Design and synthesis of novel small molecule N/OFQ receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2003.11.083

Small molecule N/OFQ receptor antagonists were designed and synthesized to further investigate the therapeutic potential of N/OFQ receptor modulators. The resulting octahydrobenzimidazol-2-ones 14 and 23 show excellent antagonistic activity towards both N/OFQ and mu receptors with high affinity to the human N/OFQ receptor.

Full text of DOI:10.1016/j.bmcl.2003.11.083

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1347–1351
Design and synthesis of novel small molecule N/OFQ
receptor antagonists
Zhengming Chen,* R. Richard Goehring, Kenneth J. Valenzano and Donald J. Kyle
Drug Discovery, Purdue Pharma L. P., 6 Cedar Brook Drive, Cranbury, NJ 08512, USA
Received 17 September 2003; revised 25 November 2003
Abstract—Small molecule N/OFQ receptor antagonists were designed and synthesized to further investigate the therapeutic
potential of N/OFQ receptor modulators. The resulting octahydrobenzimidazol-2-ones 14 and 23 show excellent antagonistic
activity towards both N/OFQ and mu receptors with high affinity to the human N/OFQ receptor.
# 2003 Elsevier Ltd. All rights reserved.
1. Introduction
produces analgesic effects in the hot plate test in mice
and the formalin test in rats.9 In addition, Ro 64-6198
elicits dose-dependent anxiolytic effects in the elevated
plus-maze, fear-potentiated startle and operant conflict
tests.12 The results of these in vivo experiments suggest
that the identification of small molecule N/OFQ receptor
agonists and antagonists with good pharmaceutical and
pharmacological profiles could lead to new medications
for pain and anxiety.6 In this paper, we present our
progress toward the design and synthesis of novel small
molecule N/OFQ receptor antagonists.
The discovery of the nociceptin/orphanin FQ (N/OFQ)
receptor (NOP, previously named ORL-1) in 1994,1 and
its endogenous ligand N/OFQ2 in 1995 has generated
considerable interest within the scientific community
due to the important roles of classical opioid receptors
in the CNS. A number of in vivo experiments have
demonstrated that N/OFQ and its peptide analogues
modulate a variety of biological functions such as food
intake, memory processes, cardiovascular functions,
locomotor activity, and control of neurotransmitter
release at peripheral and central sites.3,4 In addition, N/
OFQ modulates pain mechanisms at the level of the
spinal cord and may also play a role in CNS disorders
such as anxiety and drug abuse.4,5 However, a clear
understanding of the potential therapeutic value associated
with the modulation of the N/OFQ receptor requires
the development of selective non-peptide agonists and
antagonists with better pharmaceutical profiles than the
peptide analogues.6,7
2. Design of N/OFQ receptor antagonists
Our primary interest in a N/OFQ receptor antagonist is
its therapeutic potential as a pain modulator. Our
design of this series of N/OFQ receptor antagonists was
based on the known features of N/OFQ receptor
antagonists (Figs 1 and 2).8ꢀ11 Compound 1 (J-113397)
was reported to be a N/OFQ receptor antagonist with
an IC50 of 2.3 nM.8 We envisioned that replacing the
benzimidazol-2-one of J-113397 with an octahydrobenz-
imidazol-2-one might retain potency and antagonistic
activity. The hydrophobic cyclohexyl ring, lacking
potential p electron interaction with N/OFQ receptor,
might result in a pharmacological profile different than
the J-113397 series. In addition, the cis and trans isomers
of the disubstituted cyclohexane ring might offer potential
handles for further manipulation of pharmacological
selectivity. Based on these hypotheses, we chose com-
pounds 5 and 6 as starting points for the identification
of novel N/OFQ receptor antagonists.
Recently, several research groups have reported the
discovery of small molecule N/OFQ receptor ligands
(Fig. 1).8ꢀ11 The discovery of highly potent and selective
agonists (Ro 64-6198, Roche 5a) and antagonists (J-113397,
JTC-801) has been instrumental in further illustrating
the therapeutic potential of N/OFQ receptor modulators.
For example, in vivo studies have shown that JTC-801
antagonizes N/OFQ-induced allodynia in mice and
* Corresponding author. Tel.:+1-609-409-5126; fax:+1-609-409-6930;
e-mail: zhengming.chen@pharma.com
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.11.083

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Z. Chen et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1347–1351
Figure 1. Representative N/OFQ receptor agonists and antagonists.
Figure 2. Design of N/OFQ receptor antagonists.
3. Synthesis of N/OFQ receptor antagonists
reaction was quickly optimized by varying the ratios of
reactants. A 2:1:1.4 ratio of 7a, 8 and NaBH(OAc)3 was
found to be essential for a good isolated yield of the
desired product 9a (64% after separation). Compound
9a was further cyclized with 1,10-carbonyldiimidazole
(CDI) in acetonitrile to yield compound 10. Removal of
the Boc group under acidic conditions provided the
head group 11. This novel synthetic procedure for head
group 11 is more versatile than the previously reported
method since it has been applied to the cis-diamine
series without any modification.15,16 In contrast, the
previously reported synthetic procedure via the 1,4-
addition of 4-amino-1-benzylpiperidine to 1-nitrocyclo-
hexene affords the trans isomer as the major product.15
The synthesis of the cis-isomer via this previously
reported route would likely be difficult. Finally, alkyl-
ation of compound 10 with ethyl iodide in DMF gave
compound 12 in high yield (Scheme 2). Removal of the
Boc group provided the head group 13.
We have developed a new synthetic method for the
construction of trans- and cis-octahydrobenzimidazol-2-
one ring systems that utilizes the stereochemistry of the
readily available racemic trans- and cis-diamines 7a and
7b (Fig. 2). The synthesis starts with the preparation of
head groups 11 and 13 (Schemes 1 and 2). The reductive
amination of trans-diamine 7a and ketone 8 using
NaBH(OAc)3 in 1,2-dichloroethane provided a mixture
of mono- and dialkylated products 9a and 9b. This
Scheme 1. Synthesis of head group 11.
Scheme 2. Synthesis of head group 13.

Z. Chen et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1347–1351
1349
After the efficient synthesis of head groups 11 and 13
had been developed, we used parallel synthesis for the
construction of our final N/OFQ receptor ligands. We
utilized two high yielding reactions, namely alkylation
and reductive amination, for the parallel syntheses.
One aldehyde (cyclooctyl aldehyde) and five ketones
were chosen for the reductive amination reaction while
three halides were selected for the alkylation reaction
(Scheme 3). These reagents were carefully selected to
include alkyl, cycloalkyl and aromatic groups and also to
include better N/OFQ receptor or mu selective R groups
based on literature precedents.8,13,14
MA). Dose displacement binding assays using [3H]-
diprenophine, [3H]-U69,593 and [3H]-naltrindole, respec-
tively, were conducted according to the product inserts.
The mu functional GTPgS assay was conducted using
the commercially available membrane preparation
under conditions previously described for the N/OFQ
receptor.18 The activity of the mu agonist DAMGO was
used for data normalization (maximal effect elicited by
10 mM DAMGO=100%; background GTPgS binding
in the absence of agonist=0%).
Table 1 shows the results from N/OFQ receptor and mu
receptor binding and functional assays for the com-
pounds described above. In general, these compounds
show good affinity towards the N/OFQ receptor ranging
from 11 nM to 4.4 mM. The cyclooctylmethyl group was
found to impart the highest affinity in both series
(R1=H and ethyl). In addition, the octahydronaphthyl,
cyclooctyl and 4-isopropylcyclohexyl groups are better
groups for high N/OFQ receptor affinity (i.e., 15, 16, 19,
24, 25, and 28) while the 3,3-diphenylpropyl group
tends to enhance affinity for the mu receptor (i.e., 20). It
is interesting that the octahydrobenzimidazol-2-one series
are either antagonists or weak partial agonists through-
out the entire series with the exception of compound 20
which is a full agonist. This is in contrast to the original
J-113397 series in which 1-(1-benzyl-4-piperidyl)-1,3-
dihydro-2H-benzimidazol-2-one, a direct analogue of
compound 21, is a N/OFQ receptor agonist.8
The six reductive amination reactions (products 14–19
and 23–28) and three alkylation reactions (products 20–
22 and 29–31) were run in parallel fashion in 10 mL
vials at 0.5 mM scale. All reactions were found to have a
crude purity of 70–90% by LC/MS except for products
16 and 25 due to the high steric hindrance of the
cyclooctanone starting material. The reactions were
worked up by adding 1 N NaOH and ether in sequence.
The ether layers were pipetted into a new set of vials
and solvent was removed in a Speedvac. The residues
were dissolved in a small amount of DCM and loaded
into 20 mL tubes containing 5 g of silica gel. The tubes
were eluted in parallel first by a mixture of EtOAc/hexanes
(1:9), followed by a mixture of Et3N/EtOAc/hexanes
(5:25:70). The fractions were collected in 10 mL vials
and analyzed by LC/MS. The solvents were removed in
a Speedvac. The pure fractions (>97% pure by LC/MS
and NMR) were collected and submitted for biological
screening.17 Compounds 15, 17, 18, 19, 24, 26, 27, and
28 were screened as a mixture of isomers, no further
separation has been done.
Although the ethyl substitution on the urea position
(R1=ethyl) does not increase the resulting ligands affi-
nity towards the N/OFQ receptor in general, it does
The compounds synthesized above were tested in N/
OFQ receptor binding and GTPgS functional assays
using membrane preparations from recombinant HEK-
293 cells as previously reported.18 Recombinant human
mu, kappa and delta opioid receptor membranes were
purchased from Perkin Elmer Life Sciences (Boston,
Table 1. N/OFQ and mu receptors binding and functional assays
Compd
NOP Ki
(nM)a
NOP
functional
Mu Ki
(nM)a
Mu
functional
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
16 (ꢁ4)
51 (ꢁ18)
ANTc
PAd
ANT
PA
PA
1071(ꢁ327)
208 (ꢁ62)
1402 (ꢁ208)
426 (ꢁ239)
435 (ꢁ61)
959 (ꢁ92)
27(ꢁ9)
ANT c
ANT
ANT
ANT
ANT
ANT
PA
ND
ND
ANT
ANT
ANT
PA
125 (ꢁ31)
414 (ꢁ123)
634 (ꢁ117)
39 (ꢁ13)
PA
437 (ꢁ46)
1054 (ꢁ432)
1259 (ꢁ117)
11 (ꢁ3)
Agonistb
ANT
ND
ND
1237 (ꢁ437)
448 (ꢁ42)
279(ꢁ15)
909 (ꢁ205)
395 (ꢁ39)
2165 (ꢁ786)
421 (ꢁ84)
267 (ꢁ63)
716 (ꢁ202)
199(ꢁ47)
ANT
ANT
ANT
ANT
ND
ANT
ND
ANT
ANT
184 (ꢁ94)
111 (ꢁ33)
410 (ꢁ61)
1239 (ꢁ173)
89 (ꢁ26)
ND
ANT
ND
PA
4358 (ꢁ908)
692 (ꢁ125)
824 (ꢁ100)
ND
a Values are means of at least three experiments, standard deviation is
given in parentheses.
b A compound was considered to be a full agonist when its ability to
stimulate GTPgS binding was >75% in comparison to N/OFQ
(NOP) or DAMGO (mu).
c An antagonist (ANT) stimulated GTPgS binding with efficacy
ꢂ10% in comparison to N/OFQ (NOP) or DAMGO (mu).
d A partial agonist (PA) stimulated GTPgS binding with efficacy
>10% but <75% in comparison to N/OFQ (NOP) or DAMGO
(mu). ND=not determined.
Scheme 3. Parallel synthesis of N/OFQ receptor antagonists.

1350
Z. Chen et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1347–1351
enhance the ligands antagonistic activity at the N/OFQ
receptor. Several partial agonists in the unsubstituted
urea series (R1=H, compounds 15, 17 and 19) are con-
verted into antagonists by the ethyl substitution
(R1=Et, compounds 24, 26 and 28). In addition, both
series show general antagonism against the mu receptor
with the exception of compounds 20, 26, and 30 which
are either partial or weak partial mu agonists. For
example, compound 23 shows complete antagonistic
activity at both N/OFQ receptor and mu receptors with
40-fold selectivity over the mu receptor.
terms of mu antagonistic activity. Future work will be
focused on enhancing the selectivity and improving the
pharmaceutical profile of this novel series.
Acknowledgements
We thank Dr. Bin Shao for helpful discussions, and Ms.
Wendy S. Miller, Shen Shan, Farhana Hussain and Mr.
Leon Z. Zhou for technical assistance.
The amino acid sequence of N/OFQ receptor shares
some homology to the classical opioid receptors (about
47% identity to the mu, delta and kappa receptors).1
Hence the selectivity of our new N/OFQ receptor
ligands over mu, kappa and delta receptors was also
monitored. Table 2 shows the results for several selected
compounds in the N/OFQ receptor, mu, kappa and
delta binding assays. Most compounds in Table 2 show
good to excellent selectivity against kappa and delta
receptors. For example, compounds 23, 24 and 28 are
inactive in the kappa assay and compounds 15, 19, 23
and 28 are inactive in the delta assay. Overall, com-
pounds 14 and 23 show the best selectivity against the
three classic opioid receptors with >40-fold selectivity over
mu, >106-fold over kappa and >408-fold over delta
receptors. These compounds may have potential as phar-
maceutical tools for further studying the role of the N/OFQ
receptor in pain, drug abuse and other CNS disorders.
References and notes
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In conclusion, based on the reported N/OFQ receptor
antagonist J-113397, we designed novel N/OFQ recep-
tor antagonists by replacing the benzene ring with a
trans-cyclohexane ring. A new synthetic method for the
construction of trans-octahydrobenzimidazol-2-one ring
system has been developed in the process of synthesizing
the designed compounds. The resulting trans-octahydro-
benzimidazol-2-one series shows excellent antagonistic
activity towards both N/OFQ receptor and mu receptor
while retaining high potency at N/OFQ receptor. Our
work demonstrates that a non-aromatic hydrophobic
group replacement of the benzene ring in J-113397 is
sufficient to maintain effective interaction with N/OFQ
receptor. In addition, the non-aromatic hydrophobic
group results in a different pharmacological profile in
Table 2. Selected data for NOP, mu, kappa and delta receptor binding
assays
Compd
NOP Ki
(nM)
Mu Ki
(nM)
Kappa Ki
(nM)
Delta Ki
(nM)
12. Jenck, F.; Wichmann, J.; Dautzenberg, F. M.; Moreau,
J. L.; Ouagazzal, A. M.; Martin, J. R.; Lundstrom, K.;
Cesura, A. M.; Poli, S. M.; Roever, S.; Kolczewski, S.;
Adam, G.; Kilpatrick, G. Proc. Natl. Acad. Sci. U.S.A.
2000, 97, 4938.
13. Rover, S.; Wichmann, J.; Jenck, F.; Adam, G.; Cesura,
A. M. Bioorg. Med. Chem. Lett. 2000, 10, 831.
14. Chen, Z.; Miller, W.; Shan, S.; Valenzano, K. Bioorg.
Med. Chem. Lett. 2003, 13, 3247.
14
15
19
23
24
28
16 (ꢁ4)
51 (ꢁ18)
39 (ꢁ13)
11 (ꢁ3)
1071 (ꢁ327) 1701 (ꢁ657)
6532 (ꢁ2377)
>10,000
>10,000
>10,000
ND
208 (ꢁ62)
959 (ꢁ92)
448 (ꢁ42)
279 (ꢁ15)
421 (ꢁ84)
1394 (ꢁ136)
6212 (ꢁ2354)
>10,000
184 (ꢁ94)
89 (ꢁ26)
>10,000
>10,000
>10,000
Values are means of at least three experiments, standard deviation is
given in parentheses (>10,000=not active).

Z. Chen et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1347–1351
1351
15. Obase, H.; Nakamizo, N.; Takai, H.; Teranishi, M.;
Kubo, K.; Shuto, K.; Kasuya, Y.; Shigenobu, K.;
Hashikami, M. Chem. Pharm. Bull. 1983, 31, 3186.
16. The synthesis of the cis-octahydrobenzoimidazol-2-one
series was carried out in similar fashion to the preparation
of compounds 11 and 13, and the biological data will be
reported at a later date.
2H), 2.30 (m, 1H), 2.90 (m, 2H), 3.00 (m, 2H), 3.75 (m,
1H), 4.40 (b, 1H). HR-LC/MS: 347.29286 (Exact
Mass: 347.29366). Analytical data for 23: 1H NMR
(CDCl3) d 1.05 (t, 3H), 1.10–2.10 (m, 30H), 2.27 (m,
1H), 2.74 (m, 1H), 2.82–2.87 (m, 3H), 3.16–3.24 (m,
2H), 3.75 (m, 1H). HR-LC/MS: 375.32225 (Exact Mass:
375.32496).
17. Analytical data for 14: 1H NMR (CDCl3) d 1.20 (m, 2H),
1.30–1.70 (m, 19H), 1.78 (m, 4H), 1.95 (m, 3H), 2.05 (m,
18. Zhang, C.; Miller, W.; Valenzano, K.; Kyle, D. J. J. Med.
Chem. 2002, 45, 5280.