Structure-activity relationships of the cycloalkanol ethylamine scaffold: Discovery of selective norepinephrine reuptake inhibitors

Article abstract of DOI:10.1021/jm8002262

Further exploration of the cycloalkanol ethylamine scaffold, of which venlafaxine (1) is a member, was undertaken to develop novel and selective norepinephrine reuptake inhibitors (NRIs) for evaluation in a variety of predictive animal models. These effor

Full text of DOI:10.1021/jm8002262

4038
J. Med. Chem. 2008, 51, 4038–4049
Structure-Activity Relationships of the Cycloalkanol Ethylamine Scaffold: Discovery of
Selective Norepinephrine Reuptake Inhibitors
Paige E. Mahaney,*,§ Lori K. Gavrin,§ Eugene J. Trybulski,§ Gary P. Stack, An T. Vu,§ Stephen T. Cohn,§,‡ Fei Ye,§
Justin K. Belardi,§ Arthur A. Santilli,§ Joseph P. Sabatucci,§ Jennifer Leiter,† Grace H. Johnston,†,£ Jenifer A. Bray,†
Kevin D. Burroughs,†,£ Scott A. Cosmi,† Liza Leventhal,#,× Elizabeth J. Koury,† Yingru Zhang,§,∞ Cheryl A. Mugford,|
Douglas M. Ho,3,4 Sharon J. Rosenzweig-Lipson,# Brian Platt,# Valerie A. Smith,# and Darlene C. Deecher†
Chemical and Screening Sciences, Women’s Health and Musculoskeletal Biology, and Drug Safety and Metabolism, Wyeth Research, 500
Arcola Road, CollegeVille, PennsylVania 19426, Chemical and Screening Sciences and Department of Neuroscience, Wyeth Research, CN 8000,
Princeton, New Jersey 08543, and Department of Chemistry, Princeton UniVersity, Princeton, New Jersey 08544
ReceiVed March 3, 2008
Further exploration of the cycloalkanol ethylamine scaffold, of which venlafaxine (1) is a member, was
undertaken to develop novel and selective norepinephrine reuptake inhibitors (NRIs) for evaluation in a
variety of predictive animal models. These efforts led to the discovery of a piperazine-containing analogue,
17g (WY-46824), that exhibited potent norepinephrine reuptake inhibition, excellent selectivity over the
serotonin transporter, but no selectivity over the dopamine transporter. Synthesis and testing of a series of
cyclohexanol ethylpiperazines identified (S)-(-)-17i (WAY-256805), a potent norepinephrine reuptake
inhibitor (IC50 ) 82 nM, Ki ) 50 nM) that exhibited excellent selectivity over both the serotonin and dopamine
transporters and was efficacious in animal models of depression, pain, and thermoregulatory dysfunction.
Introduction
Norepinephrine (NEa), a major neurotransmitter, is released
from noradrenergic neurons during synaptic transmission. No-
radrenergic neurons project from the locus coeruleus to stimulate
stress responses1c,d and arousal1a,b and to regulate the sympa-
thetic nervous system.1d,e These neurons modulate nearly all
areas of the brain including the hippocampus, which is involved
in memory and learning,2 the prefrontal cortex, which is
involved in drive and motivation,3 and the hypothalamus, which
is involved in temperature regulation and sleep.4 NRIs enhance
the transmission of NE by increasing its synaptic availability
through blockage of its reuptake by the norepinephrine trans-
porter (NET), making NRIs potential treatments for a wide range
of disorders. Compounds that block both NE and serotonin (5-
HT) reuptake, such as venlafaxine (1), duloxetine (2), and
Figure 1. Chemical structures of marketed SSRIs, SNRIs, and NRIs.
milnacipran (3), Figure 1, have been approved in the U.S. and
in Europe for indications including major depressive disorder
(MDD) and pain disorders such as diabetic neuropathy. In
addition, compounds with this profile have been reported to
exhibit efficacy for other ailments such as stress urinary
incontinence (SUI),5 attention deficit hyperactivity disorder
(ADHD),6 and fibromyalgia.7 Drugs that selectively inhibit
norepinephrine reuptake, including reboxetine (4) and nisoxetine
(5), are marketed for the treatment of MDD; more recently,
atomoxetine (6) was approved for the treatment of ADHD.
Interestingly, although atomoxetine has little affinity for the
dopamine transporter (DAT), it has been shown to selectively
increase extracellular dopamine levels in the prefrontal cortex
without a concomitant increase in either the nucleus accumbens
or striatum.8 Reboxetine and atomoxetine have also been
reported to show efficacy for chronic pain disorders such as
fibromyalgia9 and low back pain.9a
* To whom correspondence should be addressed. Phone: 484-865-0627.
Fax: 484-865-9399. E-mail: mahanep@wyeth.com.
§
Chemical and Screening Sciences, Wyeth Research, PA.
Chemical and Screening Sciences, Wyeth Research, NJ.
Current address: Encysive Pharmaceuticals Inc., Houston, TX 77030.
Women’s Health and Musculoskeletal Biology, Wyeth Research, PA.
Current address: Neotropix, Inc., Malvern, PA 19355.
Department of Neuroscience, Wyeth Research, NJ.
Current address: EnVivo Pharmaceuticals, Watertown, MA 02472.
Current address: Bristol-Myers Squibb Pharmaceutical Research
‡
†
£
#
×
∞
Institute, Princeton, NJ 08543.
|
Drug Safety and Metabolism, Wyeth Research, PA.
Current address: Department of Chemistry, Harvard University,
3
Cambridge, MA 02138.
4
Department of Chemistry, Princeton University.
a Abbreviations; NRI, norepinephrine reuptake inhibitor; NE, norepi-
nephrine; NET, norephinephrine transporter; 5-HT, serotonin; MDD, major
depressive disorder; SUI, stress urinary incontinence; ADHD, attention
hyperactivity disorder; SNRI, serotonin and norepinephrine reuptake
inhibitor; SAR, structure-activity relationship; hNET, human norepineph-
rine transporter; hSERT, human serotonin transporter; cpm, counts per
minute; MDCK, Madin-Darby canine kidney; hDAT, human dopamine
transporter; BOP, benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate; THF, tetrahydrofuran; SSRI, selective serotonin
reuptake inhibitor; SRI, serotonin reuptake inhibitor; ip, intraperitoneal; sc,
subcutaneous; po, oral; AUC, area under the curve; Cmax, maximum
concentration; OVX, ovariectomized; TST, tail-skin temperature.
Background
Venlafaxine (1), a member of the cycloalkanol ethylamine
scaffold, was the first dual-acting serotonin and norepinephrine
reuptake inhibitor (SNRI) to be approved for the treatment of
10.1021/jm8002262 CCC: $40.75
2008 American Chemical Society
Published on Web 06/17/2008

SAR of the Cycloalkanol Ethylamine Scaffold
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 4039
Table 1. Characterization of Cyclohexanol Ethyl N,N′-dimethylamine Analogues at the Human Norepinephrine, Serotonin, and Dopamine Transportersa
hNET uptake,c IC50
(StDev) [rNET
uptake],10 nM
hSERT uptake,d
ratio of (hSERT
hNET binding,b Ki
IC50 (StDev) [rSERT uptake IC50, nM)/(hNET uptake
hDAT binding
compd
R1
R2
R3
(StDev), nM
uptake],10 nM
IC50, nM)e
inhibitionf (StDev), %
1
7
8
9
10
11
OCH3
H
Cl
H
H
H
H
OCH3
H
Cl
H
CF3
Cl
Cl
H
H
H
H
Cl
H
H
H
385 (103)
316 (108)
249 (121)
149 (40)
125 (49)
21 (12)
535 (64) [640]
208 (16) [620]
120 (1) [300]
121 (45) [160]
205 (119) [NR]
32 (7) [360]
46 (13)
31 (4) [210]
88 (34) [190]
123 (88) [180]
292 (115) [320]
177 (127) [NR]
534 (62) [1440]
405 (99)
0.05
0.42
1.0
45h (2)
9g
23g
28g
13g
15g
17g
0g
2.4
0.86
16.7
8.8
(R)-(-)-9
(S)-(+)-9
H
H
41 (18)
339 (100)
303 (46)
155 (78)
0.51
a Data are the average of at least two independent experiments, each run in triplicate. NR means the data were not previously reported. b 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. Ki ) IC50/(1 +
[L]/KD), where [L] equals concentration of radioligand added. c Inhibition of NE uptake in MDCK-Net6 cells stably transfected with hNET. Desipramine
(IC50 ) 3.4 ( 1.6 nM) was used as a standard. d Inhibition of 5-HT uptake in JAR cells stably transfected with hSERT. Fluoxetine (IC50 ) 9.4 ( 3.1 nM)
was used as a standard. e Unitless value as a ratio in which higher numbers represent relatively greater NET selectivity. A value of 1 represents no selectivity.
f Inhibition of radioligand 18 binding to membranes from CHO cells expressing recombinant hDAT. Mazindol (22.1 ( 6.5 nM) was used as a standard.
g Percent inhibition measured at a concentration of 1000 nM. h Percent inhibition measured at a concentration of 10 000 nM.
MDD and continues to be widely prescribed. A drug discovery
program was initiated to re-examine the structure-activity
relationship (SAR) within the cycloalkanol ethylamine scaffold
to identify novel and selective NRIs for in vivo evaluation in a
panel of predictive animal models. Our initial strategy involved
screening libraries of in-house compounds that were originally
prepared for the historical MDD program. This approach
entailed retesting compounds using a cell line expressing the
human norepinephrine transporter (hNET), since uptake inhibi-
tion of NE and 5-HT was previously evaluated in rat brain
cortical synaptosomes as reported in the original cycloalkanol
ethylamine paper.10 According to a new paradigm, compounds
were screened in vitro in MDCK-Net6 cells that were stably
transfected with hNET. These cells were then treated with the
test compound followed by [3H]NE to initiate NE uptake. Once
the cells were washed and lysed, uptake of [3H]NE was
measured by collection of counts per minute (cpm) data.
Serotonin uptake into JAR cells that were stably transfected
with the human serotonin transporter (hSERT) was similarly
measured. Binding affinity for hNET was determined using a
competition radioligand-binding assay in whole MDCK cells
with [3H]nisoxetine as the radioligand. Previous studies with
cycloalkanol ethylamine analogues revealed a significant reduc-
tion in binding affinity using purified membranes versus whole
cells.11 In addition, values from the whole cell assay more
closely correlated to inhibition of NE uptake.11 Affinity for
hNET was compared to affinity for the human dopamine
transporter (hDAT) measured using purified CHO membranes
expressing recombinant hDAT. The cocaine analogue, [3H]-
3ꢀ-(4-fluorophenyl)tropane-2ꢀ-carboxylic acid methyl ester12
(18, [3H]WIN35,428) was used as the radioligand. Target in
vitro profiles for drug candidates included an IC50 value of less
than 100 nM in the hNET uptake assay and selectivity over
hSERT and hDAT of greater than 100-fold.
electron-withdrawing nature of the aromatic ring provided more
potent inhibitors of NE uptake and improved the selectivity for
NET over SERT. For example, substitution with a p-methoxy
group (compound 1) provided the weakest NRI (535 nM) and
was 17-fold more potent at inhibiting 5-HT uptake. The
m-chloro analogue (9) had an intermediate IC50 value of 149
nM in the NE uptake assay and exhibited 2.4-fold selectivity
over SERT, while the strongest electron-withdrawing m-
trifluoromethyl analogue (11) was the most potent inhibitor of
NE uptake, with an IC50 value of 32 nM, and exhibited the best
selectivity over 5-HT uptake, nearly 17-fold. All compounds
tested exhibited only weak binding to hDAT.
The effect of stereochemistry on selectivity and potency of
the series was examined by separating compound 9 into its
enantiomers using chiral HPLC. Assignment of the stereochem-
istry was based upon analogy to (S)-(+)-venlafaxine, which was
unequivocally established previously via single-crystal X-ray
diffraction.10 The R-enantiomer of 9 exhibited an increase in
both potency for hNET and selectivity over hSERT when
compared to its racemate, while the S-enantiomer was both less
potent and selective versus the racemate. Interestingly, hSERT
had a potency preference for the S-enantiomer, which was
opposite to that observed for hNET.
Since variation of aromatic substitution in the cyclohexanol
ethyl N,N′-dimethylamine series did not provide compounds that
met selectivity criteria for hNET over hSERT, additional
examples within the cycloalkanol ethylamine scaffold with
varied amine moieties were screened. These efforts led to the
identification of a piperazine-containing analogue, 17g (WY-
46824), as a potent NRI (IC50 ) 58 nM, hNET Ki ) 46 nM)
A set of original cyclohexanol ethyl N,N′-dimethylamines,
chosen to examine the effect of aromatic ring substitution on
selectivity and potency, was characterized for activity at the
human monoamine transporters (Table 1). Good agreement was
observed between the previously reported SAR established in
rat cortical synaptosomes10 and that determined in cells trans-
fected with the human transporters; however, the absolute IC50
values differed for some compounds. In general, increasing the
that exhibited nearly 300-fold selectivity over hSERT. Unfor-
tunately, this compound had high affinity for hDAT (Ki ) 53
nM), which was an undesired target for the purposes of this

4040 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
Scheme 1. Synthesis of Cycloalkanol Ethylamine Analoguesa
Mahaney et al.
a Reagents: (a) LDA (2.5 equiv), THF, -78 °C to room temp, then cyclohexanone, -78 °C (83-99%); (b) BOP reagent, 1-Boc-piperazine, CH2Cl2
(59-87%); (c) BH3-THF, reflux, then aqueous HCl (51-69%); (d) HCHO, HCO2H, H2O, then HCl/MeOH (58-79%).
program. Therefore, new piperazine-containing analogues were
synthesized with the goal of eliminating affinity for hDAT while
maintaining the potency for hNET and selectivity over hSERT
that was exhibited by the lead compound (17g).
Chemistry
A parallel synthesis approach for the generation of new
analogues in the cyclohexanol ethylpiperazine scaffold was
evaluated. The published synthetic route10 was not deemed
suitable for parallel synthesis because of a tendency toward
retroaldol and low isolable yields obtained when the aldol
reaction was conducted on the starting phenylacetic amide.
Consequently, an improved synthesis that was amenable to a
parallel approach was developed (Scheme 1). Performing the
aldol reaction on the dianion of the starting phenylacetic acid
at -78 °C prevented the occurrence of the reverse aldol reaction
and gave the desired product in high yield. In contrast,
quenching the aldol product at 0 °C, instead of maintaining a
temperature of -78 °C, resulted in the isolation of a significant
amount of starting material, presumably due to the retroaldol
mechanism. Amine coupling to aldol product 14 to form amide
15 could be accomplished with a variety of conventional agents;
however, benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate (BOP reagent) was selected because of its
ease of handling and good yields obtained in parallel reactions.
Reduction of the amide with borane-THF complex at elevated
temperatures followed by in situ deprotection with hydrochloric
acid afforded cycloalkanol amine analogues 16, which could
be N-methylated with an aqueous solution of formaldehyde in
formic acid to afford N-methylpiperazine analogues 17. Amines
16 and 17 were converted to dihydrochloride salts using
methanolic hydrochloric acid prior to in vitro characterization.
The enantiomers of compounds 16g, 17g, 16i, and 17i were
prepared in >99.5% optical purity via separation of the Boc-
protected amide intermediates 15g and 15i using chiral super-
critical fluid chromatography. Attempts to separate the enanti-
omers of either 16g or 17g afforded products with significantly
reduced optical purity. Assignment of the absolute configuration
of (R)-(+)-17g was unequivocally established by a single-crystal
X-ray analysis (Figure 2). Assignment of the stereochemistry
of 16g, 16i, and 17i was based upon analogy.
Figure 2. ORTEP view of 1-[(1R)-1-(3-chlorophenyl)-2-(4-methylpip-
erazin-1-yl)ethyl]cyclohexanol [(R)-(+)-17g].
substitution on the SAR for reuptake inhibition at hNET in this
series was similar to that seen within the N,N′-dimethylamine
class. Again, the p-methoxy analogues (16a and 17a) were the
weakest inhibitors of NE uptake while the m-chloro (compounds
16g and 17g) and the m-trifluoromethyl analogues (16j and 17j)
were the most potent. Differing slightly, however, from the N,N′-
dimethylamine SAR, in which the m-trifluoromethyl analogue
11 was preferred over the m-chloro analogue 9 for NRI potency
and selectivity over hSERT, was the observation that within
the cyclohexanol ethylpiperazines the m-chloro substituent (16g,
17g) provided equivalent or improved potency and affinity at
hNET versus the m-trifluoromethyl analogues (16j, 17j).
Interestingly, all analogues containing the m-chloro group also
exhibited high affinity for hDAT while the trifluoromethyl
analogues did not. To further explore this observation, the
m-trifluoromethoxy analogues 16i and 17i were synthesized.
These compounds also maintained good potency and affinity
at hNET while possessing very little affinity for hDAT. In
contrast, the m-methoxy analogue (compound 17b) exhibited
significantly reduced activity at all transporters.
Results and Discussion
N-Methylation of the piperazine ring had no significant impact
in vitro on hNET potency and affinity. For example, a
comparison of the two racemic m-trifluoromethoxy analogues
revealed IC50 values for NE uptake of 147 nM for N-
In Vitro Characterization. The cyclohexanol ethylpiperazine
analogues were characterized in vitro for their activity at the
human monoamine transporters (Table 2). The effect of aromatic

SAR of the Cycloalkanol Ethylamine Scaffold
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 4041
Table 2. Characterization of Cyclohexanol Ethylpiperazine Analogues at the Human Norepinephrine, Serotonin, and Dopamine Transportersa
hNET binding,b Ki
hNET uptake,c
IC50 (StDev), nM IC50 (StDev), nM Ki (StDev), nM
hSERT uptake,d
hDAT binding,e hDAT binding inhibition
compd
17a
16a
17b
16b
17c
16c
17d
16d
17e
16e
17f
16f
R1
R2
R3
R4
(StDev), nM
at 1 µMe (StDev), %
OCH3
OCH3
H
H
H
OCH3
OCH3
H
H
H
H
H
H
H
H
H
F
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
CH3
CH3
CH3
H
34% (18)f,g
34% (1)f,g
600 (143)
870 (126)
1452 (73)
1499 (242)
240 (160)
673 (66)
19%f
11%f (13)
24%f (8)
14%f (16)
8%f (6)
13%f (4)
29%f
5
9
4
2
32
22
29g
22
519 (233)
514 (339)
745 (248)
1376 (738)
1034 (394)
364 (167)
368 (211)
34%f,g
H
F
F
H
F
F
H
H
H
H
Cl
Cl
H
H
H
H
H
H
OCF3
H
H
H
H
H
12%f
12%f
H
H
F
38%f,g
31%f,g
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
1220 (266)
717 (95)
46 (3)
187 (51)
14 (8)
18 (11)
214 (87)
17 (4)
417 (131)
91 (58)
785 (488)
50 (8)
140 (89)
544 (232)
117 (41)
61 (10)
7%f
0%f
26
35
29%f,g
1525 (322)
18795 (4600)
0%f
17g
Cl
Cl
Cl
Cl
Cl
Cl
H
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
CF3
CF3
58 (10)
577 (103)
29 (5)
38 (14)
540 (26)
25 (7)
575 (162)
147 (87)
3474 (313)
82 (21)
284 (93)
1290 (110)
140 (82)
92 (6)
53 (18)
235 (50)
73 (35)
45 (17)
340 (63)
135 (30)
(R)-(+)- 17g
(S)-(-)- 17g
16g
(R)-(-)- 16g
(S)-(+)- 16g
17h
9%f
4962 (423)
6102 (749)
2669 (175)
1214 (99)
12%f (14)
4%f
H
H
CH3
CH3
CH3
CH3
H
H
H
CH3
H
13 (9)
3% (11)
5 (7)
6 (1)
13 (5)
15 (6)
13 (12)
6
17i
(R)-(+)- 17i
(S)-(-)- 17i
16i
(R)-(-)- 16i
(S)-(+)- 16i
17j
3%f
3%f
11%f (15)
3%f
H
H
H
28%f,g
16j
58 (14)
94 (17)
13%f
4
a Data are the average of at least three independent experiments, each run in triplicate. b 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. Ki ) IC50/(1 + [L]/KD), where [L] equals concentration of radioligand
added. c Inhibition of NE uptake in MDCK-Net6 cells stably transfected with hNET. Desipramine (IC50 ) 3.4 ( 1.6 nM) was used as a standard. d Inhibition
of serotonin uptake in JAR cells stably transfected with human SERT. Fluoxetine (IC50 ) 9.4 ( 3.1 nM) was used as a standard. e Inhibition of radioligand
18 binding to membranes from CHO cells expressing recombinant hDAT. Mazindol (22.1 ( 6.5 nM) was used as a standard. f Percent inhibition measured
at a concentration of 1000 nM. g Compound gave poor dose response; therefore, no IC50 was obtained.
methylpiperazine 17i compared to 284 nM for the unsubstituted
analogue (16i). Likewise, m-trifluoromethyl analogues 16j and
17j had essentially identical IC50 values of 92 and 94 nM,
respectively.
(Ki value of 187 nM in the binding assay). This is a striking
reversal from the preferred stereochemistry seen in the cyclo-
hexanol ethyl N,N′-dimethylamine series where the R-enanti-
omer was the eutomer. A future publication will examine this
intriguing observation in detail. Unfortunately for the m-chloro
derivatives, the preferred stereochemistry for both hDAT and
hNET was the S-enantiomer; consequently, no additional
selectivity for hNET over hDAT was achieved through resolu-
tion. However, among the m-trifluoromethoxy derivatives, (S)-
(-)-17i (WAY-256805) was a potent NRI (IC50 value of 82
nM, Ki value of 50 nM) that was highly selective over both
hSERT and hDAT. This compound was selected for further
evaluation.
The potential for cross-reactivity of (S)-(-)-17i with 5-HT
and adrenergic receptors was assessed using either a calcium
mobilization assay measured on a fluorometric imaging plate
reader (FLIPR) testing for both agonists and antagonists or a
competition radioligand-binding assay. (S)-(-)-17i exhibited
IC50 and EC50 values of greater than 10 µM across all receptors.
Test receptors included 5-HT1B, 5-HT1D, 5-HT2a, 5-HT2B, 5-HT6,
5-HT7, and adrenergic receptor 1R.
While the effect of aromatic substitution on the SAR for
reuptake inhibition at hNET in the cyclohexanol ethylpiperazine
series was similar to that seen within the N,N′-dimethylamine
class, a major difference was observed for 5-HT uptake
inhibition. In general, the electronic nature of the aromatic ring
had little effect because incorporation of a piperazine ring into
the scaffold essentially eliminated 5-HT reuptake inhibitory
activity. For example, the N-methylpiperazine analogue contain-
ing a p-methoxy substituent (17a), the most potent substituent
for 5-HT uptake inhibition in the N,N′-dimethylamine series,
exhibited only 11% inhibition of hSERT at 1 µM. Among the
cyclohexanol ethylpiperazines that were synthesized, the most
potent SRIs (compounds 16f and 17h) had IC50 values of greater
than 1 µM.
The effect of stereochemistry on the selectivity and potency
of this series was examined by testing the enantiomeric pairs
of compounds 16g, 17g, 16i, and 17i. In all four examples, the
S-enantiomer was the eutomer, exhibiting an increase in both
functional potency and binding affinity at hNET compared to
the corresponding racemate while the R-enantiomer was sig-
nificantly less active. For example, in the NE uptake assay, (S)-
(-)-17g had an IC50 value of 29 nM (Ki value of 14 nM in the
binding assay) while (R)-(+)-17g exhibited an IC50 of 577 nM
In Vivo Characterization. The antidepressant-like effects
of compound (S)-(-)-17i were assessed in the mouse tail-
suspension model.13a During testing, subsequent to compound
administration, male mice were suspended upside down by their
tails and movements of the animals during a 6 min test session
were automatically recorded. These movements were classified

4042 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
Mahaney et al.
Figure 5. Effect of ip and po administration of (S)-(-)-17i in a
telemetric rat model of ovariectomized (OVX)-induced thermoregula-
tory dysfunction; / signifies a p value of <0.05 vs 2% Tween-80 /0.5%
methylcellulose in water vehicle. The onset of an effect was defined
as the first half-hour interval of two consecutive significant (p < 0.05)
half-hour intervals. The treatment effect was defined to have ended
when there are two consecutive nonsignificant (p g 0.05) half-hour
intervals. Braces mark periods of significant effects. Error bars represent
standard error of the mean of each data point.
Figure 3. Effect of (S)-(-)-17i in the tail-suspension test in male mice.
A vehicle of 2% Tween-80/0.5% methylcellulose in water was used
for delivery of test compounds. Data represent group mean ( SEM
for each dose (N ) 10 per group).
number of abdominal constrictions when compared to vehicle
(p < 0.05, ANOVA) at 10 and 30 mg/kg with similar efficacy
as desipramine at both doses. While efficacy was similar
between the two compounds, not surprisingly, desipramine was
more potent, exhibiting significant activity at doses down to 1
mg/kg,18 consistently reflecting the in vitro 25-fold difference
in potency.
NE has been reported to stimulate areas of the hypothalamus
that play an important role in temperature regulation.4 In order
to determine its effects on temperature homeostasis, compound
(S)-(-)-17i was also evaluated in a telemetric rat model of
ovariectomized (OVX)-induced thermoregulatory dysfunction.19
Intact cycling rats exhibit a diurnal rhythm during which, over
a 24 h period, their tail-skin temperature (TST) decreases during
the dark (active) phase and remains elevated during the light
(inactive) phase. Reduction in ovarian hormones as a result of
ovariectomy causes the TST of OVX-rats to remain elevated
during both the dark and light phases, and treatment with
estrogen has been reported to restore the normal diurnal
temperature pattern to that seen in an intact rat.19a Results of
the evaluation of both SSRIs and SNRIs in this model have
been previously reported;20 however, activity of a selective NRI
has not been described. According to our published paradigm,21
test compounds, administered 30 min prior to the dark phase,
were analyzed for their ability to reduce TST versus baseline
(individual baselines are measured for vehicle-dosed rats 1 day
prior to administration of compound). TST was measured with
a temperature and physical activity transmitter implanted sc with
the tip of the temperature probe tunneled 2.5 cm beyond the
base of the tail. Results for (S)-(-)-17i administered at a dose
of 30 mg/kg both ip and orally (po) are depicted in Figure 5.
Both routes of administration resulted in significant reductions
of TST; however, ip administration achieved both a larger
absolute reduction of TST and a longer duration of action when
compared to po administration (Table 3). To help understand
this difference in activity, the female rat pharmacokinetic
parameters were measured (Table 4). A significant difference
in both the maximum concentration (Cmax) and the area under
the curve (AUC) with (S)-(-)-17i in rats in the two routes of
administration was observed. The Cmax after ip administration
Figure 4. Effect of (S)-(-)-17i in the mouse p-phenylquinone model
of acute visceral (abdominal) pain. A vehicle of saline was used for
dosing. Data are presented as percent blockade compared to vehicle
(% blockade ) [(mean vehicle) - (drug)/(mean vehicle)] × 100) (
SEM for each dose (N ) 8 per group); / signifies a p value of <0.05
vs saline vehicle (ANOVA).
into periods of agitation (i.e., efforts to regain footing) and
immobility. A decrease in immobility time in this model has
been reported to correspond with antidepressant-like activity.13
As shown in Figure 3, when administered via intraperitoneal
injection (ip) (S)-(-)-17i significantly decreased immobility
time, with a p value of <0.05 (ANOVA), at 17 and 30 mg/kg,
38% and 53%, respectively, in a dose-dependent manner
indicating antidepressant activity. Desipramine, a classical
tricyclic antidepressant with selectivity for NET, has been
reported to reduce immobility time in this model 25% and 30%
when administered ip at doses of 8 and 16 mg/kg, respectively.14
The antinociceptive effects of (S)-(-)-17i were evaluated in
a mouse p-phenylquinone model of acute visceral (abdominal)
pain.15 In this model, p-phenylquinone, administered via ip
injection, acts as a localized irritant resulting in an abdominal
constriction response (i.e., writhing) which can be quantified.
The ability of a compound to reduce the number of abdominal
constrictions predicts efficacy toward attenuating visceral pain.
The role of NE, a major component of the endogenous
descending pain inhibitory system from the rostral ventral
medulla to the spinal cord,16 has been formerly characterized.17
Our dose-response data for desipramine have been previously
described in this model at doses between 1 and 30 mg/kg.18
Figure 4 shows the effect of (S)-(-)-17i compared to de-
sipramine, after subcutaneous (sc) administration between 3 and
30 mg/kg. Compound (S)-(-)-17i significantly reduced the

SAR of the Cycloalkanol Ethylamine Scaffold
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 4043
Table 3. Summary of (S)-(-)-17i in a Telemetric Rat Model of OVX-Induced Thermoregulatory Dysfunction Dosed po and ip (30 mg/kg)
route of administration
of (S)-(-)-17i (dose)
onset of activity vs
baseline (p < 0.05), h
duration of
action, h
mean reduction in
TST from baseline, °C
maximum reduction in
TST from baseline, °C
po (30 mg/kg)
ip (30 mg/kg)
1
0.5
3
6.5
-2.16
-2.75
-3.15
-4.87
Table 4. Female Sprague-Dawley (SD) Rat Pharmacokinetic
Parameters for Compound (S)-(-)-17i after ip and po Dosinga
temperature, 10% isopropanol with 0.2% DEA as CO2 modifier,
50 mL/min flow rate, 100 bar outlet pressure, 220 nm UV detection.
The chiral purity of each enantiomer was determined under the
same SFC conditions using a Chiralcel OD column (10 µm, 250
mm length × 4.6 mm i.d.) at 2.0 mL/min flow rate on a Berger
Analytical SFC instrument. The enantiomeric purity of 1-[(1R)-1-
(3-chlorophenyl)-2-(dimethylamino)ethyl]cyclohexanol was deter-
mined to be >99.9% (with <0.1% of the undesired enantiomer)
isolated as peak 2: retention time, 4.69 min; [R]2D5 +8.8° (c 10 mg/
mL, MeOH). HRMS: calcd for C16H24ClNO + H+, 282.161 92;
found (ESI, [M + H]+), 282.1634.
1-[(1S)-1-(3-Chlorophenyl)-2-(dimethylamino)ethyl]cyclohex-
anol [(S)-(+)-9]. The enantiomeric purity was determined to be
>99.9% (with <0.1% of the undesired enantiomer) isolated as peak
1: retention time, 3.91 min; [R]2D5 -10.8° (c 10 mg/mL, MeOH).
HRMS: calcd for C16H24ClNO + H+, 282.161 92; found (ESI, [M
+ H]+), 282.1628. Anal. (C16H24ClNO·HCl) C, H, N.
route of
Cmax
,
AUC0-inf,
administration
dose, mg/kg
ng/mL
t1/2, h
ng·h/mL
ipb
30
30
2686 (442)
324 (131)
2.3 (0.5)
1.6 (0)
6163 (362)
931 (310)
pob
a Numbers in parentheses are standard deviations of n ) 3 animals.
b Compound was dosed in 2% Tween-80/0.5% methylcellulose in water as
the vehicle.
was 8-fold higher than after po administration, while the AUC
values differed by nearly 7-fold.
Summary
Efforts to identify selective and potent NRIs from the
cycloalkanol ethylamine scaffold, of which venlafaxine (1) is a
member, provided a piperazine-containing class of compounds
that is highly selective for hNET over hSERT. Replacement of
the m-chloro group in the lead compound (17g) with a fluorine-
containing electron-withdrawing group such as trifluoromethoxy
or trifluoromethyl afforded potent NRIs with excellent selectivity
over both hDAT and hSERT. Further investigation of the effect
of (S)-(-)-17i in animals revealed it to be efficacious in models
of depression, acute visceral pain, and thermoregulatory
dysfunction.
General Procedure for the Synthesis of Aldol Intermediates
14a-j. A solution of diisopropylamine (7.87 mL, 56.2 mmol) in
dry tetrahydrofuran (50 mL) under nitrogen was cooled to -78 °C
and treated dropwise with a solution of n-butyllithium (2.5 M in
hexanes, 22 mL, 55 mmol). The resulting solution was warmed to
0 °C and stirred for 15 min. The solution was recooled to -78 °C
and treated, via cannula, with a solution of the appropriate
phenylacetic acid (23.4 mmol) in tetrahydrofuran (20 mL). The
mixture was then allowed to warm to 25 °C where it was stirred
for 45 min and was then recooled to -78 °C. A solution of
cyclohexanone (3.65 mL, 35.3 mmol) in tetrahydrofuran (10 mL)
was then added via cannula, and the resulting mixture was stirred
at -78 °C for 1.5 h. The reaction was then quenched by the addition
of a saturated aqueous solution of ammonium chloride, and the
tetrahydrofuran was removed in vacuo. The resulting residue was
dissolved in a 2 N aqueous solution of sodium hydroxide (30 mL)
and washed with ethyl acetate (1 × 30 mL). The aqueous layer
was then acidified to pH 1 with the addition of a 2 N aqueous
solution of hydrochloric acid. The product was extracted with ethyl
acetate (3 × 30 mL), and the combined organic extracts were dried
over magnesium sulfate and concentrated in vacuo to yield product
(83-99%) that was used without further purification.
Experimental Section
General Methods. Solvents were purchased as anhydrous grade
and were used without further purification. Melting points were
measured on a Mel-Temp II (Laboratory Device, Inc.) melting point
apparatus and are uncorrected. 1H NMR spectra were recorded on
a Varian INOVA 400 instrument, and chemical shifts are reported
in δ values (parts per million, ppm) relative to an internal standard
tetramethylsilane in CDCl3 or DMSO-d6. In all cases, NMR spectra
of enantiomers were essentially identical to those of their corre-
sponding racemates. Electrospray (ESI) mass spectra were recorded
using a Waters Alliance-ZMD mass spectrometer. Electron impact
ionization (EI, EE ) 70 eV) mass spectra were recorded on a
Finnigan Trace mass spectrometer. Combustion analyses were
performed by Robertson Microlit. Analytical thin layer chroma-
tography (TLC) was performed on precoated plates (silica gel, 60
F-254) and were visualized using UV light and/or staining with a
phosphomolybdic acid solution in ethanol. Optical rotations of
individual enantiomers were obtained on a JASCO P-1020 pola-
rimeter using a 1.0 mL microcell and methanol as the solvent. In
(1-Hydroxycyclohexyl)-4-methoxyphenylacetic Acid 14a. 1H
NMR (DMSO-d6, 400 MHz) δ 1.14-1.15 (m, 2H), 1.34-1.54 (m,
8H), 3.50 (s, 1H), 3.73 (s, 3H), 4.28 (bs, 1H), 6.85 (d, J ) 8.8 Hz,
2H), 7.32 (d, J ) 8.8 Hz, 2H), 12.35 (bs, 1H). HRMS: calcd for
C15H20O4 + NH4+, 282.169 98; found (ESI-FT/MS, [M + NH4]1+),
282.1706.
(1-Hydroxycyclohexyl)-3-methoxyphenylacetic Acid 14b. 1H
NMR (DMSO-d6, 400 MHz) δ 1.00-1.10 (m, 1H), 1.12-1.21 (m,
1H), 1.31-1.55 (m, 8H), 3.54 (s, 1H), 3.73 (s, 3H), 4.32 (bs, 1H),
6.84 (dd, J ) 2.1, 8.2 Hz, 1H), 6.96 (d, J ) 7.8 Hz, 1H), 6.99 (d,
J ) 2.1 Hz, 1H), 7.20 (t, J ) 7.9 Hz, 1H), 12.32 (bs, 1H). HRMS:
calcd for C15H20O4 + Na+, 287.1254; found (ESI-FT/MS, [M +
Na]1+), 287.1256.
1
general, compound purity was assessed by H NMR and either a
LC/UV/MS method or an analytical HPLC method as described in
Supporting Information. Biological results were obtained on
compounds of >97% chemical purity as determined by the above
methods. The syntheses of 1 and 7-11 have been previously
described.10
1-[(1R)-1-(3-Chlorophenyl)-2-(dimethylamino)ethyl]cyclohex-
anol [(R)-(-)-9]. Racemic 1-(1-(3-chlorophenyl)-2-(dimethylami-
no)ethyl)cyclohexanol 910 was dissolved in methanol at a concen-
tration of approximately 40 mg/mL, and the resulting solution was
injected onto the supercritical fluid chromatography (SFC) instru-
ment with a volume of 1.0 mL per injection. The baseline resolved
enantiomers were collected using a Berger MultiGram Prep SFC
(Berger Instruments, Inc., Newark, DE) under the following
conditions: Chiralpak AD-H SFC column (5 µm, 250 mm length
× 20 mm i.d., Chiral Technologies, Inc., Exton, PA), 35 °C column
4-Fluoro-(1-hydroxycyclohexyl)phenylacetic Acid 14c. 1H
NMR (DMSO-d6, 400 MHz) δ 1.05-1.10 (m, 2H), 1.33-1.57 (m,
8H), 3.59 (s, 1H), 4.31 (bs, 1H), 7.12 (dd, J ) 2.1, 9.0 Hz, 2H),
7.45 (dd, J ) 2.2, 8.8 Hz, 2H), 12.43 (bs, 1H). HRMS: calcd for
C14H17FO3 - H+, 251.10890; found (ESI, [M - H]-), 251.1077.
3-Fluoro-(1-hydroxycyclohexyl)phenylacetic Acid 14d. 1H
NMR (DMSO-d6, 400 MHz) δ 1.06-1.21 (m, 2H), 1.34-1.60 (m,
8H), 3.62 (s, 1H), 4.37 (bs, 1H), 7.09 (dddd, J ) 0.9, 2.6, 9.0, 9.0
Hz, 1H), 7.22 (d, J ) 7.8 Hz, 2H), 7.27-7.36 (m, 2H), 12.50 (bs,
1H). HRMS: calcd for C14H17FO3 - H+, 251.10890; found (ESI,
[M - H]-), 251.1083.

4044 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
Mahaney et al.
2-Fluoro-(1-hydroxycyclohexyl)phenylacetic Acid 14e. 1H
NMR (CDCl3, 400 MHz) δ 1.15-1.28 (m, 2H), 1.35 (d, J ) 13.5
Hz, 1H), 1.41-1.58 (m, 5H), 7.09 (ddd, J ) 3.6, 10.3, 13.4 Hz,
1H), 1.86 (dd, J ) 2.6, 14.4 Hz, 1H), 4.12 (s, 1H), 4.56 (bs, 1H),
7.04-7.13 (m, 2H), 7.25-7.30 (m, 1H), 7.61 (ddd, J ) 1.5, 7.6,
7.6 Hz, 1H). HRMS: calcd for C14H17FO3 - H+, 251.10890; found
(ESI, [M - H]-), 251.1087. Anal. Calcd for C14H17FO3 ·0.5H2O:
C, 64.35; H, 6.94; N, 0.00. Found: C, 64.40; H, 7.04; N, 0.00.
4-Chloro-(1-hydroxycyclohexyl)phenylacetic Acid 14f. 1H
NMR (DMSO-d6, 400 MHz) δ 1.05-1.21 (m, 2H), 1.33-1.60 (m,
8H), 3.59 (s, 1H), 4.34 (bs, 1H), 7.36 (ddd, J ) 2.6, 2.6, 9.1 Hz,
2H), 7.45 (ddd, J ) 2.6, 2.6, 9.2 Hz, 2H), 12.47 (bs, 1H). HRMS:
calcd for C14H17ClO3 - H+, 267.07935; found (ESI, [M - H]-),
267.0792.
(s, 1H), 7.13 (ddd, J ) 2.0, 9.0, 9.0 Hz, 2H), 7.44 (dd, J ) 5.6, 8.8
Hz, 2H). HRMS: calcd for C24H36N2O5 + H+, 433.2697; found
(ESI, [M + H]+), 433.2703.
tert-Butyl 4-[(3-Fluorophenyl)(1-hydroxycyclohexyl)acetyl]pip-
erazine-1-carboxylate 15d. 1H NMR (DMSO-d6, 400 MHz) δ
1.00-1.05 (m, 1H), 1.06-1.19 (m, 2H), 1.37 (s, 9H) 1.37-1.69
(m, 7H), 2.87-2.85 (m, 1H), 3.15-3.24 (m, 3H), 3.31-3.42 (m,
2H), 3.52-3.61 (m, 1H), 3.66-3.74 (m, 1H), 4.10 (s, 1H), 5.27
(s, 1H), 7.09 (dddd, J ) 1.3, 2.6, 9.0, 9.0 Hz, 1H), 7.25 (dd, J )
1.3, 9.0 Hz, 2H), 7.35 (m, 1H). HRMS: calcd for C23H33FN2O4 +
H+, 421.2497; found (ESI, [M + H]+), 421.2500.
tert-Butyl 4-[(2-Fluorophenyl)(1-hydroxycyclohexyl)acetyl]pip-
erazine-1-carboxylate 15e. 1H NMR (DMSO-d6, 400 MHz) δ
1.11-1.14 (m, 3H), 1.33-1.63 (m, 6H), 1.37 (s, 9H), 1.68-1.74
(m, 1H), 2.73-2.80 (m, 1H), 3.10-3.18 (m, 1H), 3.21-3.30 (m,
2H), 3.41-3.59 (m, 4H), 4.21 (s, 1H), 5.18 (s, 1H), 7.15-7.23
(m, 2H), 7.34 (ddd, J ) 1.0, 7.6, 7.6 Hz, 1H), 7.54 (ddd, J ) 1.7,
7.8, 7.8 Hz, 1H). HRMS: calcd for C23H33FN2O4 + H+, 421.2497;
found (ESI, [M + H]+), 421.2509. Anal. (C23H33FN2O4) C, H, N.
tert-Butyl 4-[(4-Chlorophenyl)(1-hydroxycyclohexyl)acetyl]pip-
erazine-1-carboxylate 15f. 1H NMR (DMSO-d6, 400 MHz) δ
0.97-1.03 (m, 1H), 1.05-1.15 (m, 2H), 1.37 (s, 9H) 1.37-1.66
(m, 7H), 2.81-2.90 (m, 1H), 3.12-3.28 (m, 3H), 3.35-3.42 (m,
2H), 3.50-3.58 (m, 1H), 3.61-3.70 (m, 1H), 4.07 (s, 1H), 5.26
(s, 1H), 7.37 (d, J ) 8.6 Hz, 2H), 7.43 (d, J ) 8.8 Hz, 2H). HRMS:
calcd for C23H33ClN2O4 + H+, 437.2202; found (ESI, [M + H]+),
437.2213.
tert-Butyl 4-[(3-Chlorophenyl)(1-hydroxycyclohexyl)acetyl]pip-
erazine-1-carboxylate 15g. 1H NMR (DMSO-d6, 400 MHz) δ
1.03-1.19 (m, 3H), 1.37 (s, 9H) 1.37-1.64 (m, 7H), 2.90-3.00
(m, 1H), 3.21-3.24 (m, 3H), 3.31-3.37 (m, 2H), 3.58-3.62 (m,
1H), 3.69-3.78 (m, 1H), 4.10 (s, 1H), 5.24 (s, 1H), 7.32-7.39
(m, 3H), 7.50 (s, 1H). HRMS: calcd for C23H33ClN2O4 + H+,
437.2202; found (ESI, [M + H]+), 437.2187.
tert-Butyl 4-{(1-Hydroxycyclohexyl)[4-(trifluoromethoxy)-
phenyl]acetyl}piperazine-1-carboxylate 15h. 1H NMR (DMSO-
d6, 400 MHz) δ 0.95-1.02 (m, 1H), 1.09-1.18 (m, 2H), 1.37 (s,
9H), 1.37-1.68 (m, 7H), 2.85-2.92 (m, 1H), 3.14-3.24 (m, 3H),
3.30-3.40 (m, 2H), 3.55-3.62 (m, 1H), 3.65-3.72 (m, 1H), 4.13
(s, 1H), 5.25 (s, 1H), 7.31 (d, J ) 8.4 Hz, 2H), 7.54 (d, J ) 8.7
Hz, 2H); MS (ESI) m/z 487. Anal. Calcd for
C24H33F3N2O5 · 0.40H2O: C, 58.38; H, 6.90; N, 5.67. Found: C,
57.91; H, 7.02; N, 6.27.
3-Chloro-(1-hydroxycyclohexyl)phenylacetic Acid 14g. 1H
NMR (DMSO-d6, 400 MHz) δ 1.06-1.23 (m, 2H), 1.33-1.56 (m,
8H), 3.61 (s, 1H), 4.37 (bs, 1H), 7.22-7.38 (m, 3H), 7.53 (s, 1H),
12.50 (bs, 1H). HRMS: calcd for C14H17ClO3 + Na+, 291.075 84;
found (ESI_FT, [M + Na]1+), 291.0748.
(1-Hydroxycyclohexyl)-(4-trifluoromethoxy)phenylacetic Acid
1
14h. H NMR (DMSO-d6, 400 MHz) δ 1.06-1.21 (m, 2H), 1.35
(d, J ) 10.4 Hz, 1H), 1.42-1.58 (m, 7H), 3.64 (s, 1H), 4.37 (bs,
1H), 7.29 (d, J ) 8.5 Hz, 2H), 7.55 (d, J ) 8.8 Hz, 2H), 12.51 (bs,
1H). HRMS: calcd for C15H17F3O4 + H+, 319.115 17; found (ESI,
[M + H]+), 319.1145. Anal. Calcd for C15H17F3O4 ·0.10H2O: C,
56.28; H, 5.41; N, 0.00. Found: C, 56.03; H, 4.99; N, 0.00.
(1-Hydroxycyclohexyl)-(3-trifluoromethoxy)phenylacetic Acid
14i. 1H NMR (DMSO-d6, 400 MHz) δ 1.10-1.21 (m, 2H),
1.34-1.54 (m, 8H), 3.67 (s, 1H), 4.40 (bs, 1H), 7.27 (d, J ) 3.6
Hz, 1H), 7.40-7.48 (m, 3H), 12.54 (bs, 1H). HRMS: calcd for
C15H17F3O4 + H+, 319.115 17; found (ESI, [M + H]+), 319.1158.
(1-Hydroxycyclohexyl)-(3-trifluoromethyl)phenylacetic Acid
14j. 1H NMR (DMSO-d6, 400 MHz) δ 1.03-1.23 (m, 2H),
1.30-1.58 (m, 8H), 3.73 (s, 1H), 4.42 (bs, 1H), 7.53 (t, J ) 7.8
Hz, 1H), 7.62 (dd, J ) 0.7, 7.8 Hz, 1H), 7.72 (d, J ) 7.7 Hz, 1H),
12.52 (bs, 1H). HRMS: calcd for C15H17F3O3 + Na+, 325.102 20;
found (ESI_FT, [M + Na]1+), 325.1024.
General Procedure for the Coupling of Aldol Intermediates
To Form boc-Protected Amides 15a-j. A solution of aldol
intermediate 14a-j (2.00 mmol), benzotriazol-1-yloxytris(dim-
ethylamino)phosphonium hexafluorophosphate (1.42 g, 3.22 mmol),
and tert-butyl 1-piperazinecarboxylate (0.60 g, 3.22 mmol) in
methylene chloride (20 mL) was treated with triethylamine (0.84
mL, 6.0 mmol). The mixture was stirred at 25 °C for 16 h, after
which time the solvent was removed in vacuo and the products
(compounds 15a-j) were purified via Biotage Horizon (FLASH
40 M, silica, gradient from 0% EtOAc/hexane to 30% EtOAc/
hexane). This reaction gave a range of yields between 59% and
87%.
tert-Butyl4-[(1-Hydroxycyclohexyl)(4-methoxyphenyl)acetyl]pip-
erazine-1-carboxylate 15a. 1H NMR (DMSO-d6, 400 MHz) δ
0.99-1.19 (m, 3H), 1.31 (s, 9H) 1.31-1.48 (m, 5H), 1.51-1.58
(m, 2H), 2.68-2.76 (m, 1H), 3.04-3.21 (m, 3H), 3.26-3.39 (m,
2H), 3.41-3.48 (m, 1H), 3.52-3.60 (m, 1H), 3.67 (s, 3H), 3.89
(s, 1H), 5.27 (s, 1H), 6.80 (d, J ) 8.8 Hz, 2H), 7.25 (d, J ) 8.7
Hz, 2H). HRMS: calcd for C23H33FN2O4 + CH3CO2, 479.2557;
found (ESI, [M + OAc]-), 479.2548.
tert-Butyl4-[(1-Hydroxycyclohexyl)(3-methoxyphenyl)acetyl]pip-
erazine-1-carboxylate 15b. 1H NMR (DMSO-d6, 400 MHz) δ
1.01-1.20 (m, 3H), 1.37 (s, 9H) 1.37-1.53 (m, 5H), 1.54-1.66
(m, 2H), 2.78-2.85 (m, 1H), 3.11-3.28 (m, 3H), 3.31-3.44 (m,
2H), 3.50-3.57 (m, 1H), 3.61-3.70 (m, 1H), 3.72 (s, 3H), 3.99
(s, 1H), 5.36 (s, 1H), 6.84 (ddd, J ) 1.9, 1.9, 7.6 Hz, 1H),
6.96-6.98 (m, 2H), 7.21 (t, J ) 7.8 Hz, 1H). HRMS: calcd for
C23H33FN2O4 + CH3CO2, 479.2557; found (ESI, [M + OAc]-),
479.2578.
tert-Butyl 4-[(4-Fluorophenyl)(1-hydroxycyclohexyl)acetyl]pip-
erazine-1-carboxylate 15c. 1H NMR (DMSO-d6, 400 MHz) δ
0.95-1.02 (m, 1H), 1.05-1.14 (m, 2H), 1.37 (s, 9H) 1.37-1.65
(m, 7H), 2.80-2.88 (m, 1H), 3.16-3.28 (m, 3H), 3.33-3.44 (m,
2H), 3.50-3.58 (m, 1H), 3.61-3.70 (m, 1H), 4.06 (s, 1H), 5.28
tert-Butyl 4-{(1-Hydroxycyclohexyl)[3-(trifluoromethoxy)-
1
phenyl]acetyl}piperazine-1-carboxylate 15i. H NMR (DMSO-
d6, 400 MHz) δ 0.96-1.04 (m, 1H), 1.10-1.19 (m, 2H), 1.37 (s,
9H), 1.38-1.69 (m, 7H), 2.72-2.80 (m, 1H), 3.10-3.30 (m, 3H),
3.39-3.54 (m, 3H), 3.62-3.70 (m, 1H), 4.16 (s, 1H), 5.24 (s, 1H),
7.27 (d, J ) 3.2 Hz, 1H), 7.41-7.49 (m, 3H). HRMS: calcd for
C24H33F3N2O5 + H+, 487.2414; found (ESI, [M + H]+), 487.2406.
tert-Butyl
4-{(1-Hydroxycyclohexyl)[3-(trifluoromethyl)-
1
phenyl]acetyl}piperazine-1-carboxylate 15j. H NMR (DMSO-
d6, 400 MHz) δ 0.90-0.96 (m, 1H), 1.02-1.12 (m, 2H), 1.31 (s,
9H) 1.32-1.60 (m, 7H), 2.82-2.90 (m, 1H), 3.08-3.18 (m, 3H),
3.28-3.35 (m, 2H), 3.51-3.59 (m, 1H), 3.64-3.71 (m, 1H), 4.18
(s, 1H), 5.17 (s, 1H), 7.49 (t, J ) 7.7 Hz, 1H), 7.57 (d, J ) 7.7 Hz,
1H), 7.68 (d, J ) 7.7 Hz, 1H), 7.75 (s, 1H). HRMS: calcd for
C24H33F3N2O4 + H+, 471.2426; found (ESI, [M + H]+), 471.2423.
General Procedure for the Separation of Enantiomers To
Form Amines (R)-(+)-15g, (S)-(-)-15g, (R)-(+)-15i, (S)-(-)-15i.
Racemic boc-protected amides 15g and 15i were dissolved in
methanol at a concentration of approximately 40 mg/mL, and the
resulting solution was injected onto the supercritical fluid chroma-
tography (SFC) instrument with a volume of 1.0 mL per injection.
The baseline resolved enantiomers were collected using a Berger
MultiGram Prep SFC (Berger Instruments, Inc., Newark, DE) under
the following conditions: Chiralpak AD-H SFC column (5 µm, 250
mm length × 20 mm i.d., Chiral Technologies, Inc., Exton, PA),
35 °C column temperature, 40% methanol as CO2 modifier for 15g
and 20% methanol as CO2 modifier for 15i, 50 mL/min flow rate,
100 bar outlet pressure, 220 nm UV detection. The chiral purity of

SAR of the Cycloalkanol Ethylamine Scaffold
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 4045
each enantiomer was determined under the same SFC conditions
using a Chiralpak AD-H column (10 µm, 250 mm length × 4.6
mm i.d.) at 2.0 mL/min flow rate on a Berger Analytical SFC
instrument.
tert-Butyl 4-[(2R)-2-(3-Chlorophenyl)-2-(1-hydroxycyclohexy-
l)acetyl]piperazine- 1-carboxylate (R)-(+)-15g. The enantiomeric
purity of was determined to be 99.5% (with 0.5% of the undesired
enantiomer) isolated as peak 1: retention time, 2.82 min; [R]2D5 +14°
(c 6.2 mg/mL, EtOH). HRMS: calcd for C23H33ClN2O4 + H+,
437.2202; found (ESI, [M + H]+), 437.2203.
tert-Butyl 4-[(2S)-2-(3-Chlorophenyl)-2-(1-hydroxycyclohexy-
l)acetyl]piperazine-1-carboxylate (S)-(-)-15g. The enantiomeric
purity of was determined to be 99.6% (with 0.4% of the undesired
enantiomer) isolated as peak 2: retention time, 3.84 min; [R]2D5 -24°
(c 4.6 mg/mL, EtOH). HRMS: calcd for C23H33ClN2O4 + H+,
437.2202; found (ESI, [M + H]+), 437.2212.
tert-Butyl 4-[(2R)-2-(1-Hydroxycyclohexyl)-2-[3-(trifluorometh-
oxy)phenyl]acetyl]piperazine-1-carboxylate (R)-(+)-15i. The enan-
tiomeric purity of was determined to be >99.9% (with <0.1% of
the undesired enantiomer) isolated as peak 1: retention time, 3.0
min; [R]2D5 +23° (c 11.6 mg/mL, EtOH). HRMS: calcd for
C23H33ClN2O4 + CH3CO2, 495.2262; found (ESI, [M + OAc]-),
495.2268.
C19H30N2O2 ·2HCl·0.5H2O: C, 57.00; H, 8.31; N, 7.00. Found: C,
57.31; H, 8.64; N, 6.89.
1-[1-(4-Fluorophenyl)-2-piperazin-1-ylethyl]cyclohexanol Di-
hydrochloride 16c. 1H NMR (DMSO-d6, 400 MHz) δ 1.02-1.16
(m, 2H), 1.20-1.24 (m, 2H), 1.32-1.35 (m, 1H), 1.44-1.64 (m,
5H), 3.29-3.80 (m, 11H), 4.62 (s, 1H) 7.18 (d, J ) 7.3 Hz, 2H),
7.39 (bs, 2H), 9.46 (bs, 2H), 10.60 (bs, 1H). HRMS: calcd for
C18H27FN2O + H+, 307.2180; found (ESI, [M + H]+), 307.2166.
Anal. Calcd for C18H27FN2O·2HCl·1.0H2O: C, 54.41; H, 7.86; N,
7.05. Found: C, 54.51; H, 7.88; N, 7.06.
1-[1-(3-Fluorophenyl)-2-piperazin-1-ylethyl]cyclohexanol Di-
1
hydrochloride 16d. H NMR (D2O, 400 MHz) δ 0.89-1.00 (m,
1H), 1.08 (d, J ) 12.2 Hz, 1H), 1.16 (dd, J ) 4.5, 10.9 Hz, 1H),
1.20-1.37 (m, 6H), 1.60 (d, J ) 13.2 Hz, 1H), 3.04 (dd, J ) 3.1,
11.1 Hz, 1H), 3.21-3.55 (m, 8H), 3.65 (dd, J ) 3.2, 13.5 Hz, 1H),
3.75 (t, J ) 11.1 Hz, 1H), 7.98 (dd, J ) 2.1, 8.3 Hz, 1H), 7.02 (t,
J ) 7.3 Hz, 1H), 7.07 (d, J ) 7.7 Hz, 1H), 7.98 (ddd, J ) 6.3, 8.1,
8.1 Hz, 1H). HRMS: calcd for C18H27FN2O + H+, 307.2180; found
(ESI,
[M
+
H]+),
307.2179.
Anal.
Calcd
for
C18H27FN2O·2HCl·1.3H2O: C, 53.68; H, 7.91; N, 6.96. Found: C,
53.97; H, 8.2; N, 7.02.
1-[1-(2-Fluorophenyl)-2-piperazin-1-ylethyl]cyclohexanol Di-
1
hydrochloride 16e. H NMR (D2O, 400 MHz) δ 0.88-0.98 (m,
1H), 1.16-1.47 (m, 8H), 1.64 (d, J ) 12.9 Hz, 1H), 3.28-3.50
(m, 9H), 3.67 (dd, J ) 3.2, 13.3 Hz, 1H), 3.70-3.80 (m, 1H), 7.05
(ddd, J ) 1.2, 8.3, 8.3 Hz, 1H), 7.13 (t, J ) 7.5 Hz, 1H), 7.13
(dddd, J ) 1.7, 5.4, 7.3, 7.3 Hz, 1H), 7.30-7.36 (m, 1H). HRMS:
calcd for C18H27FN2O + H+, 307.2180; found (ESI, [M + H]+),
307.2166. Anal. Calcd for C18H27FN2O·2.00HCl·1.0H2O: C, 54.41;
H, 7.86; N, 7.04. Found: C, 54.52; H, 8.05; N, 6.80.
tert-Butyl 4-[(2S)-2-(1-Hydroxycyclohexyl)-2-[3-(trifluorometh-
oxy)phenyl]acetyl]piperazine-1-carboxylate (S)-(-)-15i. The enan-
tiomeric purity of was determined to be 99.8% (with 0.2% of the
undesired enantiomer) isolated as peak 2: retention time, 3.58 min;
[R]D25 -19° (c 11.2 mg/mL, MeOH). HRMS: calcd for
C23H33ClN2O4 + CH3CO2, 495.2262; found (ESI, [M + OAc]-),
495.2272.
1-[1-(4-Chlorophenyl)-2-piperazin-1-ylethyl]cyclohexanol Di-
1
General Procedure for the Amide Reduction and Deprotec-
tion To Form Amines 16a-j. A solution of boc-protected amide
(15a-j) (0.46 mmol) in dry tetrahydrofuran (3 mL) under nitrogen
was treated dropwise with a solution of borane (1.0 M in
tetrahydrofuran, 1.60 mL, 1.60 mmol). The resulting solution was
heated at 70 °C for 2 h, after which time the mixture was cooled
in an ice bath and was treated dropwise with a 2 N aqueous solution
of hydrochloric acid (1 mL). The mixture was again heated at 70
°C for 1 h and was then cooled and treated with methanol (1 mL).
After the solvent was removed in vacuo, the resulting residue was
dissolved in water (5 mL) and was washed with ethyl acetate (1 ×
4 mL). The aqueous layer was basified with the addition of a 2 N
aqueous solution of sodium hydroxide until pH 10 was attained.
The product was extracted with ethyl acetate (4 × 5 mL) and the
combined organic extracts were dried over magnesium sulfate and
concentrated in vacuo to yield the amine as a colorless oil, which
was used directly in the next step or converted to the HCl salt for
pharmacological testing. The HCl salts were formed by dissolving
the product in methanol (0.5 mL) and treating with a saturated
methanolic solution of hydrochloric acid (0.5 mL) followed by
diethyl ether. After crystallizing in the refrigerator for 16 h, the
resulting solid was filtered, washed with diethyl ether, and dried in
vacuo to afford the final products (16a-k) in a yield range between
51% and 69% as white solids.
hydrochloride 16f. H NMR (D2O, 400 MHz) δ 1.09-1.12 (m,
1H), 1.24 (d, J ) 12.9 Hz, 1H), 1.30 (dd, J ) 4.8, 10.9 Hz, 1H),
1.29-1.60 (m, 6H), 1.74 (d, J ) 12.9 Hz, 1H), 3.14 (dd, J ) 3.5,
10.9 Hz, 1H), 3.41-3.49 (m, 8H), 3.71 (dd, J ) 3.5, 13.4 Hz, 1H),
3.77 (t, J ) 13.4 Hz, 1H), 7.38 (d, J ) 8.5 Hz, 2H), 7.45 (d, J )
8.7 Hz, 2H). HRMS: calcd for C18H27ClN2O + H+, 323.1885; found
(ESI,
[M
+
H]+),
323.1868.
Anal.
Calcd
for
C18H27ClN2O·2.00HCl ·1.0H2O: C, 52.24; H, 7.56; N, 6.77. Found:
C, 52.27; H, 8.0; N, 6.72.
1-[1-(3-Chlorophenyl)-2-piperazin-1-ylethyl]cyclohexanol Di-
hydrochloride 16g. 1H NMR for free base (DMSO-d6, 400 MHz)
δ 0.94-1.14 (m, 3H), 1.37-1.56 (m, 7H), 2.28 (bs, 2H), 2.33 (bs,
2H), 2.43 (dd, J ) 6.2, 12.4 Hz, 1H), 2.59 (t, J ) 4.7 Hz, 4 H),
2.93 (t, J ) 6.4, 1 H), 3.01 (dd, J ) 8.1, 12.4 Hz, 1H), 5.31 (s,
1
1H), 7.18 (d, J ) 7.3 Hz, 1H), 7.22-7.29 (m, 3H); H NMR for
dihydrochloride salt (D2O, 400 MHz) δ 0.89-1.00 (m, 1H), 1.07
(d, J ) 12.8 Hz, 1H), 1.15 (dd, J ) 4.6, 11.0 Hz, 1H), 1.20-1.41
(m, 6H), 1.60 (d, J ) 12.7 Hz, 1H), 3.00 (dd, J ) 3.2, 11.1 Hz,
1H), 3.18-3.51 (m, 8H), 3.62 (dd, J ) 3.2, 13.5 Hz, 1H), 3.71 (t,
J ) 11.1 Hz, 1H), 7.18 (d, J ) 6.8 Hz, 1H), 7.23-7.32 (m, 3H).
HRMS: calcd for C18H27ClN2O + H+, 323.1885; found (ESI, [M
+ H]+), 323.18831. Anal. Calcd for C18H27ClN2O·2.00HCl ·1H2O:
C, 52.24; H, 7.56; N, 6.77. Found: C, 52.00; H, 7.36; N, 6.78.
1-[(1R)-1-(3-Chlorophenyl)-2-piperazin-1-ylethyl]cyclohex-
anol Dihydrochloride (R)-(-)-16g. [R]2D5 -7° (c 5.1 mg/mL,
MeOH). HRMS: calcd for C18H27ClN2O + H+, 323.1885; found
(ESI, [M + H]+), 323.1873.
1-[1-(4-Methoxyphenyl)-2-piperazin-1-ylethyl]cyclohexanol Di-
1
hydrochloride 16a. H NMR (D2O, 400 MHz) δ 0.88-1.00 (m,
1H), 1.09 (d, J ) 12.4 Hz, 1H), 1.14 (dd, J ) 4.4, 10.9 Hz, 1H),
1.17-1.40 (m, 6H), 1.56 (d, J ) 12.9 Hz, 1H), 2.95 (dd, J ) 3.5,
11.3 Hz, 1H), 3.31-3.50 (m, 8H), 3.60 (dd, J ) 3.5, 13.3 Hz, 1H),
3.66-3.72 (m, 1H), 3.68 (s, 3H), 6.88 (d, J ) 9.0 Hz, 2H), 7.20
(d, J ) 8.3 Hz, 2H). HRMS: calcd for C19H30N2O2 + H+, 319.2380;
1-[(1S)-1-(3-Chlorophenyl)-2-piperazin-1-ylethyl]cyclohex-
anol Dihydrochloride (S)-(+)-16g. [R]2D5 +12.5° (c 4.5 mg/mL,
MeOH). HRMS: calcd for C18H27ClN2O + H+, 323.1885; found
(ESI, [M + H]+), 323.1867. Anal. Calcd for C18H27ClN2O·2.00
HCl·1.0 H2O: C, 52.24; H, 7.56; N, 6.77. Found: C, 52.23; H,
7.37; N, 6.65.
found (ESI, [M
C19H30N2O2 ·2HCl·1.2H2O: C, 55.26; H, 8.40; N, 6.78. Found: C,
55.48; H, 8.8; N, 6.67.
+
H]+), 319.2376. Anal. Calcd for
1-{2-Piperazin-1-yl-1-[4-(trifluoromethoxy)phenyl]ethyl}cyclo-
1
1-[1-(3-Methoxyphenyl)-2-piperazin-1-ylethyl]cyclohexanol Di-
hexanol Dihydrochloride 16h. H NMR (DMSO-d6, 400 MHz)
1
hydrochloride 16b. H NMR (D2O, 400 MHz) δ 0.90-0.99 (m,
δ1.02 (t, J ) 11.6 Hz, 2H), 1.15 (d, J ) 13.6 Hz, 1H), 1.22 (t, J
) 11.1 Hz, 1H), 1.31-1.36 (m, 1H), 1.45 (dd, J ) 2.3, 12.2 Hz,
3H), 1.55 (t, J ) 12.2 Hz, 1H), 1.67 (d, J ) 13.0 Hz, 1H),
3.10-3.81 (m, 11H), 4.53 (bs, 1H), 7.33 (d, J ) 8.1 Hz, 2H), 7.49
(d, J ) 8.5 Hz, 2H), 9.70 (bs, 2H), 11.01 (bs, 1H). HRMS: calcd
1H), 1.10-1.12 (m, 1H), 1.17-1.37 (m, 7H), 1.59 (d, J ) 12.2
Hz, 1H), 2.99 (d, J ) 10.5 Hz, 1H), 3.18 (s, 1H), 3.32-3.42 (m,
7H), 3.64 (d, J ) 13.3 Hz, 1H), 3.69 (s, 3H), 3.77 (t, J ) 12.2 Hz,
1H), 6.84-6.90 (m, 3H), 7.24 (t, J ) 7.8 Hz, 1H). Anal. Calcd for

4046 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
Mahaney et al.
for C19H27F3N2O2 + H+, 373.2097; found (ESI, [M + H]+),
373.2086. Anal. Calcd for C19H27F3N2O2 ·2.00HCl·2.10 H2O: C,
47.23; H, 6.93; N, 5.80. Found: C, 46.93; H, 6.80; N, 5.41.
1-[1-(3-Fluorophenyl)-2-(4-methylpiperazin-1-yl)ethyl]cyclo-
hexanol Dihydrocholoride 17d. 1H NMR (D2O, 400 MHz) δ
1.10-1.13 (m, 1H), 1.25 (d, J ) 12.0 Hz, 1H), 1.30-1.56 (m,
7H), 1.74 (d, J ) 12.5 Hz, 1H), 2.88 (s, 3H), 3.12 (dd, J ) 3.4,
10.9 Hz, 1H), 3.22-3.50 (m, 8H), 3.57 (dd, J ) 3.2, 13.2 Hz, 1H),
3.65 (t, J ) 13.2 Hz, 1H), 7.10-7.16 (m, 1H), 7.20 (d, J ) 7.6
Hz, 2H), 7.42 (q, J ) 8.1 Hz, 1H). HRMS: calcd for C19H29FN2O
+ H+, 321.2337; found (ESI, [M + H]+), 321.2328. Anal.
(C19H29FN2O·2.00HCl) C, H, N.
1-{2-Piperazin-1-yl-1-[3-(trifluoromethoxy)phenyl]ethyl}cyclo-
hexanol Dihydrochloride 16i. 1H NMR (D2O, 400 MHz) δ
0.86-1.00 (m, 1H), 1.08 (d, J ) 13.1 Hz, 1H), 1.14 (dd, J ) 4.9,
10.9 Hz, 1H), 1.19-1.37 (m, 6H), 1.60 (d, J ) 13.1 Hz, 1H), 3.06
(dd, J ) 3.2, 11.0 Hz, 1H), 3.31-3.49 (m, 8H), 3.66 (dd, J ) 3.0,
13.5 Hz, 1H), 3.75 (t, J ) 13.3 Hz, 1H), 7.19-7.21 (m, 2H), 7.25
(d, J ) 7.7 Hz, 1H), 7.36 (t, J ) 7.9 Hz, 1H). HRMS: calcd for
C19H27F3N2O2 + H+, 373.2097; found (ESI, [M + H]+), 373.2095.
Anal. Calcd for C19H27F3N2O2 ·2.00HCl·1.5H2O: C, 48.31; H, 6.83;
N, 5.93. Found: C, 48.45; H, 6.79; N, 5.76.
1-[1-(2-Fluorophenyl)-2-(4-methylpiperazin-1-yl)ethyl]cyclo-
hexanol Dihydrocholoride 17e. 1H NMR (D2O, 400 MHz) δ 1.03
(bs, 1H), 1.28-1.52 (m, 8H), 1.74 (d, J ) 12.6 Hz, 1H), 2.89 (s,
3H), 3.47 (bs, 9H), 3.70-3.88 (m, 2H), 7.16 (t, J ) 8.4 Hz, 1H),
7.23 (t, J ) 7.5 Hz, 1H), 7.37 (q, J ) 5.8 Hz, 1H), 7.46 (bs, 1H).
HRMS: calcd for C19H29FN2O + H+, 321.2337; found (ESI, [M
+ H]+), 321.2326. Anal. Calcd for C19H29FN2O·2.00HCl ·0.1H2O:
C, 57.74; H, 7.95; N, 7.09. Found: C, 57.61; H, 7.43; N, 6.78.
1-[1-(4-Chlorophenyl)-2-(4-methylpiperazin-1-yl)ethyl]cyclo-
hexanol Dihydrocholoride 17f. 1H NMR (D2O, 400 MHz) δ
1.09-1.12 (m, 1H), 1.24 (d, J ) 13.3 Hz, 1H), 1.28-1.56 (m,
7H), 1.73 (d, J ) 12.5 Hz, 1H), 2.89 (s, 3H), 3.10 (dd, J ) 3.5,
10.7 Hz, 1H), 3.22-3.55 (m, 8H), 3.60 (dd, J ) 3.5, 13.4 Hz, 1H),
3.65 (t, J ) 13.4 Hz, 1H), 7.37 (d, J ) 8.4 Hz, 2H), 7.44 (d, J )
8.7 Hz, 1H). HRMS: calcd for C19H29ClN2O + H+, 337.2041; found
1-{(1R)-2-Piperazin-1-yl-1-[3-(trifluoromethoxy)phenyl]ethyl}-
cyclohexanol Dihydrochloride (R)-(-)-16i. [R]2D5 -4.5° (c 10.2
mg/mL, MeOH). HRMS: calcd for C19H27F3N2O2 + H+, 373.2097;
found (ESI, [M + H]+), 373.2102.
1-{(1S)-2-Piperazin-1-yl-1-[3-(trifluoromethoxy)phenyl]ethyl}-
cyclohexanol Dihydrochloride (S)-(+)-16i. [R]2D5 +2.2° (c 9.9 mg/
mL, MeOH). HRMS: calcd for C19H27F3N2O2 + H+, 373.2097;
found (ESI, [M + H]+), 373.2094.
1-{2-Piperazin-1-yl-1-[3-(trifluoromethyl)phenyl]ethyl}cyclo-
hexanol Dihydrochloride 16j. 1H NMR (D2O, 400 MHz) δ
0.89-1.00 (m, 1H), 1.05 (d, J ) 11.5 Hz, 1H), 1.12-1.37 (m,
7H), 1.62 (d, J ) 13.1 Hz, 1H), 3.10 (d, J ) 10.6 Hz, 1H),
3.30-3.39 (m, 8H), 3.61-3.68 (m, 1H), 3.64 (dd, J ) 3.0, 13.5
Hz, 1H), 3.77 (dd, J ) 11.4, 13.2 Hz, 1H), 7.43-7.49 (m, 2H),
7.57-7.59 (m, 2H). HRMS: calcd for C19H27F3N2O + H+,
357.2148; found (ESI, [M + H]+), 357.2155.
General Procedure for N-Methylation To Form N-Meth-
ylpiperazines 17a-j. A solution of the appropriate amine (16a-j)
(0.17 mmol) in formic acid (0.33 mL) at 50 °C was treated with
an aqueous solution of formaldehyde (37% in water, 0.14 mL, 0.21
mmol). The mixture was heated at 70 °C for 1.5 h, after which
time the mixture was poured into water (5 mL) and basified to pH
10 with the addition of a 2 N aqueous solution of sodium hydroxide.
The product was then extracted with ethyl acetate (3 × 4 mL), and
the combined organic extracts were dried over magnesium sulfate
and concentrated to yield the methylamine as a colorless oil. The
product was dissolved in methanol (0.5 mL), and the resulting
solution was treated with a saturated methanolic solution of
hydrochloric acid (0.5 mL) followed by diethyl ether (2 mL). The
solution was stored in the refrigerator for 16 h. The resulting
precipitate was filtered and washed with diethyl ether to afford the
dihydrochloride salt (compounds 17a-j) in a yield range of
58-79% as a white solid.
(ESI,
[M
+
H]+),
337.2060.
Anal.
Calcd
for
C19H29ClN2O·2.00HCl ·0.3H2O: C, 54.96; H, 7.67; N, 6.75. Found:
C, 55.26; H, 7.37; N, 6.68.
1-[1-(3-Chlorophenyl)-2-(4-methylpiperazin-1-yl)ethyl]cyclo-
1
hexanol Dihydrocholoride 17g. H NMR for free base (DMSO-
d6, 400 MHz) δ 0.96-1.07 (m, 1H), 1.08-1.14 (m, 2H), 1.31-1.59
(m, 7H), 2.10 (s, 3H), 2.22 (bs, 4H), 2.38 (bs, 4H), 2.47 (dd, J )
6.8, 12.5 Hz, 1H), 2.90 (t, J ) 7.0 Hz, 1 H), 3.04 (ddd, J ) 7.5,
9.5, 12.6 Hz, 1 H), 5.12 (s, 1H), 7.18 (dd, J ) 1.5, 7.2 Hz, 1H),
7.21-7.28 (m, 3H). HRMS: calcd for C19H29ClN2O + H+,
337.2041; found (ESI, [M
(C19H29ClN2O·2.00HCl) C, H, N.
+
H]+), 337.2036. Anal.
1-[(1R)-1-(3-Chlorophenyl)-2-(4-methylpiperazin-1-yl)ethyl-
]cyclohexanol Dihydrocholoride (R)-(+)-17g. The enantiomeric
purity was determined to be >99.9% (with <0.1% of the undesired
enantiomer): retention time, 2.49 min; [R]2D5 +29° (c 9.9 mg/mL,
MeOH). HRMS: calcd for C19H29ClN2O + H+, 337.204 12; found
(ESI, [M + H]+), 337.2032. Anal. (C19H29ClN2O·2.00HCl) C, H,
N.
1-[(1S)-1-(3-Chlorophenyl)-2-(4-methylpiperazin-1-yl)ethyl-
]cyclohexanol Dihydrocholoride (S)-(-)-17g. The enantiomeric
purity was determined to be >99.9% (with <0.1% of the undesired
enantiomer): retention time, 3.58 min; [R]2D5 -21.4° (c 10 mg/mL,
MeOH). HRMS: calcd for C19H29ClN2O + H+, 337.2041; found
1-[1-(4-Methoxyphenyl)-2-(4-methyl-1-piperazinyl)ethyl]cy-
(ESI,
[M
+
H]+),
337.2032.
Anal.
Calcd
for
1
clohexanol Dihydrochloride 17a. H NMR (D2O, 400 MHz) δ
C19H29ClN2O·2.00HCl ·0.2H2O: C, 55.20; H, 7.66; N, 6.78. Found:
C, 55.24; H, 7.85; N, 6.67.
0.99-1.08 (m, 1H), 1.19 (d, J ) 12.9 Hz, 1H), 1.20-1.50 (m,
7H), 1.65 (d, J ) 13.1 Hz, 1H), 2.88 (s, 3H), 3.04 (dd, J ) 3.3,
11.0 Hz, 1H), 3.47 (bs, 8H), 3.70 (dd, J ) 3.4, 12.9 Hz, 1H),
3.75-3.81 (m, 1H), 3.78 (s, 3H), 6.97 (d, J ) 8.7 Hz, 2H), 7.29
(d, J ) 8.3 Hz, 2H). Anal. (C20H32N2O2 ·2.00HCl) C, H, N.
1-{2-(4-Methylpiperazin-1-yl)-1-[4-(trifluoromethoxy)phenyl]-
1
ethyl}cyclohexanol Dihydrocholoride 17h. H NMR (D2O, 400
MHz) δ 1.00-1.09 (m, 1H), 1.20-1.50 (m, 7H), 1.68 (d, J ) 12.9
Hz, 1H), 2.89 (s, 3H), 3.12 (dd, J ) 2.8, 10.9 Hz, 1H), 3.47 (bs,
8H), 3.72 (d, J ) 13.2 Hz, 1H), 3.80 (t, J ) 11.1 Hz, 1H), 7.30 (d,
J ) 8.1 Hz, 2H), 7.42 (d, J ) 8.3 Hz, 2H). HRMS: calcd for
C20H29F3N2O2 + H+, 387.2254; found (ESI, [M + H]+), 387.2245.
1-{2-(3-Methylpiperazin-1-yl)-1-[3-(trifluoromethoxy)phenyl]-
1-[1-(3-Methoxyphenyl)-2-(4-methyl-1-piperazinyl)ethyl]cy-
1
clohexanol Dihydrochloride 17b. H NMR (D2O, 400 MHz) δ
1.01-1.10 (m, 1H), 1.20-1.22 (m, 1H), 1.25-1.53 (m, 7H), 1.69
(d, J ) 12.5 Hz, 1H), 2.90 (s, 3H), 3.08 (dd, J ) 2.8, 11.5 Hz,
1H), 3.30-3.52 (m, 8H), 3.74 (dd, J ) 2.9, 12.1 Hz, 1H), 3.77-3.83
(m, 1H), 3.79 (s, 3H), 6.95-7.01 (m, 3H), 7.36 (t, J ) 8.1 Hz,
1H). Anal. (C20H32N2O2 ·2.00HCl) C, H, N.
1
ethyl}cyclohexanol Dihydrocholoride 17i. H NMR (D2O, 400
MHz) δ 0.96-0.98 (m, 1H), 1.09-1.43 (m, 8H), 1.62 (d, J ) 12.6
Hz, 1H), 2.81 (s, 3H), 3.06 (dd, J ) 3.2, 10.9 Hz, 1H), 3.38 (bs,
8H), 3.64 (dd, J ) 3.2, 13.5 Hz, 1H), 3.74 (dd, J ) 11.0, 13.4 Hz,
2H), 7.21-7.28 (m, 3H), 7.38 (t, J ) 8.5 Hz, 1H). HRMS: calcd
for C20H29F3N2O2 + H+, 387.2254; found (ESI, [M + H]+),
387.2263.
1-{(1R)-2-(4-Methylpiperazin-1-yl)-1-[3-(trifluoromethoxy)phenyl]-
ethyl}cyclohexanol Dihydrocholoride (R)-(+)-17i. The enantio-
meric purity was determined to be >99.8% (with 0.2% of the
undesired enantiomer): retention time, 9.52 min; [R]2D5 +20.6° (c
10.8 mg/mL, MeOH). HRMS: calcd for C20H29F3N2O2 + H+,
1-[1-(4-Fluorophenyl)-2-(4-methylpiperazin-1-yl)ethyl]cyclo-
hexanol Dihydrocholoride 17c. 1H NMR (D2O, 400 MHz) δ
1.09-1.19 (m, 1H), 1.24 (d, J ) 13.2 Hz, 1H), 1.28-1.56 (m,
7H), 1.73 (d, J ) 12.5 Hz, 1H), 2.87 (s, 3H), 3.10 (dd, J ) 3.6,
10.9 Hz, 1H), 3.22-3.50 (m, 8H), 3.58 (dd, J ) 3.5, 13.3 Hz, 1H),
3.65 (t, J ) 13.2 Hz, 1H), 7.16 (t, J ) 8.9 Hz, 2H), 7.39 (dd, J )
5.6, 8.5 Hz, 2H). HRMS: calcd for C19H29FN2O + H+, 321.2337;
found (ESI, [M + H]+), 321.2332. Anal. (C19H29FN2O·2.00HCl)
C, H, N.

SAR of the Cycloalkanol Ethylamine Scaffold
H]+), 387.2269. Anal.
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 4047
387.2254; found (ESI, [M
+
[7,8-3H]norepinephrine, ∼30 Ci/mmol from Amersham Bioscienc-
es) was diluted in assay buffer (50 nM final assay concentration)
and delivered in 10 µL aliquots to each well and the plates were
incubated for 10 min at 37 °C. Medium was then removed from
wells, and cells were washed twice with 200 µL assay buffer to
remove unincorporated [3H]NE label. The wells containing cells
were then incubated for 1 h in 80 µL of Microscint20 scintillation
fluid (PerkinElmer Life and Analytical Sciences, Boston, MA), and
the [3H]NE accumulation was quantified using a Topcount scintil-
lation counter (PerkinElmer). By use of the above detailed assay
conditions, the typical Km value for [3H]NE was 534 ( 92 nM and
the Vmax value was 5.5 ( 0.9 pmol/well. The total and nonspecific
cpms obtained for these assays are 9415 ( 191 and 213 ( 7.1,
respectively. Each compound was tested in two separate experi-
ments at a minimum and up to four times in some cases.
(C20H29F3N2O2 ·2.00HCl) C, H, N.
1-{(1S)-2-(4-Methylpiperazin-1-yl)-1-[3-(trifluoromethoxy)phenyl]-
ethyl}cyclohexanol Dihydrocholoride (S)-(-)-17i. The enantio-
meric purity was determined to be >99.9% (with <0.1% of the
undesired enantiomer): retention time, 7.85 min; [R]2D5 -25.1° (c
10 mg/mL, MeOH). HRMS: calcd for C20H29F3N2O2 + H+,
387.2254; found (ESI, [M
(C20H29F3N2O2 ·2.00HCl) C, H, N.
1-{2-(3-Methylpiperazin-1-yl)-1-[3-(trifluoromethyl)phenyl]ethyl}-
cyclohexanol Dihydrocholoride 17j. H NMR (D2O, 400 MHz)
+
H]+), 387.2249. Anal.
1
δ 0.93-0.99 (m, 1H), 1.05 (d, J ) 12.6 Hz, 1H), 1.13 (dd, J )
4.6, 11.1 Hz, 1H), 1.17-1.37 (m, 6H), 1.62 (d, J ) 12.9 Hz, 1H),
2.79 (s, 3H), 3.09 (dd, J ) 3.1, 10.9 Hz, 1H), 3.38 (bs, 8H), 3.66
(d, J ) 13.5 Hz, 1H), 3.78 (t, J ) 13.3 Hz, 1H), 7.45 (t, J ) 7.7
Hz, 1H), 7.50 (d, J ) 7.6 Hz, 2H), 7.57-7.59 (m, 2H). HRMS:
calcd for C20H29F3N2O + H+, 371.2305; found (ESI, [M + H]+),
371.2290. Anal. Calcd for C20H29F3N2O·2.00HCl·0.3H2O: C,
53.53; H, 7.10; N, 6.24. Found: C, 53.60; H, 7.22; N, 6.21.
General Procedure for Whole Cell Radioligand Binding
Assay in Cells Expressing Human Norepinephrine Transporter
(hNET). Twenty-four hours prior to assay, MDCK-Net6 cells stably
transfected with human NET21 were plated in 96-well plates at
3000-5000 cells/well in growth medium and maintained in a cell
incubator (37 °C, 5% CO2). On day 2, growth medium was replaced
with 80 µL of assay buffer (25 mM HEPES, 120 mM NaCl, 5 mM
KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 2 mg/mL glucose (pH 7.4,
37 °C)) containing 0.2 mg/mL ascorbic acid and 1 µM pargyline.
Ten microliter aliquots of test compound in assay buffer were added
directly to triplicate wells to yield final test concentrations of
10-10000 nM. Data from wells containing desipramine (1 µM)
were used to define nonspecific hNET binding (minimum hNET
binding in the presence of an NRI). Total radioligand bound was
defined by addition of 5 µL of binding buffer alone in the presence
of [3H]nisoxetine. The radioligand binding reaction was initiated
by addition of [3H]nisoxetine in 25 µL of assay buffer to each well
for a final concentration of 3 nM. The total and nonspecific cpm
values obtained for this assay were 2849 ( 69 and 188 ( 3.5,
respectively. The KD value estimated for [3H]nisoxetine was 10
nM using intact whole cells. Test compounds and cells were
preincubated at 37 °C for 15 min prior to initiating the binding
reaction by addition of radioligand. The cells in the assay buffer
with test compound and radioligand were incubated for 2 h at 37
°C. The cells were washed twice with 200 µL of assay buffer at
room temperature to remove unbound radioligand. After binding,
cells were incubated for 1 h in Microscint20 scintillation fluid
(PerkinElmer Life and Analytical Sciences, Boston, MA) and
radiolabeled ligand binding quantified using a TopCount plate
scintillation counter (PerkinElmer). Each compound was tested in
two separate experiments at a minimum and up to four times in
some cases.
General Procedure for Norepinephrine (NE) Uptake Assay
in Cells Expressing Human Norepinephrine Transporter
(hNET). On day 1, MDCK-Net6 cells, stably transfected with
human NET,22 were plated at 3000 cells/well in a 96-well plate in
growth medium and maintained in a cell incubator (37 °C, 5% CO2).
On day 2, growth medium was replaced with 80 µL of assay buffer
(25 mM HEPES, 120 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 1.2
mM MgSO4, 2 mg/mL glucose [pH 7.4, 37 °C]) containing 0.2
mg/ml ascorbic acid and 10 µM pargyline. Cells were equilibrated
in 90 µL of assay buffer for 10 min at 37 °C prior to addition of
compounds. A stock solution of desipramine was prepared in
DMSO (10 mM) and delivered to triplicate wells containing cells
for a final test concentration of 200 nM. Data from these wells
were used to define nonspecific NE uptake (minimum NE uptake).
Test compound stock solutions were prepared in DMSO/water (1:
1) (10 mM) and diluted in assay buffer according to test range
(1-10000 nM). A 10 µL aliquot of vehicle or various concentrations
of antagonist was added directly to triplicate wells containing cells
in 80 µL of assay buffer. The cells were preincubated with test
compound for 15 min at 37 °C. To initiate uptake, [3H]NE (1-
General Procedure for Serotonin (hSERT) Uptake Assay
in Cells Expressing Human Serotonin Transporter. On day 1,
JAR cells (human placental choriocarcinoma), purchased from
ATCC (catalog no. HTB-144), were plated at 15 000 cells/well in
96-well plates containing growth medium (RPMI 1640 with 10%
FBS) and maintained in a cell incubator (37 °C, 5% CO2). On day
2, cells were stimulated with staurosporine (40 nM) to increase
the expression of the 5-HT transporter.23 On day 3, cells were
removed from the cell incubator 2 h prior to assay and maintained
at room temperature to equilibrate the growth medium to ambient
oxygen concentration. Subsequently, the growth medium was
replaced with 200 µL of assay buffer (25 mM HEPES, 120 mM
NaCl, 5 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 2 mg/mL
glucose (pH 7.4, 37 °C)) containing 0.2 mg/mL ascorbic acid and
1 µM pargyline, and the cells were incubated for 5 min at 37 °C.
A stock solution of paroxetine (AHR-4389-1) was prepared in
DMSO (10 mM) and delivered to triplicate wells containing cells
for a final test concentration of 1 µM. Data from these wells were
used to define nonspecific 5-HT uptake (minimum 5-HT uptake in
the presence of a SRI). Test compounds were prepared in DMSO/
water (1:1) (10 mM) and diluted in assay buffer according to test
range (1-1000 nM). An amount of 25 µL of assay buffer
(maximum 5-HT uptake) or test compound was added directly to
triplicate wells containing cells in 200 µL of assay buffer. The cells
were incubated with the compound for 10 min (37 °C). To initiate
the reaction, [3H]hydroxytryptamine creatinine sulfate diluted in
assay buffer was delivered in 25 µL aliquots to each well for a
final test concentration of 15 nM. The cells were incubated with
the reaction mixture for 9 min at 37 °C. Decanting the supernatant
from the plates terminated the reaction. The cells were washed twice
with 200 µL of assay buffer (37 °C) to remove free radioligand.
The plates were inverted and left to dry for 2 min, then reinverted
and air-dried for an additional 10 min. Subsequently, the cells were
lysed in 25 µL of 0.25 N NaOH (4 °C) and then placed on a shaker
table and shaken vigorously for 5 min. After cell lysis, 75 µL of
scintillation cocktail was added to the wells and the plates were
sealed with film tape and replaced on the shake table for a minimum
of 10 min. The plates were counted in a Wallac Microbeta counter
(PerkinElmer) to collect the raw cpm data. By use of the above
detailed assay conditions, the typical Km value for [3H]hydrox-
ytryptamine creatinine sulfate was 431 ( 86.2 nM and the Vmax
value was 11.5 ( 1.2 pmol/well. The total and nonspecific cpm
values obtained for this assay are 4282 ( 206 and 79 ( 5.5,
respectively. Each compound was tested in two separate experi-
ments at a minimum and up to four times in some cases.
General Procedure for the Dopamine Transporter (hDAT)
Membrane Binding Assay. Frozen membrane samples from CHO
cells expressing recombinant hDAT, purchased from Perkin-Elmer,
Boston, MA (catalog no. RBHDATM, lot no. 2227), were diluted
to 7.5 mL in binding buffer (50 mM Tris-HCl, pH 7.4, 100 mM
NaCl), homogenized with a tissue-tearer (Polytron PT 1200C,
Kinematica AG), and delivered at a volume of 75 µL to each well
of a polypropylene 96-well plate. The binding reaction was carried
out in polypropylene 96-well plates (Costar General Assay Plate,
catalog no. 3359; Lid, catalog no. 3930). Homogenized membrane
preparation was delivered at a volume of 75 µL to each well of a

4048 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
Mahaney et al.
reaction plate. A stock solution of mazindol was prepared in DMSO
(10 mM) and delivered to triplicate wells containing membranes
for a final test concentration of 10 µM. Mazindol has been reported
to be a dopamine transporter inhibitor (DRI)24 with an IC50 value
of 20.5 nM in our assays. Data from wells containing mazindol
(10 µM) were used to define nonspecific (NSB) hDAT binding
(minimum hDAT binding in the presence of a DRI). Total
radioligand bound was defined by addition of 5 µL of binding buffer
alone in the presence of radioligand 18. Stock solutions of test
compounds were prepared in DMSO/water (1:1) at a concentration
of 10 mM. On day of assay, the test compound stock solution was
diluted in assay buffer according to test range (3000-100000 nM)
ensuring a maximal DMSO concentration of less than 0.5% in the
assay reaction wells. Homogenized membranes were preincubated
with test compound for 20 min at 4 °C before the initiation of the
binding reaction. The binding reaction was initiated by addition of
25 µL of radioligand 18 diluted in binding buffer. The final
concentration of radioligand 18 delivered was 32 nM. The KD value
estimated for radioligand 18 (lot no. 2227) in hDAT membranes
was 29.7 nM. The plates containing the radioligand binding
reactions were incubated for 2 h at 4 °C on a shaking table (Bellco,
Vineland, NJ) at 3 rpm. The MultiScreen-FB opaque 96-well
filtration plates containing Millipore glass fiber filters (Millipore
glass fiber B, catalog no. MAFBN0B) were used to terminate the
binding reactions and to separate bound from free radioligand. The
plates were presoaked with 0.5% polyethylenimine (PEI, Sigma
catalog no. P-3143) in water for a minimum of 2 h at room
temperature to reduce nonspecific binding of radioligand 18 during
the harvest procedure. Prior to harvesting of the reaction plates,
the PEI solution was aspirated from the filter plates using a vacuum
manifold. Aliquots of each mixture (90 µL of each 100 µL reaction
well) were transferred from the reaction plates to the filter plates
using a Zymark Rapid Plate-96 automated pipet station. The binding
reaction was terminated by vacuum filtration through the glass fiber
filters. The filter plates were aspirated at 5-10 in. Hg, and the wells
are washed 9 times with 200 µL of wash buffer (50 mM Tris-HCl,
0.9% NaCl, pH 7.4, 4 °C) using a 12-channel aspiration/wash
system. Plastic bottom supports were removed from the filter plates,
and the plates were placed in plastic liners. A 100 µL aliquot of
scintillation fluid was added to each well, and the top of each plate
was sealed with adhesive film. The plates were vigorously shaken
on an orbital shake table (Bellco) at 5 rpm for 10-15 min to ensure
adequate equilibration of aqueous to solvent partitioning. The
collection of raw cpm data was done using a Wallac Microbeta
counter (Perkin-Elmer). The total and nonspecific cpm values
obtained for this assay were 1976.3 ( 52.8 and 95.7 ( 3.2,
respectively.
Procedure for the Tail Suspension Test in Mice. Male Swiss
Webster mice (Charles River Laboratories, MA) weighing 20-35
g (2-3 months of age) were used for the study. Experimental groups
consisted of 10 mice, randomly assigned to drug or vehicle (2%
Tween-80/0.5% methylcellulose) treatment. The procedure followed
in this study was a variant to the original report by Steru et al.13a
Thirty minutes following administration of (S)-(-)-17i (10, 17, and
30 mg/kg, ip) the mice were suspended upside down by the tail
using adhesive laboratory tape (VWR International, PA) to a flat
metal bar connected to a strain gauge within a tail suspension
chamber (Med Associates, VT). The time spent immobile during a
6 min test session was automatically recorded. Eight mice were
simultaneously tested in separate chambers. Data collected were
expressed as a mean of immobility time, and statistical analysis
was performed using a one-way ANOVA with least significant
difference (LSD) post hoc test. Data represent group mean values
( SEM for each dose (N ) 10 per group).
Procedure for the p-Phenylquinone (PPQ) Model of Acute
Visceral Pain. Male CD-1 mice (Charles River; Kingston/Ston-
eridge, NY) weighing 20-25 g were maintained on a 12 h light/
dark cycle (lights on at 0630) in groups of five on corncob bedding,
and food and water were available ad libitum. Experimental groups
consisted of eight mice randomly assigned to drug or vehicle (saline)
treatment. The ability of compounds to attenuate acute visceral
(abdominal) pain was assessed following an ip injection of 2 mg/
kg PPQ (dissolved in 4-6% ethanol/distilled water). Following PPQ
injection, mice were individually placed in a Plexiglas cage and
the number of stretching movements was recorded during two 1
min periods, 5 and 10 min after injection. Compound (S)-(-)-17i
(3, 10, and 30 mg/kg, sc) and desipramine (3, 10 and 30 mg/kg,
sc) were administered 60 min prior to PPQ injection. Statistical
analysis was done using a one-way ANOVA on raw data (total
number of stretches). Significant main effects were analyzed further
by subsequent least significant difference analysis. The criterion
for significant differences was p < 0.05. Data are presented as
percent blockade compared to vehicle (% blockade ) {[(mean
vehicle) - (drug)]/(mean vehicle)} × 100).
Procedure for the OVX-Induced Thermoregulatory
Dysfunction Telemetry Rat Model. Tail-skin temperature (TST)
was measured by a temperature and physical activity transmitter
(PhysioTel TA10TA-F40, Data Sciences International) that was
implanted subcutaneously in the dorsal scapular region of ovariec-
tomized female SD rats. The tip of the temperature probe was
tunneled subcutaneously 2.5 cm beyond the base of the tail. After
a 7-day recovery period, rats were administered vehicle (2% Tween-
80/0.5% methylcellulose), and TST was monitored continuously
for 12 h (during the dark cycle). Twenty-four hours later rats were
administered either vehicle or test compound and TST was
monitored continuously for 12 h (during the dark cycle). All vehicle
and test compounds were administered 1 h prior to the onset of the
dark cycle. Test compound alterations in TST were analyzed using
a 2 day paradigm. For both the vehicle (baseline) and compound
testing, TST was recorded at 5 min intervals and an average TST
was calculated for every 30 min time point. On day 1, an overall
average baseline TST was established for each individual animal
by taking the mean over the 12 h observation period. On day 2,
test compound was administered and TST readings were reported
as an average over every 30 min as described above. All data were
analyzed as ∆TST (TST for each time point on day 2 minus average
baseline TST for day 1). A one-way ANOVA was performed to
obtain the average within-group standard deviation. For each 30
min interval, a t test was performed and evaluated to determine if
the average ∆TST was statistically different (p g 0.05) from
baseline.
Acknowledgment. The authors thank members of the
Discovery Analytical Chemistry group in Chemical Technolo-
gies at Wyeth, particularly Scott Brecker, Christopher Petucci,
Rebecca Dooley, Diane Andraka, and Oliver McConnell for
compound analysis. Their efforts and dedication are greatly
appreciated. We also thank our management, Drs. M. Abou-
Gharbia, R. Magolda, L. Freedman, and M. Pangalos for their
support of this work.
Supporting Information Available: Crystallographic informa-
tion in cif format, elemental analysis and purity results for all
compounds tested in biological assays, raw data from rat telemetry
studies, and data and procedures for pharmacokinetic studies. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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