Azepines and piperidines with dual norepinephrine dopamine uptake inhibition and antidepressant activity

Article abstract of DOI:10.1021/ml300262e

Herein, we describe the discovery of inhibitors of norepinephrine (NET) and dopamine (DAT) transporters with reduced activity relative to serotonin transporters (SERT). Two compounds, 8b and 21a, along with nomifensine were tested in a rodent receptor occupancy study and demonstrated dose-dependent displacement of radiolabeled NET and DAT ligands. These compounds were efficacious in a rat forced swim assay (model of depression) and also had activity in rat spontaneous locomotion assay.

Full text of DOI:10.1021/ml300262e

Letter
pubs.acs.org/acsmedchemlett
Azepines and Piperidines with Dual Norepinephrine Dopamine
Uptake Inhibition and Antidepressant Activity
Dean G. Brown,*,†,§ Peter R. Bernstein,† Ye Wu,† Rebecca A. Urbanek,† Christopher W. Becker,†
Scott R. Throner,† Bruce T. Dembofsky,† Gary B. Steelman,† Lois A. Lazor,† Clay W. Scott,†
Michael W. Wood,† Steven S. Wesolowski,† David A. Nugiel, Stephanie Koch,† Jian Yu,†
Donald E. Pivonka,† Shuang Li,† Carol Thompson,† Anna Zacco,† Charles S. Elmore,†
Patricia Schroeder,† JianWei Liu,† Christopher A. Hurley,‡ Stuart Ward,‡ Hazel J. Hunt,‡ Karen Williams,‡
Joseph McLaughlin,† Valerie Hoesch,† Simon Sydserff,† Donna Maier,† and David Aharony†
†CNS Discovery Research, AstraZeneca Pharmaceuticals, 1800 Concord Pike, Wilmington, Delaware 19850-5437, United States
‡Argenta, 8/9 Spire Green Centre, Flex Meadow, Harlow, Essex, CM19 5TR, United Kingdom
S
* Supporting Information
ABSTRACT: Herein, we describe the discovery of inhibitors of norepinephrine
(NET) and dopamine (DAT) transporters with reduced activity relative to
serotonin transporters (SERT). Two compounds, 8b and 21a, along with
nomifensine were tested in a rodent receptor occupancy study and demonstrated
dose-dependent displacement of radiolabeled NET and DAT ligands. These
compounds were efficacious in a rat forced swim assay (model of depression) and
also had activity in rat spontaneous locomotion assay.
KEYWORDS: norepinephrine, dopamine, uptake inhibitors, antidepressant
Dual NET/DAT reuptake inhibitors (NDRIs) have also
been utilized for the treatment of depression. The only current
clinically utilized compound with NDRI activity is buproprion,
but the mechanism of action is complex and may involve active
metabolites.3 Nomifensine 3 was a clinically useful NDRI but
was withdrawn from the market in 1986 due to increased
incidence of hemolytic anemia.7−11
Published reports on nomifensine 3 have hypothesized that
this hemolytic anemia might have been associated with a
reactive metabolite, specifically formation of a dihydroisoqui-
nolinium ion.12 With this in mind, we sought to identify a novel
scaffold with dual NET and DAT inhibition, which would not
form a dihydroisoquinolinium ion posing a potential reactive
metabolite risk.
On the basis of the known SAR from the literature and in-
house examples,13 we hypothesized that the aniline was not
necessary for activity, and the fused aromatic ring could be
replaced in nomifensine with an cyclic aliphatic ring. Several
scaffolds were investigated, but the 2,3,4,7-tetrahydro-1H-
azepine proved to be useful in having the desired SAR as
well as synthetic handles for further optimization.
espite new drugs that have helped many patients manage
Ddepressive disorders, there remains a large portion of
patients who do not respond to current therapies.1,2 The most
frequently prescribed drugs work by blocking uptake of
monoamine neurotransmitters such as serotonin (SERT).3 A
well-known example of a selective serotonin reuptake inhibitor
(SSRI) with clinical efficacy is illustrated by fluoxetine 1 (Figure
1).4 Dual monoamine reuptake inhibitors such as duloxetine 2
have gained clinical acceptance and work by blocking serotonin
as well as norepinephrine reuptake (SNRI).5 Triple reuptake
inhibitors (TRI) such as amitifadine 4 are currently in clinical
studies and demonstrate the ability to block SERT,
norepinephrine (NET), and dopamine (DAT) reuptake.6
Received: August 31, 2012
Accepted: November 12, 2012
Figure 1. Examples of SSRI, SNRI, NDRI, and TRI.
© XXXX American Chemical Society
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a
Preparation of 2,3,4,7-tetrahydro-1H-azepines is illustrated in
Scheme 1. Reaction of acid 5a−j and N-methylprop-2-en-1-
Scheme 3. Quaternary Substituted Analogues
a
Scheme 1. Preparation of Azepine Analogues
a
a
Reagents and conditions: (a) BrCH2CH2OMOM, NaH, THF, 42%.
(b) CHCHCH2Br, KOH, TBAI, MeCN, 77%. (c) DIBAL-H,
PhMe. (d) CHCHCH2NH2, NaBH(OAc)3, 1,2-dichloroethane,
37% for steps c and d. (e) Boc2O, CH2Cl2, 78%. (f) Grubbs II
catalyst,15 CH2Cl2, reflux, quan. (g) HCl, MeOH, quan., chiral SFC.
Reagents and conditions: (a) CHCHCH2NHMe, 2-chloro-1-
methyl pyridinium iodide, CH2Cl2, 25−85%. (b) Grubbs II
catalyst,14,15 PhMe, 40 °C, 40−70%. (c) LiAlH4, THF, 20−75%. (d)
ACE-Cl, PhMe, reflux, 30−62%.
amine followed by ring closing metathesis gave the lactams
6a−j.14,15 Reduction of 6a−j with LAH provided N-Me
analogues 7a−e and intermediates 7f−j. Demethylation of
7a−j provided compounds 8a−j.
Enantiomerically pure 2,3,4,7-tetrahydro-1H-azepines were
prepared by starting with enantiomerically pure acid to give the
(R) stereoisomers 7b and 8b and (S) stereoisomers 7c and
8c.16 Analogues 7a and 8a could be hydrogenated to give the
azepanes 9 and 10 (Scheme 2). Alkylation of azepine 8b
provided N-alkyl derivates such as 11 and 12.
confirmed by comparison of measured and computed vibra-
tional circular dichroism (VCD) spectra.17,18
Our investigation also examined piperidine analogues with
substitution similar to those in the azepine series. Several 2-(3-
phenylpiperidin-3-yl)ethanol type derivatives had previously
been synthesized in a different project19 and were tested for
activity in the NET, DAT, and SERT assays as a direct
comparison to the azepines 16a and 16b. From this screening
effort, 17a and 17b were identified.
The synthesis of a benzofuran analogue was accomplished in
analogous fashion (Scheme 4).20 The enantiomers were
separated using chiral SFC to give 21a and 21b and assigned
by measured and computed VCD spectra.
a
Scheme 2. Preparation of Azepine and Azepane Analogues
a
Scheme 4. Quaternary Substituted Piperidines
a
Reagents and conditions: (a) 7a, H2, Pd/C, HCl, 79%. (b) 8a, PtO2,
H2, AcOH. (c) KOH, MeCN/H20, 150 °C, microwave, 75% for 11,
35% for 12.
The preparation of the quaternary substituted 2,3,4,7-
tetrahydro-1H-azepines is illustrated in Scheme 3. The
hydroxyethyl substituent was selected based on parallel SAR
emerging from studies on a piperidine scaffold. The reaction
sequence began with 13, which was alkylated with methox-
ymethyl ether (MOM) protected bromoethanol. A second
deprotonation using KOH and tetrabutylammonium iodide
(TBAI) as a phase transfer catalyst installed the quaternary
center in 14. Reduction of the nitrile with diisobuytlaluminum
hydride (DIBAL)-H, followed by hydrolysis, led to an
intermediate aldehyde. Reductive amination and protection
led to 15, which was subjected to ring-closing metathesis and
then deprotected. Chiral supercritical fluid chromatography
(SFC) provided enantiomers 16a and 16b. Assignment of
absolute configuration was done by X-ray crystallography and
a
Reagents and conditions: (a) NaH, Br(CH2)2OMOM. (b) NaH,
Br(CH2)3Cl. (c) Ra Ni, NH4OH, NaCl (aq); HCl, MeOH; chiral
HPLC.
Results of NET, DAT, and SERT testing are reported in
Table 1, along with cardiovascular ion channel effects (hERG),
hClint, and CYP2D6 profiles. A notable observation in these
results is a pronounced difference between the nomifensine
stereoisomers [e.g., 3-(S) ≫ 3-(R)], which was confirmed to be
consistent with the literature.21 All activity for nomifensine
resides in the (S) stereoisomer, although the nomifensine
racemate was used clinically. This large dependence on
B
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Table 1. Summary Results of NET, DAT, and SERT Uptake and Selected in Vitro Properties
Ki (nM)
a
a
a
b
c
d
compd
core
R1
R2
NET
DAT
SERT
hERG IC50 (μM)
hClint
CYP2D6 IC50 (μM)
3
A
A
A
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
C
C
B
B
D
D
E
E
E
E
5.0
4.0
10
1400
1200
800
100
83
>27
NT
NT
7.6
10
37
>20
NT
NT
10.2
7.6
3 (S)
3 (R)
7a
7.0
450
5.0
3.0
13
NT
NT
57
1200
57
3,4-dichlorophenyl
(R)-3,4-dichlorophenyl
(S)-3,4-dichlorophenyl
3,4-dichlorophenyl
(R)-3,4-dichlorophenyl
(S)-3,4-dichlorophenyl
2-napthyl
Me
Me
Me
H
7b
12
51
7c
54
130
220
220
420
63
NT
11
86
11
8a
66
6.0
6.0
8.0
10
30
5.1
8b
H
17
21
33
3.9
8c
H
24
13.7
15.4
>27
26
50
3.1
8d
8e
H
25
94
6.6
3-Cl-4-Me phenyl
4-Cl, 3-Me phenyl
4-Cl, 3-Me phenyl
3-Cl, 4-F phenyl
H
83
49
600
270
NT
1500
NT
NT
800
240
260
140
150
540
31
100
85
5.6
7d
8f
Me
H
130
170
130
250
430
120
46
23
18
24
26
NT
15
NT
6.4
8g
H
68
23
8h
8i
4-Cl phenyl
H
25
>31
>33
15
NT
NT
97
NT
NT
6.7
3-Cl phenyl
H
240
60
7e
2-benzofuran
Me
H
8j
2-benzofuran
23
19
83
7.8
9
3,4-dichlorophenyl
3,4-dichlorophenyl
3,4-dichlorophenyl
3,4-dichlorophenyl
(S)-3,4-dichlorophenyl
(R)-3,4-dichlorophenyl
(S)-3,4-dichlorophenyl
(R)-3,4-dichlorophenyl
(R)-2-benzofuran
(S)-2-benzofuran
Me
H
74
8.0
11
10
NT
35
NT
2.3
10
130
12
6.1
10
11
-(CH2)2OH
37
52
>20
3.9
12
-(CH2)2OMe
15
9.0
4.2
91
5.3
>33
>33
>33
>33
>33
NT
150
<4
NT
<4
<4
22
16a
16b
17a
17b
21a
21b
H
H
H
H
H
H
5.1
49
18
230
42
NT
>20
>20
>20
NT
3.3
250
4.0
430
9.3
140
6.0
400
250
230
NT
NT
a
Uptake inhibition measured using HEK cells overexpressed with human recombinant NET, DAT, or SERT receptors with Molecular Devices
neurotransmitter kit (inhibition of a fluorescent substrate).25 Results of NET, DAT, and SERT are an average of at least n = 3 with up to 2-fold
variability. NT, not tested. Values determined in CHOK1-hERG cells using electrophysiological measurements. Human liver microsomal clearance
(μL/min/mg). In vitro inhibition of cytochrome P450 CYP2D6 isoforms.
b
c
d
stereochemistry was not observed with the 3,4-dichlorophenyl
2,3,4,7-tetrahydro-1H-azepines such as 7b versus 7c or 8b
versus 8c, where only a minor difference was observed.
Replacement of the 3,4-dichlorophenyl moiety resulted in a loss
of potency at all transporters with the exception of the napthyl
analogue 8d (an expected bioisotersic replacement of 3,4-
dichlorphenyl). This change led to similar potency at NET and
DAT but was more potent at SERT when compared to racemic
3,4-dichlorophenyl 8a. The N−H benzofuran analogue 8j was
similar in NET activity to the 3,4-dichlorophenyl analog 8a but
showed a slight drop-off in DAT affinity.
The activities of racemic saturated azepanes 9 and 10 were
similar when compared to the azepine counterparts 7a and 8a.
The introduction of groups on the azepine N atom was
generally well tolerated for NET and DAT activity. Compounds
11 and 12 illustrate that hydrophilic substituents on the N atom
gave the desired NET = DAT > SERT ratio. However, N-
alkylation appeared to give rise to more potent activity at hERG
(cf. 12 hERG IC50 = 5.3 μM vs 8b = 21 μM) as well as an
increase in hClint. Incorporation of a hydroxyethyl substituent
(16a, 17a, and 21a) at the 3-position led to an increase in NET
but also an increase in SERT. In these cases, a more
pronounced difference in stereochemical activity was seen,
similar to nomifensine 3.
Incorporation of the hydroxyethyl substituent also improved
metabolic stability, decreased hERG, and improved CYP2D6
liability relative to 8b. The benzofuran quaternary substituted
piperidine 21a provided the closest match to the nomifensine
profile with NET ≅ DAT (Ki = 4.2 and 6.3 nM, respectively)
and a large separation at SERT (Ki = 230 nM). Compounds 8b
and 21a were also tested for reactive metabolites with GSH
under in vitro metabolic activation and did not reveal any
adduct formation.
In vivo receptor occupancy was selected as a pharmacody-
namic assay to verify that selected ligands could block
accumulation of labeled NET/DAT ligands in respective
brain regions (Table 2). Test compounds were administered
to the rats, followed 30 min later by administration of a
radioligand specific for NET or DAT. This assay is described in
more detail in the Supporting Information.
Nomifensine was chosen as the benchmark comparator
compound, and [3H]-MeNER and [3H]-PE2I were used as the
C
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ACS Medicinal Chemistry Letters
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the receptor occupancy provided rank order in NET and
demonstrated potent and dose-dependent receptor occupancy
at both receptors at pharmacologically relevant doses used in
behavioral assays.
These compounds were then tested in a rat forced-swim
assay (FST) for antidepressant effects. Results are shown in
Figure 2. Nomifensine 3 demonstrated efficacy at 10, 3, and 1
mg/kg but not at 0.3 mg/kg. It should be noted that doses for
the rat forced-swim were selected based on systemic exposure
and receptor occupancy, described above. The 2,3,4,7-
tetrahydro-1H-azepines 8b demonstrated activity at 10 and 3
mg/kg but not at 1 mg/kg.
Table 2. NET and DAT Receptor Occupancy Studies in Rats
NET
DAT
EC50
(nM)
EC
NET ED50 DAT ED50
%
free
B/P
f
50a
d
d
e
compd
(nM)
(mg/kg)
(mg/kg)
ratio
c
c
3
3.1
46
31
0.05
0.5
0.7
60.5
7.7
75
10.1
4.8
b
c
8b
21a
220
2.1
c
d
5.6
33
0.17
0.8
5.8
a
EC50 = unbound plasma levels equaling 50% occupancy. Utilized
b
[3H]MeNER as NET ligand (sc) and quantified in thalamus. Utilized
[3H]WIN 35,428 as DAT radioligand (sc), quantified in striatum.
c
d
Utilized [3H] PE2I (sc) and quantified in striatum. ED50 = dose to
give 50% occupancy at the receptor. The mean value is reported. The
95% confidence interval was typically half of the mean value
The quaternary piperidine 21a demonstrated efficacy at 7.3
and 2.4 mg/kg but not at 0.7 mg/kg. The compounds were also
examined in a rat spontaneous locomotion assay (LMA) as a
behavioral measure of activation of the DAT system. In
contrast, selective NET inhibitors have been reported to
suppress locomotor activity.26 The compounds were evaluated
for both distance traveled and stereotypic behaviors in the last
15 min of the assay. Nomifensine 3 dose dependently induced
both locomotor activity and stereotypic behavior with an
inverted U-shaped curve. The reductions in LMA could be
related to hyperactivation of the dopaminergic system and a
shift toward stereotypic behaviors. Alternatively, LMA
suppression may be driven by increasing activation of the
NET system. Compound 8b exhibited a profile similar to that
of nomifensine 3. Although dose-dependent increases in LMA
and stereotypy were observed, dose-dependent decreases in
LMA and stereotypy were not observed with compound 21a.
Perhaps, declinations in these measures might be observed at
higher doses. Given the overall precision of the FST, LMA, and
stereotypy assays and the challenge in interpreting the
behavioral manifestations of activating two major neuro-
modulatory systems, it can be argued that nomifense 3 and
compounds 8b and 21a displayed similar overall behavioral
profiles. No severe stereotype such as self-biting was observed
reported. Scatter plots are presented in the Supporting Information to
e
give the reader a more detailed view of the error in the assay. Percent
f
free plasma concentrations in rat. Brain to plasma ratio.
NET and DAT ligands, respectively.22−24 It should also be
noted that early in the program, [3H]-WIN35,458 was used as
the DAT ligand,22 but it was found that [3H]-PE2I gave
smoother curves and a better correlation with our in vitro
assays. Compound 8b was tested using [3H]-WIN35,458 but
was not repeated with the new ligand. The calculated EC50 in
these assays demonstrated a slight disconnect with the human
recombinant transporter assays in Table 1, most notably against
DAT. This was not unexpected given the differences between
rat and human orthologues and the difference between a
human recombinant assay and a rat receptor occupancy assay
with the inherent errors therein. It is not clear why this
discrepancy was more prominent in the DAT measurement.
The values of 3.1 and 31 nM observed for nomifensine 3 for
NET and DAT, respectively, are consistent with the values
reported in a rat synaptosomal uptake assay (NET Ki = 4 nM,
DAT Ki = 26 nM).8 This observation led us to believe that the
rat receptor occupancy study could be used as a key tool in
dose selection for preclinical efficacy testing in rodents. Overall,
Figure 2. In vivo activity in FST, spontaneous LMA. Top row: Immobility time in FST for compounds 3, 8b, and 21a following dosing (sc) in
Sprague−Dawley rats. Middle row: Distance traveled in spontaneous locomotive activity (LMA) in rats as measured by beam breaks. Bottom row:
Stereotypic behavior in the locomotive activity assay as measured by number of times the same beam is broken. DMI = desipramine (positive
control, dosed 15 mg/kg ip). Data presented as group means SEMs. An asterisk indicates statistical significance from vehicle (Veh), using one-way
ANOVA analysis.
D
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with any compound at any dose. Because the objective of the
project was to identify an “activating” antidepressant with a
DAT uptake inhibition profile, increases in LMA were
expected. However, it remains to be determined whether
increased activation of the DAT system will afford an adequate
therapeutic index.
Plasma levels of 8b were measured at all doses in the FST
experiment. On the basis of an assumption that free plasma
would equal free brain levels, it was estimated that the free
plasma concentration at the lowest effective dose in FST (3
mg/kg) was equivalent to the free concentration that gave
between 50 and 70% occupancy at DAT and >90% at NET
(based on receptor occupancy studies, see the Supporting
Information for more details).
In summary, several potent dual NET/DAT uptake
inhibitors have been reported, which have in vivo efficacy
similar to nomifensine in a rat model of depression. Further
work will need to be done to understand clinical doses and
potential dose-limiting side effects such as abuse liability.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthetic procedures and assay details. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: 781-839-4101. E-mail: dean.brown@astrazeneca.com.
Present Address
§AstraZeneca Pharmaceuticals, 35 Gatehouse Drive, Waltham,
Massachusetts 02451.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Amy Hehman, Lynne Neveras, Lisa Leon, Laurie
Ross, Donna Sottung, John Zysk, Yan Li, William Potts,
Chengwei Fang, and Peter Dorff.
ABBREVIATIONS
■
TBAI, tetrabutylammonium iodide; DIBAL, diisobuytlalumi-
num hydride; MOM, methoxymethyl ether protecting group;
CYP, cytochrome P450 metabolic enzymes; hERG, human
ether a-go-go related gene; SFC, supercritical fluid chromatog-
raphy; ACE, 1-chloroethyl chloroformate
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