Synthesis and SAR of 4-aminocyclopentapyrrolidines as N-type Ca 2+ channel blockers with analgesic activity

Article abstract of DOI:10.1016/j.bmc.2012.04.057

A novel 4-aminocyclopentapyrrolidine series of N-type Ca²⁺ channel blockers have been discovered. Enantioselective synthesis of the 4-aminocyclopentapyrrolidines was enabled using N-tert-butyl sulfinamide chemistry. SAR studies demonstrate selectivity over L-type Ca²⁺ channels. N-type Ca²⁺ channel blockade was confirmed using electrophysiological recording techniques. Compound 25 is an N-type Ca ²⁺ channel blocker that produces antinociception in inflammatory and nociceptive pain models without exhibiting cardiovascular or motor liabilities.

Full text of DOI:10.1016/j.bmc.2012.04.057

Bioorganic & Medicinal Chemistry 20 (2012) 4128–4139
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and SAR of 4-aminocyclopentapyrrolidines as N-type Ca2+ channel
blockers with analgesic activity
⇑
Xenia Beebe , Daria Darczak, Rodger F. Henry, Timothy Vortherms, Richard Janis, Marian Namovic,
Diana Donnelly-Roberts, Karen L. Kage, Carol Surowy, Ivan Milicic, Wende Niforatos, Andrew Swensen,
Kennan C. Marsh, Jill M. Wetter, Pamela Franklin, Scott Baker, Chengmin Zhong, Gricelda Simler,
Erica Gomez, Janel M. Boyce-Rustay, Chang Z. Zhu, Andrew O. Stewart, Michael F. Jarvis, Victoria E. Scott
Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064-6100, United States
a r t i c l e i n f o
a b s t r a c t
A novel 4-aminocyclopentapyrrolidine series of N-type Ca2+ channel blockers have been discovered.
Enantioselective synthesis of the 4-aminocyclopentapyrrolidines was enabled using N-tert-butyl sulfin-
amide chemistry. SAR studies demonstrate selectivity over L-type Ca2+ channels. N-type Ca2+ channel
blockade was confirmed using electrophysiological recording techniques. Compound 25 is an N-type
Ca2+ channel blocker that produces antinociception in inflammatory and nociceptive pain models with-
out exhibiting cardiovascular or motor liabilities.
Article history:
Received 14 March 2012
Revised 20 April 2012
Accepted 27 April 2012
Available online 4 May 2012
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
N-type calcium channel
L-type calcium channel
Analgesic activity
SAR
N-tert-Butyl sulfonamide
Enantioselective synthesis
1. Introduction
not cause tolerance, but it exhibits CNS side-effects such as seda-
tion and hypotension.7,8 Small molecule N-type Ca2+ channel
Voltage gated Ca2+ channels (VGCC) are involved in numerous
neuronal functions including regulation of membrane ion conduc-
tance, neurotransmitter release, and cellular excitability. VGCCs
control Ca2+ ion flow in and out of the cell in response to changes
in membrane potential. The VGCC family includes both low voltage
activated T-type (Cav3.1-3.3) Ca2+ channels and high voltage acti-
vated L-type (Cav1.1-1.4), N-type (Cav2.2), P/Q-type (Cav2.1) and
R-type (Cav2.3) Ca2+ channels.1,2 Ten distinct mammalian genes
encode the pore forming a1 subunit of the Ca2+ channel.3 L-type
Ca2+ channels are widely distributed in skeletal, cardiac, and
smooth muscle tissue and are responsible for attenuating cardio-
vascular responses.4 The N-type and P/Q-type Ca2+ channels are lo-
cated at presynaptic nerve terminals. N-type Ca2+ channels are
responsible for the release of the neurotransmitters glutamate,
substance P, and calcitonin gene-related peptide (CGRP) involved
inhibitors that maintain analgesic efficacy with an improved ther-
apeutic window compared to Prialt™ are an attractive pursuit for
the treatment of chronic pain.
Research in the area of small molecule N-type Ca2+ channels
inhibitors has been progressing in the last few years. Selectivity of
N-type over L-type Ca2+ channels is key to discovering compounds
with reduced cardiovascular liabilities.9–18 Additionally, identifica-
tion of compounds that block the channel in a state-dependent
manner (i.e., bind to the inactivated state of the channel more
potently than the resting/closed state) may also enhance the thera-
peutic margins relative to Prialt™17 which lacks state-dependence.
Herein we describe the discovery of a novel series of small molecule
N-type Ca2+ blockers identified from screening the Abbott com-
pound collection. The compounds were designed for N-type Ca2+
channel inhibition with selectivity over L-type Ca2+ channels with
analgesic activity at doses that do not cause significant cardiovascu-
lar or motor coordination side-effects in animal models.
in pain signaling.5,6 The peptide
x-conotoxin GVIA is a potent
and selective blocker of N-type Ca2+ channels and its derivative
ziconotide (Prialt™) is used for the treatment of severe, chronic
pain following intrathecal administration.7 Ziconotide is effective
in patients that do not respond to other pain therapies and does
2. Chemistry
The synthesis of the 4-aminocyclopentapyrrolidines is outlined
in Scheme 1. The core ketone 4 was constructed using a 1,3-dipolar
cycloaddition via an azomethine ylid precursor 3. The desired
⇑
Corresponding author. Tel.: +1 847 937 8291; fax: +1 847 937 9195.
E-mail address: xenia.b.searle@abbott.com (X. Beebe).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.04.057

X. Beebe et al. / Bioorg. Med. Chem. 20 (2012) 4128–4139
4129
O
O
CF3
H
O
H
a
c
b
NH2
N
Si
N
Si
F3C
F3C
F3C
N
4
2
H
1
3
O
HO
O
NH2
R
N
R
CF3
NH
H
e
CF3
d
H
f
OH
CF3
H
N
H
N
H
N
H
6
5
7 R = Et2CH
8 R = Pr2CH
12 R = cycloheptyl
13 R = Ph2CH
9 R = cyclohexyl2CH
10 R = cyclopentyl
11 R = cyclohexyl
14 R = (p-FPh)2CH
15 R = (iPr)PhCH
Scheme 1. Reagents and conditions: (a) ClCH2Si(CH3)3, Et3N, 90 °C 16 h; (b) H2CO, MeOH, K2CO3, 0 °C, 3 h; (c) TFA, CH2Cl2, rt, 45 min; (d) H2NOH-HCl, NaOAc, H2O/EtOH,
70 °C, 1 h; (e) H2, RaÒ-Ni, 20% NH3/MeOH, rt, 16 h; (f) EDC, HOBt, CH2Cl2, rt, 16 h.
m-trifluoromethylbenzyl group was installed at the onset by
combining m-trifluoromethylbenzylamine 1 with chloromethyltri-
methylsilane to give amine 2 followed by reaction with formalde-
hyde and methanol to provide the azomethine ylid precursor 3.19
Acid catalyzed azomethine ylid cyclization with cyclopenten-2-
one proceeded smoothly to give the desired ketone 4 with syn ste-
reochemistry at the ring juncture.20,21 Subsequent condensation
with hydroxylamine gave a mixture of E,Z isomers of the oxime
5. Reduction of the oxime 5 with RaneyÒ-nickel catalyzed hydroge-
nation provided the amine 6 with all cis stereochemistry as the
major product. Coupling of the amine with the appropriate acid
provided amides 7–15.
Enantiomeric resolution was performed as shown in Scheme 2 by
condensing the ketone 16 with (R)-tert-butanesulfinamide 17 using
titanium tetraethoxide.22,23 The resulting diastereomers were sepa-
rated using silica gel chromatography with the R,R,S-diastereomer
18 eluting first and the R,S,R-diasteremer 19 eluting second.
Figure 1. Ortep drawing of the X-ray crystal structure of sulfinamide 20.
Reduction of the R,R,S-diastereomer sulfinamide 18 with
sodium borohydride at low temperature gave the all cis stereo-
chemistry of the sulfinamide 20 as shown in Scheme 3. The
absolute stereochemistry of the reduction product was determined
by single crystal X-ray crystallography on compound 20 as shown
in Figure 1.
The stereochemistry of the hydride addition in this case is con-
trolled by the cup-shaped bicyclic structure of the core and not by
the chirality of the sulfinamide used. Other reducing agents such as
L-selectride were used in an attempt to control the stereochemistry
with the chiral auxiliary but only the all cis stereochemical product
was obtained. When the opposite enantiomer (S)-tert-butanesulfi-
namide was used, the stereochemical outcome of the reduction
was the same. Deprotection of the sulfinamide with acid gave
the desired enantiomerically pure primary amine 21.
Synthesis of a single enantiomer of compound 25 and diversify-
ing the N-benzyl group was accomplished as shown in Scheme 4.
The enantiomerically pure amine was coupled with 2,2-dic-
yclohexylacetic acid 22 to give 2,2-dicyclohexylacetamide 23. Sub-
sequent debenzylation using Degussa catalyst provided pyrrolidine
24. Reductive alkylation with the appropriate aldehyde using poly-
mer-supported cyanoborohydride provided enantiomerically pure
benzyl substituted compounds 25–31.
The enantiomer of compound 25 was synthesized as shown in
Scheme 5 and Scheme 6 starting with (S)-tert-butanesulfinamide
provides the condensed products with the reversed order of
elution; the S,R,S-diastereomer 33 eluting first followed by the
S,S,R-diastereomer 34.
The desired isomer 33 was then reduced with sodium borohy-
dride at low temperature in methanol to give 35 and the
sulfinamide group deprotected to give the amine 36 with all cis
O
S
O
S
O
N
N
H
O
S
a
H
H
N
NH2
H
N
N
H
H
16
17
18
19
Scheme 2. Reagents and conditions: Ti(OEt)4, THF, 60 °C, 16 h.
O
S
O
S
NH
H
NH2
H
N
a
b
H
N
N
H
H
N
H
18
20
stereochemistry determined by X-ray
21
Scheme 3. Reagents and conditions: (a) NaBH4, MeOH, ꢀ78 °C to rt, 16 h; (b) 2N
HCl(aq), MeOH, rt, 30 min.

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X. Beebe et al. / Bioorg. Med. Chem. 20 (2012) 4128–4139
R
O
O
O
O
O
NH2
H
a
c
H
b
OH
NH
NH
NH
H
H
H
R
N
H
N
N
NH
25 R = m-CF3
26 R = o-F
27 R = m-F
28 R = p-F
H
H
H
21
22
23
24
29 R = o-CF3
30 R = m-OCF3
31 R = m-CH3
Scheme 4. Reagents and conditions: (a) EDC, HOBt, CH2Cl2, rt, 16 h; (b) 20% Pd(OH)2/carbon, MeOH, 30 psi H2, rt, 16; (c) PS-CNBH3, HOAc, CH2Cl2, rt, 16 h.
O
S
O
S
Table 1
O
Ca2+ channel activity of racemic n-benzyl amide analogues
N
N
CF3
H
a
O
CF3
CF3
H
H
O
S
N
NH2
H
N
33
N
34
R
H
H
NH
4
32
H
Scheme 5. Reagents and conditions: Ti(OEt)4, THF, 60 °C, 16 h.
N
H
stereochemistry. Coupling the amine with 2,2-dicyclohexylacetic
acid gave the desired enantiomer 37.
relative stereochemistry (racemic)
Compound R
N-type Ca2+ channels L-type Ca2+ channels
3. Results and discussion
IC50
,
l
M pIC50 SEMa IC50
,
l
M pIC50 SEMa
The N-type Ca2+ channel activity was determined using IMR32
human neuroblastoma cells that endogenously express the chan-
nels, in a functional fluorescent Ca2+ flux assay (FLIPR).24 All com-
pounds were also assessed for L-type Ca2+ channel activity using a
similar FLIPR-based Ca2+ flux assay in ND7/23 cells.
Initially, racemic compounds of the structure type shown in
Table 1 were screened in the N-type and L-type Ca2+ channel assay.
This series of compounds blocked N-type Ca2+ channels with
38
39
40
41
42
43
(Cyclohexyl)2CH
(iPr)PhCH
4.67
1.46
5.33 0.04 >30
5.83 0.23 7.21
6.03 0.05 5.15
5.97 0.17 >30
5.71 0.05 4.84
5.99 0.05 5.56
5.14 0.08
5.29 0.03
(Cyclopentyl) PhCH 0.92
(Cyclohexyl)PhCH 1.06
1-Phenylcyclopentyl 1.93
1-Phenylcyclohexyl 1.04
5.32 0.10
5.25 0.03
pound 10 with an IC50 value of 2.31
11 with an IC50 value of 1.26 M and cycloheptyl compound 12
with an IC50 value of 0.56 M. The cyclic and branched acetamides
were equipotent: the bis-propyl acetamide 8 with an IC50 value of
lM, cyclohexyl compound
potencies ranging from 1–3 lM and were generally less potent at
L-type Ca2+ channels. The first modification made was to replace
the N-benzyl substituent with a 3-trifluoromethylbenzyl substitu-
ent shown in Table 2. This single modification gave rise to
compounds that were approximately fivefold more potent at N-
type Ca2+ channels. Examples include comparing compound 38
l
l
0.95
0.56
l
l
M and cycloheptyl acetamide 12 with an IC50 value of
M as well as the bis-ethyl 7 with an IC50 value of 2.07
M.
l
M
and cyclopentyl 10 with an IC50 value of 2.31
l
with an IC50 value of 4.67
of 1.07 M and compound 39 with an IC50 value of 1.46
compound 15 with an IC50 value of 0.36 M.
l
M to compound 9 with an IC50 value
Upon resolving the chirality of the bis-cyclohexyl acetamide
analog, we found one enantiomer 25, the S,S,R-4-aminocyclopenta-
pyrrolidine to be 4-fold more potent than the R,R,S-4-aminocyclo-
pentapyrrolidine 37 for blocking N-type Ca2+ channels ( Fig. 2).
Further SAR studies were then explored at the N-benzyl substituent
for this single enantiomer as shown in Table 3. The meta- and para-
substituted aryls were submicromolar in potency. The most potent
and selective compound was found to be compound 25.
l
lM to
l
The amide substituent was also varied to accommodate achiral
acids in order to decrease the number of stereocenters in the final
product. Not surprisingly, submicromolar potencies were obtained
for compounds with more lipophilic amide substituents like bis-
cyclohexyl amide 9 with an IC50 value of 1.07
lM, diphenyl acet-
amide 13 with an IC50 value of 0.67 M, and the isopropyl phenyl
l
The activity of compound 25 was also assessed in a rat aorta
ring tissue relaxation assay that measures L-type Ca2+ channel
activity in native vascular tissue.17 Compound 25 had an EC50 value
acetamide 15 with an IC50 value of 0.36 lM. In Table 2 the potency
to block N-type Ca2+ channels increases as lipophilicity increases
for smaller substituents. Examples include the cyclopentyl com-
O
S
O
S
O
O
NH2
H
CF3
a
NH
N
NH
H
b
c
OH
CF3
CF3
CF3
H
H
N
H
N
N
N
H
H
H
33
35
36
22
37
Scheme 6. Reagents and conditions: (a) NaBH4, MeOH, ꢀ78 °C to rt, 16 h; (b) 2N HCl(aq), MeOH, rt, 30 min; (c) EDC, HOBt, CH2Cl2, rt, 16 h.

X. Beebe et al. / Bioorg. Med. Chem. 20 (2012) 4128–4139
4131
Table 2
120
Ca2+ channel activity of racemic m-trifluoromethylbenzyl analogues
O
100
80
60
40
20
0
R
NH
CF3
H
N
H
relative stereochemistry (racemic)
N-type Ca2+ channels L-type Ca2+ channels
Compound
R
IC50
l
M
pIC50 SEM IC50
lM
pIC50 SEM
7
8
(Ethyl)2CH
2.07
0.95
5.68 0.14 19.0
6.02 0.02
5.97 0.08
4.72 0.16
4.69 0.12
(Propyl)2CH
20.1
>30
9
(Cyclohexyl)2CH 1.07
-10
-9
-8
-7
-6
-5
-4
10
11
12
13
14
15
Cyclopentyl
Cyclohexyl
Cycloheptyl
Ph2CH
(p-F-Ph)2CH
(iPr)PhCH
2.31
1.26
0.56
0.67
0.64
0.36
5.64 0.09 12.8
5.90 0.07 16.1
6.25 0.11 9.3
6.18 0.06 6.3
6.19 0.08 7.1
6.45 0.12 5.0
4.89 0.16
4.79 0.13
4.86 0.17
5.2 0.05
5.15 0.06
5.3 0.02
Concentration [-log M]
Figure 3. Assessment of Compound 25 in the rat aorta ring tissue relaxation assay.
Compound 25 (N) dose-dependently relaxed KCl-evoked contraction of rat aorta
rings. The selective L-type Ca2+ channel blocker nifedipine (d) was included as a
positive control. Rat aorta rings were precontracted to 1 g tension with 80 mM KCl
and the ability of compound 25 to relax the tissue was measured following
cumulative additional of the compound over 15 min intervals. The data is presented
as % of contraction (n = 3).
All values are means SEM of at least three separate experiments.
The activity of compound 25 at N-type Ca2+ channels was con-
firmed using whole cell patch-clamp recordings of rat N-type Ca2+
channels overexpressed in HEK293 cells (Fig. 4). Cells were held
depolarized to partially inactivate the channels to allow for inhibi-
tion of multiple Ca2+ channel states to be detected. Under these
conditions, compound 25 inhibited N-type Ca2+ channels with an
O
O
N
N
R
S
CF3
CF3
H
H
R
S
N
N
H
H
S
R
IC50 value of 0.041
ized conditions compound 25 was less potent, inhibiting only
49 6% of the current at 0.3 M versus 77 6% under inactivating
conditions (p <0.05, mean SEM, n = 5,4 cells, respectively) sug-
gesting that compound 25 blocks N-type Ca2+ channels in a
state-dependent manner. Although compound 25 was less potent
in the FLIPR assay, the electrophysiological assay is a more direct
measure of channel inhibition and is assessed at a membrane po-
tential and inactivation level that may not be reflected in the cal-
cium influx FLIPR assay.24
lM (pIC50 7.39 0.05). Under more hyperpolar-
absolute stereochemistry
25
37
l
N-Type IC50= 0.46µM
L-type IC50 >30µM
N-Type IC50 =1.4µM
L-type IC50 >30µM
Figure 2. In vitro comparison of enantiomeric pair.
Table 3
Ca2+ channel activity of enantiomerically pure n-benzyl analogues
The bis-cyclohexyl moiety, while imparting the desired Ca2+
channel activity, contributes to the poor physiochemical properties
of these analogs. The aqueous solubility is low (<3 lM) and the
plasma protein binding is high (>99%). Additionally the in vitro
metabolic stability, determined in incubations with human and
rat liver microsomes is low (<5% parent remaining after 30 min).
Therefore, compound 25 was assessed in a variety of preclinical
pain models following intraperitoneal (ip) dosing where the bio-
availability was 41%.
O
N
S
H
R
S
N
R
H
absolute stereochemistry
The antinociceptive activity of compound 25 was examined in
rat models of carrageenan-induced acute inflammatory, mono-
iodoacetate-induced osteoarthritis (MIA-OA), and formalin-in-
duced persistent pain. Compound 25 was administered ip 30 min
prior to behavioral assessment. In the carrageenan model of acute
inflammatory pain, significant thermal hypersensitivity (PWL:
3.1 0.3 s) developed 2 h following carrageenan injection, com-
pared to contralateral side (PWL: 10.8 0.6 s). Compound 25
dose-dependently reversed thermal hypersensitivity, with a maxi-
mum effect of 67% at 30 mg/kg (Figure 5A, p<0.01, n = 6 per group).
In the MIA model of osteoarthritis pain, grip force is reduced
(501 50 g) 21 days following MIA injection, compared to the
naïve rats (1090 31 g). Compound 25 dose-dependently returned
the grip force to normalcy in MIA-OA rats, with a maximum effect
of 87% at 30 mg/kg (Figure 5B, p<0.01, n = 12 per group). In the for-
malin model of persistent pain, the formalin injected paw
N-type Ca2+ channel
L-type Ca2+ Channel
IC50
,
l
Ma
pIC50 SEMa
IC50
,
l
Ma
pIC50 SEMa
23
26
27
28
29
25
30
31
H
o-F
m-F
p-F
o-CF3
m-CF3
m-OCF3
m-CH3
16.5
1.12
0.99
0.74
2.43
0.46
0.51
0.86
4.78 0.07
5.95 0.07
6.01 0.04
6.13 0.06
5.61 0.07
6.33 0.06
6.38 0.05
6.07 0.09
>30
>30
>30
22.9
>30
>30
>30
22.3
4.64 0.04
4.65 0.06
a
All values are means SEM of at least three separate experiments.
of 46
channel blocker nifedipine which had a EC50 value of 12 nM (95%
CI: 30–71
M) (Fig. 3).17
l
M (95% CI: 10.7–15 nM) compared with the L-type Ca2+
l

4132
X. Beebe et al. / Bioorg. Med. Chem. 20 (2012) 4128–4139
A
B
100
1 µM
80
0.3 µM
500 pA
60
25 ms
40
control
20
0
0 mV
-8.5
-7.5
-6.5
-5.5
-4.5
log [Compound 25] (M)
-80 mV
Figure 4. Inhibition of N-type Ca2+ channel current by Compound 25 as assessed by manual electrophysiology. (A) Representative current traces illustrating the inhibition of
N-type Ca2+ channel current by 0.3
M and 1 M compound 25 under inactivating conditions as described in the methods. (B) Concentration-response curve for compound 25
yielded an IC50 value of 0.041 M (pIC50 7.39 0.05). For determining the 50% inhibition concentration (IC50) value, data were fitted to a four parameter logistic equation with
the minimum fixed at zero and maximum fixed to 100% of control. n = 4–6 for each data point.
l
l
l
displayed persistent paw flinching behaviors. Compound 25
significantly reduced flinching behavior by 51% at 10 and 30 mg/
kg, respectively (Figure 5C, p <0.05, n = 6 per group).
In the rat accelaerating rotarod assay, compound 25 did not
produce rotarod performance deficits at doses up to 30 mg/kg in
naïve rats (Figure 6, p >0.05, n = 6 per group). In cardiovascular
safety studies, compound 25 did not significantly alter hemody-
namic function (MAP, HR, or dp/dt) in the anesthetized rat cardio-
AnalogixÒ cartridges. Elemental analyses were performed by
Quantitative Technologies, Inc. Unless otherwise specified, all final
compounds were homogeneous with a purity of >95%, as deter-
mined by LCMS analysis conducted on a Finnigan Navigator mass
spectrometer and Agilent 1100 HPLC system operating under posi-
tive APCI ionization conditions. The column used was a Phenome-
nexÒ LunaÒ Combi-HTS C8(2) 5
l
m 100 A° (2.1 ꢁ 30 mm) with a
gradient of 10–100% acetonitrile and 0.1% TFA in water or a gradi-
ent of 10–100% acetonitrile and 10 mM NH4OAc in water. Optical
rotations were measured with an Autopol IV polarimeter at 20 °C
and 589 nm (sodium D line).
vascular model at plasma concentrations up to 17
kg, iv). Based on an EC50 plasma concentration in the osteoarthritis
pain model of approximately g/ml (2 M), compound 25
lg/ml (10 mg/
1
l
l
showed a greater than 15-fold therapeutic margin for antinocicep-
tion versus cardiovascular liabilities.
5.1.1. N-(3-(Trifluoromethyl)benzyl)-1-(trimethylsilyl)methan
amine (2)
(3-(Trifluoromethyl)phenyl)methanamine (25 g, 143 mmol),
(chloromethyl)trimethylsilane (19.92 mL, 143 mmol), and triethyl-
amine (23.87 mL, 171 mmol) were combined neat and the resultant
mixture was refluxed overnight. The reaction was cooled to room
temperature and 150 mL of heptane was added. The HCl salts were
removed by filtration and washed with heptane. The solvent was re-
moved by placing under house vacuum and then high vacuum. N-(3-
(Trifluoromethyl)benzyl)-1-(trimethylsilyl)methanamine (29.89 g,
80%) was isolated by vacuum distillation (bp 70–90 °C/3.2 torr):
1H NMR (300 MHz, CDCl3) d ppm 7.59 (s, 1H), 7.53–7.38 (m, 3H),
3.86 (s, 2H), 2.04 (s, 2H), 1.34 (s, 1H), 0.06 (s, 9H).
4. Conclusion
In conclusion, we have discovered a novel potent N-type
calcium channel blocker with selectivity over the L-type Ca2+ chan-
nel. This compound has analgesic activity in nociceptive pain
models without affecting hemodynamic function or motor coordi-
nation. These types of compounds are promising new chemical
matter for the treatment of pain. Future studies include the discov-
ery of potent and selective N-type Ca2+ channel blockers that have
enhanced oral bioavailability.
5. Experimental section
5.1. Synthesis
5.1.2. 1-Methoxy-N-(3-(trifluoromethyl)benzyl)-N-((trimethyl
silyl)methyl)methanamine (3)
Formaldehyde (4.08 g, 50.2 mmol) and methanol (2.032 mL,
50.2 mmol) were combined, and the mixture was cooled to 0 °C
(ice bath). N-(3-(trifluoromethyl)benzyl)-1-(trimethylsilyl)meth-
anamine (10.94 g, 41.9 mmol) was added dropwise via addition
funnel over 30 min. Potassium carbonate (4.63 g, 33.5 mmol) was
added, and the reaction stirred at 0 °C for 2 additional hours. The
reaction product was decanted from the potassium carbonate,
treated with more potassium carbonate, and decanted again. The
potassium carbonate solids were washed several times with ether,
and these washes were added to the product solution. The solvent
was removed in vacuo to give 1-methoxy-N-(3-(trifluoro-
methyl)benzyl)-N-((trimethylsilyl)methyl)methanamine (12.33 g,
96%) which was used directly in the next step.
NMR spectra were obtained on Varian M-300, BrukerAMX-400,
Varian U-400, or Varian Unity Inova 500 magnetic resonance spec-
trometers with indicated solvent and tetramethylsilane as the
internal standard. Chemical shifts are given in delta (d) values
and coupling constants (J) in Hertz (Hz). The following abbrevia-
tions are used for peak multiplicities: s, singlet; d, doublet; t,
triplet; q, quartet; m, multiplet; and br, broadened. Standard pulse
sequences and phase cycling were used for all correlation spectros-
copy (COSY), heteronuclear single-quantum coherence (HSQC),
heteronuclear multiple bond coherence (HMBC), and nuclear Over-
hauser enhancement spectroscopy (NOESY) spectra. ESI (electro-
spray ionization) mass spectra were performed on a Finnigan
SSQ7000 MS run as a flow injection acquisition. All manipulations
were performed under nitrogen atmosphere unless otherwise
noted. All solvents and reagents were obtained from commercial
sources and used without further purification. Silica gel chroma-
tography was carried out using an AnalogixÒ IntelliFlash 280 with
5.1.3. 2-(3-(Trifluoromethyl)benzyl)hexahydrocyclopenta[c]pyr
rol-4(5H)-one (4)
Cyclopent-2-enone (3.31 g, 40.4 mmol) and trifluoroacetic acid
(0.031 mL, 0.404 mmol) were combined in dichloromethane

X. Beebe et al. / Bioorg. Med. Chem. 20 (2012) 4128–4139
4133
80
A
B
++
12
10
8
++
++
60
**
40
20
0
**
6
**
4
Vehicle
3
10
30
2
Compound 25 (mg/kg, i.p.)
V
3
10
30
Diclo
Compound 25 (mg/kg, i.p.)
Figure 6. The effect of compound 25 on Rotarod performance in naïve rats.
Compound 25 (3, 10 and 30 mg/kg) was administered i.p., fall latency (seconds) was
assessed 30 min after compound treatment in the accelerating rotarod assay. Data
are shown as mean SEM (n = 6 per group).
1200
1100
1000
900
**
**
5.1.4. 2-(3-(Trifluoromethyl)benzyl)hexahydrocyclopenta[c]pyr
rol-4(5H)-one oxime (5)
800
**
700
Hydroxylamine hydrochloride (3.47 g, 50.0 mmol) and sodium
acetate (4.27 g, 52.0 mmol) were dissolved in 15 mL of water and
added to a solution of 2-(3-(trifluoromethyl)benzyl)hexahydrocy-
clopenta[c]pyrrol-4(5H)-one (11.33 g, 40 mmol) in ethanol
(80 mL). The reaction was brought to reflux for 5 min and allowed
to cool to 70 °C. After 1 h, the reaction mixture was cooled, and the
solvent was removed in vacuo to give 2-(3-(trifluoromethyl)
benzyl)hexahydrocyclopenta[c]pyrrol-4(5H)-one oxime (11.93 g,
100%) which was used without additional purification in the next
step. ; MS (ESI+) m/z 299 (M+H)+.
600
500
400
N
V
3
10
30
Celecox
Compound 25 (mg/kg i.p.)
C
70
60
50
40
30
20
10
0
5.1.5. (3aS⁄,4S⁄,6aR⁄)-2-(3-(Trifluoromethyl)benzyl)octahydro
cyclopenta[c]pyrrol-4-amine (6)
*
*
2-(3-(Trifluoromethyl)benzyl)hexahydrocyclopenta[c]pyrrol-
4(5H)-one oxime (11.9 g, 39.9 mmol) in 20% ammonia–methanol
(115 mL) was added to methanol-washed RaneyÒ-nickel, (water-
wet, 38.52 g, 295 mmol) in a 500 mL pressure bottle. The vessel
was pressurized with hydrogen (30 psi), and the mixture was sha-
ken for 16 h at ambient temperature. The mixture was filtered
through a nylon membrane, the solvent was removed in vacuo,
and the crude material was adsorbed onto silica gel. Silica gel chro-
matography using a 400 g silica gel cartridge eluting with 1–10%
methanol (2N ammonia)/dichloromethane gave the following dia-
stereomers: (3aS⁄,4S⁄,6aR⁄)-2-(3-(trifluoromethyl)benzyl)octahy-
drocyclopenta[c]pyrrol-4-amine (6) (3.946 g, 35%): 1H NMR
(300 MHz, pyridine-d5) d ppm 7.58 (s, 1H), 7.50 (t, J = 7.2, 2H),
7.45–7.37 (m, 1H), 3.59 (s, 2H), 3.09 (dd, J = 4.9, 11.4, 1H), 2.77–
2.57 (m, J = 3.9, 1H), 2.52–2.38 (m, 3H), 2.34 (dd, J = 3.7, 9.0, 1H),
2.22–2.09 (m, 1H), 2.03–1.85 (m, J = 4.7, 7.0, 10.8, 2H), 1.42–1.21
(m, 4H); MS (ESI+) m/z 285 (M+H)+ and (3aS⁄,4R⁄,6aR⁄)-2-(3-(tri-
fluoromethyl)benzyl)octahydrocyclopenta[c]pyrrol-4-amine (2.99
g g, 26%): 1H NMR (300 MHz, pyridine-d5) d ppm 7.58 (s, 1H), 7.50
(t, J = 6.7, 2H), 7.45–7.37 (m, 1H), 3.59 (s, 2H), 3.26 (dt, J = 6.3,
12.6, 1H), 2.67–2.50 (m, 4H), 2.38 (dd, J = 6.6, 8.4, 1H), 2.31–2.20
(m, 1H), 1.79–1.62 (m, 2H), 1.60–1.33 (m, 4H); MS (ESI+) m/z 285
(M+H)+.
**
Vehicle
3
10
30 Morphine
Compound 25 (mg/kg, i.p.)
Figure 5. Antinociceptive effects of compound 25 on inflammatory, osteoarthritic
and persistent pain. Compound 25 (3, 10 and 30 mg/kg) was administered ip 30 min
prior to behavioral assessment in each pain model. (A) Effect of compound 25 on
carrageenan-induced inflammatory thermal hypersensitivity. Paw withdrawal
latency (PWL) was measured 2 h following carrageenan injection of right hind
paw. Diclo: diclofenac (30 mg/kg, ip) as positive control. (B) Effect of compound 25
on MIA-induced osteoarthritic pain. Grip force was assessed at least 21 days after
MIA-injection. V: vehicle; N: naïve; Celecox: celecoxib (30 mg/kg, ip) as positive
control. (C) Compound 25 on formalin-induced persistent pain. Paw flinching was
assessed for a period of 20 min (30–50 min following formalin injection, that is,
phase II). Data are shown as mean SEM. ⁄p <0.05, ⁄⁄p <0.01, compared to vehicle-
treated rats (n = 6–12 per group).
(40 mL). 1-Methoxy-N-(3-(trifluoromethyl)benzyl)-N-((trimethyl-
silyl)methyl)methanamine (12.33 g, 40.4 mmol) was added as a
solution in 10 mL of dichloromethane dropwise via addition funnel
over 45 min at room temperature under nitrogen. The reaction was
quenched with aqueous sodium bicarbonate and extracted with
2 ꢁ 100 mL of dichloromethane. The organic washes were com-
bined and washed with brine. The solvent was removed in vacuo
to give 2-(3-(trifluoromethyl)benzyl)hexahydrocyclopenta[c]pyr-
rol-4(5H)-one (11.7 g, 100%): 1H NMR (300 MHz, CDCl3) d ppm
7.54–7.34 (m, 4H), 3.66 (d, J = 13.4, 1H), 3.52 (d, J = 13.4, 1H), 3.04
(dd, J = 1.8, 8.9, 1H), 2.91 (ddd, J = 2.8, 7.4, 11.7, 1H), 2.72–2.65 (m,
1H), 2.62 (d, J = 8.9, 1H), 2.52–2.23 (m, 4H), 2.15 (ddd, J = 8.1, 12.9,
17.1, 1H), 1.85–1.70 (m, 1H) ; MS (ESI+) m/z 284 (M+H)+.
5.1.6. (R,E)-N-((3aR,6aS)-2-Benzylhexahydrocyclopenta[c]pyr
rol-4(5H)-ylidene)-2-methylpropane-2-sulfinamide (18) and
(R,E)-N-((3aS,6aR)-2-benzylhexahydrocyclopenta[c]pyrrol-
4(5H)-ylidene)-2-methylpropane-2-sulfinamide (19)
2-Benzylhexahydrocyclopenta[c]pyrrol-4(5H)-one (8.88 g, 41.3
mmol), titanium tetraethoxide (18.82, 83 mmol), and (R)-2-methyl-
propane-2-sulfinamide (5.00 g, 41.3 mmol) were combined in tet-
rahydrofuran (138 mL). The reaction was heated at 60 °C

4134
X. Beebe et al. / Bioorg. Med. Chem. 20 (2012) 4128–4139
overnight. The reaction was then reduced in volume to about half
and poured into 75 mL of saturated aqueous ammonium chloride.
The precipitate was collected by filtration and washed with ethyl
acetate. The filtrate was poured into a separatory funnel and the or-
ganic layer was removed. The aqueous layer was extracted with
ethyl acetate (3 ꢁ 200 mL). The organic washes were combined
and washed with brine, dried (Na2SO4) and concentrated. The crude
material was chromatographed using silica gel cartridge (Analogix,
RS65–400) eluting with 5–100% ethyl acetate/hexanes to give (R,
E)-N-((3aR,6aS)-2-benzylhexahydrocyclopenta[c]pyrrol-4(5H)-yli-
dene)-2-methylpropane-2-sulfinamide (18) (4.12 g, 31% yield) and
(R,E)-N-((3aS,6aR)-2-benzylhexahydrocyclopenta[c]pyrrol-4(5H)-
ylidene)-2-methylpropane-2-sulfinamide (19) (2.88 g, 22% yield).
10.1, 1H), 2.89 (dd, J = 7.8, 9.7, 1H), 2.78 (dd, J = 7.4, 10.1, 1H),
2.71 (ddd, J = 3.2, 7.5, 16.7, 1H), 2.58 (dd, J = 3.2, 9.8, 1H), 2.50–
2.39 (m, J = 8.3, 1H), 1.98–1.89 (m, 3H), 1.89–1.80 (m, 3H), 1.79–
1.67 (m, J = 6.4, 14.9, 7H), 1.66–1.56 (m, 3H), 1.49–1.36 (m, 2H),
1.36–1.28 (m, 1H), 1.28–1.02 (m, 8H); MS (ESI+) m/z 333 (M+H)+.
5.2. General procedure for reductive alkylation for compounds
25–31
5.2.1. 2,2-dicyclohexyl-N-{(3aS,4S,6aR)-2-[3-(trifluoromethyl)
benzyl]octahydrocyclopenta[c]pyrrol-4-yl}acetamide (25)
2,2-Dicyclohexyl-N-[(3aS,4S,6aR)-octahydrocyclopenta[c]pyrrol-
4-yl]acetamide (154 mg, 0.463 mmol) 24 and 3-(trifluoromethyl)
benzaldehyde (0.062 mL, 0.463 mmol) were combined in dichloro-
methane (5 mL), and then 2 mL of acetic acid was added. The reaction
was stirred atroomtemperature for 20 min, andthenPS-cyanoborohy-
dride (396 mg, 0.926 mmol) was added. The reaction was stirred at
room temperature overnight, then filtered and the resin was washed
with dichloromethane. The solvent was removed in vacuo, and the
crude material was purified using a silica gel cartridge (Analogix, RS-
25) eluting with 1–10% methanol (2N ammonia)/dichloromethane to
5.1.7. (R)-N-((3aS,4S,6aR)-2-Benzyloctahydrocyclopenta[c]pyr
rol-4-yl)-2-methylpropane-2-sulfinamide (20)
A mixture of (R,E)-N-((3aS,6aR)-2-benzylhexahydrocyclopen-
ta[c]pyrrol-4(5H)-ylidene)-2-methylpropane-2-sulfinamide
(4.128 g, 12.96 mmol) in methanol (40 mL) was cooled to ꢀ78 °C
in dry ice/acetone bath. Sodium borohydride (1.471 g,
18
a
38.9 mmol) was added, and the reaction was allowed to warm to
room temperature overnight. The reaction was quenched with sat-
urated aqueous ammonium chloride, diluted with water, and ex-
tracted with 3 ꢁ 100 mL of ethyl acetate. The extracts were dried
(Na2SO4) and filtered. The solvent was removed in vacuo to give
(R)-N-((3aS,4S,6aR)-2-benzyloctahydrocyclopenta[c]pyrrol-4-yl)-
2-methylpropane-2-sulfinamide 20 (4.50 g, 100% yield) as a yellow
crystalline solid: 1H NMR (500 MHz, pyridine-d5) d ppm 7.39 (dt,
J = 13.1, 7.3, 4H), 7.30 (t, J = 7.2, 1H), 5.15 (d, J = 7.4, 1H), 3.80–
3.72 (m, 1H), 3.51 (s, 2H), 2.86 (dd, J = 9.4, 3.6, 1H), 2.65 (qd,
J = 7.9, 3.6, 1H), 2.46–2.35 (m, 2H), 2.32–2.25 (m, 2H), 2.06 (ddd,
J = 14.7, 12.1, 7.8, 1H), 1.69 (td, J = 11.9, 5.9, 1H), 1.63–1.54 (m,
1H), 1.38–1.29 (m, 1H), 1.19 (s, 9H) ; MS (ESI+) m/z 321(M+H)+.
give
2,2-dicyclohexyl-N-{(3aS,4S,6aR)-2-[3-(trifluoromethyl)
benzyl]octahydrocyclopenta[c]pyrrol-4-yl}acetamide 25 (160 mg,
70% yield): ½a 2D0
ꢂ
ꢀ70.2o (c 1.03, CH3OH). 1H NMR (500 MHz, pyridine-
d5) d ppm 7.73 (s, 1H), 7.66 (d, J = 7.7, 1H), 7.56 (s, 1H), 7.50 (t, J = 7.7,
1H), 4.51–4.43 (m, 1H), 3.55 (d, J = 12.9, 1H), 3.46 (d, J = 12.9, 1H),
2.75 (d, J = 8.6, 2H), 2.52–2.41 (m, J = 8.5, 19.1, 2H), 2.34–2.29 (m,
1H), 2.20 (dd, J = 7.7, 9.8, 1H), 2.14 (s, 3H, OAc), 1.96–1.54 (m, 15H),
1.49–1.05 (m, 12H), 1.03–0.93 (m, J = 11.7, 21.1, 1H); MS (ESI+) m/z
491 (M+H)+ ; Anal. (C29H41F3N2O) C, H, N.
5.2.2. 2,2-Dicyclohexyl-N-[(3aS,4S,6aR)-2-(2-fluorobenzyl)octa
hydrocyclopenta[c]pyrrol-4-yl]acetamide (26)
(89 mg, 56% yield): 1H NMR (500 MHz, pyridine-d5) d ppm 7.55
(s, 1H), 7.36 (dt, J = 6.6, 24.8, 2H), 7.20–7.15 (m, 2H), 4.44 (s, 1H),
3.68 (d, J = 12.6, 1H), 3.44 (d, J = 12.6, 1H), 2.77 (d, J = 9.6, 1H),
2.64 (dd, J = 7.5, 15.0, 1H), 2.53 (d, J = 9.0, 1H), 2.49–2.40 (m, 1H),
2.29 (t, J = 8.3, 1H), 2.16–2.09 (m, 1H), 1.98–1.64 (m, 12H), 1.61
(d, J = 11.4, 2H), 1.55–1.37 (m, J = 13.8, 20.5, 31.3, 3H), 1.34–1.08
5.1.8. (3aS,4S,6aR)-2-Benzyloctahydrocyclopenta[c]pyrrol-4-
amine (21)
(R)-N-((3aS,4S,6aR)-2-Benzyloctahydrocyclopenta[c]pyrrol-4-yl)-
2-methylpropane-2-sulfinamide20 (4.15 g, 12.95 mmol) and 2 N HCl
(10 mL, 40.0 mmol) were combined in methanol (10 mL). After
30 min, the solvent was removed and the crude material was purified
on a silica gel cartridge (Analogix, RS-40) loading with 10% metha-
nol(2 N ammonia)/dichloromethane solution and eluting with 2–
10% methanol (2 N ammonia)/dichloromethane to give (3aS,4-
S,6aR)-2-benzyloctahydrocyclopenta[c]pyrrol-4-amine 21 (2.69 g,
96% yield): 1H NMR (500 MHz, pyridine-d5) d ppm 7.44 (d, J = 7.4,
2H), 7.36 (t, J = 7.5, 2H), 7.28 (t, J = 7.3, 1H), 3.52 (q, J = 13.0, 2H),
3.29 (q, J = 7.3, 1H), 2.84 (dd, J = 3.7, 9.3, 1H), 2.55–2.43 (m, 3H),
2.37–2.30 (m, 1H), 2.24 (d, J = 5.3, 1H), 1.74–1.64 (m, 2H), 1.62–1.53
(m, 1H), 1.34 (dd, J = 5.0, 10.1, 1H) ; MS (ESI+) m/z 217(M+H)+.
(m, 9H), 1.00 (dd, J = 13.4, 22.1, 1H); MS (ESI+) m/z 441 (M+H)+
;
Anal. (C28H41FN2O.1HCl.2.2H2O) C, H, N.
5.2.3. 2,2-Dicyclohexyl-N-[(3aS,4S,6aR)-2-(3-fluorobenzyl)
octahydrocyclopenta[c]pyrrol-4-yl]acetamide (27)
(84 mg , 53% yield): 1H NMR (500 MHz, pyridine-d5) d ppm 7.36
(dd, J = 7.8, 13.9, 1H), 7.19–7.11 (m, 3H), 4.49–4.42 (m, 1H), 3.55 (d,
J = 12.8, 1H), 3.34 (d, J = 12.8, 1H), 2.78–2.69 (m, J = 8.0, 15.6, 2H),
2.51–2.43 (m, J = 10.6, 2H), 2.29 (t, J = 8.4, 1H), 2.13 (dd, J = 7.2,
9.5, 1H), 1.97–1.77 (m, 7H), 1.77–1.64 (m, 5H), 1.63–1.47 (m,
4H), 1.42 (ddd, J = 3.0, 12.2, 23.0, 1H), 1.33 (dd, J = 9.7, 23.2, 1H),
1.29–1.08 (m, 8H), 1.02 (dd, J = 11.8, 22.6, 1H); MS (ESI+) m/z
441(M+H)+ ; Anal. (C28H41FN2O.1HCl.1.7H2O) C, H, N.
5.1.9. 2,2-Dicyclohexyl-N-[(3aS,4S,6aR)-octahydrocyclopenta[c]
pyrrol-4-yl]acetamide (24)
5.2.4. 2,2-Dicyclohexyl-N-[(3aS,4S,6aR)-2-(4-fluorobenzyl)octa
hydrocyclopenta[c]pyrrol-4-yl]acetamide (28)
N-[(3aS,4S,6aR)-2-Benzyloctahydrocyclopenta[c]pyrrol-4-yl]-2,
2-dicyclohexylacetamide (2.65 g, 6.27 mmol) 23 and methanol
60 mL were added to 20% palladium hydroxide on carbon, (wet,
0.530 g, 3.77 mmol) in a 250 mL pressure bottle. The reaction
was stirred for 16 h under hydrogen (30 psi) at room temperature.
The mixture was filtered through a nylon membrane and the sol-
vent removed in vacuo. The crude material was chromatographed
on a silica gel cartridge (Analogix, RS25–25) eluting with 1–10%
methanol (2 N ammonia)/dichloromethane to give 2,2-dicyclo-
hexyl-N-[(3aS,4S,6aR)-octahydrocyclopenta[c]pyrrol-4-yl]acetam-
ide 24 (1.69 g, 81% yield): 1H NMR (500 MHz, pyridine-d5) d ppm
7.97 (d, J = 7.1, 1H), 4.57 (dt, J = 7.0, 13.8, 1H), 3.03 (dd, J = 3.2,
(103 mg, 65% yield): 1H NMR (500 MHz, pyridine-d5) d ppm
7.54 (d, J = 6.0, 1H), 7.40–7.29 (m, 2H), 7.18 (d, J = 8.7, 2H), 4.53–
4.38 (m, 1H), 3.55 (d, J = 12.5, 1H), 3.29 (d, J = 12.6, 1H), 2.72 (dd,
J = 8.6, 17.7, 2H), 2.56–2.39 (m, 2H), 2.29 (t, J = 8.3, 1H), 2.10 (t,
J = 8.1, 1H), 1.98–1.07 (m, 26H), 0.99 (dd, J = 10.2, 22.0, 1H); MS
(ESI+) m/z 441 (M+H)+; Anal. (C28H41FN2O.1HCl.3.0H2O) C, H, N.
5.2.5. 2,2-Dicyclohexyl-N-{(3aS,4S,6aR)-2-[2-(trifluoromethyl)
benzyl]octahydrocyclopenta[c]pyrrol-4-yl}acetamide (29)
(72 mg, 44% yield): 1H NMR (500 MHz, pyridine-d5) d ppm 7.74
(d, J = 7.8, 1H), 7.64 (d, J = 7.7, 1H), 7.55 (d, J = 7.6, 1H), 7.43 (dd,

X. Beebe et al. / Bioorg. Med. Chem. 20 (2012) 4128–4139
4135
J = 7.0, 13.9, 2H), 4.51–4.43 (m, 1H), 3.82 (d, J = 13.7, 1H), 3.54 (d,
J = 13.7, 1H), 2.81–2.70 (m, 2H), 2.53–2.43 (m, 2H), 2.35 (t, J = 8.1,
1H), 2.23–2.17 (m, 1H), 1.95–1.68 (m, 10H), 1.67–1.52 (m, 5H),
1.46–1.04 (m, 11H), 0.97–0.86 (m, 1H); MS (ESI+) m/z 491
(M+H)+; Anal. (C29H41F3N2O.1HCl.1.7H2O) C, H, N.
hexanes to give (S)-2-methyl-N-((3aR,4R,6aS)-2-(3-(trifluoro-
methyl)benzyl)octahydrocyclopenta[c]pyrrol-4-yl)propane-2-sul-
finamide (35) (975 mg, 73% yield) as a pale yellow oil: 1H NMR
(500 MHz, pyridine-d5) d ppm 7.75 (s, 1H), 7.60 (d, J = 7.9, 2H),
7.46 (t, J = 7.7, 1H), 5.18 (d, J = 6.8, 1H), 3.81–3.73 (m, 1H), 3.54
(s, 2H), 2.86 (dd, J = 9.4, 3.9, 1H), 2.72–2.65 (m, 1H), 2.48–2.39
(m, 2H), 2.32 (dd, J = 9.2, 7.9, 1H), 2.27 (d, J = 5.5, 1H), 2.07–1.98
(m, 1H), 1.70 (dt, J = 11.6, 5.7, 1H), 1.64–1.53 (m, 1H), 1.32 (dd,
J = 15.8, 5.3, 1H), 1.18 (s, 9H). MS (ESI+) m/z 389 (M+H)+.
5.2.6. 2,2-Dicyclohexyl-N-{(3aS,4S,6aR)-2-[3-(trifluorometh
oxy)benzyl]octahydrocyclopenta[c]pyrrol-4-yl}acetamide (30)
(109 mg, 60% yield): 1H NMR (500 MHz, pyridine-d5) d ppm 7.60
(d, J = 7.3, 1H), 7.42 (t, J = 7.9, 1H), 7.37 (s, 1H), 7.30 (dd, J = 7.9, 16.8,
2H), 4.51–4.44 (m, 1H), 3.50 (d, J = 13.0, 1H), 3.45 (d, J = 13.0, 1H),
2.80–2.72 (m, J = 4.8, 16.2, 2H), 2.51–2.42 (m, J = 8.7, 2H), 2.29 (t,
J = 8.3, 1H), 2.20 (dd, J = 7.2, 9.2, 1H), 1.96–1.89 (m, 2H), 1.89–1.78
(m, 4H), 1.77–1.65 (m, 5H), 1.64–1.51 (m, 4H), 1.42 (dd, J = 9.4,
23.0, 1H), 1.36–1.09 (m, 10H), 1.08–0.98 (m, 1H).; MS (ESI+) m/z
5.2.10. (3aR,4R,6aS)-2-(3-(Trifluoromethyl)benzyl)octahydrocy
clopenta[c]pyrrol-4-amine (36)
(S)-2-Methyl-N-((3aR,4R,6aS)-2-(3-(trifluoromethyl)benzyl)octa-
hydrocyclopenta[c]pyrrol-4-yl)propane-2-sulfinamide 35 (765 mg,
1.969 mmol) dissolved in 4 N HCl in dioxane (10 ml, 40.0 mmol)
and MeOH (10 ml) to give a yellow solution. After 30 min the solvent
was removed and the crude material purified (loaded on with meth-
anol (2N ammonia) using an 24 g silica gel cartridge and 2–10%
methanol (2 N ammonia)/dichloromethane to give (3aR,4R,6aS)-2-
(3-(trifluoromethyl)benzyl)octahydrocyclopenta[c]pyrrol-4-amine
(36) (492 mg, 88% yield): 1H NMR (500 MHz, pyridine-d5) d ppm 7.78
(s, 1H), 7.59 (d, J = 11.1, 2H), 7.45 (t, J = 7.7, 1H), 4.94 (br s, 2H), 3.14
(dd, J = 5.2, 11.5, 1H), 2.58 (tt, J = 5.1, 10.1, 1H), 2.49 (dd, J = 3.3, 9.0,
1H), 2.35 (dd, J = 7.5, 9.0, 1H), 2.31 (d, J = 5.8, 2H), 2.21–2.14 (m,
1H), 2.00–1.92 (m, 1H), 1.88 (td, J = 4.2, 9.9, 1H), 1.39–1.29 (m, 2H);
MS (ESI+) m/z 284(M+H)+.
507 (M+H)+ ; Anal. (C29H41F3N2O2 1HCl.2.6H2O) C, H, N.
.
5.2.7. 2,2-Dicyclohexyl-N-[(3aS,4S,6aR)-2-(3-methylbenzyl)octa
hydrocyclopenta[c]pyrrol-4-yl]acetamide (31)
(126 mg, 80% yield): 1H NMR (500 MHz, pyridine-d5) d ppm 7.53
(d, J = 5.8, 1H), 7.32 (t, J = 7.5, 1H), 7.24–7.22 (m, 1H), 7.20–7.14 (m,
2H), 4.50–4.42 (m, 1H), 3.57 (d, J = 12.4, 1H), 3.34 (d, J = 12.3, 1H),
2.78 (d, J = 9.4, 1H), 2.66 (dd, J = 7.5, 15.1, 1H), 2.55 (d, J = 8.8, 1H),
2.50–2.42 (m, 1H), 2.33 (s, 3H), 2.31–2.24 (m, 1H), 2.14–2.07 (m,
1H), 1.98–1.57 (m, 13H), 1.53 (ddd, J = 6.4, 11.9, 18.4, 1H), 1.42 (t,
J = 10.0, 2H), 1.38–1.08 (m, 10H), 1.02–0.92 (m, 1H); MS (ESI+) m/z
437 (M+H)+ ; Anal. (C29H44N2O.1HCl.2.6H2O) C, H, N.
5.3. General procedure for amide synthesis 38–43, 7–15, 23, 37
5.2.8. (S,E)-2-Methyl-N-((3aR,6aS)-2-(3-(trifluoromethyl)
benzyl)hexahydrocyclopenta[c]pyrrol-4(5H)-ylidene)propane-
2-sulfinamide (33) and (S,E)-2-methyl-N-((3aS,6aR)-2-(3-
(trifluoromethyl)benzyl)hexahydrocyclopenta[c]pyrrol-4(5H)-
ylidene)propane-2-sulfinamide (34)
5.3.1. N-[(3aS⁄,4S⁄,6aR⁄)-2-Benzyloctahydrocyclopenta[c]pyrrol-
4-yl]-2,2-dicyclohexylacetamide (38)
(3aS⁄,6aR⁄)-2-Benzyloctahydrocyclopenta[c]pyrrol-4-amine
(500 mg, 2.311 mmol), 2,2-dicyclohexylacetic acid (570 mg,
2.54 mmol), and 1-hydroxybenzotriazole (389 mg, 2.54 mmol)
were combined in dichloromethane (20 mL). The reaction was
stirred at room temperature for 10 min, then N-(3-dimethylamino-
propyl)-N0-ethylcarbodiimide (0.449 mL, 2.54 mmol) was added
dropwise. The reaction was stirred at room temperature for 20 h,
and then the reaction was quenched with 10 mL of water. The reac-
tion was extracted with 2 ꢁ 20 mL of dichloromethane, the solvent
was removed in vacuo, and the crude material was chromato-
graphed over a 40 g silica gel cartridge eluting with 30–50% ethyl
acetate/hexanes to give N-[(3aS⁄,4S⁄,6aR⁄)-2-benzyloctahy-
drocyclopenta[c]pyrrol-4-yl]-2,2-dicyclohexylacetamide (299 mg,
0.707 mmol, 30% yield): 1H NMR (500 MHz, pyridine-d5) d ppm
7.60 (d, J = 5.1, 1H), 7.45–7.38 (m, 4H), 7.35 (m, 1H), 4.49–4.41 (m,
1H), 3.57 (d, J = 12.6, 1H), 3.37 (d, J = 12.5, 1H), 2.76 (d, J = 9.6, 1H),
2.69 (dd, J = 7.9, 15.7, 1H), 2.50 (d, J = 9.1, 1H), 2.48–2.41 (m, 1H),
2.28 (t, J = 8.2, 1H), 2.15–2.08 (m, 1H), 1.99–1.06 (m, 26H), 1.04–
0.92 (m, 1H); MS (ESI+) m/z 423 (M+H)+; Anal. (C28H42N2O) C, H, N.
2-(3-(Trifluoromethyl)benzyl) hexahydrocyclopenta[c]pyrrol-
4(5H)-one
4
(3.672 g, 12.96 mmol), titanium tetraethoxide
(5.11 ml, 24.63 mmol), and (S)-2-methylpropane-2-sulfinamide
(1.885 g, 15.55 mmol) were combined in THF (200 ml) to give an
orange solution. The reaction was heated at 60 °C overnight to give
a brown solution. The reaction was then reduced in volume to
about half and poured into 75 mL brine and filtered, washing the
dark pink precipitate with EtOAc. The filtrate was poured into a
separatory funnel and the organics removed and the solvent re-
duced in volume. The crude material was chromatographed using
an RS65–200 with 5–100% EtOAc/hexanes to give (S,E)-2-methyl-
N-((3aR,6aS)-2-(3-(trifluoromethyl)benzyl)hexahydrocyclopenta
[c]pyrrol-4(5H)-ylidene)propane-2-sulfinamide (33) (1.327 g, 26%
yield): MS (ESI+) m/z 387 (M+H)+ and (S,E)-2-methyl-N-((3aS,
6aR)-2-(3-(trifluoromethyl)benzyl)hexahydrocyclopenta[c]pyrrol-
4(5H)-ylidene)propane-2-sulfinamide (34) (1.188 g, 24% yield): 1H
NMR (300 MHz, CDCl3) d ppm 7.57–7.36 (m, 5H), 3.59 (s, 2H),
3.13–2.99 (m, 1H), 2.98–2.62 (m, 4H), 2.57–2.43 (m, 2H), 2.12
(ddd, J = 16.9, 13.2, 8.2, 1H), 1.84–1.69 (m, 1H), 1.19 (s, 9H); MS
(ESI+) m/z 387 (M+H)+.
5.3.2. N-[(3aR⁄,4R⁄,6aS⁄)-2-Benzyloctahydrocyclopenta[c]pyrrol
-4-yl]-3-methyl-2-phenylbutanamide (39)
(22.6 mg, 23% yield): 1H NMR (500 MHz, pyridine-d5) d ppm
8.13–8.03 (m, 1H), 7.65 (d, J = 7.2, 1H), 7.55 (d, J = 7.3, 1H), 7.44
(d, J = 4.3, 2H), 7.41 (d, J = 7.0, 1H), 7.36 (t, J = 6.9, 3H), 7.28 (ddd,
J = 7.3, 14.7, 17.5, 2H), 4.40 (dd, J = 6.7, 13.3, 0.5H), 4.35–4.27 (m,
0.5H), 3.55 (d, J = 12.7, 0.5H), 3.40 (dd, J = 4.5, 12.8, 1H), 3.26 (d,
J = 12.9, 0.5H), 3.16 (d, J = 10.5, 0.5H), 3.05 (d, J = 10.5, 0.5H), 2.78
(dd, J = 8.1, 15.8, 1H), 2.69–2.54 (m, 1.5H), 2.43–2.26 (m, 3H),
2.23–2.13 (m, 1H), 1.94–1.82 (m, 1H), 1.73–1.57 (m, 1.5H), 1.50
(dt, J = 7.1, 15.0, 0.5H), 1.38 (td, J = 6.4, 12.2, 0.5H), 1.27 (m,
0.5H), 1.19 (d, J = 6.4, 1.5H), 1.11 (dd, J = 5.7, 12.0, 0.5H), 1.03 (d,
J = 6.5, 1.5H), 0.73 (dd, J = 6.7, 10.7, 3H); MS (ESI+) m/z 377
(M+H)+; Anal. (C25H32N2O.0.07CH2Cl2) C, H, N.
5.2.9. (S)-2-Methyl-N-((3aR,4R,6aS)-2-(3-(trifluoromethyl)ben zyl)
octahydrocyclopenta[c]pyrrol-4-yl)propane-2-sulfinamide (35)
(S,E)-2-Methyl-N-((3aR,6aS)-2-(3-(trifluoromethyl)benzyl)hexa-
hydrocyclopenta[c]pyrrol-4(5H)-ylidene)propane-2-sulfinamide
33 (1.327 g, 3.43 mmol) in MeOH (25 mL) was cooled to -78 °C in a
dry ice/acetone bath to give a yellow solution. NaBH4 (0.390 g,
10.30 mmol) was added and the reaction was allowed to warm
to ambient temperature overnight. The reaction was quenched
with ammonium chloride, diluted with water, and extracted with
3 ꢁ 100 mL of ethyl acetate. The extracts were dried (Na2SO4)
and filtered. The solvent was removed in vacuo and the crude
material chromatographed using an RS-40 with 5–50% acetone/

4136
X. Beebe et al. / Bioorg. Med. Chem. 20 (2012) 4128–4139
5.3.3. N-[(3aS⁄,4S⁄,6aR⁄)-2-Benzyloctahydrocyclopenta[c]pyrrol
-4-yl]-2-cyclopentyl-2-phenylacetamide (40)
1H), 1.90 (dt, J = 7.3, 11.5, 1H), 1.84–1.75 (m, 2H), 1.73–1.61
(m, 2H), 1.48–1.27 (m, 6H), 1.20–1.08 (m, 1H), 0.88 (t, J = 7.0,
(29 mg, 30% yield): 1H NMR (500 MHz, pyridine-d5) d ppm 8.06
(dd, J = 6.8, 23.5, 1H), 7.67 (d, J = 7.2, 1H), 7.46–7.39 (m, 3H), 7.40–
7.29 (m, 4H), 7.29–7.22 (m, 1H), 4.45–4.37 (m, 0.5H), 4.35–4.27 (m,
0.5H), 3.54 (d, J = 12.7, 0.5H), 3.42 (dd, J = 4.0, 12.8, 1H), 3.34–3.19
(m, 1.5H), 2.97–2.77 (m, 2.5H), 2.63 (d, J = 7.2, 0.5H), 2.46– 2.27 (m,
3H), 2.26–2.05 (m, 1.5H), 2.03–1.84 (m, 1.5H), 1.71 (dt, J = 7.0, 14.1,
0.5H), 1.65–1.36 (m, 8H), 1.33–1.22 (m, 0.5H), 1.20–0.98 (m, 2H);
MS (ESI+) m/z 403(M+H)+ ; Anal. (C27H34N2O) C, H, N.
3H), 0.83 (t, J = 7.1, 3H); MS (ESI+) m/z 411(M+H)+
; Anal.
(C23H33F3N2O) C, H, N.
5.3.9. 2,2-Dicyclohexyl-N-{(3aS⁄,4S⁄,6aR⁄)-2-[3-(trifluorometh
yl)benzyl]octahydrocyclopenta[c]pyrrol-4-yl}acetamide (9)
(176 mg, 97% yield): 1H NMR (400 MHz, pyridine-d5) d ppm 7.73
(s, 1H), 7.65 (d, J = 7.6, 1H), 7.57–7.44 (m, 3H), 4.46 (dt, J = 6.4, 12.6,
1H), 3.55 (d, J = 12.9, 1H), 3.47 (d, J = 12.8, 1H), 2.75 (d, J = 8.1, 2H),
2.53–2.39 (m, 2H), 2.32 (t, J = 8.2, 1H), 2.21 (t, J = 8.3, 1H), 1.97–
1.53 (m, 15H), 1.50–1.04 (m, 11H), 0.98 (dd, J = 11.3, 23.1, 1H); MS
(ESI+) m/z 491(M+H)+ ; Anal. (C29H41F3N2O) C, H, N.
5.3.4. N-[(3aR⁄,4R⁄,6aS⁄)-2-Benzyloctahydrocyclopenta[c]pyrrol
-4-yl]-2-cyclohexyl-2-phenylacetamide (41)
(41 mg, 41% yield): 1H NMR (500 MHz, pyridine-d5) d ppm 8.10
(s, 1H), 7.67 (d, J = 7.2, 1H), 7.44 (dd, J = 5.7, 12.3, 3H), 7.40–7.35
(m, 3H), 7.33 (t, J = 7.4, 1H), 7.27 (dd, J = 7.6, 16.2, 1H), 4.43–4.37
(m, 0.5H), 4.36–4.29 (m, 0.5H), 3.56 (d, J = 12.6, 0.5H), 3.42 (t,
J = 11.7, 1H), 3.26 (dd, J = 6.6, 11.7, 1H), 3.14 (d, J = 10.5, 0.5H),
2.80 (d, J = 8.3, 1H), 2.63 (d, J = 6.9, 0.5H), 2.24–2.13 (m, 1.5H),
2.02 (d, J = 12.5, 0.5H), 1.91 (d, J = 7.0, 1H), 1.74–1.59 (m, 2.5H),
1.58–1.34 (m, 4H), 1.33–1.19 (m, 1.5H), 1.18–0.99 (m, 3H), 0.87
5.3.10. N-{(3aS⁄,4S⁄,6aR⁄)-2-[3-(Trifluoromethyl)benzyl]octahy
drocyclopenta[c]pyrrol-4-yl}cyclopentanecarboxamide (10)
(120 mg, 75% yield): 1H NMR (500 MHz, pyridine-d5) d ppm
7.82 (d, J = 6.9, 1H), 7.71 (s, 1H), 7.61 (d, J = 7.7, 1H), 7.55 (d,
J = 7.7, 1H), 7.46 (t, J = 7.7, 1H), 4.48–4.41 (m, 1H), 4.07 (q, J = 7.1,
1H), 2.82 (ddd, J = 2.9, 7.8, 16.7, 1H), 2.72 (dd, J = 2.9, 9.5, 1H),
2.66 (p, J = 7.9, 1H), 2.50–2.42 (m, 1H), 2.37 (dd, J = 2.9, 9.0, 1H),
2.34–2.29 (m, 1H), 2.21 (dd, J = 7.5, 9.4, 1H), 2.03 (dt, J = 7.0, 15.3,
1H), 1.86 (dddd, J = 7.4, 12.3, 20.5, 23.4, 3H), 1.75–1.60 (m, 5H),
1.53–1.41 (m, 2H), 1.30 (td, J = 4.7, 11.1, 1H), 1.11 (t, J = 7.1, 1H);
MS (ESI+) m/z 381(M+H)+ ; Anal. (C21H27F3N2O) C, H, N.
(d, J = 14.3, 0.5H), 0.81–0.68 (m, 1H); MS (ESI+) m/z 417 (M+H)+
;
Anal. (C28H36N2O) C, H, N.
5.3.5. N-[(3aS⁄,4S⁄,6aR⁄)-2-Benzyloctahydrocyclopenta[c]pyrrol
-4-yl]-1-phenylcyclopentanecarboxamide (42)
5.3.11. N-{(3aS⁄,4S⁄,6aR⁄)-2-[3-(Trifluoromethyl)benzyl]octahy
drocyclopenta[c]pyrrol-4-yl}cyclohexanecarboxamide (11)
(127 mg, 76% yield): 1H NMR (500 MHz, pyridine-d5) d ppm
7.74 (d, J = 6.1, 1H), 7.71 (s, 1H), 7.62 (d, J = 7.7, 1H), 7.57 (d,
J = 9.2, 1H), 7.47 (t, J = 7.7, 1H), 4.48–4.41 (m, 1H), 3.53 (d,
J = 13.2, 1H), 3.47 (d, J = 13.2, 1H), 2.84 (ddd, J = 2.8, 7.9, 10.3,
1H), 2.75 (dd, J = 2.8, 9.5, 1H), 2.51–2.43 (m, 1H), 2.37 (dd, J = 2.8,
9.1, 1H), 2.34–2.29 (m, 1H), 2.27–2.19 (m, 2H), 1.94–1.83 (m,
2H), 1.80 (d, J = 12.9, 1H), 1.73–1.60 (m, 6H), 1.57–1.51 (m, 1H),
(40 mg, 42% yield): 1H NMR (500 MHz, pyridine-d5) d ppm
7.52–7.28 (m, 9H), 7.25 (d, J = 7.7, 1H), 4.43–4.34 (m, 1H), 3.56
(d, J = 12.9, 1H), 3.17 (d, J = 12.9, 1H), 2.83–2.67 (m, 2H), 2.59–
2.50 (m, 1H), 2.50–2.44 (m, 1H), 2.35–2.29 (m, 2H), 2.04–1.87
(m, 4H), 1.84–1.54 (m, 6H), 1.43–1.26 (m, 1H), 1.04–0.94 (m,
1H); MS (ESI+) m/z 389 (M+H)+; Anal. (C26H32N2O) C, H, N.
5.3.6. N-[(3aS⁄,4S⁄,6aR⁄)-2-Benzyloctahydrocyclopenta[c]pyrrol
-4-yl]-1-phenylcyclohexanecarboxamide (43)
1.35–1.27 (m, 1H), 1.22–1.10 (m, 3H); MS (ESI+) m/z 395(M+H)+
;
(34 mg, 35% yield): 1H NMR (500 MHz, pyridine-d5) d ppm
7.57–7.54 (m, 2H), 7.45 (t, J = 7.3, 2H), 7.37 (dd, J = 7.6, 15.3, 5H),
7.29 (d, J = 6.9, 1H), 7.25 (t, J = 7.3, 1H), 4.41 (m, 1H), 3.59 (d,
J = 12.9, 1H), 3.18 (d, J = 12.9, 1H), 2.52 (dd, J = 8.9, 17.6, 4H), 2.34
(dd, J = 8.5, 15.2, 2H), 2.01–1.92 (m, 2H), 1.91–1.80 (m, 2H),
1.79–1.68 (m, 3H), 1.61 (d, J = 25.2, 4H), 1.43–1.33 (m, 1H), 1.27
(d, J = 8.7, 1H), 1.04–0.93 (m, 1H); MS (ESI+) m/z 403 (M+H)+ ; Anal.
(C27H34N2O) C, H, N.
Anal. (C22H29F3N2O) C, H, N.
5.3.12. N-{(3aS⁄,4S⁄,6aR⁄)-2-[3-(Trifluoromethyl)benzyl]octahy
drocyclopenta[c]pyrrol-4-yl}cycloheptanecarboxamide (12)
(104 mg, 60% yield): 1H NMR (500 MHz, pyridine-d5) d ppm
7.79 (d, J = 6.9, 1H), 7.71 (s, 1H), 7.62 (d, J = 7.7, 1H), 7.56 (s, 1H),
7.47 (t, J = 7.7, 1H), 4.48–4.40 (m, 1H), 3.54 (d, J = 13.2, 1H), 3.46
(d, J = 13.2, 1H), 2.86 (ddd, J = 3.0, 7.8, 16.6, 1H), 2.76 (dd, J = 3.0,
9.5, 1H), 2.51–2.39 (m, 2H), 2.37–2.30 (m, 2H), 2.26 (dd, J = 7.5,
9.4, 1H), 2.00–1.80 (m, 5H), 1.75–1.60 (m, 4H), 1.49–1.41 (m,
5.3.7. 2-ethyl-N-{(3aS⁄,4S⁄,6aR⁄)-2-[3-(Trifluoromethyl)benzyl]
octahydrocyclopenta[c]pyrrol-4-yl}butanamide (7)
4H), 1.33 (ddt, J = 7.2, 8.9, 12.1, 3H); MS (ESI+) m/z 409(M+H)+
;
(158 mg, 98% yield): 1H NMR (500 MHz, pyridine-d5) d ppm
7.90 (d, J = 6.7, 1H), 7.71 (s, 1H), 7.61 (d, J = 7.7, 1H), 7.54 (d,
J = 7.7, 1H), 7.46 (t, J = 7.7, 1H), 4.51–4.43 (m, 1H), 3.49 (s, 2H),
2.83 (ddd, J = 2.9, 7.8, 16.8, 1H), 2.75 (dd, J = 2.9, 9.5, 1H), 2.52–
2.43 (m, 1H), 2.37 (dd, J = 2.9, 9.1, 1H), 2.35–2.30 (m, 1H), 2.25
(dd, J = 7.6, 9.4, 1H), 2.01 (tt, J = 4.8, 9.5, 1H), 1.89 (dt, J = 7.3,
11.6, 1H), 1.77 (ddt, J = 7.5, 9.3, 15.2, 2H), 1.70–1.60 (m, 2H),
1.51–1.37 (m, 2H), 1.34–1.26 (m, 1H), 0.95 (t, J = 7.4, 3H), 0.83
Anal. (C23H31F3N2O) C, H, N.
5.3.13. 2,2-Diphenyl-N-{(3aS⁄,4S⁄,6aR⁄)-2-[3-(trifluoromethyl)
benzyl]octahydrocyclopenta[c]pyrrol-4-yl}acetamide (13)
(100 mg, 39% yield): 1H NMR (500 MHz, pyridine-d5) d ppm
8.50 (s, 1H), 7.71 (d, J = 7.5, 2H), 7.63 (d, J = 7.2, 2H), 7.45 (d,
J = 7.4, 2H), 7.39–7.28 (m, 5H), 7.25 (dd, J = 5.8, 8.9, 3H), 4.51–
4.42 (m, 1H), 3.44 (d, J = 13.3, 1H), 3.25 (d, J = 13.3, 1H), 2.85 (qd,
J = 3.2, 7.9, 1H), 2.59 (dd, J = 3.4, 9.5, 1H), 2.47–2.37 (m, 1H), 2.26
(t, J = 8.3, 1H), 2.21–2.13 (m, 2H), 1.85–1.76 (m, 1H), 1.67–1.52
(m, 3H), 1.27–1.18 (m, 1H); MS (ESI+) m/z 479(M+H)+ ; Anal.
(C29H29F3N2O.1.4HCl) C, H, N.
(t, J = 7.4, 3H); MS (ESI+) m/z 383(M+H)+.
;
Anal.
(C21H29F3N2O.0.06CH2Cl2) C, H, N.
5.3.8. 2-Propyl-N-{(3aS⁄,4S⁄,6aR⁄)-2-[3-(trifluoromethyl)ben
zyl]octahydrocyclopenta[c]pyrrol-4-yl}pentanamide (8)
(130 mg, 75% yield): 1H NMR (500 MHz, pyridine-d5) d ppm
7.94 (d, J = 6.9, 1H), 7.72 (s, 1H), 7.62 (d, J = 7.7, 1H), 7.56 (d,
J = 7.7, 1H), 7.47 (t, J = 7.7, 1H), 4.48 (dt, J = 7.3, 14.6, 1H), 3.52
(d, J = 13.0, 1H), 3.48 (d, J = 13.0, 1H), 2.84 (ddd, J = 3.0, 7.7,
16.7, 1H), 2.74 (dd, J = 3.1, 9.5, 1H), 2.52–2.45 (m, 1H), 2.38–
2.34 (m, 2H), 2.27 (dd, J = 7.6, 9.5, 1H), 2.22 (td, J = 4.6, 9.5,
5.3.14. 2,2-Bis(4-fluorophenyl)-N-{(3aS⁄,4S⁄,6aR⁄)-2-[3-(trifluo
romethyl)benzyl]octahydrocyclopenta[c]pyrrol-4-yl}acetamide
(14)
(51 mg, 22% yield): 1H NMR (500 MHz, pyridine-d5) d ppm 8.68
(d, J = 7.0, 1H), 7.64 (s, 1H), 7.60 (t, J = 6.4, 1H), 7.57–7.54 (m, 2H),
7.52–7.43 (m, 4H), 7.17–7.07 (m, 4H), 4.48–4.41 (m, 1H), 3.46 (d,

X. Beebe et al. / Bioorg. Med. Chem. 20 (2012) 4128–4139
4137
J = 13.3, 1H), 3.30 (d, J = 13.3, 1H), 2.90 (qd, J = 3.5, 8.0, 1H), 2.59
5.5. Biological assays
(dd, J = 3.5, 9.5, 1H), 2.50–2.42 (m, 1H), 2.32 (t, J = 8.3, 1H), 2.25–
2.19 (m, 3H), 1.87–1.77 (m, 1H), 1.66 (td, J = 5.7, 11.4, 1H), 1.62–
1.54 (m, 1H), 1.29–1.20 (m, 1H); MS (ESI+) m/z 515(M+H)+ ; Anal.
(C29H27F5N2O.1HCl) C, H, N.
5.5.1. N-type FLIPR Assay
The N-type FLIPR assay was performed as described previ-
ously.17,24 Briefly, IMR32 human neuroblastoma cells were plated
at a density of 120,000 cells per well and incubated for 48 h at
37 °C with 5% CO2. Cells were incubated with 100
cium 4 dye prepared in HBSS containing 1 mM HEPES, pH 7.4 for
1.5 h at room temperature. Antagonists (50 l) were pre-incubated
during dye loading for 1 h prior to the addition of depolarizing buf-
fer (50 l, HBSS) containing 80 mM KCl in the presence of 5 mM
CaCl2. Experiments were completed in the presence of 2 M nifed-
ipine to block endogenous L-type calcium channels. Data are plot-
ted as the percent of control response in the absence of antagonist
and IC50 values were determined following nonlinear regression
analysis.
ll of diluted Cal-
5.3.15. 3-Methyl-2-phenyl-N-{(3aS⁄,4S⁄,6aR⁄)-2-[3-(trifluoro
methyl)benzyl]octahydrocyclopenta[c]pyrrol-4-yl}butanamide
(15)
l
(745 mg, 95% yield, white solid): 1H NMR (500 MHz, pyridine-
d5) d ppm 8.23 (d, J = 6.9, 0.5H), 8.13 (d, J = 7.1, 0.5H), 7.73 (s,
0.5H), 7.67 (s, 0.5H), 7.63 (dd, J = 6.7, 8.0, 2H), 7.56–7.53 (m, 1H),
7.50 (t, J = 8.5, 2H), 7.38–7.22 (m, 3H), 4.45–4.38 (m, 0.5H), 4.33–
4.26 (m, 0.5H), 3.50 (s, 1H), 3.44 (d, J = 13.4, 0.5H), 3.21 (d,
J = 13.4, 0.5H), 3.17 (d, J = 10.5, 0.5H), 3.10 (d, J = 10.5, 0.5H), 2.91
(qd, J = 3.4, 7.9, 0.5H), 2.79 (dd, J = 3.4, 9.5, 0.5H), 2.74–2.56 (m,
1.5H), 2.47–2.31 (m, 2.5H), 2.27 (dd, J = 3.1, 8.9, 0.5H), 2.21 (d,
J = 2.7, 0.5H), 1.98–1.93 (m, 0.5H), 1.89–1.79 (m, 0.5H), 1.74–1.56
(m, 1.5H), 1.53–1.46 (m, 0.5H), 1.46–1.39 (m, 0.5H), 1.30–1.23
(m, 1H), 1.19 (d, J = 6.5, 1.5H), 1.17–1.11 (m, 0.5H), 1.08 (d,
J = 6.5, 1.5H), 0.72 (dd, J = 6.7, 9.6, 3H); MS (ESI+) m/z 445
(M+H)+; Anal. (C26H31F3N2O) C, H, N.
l
l
5.5.2. L-type FLIPR assay
ND7/23 cells were maintained in Dulbecco’s Modified Eagle’s
Medium (DMEM) containing 10% (v/v) heat-inactivated FBS. Fol-
lowing dissociation with enzyme-free PBS-based buffer, cells were
resuspended in growth media and seeded at a density of 5.0 ꢁ 104
cells per well in 96-well poly-D-Lysine coated plates. Cells were
incubated for 24 h at 37 °C in a humidified incubator with 5%
CO2 prior to use in the calcium mobilization assay. Calcium dye
(10ꢁ) (Molecular Devices) was diluted 1:10 with HBSS containing
20 mM HEPES, pH 7.4 (buffer A). The growth media was removed
using gently aspiration and the cells were then incubated with
5.3.16. N-[(3aS,4S,6aR)-2-Benzyloctahydrocyclopenta[c]pyrrol-
4-yl]-2,2-dicyclohexylacetamide (23)
(468 mg, 77% yield): 1H NMR (500 MHz, pyridine-d5) d ppm
7.44–7.38 (m, 4H), 7.37–7.32 (m, 1H), 4.49–4.38 (m, 1H), 3.57
(d, J = 12.6, 1H), 3.37 (d, J = 12.5, 1H), 2.77 (dd, J = 1.7, 9.6, 1H),
2.68 (td, J = 2.0, 9.3, 1H), 2.51 (dd, J = 1.9, 9.0, 1H), 2.45 (dd,
J = 7.1, 15.9, 1H), 2.30–2.25 (m, 1H), 2.11 (dd, J = 7.1, 9.6, 1H),
1.98–1.83 (m, 3H), 1.83–1.58 (m, 12H), 1.57–1.51 (m, 1H), 1.48
(d, J = 13.0, 1H), 1.44–1.37 (m, 1H), 1.36–1.09 (m, 9H), 1.04–
0.94 (m, 1H); MS (ESI+) m/z 423(M+H)+; Anal. (C28H42N2O-
0.5H2O) C, H, N.
100
lL of diluted calcium dye for 1.5 h at room temperature. The
compounds (50
ll) prepared in buffer A were incubated with the
cells for 3 min at room temperature prior to activation of L-type
Ca2+ channels by addition of a high concentration of KCl and CaCl2,
50 mM and 2.5 mM, respectively in HBSS in 50 ll. Data are plotted
as the percent of control response in the absence of antagonist and
IC50 values were determined using nonlinear regression analysis.
5.3.17. 2,2-Dicyclohexyl-N-{(3aR,4R,6aS)-2-[3-(trifluoromethyl)
benzyl]octahydrocyclopenta[c]pyrrol-4-yl}acetamide (37)
5.5.3. Whole cell patch clamp studies
The N-type cell line stably co-expressing rat recombinant CaV2.2
exons [D18a, D24a, 31a, 37b], CaVb3, and a2d1 subunits was ob-
tained from Dr. Diane Lipscombe (Brown University). Cells were
maintained at 37 °C and 5% CO2 in growth media containing Dul-
becco’s modified Eagle’s medium supplemented with 10% fetal bo-
vine serum, 1% GlutaMAX, 100 IU/mL penicillin, 100 mg/mL
(0.780 g, 45% yield): ½a D20
ꢂ
+68.9o (c 1.02, CH3OH) 1H NMR
(500 MHz, pyridine-d5) d ppm 7.73 (s, 1H), 7.66 (d, J = 7.7, 1H),
7.55 (d, J = 7.3, 2H), 7.50 (t, J = 7.7, 1H), 4.50–4.43 (m, 1H), 3.54
(d, J = 12.9, 1H), 3.46 (d, J = 12.9, 1H), 2.74 (td, J = 2.6, 10.5, 2H),
2.51–2.42 (m, 2H), 2.31 (t, J = 8.3, 1H), 2.20 (dd, J = 7.8, 9.8, 1H),
1.94–1.54 (m, 15H), 1.46 (d, J = 13.0, 1H), 1.40 (d, J = 14.4, 1H),
1.35–1.07 (m, 9H), 0.98 (qd, J = 3.0, 12.3, 1H); MS (ESI+) m/z
491(M+H)+; Anal. (C29H41F3N2O.1HCl.2.3H2O) C, H, N.
streptomycin, 10
lg/mL blasticidin, 50 lg/mL hygromycin B, and
50 g/mL zeocin. All cell culture reagents were purchased from
l
Invitrogen (Carlsbad, CA). Assessment of the activity of compound
25 at N-type calcium channels was performed using whole cell
patch clamp analysis as described previously.17 Briefly, on the day
of the experiment, cells plated in a T25 cell culture flasks and grown
to 60–80% confluency were rinsed once in DPBS and dissociated
with 5 ml Detachin (Genlantis, San Diego, CA) at 37 °C for 4–
5 min. Dissociated cells were then centrifuged at 1200 rpm for
4 min at room temperature, the supernatant was removed, and
the pellet was re-suspended in growth media. Membrane currents
were recorded from the stable N-type HEK293 cell line using the
whole-cell patch clamp technique. Fire-polished borosilicate glass
5.4. Single crystal X-ray diffraction studies
A single crystal of compound 20 was mounted on a glass fiber.
Intensity data were collected on a Bruker SMART system equipped
with an APEX CCD camera. Data were collected at 173 K with
graphite-monochromated MoKa radiation (k = 0.71073 Å). Data
were collected in four sets using omega-phi scans with omega
steps of 0.3° and phi steps of 90°. A total of 2350 frames were col-
lected with 20 sframe exposures. Data were processed using
SaintPlus.25 Corrections for Lorentz-polarization effects were ap-
plied. Absorption was negligible. All structures were solved using
direct methods that yielded the non-hydrogen atoms. All pre-
sented hydrogen atoms were located in Fourier-difference elec-
tron density maps. All non-hydrogen atoms were refined
anisotropically. Hydrogen atoms associated with carbon atoms
were refined in geometrically constrained riding positions. The
hydrogen atom associated with the sulfonamide nitrogen atom
was included in the located positions. Refinement was achieved
with the use of SHELX-97.26
electrodes had resistances from 1–3 M
X when filled with internal
solution which contained (in mM): 101 CsCl, 24 CsF, 1.8 NaCl. , 7.4
EGTA, 9 HEPES, 4 Mg2ATP, 14 Creatine phosphate, and 0.3 GTP, pH
7.4 adjusted with CsOH and an osmolarity of approximately 290
mOsM. Cell were superfused with extracellular solution which con-
tained (in mM): 87.5 CsCl, 40 (TEA)-Cl, 10 HEPES, 10 glucose, 5
BaCl2, and 1 MgCl2, pH 7.2, 310 mOsm. To evaluate compound po-
tency, cells were first voltage clamped at ꢀ115 mV and stepped to
0 mV once every 15 s to determine the cells maximal current. Cells

4138
X. Beebe et al. / Bioorg. Med. Chem. 20 (2012) 4128–4139
were then voltage-clamped at ꢀ80 to ꢀ95 mV and stepped to 0 mV
once every 5 s to induce a degree of channel inactivation. After a sta-
ble control baseline current was obtained, compound was added in
escalating concentrations of 1 to 3 concentrations per cell and per-
cent control values were determined after steady state inhibition
was achieved at each concentration. GraphPad Prism (GraphPad
Software, San Diego, CA) was used for data plotting and data fitting.
hypersensitivity of paw withdrawal latency (PWL) was calculated
as the mean of the two shortest latencies. Compound 25 (3, 10,
30 mg/kg, ip, n = 6) was administered 30 min prior to the assess-
ment of thermal hypersensitivity.
5.6.3. Monoiodoacetatic acid (MIA) induced osteoarthritis pain
Under light (2–4%) isoflurane (Baxter, IL, USA) anesthesia, MIA
(3 mg/rat in 50 ll sterile isotonic saline) was intra-articularly in-
5.5.4. Rat aorta ring tissue relaxation assay
jected into right knee joint cavity of the hind leg using a 26G needle.
Measurements of peak hind limb grip force were conducted at least
21 days following MIA-injection as described previously.28 Each rat
was sequentially tested twice at approximately 2-minute intervals
to obtain a maximum compressive grip force (CFmax) exerting on
the hindlimb strain gauge, in a commercially available grip force
measurement system (Columbus Instruments, Columbus, OH,
USA). The impact of body weight on grip force was calculated using
the formula: g (CFmax)/kg (body weight). Compound 25 (3, 10, and
30 mg/kg, ip) was administered 30 min prior to grip force assess-
ment. The vehicle control group was taken as 0%, whereas an age-
matched naïve group was designated as 100% (normal).
The activity of compound 25 at native L-type calcium channels
was assessed using rat aorta ring tissue as described previously.17
Briefly, male Sprague–Dawley rats (200–350 g; Charles River Labo-
ratories, Wilmington, MA) were euthanized with CO2 and decapi-
tated. The entire thoracic aorta was removed and immediately
placed into Krebs Ringer bicarbonate solution and equilibrated
with 5% CO2; 95% O2. Propranolol (Sigma–Aldrich, 0.004 mM was
included in all of the assays to block b adrenoreceptors. The aorta
was cut into 3–4 mm rings and mounted in 10 ml isolated tissue
baths at 37 °C. The aorta from each rat supplied 8 tissue rings with
one end of the aorta fixed to a stationary glass rod and the other to
a Grass FT03 transducer at a basal preload of 1.0 g. The aorta was
primed once with 80 mM KCl, rinsed and allowed to return to basal
5.6.4. Formalin induced persistent pain
tension and primed again with 10
l
M phenylephrine (PE). After an
The formalin assay was performed following the procedure orig-
additional 30 min equilibration period tension was established
using 80 mM KCl solution. A relaxation concentration response
curve was generated for each tissue. Data collection was by
Power-Lab/800 data acquisition system (Castle Hill, Australia).
The amount of compound 25 necessary to cause a 50% reversal
(ED50) of 80 mM KCl evoked contraction was calculated by nonlin-
ear regression analysis using the Levenberg–Marquardt curve fit-
ting algorithm in GraphPad Prism.
inally described by Dubuisson and Dennis29; 50
ll of a 5% formalin
solution was injected subcutaneously (s.c.) into the dorsal aspect of
the right hind paw and the rats were then individually placed into
clear observation cages. Rats were observed for a period of 20 min
(30–50 min following formalin injection).30 The number of flinching
behaviors of the injected paw was recorded using a sampling tech-
nique in which each animal was observed for one 60-second period
during each 5-minute interval; thus twelve 60-second observation
periods were recorded. Compound 25 (3, 10, 30 mg/kg, ip, n = 6)
was administered 30 min prior to formalin injection.
5.6. In vivo characterization
5.6.1. Animals
5.6.5. Rotarod
Male Sprague–Dawley rats (200–350 g, Charles River Laborato-
ries, Wilmington, MA, USA) were used for behavioral assessment.
All procedures were performed in an Association for Assessment
and Accreditation of Laboratory Animals Care (AAALAC) approved
facility and approved by the Institutional Animals Care and Use
Committee (IACUC) at Abbott Laboratories. Compound 25 was
administered by intraperitoneal injection (ip), prepared in 10%
dimethylsulfoxide (DMSO)/polyethylene glycol 400 (PEG) (Sigma-
Aldrich, St. Louis, MO, USA) in a volume of 2.0 ml/kg. One way anal-
ysis of variance (one-way ANOVA) was performed for all data
analysis. Post hoc analyses were performed using Bonferroni: com-
pared selective pairs of columns for multiple comparisons (Graph-
Pad Prism, GraphPad Software. Inc., USA).
Rotarod performance was measured using an accelerating rota-
rod apparatus (Omnitech Electronics, Inc. Columbus, OH, USA) as
described previously.17 Each rat was given three training sessions
by being placed on a 9 cm diameter rod that increased in speed
from 0 to 20 rpm over a 60 s period. The time required for the
rat to fall from the rod was recorded, with a cut off score of 60 s.
Compound 25 (3, 10 and 30 mg/kg, ip, n = 6 per group) was then
administered, and the animals were placed back on the rotarod
apparatus for 3 consecutive testing trials 30 min following the
compound administration.
5.6.6. Anesthetized rat cardiovascular studies
The effects of Compound 25 on hemodynamic and cardiovascu-
lar function in the anesthetized rat were performed as described
previously.31
5.6.2. Carrageenan thermal hypersensitivity
Acute thermal hypersensitivity was induced by injecting 100 ll
of a 1% solution of k-carrageenan (Sigma Chemical Co., St. Louis,
MO) in physiological saline into the plantar surface of the right
hind paw. Thermal hypersensitivity was determined 2 h following
carrageenan injection, using a commercially available thermal paw
stimulator (UARDG, University of California, San Diego, CA) as de-
scribed by Hargreaves et al.27 Rats were placed into individual
plastic cubicles mounted on a glass surface maintained at 30 °C,
where a thermal stimulus was then applied to the plantar surface
of each hind paw. The stimulus current was maintained at
4.50 0.05 amp, and the maximum time of exposure was set at
20.48 s to limit possible tissue damage. The elapsed time until a
brisk withdrawal of the hind paw from the thermal stimulus was
recorded automatically using photodiode motion sensors. The right
and left hind paw of each rat was tested in 3 sequential trials at
approximately 5 min intervals. Carrageenan-induced thermal
Acknowledgements
The authors would like to thank the HT-ADME group for micro-
somal stability and solubility data, the structural chemistry group
for 1H NMR and MS on all compounds and Paul Wiedeman for
careful reading of the manuscript.
References and notes
1. Kisilevsky, A. E.; Zamponi, G. W. Handb. Exp. Pharmacol. 2008, 184, 45.
2. Cao, Y. Pain 2006, 126, 5.
3. Ertel, E. A.; Campbell, K. P.; Harpold, M. M.; Hofmann, F.; Mori, Y.; Perez-Reyes,
E.; Schwartz, A.; Snutch, T. P.; Tanabe, T.; Birnbaumer, L.; Tsien, R. W.; Catterall,
W. A. Neuron 2000, 25, 533.
4. Barrett, T. D.; Triggle, D. J.; Walker, M. J. A.; Maurice, D. H. Mol. Interv. 2005, 5,
84.

X. Beebe et al. / Bioorg. Med. Chem. 20 (2012) 4128–4139
4139
5. McGivern, J. G. Drug Discovery Today 2006, 11, 245.
Jochnowitz, N.; Liang, A.; MacIntyre, D. E.; McGowan, E.; Mistry, S.; White, V.
V.; Hoyt, S. B.; London, C.; Lyons, K. A.; Bunting, P. B.; Volksdorf, S.; Duffy, J. L. J.
Pharm. Exp. Ther. 2010, 334, 545.
6. Yamamoto, T.; Takahara, A. Curr. Top. Med. Chem. 2009, 9, 377.
7. Schmidtko, A.; Lotsch, J.; Freynhagen, R.; Geisslinger, G. Lancet 2010, 375, 1569.
8. Rauck, R. L.; Wallace, M. S.; Leong, M. S.; MineHart, M.; Webster, L. R.;
Charapata, S. G.; Abraham, J. E.; Buffinton, D. E.; Ellis, D.; Kartzinel, R. J. Pain
Symptom Manage 2006, 31, 393.
9. Pajouhesh, H.; Feng, Z.-P.; Ding, Y.; Zhang, L.; Pajouhesh, H.; Morrison, J.-L.;
Belardetti, F.; Tringham, E.; Simonson, E.; Vanderah, T. W.; Porreca, F.;
Zamponi, G. W.; Mitscher, L. A.; Snutch, T. P. Bioorg. Med. Chem. Lett. 2010,
20, 1378.
17. Scott, V. E.; Vortherms, T. A.; Niforatos, W.; Swensen, A. M.; Neelands, T.;
Milicic, I.; Banfor, P. N.; King, A.; Zhong, C.; Simler, G.; Zhan, C.; Bratcher, N.;
Boyce-Rustay, J. M.; Zhu, C. Z.; Bhatia, P.; Doherty, G.; Mack, H.; Stewart, A. O.;
Jarvis, M. F. Biochem. Pharmacol. 2012, 83, 406.
18. Doherty, G.; Bhatia, P.; Vortherms, T. A.; Marsh, K. C.; Wetter, J. M.; Mack, H.;
Scott, V. E.; Jarvis, M. F.; Stewart, A. O. Bioorg. Med. Chem. Lett. 2012, 22, 1716.
19. Padwa, A.; Dent, W. J. Org. Chem. 1987, 52, 235.
10. Zamponi, G. W.; Feng, Z.-P.; Zhang, L.; Pajouhesh, H.; Ding, Y.; Belardetti, F.;
Pajouhesh, H.; Dolphin, D.; Mitscher, L. A.; Snutch, T. P. Bioorg. Med. Chem. Lett.
2009, 19, 6467.
11. Yamamoto, T.; Niwa, S.; Ohno, S.; Tokumasu, M.; Masuzawa, Y.; Nakanishi, C.;
Nakajo, A.; Onishi, T.; Koganei, H.; Fujita, S.; Takeda, T.; Kito, M.; Ono, Y.; Saitou,
Y.; Takahara, A.; Iwata, S.; Shoji, M. Bioorg. Med. Chem. Lett. 2008, 18, 4813.
12. Yamamoto, T.; Niwa, S.; Iwayama, S.; Koganei, H.; Fujita, S.; Takeda, T.; Kito, M.;
Ono, Y.; Saitou, Y.; Takahara, A.; Iwata, S.; Yamamoto, H.; Shoji, M. Bioorg. Med.
Chem. 2006, 14, 5333.
13. Knutsen, L. J.; Hobbs, C. J.; Earnshaw, C. G.; Fiumana, A.; Gilbert, J.; Mellor, S. L.;
Radford, F.; Smith, N. J.; Birch, P. J.; Ward, S. D.; Russell Burley, J.; James, I. F.
Bioorg. Med. Chem. Lett. 2007, 17, 662.
14. Hu, L.-Y.; Ryder, T. R.; Rafferty, M. F.; Cody, W. L.; Lotarski, S. M.; Miljanich, G.
P.; Millerman, E.; Rock, D. M.; Song, Y.; Stoehr, S. J.; Taylor, C. P.; Weber, M. L.;
Szoke, B. G.; Vartanian, M. G. Bioorg. Med. Chem. Lett. 1999, 9, 907.
15. Hu, L.-Y.; Ryder, T. R.; Nikam, S. S.; Millerman, E.; Szoke, B. G.; Rafferty, M. F.
Bioorg. Med. Chem. Lett. 1999, 9, 1121.
20. Terao, Y.; Kotaki, H.; Imai, N.; Achiwa, K. Chem. Pharm. Bull. 1985, 8, 2762.
21. Santora, V. J.; Covel, J. A.; Hayashi, R.; Hofilena, B. J.; Ibarra, J. B.; Pulley, M. D.;
Weinhouse, M. I.; Sengupta, D.; Duffield, J. J.; Semple, G.; Webb, R. R., et al
Bioorg. Med. Chem. Lett. 2008, 18, 1490.
22. Robak, M. T.; Herbage, M. A.; Ellman, J. A. Chem. Rev. 2010, 110, 3600.
23. Tanuwidjaja, J.; Peltier, H. M.; Ellman, J. A. J. Org. Chem. 2007, 72, 626.
24. Vortherms, T. A.; Swensen, A. M.; Niforatos, W.; Limberis, J. T.; Neelands, T. R.;
Janis, R. S.; Thimmapaya, R.; Donnelly-Roberts, D. L.; Namovic, M. T.; Zhang, D.;
Putman, C. B.; Martin, R. L.; Surowy, C. S.; Jarvis, M. F.; Scott, V. E. Inflamm. Res.
2011, 60, 683.
25. Bruker AXS Inc., SaintPlus, version 6.02, Madison Wisconsin, 1999.
26. Sheldrick, G. M. University of Göttingen, Germany, 1998.
27. Hargreaves, K.; Dubner, R.; Brown, F.; Flores, C.; Joris, J. Pain 1988, 32, 77.
28. Chandran, P.; Pai, M.; Blomme, E. A.; Hsieh, G. C.; Decker, M. W.; Honore, P. Eur.
J. Pharmacol. 2009, 613, 39.
29. Dubuisson, D.; Dennis, S. G. Pain 1977, 4, 161.
30. Abbott, F. V.; Franklin, K. B.; Westbrook, R. F. Pain 1995, 60, 91.
31. Liu, G.; Zhao, H.; Liu, B.; Xin, Z.; Liu, M.; Serby, M. D.; Lubbers, N. L.; Widomski,
D. L.; Polakowski, J. S.; Beno, D. W. A., et al Bioorg. Med. Chem. Lett. 2007, 17,
495.
16. Abbadie, C.; McManus, O. B.; Sun, S.-Y.; Bugianesi, R. M.; Dai, G.; Haedo, R. J.;
Herrington, J. B.; Kaczorowski, G. J.; Smith, M. M.; Swensen, A. M.; Warren, V.
A.; Williams, B.; Arneric, S. P.; Eduljee, C.; Snutch, T. P.; Tringham, E. W.;