Discovery and Structure-Activity Relationships of Nociceptin Receptor Partial Agonists That Afford Symptom Ablation in Parkinson's Disease Models

Article abstract of DOI:10.1021/acs.jmedchem.9b02134

A novel series of C(3)-substituted piperdinylindoles were developed as nociceptin opioid receptor (NOP) partial agonists to explore a pharmacological hypothesis that NOP partial agonists would afford a dual pharmacological action of attenuating Parkinson'

Full text of DOI:10.1021/acs.jmedchem.9b02134

Subscriber access provided by The Univ of Iowa Libraries
Article
Discovery and Structure-activity Relationships of Nociceptin Receptor Partial
Agonists that Afford Symptom Ablation in Parkinson's Disease Models
Uma Gayathri Kamakolanu, Michael E Meyer, Dennis Yasuda, Willma E Polgar, Matteo Marti,
Daniela Mercatelli, Clarissa Anna Pisanò, Alberto Brugnoli, Michele Morari, and Nurulain T. Zaveri
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b02134 • Publication Date (Web): 17 Jan 2020
Downloaded from pubs.acs.org on January 19, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
TITLE PAGE:
Discovery and Structure-activity Relationships of Nociceptin Receptor Partial Agonists
that Afford Symptom Ablation in Parkinson's Disease Models
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1#
1#
1
1
Uma Gayathri Kamakolanu, Michael E. Meyer, Dennis Yasuda, Willma E. Polgar,
2
3
3
3
3
Matteo Marti, Daniela Mercatelli, Clarissa Anna Pisanò, Alberto Brugnoli, Michele Morari,
Nurulain T. Zaveri1*
1
Astraea Therapeutics, 320 Logue Avenue, Suite 142, Mountain View, CA 94043, USA
2
Department of Morphology, Surgery and Experimental Medicine, Section of Legal Medicine,
University of Ferrara, Ferrara, Italy 44100.
3
Department of Medical Sciences, Section of Pharmacology, University of Ferrara, Ferrara, Italy
4
4100.
#
Both authors contributed equally to this work.
*Address correspondence to:
Dr. Nurulain T. Zaveri
Astraea Therapeutics,
3
20 Logue Avenue, Suite 142,
Mountain View, CA 94043, USA.
Tel: 650-254-0786
Email: nurulain@astraeatherapeutics.com
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ABSTRACT
A novel series of C(3)-substituted-piperdinylindoles were developed as nociceptin opioid
receptor (NOP) partial agonists to explore a pharmacological hypothesis that NOP partial
agonists would afford a dual pharmacological action of attenuating Parkinson’s Disease (PD)
motor symptoms and development of levodopa-induced dyskinesias. SAR around the C-3
substituents investigated effects on NOP binding, intrinsic activity and selectivity and showed
that, while the C(3)-substituted indoles are selective, high affinity NOP ligands, the steric, polar
and cationic nature of the C-3 substituents affected intrinsic activity, to afford partial agonists
with a range of efficacies. Compounds 4, 5, and 9 with agonist efficacies between 25-35%
significantly attenuated motor deficits in the 6-OHDA-hemilesioned rat model of PD. Further,
unlike NOP antagonists, which appear to worsen dyskinesia expression, these NOP partial
agonists did not attenuate or worsen dyskinesia expression. The NOP partial agonists and their
SAR reported here may be useful to develop nondopaminergic treatments for PD.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
ACS Paragon Plus Environment

Page 3 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
INTRODUCTION
Parkinson’s Disease (PD) is the second most common neurodegenerative disorder after
1
Alzheimer’s disease affecting over 6 million individuals worldwide. PD is clinically
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
characterized as having classic motor symptoms (hypo/akinesia, rigidity, gait disturbance and
resting tremor), as well as non-motor complications such as depression and cognitive decline.2
The dopamine (DA) precursor levodopa (L-3,4-dihydroxyphenylalanine; L-DOPA) is the
cornerstone of PD therapy, often given in combination with COMT and MAO inhibitors to
2
extend its bioavailability and therapeutic action. However, chronic L-DOPA therapy is
associated with the eventual appearance (within 10 years in ~80% of patients) of motor
fluctuations and dyskinesias (involuntary movements) that limit its clinical effectiveness and
3
greatly reduce the quality of life of patients. The pathogenic hallmark of PD is the
neurodegeneration of dopamine producing neurons in the substantia nigra pars compacta leading
to striatal dopamine depletion, which leads to the classic motor features. On the other hand, long-
term manifestations of PD treatment, such as the levodopa-induced dyskinesias (LID) are the
result of an irreversible brain sensitization to L-DOPA. There is only one marketed
antidyskinetic drug, amantadine, a glutamate antagonist, which has poor and short-lasting
4
clinical efficacy. Current strategies to minimize LID involve reducing the L-DOPA dosage and
combining it with a dopaminomimetic (e.g. DA receptor agonists) to maintain symptomatic
benefit. Thus, newly developed drugs that can effectively treat PD symptoms, and delay or even
prevent the development of dyskinesia, would provide significant relief to PD patients, and fill in
a gap of unmet medical need in PD therapy.
Previous studies by Morari and co-workers have demonstrated the involvement of the
nociceptin/orphanin FQ peptide receptor (NOP, known previously as ORL-1 (opioid receptor-
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
like-1 receptor)) in Parkinson’s disease, particularly in development of motor symptoms.5,6 The
NOP receptor, a G protein coupled receptor (GPCR) is the fourth member of the opioid receptor
7
family having close homology to the classical opioid receptors (mu, delta, kappa). The
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
endogenous ligand for the NOP receptor is the heptadecapeptide nociceptin/orphanin FQ
(N/OFQ), which is similar to the endogenous kappa opioid peptide dynorphin, but has no binding
affinity to the three classic opioid receptors. NOP activation by endogenous N/OFQ is implicated
in many physiological functions and pathologies, including pain and analgesia, anxiety, learning
8
and memory, as well as modulation of tolerance development and reward. The NOP receptor is
found throughout the brain, specifically, in brain cortical and subcortical areas, particularly in
9
striatum, globus pallidus and substantia nigra (SNr) neurons. Morari et al. have shown that
endogenous N/OFQ contributes to the development of PD symptoms based on the following
findings: 1) increased levels of N/OFQ found in the SNr following dopamine (DA) cell loss or
impairment of DA transmission,10,11 2) NOP receptor antagonists reverse parkinsonian symptoms
in neurodegenerative (6-OHDA hemilesioned rat, MPTP-treated mouse and macaque) and
functional (reserpinized or haloperidol-treated animals) models of PD,10,12,13 and 3)
genetic deletion of the N/OFQ or NOP genes, or pharmacological blockade of the NOP
receptor protects mice from the neurotoxic action of MPTP.10,14 Mechanistic studies
revealed that the antiparkinsonian action of NOP antagonists is accomplished through
normalization of the imbalance between excitatory (Glu) and inhibitory (GABA) inputs
to the nigro-thalamic neurons, generated by striatal DA deafferentation.15,16 This
suggests that NOP antagonists may provide a non-dopaminergic approach for the
symptomatic and neuroprotective therapy of PD.
4
ACS Paragon Plus Environment

Page 5 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Morari and colleagues also showed that NOP receptor agonists attenuate the expression
of abnormal involuntary movements (AIMs, a rodent correlate of LID) in dyskinetic rats and
nonhuman primates challenged with L-DOPA, by acting in the striatum where, contrary to SNr,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
the N/OFQ tone is reduced and NOP receptors are up-regulated following DA cell loss.17,18 This
action can be dissociated from the typical motor inhibiting effects of NOP agonists since
antidyskinetic doses were 100-fold lower than doses that caused hypolocomotion. Morari et al.
showed that N/OFQ and NOP agonists specifically target dyskinesia pathways, preventing D1
receptor-mediated correlates of LID such as pERK phosphorylation and loss of depotentiation of
synaptic plasticity in striatal GABA neurons.17,18
Based on the above evidence from NOP antagonists targeting PD symptoms by blocking
nigral NOP receptors, and NOP agonists attenuating LID symptoms by stimulating up-regulated
striatal NOP receptors, we hypothesized that NOP partial agonists could provide a balanced
action in both areas affected in PD. A NOP partial agonist would be expected to function as an
antagonist under conditions of high extracellular levels of endogenous N/OFQ (as in SNr) and
alleviate parkinsonian motor dysfunction. On the other hand, in areas where the endogenous
N/OFQ tone is low or absent and NOP receptors are upregulated (i.e. in striatum during LID
development), NOP partial agonists would function as agonists. Based on this, we explored the
discovery and structure-activity relationships of NOP partial agonists to identify suitable
compounds to validate our hypothesis that NOP partial agonists may be a viable approach for PD
and LID treatment, and to gain further insights into the role of the NOP-N/OFQ system in the
pathophysiology of PD and dyskinesias.
RESULTS AND DISCUSSION
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Chemistry. Zaveri et al previously disclosed NOP ligands with an indolinone motif
(
piperidinyl-indolinones) that provide both full and partial agonist efficacy and modest
selectivity over the mu opioid receptor (MOP).19-21 An SAR exploration was initiated with other
similar bicyclic heterocycles, such as an indole and indoline ring in place of the indolinone
moiety. This resulted in two new lead compounds 4 (AT-001) and 5 (AT-004), which were
found to have partial agonist efficacy at NOP and >30-fold selectivity over the classical opioid
receptors (Table 1). Further SAR investigation of these lead compounds to improve binding
affinity and modulate intrinsic activity focused on the C(3) substituent of the indole ring is
discussed below.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The synthesis of lead compounds indoline 4 and indole 5 is shown in Scheme I.
Commercially available indoline 1 and N-Boc-4-piperidone 2 were subjected to standard
reductive animation to provide intermediate 3 in 96% yield. Subsequent Boc removal, followed
by a reductive alkylation of the piperidine nitrogen with 4-iPr-cyclohexanone provided indoline
4
as a mixture of ca. 1.5:1 cis:trans isomers (with respect to the disubstituted cyclohexyl ring).
The diastereomers could be separated via column chromatography to provide indoline 4 as a
single diastereomer in 28% yield over two steps. Oxidation of 4 with MnO in dichloromethane
2
smoothly provided indole 5 in 79% yield.
To explore the SAR of different functionalities at the C-3 of the indole ring, several C-3
substituted indole analogs were synthesized using chemical transformations of indole 5.
Treating 5 with chlorosulfonylisocyanate (ClSO NCO) in MeCN provided C(3)-substituted
2
amide 6 in modest yield. Aldehyde 7 was synthesized from indole 5 using Vilsmeier-Haack
conditions (Scheme I). Conversion of aldehyde 7 to oxime 8 using standard conditions occurred
readily in 96% yield. Oxime 8 was subsequently reduced using Ra (Ni) hydrogenation to afford
6
ACS Paragon Plus Environment

Page 7 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
amine 9 (AT-035) in 78% yield, and dimethylamine 10 in low yield as a minor byproduct
(Scheme I). In addition, reaction of amine 9 with benzoylthioisocyanate (BzNCS), followed by
exposure to K CO in MeOH readily provided thiourea 11 in moderate yield. Lastly, aldehyde 7
2
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
was smoothly converted to alcohol 12 via NaBH reduction in 86% yield.
4
Scheme I
Reagents and Conditions: (a) AcOH, STAB, DCE, rt, 16 h, 96%; (b) i. TFA, CH Cl , 1h, ii. 4-
2
2
iPr-cyclohexanone, STAB, AcOH, DCE, rt, 1-2 days, 28% over 2 steps; (c) MnO , CH Cl , rt, 16 h, 79%;
2
2
2
(
d) ClSO NCO, MeCN, 0˚C, 30 min, 37%; (e) POCl , DMF, 0˚C, 1 h, 74%; (f) NH OH·HCl,
2
3
2
NaOAc·3H O, EtOH:H O (2:1), 110 ˚C, 20 min, 96%; (g) H (g) (55 psi), 100 wt% Ra-Ni, MeOH, 16 h,
2
2
2
78% for 9, 10-15% for 10; (h) i. BzNCS, CH Cl , 0˚C, ii. K CO , MeOH, rt, 58%; (i) NaBH , EtOH, rt, 2
2
2
2
3
4
h, 86%.
To explore longer alkyl substitution at the indole C(3) position (e.g. 21-24), the route was
amended so that the indole ring was constructed using a Larock heteroannulation reaction
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
22
between a terminally silated alkyne and a N-substituted iodoaniline (Scheme II). The N-Boc
protected amino alkynes 15 and 16 were obtained from the corresponding alkynyl alcohols (13
and 14, respectively) in four straight-forward steps: 1) the alcohol was converted into a tosylate
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
group, 2) S 2 displacement of the tosylate group with phthalimide, 3) removal of the phthalimide
N
group using hydrazine, and 4) Boc-protection of the amine.
Scheme II
Reagents and Conditions: (a) TsCl, TEA, cat. DMAP, CH Cl , rt, 16 h, 93-99%; (b) phthalimide,
2
2
K CO , DMF, 50˚C, 6 h, 67-94%; (c) NH NH , MeOH, rt, 16 h, 59-70%; (d) (Boc) O, cat. DMAP, THF,
2
3
2
2
2
rt, 85-88%; (e) AcOH, STAB, DCE, rt, 16 h; (f) TFA, CH Cl , 0 ˚C⟶rt, 1 h; (g) 4-iPr-cyclohexanone,
2
2
AcOH, STAB, DCE, rt, 1-2 days, 39% over 3 steps; (h) alkyne 15 or 16, cat. Pd(OAc) , LiCl, K CO ,
2
2
3
DMF, 102˚C, 2.5 h, 65-76%; (i) AcCl, MeOH, 0˚C⟶rt, then EtOAc, 78-80%; (j) i. BzNCS, CH Cl , 0˚C,
2
2
ii. K CO , MeOH, rt, 47-50%.
2
3
The synthesis of the requisite iodoaniline 18 and the formation of the indole ring is
shown in Scheme II. Commercially available 2-iodoaniline (17) was converted to intermediate
18 in a 3-step sequence consisting of 1) reductive amination with N-Boc-4-piperidone (2), 2)
8
ACS Paragon Plus Environment

Page 9 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
removal of the Boc group, and 3) reductive amination of the piperidine nitrogen with 4-
isopropylcyclohexanone in 39% yield over the three steps. At this stage, the key Larock
heteroannulation reaction between iodoaniline 18, and either alkyne 15 or 16, proceeded in good
yield to provide indole intermediates 19 and 20, respectively. When indoles 19 and 20 were
separately exposed to AcCl/MeOH conditions, clean removal of both the silyl group and the Boc
moiety occurred to provide amines 21 and 22 in situ as bis HCl salts (Scheme II). Finally, ethyl-,
and propyl-linked amines 21 and 22 each reacted with benzoylthioisocyanate (BzNCS) to give
the respective ethyl-, and propyl-linked thioureas 23 and 24 in moderate yields (Scheme II).
Other C-3 substituents were introduced through further functionalization of aldehyde 7
via reductive amination with various nitrogen nucleophiles to provide C(3)-substituted cyclic and
acyclic amines (25, 27-31) in moderate to good yields (Scheme III).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme III
NHR2
N
2
2
6 R = Boc
N
R1
b
27 R2
= H
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
OH
N
N
a
25
N
R1
28
N
R1
a
a
a
a
CHO
N
1
R =
N
7
R1
N
H
OH
N
a
OH
N
29
N
31
R1
OH
R1
N
N
R1
30
Reagents and Conditions: (a) amine, AcOH, STAB, DCE or MeOH, rt, 1-2 days, 22-98%; (b) TFA,
CH Cl , 0˚C⟶rt, 20 min, 78%.
2
2
The synthesis of amides 33, 35, and 37 was accomplished in good yield from amine 9 via
EDCI coupling with the corresponding N-Cbz-protected acid, followed by removal of the Cbz
protecting group (Scheme IV). The use of the Cbz protecting group was critical in synthesizing
amides 33, 35, and 37, as the Boc-protected substrate degraded upon exposure to acidic
conditions during deprotection.
1
0
ACS Paragon Plus Environment

Page 11 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Scheme IV
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Reagents and Conditions: (a) HOBT, EDCI, TEA, N-Cbz acid, CH Cl , 0˚C, 67-88%; (b) H
2
2
2
balloon, 10 wt% Pd/C (10%), THF or EtOH, 3 h, 39-87%.
In Vitro Binding and Functional Efficacy. The compounds were tested in vitro for
binding affinity at the four opioid receptor subtypes in radioligand displacement assays in
membranes from human NOP, MOP, KOP and DOP receptor-transfected chinese hamster ovary
(
CHO) cells, using methods we have described in detail previously.21,23 Binding affinity was
calculated as the binding constant Ki (nM), shown in Table 1. The intrinsic activity (functional
3
5
efficacy) was determined using the [ S]GTPS binding assay in membranes prepared from the
opioid receptor-transfected CHO cells, using methods we have previously published.21,23,24 The
intrinsic activities shown in Table 1 are reported as the % stimulation compared to that of the full
agonist N/OFQ taken as 100%. The agonist potencies are reported as the EC (Table 1). All
5
0
compounds showed weak binding affinity for the  opioid receptor (DOP) and  opioid receptor
(
KOP), and were not further tested for intrinsic activity at these receptors.
Compounds in Table 1 were designed to explore the SAR of the indole moiety on the N-
substituted piperidine scaffold, the latter of which is a common motif in most NOP ligands.25,26
SAR for small-molecule NOP partial agonists have not been extensively explored previously,
1
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
with respect to the effects of structure modification on NOP binding affinity, opioid selectivity or
intrinsic efficacy.27,28 To explore the SAR of the N-piperidinylindoles for high affinity NOP
partial agonists, we focused on the C-3 substituent and maintained the N-
isopropylcyclohexylpiperidinyl moiety in all the analogs synthesized, as this piperidinyl
substituent has afforded high NOP binding affinity in other scaffolds we have investigated.20,21,29
INSERT TABLE 1 HERE
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The C(3)-unsubstituted lead compounds indoline 4 and indole 5 show nanomolar affinity
for NOP (10 nM), and weak affinity for the three classical opioid receptors (Table 1, entries 1
and 2), giving at least >40-fold selectivity for the NOP receptor. Both compounds show partial
3
5
agonist efficacy at NOP in the [ S]GTPS functional assay. Indoline 4 showed a three-fold
higher potency (EC ) as an agonist at NOP than indole 5. From the C(3)-substituted indoles
5
0
bearing a polar substituent on a single methylene linker (compounds 9, 10, and 12), primary
amine 9 has high binding affinity (NOP Ki = 3 nM), ~20-fold selectivity versus MOP, and
modest potency as a NOP partial agonist. Alcohol 12 and dimethylamino (tertiary amine
substituent) 10 have 10-fold lower affinity than primary amine 9, although alcohol 12 has
comparable intrinsic activity to amine 9 whereas dimethylamine 10 showed significantly reduced
agonist potency. We performed a chain-length SAR of amine 9, to the ethyl-, and propyl-linked
amines 21 and 22, respectively. Both methyl- (9), and ethyl-linked (21) amines are optimal for
NOP affinity and the increase in chain length to the ethyl-linked amine 21 also results in a 10-
fold higher NOP selectivity compared to 9. Functionally, the two compounds are both partial
agonists at NOP, with similar potency. However, a further extension of the chain length to the
propylamine 22 was not optimal for NOP binding.
1
2
ACS Paragon Plus Environment

Page 13 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
There appears to be a steric size tolerance for a cationic amino group at the C(3) indole
position. The pyrrolidinomethylene at C(3), as in compound 25, showed high binding affinity for
NOP (Ki 1.47 nM) and >200-fold selectivity versus the other opioid receptors. Interestingly,
amine 25 also had high intrinsic activity, to a nearly full agonist (Table 1). However, a larger 4-
amino-substituted piperidine moiety (as in compound 27) resulted in a significant drop in
affinity, although the 4-hydroxypiperidine analog 28 showed higher binding affinity than 27.
Interestingly however, 2-hydroxypyrrolidine 29 showed an even higher NOP binding affinity
than hydroxypiperidine 28, and an increase in intrinsic activity. It appears that pyrrolidine 25 and
hydroxypyrrolidine 29 show higher agonist efficacy than the open-chain primary amines 9, 21
and 22, suggesting that these compounds may be binding in different orientations to the receptor
due to steric reasons, differentially affecting the activation of the receptor towards higher
intrinsic activity. Further contraction of the hydroxypyrrolidine ring of 29 to azetidine 30, retains
modest binding affinity at NOP (Ki = 23 nM), but shows very low agonist potency in the
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
[35
S]GTPS functional assay (EC = 1559 nM). In addition, acyclic alkyl amine 31, an acyclic
5
0
variant of hydroxypyrrolidine 29 showed weak binding affinity toward all four opioid receptors.
When compounds 9, 10, 12, 21, and 22, are compared to compounds 25 and 29, it appears that
cationic cyclic C(3) substituents, particularly pyrrolidinyl, show higher intrinsic activity (towards
full agonist efficacy) compared to cationic open-chained C(3) substituents which show partial
agonist activity.
Thiourea analogs 11, 23, 24 and amide analogs 33, 35, 37 were synthesized to investigate
the importance of a cationic amine moiety at the C-3 substituent of the piperidinylindoles. The
thiourea analogs 11, 23, and 24 showed only a modest (2-3-fold) drop in NOP binding affinity
1
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
and agonist potency, suggesting that it may be the hydrogen bonding or polar interactions of the
C-3 substituent that are important for high affinity rather than a charged interaction.
Amides at the C-3 substituent containing an arginine unit (as in 33), a lysine-like 6-
aminohexanamide sidechain (as in 35), or a ethylaminomethylamide (as in 37) were also found to
have only modest binding affinities (11–23 nM) at NOP, suggesting that a charged interaction of
the guanidinium group in 33 or the amino group in 35 is likely not an important contributor to
the binding affinity at NOP. Further, these compounds had poor agonist potencies and showed
low intrinsic activity.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
Compound 6, containing an sp hybridized C(3) substituent in the form of an amide,
showed weak binding affinity for all the opioid receptors (Ki > 100 nM). However, the C(3)-
oxime 8, on the other hand, was found to be a high-affinity (Ki = 2 nM), NOP-selective
(NOP/MOP = ca. 25 fold) potent full agonist.
Overall, our SAR showed that the C(3)-substituted piperidinylindoles afforded selective
NOP agonists of varying efficacies, ranging from partial agonists (>20%) to nearly full agonist
efficacy (>80%). It is possible that high intrinsic efficacy seen with C(3)-substituted indoles 8,
2
5, and 29 may arise from a different binding mode of these indoles (bearing a bulkier C(3) polar
substituent, as for 25 and 29) than the smaller alkyl-linked or unsubstituted indoles 5 or 9. We
have shown, using molecular dynamics simulations, that polar interactions between a NOP
agonist ligand and NOP receptor residues at the extracellular end of the transmembrane helices
and EL2 loop, result in agonist-induced reorganization of polar networks, during receptor
activation.30 Further SAR development supported by molecular docking and site-directed
mutagenesis is needed to explain the observed NOP full agonism for compounds 8 and 25.
1
4
ACS Paragon Plus Environment

Page 15 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
To explore our hypothesis that NOP partial agonists would have dual efficacy in blocking
PD symptoms and attenuating LID symptoms, four compounds were selected for further testing,
based on the criteria of having high NOP binding affinity (Ki less than or equal to 10 nM) and
partial agonist efficacy (20-35%) with agonist potency EC50 < 150 nM. Based on our in vitro
pharmacological characterization, compounds 4, 5, 9 and 21 were selected for further in vitro
ADME characterization and compounds 4, 5 and 9 were advanced to in vivo pharmacokinetic
evaluation for further investigation in vivo in PD models.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In Vitro ADME characterization and In vivo PK. Selected compounds were evaluated in
vitro in ADME assays to characterize intestinal permeability, blood brain barrier permeability,
and liver microsomal stability (Table 2). Indoline 4 and indole 5 show high permeability and low
efflux in the Caco-2 intestinal permeability assay as well as in the MDR1-MDCK assay for BBB
permeability (Table 2). Primary amines 9 and 21 showed lower permeability in the Caco-2 and
BBB assay and high efflux in the BBB permeability assay. However, both primary amines 9 and
2
1 show excellent stability in rodent liver microsomes (Table 2), whereas indole 5 has a short
half-life in rat liver microsomes. From the in vitro microsomal stability of the amine 9 compared
to that of unsubstituted indole 5, it appears that the unsubstituted C(3)-position in indole 5 may
be a site of metabolic liability, and that the high permeability of indole 5, which arises from its
high lipophilicity, comes at the expense of solubility and metabolism.
INSERT TABLE 2 HERE
The plasma and brain concentrations of compounds 4, 5 and 9 were determined in an in
vivo pharmacokinetic study after subcutaneous administration to rats. As seen in Table 3,
compounds 4 and 5 showed high total plasma and brain concentrations, with B/P ratios >6,
suggesting high brain permeability of these compounds. Compound 9, which showed poor BBB
1
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
permeability and high efflux in the in vitro BBB assay (Table 2) also showed a B/P ratio >1,
although it showed overall lower concentrations than 4 or 5, consistent with its lower
permeability.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
INSERT TABLE 3 HERE
Assessment of In vivo efficacy of NOP partial agonists in rat models of Parkinson’s disease
and levodopa-induced dyskinesia. NOP receptor partial agonists 4, 5 and 9 were evaluated in
vivo for their efficacy in improving parkinsonian motor disabilities in a rodent neurodegenerative
model of PD, i.e. the 6-hydroxydopamine-induced hemilesioned rat model, that we have
extensively used to characterize NOP antagonists.10,15,16 The compounds were administered
intraperitoneally (i.p.) in a range of doses.
Figure 1 shows that NOP partial agonists 4, 5 and 9 attenuate parkinsonian disabilities in
6
-OHDA hemilesioned rats. Parkinsonian motor deficits were assessed using three different
measures of motor activity.10,15,16,31 Acute administration of 5 (0.1-3 mg/kg, i.p.) improved motor
deficits, partially reversing (~25%) the increase of immobility time (a measure of akinesia) at the
contralateral (parkinsonian) paw in the bar test (Figure 1 panel D), and doubling stepping activity
at the contralateral paw (a measure of akinesia/bradykinesia) in the drag test (Figure 1 panel E).
Moreover, it enhanced by ~20%, the time spent on the rotarod (a measure of overall gait ability)
(Figure 1B panel F). This profile is predictive of antiparkinsonian activity since it is replicated by
L-DOPA10, 16 and by NOP antagonists.12,15,16 Compound 4 and 9 also showed similar patterns in
improving parkinsonian motor deficits, although with some differences in efficacy in the three
tests, at doses of 0.01-1 mg/kg (Figure 1, panels A-C and G-I), suggesting that the NOP partial
agonists may actually be counteracting endogenous N/OFQ action at the NOP receptor, as
predicted by our working hypothesis.
1
6
ACS Paragon Plus Environment

Page 17 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
INSERT FIGURE 1 HERE
However, in the same dose ranges, all three compounds had no effect on the ‘expression’
of LID in fully dyskinetic rats challenged with L-DOPA, using methods previously reported by
us (data not shown).17,18 It is possible that the partial agonist efficacy of about 25-35% in all
three compounds is too low to elicit an anti-dyskinetic effect seen with NOP full agonists that we
have previously reported.17,18 SAR studies to obtain suitable higher efficacy NOP partial
agonists from the piperidinylindole and other novel NOP ligand scaffolds are underway to
address this hypothesis and will be reported in due course.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CONCLUSIONS
In summary, we report the discovery and SAR of a novel series of 4-piperidinyl-1H-
indoles as NOP receptor partial agonists. The SAR studies reported here represent the first
systematic SAR study around NOP partial agonists and a novel NOP ligand scaffold, the
piperidinyl-indoles. SAR analysis showed that the C(3)-substituted piperidinylindoles show
significantly lower affinity for the classical opioid receptors mu, delta and kappa, resulting in
selective NOP ligands with a range of intrinsic efficacies, ranging from partial agonists to nearly
full agonists. Our SAR further indicates that size and polar nature of the C(3) substituent of the
indole moiety influences the binding affinity and intrinsic activity of these ligands, such that
smaller alkyl-linked C(3) substituents on the indole have partial agonist efficacy (e.g. compound
9, 21) whereas bulkier polar substituents have higher agonist efficacy (e.g. compound 25),
possibly arising from different binding modes favoring receptor activation. Selected NOP partial
agonists 4, 5 and 9 also reduced parkinsonian motor symptoms in the 6-OHDA-hemilesioned rat
model but were ineffective in reducing LID symptoms at the same dose range. Together with our
1
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
previous studies, these results provide further evidence that the NOP receptor is a possible non-
dopaminergic target for the symptomatic treatment of PD and that NOP receptor partial agonists
are promising for development of new symptomatic PD therapies. The SAR of novel NOP
partial agonists reported here will assist in future lead optimization efforts in this regard.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
8
ACS Paragon Plus Environment

Page 19 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Table 1. Binding affinities and functional efficacies of C(3)-substituted piperidinylindoles at human opioid receptors
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
a
35
b
Binding Affinities, K (nM)
[S] GTPS Functional Assay (nM)
NOP MOP
i
Compound number
NOP
MOP
DOP
KOP
EC50
% Stim
EC50
% Stim
4
10.3 ± 0.03
604 ± 5
3070 ± 747
562 ± 150
54.5 ± 17.6
33.0 ± 2.8
ND
––––
R
5
9
H
9.80 ± 0.86
3.27 ± 0.30
44.7 ± 9.1
376 ± 37
65.3 ± 2.4
716 ± 21
923 ± 413
>10K
1594 ± 57
1737 ± 172
>10K
160 ± 64
121 ± 51.7
117 ± 13.7
29.3 ± 9.5
35.9 ± 5.7
18.9 ± 0.70
232 ± 12.6c
410 ± 105
ND
16.3 ± 7.6c
11.8 ± 2.7
––––
12
>10K
1
0
1
43.8 ± 7.4
504 ± 173
>10K
>10K
532 ± 185
28.1 ± 2.9
ND
––––
2
2.27 ± 0.11
20.9 ± 7.6
437 ± 118
46.2 ± 6.1
>10K
>10K
2750 ± 383
1320 ± 411
139 ± 30.3
249 ± 9
27.8 ± 7.2
18.6 ± 2.8
ND
––––
22
1764 ± 118
15.7 ± 3.2
2
5
7
1.47 ± 0.57
216 ± 12
453 ± 107
1308 ± 18
>10K
>10K
461 ± 135
97.4 ± 7.4
ND
87.9 ± 11.8
––––
ND
ND
––––
––––
2
3456 ± 300
1
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
Table 1. Binding affinities and functional efficacies of C(3)-substituted piperidinylindoles at human opioid receptors
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
a
35
b
Binding Affinities, K (nM)
[S] GTPS Functional Assay (nM)
NOP MOP
i
Compound number
NOP
MOP
DOP
KOP
EC50
% Stim
EC50
% Stim
28
64.0 ± 21.3
9.46 ± 1.01
>10K
6028 ± 652
>10K
3840 ± 1887
>10K
446 ± 130
10.6 ± 1.2
ND
––––
29
306 ± 77
575 ± 157
69.9 ± 12.1
ND
––––
30
31
23.6 ± 3.1
136 ± 7.50
>10K
>10K
>10K
>10K
1559 ± 398
ND
34.3 ± 9.3
ND
ND
ND
––––
––––
973 ± 211
1490 ± 52
1
1
3
10.5 ± 1.25
24.4 ± 0.30
71.3 ± 16.2
227 ± 50.0
4067 ± 68
>10K
1328 ± 288
81.0 ± 2.8b
343 ± 18
>10K
51.2 ± 9.7
5.6 ± 3.0
>10K
ND
9.8 ± 2.1
––––
2
2
0
ACS Paragon Plus Environment

Page 21 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Table 1. Binding affinities and functional efficacies of C(3)-substituted piperidinylindoles at human opioid receptors
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
a
35
b
Binding Affinities, K (nM)
[S] GTPS Functional Assay (nM)
NOP MOP
i
Compound number
NOP
MOP
DOP
KOP
EC50
% Stim
EC50
% Stim
2
4
31.8 ± 1.7
11.0 ± 2.7
23.1 ± 8.5
382 ± 129
857 ± 109
949 ± 74
>10K
>10K
>10K
830 ± 181
674 ± 85
>10K
175 ± 23
34.5 ± 3.6
ND
––––
33
>10K
11.1 ± 1.5
37.7 ± 8.9
ND
ND
––––
––––
3
5
7
464 ± 124
3
15.9 ± 3.7
129 ± 41.4
269 ± 12
453 ± 8
>10K
1440 ± 342
446 ± 24
277 ± 144
ND
15.9 ± 4.9
ND
ND
ND
––––
––––
6
2712 ± 300
8
2.17 ± 0.35
56.4 ± 1.7
1690 ± 870
>10K
67.0 ± 11.0
85.9 ± 9.8
3199 ± 260
37.5 ± 2.9
2
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
a
Binding affinities were determined using radioligand displacement assays performed in membranes of CHO cells stably expressing the
3
3
3
human NOP, MOP, KOP and DOP receptors, and their respective ra-dioligands, [ H]N/OFQ–NOP, [ H]U69,593–KOP, [ H]DAMGO–MOP and
3
[ H]DPDPE–DOP receptor. Equilibrium dissociation constants (K ) were derived from IC values using the Cheng−Prusoff equation. Each K value
i
50
i
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
represents the arithmetic mean ± SEM from at least three independent experiments, each performed in triplicate. ND = not determined.
b
Compounds with K values >100nM were not tested in functional assays (N.D.). At the DOP and KOP receptor, all compounds showed
i
binding affinity K >100nM, hence functional efficacy at KOP and DOP not determined. Functional activity was determined by stimulation of
i
[35
S]GTPγS binding to cell membranes, EC is the ligand concentration producing half maximal stimulation, % stimulation was obtained as a
50
percentage of stimulation of the standard full agonists, N/OFQ (for NOP), and DAMGO (for MOP), which showed at least 2- to 5-fold stimulation
over basal. Results are the means ± SEM for at least 3 independent experiments each performed in triplicate.
2
2
ACS Paragon Plus Environment

Page 23 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Table 2. In Vitro ADME data for indoles 4, 5, 9 and 21
Caco-2 Bidirectionala
MDR1-MDCK Permeabilityb
Human
Liver
Microsomal
Stability
Rat Liver Mouse Liver
Microsomal Microsomal
-
6
-6
P
app (x 10 cm/s)
Efflux Ratio
P
app(x 10 cm/s)
Efflux Ratio
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Stability
Stability
A–B
B–A
(Perm/Efflux)
A–B
B–A
(Perm/Efflux)
(t½ min)
(t½ min)
(t½ min)
4
5
9
30
24
25
19.1
4.92
6.49
-6
0.8 (Hi/No)
0.8 (Hi/No)
1.1 (Hi/No)
1.5 (Hi/No)
26.8
28.8
0.34
0.60
33.8
15.2
14.4
22.1
1.3 (Hi/No)
0.5 (Hi/No)
43 (Low/Hi)
37 (Low/Hi)
b
ND
ND
5.8
>60
ND
2.8
5.3
4.64
4.36
>60
ND
21
>60
a
(
P >1.0x10 cm/s and efflux ratio<3, high perm/no efflux), (P A–B >3.0 and efflux ratio <3 = high
app
app
BBB permeability, efflux ratio>3 = high efflux). These experiments were conducted according to
methods described briefly in the Experimental section.
Table 3. In vivo pharmacokinetics for selected indoles 4, 5 and 9 after s.c. administration to
Sprague Dawley rats.a
Plasma
Brainc
Dose
mg/kg)
B/P Ratio
C
max
AUClast
C
max
AUClast
(
(at Cmax)
Tmax (h)
t ½ (h)
Tmax(h)
t ½ (h)
(nM)
(nM.h)b
(nM)
(nM.h)
4
5
9
3 (s.c.)
3 (s.c.)
3 (s.c.)
456
626
529
0.5
1
1246
1960
1004
2.13
2.19
2.61
3069
4166
956
0.5
0.5
6695
10104
6129
2.71
2.43
10.8
6.73
6.65
1.81
0.5
4.00
a
b
c
These experiments were conducted according to methods described briefly in the Experimental section.
Timepoint for AUClast is 8h
Concentrations represent total brain concentrations
2
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 1. Effect of NOP partial agonists 4 (AT-001), 5 (AT-004) and 9 (AT-035) on
parkinsonian disabilities in the 6-OHDA hemilesioned rat model of PD. Acute systemic
administration of 4, 5 and 9 in a range of doses (panels A–C, 4, AT-001, 0.001-3 mg/kg, i.p.)
(panels D–F, 5, AT-004, 0.1-3 mg/kg, i.p.) or (panels G–I, 9, AT-035, 0.01 – 3 mg/kg, i.p.)
attenuated motor deficits in three behavioural measures, (A) the bar test (left graphs), (B) the
drag test (middle graphs) and (C) rotarod test (right graphs). Data are means ± SEM of 6-7 (AT-
2
4
ACS Paragon Plus Environment

Page 25 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
0
01 and AT-035) or 17-23 (AT-004) determinations per group. AT-001 data (panels A-C) are
means of 17-23 mice per group, AT-004 data (panels D-F) are means of 6-12 mice per group, and AT-035
data (G-I) are means of 6-7 mice per group. Essential statistical values: treatment effect panels A-C
(F =4.60, p=0.0007, F =4.52, p=0.0008, F =13.72, p<0.0001, respectively), panels D-F
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
,2
5,2
5,1
(F =4.39, p=0.0051, F =8.45, p<0.0001, F =7.59, p<0.0001, respectively), panels G-I
3,2 3,2 3,1
(F =14.31, p<0.0001, F =3.79, p=0.0071, F =11.51, p<0.0001, respectively) *P < 0.05, **P
4
,2
4,2
4,1
< 0.01 different from vehicle (saline + 5% Tween 80 and 1% DMSO).
2
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
EXPERIMENTAL SECTION
CHEMISTRY METHODS
Thin layer chromatography was performed on Analtech silica gel GF 250 micron TLC
plates. The plates were visualized with a 254 nm UV light and iodine. Flash chromatography was
carried out on F60 silica gel, 43-60 m (230-400 mesh), 60 Å (Silicycle SiliaFlash). All solvents
and chemicals were purchased from commercial suppliers and used without further purification.
All reactions were capped from the atmosphere unless otherwise stated. NMR was recorded on a
Varian Mercury Plus NMR (300 MHz) using CDCl (7.27 ppm standard) or DMSO-d (2.50
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
6
1
ppm standard). Data for H NMR were recorded as follows:  chemical shift (ppm), multiplicity
(s, singlet; d, doublet; t, triplet; dd, doublet of doublets; dt, doublet of triplets; q, quartet; dq,
doublet of quartets; m, multiplet; br, broad; etc.), coupling constant (Hz), integration. Mass
spectra were obtained on a LCQ Fleet Ion Trap LCMS, a micromass ZMD 1000 or PE Sciex API
1
50EX LCMS using electrospray ionization (ESI), or APCI mode. Elemental analyses were
performed by Atlantic Microlabs, Norcross, GA. HRMS analyses were performed by the Mass
Spectrometry Service Laboratory, University of Minnesota Department of Chemistry,
Minneapolis, MN on a Bruker BioTOF II HRMS using ESI mode. HPLC analysis was
performed on a reverse phase Agilent Zorbax SB-Phenyl column (5 m, 2.1 x150 mm), using a
binary gradient of 95:5→5:95 solvent A (95/5 H O/ACN + 0.1% formic acid): solvent B (5/95
2
H O/ACN + 0.1% formic acid) for 12 minutes, at a flow rate of 0.40 mL/min. Eluted peaks were
2
monitored at 254 nm with a Shimadzu SPD-10AVP UV-Vis detector. All final compounds were
confirmed to be of ≥95% purity by the HPLC method described above. Purity of final
compounds was confirmed by elemental analysis and was within ±0.4% of the theoretical value.
Molecular weights of selected compounds were also confirmed by HRMS, as indicated in the
2
6
ACS Paragon Plus Environment

Page 27 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
experimental section. Based on H NMR, HPLC, combustion analysis data and HRMS, all final
compounds were ≥95% pure.
SYNTHESIS
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
General Procedure A: Reductive Amination. The aniline or amine substrate (1.00-2.00
equiv) and carbonyl compound (1.00-1.50 equiv) were charged into a round bottom flask. 1,2-
DCE (0.030-0.25M) was added, and the mixture was stirred until both components dissolved.
NOTE: If amine was a salt, iPr NEt (2.30 equiv) was added at this stage to freebase. To this
2
solution was added glacial AcOH (1.00-2.30 equiv) at ambient temperature, and the solution was
stirred for 60 minutes. At this stage, sodium triacetoxyborohydride (STAB) (1.50-3.00 equiv)
was added. The reaction was allowed to stir at room temperature and monitored by TLC
(
EtOAc:Hexanes). After 1-2 days, the reaction was ≥90% complete by TLC analysis. The
reaction was quenched with saturated NaHCO (aq.) and stirred until the reaction mixture was
3
basic and bubbling had ceased. The biphasic layer was separated, and the organic layer was
washed 2x with H O, brine, dried with MgSO , filtered and concentrated in vacuo to provide a
2
4
crude product that was purified via flash chromatography to provide the desired product.
General Procedure B: Boc Removal (Step 1) and Reductive Amination with 4-
isopropylcylohexanone (Step 2). Step 1. A solution of N-Boc intermediate (1.00 equiv) in
CH Cl (0.25-0.30M) was cooled to 0˚C, and then TFA (6-30 equiv) was added over several
2
2
minutes. Upon completion of addition, the ice-bath was removed and the reaction was allowed to
warm to room temperature and monitored by TLC (EtOAc:Hexanes). After 2 hours, the reaction
was complete. The reaction was concentrated in vacuo, followed by the addition of EtOAc,
which was consequently removed in vacuo to ensure the majority of excess TFA was removed.
The resulting oily residue was dissolved in EtOAc and was stirred as saturated NaHCO (aq.)
3
2
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 64
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
was added until the aqueous layer remained basic. The layers were separated, and the aqueous
layer was extracted with EtOAc until UV activity in the aqueous layer was minimal (3-8x). The
EtOAc layers were combined, washed with brine, dried with MgSO , filtered, and concentrated
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
in vacuo to provide the piperidine intermediate.
Step 2. The piperidine intermediate from the previous step (1.00 equiv) and 4-iPr-
cyclohexanone (1.00-1.50 equiv) were dissolved in 1,2-DCE (0.070M). To the reaction was
added glacial AcOH (1.00-2.30 equiv), and the reaction was stirred for 30 minutes. After 30
minutes, STAB (1.50-2.30 equiv) was added. An Ar balloon was fitted on top of the reaction,
and the reaction was monitored by TLC (MeOH:CH Cl :NH OH (aq.)). After 2-3 days, the
2
2
4
reaction was ≥95% complete. Saturated NaHCO (aq.) was added until the aqueous layer
3
remained basic. At this stage, the layers were separated, and the aqueous layer was extracted 2x
with CH Cl . The organic layers were combined, and washed 2x with H O, brine, dried with
2
2
2
MgSO , filtered, and concentrated in vacuo to provide a crude residue that was purified via flash
4
chromatography using EtOAc:Hexanes:NH OH (aq.).
4
General Procedure C: Formation of thiourea from amine. The amine (1.00 equiv)
was dissolved in CH Cl (0.10 M) at room temperature, and then the reaction was placed in an
2
2
ice-bath and cooled to 0˚C. Benzoyl isothiocyanate (1.05-1.10 equiv) was added, and after
several minutes of stirring at 0˚C, the reaction was allowed to warm to room temperature and
monitored by TLC (EtOAc:Hexanes). After 1-3 hours, the reaction was complete by TLC. The
reaction was concentrated in vacuo, the remaining residue was dissolved in MeOH (0.050 M),
and to this was added K CO (7.00 equiv). The reaction was stirred at room temperature and
2
3
monitored by TLC (EtOAc:Hexanes). Once the reaction was complete, it was filtered over a pad
of Celite, rinsed with MeOH, and then concentrated in vacuo to provide a crude material that was
2
8
ACS Paragon Plus Environment

Page 29 of 64
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
purified via flash chromatography using EtOAc:Hexanes:NH OH (aq.) to provide the desired
4
thiourea product.
tert-butyl 4-(indolin-1-yl)piperidine-1-carboxylate (3). See General Procedure A:
Indoline 1 (10.0 g, 83.9 mmol, 1.00 equiv), N-Boc piperidone 2 (17.6 g, 88.1 mmol, 1.05 equiv),
AcOH, 4.80 mL, 83.9 mmol, 1.00 equiv), STAB (26.7 g, 12.6 mmol, 1.50 equiv), DCE (352 mL,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0
.25M). The crude oil was purified via flash chromatography using 10:90 EtOAc:Hexanes to
1
provide indoline 3 as a white solid (24.3 g, 96% yield). H NMR (300 MHz, CDCl )  7.06 (t, J
3
=
6.0 Hz, 2H), 6.03 (t, J = 6.0 Hz, 1H), 6.43 (d, J = 6.0 Hz, 1H), 4.25 (m, 2H), 3.52 (m, 1H),
3.35 (t, J = 6.3 Hz, 2H), 2.79 (m, 2H), 1.80 (d, J = 9.3 Hz, 2H), 1.60 (m, 4H), 1.49 (s, 9H); MS
(APCI) m/z: 303.06 [M+H]+.
cis-1-(1-(-4-isopropylcyclohexyl)piperidin-4-yl)indoline (4). See General Procedure B:
Step 1 (Boc Removal): Indoline 3 (24.4 g, 80.5 mmol, 1.00 equiv), TFA (38.0 mL, 496 mmol,
6.20 equiv), CH Cl (300 mL, 0.27M). NOTE: Combined EtOAc layers were dried immediately
2
2
with MgSO , and were not washed with water or brine, as the product is water-soluble. The
4
product was obtained as a grey solid (13.6 g, 84% yield); Step 2 (Reductive Amination): N-H
piperidine from the previous step (13.6 g, 67.2 mmol, 1.00 equiv), iPr-cyclohexanone (9.40 g,
6
1
7.2 mmol, 1.00 equiv), AcOH (3.85 mL, 67.2 mmol, 1.00 equiv), STAB (21.3 g, 101 mmol,
.50 equiv), DCE (960 mL, 0.070M). Purified via flash chromatography using 10:90:1.5
EtOAc:Hexanes:NH OH (aq.) to provide compound 4 as a light-gold oil (33% yield). R = 0.25
4
f
1
(
20:80:3 drops EtOAc:Hexanes:NH OH (aq.), UV, pAA, I ); H NMR (300 MHz, CDCl )  7.05
4
2
3
(t, J = 5.7 Hz, 2H), 6.60 (t, J = 5.7 Hz, 1H), 6.41 (d, J = 5.7 Hz, 1H), 3.37 (m, 3H), 3.10 (d, J =
8.7 Hz, 2H), 2.94 (t, J = 6.3 Hz, 2H), 2.27 (m, 1H), 2.14 (t, J = 8.7 Hz, 2H), 1.82-1.54 (m, 11H),
.38 (m, 2H), 1.13 (m, 1H), 0.88 (d, J = 5.1 Hz, 6H); MS(ESI) m/z: 327.4 [M+H]+. The HCl salt
1
2
9
ACS Paragon Plus Environment