Discovery of novel selective norepinephrine inhibitors: 1-(2-morpholin-2-ylethyl)-3-aryl-1,3-dihydro-2,1,3-benzothiadiazole 2,2-dioxides (WYE-114152)

Article abstract of DOI:10.1021/jm200733r

Sequential modification of the previously identified 4-[3-aryl-2,2-dioxido- 2,1,3-benzothiadiazol-1(3H)-yl]-1-(methylamino)butan-2-ols led to the identification of a new series of 1-(2-morpholin-2-ylethyl)-3-aryl-1,3-dihydro- 2,1,3-benzothiadiazole 2,2-dioxides that are potent and selective inhibitors of the norepinephrine transporter over both the serotonin and dopamine transporters. One representative compound 10b (WYE-114152) had low nanomolar hNET potency (IC₅₀ = 15 nM) and good selectivity for hNET over hSERT (>430-fold) and hDAT (>548-fold). 10b was additionally bioavailable following oral dosing and demonstrated efficacy in rat models of acute, inflammatory, and neuropathic pain.

Full text of DOI:10.1021/jm200733r

ARTICLE
pubs.acs.org/jmc
Discovery of Novel Selective Norepinephrine Inhibitors:
1-(2-Morpholin-2-ylethyl)-3-aryl-1,3-dihydro-2,1,3-benzothiadiazole
2,2-Dioxides (WYE-114152)
David J. O’Neill,*,† Adedayo Adedoyin,§ Jenifer A. Bray,|| Darlene C. Deecher,‡ Andrew Fensome,†
Joel A. Goldberg,† Jim Harrison,|| Liza Leventhal,|| Charles Mann,† Lilly Mark,|| Lisa Nogle,†
Nicole R. Sullivan,|| Taylor B. Spangler,|| Eugene A. Terefenko,† Eugene J. Trybulski,†
Albert J. Uveges,|| An Vu,† Garth T. Whiteside,|| and Puwen Zhang†
†Chemical Sciences, ‡Musculoskeletal Biology, and §Drug Safety and Metabolism, Pfizer, 500 Arcola Road, Collegeville,
Pennsylvania 19426, United States
Neuroscience, Pfizer, CN 8000, Princeton, New Jersey 08543, United States
S Supporting Information
b
ABSTRACT: Sequential modification of the previously identified 4-[3-aryl-2,2-dioxido-2,1,3-benzothiadiazol-
1(3H)-yl]-1-(methylamino)butan-2-ols led to the identification of a new series of 1-(2-morpholin-2-ylethyl)-3-
aryl-1,3-dihydro-2,1,3-benzothiadiazole 2,2-dioxides that are potent and selective inhibitors of the norepinephr-
ine transporter over both the serotonin and dopamine transporters. One representative compound 10b
(WYE-114152) had low nanomolar hNET potency (IC50 = 15 nM) and good selectivity for hNET over hSERT
(>430-fold) and hDAT (>548-fold). 10b was additionally bioavailable following oral dosing and demonstrated
efficacy in rat models of acute, inflammatory, and neuropathic pain.
’ INTRODUCTION
The norepinephrine transporter (NET) is a membrane bound
protein that regulates the uptake of the neurotransmitter nor-
epinephrine (NE) from the synaptic cleft of noradrenergic
neurons during synaptic transmission.1 It therefore plays an
important role in regulating the physiological functions of NE,
the deficiency of which has been implicated in a number of
neurological disorders. Norepinephrine reuptake inhibitors
(NRIs) block the return of NE to the axon terminal, thereby
increasing its synaptic concentration, resulting in a treatment for
a range of CNS disorders.2 In the past 20 years a number of
monoamine neurotransmitter reuptake inhibitors have been
approved for the treatment of neurological disorders such as
depression and pain.3 More recently, selective NRIs such as
reboxetine 1 and atomoxetine 2 have been used clinically for the
alleviation of major depressive disorder and attention deficit
transporter and were selective against other human transporters.8
Conversion of the alcohol to the methyl ether resulted in a divergence
in potency with the R-enantiomer over 5-fold more potent at hNET
than the corresponding S-enantiomer. Both compounds, however,
demonstrated weaker hNET potency than the starting alcohol. In an
effort to reduce molecular flexibility and improve compound
properties,9 the methyl groups of compounds 6 and 7 were tethered
together to form morpholines 8À10a. These morpholines 8À10a
restored the hNET activity (IC50 = 11À18 nM) and were equally
potent and selective over hDAT; however, the R-enantiomer 10a was
6-fold more selective over hSERT than the S-enantiomer 9.10
hyperactivity disorder, respectively.4 There is also evidence that
these compounds may have efficacy in the treatment of chronic
pain including fibromyalgia and lower back pain.5
Our previous reports have detailed efforts that resulted in the
discovery of selective NRIs including benzimidazol-2-ones6
(e.g., 3) and 4-[3-aryl-2,2-dioxido-2,1,3-benzothiadiazol-1(3H)-yl]-
(methylamino)butan-2-ols such as compound 4.7
’ RESULTS AND DISCUSSION
In Vitro Characterization. We have previously discussed7 the
structureÀactivity relationship of the chiral amino alcohols 4 and
5 and were interested in further exploring this scaffold (Table 1).
Both enantiomers were potent inhibitors of the norepinephrine
Received: June 8, 2011
Published: September 14, 2011
r
2011 American Chemical Society
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Table 1. Monoamine Reuptake Inhibition of 4À15a
a Data are reported as the mean ( SEM. Compounds without standard errors are reported as having an n of 1. b Inhibition of norepinephrine uptake in
MDCK-Net6 cells stably transfected with human norepinephrine transporter (hNET). Desipramine (IC50 = 3.4 ( 1.6 nM) was used as a standard.
c Inhibition of [3H]nisoxetine binding to MDCK-Net6 cells stably transfected with hNET. Desipramine (Ki = 2.1 ( 0.6 nM) was used as a standard.
d Inhibition of serotonin uptake in JAR cells, natively expressing human serotonin transporter (hSERT). Fluoxetine (IC50 = 9.4 ( 3.1 nM) was used as a
e
standard. Inhibition of [3H]WIN-35,428 binding to membranes from CHO cells expressing recombinant human dopamine transporter (hDAT).
Mazindol (Ki = 22.1 ( 6.5 nM) was used as a standard. f Values in the parentheses are IC50 (nM). g ND: not determined.
Decreasing the length of the carbon chain between the core and the
morpholine moiety, 11À13, resulted in reduced hNET potency
(IC50 = 147À207 nM). As expected, alkylation of the morpholine
amine further attenuated the hNET potency (IC50 = 4310 nM).
Conversion of racemic morpholine 8 to the corresponding piperidine
15 illustrates the impact of the morpholine oxygen on hNET potency
with a decrease of 3-fold (IC50 = 55 nM).
Substitution on the benzo ring was investigated next (Table 3).
Incorporation of a F group at positions 4À7 had little impact on
hNET potency except for compound 18 (6-F) where there was a
10-fold decrease. Compound 18 also showed decreased hSERT
selectivity as did compound 16 containing the 4-F group. Com-
pounds 17 and 19 with 5-F and 7-F substitution, respectively, had
a profile similar to that of desfluoro compound 10b.
The SAR of substitution around the pendent aryl ring of 10a
was also examined (Table 2). 2-F substitution, 10b, increased
hDAT selectivity (IC50 = 2190 nM) while maintaining hNET
potency and hSERT selectivity. Addition of a 3-OMe moiety,
10c, decreased hNET potency (IC50 = 89 nM) as did incorpora-
tion of a 4-F group (IC50 = 611 nM). 2,4-di-F substitution, 10e,
resulted in reduced potency at both hDAT and hNET. Com-
pound 10f containing the 2,4,6-tri-F aryl ring again showed
decreased hNET potency.
Chemistry. A synthetic route toward the preparation of the
1-(2-morpholin-2-ylethyl)-3-aryl-1,3-dihydro-2,1,3-benzothia-
diazole 2,2-dioxides is described in Scheme 1. Commercially
available (4-benzylmorpholin-2-yl)acetic acid ethyl ester 20 was
debenzylated using palladium on carbon and ammonium for-
mate, then reduced with lithium aluminum hydride to the alcohol
before being protected as the tert-butyl carbamate 22. Mitsonobu
N-alkylation of 22 with the 2,2-dioxido-2,1,3-benzothiadiazol-1(3H)-yl
cores 23aÀf followed by treatment with HCl provided racemic
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Table 2. Monoamine Reuptake Inhibition of 1-(2-Morpholin-2-ylethyl)-3-aryl-1,3-dihydro-2,1,3-benzothiadiazole 2,2-Dioxides
10aÀga
a Data are reported as the mean ( SEM. Compounds without standard errors are reported as having an n of 1. b Inhibition of norepinephrine uptake in
MDCK-Net6 cells stably transfected with human norepinephrine transporter (hNET). Desipramine (IC50 = 3.4 ( 1.6 nM) was used as a standard.
c Inhibition of [3H]nisoxetine binding to MDCK-Net6 cells stably transfected with hNET. Desipramine (Ki = 2.1 ( 0.6 nM) was used as a standard.
Compounds without standard errors are reported as having an n of 1. d Inhibition of serotonin uptake in JAR cells, natively expressing human serotonin
transporter (hSERT). Fluoxetine (IC50 = 9.4 ( 3.1 nM) was used as a standard. e Inhibition of [3H]WIN-35,428 binding to membranes from CHO cells
expressing recombinant human dopamine transporter (hDAT). Mazindol (Ki = 22.1 ( 6.5 nM) was used as a standard. f ND: not determined.
morpholines 24aÀf. Chiral HPLC was then utilized to furnish the
desired target compounds 10aÀf. Compounds 6, 7, and 11À15
were prepared in a similar fashion.
and/or maintenance of chronic pain states.14 It was reported that by
blocking reuptake of NE, NRIs increase NE levels which subse-
quently activate descending inhibitory pathways.13 The rat hot plate
assay of acute analgesia was used to investigate the efficacy of 10b on
alleviating pain.15 In this assay, rats are placed on a metal plate
maintained at 52 °C. The latency to exhibit a response to this
thermal stimulus, such as hind paw lift, flutter, licking, or escape
behavior, was measured with a cutoff of 30 s to avoid tissue damage.
Upon oral administration, 10b significantly increased latency at both
10 and 30 mg/kg 1À5 h postdosing (Figure 1). These data suggest
that compound 10b was efficacious in treating acute pain.
Compound 10b was then evaluated in the carrageenan-induced
thermal hyperalgesia model of acute inflammatory pain.16 In this
assay, inflammation is induced in the left hind paw of a rat by injection
of 50 μL of 2% carrageenan. Upon oral administration 10b again
increased latency at both 10 and 30 mg/kg 4 h postdosing (Figure 2).
These data suggest that compound 10b was efficacious in treating
acute inflammatory pain.
In Vivo Characterization. Compound 10b was selected based
on its in vitro profile for in vivo characterization.11 Table 4 highlights
the pharmacokinetic parameters of 10b in the male rat using both
intravenous and oral administration. In general, 10b showed high to
moderate clearance, moderate volume of distribution, and moderate
terminal half-life. The compound showed an oral bioavailability of
28%. In addition, 10b had good brain penetration with a brain/
plasma ratio of 3.9 in male rats. Furthermore, microdialysis studies in
male SpragueÀDawley rats demonstrated a 390% increase over
baseline for norepinephrine in the medial prefrontal cortex without
significant effect on serotonin at 30 mg/kg po.
Compound 10b was profiled in a series of in vivo models12 to
assess its effects on norepinephrine neurobiology. NE has been
implicated in the modulation of nociceptive processing13 which is a
component of the endogenous descending pain inhibitory system.
Reduced levels of NE may in part contribute to the establishment
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Table 3. Monoamine Reuptake Inhibition of 16À19a
compd
substitution
hNET uptake, IC50 (nM)b
hNET binding, IC50 (nM)c
hSERT uptake, IC50 (nM)d
hDAT binding % inh at 1 μMe
16
17
18
19
4-F
5-F
6-F
7-F
22 ( 0.4
14 ( 10
120 ( 52
7 ( 3
5 ( 3
8
992 ( 887
50
43
23
20
8347 ( 2447
806 ( 409
209
13
5790 ( 2619
a Data are reported as the mean ( SEM. Compounds without standard errors are reported as having an n of 1. b Inhibition of norepinephrine uptake in
MDCK-Net6 cells, stably transfected with human norepinephrine transporter (hNET). Desipramine (IC50 = 3.4 ( 1.6 nM) was used as a standard.
c Inhibition of [3H]nisoxetine binding to MDCK-Net6 cells stably transfected with hNET. Desipramine (Ki = 2.1 ( 0.6 nM) was used as a standard.
d Inhibition of serotonin uptake in JAR cells, natively expressing human serotonin transporter (hSERT). Fluoxetine (IC50 = 9.4 ( 3.1 nM) was used as a
e
standard. Inhibition of [3H]WIN-35,428 binding to membranes from CHO cells expressing recombinant human dopamine transporter (hDAT).
Mazindol (Ki = 22.1 ( 6.5 nM) was used as a standard.
’ CONCLUSION
Scheme 1. Synthesis of 1-(2-Morpholin-2-ylethyl)-3-aryl-
1,3-dihydro-2,1,3-benzothiadiazole 2,2-Dioxides 10aÀfa
Modification of the previously identified 4-[3-aryl-2,2-dioxi-
do-2,1,3-benzothiadiazol-1(3H)-yl]-1-(methylamino)butan-2-
ols led to the identification of a new series of 1-(2-morpholin-2-
ylethyl)-3-aryl-1,3-dihydro-2,1,3-benzothiadiazole 2,2-dioxides
that are potent and selective for the inhibition of norepinephrine
transporter versus serotonin and dopamine transporters. The
lead compound demonstrated good pharmacokinetic and phar-
maceutical profiles. Compound 10b was orally efficacious toward
alleviating behavioral symptoms associated with NE deficiency in
multiple in vivo models including acute, acute inflammatory,
chronic inflammatory, and neuropathic pain. On the basis of this
profile, 10b (WYE-114152)19 was identified as a candidate for
advancement into development.
’ EXPERIMENTAL SECTION
1H NMR spectra were recorded on a Varian INOVA 400 or Bruker
AVANCE II 400 instrument. Chemical shifts are reported in δ values
(parts per million, ppm) relative to an internal standard of tetramethyl-
silane in CDCl3 or DMSO-d6. Electrospray (ESI) mass spectra were
recorded using a Hewlett-Packard 5989B MS engine or Waters Alliance-
ZMD mass spectrometer. Electron impact ionization (EI, EE = 70 eV)
mass spectra were recorded on a Finnigan Trace mass spectrometer.
Analytical thin-layer chromatography (TLC) was carried out on pre-
coated plates (silica gel, 60 F-254), and spots were visualized with UV
light and stained in iodine. Solvents were purchased as anhydrous grade
and were used without further purification. Crude reaction products
were purified using the ISCO Rf purification system. Preparative HPLC
purifications were performed on a preparative Gilson HPLC system
using a CombiPrep Pro C18 column with acetonitrile (0.1% TFA) and
water (0.1% TFA) as solvents at a flow rate of 20 mL/min. Chiral HPLC
separations were carried out using supercritical fluid chromatography
conditions using a 250 mm  4.6 mm i.d. column at 2.0 mL/min flow
rate using a Chiralpak AS-H 5 analytical supercritical fluid chromato-
graph (Berger Instruments, Inc. Newark, DE). Compound purity was
assessed by 1H NMR and analytical HPLC as described in the Support-
ing Information. Biological results were obtained on compounds of
>95% chemical purity as determined by the above methods.
a Reagents and conditions: (a) Pd/C, NH4HCO2, EtOH, 23 °C; (b)
LiAlH4, Et2O, 0 °C, then Boc2O, NaOH, DCM; (c) 12, DIAD, PPh3,
THF, 0 °C; (d) HCl (4 M in dioxane), DCM; (e) chiral HPLC.
Compound 10b also demonstrated efficacy in the FCA-induced
mechanical hyperalgesia model of chronic inflammatory pain
(Figure 3).17 In this assay, rodents were injected in the left hind
paw with 50 uL of 50% FCA and were given 10b, celecoxib, or
vehicle (po) 24 h later. Threshold to paw withdrawal was measured.
Upon oral administration 10b showed efficacy at 3 mg/kg 3 and 5 h
postdosing (Figure 2). These data suggest that compound 10b was
efficacious in treating chronic inflammatory pain.
Finally, the activity of compound 10b was evaluated in a rat spinal
nerve ligation (SNL) model of neuropathic pain.18 After ligation,
assessment of mechanical thresholds was measured as the hind paw
withdrawal threshold to a noxious mechanical stimulus. Compound
10b significantly and dose-dependently reversed mechanical hyper-
algesia at 3, 10, and 30 mg/kg, suggesting efficacy in treating
neuropathic pain (Figure 4).
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Table 4. Pharmacokinetic Parameters of Compound 10b in Male Rats after Intravenous and Oral Administrationsa
dose (mg/kg)
Clp ((mL/min)/kg)
38
Vss (L/kg)
Cmax (ng/mL)
Tmax (h)
t1/2 (h)
AUC0Àinf(h ng/mL)
F (%)
3
2 (iv)
5
1.8
1.8
958
10 (po)
373
1.0
1322
28
a 2% Tween-80/0.5% methylcellulose in water and DMSO/80%PEG200 were used as vehicles for oral and intravenous administrations, respectively.
Three rats were used in each study.
Figure 3. Oral activity of compound 10b on FCA-induced mechanical
Figure 1. Oral activity of compound 10b on hot plate latency. Male
hyperalgesia. Male SpragueÀDawley rats (210À242 g, 9À10/group)
SpragueÀDawley rats, wt = 190À220 g (n = 10À11/group) were used.
were injected in the left hind paw with 50 μL of 50% FCA and were given
The hot plate was set at 52 °C and cutoff was set at 30 s. Latency to
nocicfensive response was measured. 10b was administered (po) as
a solution in 2% Tween/0.5% methylcellulose (vehicle). Morphine
was administered (sc) as a solution in 0.9% saline. Data shown are the
mean ( SEM.
10b, celecoxib, or vehicle (po) 24 h later. Threshold to paw withdrawal
(PWT) was measured. 10b and celecoxib, the positive control, were
administered (po) as suspensions in 2% Tween/0.5% methylcellulose
(vehicle). Data shown are the mean ( SEM.
Figure 4. Oral activity of compound 10b on SNL-induced mechanical
hyperalgesia. Results are for male SpragueÀDawley rats (249À305 g,
9À10/group), 3 weeks postsurgery. Threshold to paw withdrawal
(PWT) was measured. The compound was administered orally as a
suspension of 0.5% methylcellulose plus 2% Tween in water. Gabapentin
was used as a positive control and administered (ip) as a solution in
0.9% saline. Data shown are the mean ( SEM. The asterisk (/) indicates
p e 0.05 vs SNL/vehicle (ANOVA).
Figure 2. Oral activity of compound 10b on carrageenan-induced
thermal hyperalgesia. Male SpragueÀDawley rats, wt =197À238 g
(n = 8/group) were used. Rats were injected in the left hind paw with
50μL of2% carrageenan. Paw withdrawallatencywas measured. 10b (po)
or indomethacin (po) was administered as a solution in 0.5% methylcellu-
lose + 2% Tween 3 h after carrageenan. Data shown are the mean
( SEM.
The reaction mixture was concentrated and then loaded directly onto silica
gel and purified via chromatography (Redisep, silica, gradient 0À50% ethyl
acetate in hexane) to afford 0.08 g of 4-[3-phenyl-2,2-dioxido-2,1,3-
benzothiadiazol-1(3H)-yl]-1-(methylamino)butan-2-ol-carbamic acid tert-
butyl ester as a clear oil. To this was added trimethyloxonium tetrafluor-
oborate (71 mg, 0.483 mmol), proton sponge (121 mg, 0.564 mmol), and
4 Å molecular sieves. This was stirred in dichloromethane (5 mL) in a sealed
vial at room temperature for 18 h. The reaction mixture was concentrated
and then loaded directly onto silica gel and purified via chromatography
(Redisep, silica, gradient 0À50% ethyl acetate in hexane) to afford 0.065 g
of (2S)-4-[3-phenyl-2,2-dioxido-2,1,3-benzothiadiazol-1(3H)-yl]-2-meth-
oxy-N-methylbutan-1-aminecarbamic acid tert-butyl ester as a clear oil. This
material was dissolved in diethyl ether/methanol, and 4 N HCl in dioxane
was added. A precipitate formed and the mixture was filtered to afford 0.05 g
(2S)-(4-[3-(2-Fluorophenyl)-2,2-dioxido-2,1,3-benzothiadia-
zol-1(3H)-yl]-1-(methylamino)butan-2-ol (4) and (2R)-(4-[3-(2-
Fluorophenyl)-2,2-dioxido-2,1,3-benzothiadiazol-1(3H)-yl]-
1-(methylamino)butan-2-ol (5). These compounds were prepared
as previously described.7
(2S)-4-(2,2-Dioxido-3-phenyl-2,1,3-benzothiadiazol-1(3H)-
yl)-2-methoxy-N-methylbutan-1-amine (6) and (2R)-4-(2,2-
Dioxido-3-phenyl-2,1,3-benzothiadiazol-1(3H)-yl)-2-methoxy-
N-methylbutan-1-amine (7). 4-[3-(3-Phenyl)-2,2-dioxido-2,1,3-ben-
zothiadiazol-1(3H)-yl]-1-(methylamino)butan-2-ol (113 mg, 0.325 mmol)
and di-tert-butyl dicarbonate (71 mg, 0.325 mmol) were stirred in
dichloromethane (5 mL) in a sealed vial at room temperature for 18 h.
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of 4-(2,2-dioxido-3-phenyl-2,1,3-benzothiadiazol-1(3H)-yl)-2-methoxy-N-
methylbutan-1-amine as a solid. This was dissolved in methanol. Then
200 μL of the resulting solution was repetitively injected onto the supercritical
fluid chromatography instrument, and the baseline resolved enantiomers
were separately collected. The chiral purity of each enantiomer was
determined under the same supercritical fluid chromatography conditions
using a Chiralpak AS-H 5 μm, 250 mm  4.6mmi.d. columnat2.0 mL/min
flow rate using analytical supercritical fluid chromatography (Berger
Instruments, Inc. Newark, DE). Both enantiomers were found to be >99.9%
enantiomerically pure.
10b was prepared following the procedure for compound 8 with 1-phenyl-
1,3-dihydro-2,1,3-benzothiadiazole 2,2-dioxide being replaced with 1-(2-
fluorophenyl)-1,3-dihydro-2,1,3-benzothiadiazole 2,2-dioxide and was iso-
lated via chiral HPLC as for compound 10a. MS (ESI) m/z 378.1. HRMS:
calcd for C18H20FN3O3S + H+, 378.1282; found (ESI, [M + H]+ obsd),
378.1284. 1H NMR (DMSO-d6): δ 9.09 (br s, 2H), 7.70 (m, 1H), 7.59 (m,
2H), 7.47 (m, 1H), 7.11 (m, 2H), 6.98 (m, 1H), 6.56 (d, J = 7.87 Hz, 1H),
4.01 (m, 2H), 3.92 (m, 1H), 3.82 (m, 1H), 3.70 (m, 1H), 3.28 (d, J = 12.69
Hz, 1H), 3.17 (d, J = 12.96 Hz, 1H), 2.99 (m, 1H), 2.81 (m, 1H), 2.04 (m,
1H), 1.85 (m, 1H).
6: MS (ES) m/z 362.1 ([M + H]+). HRMS: calcd for C18H23N3O3S +
H+, 362.1533; found (ESI, [M + H]+ calcd), 362.1533. 1H NMR (DMSO-
d6) δ 8.66 (br s, 2H), 7.61 (m, 3H), 7.50 (m, 2H), 7.18 (m, 1H), 7.09 (m,
1H), 6.98 (m, 1H), 6.67 (d, J = 7.85 Hz, 1H), 3.92 (m, 2H), 3.71 (m, 1H),
3.38 (s, 3H), 3.20 (m, 1H), 3.09 (m, 1H), 2.56 (s, 3H), 2.07 (m, 2H).
7:MS(ES) m/z362.1 ([M + H]+). HRMS: calcd for C18H23N3O3S+H+,
362.1533; found (ESI, [M + H]+ calcd), 362.1533. 1H NMR (DMSO-
d6) δ 8.66 (br s, 2H), 7.61 (m, 3H), 7.50 (m, 2H), 7.18 (m, 1H), 7.09
(m, 1H), 6.98 (m, 1H), 6.67 (d, J = 7.85 Hz, 1H), 3.92 (m, 2H), 3.71
(m, 1H), 3.38 (s, 3H), 3.20 (m, 1H), 3.09 (m, 1H), 2.56 (s, 3H), 2.07
(m, 2H).
1-(3-Methoxyphenyl)-3-{2-[(2R)-morpholin-2-yl]ethyl}-1,
3-dihydro-2,1,3-benzothiadiazole 2,2-Dioxide (10c). Com-
pound 10c was prepared following the procedure for compound 8 with
1-phenyl-1,3-dihydro-2,1,3-benzothiadiazole 2,2-dioxide being replaced
with 1-(3-methoxyphenyl)-1,3-dihydro-2,1,3-benzothiadiazole 2,2-di-
oxide and was isolated via chiral HPLC as for compound 10a. MS
(ESI) m/z 389.9. HRMS: calcd for C19H23N3O4S + H+, 390.1482;
found (ESI, [M + H]+ calcd), 390.1482. 1H NMR (DMSO-d6): δ 9.11
(br s, 2H), 7.54 (t, J = 8.15 Hz 1H), 7.12 (m, 4H), 6.99 (m, 2H), 6.3 (d,
J = 7.73 Hz, 1H), 3.96 (m, 3H), 3.81 (s, 3H), 3.60 (m, 1H), 3.24 (d, J =
12.10 Hz, 1H), 3.16 (d, J = 12.91 Hz, 1H), 3.00 (m, 1H), 2.81 (m, 1H),
2.03 (m, 1H), 1.83 (m, 1H).
1-(2-Morpholin-2-ylethyl)-3-phenyl-1,3-dihydro-2,1,3-ben-
zothiadiazole 2,2-Dioxide (8). To a solution of 1-phenyl-1,3-dihy-
dro-2,1,3-benzothiadiazole 2,2-dioxide (0.2 g, 0.8 mmol) in THF (10 mL)
was added triphenylphosphine (0.26 g, 1 mmol), tert-butyl 2-(2-hydro-
xyethyl)morpholine-4-carboxylate 20 (0.2 g, 0.9 mmol), and DIAD (0.2 g,
1 mmol) at 0°C. The mixture was allowed to warm to ambient temperature
overnight and then concentrated and chromatographed on silica gel
(0À40% EtOAC in hexane). The resulting mostly pure carbamate was
dissolved in dichloromethane (10 mL) and treated with HCL (4 mL, 4 M in
dioxane). The resulting salt was chromatographed on silica (0À100% of
(7NNH3/MeOH) in dichloromethane), giving the desired product as a clear
oil (0.23 g). MS (ES) m/z 359.8. HRMS: calcd for C18H21N3O3S + H+,
360.1376; found (ESI, [M + H]+ obsd), 360.1377 . 1H NMR (DMSO-d6)
δ 9.17 (br s, 1H), 9.09 (br s, 1H), 7.61 (m, 3H), 7.50 (m, 2H), 7.11 (m,
2H), 6.98 (m, 1H), 6.67 (d, J = 7.57 Hz, 1H), 4.00 (m, 2H), 3.92 (m, 1H),
3.82 (m, 1H), 3.60 (m, 1H), 3.26 (d, J = 12.65 Hz, 1H), 3.18 (d, J = 12.78
Hz, 1H), 3.00 (m, 1H), 2.81 (m, 1H), 2.03 (m, 1H), 1.87 (m, 1H).
1-{2-[(2S)-Morpholin-2-yl]ethyl}-3-phenyl-1,3-dihydro-2,1,3-
benzothiadiazole 2,2-Dioxide (9) and 1-{2-[(2R)-Morpholin-2-
yl]ethyl}-3-phenyl-1,3-dihydro-2,1,3-benzothiadiazole 2,2-
Dioxide (10a). 8 was dissolved in methanol, and 200 μL of the
resulting solution was repetitively injected onto the supercritical fluid
chromatography instrument. The baseline resolved enantiomers were
separately collected. The chiral purity of each enantiomer was deter-
mined under the same supercritical fluid chromatography conditions
using a Chiralpak AS-H 5 μm, 250 mm  4.6mmi.d. columnat2.0 mL/min
flow rate using an analytical supercritical fluid chromatograp (Berger
Instruments, Inc. Newark, DE). Both enantiomers were found to be
>99.9% enantiomerically pure.
1-(4-Fluorophenyl)-3-{2-[(2R)-morpholin-2-yl]ethyl}-1,3-
dihydro-2,1,3-benzothiadiazole 2,2-Dioxide (10d). Compound
10d was prepared following the procedure for compound 8 with 1-phenyl-
1,3-dihydro-2,1,3-benzothiadiazole 2,2-dioxide being replaced with 1-(4-
fluorophenyl)-1,3-dihydro-2,1,3-benzothiadiazole 2,2-dioxide and was iso-
lated via chiral HPLC as for compound 10a. MS (ESI) m/z 379.2. HRMS:
calcd for C18H20FN3O3S + H+, 378.12822; found (ESI, [M + H]+ obsd),
378.1272. 1H NMR (DMSO-d6): δ 9.12 (br s, 2H), 7.56 (m, 2H), 7.48 (m,
2H), 7.11 (m, 2H), 6.98 (m, 1H), 6.64 (d, J = 7.76 Hz, 1H), 4.00 (m, 2H),
3.90 (m, 1H), 3.82 (m, 1H), 3.69 (m, 1H), 3.26 (d, J = 12.18 Hz, 1H), 3.18
(d, J = 12.54 Hz, 1H), 3.00 (m, 1H), 2.81 (m, 1H), 2.03 (m, 1H), 1.84 (m,
1H).
1-(2,4-Difluorophenyl)-3-{2-[(2R)-morpholin-2-yl]ethyl}-1,
3-dihydro-2,1,3-benzothiadiazole 2,2-Dioxide (10e). Com-
pound 10e was prepared following the procedure for compound 8 with
1-phenyl-1,3-dihydro-2,1,3-benzothiadiazole 2,2-dioxide being replaced
with 1-(2,4-difluorophenyl)-1,3-dihydro-2,1,3-benzothiadiazole 2,2-diox-
ide and was isolated via chiral HPLC as for compound 10a. MS (ESI) m/z
395.8. HRMS: calcd for C18H19F2N3O3S + H+, 396.1188; found (ESI,
[M + H]+ obsd), 396.1178. 1H NMR (DMSO-d6): δ 9.20 (br s, 2H), 7.69
(m,2H),7.36(m,1H),7.11(m,2H),6.98(m,1H),6.61(d,J=7.82Hz,1H),
4.00 (m, 2H), 3.92 (m, 1H), 3.81 (m, 1H), 3.70 (m, 1H), 3.26 (d, J =
12.30 Hz, 1H), 3.18 (d, J = 12.55 Hz, 1H), 2.99 (m, 1H), 2.81 (m, 1H),
2.04 (m, 1H), 1.85 (m, 1H).
1-{2-[(2R)-Morpholin-2-yl]ethyl}-3-(2,4,6-trifluorophenyl)-1,
3-dihydro-2,1,3-benzothiadiazole 2,2-Dioxide (10f). Compound
10f was prepared following the procedure for compound 8 with
1-phenyl-1,3-dihydro-2,1,3-benzothiadiazole 2,2-dioxide being replaced
with 1-(2,4,6-trifluorophenyl)-1,3-dihydro-2,1,3-benzothiadiazole
2,2-dioxide and was isolated via chiral HPLC as for compound 10a.
MS (ESI) m/z 413.8. HRMS: calcd for C18H18F3N3O3S + H+, 414.10937;
found (ESI, [M + H]+ obsd), 414.1080. 1H NMR (DMSO-d6): δ 9.14
(br s, 2H), 7.62 (m, 2H), 7.14 (m, 2H), 6.99 (m, 1H), 6.73 (d, J = 7.82 Hz,
1H), 4.02 (m, 2H), 3.92 (m, 1H), 3.82 (m, 1H), 3.70 (m, 1H), 3.27 (d,
J = 13.07 Hz, 1H), 3.18 (d, J = 12.68 Hz, 1H), 2.98 (m, 1H), 2.81 (m,
1H), 2.05 (m, 1H), 1.85 (m, 1H).
1-(Morpholin-2-ylmethyl)-3-phenyl-1,3-dihydro-2,1,3-ben-
zothiadiazole 2,2-Dioxide (11). Compound 11 was prepared
following the procedure for compound 8 with tert-butyl 2-(hydroxyethyl)
morpholine-4-carboxylate being replaced with tert-butyl 2-(hydroxymethyl)
morpholine-4-carboxylate. MS (ES) m/z 345.9. HRMS: calcd for
C17H19N3O3S + H+, 346.1219; found (ESI, [M + H]+), 346.1194. 1H
9: MS (ESI) m/z 359.8. HRMS: calcd for C18H21N3O3S + H+,
360.1376; found (ESI, [M + H]+ obsd), 360.1378. δ 9.10 (br s, 2H), 7.61
(m, 3H), 7.50 (m, 2H), 7.11 (m, 2H), 6.98 (m, 1H), 6.67 (d, J = 7.57 Hz,
1H), 4.00 (m, 2H), 3.92 (m, 1H), 3.82 (m, 1H), 3.60 (m, 1H), 3.26 (d,
J = 12.65 Hz, 1H), 3.18 (d, J = 12.78 Hz, 1H), 3.00 (m, 1H), 2.81
(m, 1H), 2.03 (m, 1H), 1.87 (m, 1H).
10a: MS (ESI) m/z 359.8. HRMS: calcd for C18H21N3O3S + H+,
360.1376; found (ESI, [M + H]+ obsd), 360.1379. 1H NMR (DMSO-
d6): δ 9.19 (br s, 2H), 7.61 (m, 3H), 7.50 (m, 2H), 7.11 (m, 2H), 6.98
(m, 1H), 6.67 (d, J = 7.57 Hz, 1H), 4.00 (m, 2H), 3.92 (m, 1H), 3.82 (m,
1H), 3.60 (m, 1H), 3.26 (d, J = 12.65 Hz, 1H), 3.18 (d, J = 12.78 Hz,
1H), 3.00 (m, 1H), 2.81 (m, 1H), 2.03 (m, 1H), 1.87 (m, 1H).
1-(2-Fluorophenyl)-3-{2-[(2R)-morpholin-2-yl]ethyl}-1,3-
dihydro-2,1,3-benzothiadiazole 2,2-Dioxide (10b). Compound
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Journal of Medicinal Chemistry
ARTICLE
NMR (DMSO-d6): δ 9.38 (br s, 2H), 7.57 (m, 3H), 7.48 (m, 2H),
7.12 (d, J = 7.81 Hz, 1H), 7.03 (t, J = 7.81 Hz, 1H), 6.92 (t, J = 7.81
Hz, 1H), 6.59 (d, J = 7.81 Hz, 1H), 4.12 (m, 1H), 3.97 (m, 3H), 3.70
(m, 1H), 3.38 (d, J = 12.17 Hz, 1H), 3.13 (d, J = 12.68 Hz, 1H), 2.90
(m, 2H).
with 4-fluoro-1-(2-fluorophenyl)-1,3-dihydro-2,1,3-benzothiadiazole 2,2-
dioxide. MS (ES) m/z 396.1. HRMS: calcd for C18H19F2N3O3S + H+,
396.1188, found (ESI, [M + H]+), 396.1190. 1H NMR (DMSO-d6): δ 9.21
(br s, 2H), 7.70 (m, 1H), 7.60 (m, 2H), 7.47 (m, 1H), 7.04 (m, 2H), 6.46
(m, 1H), 4.00 (m, 3H), 3.85 (m, 1H), 3.71 (m, 1H), 3.28 (m, 1H), 3.15 (m,
1H), 2.99 (m, 1H), 2.80 (m, 1H), 2.00 (m, 6H), 1.85 (m, 1H).
1-[(2R)-Morpholin-2-ylmethyl]-3-phenyl-1,3-dihydro-2,1,
3-benzothiadiazole 2,2-Dioxide (12). Compound 12 was pre-
pared following the procedure for compound 8 with tert-butyl 2-
(hydroxyethyl)morpholine-4-carboxylate being replaced with tert-butyl
2-(hydroxymethyl)morpholine-4-carboxylate and was isolated via chiral
HPLC as for compound 10a. MS (ES) m/z 346.2. HRMS: calcd for
C17H19N3O3S + H+, 346.1220; found (ESI, [M + H]+), 346.1235. 1H
NMR (DMSO-d6): δ 9.38 (br s, 2H), 7.57 (m, 3H), 7.48 (m, 2H), 7.12
(d, J = 7.81 Hz, 1H), 7.03 (t, J = 7.81 Hz, 1H), 6.92 (t, J = 7.81 Hz, 1H),
6.59 (d, J = 7.81 Hz, 1H), 4.12 (m, 1H), 3.97 (m, 3H), 3.70 (m, 1H),
3.38 (d, J = 12.17 Hz, 1H), 3.13 (d, J = 12.68 Hz, 1H), 2.90 (m, 2H).
1-[(2S)-Morpholin-2-ylmethyl]-3-phenyl-1,3-dihydro-
2,1,3-benzothiadiazole 2,2-Dioxide (13). Compound 13 was
prepared following the procedure for compound 8 with tert-butyl
2-(hydroxyethyl)morpholine-4-carboxylate being replaced with tert-
butyl 2-(hydroxymethyl)morpholine-4-carboxylate and was isolated
via chiral HPLC as for compound 10a. MS (ES) m/z 346.2. HRMS:
calcd for C17H19N3O3S + H+, 346.1220; found (ESI, [M + H]+),
346.1229. 1H NMR (DMSO-d6): δ 9.38 (br s, 2H), 7.57 (m, 3H), 7.48
(m, 2H), 7.12 (d, J = 7.81 Hz, 1H), 7.03 (t, J = 7.81 Hz, 1H), 6.92 (t, J =
7.81 Hz, 1H), 6.59 (d, J = 7.81 Hz, 1H), 4.12 (m, 1H), 3.97 (m, 3H), 3.70
(m, 1H), 3.38 (d, J = 12.17 Hz, 1H), 3.13 (d, J = 12.68 Hz, 1H), 2.90 (m,
2H).
1-[(4-Methylmorpholin-2-yl)methyl]-3-phenyl-1,3-dihydro-
2,1,3-benzothiadiazole 2,2-Dioxide (14). To a solution of 11
(76 mg, 0.2 mmol) in methanol (2 mL) was added a solution of formaldehyde
(37% in water, 0.15 mL), and the mixture was stirred for 30 min. Sodium
cyanoborohydride (38 mg, 0.6 mmol, 3 equiv) was added portionwise, and
the mixture was stirred for an additional 3 h. Saturated aqueous sodium
bicarbonate (5 mL) was added slowly followed by the addition of water
(5 mL). The reaction mixture was extracted with ethyl acetate (2 Â 15 mL).
The combined organic extracts were washed with brine, dried (anhydrous
sodium sulfate), and concentrated. The crude liquid residue was purified by
chromatography (silica gel, 0À10% methanol/dichloromethane) to give
70 mg (98%) of 1-[(4-methylmorpholin-2-yl)methyl]-3-phenyl-1,3-dihy-
dro-2,1,3-benzothiadiazole 2,2-dioxide as a viscous colorless liquid. This free
base was dissolved in dichloromethane (3 mL) and was treated with an
ethereal solution of hydrochloric acid (1 M, 0.3 mL, 0.3 mmol). To the
resulting solution was added hexane until a white powder formed, which was
collected, washed with hexane, and dried in vacuo to yield 76 mg (96%) of
14. MS (ESI) m/z 360.2 ([M + H]+). HRMS: calcd for C18H21N3O3S +
H+, 360.1376; found (ESI, [M + H]+), 360.1362. 1H NMR (DMSO-d6): δ
11.08 (br s, 1H), 7.57 (m, 3H), 7.48 (m, 2H), 7.12 (m, 1H), 7.03 (t, J= 7.82
Hz, 1H), 6.92 (t, J = 7.81 Hz, 1H), 6.59 (d, J = 7.81 Hz, 1H), 4.18 (m, 1H),
3.98 (m, 3H), 3.78 (m, 1H), 3.56 (d, J = 12.29 Hz, 1H), 3.3 (m, 1H), 2.95
(m, 2H), 2.75 (s, 3H).
5-Fluoro-1-(2-fluorophenyl)-3-(2-morpholin-2-ylethyl)-1,
3-dihydro-2,1,3-benzothiadiazole 2,2-Dioxide (17). Compound
17 was prepared following the procedure for compound 10b with
1-(2-fluorophenyl)-1,3-dihydro-2,1,3-benzothiadiazole 2,2-dioxide being re-
placed with 5-fluoro-1-(2-fluorophenyl)-1,3-dihydro-2,1,3-benzothiadiazole
2,2-dioxide. MS (ES) m/z 396.1. HRMS: calcd for C18H19F2N3O3S + H+,
396.1188 found (ESI, [M + H]+), 396.1193. 1H NMR (DMSO-d6): δ 9.38
(br s, 2H), 7.69 (m, 1H), 7.58 (m, 2H), 7.46 (m, 1H), 7.19 (dd, J = 2.52,
9.20 Hz, 1H), 6.80 (dt, J = 2.51, 9.34 Hz, 1H), 6.60 (dd, J = 4.65, 8.68 Hz,
1H), 4.00 (m, 3H), 3.83 (m, 1H), 3.72 (m, 1H), 3.26 (d, J = 12.59 Hz, 1H),
3.16 (d, J = 12.33 Hz, 1H), 2.99 (m, 1H), 2.80 (m, 1H), 2.08 (m, 6H), 1.82
(m, 1H).
5-Fluoro-3-(2-fluorophenyl)-1-(2-morpholin-2-ylethyl)-1,
3-dihydro-2,1,3-benzothiadiazole 2,2-Dioxide (18). Com-
pound 18 was prepared following the procedure for compound 10b with
1-(2-fluorophenyl)-1,3-dihydro-2,1,3-benzothiadiazole 2,2-dioxide being
replaced with 6-fluoro-1-(2-fluorophenyl)-1,3-dihydro-2,1,3-benzothiadia-
zole 2,2-dioxide. MS (ES) m/z 396.1. HRMS: calcd for C18H19F2N3O3S +
H+, 396.1188 found (ESI, [M + H]+), 396.1200. 1H NMR (DMSO-d6):
δ 9.24 (br s, 2H), 7.70 (m, 1H), 7.60 (m, 2H), 7.46 (m, 1H), 7.16 (dd, J =
4.63, 8.78 Hz, 1H), 6.94 (dt, J = 2.58, 9.51 Hz, 1H), 6.59 (m, 1H), 4.00 (m,
3H), 3.81 (m, 1H), 3.70 (m, 1H), 3.25 (d, J = 12.43 Hz, 1H), 3.18 (d, J =
12.31 Hz, 1H), 3.00 (m, 1H), 2.80 (m, 1H), 2.04 (m, 6H), 1.82 (m, 1H).
4-Fluoro-3-(2-fluorophenyl)-1-(2-morpholin-2-ylethyl)-1,
3-dihydro-2,1,3-benzothiadiazole 2,2-Dioxide (19). Compound
19 was prepared following the procedure for compound 10b with
1-(2-fluorophenyl)-1,3-dihydro-2,1,3-benzothiadiazole 2,2-dioxide being
replaced with 7-fluoro-1-(2-fluorophenyl)-1,3-dihydro-2,1,3-benzothiadia-
zole 2,2-dioxide. MS (ES) m/z 396.1. HRMS: calcd for C18H19F2N3O3S +
H+, 396.1188 found (ESI, [M + H]+), 396.1195. 1H NMR (DMSO-d6): δ
9.04 (br s, 2H), 7.58 (m, 2H), 7.50 (m, 1H), 7.49 (m, 1H), 7.17 (m, 1H),
7.04 (d, J = 7.90 Hz, 1H), 6.60 (dd, J = 11.05, 8.65 Hz, 1H), 4.00 (m, 3H),
3.79 (m, 1H), 3.68 (m, 1H), 3.24 (m, 1H), 3.18 (m, 1H), 2.99 (m, 1H),
2.81 (m, 1H), 2.04 (m, 6H), 1.83 (m, 1H).
’ ASSOCIATED CONTENT
S
Supporting Information. Analytical HPLC purity data
b
for all tested compounds; experimental procedures, including
analytical and spectral data, for compounds 21 and 22. This
material is available free of charge via the Internet at http://pubs.
acs.org.
’ AUTHOR INFORMATION
Corresponding Author
1-Phenyl-3-(2-piperidin-3-ylethyl)-1,3-dihydro-2,1,3-ben-
zothiadiazole 2,2-Dioxide (15). Compound 15 was prepared following
the procedure for compound 8 with tert-butyl 2-(hydroxyethyl)morpholine-
4-carboxylate being replaced with 1-N-Boc-piperidine-3-ethanol. MS (ES)
m/z 358.1. HRMS: calcd for C19H23N3O2S + H+, 358.1584 found (ESI,
[M+H]+), 358.1587. 1H NMR (DMSO-d6):δ8.85 (br s, 2H), 7.57 (m, 3H),
7.45 (m, 2H), 7.12 (m, 1H), 7.04 (t, J = 7.69 Hz, 1H), 6.92 (t, J = 7.68 Hz,
1H), 6.61 (d, J = 7.81 Hz, 1H), 3.82 (m, 2H), 3.25 (m, 1H), 3.13 (d, J =
12.17 Hz, 1H), 2.71 (m, 1H), 2.59 (m, 1H), 1.73 (m, 6H), 1.20 (m, 1H).
4-Fluoro-1-(2-fluorophenyl)-3-(2-morpholin-2-ylethyl)-1,
3-dihydro-2,1,3-benzothiadiazole 2,2-Dioxide (16). Compound
16 was prepared following the procedure for compound 10b with 1-
(2-fluorophenyl)-1,3-dihydro-2,1,3-benzothiadiazole 2,2-dioxide being replaced
*Phone: (860) 715-6627. Fax: (860) 686-5256. E-mail: david.
oneill@pfizer.com. Address: Pfizer Global Research & Develop-
ment, MS 8220-3445, Eastern Point Road, Groton, CT 06340,
U.S.
’ ACKNOWLEDGMENT
The authors thank the Wyeth scientists whose work contrib-
uted to the program described in this manuscript: the Neu-
roscience department provided assay data; the Chemical
Technologies group provided stability data and performed chiral
resolutions; the Structural Biology & Computational Chemistry
6830
dx.doi.org/10.1021/jm200733r |J. Med. Chem. 2011, 54, 6824–6831

Journal of Medicinal Chemistry
ARTICLE
group provided computational support. Additional members of
the project team are acknowledged for helpful discussions.
(11) Gavrin, L. K.; Mahaney, P. E.; Jenkins, D.; Nogle, L. M.;
Mugford, C. A.; Huselton, C.; Leiter, J.; Johnston, G. H.; Bray, J. A.;
Burroughs, K. D.; Cosmi, S. A.; Alfinito, P.; Ho, D. M.; Deecher, D. C.;
Trybulski, E. J. Discovery of WAY-260022, a potent and selective
inhibitor of the norepinephrine transporter. ACS Med. Chem. Lett.
2010, 3, 91–95.
’ ABBREVIATIONS USED
NE, norepinephrine; NET, norephinephrine transporter; NRI, nor-
epinephrine reuptake inhibitor; CNS, central nervous system; SERT,
serotonin transporter; DAT, dopamine transporter; hNET, human
norepinephrine transporter; hSERT, human serotonin transporter;
hDAT, human dopamine transporter; SAR, structureÀactivity rela-
tionship; HPLC, high performance liquid chromatography; PK,
pharmacokinetics; Clp, clearance; Vss, steady state volume of distri-
bution; Cmax, maximum concentration; Tmax, time to maximum
concentration; F, percent bioavailability; AUC, area under the
curve; FCA, Freund’s complete adjuvant; SNL, spinal nerve ligation
(12) The Wyeth Institutional Animal Care and Use Committee
approved all animal procedures according to the guidelines of the Office
of Laboratory Animal Welfare. Animal care and use were conducted in
accordance with state and federal regulations and guidelines and
complied with or exceeded the Animal Welfare Act Regulation, 9 CFR
Parts 1À3, and with the Association for Assessment and Accreditation of
Laboratory Animal Care, International Standards, as set forth for by the
Guide for the Care and Use of Laboratory Animals (1996 National
Academy Press, Washington, DC). For all studies, animals were main-
tained in climate-controlled room on a 12 h light/dark cycle with food
and water available ad libitum. All assays were performed in a rando-
mized manner by individuals blinded to the experimental condition.
Detailed protocols for all in vivo studies are described in refs 8 and 16.
(13) Pertovaara, A. Noradrenergic pain modulation. Prog. Neurobiol.
2006, 80 (2), 53–83.
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R. Descending modulation in persistent pain: an update. Pain 2002,
100, 1–6. (c) Ren, K.; Ruda, M. A. Descending modulation of Fos
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7, 2186–2190.
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