepared by an identical sequence
but starting with the (S)-acid 28.
Further SAR was directed at improving SRI activity in
the (SS)-series by exploring a broader set of substituents
on the aryloxy ring 13a–17a. Increasing the size of the
group at the 4-position gave 15a (4-Br) as a potent, bal-
anced dual SNRI, although the introduction of 4-CF3
16a inverted the SNRI profile with a greater affinity
for the 5-HT transporter. Substitution on the aryl ring
was investigated by the introduction of a F or Me at
the 2-, 3-, or 4-positions of 18a–23a (see Table 1).
From these experiments, 5a emerged as having a desir-
able combination of dual NRI and SRI activity, good
selectivity over DRI, and no significant off-target phar-
macology. Furthermore, 5a was one of the least lipo-
philic structures of those presented in Table 1.
Reducing lipophilicity was an important selection crite-
ria for this programme.19 Compound lipophilicity was
initially assessed by the calculation of partion coeffi-
cients (clogP) and then confirmed for selected examples
The absolute stereochemistry of 517 was found to have a
significant effect on activity (Table 1): (SS)-5a was a po-
HO
O
O
a, b
c
HN
Ph
Ph
N
Ph
n-BuO
N
NC
O
26
O
P
24
25
O
P
O
P
O
O
O
T3P
d
O
R
O
R
O
O
f, g
e
X
N
Ph
n-BuO
N
n-BuO
N
HO
N
Ph
+
boc
boc
R
S
O
O
O
O
30: X = OLi
31: X = N(Me)OMe
29
27: 94 % yield
28: 97 % yield
100 % e.e.
100 % e.e.
Scheme 1. Reagents and conditions: (a) CH2@C(Cl)CN, Et2O, 40 ꢁC, quant.; (b) t-BuOK, DME, reflux 65%; (c) n-BuOH, c. H2SO4, reflux, 85%; (d)
Candida rugosa, t-BuOMe–H2O, rt, 42 h; (e) 2,5-dihydrotoluene, 10%-Pd/C, BOC2O, EtOH, reflux, quant.; (f) LiOH (0.95 equiv), THF-H2O, 0 ꢁC,
90%; (g) T3P, HN(Me)OMe, NEt3, CH2CH2, then aq K2CO3, 85%.

P. V. Fish et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2562–2566
2565
Me
N
O
R
O
O
R
R
R
b
a
S
N
N
N
MeO
boc
boc
boc
R
O
O
OH
31
32
33
c
O
R
O
R
O
R
R
R
R
d
R
O
e
R
O
S
NH
N
N
boc
R4
boc
OMs
R2
R4
R2
35
R3
5, 8-23
R3
34
Scheme 2. Reagents and conditions: (a) ArMgBr, THF, rt, 98%; (b) Zn(BH4)2, Et2O, rt, 87%; (c) MeSO2Cl, NEt3, CH2Cl2, 0 ꢁC, quant.; (d) ArOH,
Cs2CO3, microwave in THF or reflux in dioxane, 95%; (e) HCl in dioxane, CH2Cl2, 95%.
by the measurement of the octanol-buffer distribution
coefficient (5a: logD7.4 1.9).
determinations. P.V.F. thanks David L. Gray (Discov-
ery Chemistry) for helpful discussions and his assistance
in the preparation of this manuscript.
Additional screening20 in vitro showed 5a to have good
membrane permeability (CaCO-2 18/16) with low affin-
ity for P-gp efflux transporters (MDCK-mdr1 22/35)
suggesting the potential for good oral absorption and
CNS penetration.10 Compound 5a had good metabolic
stability in human liver microsomes (t1/2 > 120 min)
and human hepatocytes (t1/2 186 min) consistent with
low predicted clearance. Compound 5a had no signifi-
cant inhibition of CYP450 enzymes (1A2, 2C9, 2C19,
2D6, 3A4; IC50 > 29 lM) and modest ion channel activ-
ity as measured by binding to potassium hERG ([3H]-
dofetilide, Ki 3.3 lM), sodium (site 2, Ki 1.2 lM) and
calcium (L-type diltiazem site, Ki 2.0 lM) channels.
References and notes
1. (a) Montgomery, S. Int. J. Psych. Clin. Pract. 2006, 10, 5;
(b) Baldwin, D. S. Int. J. Psych. Clin. Pract. 2006, 10, 12;
(c) Wernicke, J. F.; Iyengar, S.; Ferrer-Garcia, M. D.
Curr. Drug Therapy 2007, 2, 161; (d) Jackson, S. Curr.
Med. Res. Opin. 2005, 21, 1669.
2. For recent reviews of new chemical entities, see: (a)
Walter, M. W. Drug Dev. Res. 2005, 65, 97; (b) Huang, Y.;
Williams, W. A. Expert Opin. Ther. Patents 2007, 17, 889.
3. Thor, K. B.; Kirby, M.; Viktrup, L. Int. J. Clin. Pract.
2007, 61, 1349.
4. Steers, W. D.; Herschorn, S.; Kreder, K. J.; Moore, K.;
Strohbehn, K.; Yalcin, I.; Bump, R. C. BJU Int. 2007, 100,
337.
5. (a) Fray, M. J.; Bish, G.; Brown, A. D.; Fish, P. V.; Stobie,
A.; Wakenhut, F.; Whitlock, G. A. Bioorg. Med. Chem.
Lett. 2006, 16, 4345; (b) Fray, M. J.; Bish, G.; Fish, P. V.;
Stobie, A.; Wakenhut, F.; Whitlock, G. A. Bioorg. Med.
Chem. Lett. 2006, 16, 4349.
Further pharmacological evaluation in vivo, in microdi-
alysis experiments,21,22 showed 5a to increase both NA
and 5-HT levels in interstitial fluid of the prefrontal cor-
tex of conscious rats by 170% and 160%, respectively,
above pre-drug baseline levels (10 mg/kg administered
s.c., n = 6).
6. Fish, P. V.; Fray, M. J.; Stobie, A.; Wakenhut, F.;
Whitlock, G. A. Bioorg. Med. Chem. Lett. 2007, 17, 2022.
7. Whitlock, G. A.; Blagg, J.; Fish, P. V. Bioorg. Med. Chem.
Lett. 2008, 18, 596.
8. Hajos, M.; Fleishaker, J. C.; Filipiak-Reisner, J. K.;
Brown, M. T.; Wong, E. H. F. CNS Drug Rev. 2004, 10,
23.
Based on this profile, dual SNRI 5a (PF-734629)23 was
selected as a candidate for further evaluation in pre-clin-
ical disease models. The results of these studies will be
reported in future publications.24,25
9. Fish, P. V.; Mackenny, M. C.; Stobie, A.; Wakenhut, F.;
Whitlock, G. A. WO Patent 105100, 2005.
Acknowledgments
10. Mahar Doan, K. M.; Humphreys, J. E.; Webster, L. O.;
Wring, S. A.; Shampine, L. J.; Serabjit-Singh, C. J.;
Adkison, K. K.; Polli, J. W. J. Pharm. Exp. Ther. 2002,
303, 1029.
We thank Gerwyn Bish, Timothy Buxton, Russell Cave,
David Drouard, Debbie Lovering, Christelle Pasquinet,
Bhairavi Patel, and Melanine Paul for compound syn-
thesis. We are grateful to Stephen Phillips, David Bei-
dler and Taraneh Haske (Discovery Biology) for
screening data, and to Anthony Harrison, Christopher
Kohl, Tycho Heimbach and Christopher Lepsy (PDM)
for ADME studies. We thank Brain Samas, Neil Feeder
and Cheryl Doherty for single crystal X-ray structure
11. Definition of terms: m. wt.: molecular weight; clogP:
calculated partion coefficient (BioByte software); logD7.4
:
measured octanol-buffer distribution coefficient; pKa:
measured ionisation constant; HBD: H-bond donor count
(NH+OH); HBA: H-bond acceptor count (N+O); PSA:
polar surface area.

2566
P. V. Fish et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2562–2566
12. Boot, J.; Cases, M.; Clark, B. P.; Findlay, J.; Gallagher, L.
H.; Man, T.; Montalbetti, C.; Rathmell, R. E.; Rudyk, H.;
Walter, M. W.; Whatton, M.; Wood, V. Bioorg. Med.
Chem. Lett. 2005, 15, 699.
13. For racemic syntheses, see: (a) Melloni, P.; Carniel, G.;
Della Torre, A.; Bonsignori, A.; Buonamici, M.; Pozzi, O.;
Ricciardi, S.; Rossi, A. C. Eur. J. Med. Chem. 1984, 19,
235; (b) reference 12; (c) Henegar, K. E.; Ball, C. T.;
Horvath, C. M.; Maisto, K. D.; Mancini, S. E. Org. Proc.
Res. Dev. 2007, 11, 346; For an asymmetric synthesis of
(+)-(S,S)-reboxetine, see: (d) Brenner, E.; Baldwin, R. M.;
Tamagnan, G. Org. Lett. 2005, 7, 937.
14. King, F. D.; Hadley, M. S.; Joiner, K. T.; Martin, R. T.;
Sanger, G. J.; Smith, D. M.; Smith, G. E.; Smith, P.;
Turner, D. H.; Watts, E. A. J. Med. Chem. 1993, 36, 683.
15. Lipase Candida rugosa is commercially available from
Sigma (catalogue number: L-1754; 1140 U/mg).
16. Enantioselectivities were determined by chiral HPLC
(Chiralcel OJ-H, 250 · 4.6 mm; hexane/0.1% TFA in
EtOH, 80:20 isocratic, 1 mL/min).
17. The absolute stereochemistry was determined by conver-
sion of 28 to an authentic sample of (S,S)-reboxetine and
confirmed by single crystal X-ray structure determination
of 5a PhSO3H salt (mp 182 ꢁC). Crystallographic data
(excluding structure factors) for the structures in this letter
have been deposited with the Cambridge Crystallographic
Data Centre as supplementary publication number
CCDC680331. Copies of the data can be obtained free
of charge, on application to CCDC, 12 Union Road,
Cambridge, CB2 1EZ, UK.
18. Walsh, G. M. Exp. Opin. Drug Safety 2002, 1, 225.
19. For an analysis of the influence of drug lipophilicity on
off-target pharmacology and in vivo toxicological out-
comes, see: (a) Leeson, P. D.; Springthorpe, B. Nature
Rev. Drug Dis. 2007, 6, 881; (b) Hughes, J. D.; Blagg, J.;
Bailey, S.; De Crescenzo, G. A.; Dalvie, D.; Devraj, R.
V.; Doubovetzky, M.; Ellsworth, E.; Fobian, Y. M.;
Gibbs, M. E.; Gilles, R. W.; Grant, D.; Greene, N.;
Huang, E.; Kreiger-Burke, T.; Lee, L.; Loesel, J.; Nahas,
K.; Price, D. A.; Wager, T.; Whiteley, L.; Zhang, Y.
Correlations between compound physicochemical prop-
erties and in vivo toxicological outcomes: Comprehensive
analysis of a 245 compound data set. Nat. Biotech., 2008,
in press.
20. CaCO-2: human colon adenocarcinoma cell line. MDCK-
mdr1: Madin-Darby canine kidney cell line expressing the
P-glycoprotein transporter (P-gp). Flux across cells was
measured at 10 lM substrate concentrations. Figures
quoted correspond to the flux rates (Papp · 10ꢀ6 cmꢀ1
)
for apical to basolateral (AB) and basolateral to apical
(BA) directions. The maximum measurable half-life in
human liver microsomes was 120 min, whereas the human
hepatocyte assay was able to accurately determine half-
lives of up to 240 min. See: van de Waterbeemd, H.;
Smith, D. A.; Beaumont, K.; Walker, D. K. J. Med.
Chem. 2001, 44, 1313 and references therein. Ion channel
screening (Na+, Ca2+) was performed by CEREP, France.
21. Deecher, D. C.; Beyer, C. E.; Johnston, G.; Bray, J.; Shah,
S.; Abou-Gharbia, M.; Andree, T. H. J. Pharm. Exp.
Ther. 2006, 318, 657.
22. Rat 5-HT and NA transporter inhibition was measured in
HEK293 cells expressing the rat 5-HT and NA transport-
ers. For 5a: rSRI, Ki = 59 nM (n = 2); rNRI, Ki = 6 nM
(n = 2).
23. Data for 5a HCl salt: mp 148 ꢁC; [a]D = +14.4ꢁ (MeOH;
1
c = 0.20); H NMR (CD3OD, 400 MHz) d 3.05–3.20 (m,
3H), 3.25 (d, 1H), 3.78–3.87 (m, 4H), 4.08–4.20 (m, 2H),
5.31 (d, 1H), 6.70 (m, 2H), 6.95 (s, 1H), 7.28–7.44 (m, 5H);
LRMS APCI m/z 334 (MH+); 100% e.e. by chiral HPLC.
24. For an accompanying paper, see: Xu, W.; Gray, D. L.;
Glase, S. A.; Barta, N. S. Bioorg. Med. Chem. Lett. 2008,
submitted for publication.
25. Target compounds were tested for their ability to inhibit
specific binding of selective radioligands at the human 5-
HT, NA and DA transporters utilising scintillation
proximity assay (SPA) technology and cellular membrane
preparations generated from recombinant HEK293 cells
expressing a single monoamine transporter. For experi-
mental details of the assay conditions, see: Andrews, M.
D.; Brown, A. D.; Fish, P. V.; Fray, M. J.; Lansdell, M. I.;
Ryckmans, T.; Stobie, A.; Wakenhut, F.; Gray, D. L. F.
WO Patent 064351, 2006, p 56–59.