Discovery and pharmacological characterization of aryl piperazine and piperidine ethers as dual acting norepinephrine reuptake inhibitors and 5-HT1A partial agonists

Article abstract of DOI:10.1016/j.bmcl.2009.10.014

Compounds that are both norepinephrine reuptake inhibitors (NRI) and 5-HT1_A partial agonists may have the potential to treat neuropsychiatric disorders including attention deficit hyperactivity disorder (ADHD) and depression. Targeted screening

Full text of DOI:10.1016/j.bmcl.2009.10.014

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6604–6607
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and pharmacological characterization of aryl piperazine
and piperidine ethers as dual acting norepinephrine reuptake inhibitors
and 5-HT1A partial agonists
a
a
a
b
b
David L. Gray a, , Wenjian Xu , Brian M. Campbell , Amy B. Dounay , Nancy Barta , Susan Boroski ,
*
Lynne Denny b, Lori Evans b, Nancy Stratman a, Al Probert a
a Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, United States
b Pfizer Global Research and Development, 2800 Plymouth Road, Ann Arbor, MI 48105, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Compounds that are both norepinephrine reuptake inhibitors (NRI) and 5-HT1A partial agonists may have
the potential to treat neuropsychiatric disorders including attention deficit hyperactivity disorder
(ADHD) and depression. Targeted screening of NRI-active compounds for binding to the 5-HT1A receptor
provided a series of thiomorpholinone hits with this dual activity profile. Several iterations of design, syn-
thesis, and testing led to substituted piperidine diphenyl ethers which are potent NRIs with 5-HT1A par-
tial agonist properties. In addition, optimization of these molecules provided compounds which exhibit
selectivity for NRI over the dopamine (DAT) and serotonin (SERT) reuptake transporters. Monoamine and
5-HT1A in vitro functional activities for select compounds from the developed piperidine diphenyl ether
series are also presented.
Received 4 September 2009
Accepted 5 October 2009
Available online 12 October 2009
Keywords:
Norepinephrine reuptake inhibitor
NRI
5-HT1A agonist
NET
DAT
Ó 2009 Elsevier Ltd. All rights reserved.
SERT
5-Hydroxytryptamine 1A
ADHD
Depression
A number of drugs that are effective in treating psychiatric ill-
ness are thought to act via modulation of monoamine neurotrans-
mitter levels including dopamine (DA), norepinephrine (NE), and/
or serotonin (5-HT).1 As a result of commercial success and devel-
oping science, the discovery of novel compounds that enhance
synaptic monoamine signaling remains an active area of pharma-
ceutical research.2 Compounds approved for the treatment of
ADHD include amphetamine, methylphenidate, and atomoxetine,
which all elevate monoamine synaptic neurotransmitter levels
(DA and NE in particular), albeit via distinct cellular pathways.3
Targeting the dual mechanism of norepinephrine reuptake inhibi-
tion (NRI) and 5-HT1A partial agonism could effect significant DA
elevation in key cortical regions.4,5 While NRIs increase synaptic
NE and DA concentration by slowing clearance of these compounds
from the synaptic space, stimulation of 5-HT1A receptors within the
prefrontal cortex has been shown to activate subcortical DA sys-
tems resulting in elevated cortical DA release via a reciprocal path-
way.6 This hypothesis is supported by an intriguing synergistic
increase in extracellular DA levels within the prefrontal cortex
when atomoxetine and buspirone are dosed simultaneously.7
Moreover, preclinical behavioral studies measuring antidepressant
efficacy and cognitive function suggest that compounds possessing
both NRI and 5-HT1A partial agonist properties are more effective
than NRI agents alone.8 Thus, identification of agents with dual
NRI and 5-HT1A partial agonist pharmacology may provide a new
therapeutic approach for the treatment of ADHD, depression, and
anxiety.
With these ideas in mind, we sought to identify a series of com-
pounds that had the desired dual activity (NRI and 5-HT1A agon-
ism) with minimal activity at homologous receptors, such as the
dopamine reuptake transporter (DAT) or serotonin reuptake trans-
porter (SERT). Historical broad panel screening data for ligands that
are individually potent for either NRI or 5-HT1A indicated that it
might be challenging to obtain compounds that are <50 nM at both
of the desired pharmacologies, while also displaying general selec-
tivity.9,10 We used computational techniques to query our exten-
sive broad panel screening database and define the ligand
properties and broad panel profiles of ligands which strongly inter-
acted with NRI, 5-HT1A, and SRI. In this analysis, there was a mean-
ingful relationship between SRI and 5-HT1A ligands, but a very
weak correlation between the properties and overall selectivity
profiles of NRI ligands and 5-HT1A ligands. The concern from the
outset was that any compound with potency at both NRI and
* Corresponding author.
E-mail address: david.l.gray@pfizer.com (D.L. Gray).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.014

D. L. Gray et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6604–6607
6605
Table 1
5-HT1A might contain a promiscuous basic amine which would
interact with many G-protein coupled receptor (GPCR) targets.
Consequently, we incorporated upfront selectivity screening ver-
sus SERT and DAT to get an early read on ‘promiscuity’ and placed
an emphasis on this selectivity (vs potency at either target recep-
tor) when deciding on which SAR directions to pursue.
We screened our internal collection of NRIs for compounds that
also had binding affinity for 5-HT1A receptors. Among other hits,
we identified aryl piperazine thiomorpholinone structures 1 and
2 (Fig. 1) as promising dual activity molecules.11 The aryl pipera-
zine moiety is a recurring motif in many potent 5-HT1A ago-
nists.12,13 Computational analysis of 1 and 2 in an empirical NRI
pharmacophore model guided early follow-up design in this ser-
ies.14 This modeling suggested that initial hits such as 1 might be
unnecessarily large, with the halogenated ring potentially contrib-
uting little to binding affinity at NET.
5-HT1A, NET, DAT, and SERT binding affinities for simplified aryl piperazine ethers15,16
ID R1
5-HT1A Ki
NET Ki
DAT Ki
SERT Ki
(nM)
(nM)
(nM)
(nM)
7a
H
189
116
101
466
183
104
80
701
>2000
470
780
17
781
53
7b 3-F
7c 2,3-F
7d 2-c-
Propyl
8a
8b 3-F
192
>6000
H
>1000
>1000
1710
1940
396
461
364
49
H
N
Boc
N
Boc
N
R1
a
b,c
X
OH
X
Z
X
A number of truncated variants of 1 and 2 were prepared, and
among these, a phenoxymethyl piperazine (7a, Table 1) was found
to retain much of the desired activity when tested for binding to
the transfected human NET transporter and 5-HT1A receptor.15,16
The synthesis of analogous compounds (e.g., 7b–d, 8a–b, Ta-
ble 1) was straightforward and could be executed using parallel
chemistry techniques from common intermediates as outlined in
Scheme 1. When the benzylic alcohols 317 or 418 were treated with
methanesulfonyl chloride at room temperature, a mixture of the
expected mesylate and the benzylic chloride was obtained in each
case. The mixtures (5 or 6) were used directly in phenoxide substi-
tution reactions to afford piperazine (7) and piperidine (8) benzyl
phenyl ethers after carbamate deprotection. Not surprisingly, these
compounds had generally modest selectivity for NET versus DAT
and SERT, however SERT displayed a particular sensitivity to aryl
substitution pattern (e.g., 7b and 7c, Table 1). Piperidine com-
pounds in this set (8a, 8b) had inferior NET and 5-HT1A activity rel-
ative to piperazine-based congeners.
O
Z = Cl, OMs
5: X = N
7
: X = N
3: X = N
8: X = C
4
6
: X = C
: X = C
Scheme 1. Reagents and conditions: (a) MeSO2Cl, Et3N, CH2Cl2, rt, 71–89%
combined yield of Z = Cl and Z = OMs; (b) ROH (2 equiv), Cs2CO3, toluene/tBuOH,
110 °C; (c) 4 M HCl/dioxane, rt, then ‘catch-and-release’ (MP-TsOH resin) 49–86%
yield (two steps).19
Boc
Boc
N
N
Br
OH
c
a,b
OH
OH
R1
R1
R1
A variation of this series was obtained by reversing the benzylic
ether linkage such that the oxygen atom is adjacent to the central
aryl ring. These analogs were assembled from key intermediates 12
and 1420 via phenol alkylation (Scheme 2). Suzuki–Miyura cross
coupling between 4-piperidine vinyl boronate 9 and various brom-
ophenols (10) followed by subsequent hydrogenation of the
dehydropiperidines 11 provided target phenols 12.21,22 Despite
the modest yields of the Pd0 coupling, we favored this method
for installation of the piperidine by virtue of its generality and sim-
plicity. We used TES protection of the phenols (10) with the
assumption that this would improve overall handling and coupling
yields, and nearly all of the phenolic piperidine templates 12 were
prepared with this protection scheme.
10
12
11
d,e
Boc
N
Boc
N
H
N
3
2
4
d,e
N
X
B
OH
O
O
O
3
R2
4
6
5
9
R1
13: X = C
15: X = N
14
The employed Suzuki conditions caused complete removal of
the TES protecting group; however, subsequent experimentation
showed that at least in one test case (R1 = 3-F leading to 13d, Ta-
ble 2), the protection was unnecessary and overall yield for the
Scheme 2. Reagents and conditions: (a) Et3SiCl, Et3N, CH2Cl2, rt, 71–89% yield; (b) 9
(1.3 equiv), Pd(dppf)Cl2 (0.15 equiv), CsF, DMF, 110 °C, 27–65% yield; (c) H2 (3 atm),
Pd/C, MeOH, rt, 49–94% yield; (d) BrCH2Ar (2 equiv), K2CO3, acetone, rt, 45–86%
yield; (e) 4 M HCl/dioxane, rt, then ‘catch-and-release’ (MP-TsOH resin), 45–76%
yield.19
H
N
H
N
transformation of 14 to 16 was actually improved by performing
the Pd-mediated cross coupling with 9 on the unprotected phenol.
Reversing the ether linkage afforded additional active leads
with general structures 13 and 15 including the compounds in Ta-
ble 2. Efforts to optimize the targeted activities while prospectively
designing for minimized in vitro microsomal turnover led us to fre-
quently incorporate fluoro substitution on either or both of the aryl
rings.23 As summarized in Table 2, compounds with the reversed
benzyl ether linkages often had both NET and 5-HT1A binding
activity, as well as meaningful selectivity versus DAT. Remarkably,
the modified connectivity caused the piperidine analogs (13) to ex-
hibit the superior profile. The majority of the analogs in this series
Cl
Cl
F
O
O
N
N
N
N
S
S
F
1
2
5-HT : 9 nM
NET:1A 5 nM
DAT: 87nM
SERT: 81nM
5-HT : 4 nM
NET:1A 650nM
DAT: 1360nM
SERT: 98nM
Figure 1. Structures and binding Ki’s of initial NRI/5-HT1A dual activity leads.

6606
D. L. Gray et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6604–6607
Table 2
Table 3
Binding activity profiles of aryl piperidines and piperazines reversed benzyl
5-HT1A and monoamine binding affinities of piperidines and piperazine biaryl
ethers15,16
ethers15,16
ID
R1
R2
5-HT1A Ki
NET Ki
DAT Ki
SERT Ki
ID
R1
R2
5-HT1A Ki
NET Ki
DAT Ki
SERT Ki
(nM)
(nM)
(nM)
(nM)
(nM)
(nM)
(nM)
(nM)
13a
13b
13c
13d
13e
13f
13g
13h
15a
15b
H
H
H
2-F
H
121
47
606
364
77
2210
>1000
1160
28
76
34
9
9
162
2
1000
1810
7370
1030
9120
659
5530
452
>8720
>10,000
74
56
2970
28
274
2
40
2
1140
978
16a
16b
16c
16d
16e
16f
16g
16h
16i
16j
16k
16l
17a
17b
17m
H
H
H
3-F
H
63
416
21
170
1370
11
18
13
115
97
1
170
3
554
1710
1720
284
13
45
239
166
139
310
1680
6
1760
319
706
2330
901
1040
211
427
157
260
2
117
128
20
11
21
6-F
3-F
H
4-F
4-Cl
4-F
H
3-F
6-F
H
3-F
H
5-Me
3-F
4,5-F
5-Me
H
H
H
4-F
4-F
4-F
3,4-Cl
H
2-F
2-F
2-Cl
3-F
3-F
3-F
4-F
2-Me, 5-F
H
6
5
20
168
526
>4570
156
>4570
>4570
57
128
8
9
H
2-F
31
1
1450
1080
4730
4420
>7000
26
H
H
H
1880
994
3100
3-F
4-F
were nearly equipotent at NET and SERT, although a few notable
exceptions to this trend were identified (e.g., 13c, Table 2). The
SAR at the 4-position of the central ring was sensitive (e.g., 13e–
f), but fluorine atoms on other positions on either ring were
tolerated.
Despite the promising activity profile of compounds in both
ether variants, there were several immediate medicinal chemistry
challenges to solve including general in vitro microsomal instabil-
ity and a notable lack of functional agonism in our FLIPR-based 5-
HT1A functional activity screen (vide infra).10a With a desire to
eliminate a potential benzylic metabolic ‘soft-spot’ and perhaps
introduce functional agonist properties at the 5-HT1A receptor,
we targeted a set of analogous diphenyl ethers (16 and 17) starting
from 12 or 14 according to the route in Scheme 3.
In order to enable parallel synthesis of diphenyl ethers 16 and
17, a known Ullman procedure was adapted into a very effective
one-pot coupling-deprotection sequence.24 The crude Ullman cou-
pling mixture (NMP, CuI, etc.) was ‘quenched’ with an excess of
4 M HCl in dioxane solution, which removed the Boc protecting
group. The resulting reaction mixtures were diluted with MeOH
and directly poured onto commercial MP-TsOH resin columns,
washed with portions of MeOH, and eluted from the resin with
2 N NH3 in MeOH.19 This initial ‘catch-and-release’ isolation was
followed by automated, mass-guided HPLC purification. The same
procedure was successfully scaled to multi-gram quantities using
sealed reaction vessels and larger amount of resin, followed by sil-
ica-gel based final purification.
Gratifyingly, compounds in the diphenyl ether series displayed
NET and 5-HT1A binding affinity (Table 3). Importantly, within this
series, compounds such as 16e–g had meaningful selectivity for
NET binding versus the other two monoamine transporters while
also retaining activity at 5-HT1A. Significant activity changes oc-
curred with minor variation in substitutions within this series.
For instance, compound 16a is a modest inhibitor of NET, but addi-
tion of an ortho-fluoro substituent to this compound (16e) dramat-
ically enhances NET affinity. In agreement with previous SAR (see
Table 2), certain central-ring substitutions were detrimental to 5-
HT1A activity (see 16h, j); however, central-ring-fluoro substitution
at the 3- and 6-positions (e.g., 16c, d, f, i) preserved and even en-
hanced this desired pharmacology. Within the diphenyl ethers,
the aryl piperazines (17a, b, m) were weaker inhibitors of NET than
their piperidine counterparts (16a, b).
The identification of selective and potent piperidine diphenyl
ether hits such as 16e–g (Table 3) was a breakthrough on the pri-
mary pharmacology and a few analogs from this series were se-
lected for further profiling in functional assays for 5-HT1A and
the monoamine transporters. These data, along with early
in vitro metabolic stability data are presented in Table 4.25 The ini-
tially promising leads 16d and 16g had disappointing functional 5-
HT1A potency and eroded functional monoamine selectivity. Con-
sidering functional potency measures, the ortho-fluoro substituted
compounds 16e and 16f emerged as superior because they com-
bine into a single compound 5-HT1A partial agonism and functional
norepinephrine reuptake inhibition. These molecules also satisfied
other important requirements for advancing chemical matter such
as low microsomal clearance, functional selectivity versus DAT and
SERT, and straightforward synthetic preparation.
Given the location of these targets in the CNS, we felt it was
important to confirm that our lead compound 16f was capable of
interacting with central NET and 5-HT1A receptors in vivo after sys-
temic administration. Ex vivo receptor occupancy measurements
can be a powerful tool for developing and understanding the rela-
tionship between pharmacodynamic effects and pharmacokinetic
parameters.26 In the case of central targets, this methodology can
also provide confidence that molecules of interest reach an accept-
able brain/plasma equilibrium concentration at a given dose. Using
this technique, we demonstrated that compound 16f was brain pe-
netrant and binds to both target receptors in Sprauge-Dawley rats
Boc
N
H
N
following
a 10 mg/kg subcutaneous injection. Specifically, at
30 min post-dose in prefrontal cortex, ex vivo NET and 5-HT1A
receptor occupancies were determined to be 80% and 77%, respec-
tively.27 This level of occupancy was in line with targeted values at
both receptors and consistent with expectations based on the com-
pound’s pharmacokinetic parameters.
In conclusion, rational application of NRI pharmacophore infor-
mation to an initial thiomorpholinone aryl piperazine hit provided
a lead series of aryl piperazine and aryl piperidines ethers that dis-
play both NET inhibition and 5-HT1A binding activity. An iterative
screening and re-design process that simultaneously considered
potency and selectivity criteria led to some piperidine diphenyl
ethers (e.g., 16e and 16f, Table 3) with the targeted activity as well
X
X
1
a,b
OH
O
2
3
3
4
5
R2
R1
R1
12: X = C
14: X = N
16: X = C
17: X = N
Scheme 3. Reagents and conditions: (a) ArI (2 equiv), CuI, 2,2,6,6-tetramethylhep-
tane-3,5-dione, NMP, 140 °C; (b) 4 M HCl/dioxane, rt, then ‘catch-and-release’ (MP-
TsOH resin), 25–46% yield (two steps).19

D. L. Gray et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6604–6607
6607
Table 4
5-HT1A, NET, DAT, and SERT functional activities for select analogs25
ID
5-HT1A EC50 (nM)
5-HT1A Emax (%)
NE uptake EC50 (nM)
DA uptake EC50 (nM)
5-HT uptake EC50 (nM)
HLMCL (l
L/min/mg)a
15a
16d
16e
16f
>10,000
877
219
137
1150
N/A
35
51
74
44
83
358
21
28
49
5210
982
657
498
1880
2930
1760
1020
915
32
15
18
10
17
16g
186
a
In vitro turnover in human liver microsomes.
14. Collantes, E. M.; Ortwine, D. F. Abstracts of Papers, 238th ACS National Meeting,
Washington, DC, United States, March 16–20, 2009; COMP-229.
15. All binding data are averages from (at least) duplicate runs. For reference, the
following data on standard compounds were generated in these assays:
atomoxetine (NET = 8 nM, SERT = 62 nM); GBR12909 (DAT = 4 nM), buspirone
(5-HT1A = 14 nM).
16. (a) Monoamine binding assay: Homogenized paste prepared from HEK293 cell
lines expressing human clones for NE, DA, and 5HT transporters was incubated
with drug and 50 pM [125I]-RTI-55 for 90 min at rt in PEI coated FlashPlates.
Binding was terminated by assay volume withdraw with plates subsequently
counted for beta emissions. Nonspecific binding was determined in the
presence of selective inhibitors desipramine, GBR12909 and citalopram.; 5-
HT1A binding assay according to published protocol: (b) Graham, J. M.;
Coughenour, L. L.; Barr, B. M.; Rock, D. L.; Nikam, S. S. Bioorg. Med. Chem. Lett.
2008, 18, 489.
as selectivity versus DAT and SERT. As anticipated, subtle structural
changes within this series sometimes resulted in distinct binding
profiles across the four reported assays. Compounds with promis-
ing overall profiles were further characterized by screening in
in vitro functional assays. Finally, ex vivo receptor occupancy
was used to demonstrate CNS penetration and target binding for
a prototype lead compound from this series. Additional results
describing further refinement and characterization of these dual
activity biaryl ether piperidines will be reported in due course.
Acknowledgments
17. Dyck, B.; Parker, J.; Phillips, T.; Carter, L.; Murphy, B.; Summers, R.; Hermann, J.;
Baker, T.; Cismowski, M.; Saunders, J.; Goodfellow, V. Bioorg. Med. Chem. Lett.
2003, 21, 3793.
The authors are grateful to Dan Ortwine for guidance on the use
of the NRI pharmacophore model, Mark Lovdahl for assistance with
hydrogenation reactions, Cindy Donovan, Satavisha Dutta, Kim
Zoski, and Tammy Whisman for contributions to biological screen-
ing, and Martin Pettersson for helpful discussions.
18. Aryl piperidine benzyl alcohol
4
was prepared starting from 2-
bromobenzaldehyde by the method outlined in Scheme 2.
19. Free amines were obtained (with enhanced purity) from HCl salts (following
acidic N-Boc deprotection) by loading crude deprotection mixtures onto
commercial (Argonaut) mesoporous toluenesulfonated polystyrene (MP-
TsOH) columns, washing with MeOH, and eluting with 2 N NH3 in MeOH.
20. (a) Martin, G. E.; Elgin, R. J., Jr.; Mathiasen, J. R.; Davis, C. B.; Kesslick, J. M.;
Baldy, W. J.; Shank, R. P.; DiStefano, D. L.; Fedde, C. L.; Scott, M. K. J. Med. Chem.
1989, 42, 1052; (b) Richardson, T. I.; Ornstein, P. L.; Briner, K.; Fisher, M. J.;
Backer, R. T.; Biggers, C. K.; Clay, M. P.; Emmerson, P. J.; Hertel, L. W.; Hsiung, H.
M.; Husain, S.; Kahl, S. D.; Lee, J. A.; Lindstrom, T. D.; Martinelli, M. J.; Mayer, J.
P.; Mullaney, J. T.; O’Brien, T. P.; Pawlak, J. M.; Revell, K. D.; Shah, J.; Zgombick, J.
M.; Herr, R. J.; Melekhov, A.; Sampson, P. B.; King, C.-H. R. J. Med. Chem. 2004,
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25. (a) Functional 5-HT1A (FLIPR assay) see Ref. 10a.; (b) Monoamine uptake:
HEK293 cell lines expressing human clones for NE, DA, and 5HT transporters
were used to evaluate monamine reuptake inhibition. Cell suspensions were
filter harvested following a 20 minute, RT exposure to tritiated versions of NE
(100 nM), DA (50 nM), or 5HT (25 nM) in the presence or absence of test
agents. Non-specific uptake was determined in the presence of selective
antagonists atomoxetine, GBR12909 and citalopram. (c) HLM assay expressed
as apparent intrinsic clearance (uncorrected for microsomal binding): Riley, R.
J.; McGinnity, D. F.; Austin, R. P. Drug. Metab. Dispos. 2005, 33, 1304.
26. Grimwood, S.; Hartig, P. R. Pharmacol. Therap. 2009, 122, 281.
27. Ex vivo binding studies were carried out on homogenized brain tissue derived
from animals treated with putative NRI/5-HT1A compounds (subcutaneous,
60 min post-dose). 5-HT1A binding was determined in hippocampal
preparations with or without 1 nM [3H] 8-OH-DPAT (GE Healthcare—
TRK850) as a competitive binding agent. Norepinephrine transporter binding
was performed on cortical tissue homogenates using 5 nM [3H] nisoxetine
(Perkin–Elmer—NET1084). Non-specific binding was defined using 1
lM WAY-
100635 for 5HT1A receptors or 1 M desipramine for NET. 5-HT1A SEM = 0.6%
l
12. (a) Pessoa-Mahana, H.; Araya-Maturana, R.; Claudio, S. B.; Pessoa-Mahana, C. D.
Mini-Rev. Med. Chem. 2003, 3, 77; (b) Murasaki, M.; Miura, S. Prog. Neuro-
Psychopharmacol. Biol. Psych. 1992, 16, 833.
13. Thomas, G. H.; Babbs, A. J.; Chatfield, R. E.; Krulle, T. M.; Widdowson, P. S.;
Provost, D.; McCormack, J. G. Obesity 2009, 17, 467.
(n = 4), NET SEM = 1.5% (n = 4). IACUC approved procedures were carried out in
compliance with the NIH Guide for the Care and Use of Laboratory Animals
(1985).