A new synthesis of the ORL-1 antagonist 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidinyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (J-113397) and activity in a calcium mobilization assay

Article abstract of DOI:10.1016/j.bmc.2007.10.023

A new chiral synthesis of the ORL-1 antagonist 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidinyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (2, J-113397) was developed. J-113397 has a K_e = 0.85 nM in an ORL-1 calcium mobilization assa

Full text of DOI:10.1016/j.bmc.2007.10.023

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 822–829
A new synthesis of the ORL-1 antagonist
1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidinyl]-3-
ethyl-1,3-dihydro-2H-benzimidazol-2-one (J-113397)
and activity in a calcium mobilization assay
Emilie D. Smith,a N. Ariane Vinson,a Desong Zhong,a Bertold D. Berrang,a
a
a
a
´
Jennifer L. Catanzaro, James B. Thomas, Hernan A. Navarro,
Brian P. Gilmour,a Jeffrey Deschampsb and F. Ivy Carrolla,*
aOrganic and Medicinal Chemistry, Research Triangle Institute, PO Box 12194, Research Triangle Park, NC 27709, USA
bLaboratory for the Structure of Matter, Naval Research Laboratory, 4555 Overlook Avenue, Washington, DC 20375-5341, USA
Received 19 July 2007; revised 21 September 2007; accepted 10 October 2007
Available online 13 October 2007
Abstract—A new chiral synthesis of the ORL-1 antagonist 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidinyl]-3-ethyl-
1,3-dihydro-2H-benzimidazol-2-one (2, J-113397) was developed. J-113397 has a Ke = 0.85 nM in an ORL-1 calcium mobilization
assay and is 89-, 887-, and 227-fold selective for the ORL-1 receptor relative to the l, d, and j opioid receptors.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
The first selective small organic molecule antagonist,
1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-pipe-
ridinyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (J-
113397, 2, Chart 1), was reported by Ozaki et al. in
1998.13–15 The lead compound 1-(1-benzyl-4-piperidi-
nyl)-1,3-dihydro-2H-benzimidazol-2-one (3, Chart 1)
was identified from a chemical library and showed high
affinity for ORL-1 but poor selectivity relative to l- and
j-receptors.
The opioid-like receptor (ORL-1), also known as OP4
and recently named NOP (the fourth member of the opi-
oid peptide receptor family) or nociceptin/orphanin FQ
receptor, was identified in 1994 as an orphan opioid
receptor.1,2 Even though this G-protein coupled recep-
tor displays a significant homology with the classical
l-, j-, d-opioid receptors (NOP, KOP, and DOP,
respectively), it does not bind classical opioid ligands
with high affinity. A 17-amino acid peptide was identi-
fied in 1995 as an endogenous ligand for ORL-1 and
was named nociceptin (NC) or orphanin FQ (OFQ)
(1).3 It was found to be specific to ORL-1,4 as it did
not bind other opioid receptors. Nociceptin and ORL-
1 receptors are widely distributed in the central nervous
system and appear to play an important role in anxiety,5
pain,6 cognition,7–10 and locomotion.11 Importantly,
antagonists for this system may be useful in pain man-
agement without having the undesirable side effects of
opioids such as abuse, dependence, and withdrawal.12
In this study, we report a new synthesis of J-113397 and
present its potency at the ORL-1, l, d, and j opioid
receptors using in vitro efficacy assays.
2. Chemistry
The synthesis used for 1 is shown in Scheme 1. This syn-
thesis provides the desired optical isomer 2 without the
use of a chiral column needed in the originally reported
syntheses.13–17
Several groups have reported the reduction of piperidine
b-ketoester 5 generated from 4 with Baker’s yeast to be
both diastereoselective and enantioselective.17–19 We
were pleased to find that only the cis diastereomer 6
Keywords: J-113397; ORL-1 antagonist; Chiral synthesis; NOP.
*
Corresponding author. Tel.: +1 919 541 6679; fax: +1 919 541
8868; e-mail: fic@rti.org
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.10.023

E. D. Smith et al. / Bioorg. Med. Chem. 16 (2008) 822–829
823
H
N
N
O
N
O
N
N
OH
N
H-Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-
Ser-Ala-Arg-Lys-Leu-Ala-Asn-Gin-OH
2
3
1 (N/OFQ)
Chart 1.
OH
O
O
OH
N
CO2Et
CO2Et
CO2Et
OTBDMS
a
b
c,d
N
N
H
N
BOC
BOC
BOC
5
6
4
(+/-)
7
(+/-)
100%
78%
100%
N3
N
NH2
OTBDMS
OTBDMS
e
f,g,h
i
N
BOC
BOC
8
(+/-)
9
(+)
68%
H
N
O2N
HN
N
N
N
N
O
O
O
N
OTBDMS
l,m
OTBDMS
j,k
OH
n
OH
N
N
N
N
BOC
BOC
H
10
11
12
2
88%
91%
71%
42%
Scheme 1. Reagents: (a) (BOC)2O, Et3N, CH2Cl2; (b) Baker’s yeast, sucrose, tap water; (c) LiBH4,THF; (d) TBDMSCI, DMAP Et3N, CH2Cl2; (e)
ZnN6*2 Py, PPh3, DIAD, toluene; (f) H2, Pd/C, MeOH; (g) (R)-3-bromo-8-camphorsulfonic acid, ammonium salt; (h) NaHCO3, CHCl3; (i) 3-
fluoronitrobenzene, n-BuOH, Na2CO3; (j) Raney Ni, hydrazine hydrate, EtOH; (k) N,N0-disuccimidyl carbonate, DMF; (l) NaH, Etl, DMF; (m)
HCl, MeOH; (n) NaBH(OAc)3, THF, cyclooctanecarboxaldehyde.
was isolated in good yield (78%), however, in our hands
the enantioselectivity was quite low (ee around 30%) de-
spite numerous changes to conditions. Other groups
have reported this problem.20 with the activity of the
yeast at the heart of the problem. The optical rotation
of the product 6 varies from batch to batch of yeast (from
+5ꢁ to +15ꢁ). To overcome this problem, the single dia-
stereomer was reduced with lithium borohydride and
protected as the tert-butyldimethylsilyl ether 7. The sec-
ondary alcohol was converted to the azide 8 using the
Mitsunobu conditions with zinc azide/bis pyridine com-
plex,21 resulting in complete inversion of configuration
at C4 of the piperidine ring. The primary amine, ( )-9,
obtained by hydrogenation of the azide, was resolved
to give (+)- and (ꢀ)-9 using (+)- and (ꢀ)-3-bromo-8-
camphorsulfonic acid ammonium salt. A single-crystal
X-ray analysis of the (ꢀ)-3-bromo-8-camphorsulfonic
acid salt showed that (ꢀ)-9 possessed the (3S,4S)-stereo-
chemistry and thus (+)-9 was the (3R,4R)-isomer (Fig. 1).
Thus, (+)-9 was used to complete the synthesis of 2.
From this versatile intermediate, the synthesis of 2 pro-
ceeded by coupling the chiral 4-amino-piperidine (+)-9
with 1-fluoro-2-nitrobenzene to give 10 in high yield
(88%). Reduction of the nitro-group using Raney Nickel
and hydrazine hydrate in ethanol followed by treatment
with N,N0-disuccinimidyl carbonate afforded benzimi-
dazolone 11 in 91% yield. Chiral-HPLC analysis of 11
showed a 99% ee. Alkylation of the benzimidazolone
nitrogen with sodium hydride and iodoethane in DMF,
followed by removal of the tert-butyloxycarbonyl and
tert-butyldimethylsilanyl protecting groups with hydro-
gen chloride in methanol, afforded the deprotected piper-

824
E. D. Smith et al. / Bioorg. Med. Chem. 16 (2008) 822–829
phospholipase C and the mobilization of internal cal-
cium in addition to Gi. The transfected cells were se-
lected in 400 lg/mL geneticin. The clone used for this
experiment was selected after screening the surviving
cells for functional ORL-1 receptor expression using
nociceptin and the Calcium 3 assay kit (Molecular De-
vices). These assays were run according to manufac-
turer’s specifications using a FlexStationII384 plate
reader (Molecular Devices) pre-warmed to 37 ꢁC.
3.2. Functional assays and data analysis
Compound 2 was evaluated at 10 lM for intrinsic activ-
ity and for its ability to inhibit the mobilization of inter-
nal calcium and subsequent fluorescence elicited by
nociceptin (EC80; 1.5 nM Table 1). All test conditions
were run in duplicate. For these assays, 96-well cell cul-
ture plates were seeded with 10,000 cells the day before
the assay. On the day of assay, the cells were incubated
at 37 ꢁC with the Calcium 3 fluorescent dye (1/3 the stan-
dard concentration) for 1 h. For the antagonist assays,
the test compound was added fifteen minutes before
the end of the incubation period. The plate was then
placed into the FlexStation (37 ꢁC) and basal fluores-
cence recorded for 13 s followed by the addition of test
compound or nociceptin (EC80), and fluorescence re-
corded for an additional 47 s. The effect of 2 on the mobi-
lization of internal calcium was determined using the
MAX–MIN option in the recording software. Because
2 did not possess detectable intrinsic activity and it inhib-
ited nociception-stimulated mobilization of internal cal-
cium, it had its apparent affinity (Ke) determined. For
these experiments, we assessed the ability of a single con-
centration of 2 to shift the nociceptin concentration re-
sponse curve to the right using the same assay
conditions described above. The EC50 values for noci-
ceptin (A) and nociceptin + 2 (A0) were used to calculate
the test compound Ke using the formula: Ke = [L]/
(DR ꢀ 1), where [L] equals the concentration of 2 in
the assay and DR equals the dose ratio or A0/A. The con-
centration of test compound was selected to produce at
least a twofold increase in the nociceptin EC50, and eight
different concentrations of nociceptin were used such
that clear upper and lower asymptotes were obtained
for both curves. Two different concentrations of 2 were
used to determine its Ke. Compound 2 was then evalu-
ated for its activity (agonist and antagonist) and selectiv-
ity for the human j, l, and d opioid receptors using the
[35S]GTPcS functional binding assay and membrane
homogenates prepared from CHO cells stably expressing
one of these receptors. The [35S]GTPcS binding assays
were conducted as previously described.22 A three-
parameter logistic equation (shared bottom and top)
was fitted to the concentration response data with Prism
(v4 for Macintosh, GraphPad Software; San Diego, CA)
Figure 1. The conformation of (ꢀ)-9 as determined by single-crystal
X-ray diffraction. The 3S4S conformation was determined with respect
to a reference molecule (3-bromocamphorsulfonic acid) which is not
shown in the figure. All hydrogen atoms (except those on C3 and C4)
and one of the positions for the disordered tert-butyl group off N1
have been omitted for clarity. Displacement ellipsoids are shown at the
30% level.
idine intermediate 12. Reductive alkylation of 12 with
cyclooctanecarboxaldehyde using sodium triacetoxy-
borohydride afforded 2 (J-113397) with an optical rota-
tion almost identical to that previously described
20
20
D
{observed ½aꢁ +6.25 (c 0.272, 0.1 M HCl), lit. ½aꢁ
D
+6.4 (c 1, 0.1 M HCl)}.
3. Biology
3.1. Creation of an ORL-1 stable cell line
Human ORL-1 cDNA in the mammalian expression
vector pcDNA3.1+ was purchased from the University
of Missouri-Rolla cDNA Resource Center. CHO cells
stably expressing the promiscuous Gq protein Ga16
(Molecular Devices, Sunnyvale, CA) were transfected
with the ORL-1 vector using Lipofectamine 2000 (Invit-
rogen, Carlsbad, CA). This was done in order to create a
cell line where ORL-1 is also coupled to activation of
Table 1. Apparent affinities (Ke) for J-113397 (2) at cloned human ORL-1, l, d, and j receptorsa
Compound
Ke (nM)
j
ORL-1
l
d
l/ORL-1
d/ORL-1
j/ORL-1
2
0.85 .09
75.6 15
754 193
195 34
89
887
227
a The apparent affinities (Ke) for 2 represent the mean SE from at least three independent experiments.

E. D. Smith et al. / Bioorg. Med. Chem. 16 (2008) 822–829
825
to calculate the EC50 values. These values are reported as
means SE from at least three independent experiments.
CombiFlash Companion and RediSep columns by Isco,
using the solvent system specified in the section below.
Elemental analyses were carried out by Atlantic Micro-
lab, Inc. Baker’s yeast was purchased from ICN
biomedicals.
4. Results and discussion
The original synthesis13 of 2 as well as an improved syn-
thesis described by Kawamoto et al.17 and De Risi
et al.16 committed to a choice of N-substituent in the
first step and involved the use of a chiral column. In a
more recent report, Sulima et al. reported a new ap-
proach for the synthesis of J-113397 that did not require
a chiral column.23 In this study, we developed an alter-
nate synthetic approach to 2 where the N-substituent
is added in the last step (Scheme 1). Thus, in contrast
to the Kawamoto type synthesis.17 the present route
could be used to prepare 2 as well as N-substituent ana-
logs of 2. The present synthesis involves 14 steps starting
with 4 (one, an optical resolution; another, the neutral-
ization of the resolved salt to give the freebase) with
eight isolated intermediates. Note, however, intermedi-
ate 5 is commercially available. We chose to synthesize
5 since we had a large amount of 4 in hand. The overall
yield was 6%. The Kawamoto synthesis involves nine
steps (one being a separation of diastereoisomers and
one a resolution with a chiral column) with six isolated
intermediates. The overall yield was 8.3%.
5.1. Synthesis of 1-tert-butyl-3-ethyl-4-oxopiperidine-1,3-
dicarboxylate (5)
A solution of di-tert-butyl dicarbonate (35.5 g, 0.17 mol)
in CH2Cl2 (70 mL, anhydrous) was added dropwise to a
stirred solution of ethyl 4-oxopiperidine-3-carboxylate
hydrochloride (4, 30.4 g, 0.146 mol) and triethylamine
(29 mL, 208 mmol) in CH2Cl2 (70 mL, anhydrous).
After being stirred at rt for 16 h, the reaction mixture
was washed with HCl (1 M, 2· 250 mL) and brine (2·
150 mL). The organic layer was dried over Na2SO4
and evaporated to dryness under reduced pressure to af-
ford 40 g (100%) of 5 as a white solid. The material was
1
used without further purification. H NMR (CDCl3) d
12.06 (s, 1H), 4.25 (q, 2H, J = 6.9 Hz), 4.08 (br s, 2H),
3.58 (t, 2H, J = 5.85 Hz), 2.39 (t, 2H, J = 5.85 Hz),
1.49 (s, 9H), 1.32 (t, 3H, J = 6.9 Hz); 13C NMR (CDCl3)
d 170.8, 168.05, 154.6, 96.29, 80.1, 60.6, 40.7, 40.3, 28.7,
28.4, 14.2; MS (APCI) m/z 172.1 (M+HꢀBOC)+.
5.2. 1-tert-Butyl-3-ethyl (3,4-cis)-4-hydroxypiperidine-
1,3-dicarboxylate (6)
In agreement with the originally reported data,13 we
found that 2 is a potent antagonist at the ORL-1 recep-
tor (Ke = 0.85 nM). We found that 2 was 89-, 887-, and
227-fold selective for the ORL-1 receptor relative to the
l, d, and j opioid receptors, respectively. While these
selectivities are good, they are much less than the 956-,
>4347-, and 609-fold selectivity for the ORL-1 receptor
relative to the l, d, and j opioid receptors originally re-
ported using competition binding assays using opioid
receptors heterologously expressed in CHO cells.13
These differences in selectivity between the assays could
be due to our use of functional assays that assess the
ability of 2 to disrupt receptor activation by an agonist
as opposed to interfering with the binding of a radioli-
gand. Nevertheless, the rank order potency of 2 at the
various receptors is similar in both assays.
Baker’s yeast (210 g, dry active), sucrose (210 g, pure
cane sugar), and tap water (2100 mL) were mixed in a
vat (ꢂ20 L). The mixture was stirred for 6 h at rt, after
which no rising in the yeast was observed. To the yeast
mixture was added 1-tert-butyl-3-ethyl-4-oxopiperidine-
1,3-dicarboxylate (5) (26.1 g, 0.096 mol) in EtOH
(100 mL). After being stirred at rt for 65 h, the reaction
mixture was transferred to six 250 mL-centrifuge bottles
and centrifuged at 5000 rpm for 20 min at 8 ꢁC. The
supernatant in each bottle was decanted and the solids
were mixed with water (180 mL per bottle). The mixture
was centrifuged again at 5000 rpm for 20 min at 5 ꢁC.
The supernatants were pooled and the washing process
of the solids was repeated 2 more times. All the superna-
tants were combined and extracted with CH2Cl2 (5·
800 mL). The organic phases with a small amount of
emulsion were combined and washed with brine (1 L),
dried over Na2SO4, and filtered through a membrane fil-
ter with Celite on top. The solvent was evaporated and
In summary, a new synthesis of the ORL-antagonist 2,
which proceeds without the need of a chiral column,
was developed. Coupling the human ORL-1 to the
mobilization of internal calcium via Ga16 identified J-
113397 as a potent ORL-1 antagonist, and more impor-
tantly, it provides a rapid method to evaluate com-
pounds at this receptor.
the residue was dried in vacuo to give 18 g (69%) of 6
20
D
as a white solid. ½aꢁ +10.5 (c 1.34, CH2Cl2).
The remaining solids were mixed with MeOH (150 mL
per bottle). The mixture was filtered through a Celite
pad. The filter cake was washed with MeOH (2·
20 mL). All the filtrates were combined, concentrated
to about 250 mL, and extracted with CHCl3 (4·
50 mL). The extracts were combined, washed with brine
(3· 50 mL), and dried over Na2SO4. Evaporation of the
solvent under reduced pressure afforded an additional
5. Experimental
Melting points were determined on a Thomas–Hoover
capillary tube apparatus. All optical rotations were
determined at the sodium D line using a Rudolph Re-
search Autopol III polarimeter (1 dm cell). NMR spec-
tra were recorded on a 300 MHz (Bruker AVANCE
300) spectrometer using tetramethylsilane as internal
standard. Chromatography was performed using a
1
2.11 g (8%) of 6 as an orange oil (total yield: 77%). H
NMR (CDCl3) d 4.29 (br s, 1H), 4.19 (q, 2H,
J = 7.2 Hz), 4.17 (br s, 1H), 3.73 (dt, 1H, J = 3.6 and
13.2 Hz), 3.40 (br s, 1H), 3.34–3.25 (m, 1H), 2.66–2.61

826
E. D. Smith et al. / Bioorg. Med. Chem. 16 (2008) 822–829
(m, 1H), 1.88–1.83 (m, 1H), 1.69–1.66 (br s, 2H), 1.48 (s,
9H), 1.30 (t, 3H, J = 7.2 Hz); 13C NMR (CDCl3) d
173.4, 155.1, 104.5, 80.2, 65.4, 61.4, 46.3, 41.0, 39.1,
31.8, 29.0, 28.8, 14.5; MS (ESI) m/z 274.6 (M+H)+.
20% EtOAc–hexanes (2· 25 mL). All the filtrates were
combined and concentrated to 3.4 g. The crude product
was then purified by using a RediSep column (SiO2,
40 g) eluted with 0–10% B (0–5 min) (A = hexanes,
B = EtOAc), 10% B (5–13 min), 10–20% B (13–
17 min), 20% B (17–20 min), and 20–40% B (20–
5.3. 3-(tert-Butyldimethylsilanyloxymethyl)-piperidine-1-
carboxylic acid tert-butyl ester (7)
1
25 min), to give 0.882 g (68%) of 8 as a clear oil. H
NMR (CDCl3) d 4.07 (dd, 2H, J = 3.2 and 13.1 Hz),
3.67 (br s, 2H), 3.37 (br s, 2H), 2.81–2.73 (m, 2H),
2.03–1.98 (m, 1H), 1.60–1.55 (m, 3H), 1.46 (s, 9H),
0.90 (s, 9H), 0.07 (s, 3H), 0.06 (s, 3H); 13C NMR
(CDCl3) d 155.1, 132.6, 132.5, 129.0, 128.8, 80.2, 62.1,
59.6, 43.7, 42.5, 30.5, 28.9, 28.8, 26.4, 26.3, 22.1, ꢀ5.2.
To a solution of 1-tert-butyl-3-ethyl (3,4-cis)-4-hydrox-
ypiperidine-1,3-dicarboxylate (6) (8.78 g, 0.032 mol) in
anhydrous THF (1000 mL) was added dropwise a solu-
tion of LiBH4 in THF (16.1 mL, 32.2 mmol). The reac-
tion mixture was then heated at reflux for 10 min and
allowed to cool to rt over 20 min. The mixture was
cooled to 0 ꢁC and an aqueous saturated solution of
KHSO4 (1000 mL) was slowly added. The two layers
were separated and the aqueous phase was extracted
with EtOAc (2· 500 mL). The combined organic layers
(including the THF layer from the first separation) were
washed with brine (600 mL), dried over MgSO4, and
evaporated to dryness to give 7.43 g (99.5%) of (3,4-
cis)-4-hydroxy-3-hydroxymethyl-piperidine-1-carboxylic
5.5. 4-Amino-3-(tert-butyldimethylsilanyloxymethyl)-
piperidine-1-carboxylic acid tert-butyl ester (9)
Anhydrous methanol (100 mL) was added to a mixture
of 4-azido-3-(tert-butyldimethylsilanyloxymethyl)-piper-
idine-1-carboxylic acid tert-butyl ester (8) (5.74 g,
0.015 mol) and palladium on active carbon (10% wt,
0.6 g). The reaction flask was connected to a hydrogena-
tion system and the mixture was degassed and then stir-
red under H2 for 16 h. The reaction mixture was then
filtered through a pad of Celite and the filter cake was
washed with methanol (2· 50 mL). The filtrates were
combined and then evaporated to dryness to give
5.24 g of product. Purification of the product by flash
chromatography (SiO2, 40% EtOAc/hexanes) CHCl3–
CH3OH–NH4OH (80/18/2) then afforded 3.56 g (67%)
1
acid tert-butyl ester as a clear oil. H NMR (CDCl3) d
4.23–4.19 (m, 1H), 4.21 (t, 2H, J = 3 Hz), 3.56 (dd,
2H, J = 3.9 and 13.2 Hz), 3.43–3.36 (m, 2H), 3.29 (br
s, 1H), 1.87–1.68 (m, 4H), 1.47 (s, 9H); 13C NMR
(CDCl3) d 211.2, 80.1, 69.9, 63.4, 54.2, 42.0, 32.5, 32.1,
29.6, 28.8, 25.2; MS (APCI) m/z 132.2 (M+HꢀBOC)+.
To a solution of (3,4-cis)-4-hydroxy-3-hydroxymethyl-
piperidine-1-carboxylic acid tert-butyl ester (7.39 g,
0.032 mol) in anhydrous CH2Cl2 (65 mL) were added
4-dimethylaminopyridine (0.43 g, 0.035 mol), triethyl-
amine (5.8 mL, 41.54 mmol), and finally tert-butyldi-
methylsilyl chloride (5.8 g, 0.039 mol). The mixture
was stirred at rt for 16 h, during that time the reaction
turned cloudy with some solid at the bottom and on
the walls of the flask. The reaction mixture was diluted
with 500 mL of CH2Cl2, washed with water (2·
50 mL) and brine (50 mL). The organic layer was dried
over Na2SO4, evaporated to dryness, and dried under
vacuum to give 11 g (99.7%) of 7 as a clear oil. 1H
NMR (CDCl3) d 4.15 (br s, 1H), 3.73 (br s, 1H), 3.51
(dd, 2H, J = 3.9 and 13.2 Hz), 3.38–3.29 (m, 3H),
1.82–1.64 (m, 3H), 1.43 (s, 9H), 0.89 (s, 9H), 0.06 (s,
6H); 13C NMR (CDCl3) d 148.9, 107.0, 80.2, 68.4,
42.0, 39.4, 32.4, 28.8, 26.1, 18.4, ꢀ3.2; MS (APCI) m/z
246.5 (M+HꢀBOC)+.
1
of 9 as a clear oil. H NMR (CDCl3) d 4.07–4.01 (m,
2H), 3.66 (br s, 2H), 2.66 (t, 2H, J = 12 Hz), 2.52–2.44
(m, 1H), 1.76–1.70 (m, 1H), 1.40 (s, 9H), 1.35–1.22 (m,
4H), 0.84 (s, 9H), 0.06 (s, 6H); 13C NMR (CDCl3) d
155.2, 79.9, 63.4, 50.8, 46.8, 43.0, 35.6, 28.8, 28.5, 26.3,
18.6, ꢀ5.1; MS (APCI) m/z 345.5 (M+H)+.
5.6. Resolution of racemic amine 9 using (+)-3-bromo-8-
camphorsulfonic acid ammonium salt
4-Amino-3-(tert-butyldimethylsilanyloxymethyl)-piperi-
dine-1-carboxylic acid tert-butyl ester (9) (805 mg,
2.34 mmol) was dissolved in methanol (8 mL). To the
well-stirred solution was added [(1R)-(endo, anti)]-(+)-
3-bromocamphor-8-sulfonic acid ammonium salt
(390 mg, 1.17 mmol). The mixture was stirred until a
clear mixture was observed (about 20 min). The solvent
was slowly evaporated under a stream of N2 (3.5 h). An
additional volume of methanol (2 mL) was added and
evaporated under N2 to give a thick oil that was placed
under vacuum yielding a white foam. That foam was
dissolved in tert-butyl methyl ether (TBME) (2 mL).
The solvent was slowly evaporated under a stream of
N2 (0.5 h). Toluene (6 mL) was added, followed by hex-
anes (2 mL), and the solution was left at rt for 16 h. The
resulting crystals were washed with 50% toluene–hex-
anes (5 mL), then with hexanes (5 mL). The white crys-
tals were dried under vacuum to give 357 mg of resolved
complex. Part of the crystals (22 mg) was dissolved in
CHCl3 (1 mL), and NaHCO3 (saturated, 1 mL) was
added. The mixture was stirred at rt for 30 min. The
phases were separated and the aqueous layer was ex-
tracted with CHCl3 (2· 1 mL). The combined organic
5.4. 4-Azido-3-(tert-butyldimethylsilanyloxymethyl)-
piperidine-1-carboxylic acid tert-butyl ester (8)
To a solution of 3-(tert-butyldimethylsilanyloxymethyl)-
piperidine-1-carboxylic acid tert-butyl ester (7) (1.23 g,
0.0036 mol) and triphenylphosphine (1.88 g, 0.007 mol)
in anhydrous toluene (18 mL) was added zinc azide bis
pyridine complex (830 mg, 2.68 mmol). The mixture
was stirred at rt for 5 min and then cooled to 0 ꢁC.
Diisopropylazodicarboxylate (1.38 mL, 7.14 mmol) was
then slowly added to the mixture. The ice bath was re-
moved and the reaction mixture was stirred at rt for
4 h. The reaction mixture was then filtered through a
pad of Celite (3 cm). The filter cake was washed with

E. D. Smith et al. / Bioorg. Med. Chem. 16 (2008) 822–829
827
layers were washed with brine (1 mL), dried over
Na2SO4, and evaporated to dryness to give 14 mg of free
amine. ½aꢁ +11.53 (c 0.23, CH3OH).
and dried over Na2SO4. The solvent was evaporated un-
der reduced pressure and the residue was purified by col-
umn chromatography (SiO2, 10% EtOAc/hexanes) to
afford 192 mg (88%) of pure product 10. 1H NMR
(CDCl3) d 8.18 (d, 1H, J = 8.67 Hz), 8.09 (d, 1H,
J = 8.29 Hz), 7.40 (t, 1H, J = 7.54 Hz), 7.02 (br s, 1H),
6.64 (t, 1H, J = 7.8 Hz), 4.28–3.95 (m, 2H), 3.71–3.65
(m, 3H), 3.20–2.76 (m, 2H), 2.09 (br d, 1H,
J = 12 Hz), 1.75–1.74 (m, 1H), 1.49 (s, 9H), 0.87 (s,
9H), 0.01 (s, 3H), ꢀ0.07 (s, 3H); 13C NMR (CDCl3) d
162.5, 154.9, 145.3, 136.4, 127.3, 115.6, 114.4, 80.1,
61.7, 50.1, 44.3, 28.6, 28.0, 18.7, ꢀ5.5; MS (APCI) m/z
366.9 (M+HꢀBOC)+.
20
D
The mother liquor was dissolved in CHCl3 (10 mL) and
treated with NaHCO3 (saturated, 10 mL). The mixture
was stirred at rt for 30 min. The phases were separated
and the aqueous layer was extracted with CHCl3 (2·
10 mL). The combined organic layer was washed with
brine (10 mL), dried over Na2SO4, and evaporated to
20
D
dryness to give 560 mg of free amine. ½aꢁ ꢀ5.5, (c
1.09, CH3OH).
The rest of the resolved complex was dissolved in CHCl3
(20 mL) and treated with NaHCO3 (saturated, 20 mL).
The mixture was stirred at rt for 30 min. The phases
were separated and the aqueous layer was extracted with
CHCl3 (2· 10 mL). The combined organic layer was
washed with brine (10 mL), dried over Na2SO4, and
evaporated to dryness to give 0.177 g of resolved free
amine.
5.9. 3-(tert-Butyldimethylsilanyloxymethyl)-4-(2-oxo-2,3-
dihydrobenzimidazol-1-yl)-piperidine-1-carboxylic acid
tert-butyl ester (11)
To a solution of pure nitro compound 10 (587 mg,
1.26 mmol) in EtOH (20 mL) were added Raney Nick-
el (2.2 mL) and hydrazine hydrate (0.74 mL, 15 mmol).
The mixture was stirred at rt for 2 h and then filtered
through a pad of Celite. The flask was washed with
EtOH (50 mL) and the washings were filtered through
the pad. The filtrates were combined and concentrated.
The residue was dissolved in Et2O (10 mL), washed
with brine (10 mL), and dried over Na2SO4. The
solvent was evaporated under reduced pressure and
the residue was dried in vacuo to give 510 mg (91%)
of 3-(tert-butyldimethylsilanyloxymethyl)-4-(2-amin-
ophenylamino)-piperidine-1-carboxylic acid tert-butyl
5.7. Resolution of 9 using (ꢀ)-3-bromo-8-camphorsulfonic
acid ammonium salt
From 343 mg of 9 (1 mmol) and [(1S)-endo, anti]-(ꢀ)-3-
bromocamphor-8-sulfonic acid ammonium salt (333 mg,
1.0 mmol) in methanol as described above the respective
(ꢀ)-sulfonic acid salt was prepared. The resulting glassy
solid was dissolved in 2 mL of TBME at ambient tem-
perature and allowed to crystallize undisturbed for
15 h. The solution was very slowly concentrated to half
the volume over a 4-h period. The crystalline salt was
carefully separated by filtration, washed with three
0.4 mL portions of TBME. The combined filtrate and
washings were allowed to slowly evaporate to half the
volume, which furnished additional crystalline salt.
After filtration this salt was washed with toluene–30%
TBME and combined with the first fraction to yield
217 mg of white crystalline salt. This material was dis-
solved in 1.5 mL of ethyl acetate, 3 volumes of toluene
were added, and the solution was concentrated to an
oil. A solution of the material in 2 mL of toluene was
left to crystallize undisturbed for 30 h. White clusters
of crystals were separated and washed twice with
0.5 mL toluene and air dried for 2 h. An 1H NMR spec-
trum (CDCl3) showed characteristic signals at d 7.89 (br
s, 3H), 4.46 (d, 1H, J = 3Hz), 1.32 (s, 9H), 1.08 (s, 3H),
0.83 (s, 3H), 0.79 (s, 9H), and 0.14 (s, 6H). A sample of
20 mg was used for X-ray diffraction analysis.
1
ester as orange foam. H NMR (CDCl3) d 6.77–6.68
(m, 4H), 4.14 (dd, 1H, J = 4 and 13.5 Hz), 3.75–3.72
(m, 2H), 3.38 (br s, 5H), 2.92–2.79 (m, 2H), 2.11
(dd, 1H, J = 3.15 and 13 Hz), 1.68–1.65 (m, 1H),
1.47 (s, 9H), 0.90 (s, 9H), 0.04 (s, 3H), 0.02 (s, 3H);
13C NMR (CDCl3) d 155.0, 136.4, 126.0, 120.6,
119.3, 117.1, 79.7, 62.9, 51.5, 28.6, 26.1, 18.4, ꢀ5.3;
MS (APCI) m/z 436.7 (M+H)+.
3-(tert-Butyldimethylsilanyloxymethyl)-4-(2-aminophe-
nylamino)-piperidine-1-carboxylic acid tert-butyl ester
(510 mg, 1.17 mmol) and N,N0-disuccinimidyl carbon-
ate (600 mg, 2.34 mmol) were dissolved in DMF
(20 mL). The reaction mixture was stirred at rt for 2
days. It was then treated with brine (5 mL) and water
(5 mL). The aqueous phase was extracted with Et2O
(4· 10 mL). The combined organic phase was washed
with brine (2· 5 mL) and dried over Na2SO4. Evapora-
tion of the solvent under reduced pressure afforded
600 mg (100%) of 11 as an orange oil. The chiral purity
of benzimidazolone 11 was determined by chiral-
HPLC: ChromTech Chiral-AGP column 4 · 150 mm,
5 lm, solvent A: [10 mM K2HPO4 + KH2PO4, pH
7.0], solvent B: CH3OH, 5% B, flow rate: 1 mL/min,
UV: 235 nm, retention time: 16.8 min. 1H NMR
(CDCl3) d 10.74 (s, 1H), 7.11–7.00 (m, 4H), 4.36–4.32
(m, 2H), 3.44–3.37 (m, 2H), 2.80–2.38 (m, 5H), 1.80–
1.77 (m, 1H), 1.48 (s, 9H), 0.78 (s, 9H), ꢀ0.17 (s,
3H), ꢀ0.19 (s, 3H); 13C NMR (CDCl3) d 155.6,
154.9, 128.4, 121.4, 121.0, 109.9, 79.9, 61.8, 51.9,
36.6, 28.6, 25.9, 18.2, ꢀ5.6; MS (ESI) m/z 460.9
(MꢀH)ꢀ.
5.8. 3-(tert-Butyldimethylsilanyloxymethyl)-4-(2-nitro-
phenylamino)-piperidine-1-carboxylic acid tert-butyl
ester (10)
A mixture of resolved amine (+)-9 (172 mg, 0.5 mmol),
sodium carbonate (64 mg, 0.6 mmol), n-butanol
(5 mL),
and
1-fluoro-2-nitrobenzene
(0.16 mL,
0.75 mmol) was refluxed for 4 h. The reaction mixture
was cooled to rt, filtered through a pad of Celite. The
flask was washed with ether (3· 10 mL) and the washing
was filtered through the pad. The filtrates were com-
bined, washed with water (2· 5 mL) and brine (5 mL),

828
E. D. Smith et al. / Bioorg. Med. Chem. 16 (2008) 822–829
5.10. 1-Ethyl-3-(3-hydroxymethyl-4-piperidinyl)-1,3-
dihydrobenzimidazol-2-one (12)
J = 3.7 and 12.3 Hz), 2.31–2.03 (m, 5H), 1.72–1.46 (m,
14H), 1.34 (t, 3H, J = 7.1 Hz), 1.28–1.22 (m, 2H); 13C
NMR (CDCl3) d 155.0, 129.7, 128.5, 122.2, 121.5,
121.5, 110.6, 108.3, 66.5, 62.3, 56.9, 54.0, 52.2, 41.4,
36.5, 35.3, 31.3, 31.3, 29.8, 29.2, 27.6, 27.6, 26.8, 26.0,
26.0, 25.4, 13.9; MS (ESI) m/z 400.6 (M+H)+.
To
a
solution of benzimidazolone 11 (600 mg,
1.17 mmol) in DMF (25 mL) cooled at 0 ꢁC was added
NaH (60% suspension in mineral oil, 170 mg, 4.2 mmol).
The reaction mixture was slowly warmed to rt over 1 h.
Iodoethane (0.59 mL, 7.3 mmol) was added and the
reaction mixture was stirred at rt for 90 min. It was then
cooled to 0 ꢁC and treated with water (30 mL). The
aqueous phase was extracted with EtOAc (4· 15 mL).
The combined organic layer was washed with brine
(15 mL) and dried over Na2SO4. Evaporation of the
solvent under reduced pressure afforded 580 mg
(100%) of 3-(tert-butyldimethylsilanyloxymethyl)-4-(3-
ethyl-2-oxo-2,3-dihydrobenzimidazol-1-yl)-piperidine-1-
carboxylic acid tert-butyl ester. 1H NMR (CDCl3) d
7.13–6.99 (m, 4H), 4.39–4.27 (m, 3H), 3.93 (q, 2H,
J = 7.3 Hz), 3.43–3.33 (m, 2H), 2.96–2.37 (m, 4H),
1.79 (d, 2H, J = 11.1 Hz), 1.50 (s, 9H), 1.33 (t, H,
J = 7.1 Hz), 0.81 (s, 9H), ꢀ0.13 (s, 3H), ꢀ0.15 (s, 3H);
13C NMR (CDCl3) d 154.8, 153.5, 129.2, 121.0, 120.9,
109.1, 107.6, 79.8, 62.0, 52.3, 35.8, 29.8, 29.4, 28.9,
28.5, 25.9, 18.2, 13.6, ꢀ5.7; MS (ESI) m/z 490.8
(M+H)+.
1-[(3R,4R)-1-Cyclooctylmethyl-3-hydroxymethyl-4-
piperidinyl-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one
(14.4 mg) was dissolved in 10% MeOH/HCl (1 mL). The
solvent was removed under reduced pressure and the
oily residue was then dissolved in Et2O (1 mL). Some
white solids precipitated on the flask walls. The solvent
was removed under reduced pressure and the residue
was placed under vacuum to give 13.6 mg of the hydro-
20
D
chloride salt as a white solid. ½aꢁ +6.25 (c 0.272, 0.1 M
HCl) (C24H38ClN3O2ÆH2O) C, H, N.
5.12. X-ray crystal structure of (ꢀ)-3-bromo-8-camphor-
sulfonic acid salt of (ꢀ)-9
Single-crystal X-ray diffraction data were collected at
295 K using Cu Ka radiation and a Bruker SMART
6000 CCD area detector. A 0.42 · 0.10 · 0.07 mm3 crys-
tal was mounted on a glass rod and transferred to the
diffractometer. The crystal was orthorhombic in space
group P212121 with unit cell dimensions a = 6.9722
3-(tert-Butyldimethylsilanyloxymethyl)-4-(3-ethyl-2-oxo-
2,3-dihydrobenzimidazol-1-yl)-piperidine-1-carboxylic acid
tert-butyl ester (130 mg, 0.27 mmol) was dissolved in
HCl/MeOH (10% vv, 4.5 mL). The solution was stirred
at rt for 3 h, then concentrated to about 1 mL. The res-
idue was mixed with aqueous HCl (0.5 M, 5 mL). The
aqueous solution was washed with Et2O (3· 3 mL), basi-
fied to pH 12 with NaOH (2 M), and extracted with
CHCl3 (4· 7 mL). The extracts were combined, washed
with H2O (2· 5 mL), dried over Na2SO4, evaporated in
vacuo to give 56 mg (76%) of 12 as a clear oil. 1H NMR
(CDCl3) d 7.32 (d, 1H, J = 7.5 Hz), 7.14–6.89 (m, 3H),
4.47 (dt, 1H, J = 3.7 and 12.2 Hz), 4.02–3.89 (m, 2H),
3.34–3.21 (m, 4H), 2.90–2.45 (m, 5H), 2.26 (br s, 1H),
1.92–1.91 (m, 1H), 1.34 (t, 3H, J = 7.35 Hz); 13C
NMR (CDCl3) d 154.6, 129.4, 128.3, 121.4, 121.3,
110.2, 108.1, 61.7, 51.9, 49.2, 46.6, 36.2, 30.1, 29.9,
13.7; MS (APCI) m/z 276.3 (M+H)+.
˚
˚
˚
(2) A, b = 20.3743(6) A, c = 24.2633(7) A. Corrections
were applied for Lorentz, polarization, and absorption
effects. Data were 98.1% complete to 67.16ꢁ h (approxi-
˚
mately 0.83 A) with an average redundancy of 5.0. The
structure was solved by direct methods and refined by
full-matrix least squares on F2 values using the pro-
grams found in the SHELXTL suite (Bruker, SHEL-
XTL v6.12, 2006, Bruker AXS Inc., Madison, WI).
Parameters refined included atomic coordinates and
anisotropic thermal parameters for all non-hydrogen
atoms. Hydrogen atoms on carbons were included using
a riding model [coordinate shifts of C applied to H
˚
atoms] with C–H distance set at 0.96 A. The absolute
configuration was set based on the reference molecule
(3-bromocamphorsulfonic acid) of known configura-
tion. The tert-butyl group off N1 was refined with a dis-
order over two positions with an occupancy ratio of
52:48. The tert-butyl group off the silica atom showed
a similar disorder, but the occupancy of the minor posi-
tion was less than 25% and this disorder was omitted
from the final cycles of refinement.
5.11. 1-[(3R,4R)-1-Cyclooctylmethyl-3-hydroxymethyl-4-
piperidinyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one
(2) hydrochloride
To a stirred solution of 1-ethyl-3-(3-hydroxymethyl-4-
piperidinyl)-1,3-dihydrobenzimidazol-2-one (12) (24.1 mg,
0.085 mmol) in THF (1 mL) were added cyclooctanecar-
boxaldehyde (0.01 mL, 0.1 mmol) and sodium triacet-
oxyborohydride (28 mg, 0.13 mmol). After 2.5 d at rt,
the mixture was mixed with EtOAc (1 mL) and H2O
(1 mL). The organic layer was separated, washed with
NaHCO3 (satd, 1 mL), brine (1 mL), dried over
Na2SO4, and placed under vacuum. Purification of the
crude product by prep TLC (Silica gel, 1000 lm) with
Acknowledgments
This research was supported by the National Institute
on Drug Abuse, Grants DA05477 and DA09045.
Supplementary data
Crystal data, structural refinement analysis, atomic
coordinates, bond lengths, bond angles, anisotropic dis-
placement parameters, hydrogen coordination, isotropic
displacement parameters of (ꢀ)-9-(3-bromo-8-camphor-
sulfonic acid) salt, and results from elemental analysis.
1
70% EtOAc/hexanes afforded 14.4 mg (42%) of 2. H
NMR (CDCl3) d 7.33–7.27 (m, 1H), 7.20–7.02 (m,
3H), 4.37 (dq, 1H, J = 3.8 Hz), 3.98–3.92 (m, 2H), 3.33
(br s, 2H), 3.00 (d, 3H, J = 9.42 Hz), 2.60 (dq, 1H,

E. D. Smith et al. / Bioorg. Med. Chem. 16 (2008) 822–829
829
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at http://pubs.acs.org. Supplementary data associated
with this article can be found, in the online version, at
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