A selective small molecule NOP (ORL-1 receptor) partial agonist for the treatment of anxiety

Article abstract of DOI:10.1016/j.bmcl.2014.12.015

Small molecule (1) has been identified as a selective partial agonist of Opioid Receptor Like-1 (ORL-1) with potential utility for the treatment of anxiety and other disorders. Nociceptin (orphanin FQ) is an endogenous peptide ligand that binds to ORL-1, however it does not bind the classical δ, μ and κ opioid receptors with high affinity. The synthesis of 1 involved using a molecular diversity approach, to rapidly advance a library of compounds for biological testing. A lead selective potent partial agonist (35-fold ORL-1/Mu) progressed to ORL-1 (NOP or OP4) proof of concept testing in advanced studies. The synthetic approach and biological data for the related chemical series will be presented.

Full text of DOI:10.1016/j.bmcl.2014.12.015

Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A selective small molecule NOP (ORL-1 receptor) partial agonist
for the treatment of anxiety
c
a
d
a
Tina Morgan Ross b, , Kathleen Battista , Gilles C. Bignan , Doug E. Brenneman , Peter J. Connolly ,
Jingchun Liu e, Steven A. Middleton f, Michael Orsini g, Allen B. Reitz d, Dan I. Rosenthal h,
Malcolm K. Scott a, , Anil H. Vaidya i
⇑
a Johnson & Johnson Pharmaceutical Research and Development, LLC, Spring House, PA, USA
b West Chester University, Chemistry Department, West Chester, PA, USA
c Ace Glass, Vineland, NJ, USA
d Fox Chase Chemical Diversity Center, Doylestown, PA, USA
e Sanofi-Aventis, Boston, MA, USA
f Hoffmann La-Roche, Nutley, NJ, USA
g Bristol-Myers Squibb, New Brunswick, NJ, USA
h Burlington Community College, Pemberton, NJ, USA
i Pfizer-Makro Technologies, Bridgewater, NJ, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Small molecule (1) has been identified as a selective partial agonist of Opioid Receptor Like-1 (ORL-1)
Received 21 August 2014
Revised 3 December 2014
Accepted 5 December 2014
Available online xxxx
with potential utility for the treatment of anxiety and other disorders. Nociceptin (orphanin FQ) is an
endogenous peptide ligand that binds to ORL-1, however it does not bind the classical d,
l and j opioid
receptors with high affinity. The synthesis of 1 involved using a molecular diversity approach, to rapidly
advance a library of compounds for biological testing. A lead selective potent partial agonist (35-fold
ORL-1/Mu) progressed to ORL-1 (NOP or OP4) proof of concept testing in advanced studies. The synthetic
approach and biological data for the related chemical series will be presented.
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Nociceptin
NOP receptor
Orphanin FQ
Opioid receptor-like receptor-1
Drug discovery
The ORL-1 (opioid receptor like-1) G-protein coupled receptor,
was first reported in 1994,1–4 and was discovered based on its
homology with the classic delta (OP-1), mu (OP-3) and kappa
(OP-2) opioid receptors. The ORL-1 (OP-4) G-protein coupled
receptor does not bind classical opioid ligands with high affinity
even though the natural peptide shares structural similarity with
classical opioid peptide dynorphin A.5 The endogenous ligand of
ORL-1, known as nociceptin, a highly basic 17 amino acid peptide,
was isolated from tissue extracts in 1995.5 It was named nocicep-
tion (NOP), because it increased sensitivity to pain when injected
into mouse brain and orphanin FQ (OFQ) because of the terminal
phenylalanine (F) and glutamine (Q) residues that flank the peptide
on the N–C-termini respectively. In the course of time the name of
the protein changed (ORL-1, N/OFQ-, OP-4, NOP-receptor) and dif-
ferent denotations used by diverse working groups. Within this
work, we will use NOP and ORL-1.1–5 Nociceptin binding to
ORL-1 receptors causes inhibition of cAMP synthesis, inhibition
of voltage-gated calcium channels and activation of potassium
conductance. Behavioral studies in rats and studies in mice lacking
ORL-1 suggest that an ORL-1 antagonist might enhance learning
ability and memory.6 Furthermore, several studies employing dif-
ferent behavioral measures of stress/anxiety in rodents suggest
that ORL-1 agonists will possess anxiolytic-like properties.6 An
agonist may also be useful in the treatment of opioid dependence
and withdrawal, since nociception exhibits anti-opioid activity.
Finally, recent physiological and behavioral studies in animals
suggest that agonists of ORL-1 could be useful as atypical opioid
analgesics and for alleviating abnormal (neuropathic) pain result-
ing from spinal cord and peripheral nerve injury and inflammation
(hyperalgesia and allodynia).6
In continuation of our exploration of opioid receptors of the
8-(heteroaryl)phenalkyl-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-
ones series by Jordan and coworkers, where substitution of the
thienyl-phenethylalkyl (s1 side) produced enhanced affinity for
⇑
Corresponding author.
E-mail address: tross@wcupa.edu (T.M. Ross).
Retired from J&JPRD.
http://dx.doi.org/10.1016/j.bmcl.2014.12.015
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Ross, T. M.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.12.015

2
T. M. Ross et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Chemical diversity was introduced by reacting a set of 88
OH
O
*
amines (Supplemental Table 1) selected from a larger set of >350
commercially available amines minimized time (by a quarter) in
synthesis (and cost). These amines were reacted with the chiral
epoxide functionality installed on the h3 side (Fig. 2) of the mole-
cule using simple SN2 conditions (Scheme 1). Once the desirable
biological in vitro activity was achieved, we then expanded the
‘quadrant’ of the diversity set with more examples to achieve the
selectivity or PK attributes needed.12
τ1
N
X
θ1
N
A
N
θ3
R
Figure 1. NOP (ORL-1) agonist scaffold.
O
We were able to quickly synthesize over 100 compounds using
this cassette approach. The compounds were then screened for
their binding affinity with ORL-1.12 The initial library set with 2-
(2-thienyl)phenyl ethyl as the s1 side substituent showed a differ-
ence in Mu-opioid selectivity between the R and S stereochemistry
as shown in Table 1. The R ethanolamine 6 produced a greater than
26 fold selectivity for ORL-1 over Mu-opioid, reassuring our goal to
deliver a therapeutic anxiolytic dose without the risks of respira-
tory depression, nausea and potential for abuse or dependency
associated with a Mu-opioid active compound.
S
NH
N
N
θ3
τ1
2
Figure 2. Triazasprio[4.5]dec-4-ones.
Encouraged by these results, we switched to the 4-fluoro spip-
erone core based on our Janssen colleagues’ experience from legacy
PK studies conducted within the series.11 The knowledge gained
from the diversity libraries (h3-region) was used to explore new
O
O
S
S
O
N
*
NH
Cl
N
N
*
N
O
N
NaH, DMF
0oC
s
1 side analogs, which were based on a review of literature/patent
2
3
*(S) or (R)
Epichlorohydrin
compounds (Roche,13–15 Euro-Celtique, Novo Nordisk, Banyu),16 to
generate unique selective compounds. We then expanded our h3-
region diversity set to include thiol and alcohol reagents to open
the epoxide ring.
O
S
N
*
HNR1R2 or SR1, OR1
Ethanol, 80oC
OH
N
As the project progressed we discovered while using chiral
reagents (Rc) to explore new s1 side analogs, that our epichlorohy-
N
X
X = NR1R2,
SR1, OR1
drin reagent (Method A) had a liability under the reaction condi-
tions (Scheme 2). The competing C1 versus C3 selectivity under
the reaction conditions made this an inadequate reagent (see note
1, Supplemental). This observation was not readily apparent until
we had the two chiral centers to distinguish the diastereomeric
products. We altered the synthesis by using the versatile glycidyl
nosylate (Method B), taking advantage of the lower nucleophilicity
of the nosylate leaving group.17 Interestingly we also identified a
side product as the surprisingly stable O-alkylated compound
(15–28%) which was addressed synthetically by our scale-up
group.
4
Scheme 1. Chiral diversity probe.
the Mu-opioid receptor,7 we explored the effect of chirality on the
opioid receptor for selectivity. We felt that an effective therapeutic
drug would need to counteract the Mu-opioid receptor affinity
(addiction, constipation, nausea, etc.) by improving selectivity of
NOP over the Mu-opioid receptor. We proposed introducing a chi-
ral moiety to the molecule to provide opioid selectivity for noci-
ception. For selective NOP agonists, we knew four structural
elements are necessary for mu receptor recognition of the spiro-
Our goal was to achieve greater than 1 lM in Mu-opioid bind-
ing affinity to reduce the negative side effects associated with a
piperdine ligands (e.g., h1, h3, s
1) and ring A.8 We kept the h1 proton
Table 1
accepting site, the anilido nitrogen region with the phenyl substi-
tution. In the h3-region described by Cometta-Morini and co-work-
ers,9 we explored substitution on the spirane amide nitrogen via a
diversity library to scan for possible selectivity differences
amongst the opioid receptors and arrive at a selective NOP agonist.
By manipulating the subtle difference in the overall spatial require-
3-(3-Amino-2-hydroxy-propyl)-1-phenyl-8-[2-(2-thiopen-2-yl-phenyl)ethyl]-1,3,8-
triaza-spiro[4.5]decan-4-ones
O
S
N
*
N
OH
N
Amine
ments for mu recognition (h1, h3, s1) and ring A, we were able to
discover more selective NOP agonists by introducing a chiral side
5 - 14
chain (Fig. 1).
We chose diverse reagents based on physicochemical properties
for a synthetic ‘cassette’ library approach used previously on an
internal Neuroprotection project,10 to react with the core molecule
on the h3 side for rapid synthesis. The structure activity relation-
ship (SAR) study was initially conducted around compound (2),7
utilizing the triazaspiro[4.5]decan-4-one starting material from
the Spiperone Project11 that was generously provided by Janssen
colleagues (Fig. 2). Potent affinity for ORL-1 was achieved after
introducing a substituent on the piperidine (s1 side). Selectivity
#
⁄
Amine
ORL-1
IC50
Mu
M) IC50 (lM)
Mu/ORL-
1
(l
5
6
7
8
S
R
S
R
S
R
S
4-Methoxybenzylamine
4-Methoxybenzylamine
N-Butylbenzylamine
N-Butylbenzylamine
2-Thiophenethylamine
2-Thiophenethylamine
2-Methylbenzylamine
2-Methylbenzylamine
3,4-
0.061
0.0042
0.0997
0.166
0.16
0.008
0.014
0.026
0.0039
0.029
0.11
0.036
0.047
0.029
0.08
0.069
0.103
0.079
0.5
26
0.36
0.28
0.18
10
4.9
4
20.4
9
10
11
12
13
R
R
versus
l, d, and j opioid receptors was generally accomplished
Dimethoxyphenethylamine
t-Butyl
using an aminoethylalcohol motif on the amide of the imidazoli-
din-4-one (h3 side).
14
R
0.0409
0.057
1.4
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T. M. Ross et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
O
3
OH
Method A
3
(R)
Cl
Epimerization
O
1
O
O
Cl
or
Cl
Rc
Cl
N
(S)
Major
1
Minor
NH
(R)
O
NaH, DMF, 0oC
Rc
Rc
N
(E)
N
N
O
Method B
N
N
N
+
(S)
O
O
N
3
S
O
O
O
(S)
F
1
Major (>98%)
NO2
F
F
3:1
O-Alkylated
N-Alkylated
Scheme 2. Glycidyl nosylate for improved SN2 selectivity.
R = CyclooctylCH2-
O
morphine-like opioid, for example, respiratory depression, nausea
and potential for abuse or dependency possible with a Mu–opioid
active compound. The 1 lM value was used due to legacy knowl-
OH
OH
H
N
O
(S)
N
(S)
N
N
edge within the analgesia research community as a guideline.
Our SAR trends (Table 1) led us to pursue alternative s1 side
substituents. We were pleased that the cyclooctylmethyl series
achieved high affinity for ORL-1 and displayed differences in func-
tional efficacy as well. Most cyclooctylmethyl analogs were partial
agonists (e.g., 1 and 15) (Fig. 3).
Data for the cyclooctylmethyl series showed that thio analogs
(16) were in general less selective for mu versus ORL-1 (Fig. 4).
The corresponding phenethyl ether (17) were generally less potent
in ORL-1. Coupling amino acids to the h3 side chain secondary
amino nitrogen (18) afforded greater potency however the benefit
was muted by the increase in formula weight, which may affect
Blood Brain Barrier penetration.
R
N
R
N
N
N
O
O
F
F
15
ORL-1 = 0.0088μM
Mu = 0.99μM
1
ORL-1 = 0.034 μM
Mu = 1.19 μM
kappa = 0.24 μM
μ/ORL-1 = 35
κ/ORL-1 = 7
kappa = 0.20 μM
μ/ORL-1 = 112
κ/ORL-1 = 23
Partial Agonist
Partial Agonist
Figure 3. Partial agonists.
R = CyclooctylCH2-
OH
Because compounds in the cyclooctylmethyl series had high
receptor binding affinity for ORL-1, good selectivity versus other
opioid receptors, and functional activity as partial agonists,12 we
were interested in evaluating them in several in vivo behavioral
models to measure potential anxiolytic effects and off-target CNS
activities. To evaluate desirable activity, the Elevated Plus Maze
(EPM) is a well-established model of anxiety in which benzodiaze-
pines such as Lorazepam show excellent activity. On the other
hand, the Spontaneous Locomotor Activity (SLMA) behavioral test
may be used as a selectivity screen to measure undesirable seda-
tive effects of a test compound. Lorazepam has sedation in the
OH
(S)
OH
(R)
O
O
O
(R)
N
N
(R)
NH2
N
R N
O
N
R N
O
R N
S
N
N
N
O
F
O
F
F
16
17
ORL-1 = 0.082
18
μ
ORL-1 = 0.018 M
μ
μ
ORL-1 = 0.037
M
M
μ
μ
Mu
= 2.6
M
μ
M
Mu
= 0.54
M
Mu
= 0.82
μ
/ORL-1 = 46
μ
μ
/ORL-1 = 14
/ORL-1 = 28
Figure 4. Selectivity of ORL-1 with other opioid receptors.
THE EFFECTS OF Compound #1 (IP) IN LONG-EVANS HOODED
RATS IN THE ELEVATED-PLUS MAZE MODEL
35
30
25
20
15
10
5
P=0.0015
Mann Whitney U Test (One-Tailed)
222.4 % N=29
P=0.0089
P=0.0262
57.8 %
P=0.0235
72.5 % N=32
N=32
54.4 %
N=32
24.0 %
N=16
N=16
5.9 %
N=32
0
PEG
0.5
0.03
0.1
0.3
1.0
3
# (ORL-1 Agonist)
Lorazepam
PRE-TREATMENT (mg/kg, ip, 30 min)
Figure 5. Elevated Plus Maze.
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4
T. M. Ross et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Figure 6. Spontaneous Locomotor Activity Test.
SLMA model at doses that are anxiolytic in the EPM model. The
details of these advanced tests are in the Supplemental section.12
Partial agonist 1 was tested in the EPM model of anxiety in rats
at doses ranging from 0.03 to 3 mg/kg (ip, 30 min pretreatment). At
0.1, 0.3, and 1.0 mg/kg rats showed 54% to 75% increase in open
arm time [(P = 0.009–0.026) Fig. 5]. Although this statistically sig-
nificant response was not as robust as Lorazepam (222%,
P = 0.0015), no detrimental side effects were observed after treat-
ment with 1.12
reduction in rearing, P <0.001) (Fig. 6). Compound 1 was not orally
active.12
The final synthesis to scale up compounds for efficacy studies is
shown in Scheme 3. The spiro core was synthesized by cyanide
addition to the ketone (19) followed by hydrolysis to the acid.
Nucleophilic addition of the aniline (20) followed by ring forma-
tion with paraformaldehyde under protic conditions afforded spip-
erone core 21. Cyclooctylmethylcarboxaldehyde (22) was made
from cyclooctanone under Vilsmeyer conditions (POCl3, DMF) fol-
lowed by reduction of double bond (10% Pd/C/H2).18 Reaction of
22 with the spiperone core (21) using reductive amination condi-
tions19 yielded the cyclooctylmethyl spiperone 23. Compound 23
was reacted with the S-glycidyl nosylate under Cs2CO3/THF condi-
tions with an optimized yield of greater than 68% (25); less than
16% of the O-alkylated side-product 24 was observed.
In the Spontaneous Locomotor Activity Test at (0.03–3.0 mg/kg,
ip, pretreatment 30 min), Partial agonist 1 showed no sedation
compared to Lorazepam, which caused significant sedation (70%
NH2
O
EtO
+
N
O
i
In conclusion, an extensive SAR study related to the spiro-imi-
dazolidinone series was conducted and generated numerous com-
pounds with potent ORL-1 affinity such as 1 (IC50 = 34 nM) that
F
19
20
O
were selective versus l, d, and j opioid receptors and had partial
O
O
NH
HN
iii
ii
agonist activity in a cellular functional assay. Modifications to
the A ring (s1 side) in addition to the functionalization of the h3
side with a chiral group capable of hydrogen bonding with an elec-
trophilic site provided compounds with diminished Mu opioid
affinity and increased selectivity for ORL-1. As explained more
recently in molecular modeling studies by Broer, et al.20 the
ORL-1 receptor sequence differs from other opioid receptors at
key amino acid residues that are important partners in hydrophilic
interactions with ligands.
This approach achieved selectivity with opioid receptor as par-
tial agonists by using the molecular diversity cassette synthetic
approach in a reasonable amount of time (ꢀthree months). This
allowed the team to progress in a restricted timeline to proof of
concept in collaboration with our eager biologists. Thus demon-
strating an additional strategy that medicinal chemists can employ
in the future with other innovative targets.
N
+
H
21
22
F
O
NO2
O
NH
iv
N
N
+
O
S
O
(S)
O
23
F
(S)
O
O
O
(R) O
v
N
N
N
N
N
N
1
+
Acknowledgments
24
25
The authors would like to thank Dave Demeter for his contribu-
tion with the initial diversity libraries, Michael Reuman for per-
forming scale-ups, Frank Villani (Chemical Development) for
coordinating the improved alkylation conditions on the alternate
s1 side scaffold and John Moyer for championing the project
beyond Drug Discovery.
F
F
1:4
Scheme 3. Reagents and conditions: (i) (1) NaCN, AcOH; (2) (a) H2SO4, toluene, (b)
AcOH, (c) NaOH; (3) aq (CH2O)n, AcOH, DMF; (4) (a) KOH, iPrOH, (b) H2O; (ii) (a)
POCl3, DMF (b) H2, 10% Pd/C, (c) HCl; (iii) DCE, NaBH(OAc)3; (iv) NaH, DMF, 0 °C or
Cs2CO3, THF; (v) N-benzyl-N-butylamine, EtOH, 80 °C.
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T. M. Ross et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
5
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357.
Supplementary data
9. Cometta-Morini, C.; Maguire, P. A.; Loew, G. H. Mol. Pharmacol. 1992, 41,
185.
10. Ross, T. M.; Zivin, R.; Burnett, P.; Scott, M. K.; Grant, E.; Brenneman, D. E.; Reitz,
A. B. Aminopyrimidines as Neuroprotective Agents. 230th ACS National Meeting;
American Chemical Society: Washington, D.C., 2005.
11. Janssen, P. A. J. U.S. Patent 3, 238, 216. 1964.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.12.
015.
12. Battista, K., Bignan, G., Connolly, P. J., Reitz, A. B., Ross, T. M., Scott, M. K.,
Middleton, S. A., Orsini, M. U.S. Patent 7,582,649 B2, 2009.
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