Synthesis and structure-activity relationships of selective norepinephrine reuptake inhibitors (sNRI) with improved pharmaceutical characteristics

Article abstract of DOI:10.1016/j.bmcl.2008.10.013

The design synthesis and SAR of a series of chiral ring-constrained norepinephrine reuptake inhibitors with improved physicochemical properties is described. Typical compounds are potent (IC₅₀s <10 nM), selective against the other monoamine tra

Full text of DOI:10.1016/j.bmcl.2008.10.013

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6151–6155
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and structure–activity relationships of selective norepinephrine
reuptake inhibitors (sNRI) with improved pharmaceutical characteristics
Joseph Pontillo a, Dongpei Wu a, Brett Ching a, Sarah Hudson a, Marc J. Genicot a, Yinghong Gao b,
Todd Ewing b, Beth A. Fleck c, Kathleen Gogas d, Anna Aparicio e, Hua Wang e, Jenny Wen e,
Warren S. Wade a,
*
a Department of Medicinal Chemistry, Neurocrine Biosciences, Inc., 12790 El Camino Real, San Diego, CA 92130, USA
b Department of Computational Chemistry, Neurocrine Biosciences, Inc., 12790 El Camino Real, San Diego, CA 92130, USA
c Department of Pharmacology, Neurocrine Biosciences, Inc., 12790 El Camino Real, San Diego, CA 92130, USA
d Department of Neuroscience, Neurocrine Biosciences, Inc., 12790 El Camino Real, San Diego, CA 92130, USA
e Department of Preclinical Development, Neurocrine Biosciences, Inc., 12790 El Camino Real, San Diego, CA 92130, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The design synthesis and SAR of a series of chiral ring-constrained norepinephrine reuptake inhibitors
with improved physicochemical properties is described. Typical compounds are potent (IC50s <10 nM),
selective against the other monoamine transporters, weak CYP2D6 inhibitors (IC50s > 1 lM) and stable
Received 14 August 2008
Revised 30 September 2008
Accepted 2 October 2008
Available online 7 October 2008
to oxidation by human liver microsomes. In addition, the compounds exhibit a favorable polarity profile.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Norepinephrine
Serotonin
Monoamine reuptake inhibition has been an effective therapeu-
tic intervention in a variety of CNS diseases starting with depres-
sion, and recently expanding to include chronic pain, ADHD, and
stress urinary incontinence.1–4 We were interested in multiple
indications in the therapeutic spectrum of sNRI compounds and
initiated work directed toward finding potent and selective com-
pounds with good pharmaceutical properties and minimal risk of
drug–drug interactions.5–7 In the previous letter,7 a series of chiral
sNRIs with improved stability was reported. However, the overall
hydrophobicity of the best compounds severely limited the ability
to avoid CYP2D6-related drug–drug interactions with marketed
pharmacotherapies. Further work directed at providing a larger
range of potential drug candidates is reported here.
Compounds in the previously discovered series, shown in Fig-
ure 1, were generally more hydrophobic than atomoxetine, and
unfortunately, hydrophobicity correlated with greater CYP2D6
inhibition. Substitution on the aryloxy ring resulted in large
changes in NET/SERT selectivity, and therefore was limited in
scope. Consequently, it was necessary to look for modifications of
the core structure that maintained potency at NET while being
intrinsically more hydrophilic. In addition, synthesis of the more
potent cis diastereomers like 3 and 4 was difficult, especially for
electron-rich aromatic rings that gave the best potency and selec-
tivity. If possible, a series where synthesis of the cis diastereomer
was straightforward, even for electron-rich aryl groups, and with
no additional stereocenters would be preferred. These require-
ments were met with the hydroxyindane core shown in Scheme 1.
Synthesis of the hydroxyindane compounds proceeded from the
known hydroxymethylindene 5.8 Following protection as the TBS
ether, epoxidation was performed with mCPBA, and the resulting
epoxide 6 was subsequently opened with the anion of a phenol
or thiophenol, illustrated in Scheme 1 for the direct analogs of ato-
moxetine. During the epoxide opening, concomitant partial to full
deprotection occurred. In the cases of partial deprotection, tetrabu-
tylammonium fluoride was added to the crude mixtures to gener-
ate diol 7 in good yields. Installation of the amine followed a three
step procedure that was best accomplished in one pot. Formation
of the primary mesylate was followed by conversion to the exo
epoxide that was then opened with a primary or secondary amine
to give predominantly the aminomethyl regioisomer. For the car-
bon analog, epoxide 6 was opened with an in situ generated cup-
rate, and the resultant diol 10 was converted to the amine under
similar conditions. This sequence ensured the relative stereochem-
istry observed for previous compounds,7 where the aryloxy
group and aminomethylene groups are cis relative to each other.
A crystal structure of 26-II,9 the inactive enantiomer of 26-I in
Table 2, established that the two oxygen substituents are in the
* Corresponding author. Tel.: +1 858 617 7600; fax: +1 858 617 7601.
E-mail addresses: shudson@neurocrine.com (S. Hudson), warrenswade@gmail.
com (W.S. Wade).
URL: http://www.neurocrine.com (W.S. Wade).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.013

6152
J. Pontillo et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6151–6155
O
O
O
O
O
N
H
N
H
N
H
N
H
O
2
3
4
1
Figure 1. Structures of the active enantiomers of four ring-constrained isomers of atomoxetine reported previously.5–7
pseudoaxial position, as had been observed previously for the aryl-
oxy ring.7 The less active enantiomer was produced in reasonable
quantity during the chiral separation of 26-I for in vivo studies.
The hydroxyl anion of Boc-protected 8-I could be alkyated with
methyl iodide then deprotected under acidic conditions to gener-
ate 12-I.
Two variants of this sequence were performed, as shown in
Scheme 2. For the primary amine products (e.g., 16), azide anion
was used as the nucleophile to give 15 that was then reduced using
a Staudinger reduction.10 The alternative diastereomer could most
easily be synthesized starting with diol 7 and reversing the direc-
tion of formation of the exo epoxide. After protection of the pri-
mary alcohol as the TBS ether and formation of the tertiary
mesylate 17, deprotection of the primary alcohol, anionic epoxide
formation and ring opening with methylamine generated 18. The
intermediates in this sequence were sensitive to elimination. Con-
sequentially, yields were considerably lower.
The synthesis of compounds 21 and 23 is shown in scheme 3. A
tandem alkylation/aldol reaction generated 19 as the major diaste-
reomer that could be selectively crystallized from hexane/ethyl
acetate. Aryl substitution proceeded normally to generate both dia-
stereomers of the methyl dihydrofuran core. The relative stereo-
chemistry of 23 was established by NOE.
Compound enantiomers were initially separated by chiral HPLC,
but it was found that chiral 7 could be produced in high optical
purity (85–90%ee) under Jacobsen epoxidation conditions.11
Recrystallization of the final product salts then gave material with
95–99%ee. Compound 24 proved difficult to separate via chiral
HPLC, so synthesis of 24-I was accomplished by the chiral route.
The more active enantiomer consistently showed a positive optical
X
X
O
a, b
c, d
OTBS
OH
e, f, g
e, f, g
OH
OH
OH
N
H
6
8 X = O
9 X = S
5
7
h
OH
OH
OH
N
H
10
11
Scheme 1. Reagents and conditions: (a) TBSCl, TEA, DMAP, DCM, rt, 3 h, 90%; (b) mCPBA, NaHCO3, DCM, 0 °C, 6 h; (c) ArylXH, NaH, DMSO, then 6, 75 °C, 18 h; (d) TBAF, THF, rt,
10 min, 60–70% for combined steps (b–d); (e) Ms2O, TEA, DCM, À30 to 0 °C, 1 h; (f) K2CO3, DMF, rt, 1.5 h; (g) methylamine, THF, 70 °C, 18 h, 30–70% for three steps;
(h) 2-MeBnMgBr, CuI, THF, À78 °C, 0.25 h, then 6, À40 to À20 °C, 3 h, 74%.
O
O
OH
N3
OH
NH2
c
a, b
d, e
O
15
17
16
18
OH
OH
O
O
f, g
OMs
OTBS
OH
7
N
H
Scheme 2. Reagents and conditions: (a) MsCl, TEA, DCM, 0 °C, 1 h; (b) K2CO3, DMF, rt, 2 h, then NaN3, 15-crown-5, 70 °C, 18 h, 75% for two steps; (c) PPh3, THF, rt, 1 h then
H2O, 65 °C, 1 h, 78%; (d) TBSCl, TEA, DCM, 0 °C–rt, 16 h, 50%; (e) MsCl, TEA, DCM, 0 °C–rt, 18 h, 21%; (f) TBAF, THF, rt, 1 h, 63%; (g) NaH, DMA, rt, 1.5 h, then methylamine, 55 °C,
18 h, 7%.

J. Pontillo et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6151–6155
6153
OH
O
d
b, c
O
N
H
O
N
H
O
OH
20
22
21
23
a
CO2Me
e
O
OH
O
O
b, f
19
CO2Me
O
N
H
O
Scheme 3. Reagents and conditions: (a) Methyl 2-bromopropionate, K2CO3, DMF, 100 °C, 16 h, 60%; (b) DiBAlH, DCM, À78 °C, 2 h; (c) Methylamine, BH3.pyr, THF, rt, 16 h, 10%
(two steps); (d) 2-Fluorotoluene, NaH, DMSO, 80 °C, 16 h, 10%; (e) 2-Methylphenol, PPh3, DEAD, THF, rt, 18 h, 32%; (f) Methylamine, Na(OAc)3BH, THF, rt, 16 h, 50%
(two steps).
rotation. Absolute conformations were determined by analogy to
the aforementioned crystal structure of 26-II.
for NET active compounds.13 As expected, no significant inhibition
of CYP3A4 was observed for any of these compounds.
All compounds were tested for their ability to inhibit norepi-
nephrine, serotonin and dopamine uptake in HEK cell lines that
had been stably transfected with the human transporters. Com-
pounds were initially tested in two independent dose–response
experiments, and those with reasonable potency at NET were re-
tested multiple times. Atomoxetine was included on all assay
plates as a standard control, and assay variability was reasonable
for a functional assay with SEM values typically below 0.2 log
units. Potency values are reported as IC50, though with the neuro-
transmitter concentrations in each assay well below their respec-
tive Km values, little difference would be expected between the
measured IC50 and Ki of the compounds. Active compounds were
further tested in transporter binding assays by competition with
the appropriate radioligand. For all compounds, active functional
inhibition at NET (IC50) correlated well with displacement of 3H-
nisoxetine from its NET binding site (Ki). All compounds were
tested for inhibition of CYP3A4 and CYP2D6 activity,12 and the
metabolic stability in human liver microsomes was also measured
Initial testing of the hydroxyindane racemates indicated that
the activity observed with 2–4 and atomoxetine was retained in
the hydroxyindanes. SAR (Table 1) matched that observed for pre-
vious series at the amine site,6 with 8 being the most potent, the
primary amine 16 being slightly less active and the ethyl amine
13 20-fold less potent. In this series, the sulfur and carbon analogs
at the ring junction, 9 and 11, were synthetically accessible and
maintained potency at NET. Upon separation of the racemate, the
direct analog of atomoxetine in this series, 8-I, was indistinguish-
able from atomoxetine in the inhibition of all three transporters
and CYP2D6. Consistent with previous series,7 the absolute config-
uration of the active enantiomer 8-I matches atomoxetine and
reboxetine.
Positioning and substitution of the hydroxyl group was critical
for activity (Fig. 2). The alternative diastereomer, 18, was >100-fold
less active, and both diastereomers of the regioisomeric 2-methyl-
benzofuran 21 and 23 were completely inactive. Additionally,
methylation of 8-I to generate 12-I removed all transporter
Table 1
SAR of the hydroxyindane corea
X
Z
N
H
R1
Compound
Isomer
X
Z
R1
NETb
SERTb
DATb
3000
3500
2700
1600
2300
CYP2D6c
cLogPd
Atomoxetine
2
8
R
Me
Me
Me
Me
Me
Me
Me
Me
5
7
180
1,400
700
2000
800
2300
2000
2500
400
3.3
3.9
3.1
3.1
3.1
3.6
3.7
3.6
3.6
2.5
2.8
2.8
R,S
rac
S,R
R,S
rac
rac
S,R
rac
rac
rac
S,R
O
O
O
O
S
H
OH
OH
OH
OH
OH
OMe
OH
OH
OH
OH
30
3
770
10
30
>10,000
700
2,800
75
8-I
8-II
9
11
12-I
13
14
16
16-I
184
5500
1400
2500
>10,000
5700
3500
4000
3000
4700
C
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
900
O
O
O
O
O
5000
1300
>10,000
4000
3300
Et
CH2CH2OH
H
H
77
a
Transporter data are the average of two or more independent measurements.
IC50 (nM) for inhibition of monoamine uptake.
IC50 (nM) for inhibition of CYP2D6.12
b
c
d
Calculated LogP.14

6154
J. Pontillo et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6151–6155
micromolar range for all fluoro isomers, an improvement over pre-
vious series.7 Halogen substitution at the 2 position (25-I and 26-I)
also generated potent and selective compounds with reasonable
CYP2D6 activity. In this series as well, 2-methoxy substitution re-
sulted in both a decrease in activity at NET and an increase in activ-
ity at CYP2D6. Overall the most potent and selective compound
was 27-I, though the CYP2D6 inhibition of this compound was just
O
O
O
OH
O
O
N
N
H
N
H
H
18
21
23
below 1 lM. Oxidative clearance in microsomes was significantly
lower than atomoxetine for this series, with several compounds
exhibiting no measurable loss of parent in a 60-min incubation.
The only exceptions were methyl substitution of 8-I to give
compounds 31-I and 32-I, where there is the potential for benzylic
oxidation and the naphthyl compound, 24-I.
Figure 2. Inactive isomers of 8.
activity, as did adding an additional hydroxyl group near the amine
position (see 14).
Selectivity against an in-house panel of receptors for amine
neurotransmitters (selected dopamine, serotonin, adrenergic, and
muscarinic receptors) revealed no activity for these compounds ex-
To further ameliorate the CYP2D6 inhibition, more polar substi-
tution of the aryloxy ring was evaluated. Hydroxyl substitution on
the methyl of 8-I to generate 30-I was not tolerated. However, as
had been observed previously,7 phenolic substitution at the 4-po-
sition generated the potent yet nonselective transporter inhibitor
38-I. Neither compound displayed significant CYP2D6 inhibition.
Like the fluorine substituted compounds, 3-hydroxy substitution
(37-I) improved the selectivity over SERT to 20-fold and main-
tained minimal CYP2D6 inhibition. Oxidative clearance remained
low for these compounds, though the phase II clearance is un-
known. The equivalent metabolite in atomoxetine is converted
rapidly to the glucoronide in vivo.14
Overall, this series produced a number of potent and selective
compounds with advantageous stability and similar or better
CYP2D6 inhibition compared with atomoxetine. Accordingly, a
number of compounds identified were tested for in vivo activity
and the results will be reported in future publications.
cept at the 5HT2b receptor (500 nM–2 lM). This represents >100-
fold selectivity against other proteins that interact with the same
substrates and is an improvement over the 30-fold selectivity of
both 1 and 2 against 5HT2b. Similar selectivity is observed for ato-
moxetine against this receptor.14
CYP2D6 inhibition maintained the trend observed previously,7
where hydrophobicity contributes to increased inhibition. Com-
pared to 2, 8-I was 3-fold less potent at CYP2D6 and the calculated
logP15 was about 0.8 log units lower. As the hydrophobicity of the
ring attachment atom increased from O to C to S, CYP2D6 activity
also increased to IC50 values below 1 lM. Unlike previous com-
pounds, there was no significant difference in CYP2D6 inhibition
between enantiomers (e.g., 8-I/II).7
SAR for aryloxy ring substitution for the active enantiomers is
shown in Table 2. Similar activity trends were observed for both
methyl and fluoro substitution of 8-I, with 4-substitution generat-
ing compounds nearly equipotent on SERT (32-I and 34-I). Selec-
tivity improved to P12-fold with 3-substitution, and the other
isomers were more selective. CYP2D6 inhibition remained in the
Acknowledgments
The authors are indebted to Mr. Brock Brown and Ms. Kathy
Barrett for technical support in the transporter assays and Dr. Tao
Table 2
SAR of the aryloxy ring in the hydroxyindanesa
R1
R2
O
OH
N
H
Compound
Isomer
R1
R2
NETb
SERTb
DATb
1600
10,000
4000
10,000
>10,000
>10,000
>10,000
>10,000
3500
8000
3000
5000
>10,000
>10,000
4000
CYP2D6c
Clearanced
8-I
S,R
S,R
S,R
S,R
S,R
S,R
S,R
S,R
S,R
S,R
S,R
S,R
S,R
S,R
S,R
S,R
Me
1-Napthyl
F
Cl
Et
OMe
SMe
CH2OH
Me
Me
Me
3
3
11
5
1.5
17
2
300
3
2
3
5
7
184
10
2000
600
4000
2000
800
10
23
63
10
63
63
43
ND
43
32
63
63
7
24-I
25-I
26-I
27-I
28-I
29-I
30-I
31-I
32-I
33-I
34-I
35-I
36-I
37-I
38-I
700
700
800
2200
500
1800
50
5
36
6
500
1400
>10,000
4000
6500
1500
1500
1000
1000
>10,000
>10,000
3-Me
4-Me
3-F
4-F
5-F
6-F
3-OH
4-OH
Me
Me
Me
Me
200
150
100
14
5
4
4
17
6
10
Me
5000
a
Transporter data are the average of two or more independent measurements.
IC50 (nM) for inhibition of monoamine uptake.
b
c
IC50 (nM) for inhibition of CYP2D6.12
d
Scaled intrinsic clearance (mL/min/kg) in human liver microsomes,13 clearance of 63 indicates no detectable compound loss. ND, not done.

J. Pontillo et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6151–6155
6155
stereochemistry was established by Flack parameter refinement. All non-
hydrogen atoms were refined anisotropically and hydrogen atoms were placed
in idealized locations. All software was contained in the APEX, SAINT, and
SHELX software packages distributed by Bruker-AXS, Madison, WI. Crystal data
for 26-II: C15H19Cl2NO2, monoclinic, P21, a = 7.8168(12), b = 5.8834(8),
c = 18.473(3) Å, b = 102.160(2)°, V = 830.5(2) Å3, Z = 2, T = 208 K. R(F) = 5.89%
based on 2172 independent reflections.
Hu and Ms. Mila Lagman for chiral chromatography development
and purification.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.10.013.
10. Gololobov, Y. G.; Zhmurova, I. N.; Kasukhin, L. F. Tetrahedron 1981, 37, 437.
11. Hughes, D. L.; Smith, G. B.; Liu, J.; Dezeny, G. C.; Senanayake, C. H.; Larsen, R. D.;
Verhoeven, T. R.; Reider, P. J. J. Org. Chem. 1997, 62, 2222.
12. Recombinant CYP2D6 or CYP3A4 was incubated with a marker substrate and a
NADPH-generating system for 30 min at 37 °C in the presence of varying
concentrations of test compound. CYP3A4 activity was quantitated from the
cleavage of substrate BFC to its fluorescent product 7-HFC. Ketoconazole was
used as a positive control. CYP2D6 activity was quantitated from the cleavage
of AMMC to its fluorescent product AHMC. Quinidine was used as positive
control.
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13. Test compounds were incubated at 1 lM concentration in the presence of
pooled human liver microsomes (0.5 mg/mL; n > 10; mixed gender) in the
presence of an NADPH-generating system. The rate of disappearance of the
parent compound was quantitated by LC/MS/MS at five time points over
60 min and the measured rate scaled to human body mass.
14. Sauer, J.-M.; Ring, B. J.; Witcher, J. W. Clin. Pharmacokinet. 2005, 44, 571.
15. Calculated logPs were determined using ACD/LogP DB, version 9.02, Advanced
Chemistry Development, Inc., Toronto, ON, Canada, http://www.acdlabs.com,
2005. Calculated and measured values for atomoxetine, 8-I, and 16-I were the
same within experimental error.
8. Palandoken, H.; McMillen, W. T.; Nantz, M. H. Org. Prep. Proceed. Int. 1996, 28, 702.
9. Generated at the UCSD Small-Molecule Crystallography Facility. The crystal
system and space group assignments were unambiguous, and the absolute