Development of LC-MS/MS-based receptor occupancy tracers and positron emission tomography radioligands for the nociceptin/orphanin FQ (NOP) receptor

Article abstract of DOI:10.1021/jm201629q

Currently, a lack of sufficient tools has limited the understanding of the relationship between neuropsychiatric disorders and the nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor. Herein, we describe the discovery and development of an antagonist NOP receptor occupancy (RO) tracer and a novel positron emission tomography (PET) radioligand suitable to probe the NOP receptor in human clinical studies. A thorough structure-activity relationship (SAR) around the high-affinity 3-(2′-fluoro-4′,5′- dihydrospiro[piperidine-4,7′-thieno[2,3-c]pyran]-1-yl)-2-(2-halobenzyl) -N-alkylpropanamide scaffold identified a series of subnanomolar, highly selective NOP antagonists. Subsequently, these unlabeled NOP ligands were evaluated in vivo by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in rat to determine brain uptake, kinetics and specific binding. (S)-27 was identified as a suitable unlabeled preclinical RO tracer to accurately quantify NOP receptor engagement in rat brain. Three compounds were selected for evaluation in nonhuman primates as PET tracers: (-)-26, (-)-30, and (-)-33. Carbon-11 labeling of (+)-31 yielded [¹¹C]-(S)-30, which exhibited minimal generation of central nervous system (CNS) penetrant radiometabolites, improved brain uptake, and was an excellent PET radioligand in both rat and monkey. Currently [¹¹C]-(S)-30 is being evaluated as a PET radiotracer for the NOP receptor in human subjects.

Full text of DOI:10.1021/jm201629q

Subscriber access provided by Penn State | University Libraries
Article
Development of LC-MS/MS-Based Receptor Occupancy
Tracers and Positron Emission Tomography Radioligands
for the Nociceptin/Orphanin FQ (NOP) Receptor
Concepcion Pedregal, Elizabeth Joshi, Miguel A Toledo, Celia Lafuente, Nuria Diaz, Maria
Angeles Martinez-Grau, Alma Jimenez, Ana Benito, Antonio Navarro, Zhaogen Chen,
Daniel R Mudra, Steven D Kahl, Karen S Rash, Mike A Statnick, and Vanessa N Barth
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm201629q • Publication Date (Web): 30 Apr 2012
Downloaded from http://pubs.acs.org on May 8, 2012
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 free 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 accessible to all
readers and 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.
Journal of Medicinal Chemistry 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 45
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
Development of LC-MS/MS-Based Receptor Occupancy Tracers and Positron
Emission Tomography Radioligands for the Nociceptin/Orphanin FQ
(NOP) Receptor
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Concepción Pedregal,a* Elizabeth M. Joshi,b Miguel A. Toledo,a Celia Lafuente,a Nuria Diaz,a Maria
A. MartinezꢀGrau,a Alma Jiménez,a Ana Benito,a Antonio Navarro,b Zhaogen Chen,b Daniel R.
Mudra,b Steven D. Kahl,b Karen S. Rash,b Michael A. Statnick,b Vanessa N. Barthb*
[a] Centro de Investigación Lilly, Avda. de la Industria 30, 28108-Alcobendas, Madrid, Spain
[b] Eli Lilly & Co., Lilly Research Laboratories, Indianapolis, IN 46285, USA
*Authors for correspondence: Tel: 34ꢀ91ꢀ6633426. Fax: 34ꢀ91ꢀ6633411. eꢀmail:
conchipe@lilly.com and Tel: 1ꢀ317ꢀ433ꢀ8151. Fax: 1ꢀ317ꢀ277ꢀ0778. eꢀmail: vparziale@lilly.com
Abbreviations: BOC, tertꢀButoxycarbonyl; BBB, bloodꢀbrain barrier; BP, binding potential; CNS, central nervous
system; DEA, diethylamine; DIPEA, diꢀisopropylethylamine; DMEA, dimethylethylamine; EDCI, 1ꢀethylꢀ3ꢀ
(dimethylaminopropyl)ꢀcarbodiimide; HOBt, 1ꢀhydroxybenzotriazole; LCꢀMS/MS, liquid chromatographyꢀtandem
mass spectrometry; MTBE, methyl tertꢀbutyl ether; NCS, Nꢀchlorosuccinimide; N/OFQ, nociceptin/orphanin FQ; NOP,
nociceptin opioid peptide; ORL1, opioid receptorꢀlike 1; PET, positron emission tomography; RO, receptor occupancy;
rt, room temperature; SAR, structureꢀactivity relationship; SPA, scintillation proximity assay; SPECT, single photon
emission computed tomography; SUV, standarized uptake value; TLC, thinꢀlayer chromatography; TEA, triethylamine;
TFA, trifluoracetic acid.
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 45
1
2
3
Abstract
4
5
6
7
8
Currently, a lack of sufficient tools has limited the understanding of the relationship between
neuropsychiatric disorders and the nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor.
Herein, we describe the discovery and development of an antagonist NOP receptor occupancy (RO)
tracer and a novel positron emission tomography (PET) radioligand suitable to probe the NOP
receptor in human clinical studies. A thorough structureꢀactivity relationship (SAR) around the
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
highꢀaffinity
3ꢀ(2’ꢀfluoroꢀ4’,5’ꢀdihydrospiro[piperidineꢀ4,7’ꢀthieno[2,3ꢀc]pyran]ꢀ1ꢀyl)ꢀ2ꢀ(2ꢀ
halobenzyl)ꢀNꢀalkylpropanamide scaffold identified a series of subnanomolar, highly selective,
NOP antagonists. Subsequently, these unlabeled NOP ligands were evaluated in vivo by liquid
chromatographyꢀtandem mass spectrometry (LCꢀMS/MS) in rat to determine brain uptake, kinetics
and specific binding. (S)-27 was identified as a suitable unlabeled preclinical RO tracer to
accurately quantify NOP receptor engagement in rat brain. Three compounds were selected for
evaluation in nonhuman primates as PET tracers; (-)-26, (-)-30 and (-)-33. Carbonꢀ11 labeling of
(+)-31 yielded [11C]-(S)-30, which exhibited minimal generation of central nervous system (CNS)
penetrant radiometabolites, improved brain uptake, and was an excellent PET radioligand in both
rat14 and monkey.15 Currently [11C]-(S)-30 is being evaluated as a PET radiotracer for the NOP
receptor in human subjects.
Keywords: Nociceptin/Orphanin FQ (N/OFQ) Peptide (NOP) Receptor, Receptor Occupancy (RO),
StructureꢀActivity Relationship (SAR), Positron Emission Tomography (PET), Binding Potential
(BP), Standarized Uptake Value (SUV).
2
ACS Paragon Plus Environment

Page 3 of 45
Journal of Medicinal Chemistry
1
2
3
Introduction
4
5
6
7
8
9
Nociceptin/Orphanin FQ1, (N/OFQ) is a 17 amino acid peptide, and was the first
neuropeptide discovered by screening the orphan G proteinꢀcoupled receptor LC132 using
fractionated brain extracts. The receptor for N/OFQ is the opioid receptorꢀlike 1 (ORL1) or
nociceptin opioid peptide (NOP) receptor, a Class A GPCR that is widely expressed in the central
nervous system (CNS). ORL1/NOP receptors are also expressed in the peripheral nervous system,
gastrointestinal tract, smooth muscle, and immune system. NOP receptors are coupled to the cAMP
cascade, as well as to voltageꢀgated Ca2+ and K+ channels.3 While N/OFQ is similar to opioid
peptides, it exhibits no significant cross reactivity at the classical opioid receptors mu, kappa and
delta. Moreover, the classical opioid peptides (enkephalins, endorphin, and dynorphin) do not bind
to ORL1/NOP receptors. In vivo, N/OFQ modulates several physiological functions and behaviors
in animal models including depression, stress, anxiety, feeding, locomotor activity, body
temperature, substance abuse, memory and pain.4, 5, 6 Accordingly, the brain NOP receptor is a
target of interest to study physiological mechanisms of CNS function, and is also a potential target
for the discovery of novel drug therapies.4
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Positron emission tomography (PET)7 is a powerful nonꢀinvasive in vivo imaging technique
that measures radiolabeled molecules permitting measurements of receptor occupancy (RO), target
expression, and provides indirect pharmacodynamic measures such as endogenous transmitter tone.
Molecular imaging affords data that can aid in selecting drug candidates, identifying optimal dosing
regimens, and validating targets. However, investigation of these opportunities is predicated on
having the tool, or tracer, available to do so. The development of PET tracers is challenging in the
context of balancing physicochemical properties to achieve specific target binding (reduced
nonspecific binding), suitable brain uptake/penetration, and appropriate brain and plasma kinetics,
while maintaining in vivo activity, selectivity and avoiding radiometabolite interference. 8, 9, 10, 11
In vivo RO can be calculated based on blockade of the target specific binding of the tracer,
as is the case with PET and single photon emission computed tomography (SPECT) occupancy
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 45
1
2
3
4
5
6
7
8
9
studies. The use of the high performance liquid chromatography coupled to tandem mass
spectrometry (LCꢀMS/MS) method to conduct in vivo target occupancy experiments has a number
of advantages, the most obvious being avoidance of the environmental, regulatory, and purchase
costs of working with radiopharmaceuticals. In addition, use of LCꢀMS/MS permits one the ability
to run target engagement experiments more quickly, achieving highꢀthroughput in vivo screening,
the ability to establish occupancyꢀexposure relationships by measuring the test compound along
with the tracer within the same animal, and the ability to use multiple tracers in the same animal to
evaluate receptor selectivity. Most importantly, the LCꢀMS/MS evaluation of unlabeled molecules
after intravenous microdosing enables the evaluation of new potential tracers much faster than is
feasible using traditional radiolabeled screening paradigms. 12, 13
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Finding tracers for new targets is the rate limiting step in generating both preclinical
unlabeled target occupancy assays as well as for PET/SPECT imaging of those targets. Since few
small molecules possess the physicochemical properties of a good tracer (i.e. high potency, high
selectivity, high brain uptake and the ability to differentially distribute between target rich and
target poor tissues), one typically needs to examine many candidate molecules. Instead of the weeks
to months required to label and test a new tracer using traditional radiolabeled techniques, a
unlabeled tracer candidate can be tested in a day using the LCꢀMS/MS methodology. Employing
LCꢀMS/MS evaluation of unlabeled tracer candidates permits increased iterations and learning
within the structureꢀactivity relationship (SAR), and expands the chemical space to include
molecules without labeling sites, or with challenging labeling chemistry. The desirable properties
for a candidate unlabeled LCꢀMS/MS tracer include high binding affinity and selectivity for the
receptor, and low nonspecific binding. Tracers enabling LCꢀMS/MS based RO assays do not
require a label site, nor do they require as high a brain uptake, or standardized uptake value (SUV),
that a PET tracer requires. Radiometabolites are not generated, since only the parent tracer molecule
is measured by LCꢀMS/MS, eliminating a significant source of interference. Removal of both active
and inactive radiometabolites increase the chance of seeing a differential distribution and
4
ACS Paragon Plus Environment

Page 5 of 45
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
identification of a chemical starting point for tracer discovery. Thus, using our LCꢀMS/MS methods
it is easier to identify a tracer suitable for preclinical studies. More often, than not, the identification
of such a tool represents a starting point to design an optimized PET tracer suitable for clinical
investigation. Throughout the entire tracer discovery process the identification of a compound with
low enough nonspecific binding to see a differential distribution towards a receptor rich tissue,
versus a tissue devoid of or containing very little, target occurs earlier. Once a tracer is identified,
investigators can use the RO data for dose selection in more labor intensive behavioral and
neurochemical assays. In parallel, the development and optimization process for the PET tracer can
be initiated.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Herein, we describe the discovery of a new series of highꢀaffinity small molecules based on
a dihydrospiro[piperidineꢀ4,7’ꢀthieno[2,3ꢀc]pyrane] scaffold (5) (Chart 2) as unlabeled tracers for
the NOP receptor. The identification of this scaffold has enabled the development of a preclinical
receptor occupancy assay to prioritize SAR development. Furthermore, optimization of the
preclinical tracer scaffold was undertaken to improve SUV and to mitigate against potential CNS
penetrant radiometabolites. These efforts have led to the discovery of a successful nonhuman
primate PET 11Cꢀlabeled radioligand (2S)ꢀ[(2ꢀfluorophenyl)methyl]ꢀ3ꢀ(2ꢀfluorospiro[4,5ꢀ
dihydrothieno[2,3ꢀc]pyranꢀ7,4'ꢀpiperidine]ꢀ1'ꢀyl)ꢀN-methylpropanamide ([11C]ꢀ(S)-30)14, 15 which is
currently under evaluation in human subjects.
Results and Discussion
Chemistry
The synthetic route for generation of NOP ligands is outlined in Scheme 1. The synthesis of
spiro[4,5ꢀdihydrothieno[2,3ꢀc]pyranꢀ7,4'ꢀpiperidine] (6a) and 2ꢀfluorospiro[4,5ꢀdihydrothieno[2,3ꢀ
c]pyranꢀ7,4'ꢀpiperidine] (6b) is described in ref 14. Excellent yield of 2ꢀchlorospiro[4,5ꢀ
dihydrothieno[2,3ꢀc]pyranꢀ7,4'ꢀpiperidine] (6c) was obtained by chlorination of Nꢀtertꢀ
butoxycarbonyl (BOC) protected piperidine 6a with Nꢀchlorosuccinimide followed by Nꢀ
deprotection. Michael addition between the piperidine intermediates 6aꢀc and tertꢀbutyl acrylate in
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 45
1
2
3
4
5
6
7
8
9
the presence of triethylamine (TEA) yielded the common propanoate intermediates 7aꢀc. αꢀ
Alkylation of the propanoates 7aꢀc with the corresponding substituted benzyl bromides was
achieved with LiN(TMS)2 as base. Ester hydrolysis followed by conventional amide formation with
the appropriate amine yielded the final racemic amides 9-14 and 20-33. Racemic amides 10 and 20ꢀ
33 were separated into individual enantiomers by chiral HPLC. The Lꢀtartaric acid salt was
prepared when the final compound was an oil.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In the same way, Michael addition of the piperidine intermediates 6a and 6b to ethyl 2ꢀ((1Hꢀ
pyrazolꢀ1ꢀyl)methyl)acrylate gave rise to the corresponding Michael adducts. Ester hydrolysis
under basic conditions followed by amide formation with dimethylamine hydrochloride afforded
the final racemic amides (±)-8 and (±)-19. Chiral chromatography was used to isolate single
enantiomers.
Reduction of the tertꢀbutyl esters with lithium aluminum hydride yielded the corresponding
free alcohol. The alcohol was converted under basic conditions to the methyl ether compound (±)-
16 by treatment with dimethylsulfate. Alternatively, methanesulfonyl chloride was used in the
transformation of the alcohol into the chlorinated product (±)-18. The fluorinated derivative (±)-15
was synthesized from (±)-18 following addition of Nꢀtetrabutylammonium fluoride. Finally, the
nitrile (±)-17 was obtained by dehydration of the corresponding primary carboxamide.
[Scheme 1]
Tracer evaluation of known NOP ligands.
The distribution of NOP receptors in rat brain has been studied by receptor binding
autoradiography using peptide radioligands.16, 17, 18, 19 In the rat brain, NOP binding sites are widely
distributed throughout the neural axis. A relatively high density of NOP binding sites is present in
the rat cortex, hypothalamus, thalamus and amygdala, while low levels of NOP binding were found
in the caudate putamen.16, 17 The estimated density of NOP receptors in rat brain membranes is 237
fmol/mg protein (about 24 nM in whole brain assuming 100 mg protein per mL of tissue).17 Data on
the distribution and density of NOP receptors in the brain of higher species are sparse, except for a
6
ACS Paragon Plus Environment

Page 7 of 45
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
single study in macaque.20 In macaques the distribution of NOP was similar to that of the rat but
with some notable differences, particularly in the straitum. In nonhuman primate, a NOP Bmax of
greater than 6 fmol/mg of tissue (equating to > 6 nM in whole brain) was found in NOP receptorꢀ
rich regions.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Using our LCꢀMS/MS methodology, specific binding is expressed as the binding potential
(BP) and is defined as the ratio of the tracer concentration in a brain area with high receptor density
divided by the tracer concentration detected in an area with little to no receptor expression, minus
one. Additionally, brain uptake or standard uptake value (SUV) is calculated as the ratio of the
tissue tracer levels divided by the injected tracer dose. Binding potential is mathematically defined
as the Bmax/Kd, and can be modelled or directly determined through PET/SPECT imaging studies. A
guideline for tracer screening in vitro is that the product of receptor density (Bmax, nM) and ligand
affinity (1/Kd, nMꢀ1) should exceed a value of 5 in targetꢀrich regions of brain of interest (Equation
1). As one can see from eq 1, tracer identification becomes more difficult for receptors expressed at
low density in the tissue of interest.
Bmax / Kd > 5
(Equation 1)
Therefore, with an estimated Bmax in rat brain of approximately 200 fmol/mg of tissue for
NOP binding sites, we hypothesized that NOP RO tracer candidates should have a Kd value in the
low nanomolar to subnanomolar range. Using these criteria we have successfully identified several
novel LCꢀMS/MS tracers that were optimized to generate PET ligands.21, 22
Two nonꢀpeptide imaging agents have been previously reported for the NOP receptor: the
agonist
triazaspiro[4.5]decanꢀ1ꢀone ([11C]ꢀMethyl Ro 64-6198)23 by HoffmannꢀLa Roche and the
1ꢀ[(3R,4R)ꢀ1ꢀcyclooctylꢀ3ꢀhydroxymethylꢀ4ꢀpiperidy]ꢀ3ꢀ[11C]ethylꢀ1,3ꢀdihydroꢀ2Hꢀ
8ꢀ[(1S,3aS)ꢀ2,3,3a,4,5,6ꢀhexahydroꢀ1Hꢀphenalenꢀ1ꢀyl]ꢀ2ꢀmethylꢀ4ꢀphenylꢀ2,4,8ꢀ
antagonist
benzimidazolꢀ2ꢀone ([11C]ꢀCPEB)24 by Merck (Chart 1). We prepared the respective unlabeled
analogues and demonstrated high affinity binding for the NOP receptor as well as high selectivity
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 45
1
2
3
4
5
6
7
8
versus the classical mu, kappa and delta opioid receptors as reported (Table 1), however, neither
compound was suitable as a tracer because of the high level of nonspecific binding reported.23, 24
[Chart 1]
9
NOP tracer identification was initiated by applying our previously published in vivo/ex vivo
LCꢀMS/MS method,12, 13 to evaluate known small molecule NOP ligand agonists 1 (W-212393),25 2
(NNC 63-0532)26 and the antagonist 3 (SB-612111)27 as potential unlabeled RO tracers in rat
(Chart 2).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[Chart 2]
While differences in receptor binding interactions between agonist and antagonist ligands
exist, we were interested in evaluating compounds for their ability to behave as tracers, regardless
of functional activity, since they are ligands with known affinity for the NOP receptor and exhibit
CNS activity following systemic administration. The opioid receptor binding affinities and
functional activities for 1, 2 and 3 can be found in Table 1. Consistent with previous reports, we
found all three compounds were potent ligands at the NOP receptor, while exhibiting high
selectivity versus the classical mu, kappa and delta opioid receptors. We tested these molecules in
vivo in rat by LCꢀMS/MS to establish any ability to differentially distribute between NOP receptor
rich and poor brain regions. The first molecule evaluated, 1 showed no SUV or BP due to high
nonspecific binding (Table 2). Dispite its lower affinity, 2 exhibited differential distribution
between the NOP receptor rich region, hypothalamus, as compared to the striatum (a region with
low NOP binding), giving BP = 3. However, 2 showed low brain uptake (SUV = 6%) preventing its
use as a suitable LCꢀMS/MS tracer. On the contrary, 3 was found to have a significantly higher
brain uptake (SUV = 130%) suggesting suitable penetration of the bloodꢀbrain barrier (BBB), but
exhibited a low BP (0). Because of the measureable BP exhibited by 2, this compound was
subsequently studied as a tracer via blocking experiments with the antagonist 3. No reduction in
brain concentrations of 2 were observed following pretreatment with 3 in the hypothalamus,
8
ACS Paragon Plus Environment

Page 9 of 45
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
indicating that the differential distribution was likely due to nonspecific binding and was not
attributed to specific binding of the NOP receptor. No additional studies were performed using 2.
Next, we prepared the racemate 2ꢀ[(5ꢀfluorospiro [1Hꢀisobenzofuranꢀ3,4'ꢀpiperidine]ꢀ1'ꢀ
yl)methyl]ꢀN,Nꢀdimethylꢀ3ꢀthiazolꢀ4ꢀylꢀpropanamide ((±)-4) described in patent WO 05092858
(Chart 2).28 Following chiral chromatography of (±)-4, the active enantiomer was identified as (-)-
4. Although less potent than the other antagonist molecules tested, (-)-4 exhibited a measureable BP
(1.3) and SUV (25%) (Table 2). Next, we explored substitution of the 5ꢀfluorospiro[1Hꢀ
isobenzofuranꢀ3,4'ꢀpiperidine] piece of compound (-)-4 with the spiro[4,5ꢀdihydrothieno[2,3ꢀ
c]pyranꢀ7,4'ꢀpiperidine] motif and improved BP and SUV was retained as exemplified by
compound (+)-19. Subsequently, we initiated an SAR study to optimize this novel NOP tracer
scaffold.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[Table 1]
[Table 2]
Ligand Pharmacology.
Table 3 and 4 contain compounds which were prepared for tracer evaluation with binding
affinities and functional antagonism for the NOP receptor. We began the SAR by increasing the
lipophilicity of the compounds by replacement of the pyrazyl heterocycle with a phenyl ring in an
effort to increase the permeability and subsequent brain uptake of the compounds into the CNS.
Fortunately, we were able to mantain high affinity binding following phenyl ring substitution ((±)-8
versus (±)-9, Table 3). In addition, further increase of lipohilicity by incorporation of a chlorine
atom in the ortho position of the phenyl ring increased the affinity five fold (compare (±)-9 with
(±)-10). Introduction of one fluorine atom in the ortho position ((±)-11) also maintained the affinity
as compared to the chloro substitution. Meanwhile, the 2,6ꢀdifluorophenyl ((±)-12), the 2ꢀpyridine
((±)-13) and methoxy derivatives ((±)-14) exhibited a significant reduction in affinity (Ki higher
than 10 nM). Additionally, the influence of the carboxamide on receptor affinity was evaluated by
replacing this group by fluoromethyl ((±)-15), methoxymethyl ((±)-16), nitrile ((±)-17), and
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 45
1
2
3
4
5
6
7
8
9
chloromethyl ((±)-18). However, substitution of the carboxamide group resulted in considerable
loss of affinity (Ki higher than 10 for (±)-15 and 40 nM for (±)-16ꢀ(±)-18). Thus, the racemic (±)-10
exhibited the highest affinity in this early phase of the SAR exploration, while maintaining
physicochemical properties associated with good brain uptake. Asymmetric chiral chromatography
was used to separate the individual enantiomers of (±)-8 and (±)-10, resulting in the identification
of (-)-10 as the first subnanomolar molecule in the series. The affinity of the (+) enantiomers was
significantly less potent, therefore, the high binding affinity of the racemates (±)-8 and (±)-10
resided exclusively in the (-) enantiomer (Table 3).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Next, to further explore the SAR in this scaffold a chlorine or fluorine atom was introduced
at the 2ꢀposition of the thiophenyl ring of the spiro system (Table 4 where data is shown for the
most active enantiomer in most cases). This modification was shown to dramatically increase
(around 10ꢀfold) the affinity of (+)-19 in Table 4 versus (-)-8 in Table 3 and in a lesser extent
(around 3ꢀfold) in (-)-20 and (+)-25 in Table 4 versus (-)-10 in Table 3. A wide range of
substituted carboxamides (primary, secondary and tertiary) were also synthesized, and found to
maintain high affinity for the NOP receptor (Table 4). Finally, it should be pointed out that the
selectivity versus classical opioid receptors (mu, delta and kappa) of this novel scaffold for NOP
receptor ligands is excellent given that no specific binding was observed at concentrations up to 400
nM.
[Table 3]
[Table 4]
Following chiral chromatographic resolution of (±)-31 and (±)-32, and singleꢀcrystal Xꢀray
crystallography, the enantiomer with high binding affinity, (+)-31 was found to have S-
configuration (see Supporting Information in reference 14). Conversely, the Rꢀenantiomer, (-)-31
exhibited a markedly lower affinity for the NOP receptor (Table 4). Consistent data were generated
with the (+)-32 enantiomer, that exhibited low NOP receptor binding affinity and has the R-
configuration (see Supporting Information in reference 14). To assess the stereochemical stability
10
ACS Paragon Plus Environment

Page 11 of 45
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
of the chiral center the compounds (+)-31 and (-)-32 were treated at 25°C with sodium hydride and
methyl iodide in dimethyl sulfoxide (Scheme 2). Chiral HPLC resolved the Nꢀmethylated products
(-)-30 and (-)-33, respectively, in addition to the dimethylated compound (-)-27, and unchanged
starting material (-)-32. Therefore, under basic alkylation conditions racemization of the
stereocenter does not occur, and all products maintain the Sꢀconfiguration (i.e., the products are (-)-
30, (-)-27 and (-)-33). Thus, we have tentatively assigned the S configuration to all high affinity
NOP enantiomers.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[Scheme 2]
LC-MS/MS Tracer Evaluation in Rats.
BP, SUV and brain kinetics were evaluated with several analogues from the [4,5ꢀ
dihydrothieno[2,3ꢀc]pyranꢀ7,4'ꢀpiperidine] scaffold. As it is shown, Table 5, the pyrazoles 8 and 19
differentially distributed in the CNS (indicated by the BP values) when administered in vivo.
However, the reduced lipophilicity of 8 and 19, impaired brain penetration, as measured by SUV, in
vivo. Therefore, the correlation we have established between the calculated lipophilicities (clogD)
and the measured logD, was overall predictive of good brain penetration for the majority of
compounds. Previous studies have reported a similar relationship between CNS permeability and
logD.29 Thus, in an effort to maximize CNS penetration, the tracer SAR evolved to increase
compound lipophilicity. Promising PET ligand candidates were identified from the tracer SAR
using selection criteria of a BP greater than 2 following 3 ꢀg/kg, iv at 40 minutes and a %SUV
greater than 100 at 10 minutes post dose. These two data points were found to select the best tracer
ligands candidates for further evaluation as radiolabeled ligands. (+)-25, (-)-26, (-)-27, (-)-30 and (-
)-33 exhibited specific binding in the hypothalamus (BP >1) and improved uptake, as measured by
SUV. Interesting structural observations were noted when the thiophene ring was substituted by a
chloro atom at the 2 position (R1=Cl) as in (-)-20 to (-)-24, with the difference being halogen atom
at the ortho position of the aryl group (R3=Cl or F) and the carboxamide. We found that while the
signal to noise varied for the ligands, both BP and SUV were maintained. However, when the ortho
11
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 45
1
2
3
4
5
6
7
8
9
substituent in the thiophene was a flourine atom, as in (+)-25 to (-)-30 and (-)-33, the binding
potentials were greater than 2 and SUV was greater than 100. Two notable exceptions were (-)-28
and (-)-29, that exhibited the highest measured logD but a low BP, due to higher nonspecific
binding. Our evaluation led us to conclude that the best potential tracers contain two fluorine atoms.
Therefore, compound (-)-27 was selected as a preclinical tracer for further evaluation. Data from
the rodent tracer evaluation for (-)-27 can be seen in Figure 1A, where differential distribution was
measured in hypothalamus, thalamus and striatum. Higher specific binding can be seen in the
receptor rich hypothalamus versus striatum producing a BP = 3.5. The brain concentration of (-)-27
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
diminishes after 60 min. The ratio of tissue concentrations of (-)-27 in hypothalamus and thalamus
17
versus striatum demonstrated a consistent BP in receptor rich regions16,
over the 60 min
evaluation period (Figure 1B).
To complete our characterization, (-)-27 was evaluated in a blockade study with the known
NOP receptor antagonist 3. Dose dependent blockade of tracer (-)-27 was demonstrated with 3, as
shown in Figure 1C. Increasing doses of 3 were seen to dose dependently reduce brain
concentrations of tracer compound (-)-27 in the hypothalamus, one hour following oral
administration of 3, the time at which the tracer was administered. Little tracer blocking was
observed in the striatum, low density NOP receptor region, supporting that the binding of (-)-27 is
selective for the NOP receptor. Subsequently, receptor occupancy of compound 3 was determined
using a 3ꢀg/kg, iv, dose of the unlabeled tracer compound (-)-27 following the oral administration
of 3. Interestingly, compound 3 exhibited good in vivo potency, with a calculated ED50 for NOP
RO of 6.9 mg/kg (Figure 1D).
[Table 5]
[Figure 1]
An optimized tracer suitable for PET ligand development needs high brain and plasma
clearance for the desired kinetics amenable for carbonꢀ11 imaging, and should have limited CNS
penetrant radiometabolites. In an effort to understand the mechanism of clearance associated with (-
12
ACS Paragon Plus Environment

Page 13 of 45
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
)-27, in vitro metabolic stability studies were conducted in rat, dog, monkey and human
microsomes. Moreover, metabolite profiling was conducted using cryopreserved hepatocytes
isolated from the same species, since oxidative metabolism was thought to be a predominant
clearance pathway for the molecule. The LCꢀMS/MS profiling of these samples showed oxidative
Nꢀdemethylation to be a primary pathway of clearance for (-)-27 (data not shown). To reduce the
potential formation of radiometabolites resulting from Nꢀdemethylation, we evaluated the monoꢀ
methylated high affinity ligands, (-)-26, (-)-30 and the isopropyl derivative (-)-33. All three
compounds possess a potential site for radioꢀlabeling as well as excellent binding potential and
brain penetration in vivo (Table 5). Of the three compounds, [11C]ꢀ(S)-30 was identified as an
efficient PET radioligand due to its good brain entry, most favorable kinetics, and large receptorꢀ
specific signal in rat and monkey brain in vivo as previously reported.14, 15
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Summary
We have identified the 3ꢀ(2’ꢀfluoroꢀ4’,5’ꢀdihydrospiro[piperidineꢀ4,7’ꢀthieno[2,3ꢀc]pyran]ꢀ
1ꢀyl)ꢀ2(2ꢀhalobenzyl)ꢀNꢀalkylpropanamides as a chemotype with highꢀaffinity and selectivity for
the NOP receptor. In rats we optimized the SAR to produce compounds with subnanomolar binding
affinities, high selectivity, low nonspecific binding, and high brain uptake. Consequently, we have
identified (-)-27 as a novel unlabeled receptor occupancy tracer which allows preclinical high
throughput determination of rat brain NOP occupancy, and [11C]ꢀ(S)-30 as a PET radiotracer.
Experimental Section
Materials and Methods. All reagents used were obtained from commercial sources
(SigmaꢀAldrich, unless otherwise stated). All solvents were of an analytical grade. The NOP
agonist ligands Ro 64-6198,23 W-212393,25 NNC 63-053226 and the antagonist ligands J-113397,24
SB-61211127 and (-)-428 were synthesized as reported. [35S]ꢀGTPγS was obtained commercially.
Preparation of racemic and enantiomerically pure 26, 30, 31, 32 and 33 was according to previously
reported methods.14
13
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 45
1
2
3
4
5
Isolation of compounds by column chromatography was performed on silica gel columns
(SP1a HPFC system, Biotage Inc., Charlottesville, VA).
6
7
8
9
LCꢀMS was performed with an 1100 Series LCꢀMSD single quadrupole instrument
(Agilent; Santa Clara, CA) with an ESI interface.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The purity of synthesized ligands was found to be ≥ 95%, by the following described LCꢀ
MS methods 1ꢀ5 as specified in each case.
METHOD 1: Used a heated (50 ºC) XBridge C18 column (3.5 ꢀm; 2.1 mm x 50 mm;
Waters, Milford, MA) eluted at 1 mL/min with a gradient of A (10 mM aq NH4HCO3; pH 9) and B
(MeCN), with B increased linearly from 10% to 100% (v/v) over 3.5 min. Ions between m/z 100ꢀ
750 were captured after electrospray ionization of the eluted test sample.
METHOD 2: Used a heated (45 ºC) CAPCELL PAK C18 MG column (3.0 ꢀm; 4.6 mm x
35 mm), eluted at 1.2 mL/min with a gradient of H2O (0.05% trifluoracetic acid (TFA)) (A)/ MeCN
(0.05% TFA) (B), with B increased linearly from 10% to 95% (v/v) over 2.4 min. Detection: UV
(214.4 nm). Ions between m/z 80ꢀ800 were captured after electrospray ionization of the eluted test
sample.
METHOD 3: Used a heated (50 ºC) Gemini C18 column (3.0 ꢀm; 2.0 mm x 50 mm), eluted
at 1.0 mL/min, with a gradient of H2O (0.05%TFA) (A)/ MeCN (0.05%TFA) (B), with B increased
linearly from 5% to 100% over 7 min. Detection: UV (214.4 nm). Ions between m/z 70ꢀ800 were
captured after electrospray ionization of the eluted test sample.
METHOD 4: Used a heated (50 ºC) Gemini C18 (3.0 ꢀm; 2.0 mm x 50 mm), eluted at 1.0
mL/min with a gradient of H2O (0.1% Formic acid) (A)/ MeCN (0.1% formic acid) (B), with B
increased from 5% to 100% over 7 min. Detection: UV (214.4 nm). Ions between m/z 70ꢀ800 were
captured after electrospray ionization of the eluted test sample.
METHOD 5: Used a heated (50 ºC) SepaxBRꢀC18 column (3.0 ꢀm; 2.1 mm x 50 mm),
eluted at 1.0 mL/min with a gradient of H2O (10 mmol NH4HCO3) (A)/ MeCN (B), with B
14
ACS Paragon Plus Environment

Page 15 of 45
Journal of Medicinal Chemistry
1
2
3
4
5
increased from 10% to 100% over 7 min. Detection: UV (214.4 nm). Ions between m/z 70ꢀ800
were captured after electrospray ionization of the eluted test sample.
6
7
8
9
Chiral HPLC Method 1: HPLC, Chiralpak AD 20 mm x 250 mm, 0.2% dimethylethylamine
(DMEA) in hexane/EtOH (90:10 v/v). Flow rate of 12 mL/min. λ = 254 nm.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Chiral HPLC Method 2: HPLC, Chiralpack ADꢀH 4.6 mm x 250 mm, 0.1% diethylamine
(DEA) in hexane/EtOH (90:10 v/v). Flow rate of 15 mL/min. λ = 214 and 254 nm.
Highꢀresolution mass spectrometry (HRMS) was performed on an TOF/QꢀTOF instrument
(Agilent). Data were acquired in dual ESI mode (+ and ꢀ) in the range of 95−1700 amu. The sample
(~ 0.1 mg/mL in MeCNꢀH2O, 1: 1 v/v; 2 ꢁL) was injected in loop mode onto a Zorbax SBꢀC8
column (3.5 ꢁm; 3.0 × 150 mm; Agilent) eluted at 0.5 mL/min with a mixture of 0.1% formic acid
in H2O (A) and 0.1% formic acid in MeCN (B) with B increasing from 5 to 100% over 15 min and
then held at 100% for 8 min. Eluate was monitored with a diode array detector operating in the
range 190 to 700 nm.
1H NMR (300 MHz) spectra were acquired at 20 °C on a System 300 instrument (Varian;
Palo Alto, CA). Abbreviations s, d, dd, ddd, m, and br denote singlet, doublet, double doublet,
double double doublet, multiplet, and broad, respectively.
o
Optical rotations were measured with a 341 polarimeter (Perkin Elmer) at 20 C and at 589
nm (sodium lamp).
Statistics. Values are given as means ± the standard deviation of the mean, unless otherwise
stated.
Animals. Male SpragueꢀDawley rats (Harlan SpragueꢀDawley, Indianapolis, IN), weighing
between 230 and 280 g, were used for in vivo experiments. All rats were housed in rooms using a
12 h light/dark cycle and had ad lib access to normal rat chow and H2O until the beginning of the 3
h experimental protocol. All experiments with rats were performed in accord with the National
Research Council Guide under protocols approved by the Animal Care and Use Committee of Eli
Lilly and Company.
15
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 45
1
2
3
Chemistry.
4
5
6
7
8
2-Chlorospiro[4, 5-dihydrothieno[2,3-c]pyran-7,4'-piperidine] (6c). Step 1: Nꢀ
chlorosuccinimide (2.63 g, 19.39 mmol) was added to a solution of tertꢀbutyl spiro[4, 5ꢀ
dihydrothieno[2,3ꢀc]pyranꢀ7,4'ꢀpiperidine]ꢀ1'ꢀcarboxylate14 (5 g, 16.16 mmol) in acetonitrile (6
mL). The resulting mixture was heated to 70 ºC overnight. The reaction was allowed to reach room
temperature (rt) and the solvent was evaporated. The oily residue was dissolved in methyl tertꢀbutyl
ether (MTBE) (20 mL). Hexane (200 mL) was added and the resulting mixture was stirred at rt for
1 h. The solid was filtered off and the filtrate was concentrated to give tertꢀbutyl 2ꢀchlorospiro[4,5ꢀ
dihydrothieno[2,3ꢀc]pyranꢀ7,4’ꢀpiperidine]ꢀ1’ꢀcarboxylate as a yellow solid (5.5 g, 99%). LCꢀMS
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
(ESI): m/z 287 (MH)+, 243, tR= 2.84 min, purity 100%, method 1. H NMR (300 MHz, CDCl3):
6.59 (s, 1H), 3.99ꢀ3.89 (m, 4H), 3.11 (td, J= 13.0, 2.5 Hz, 2H), 2.60 (t, J= 5.5 Hz, 2H), 1.97 (dd, J=
1.9, 14.3 Hz, 2H), 1.74ꢀ1.60 (m, 2H), 1.48 (s, 9H). HRMS (ESI): calcd for C16H22ClNO3S,
343.1008; found, 343.1009. Step 2: HCl (4M) solution in dioxane (2.5 mL, 10 mmol) was added to
a solution of the compound obtained in step 1 (380 mg, 1.1 mmol) in dioxane (2.5 mL), and the
resulting mixture was stirred at rt. When the reaction was completed, the solvent was evaporated
under vacuum and the residue partitioned in CH2Cl2 and NaOH (1N). The organic layer was
separated, dried over MgSO4 and evaporated to yield 6c (264 mg, 98%) as a yellow solid. LCꢀMS
(ESI): m/z 243 (MH)+, tR= 2.65 min, purity 100%, method 1. 1H NMR (300 MHz, CDCl3): 6.57 (s,
1H), 3.90 (t, J= 5.5 Hz, 3H), 3.08ꢀ2.90 (m, 5H), 2.59 (t, J= 5.4 Hz, 3H), 2.00 (d, J= 13.3 Hz, 3H),
1.80ꢀ1.68 (m, 3H). HRMS (ESI): calcd for C11H14ClNOS, 243.049; found, 243.0485.
General Michael Addition Procedure: Triethylamine (TEA) (2.48 g, 19.34 mmol) and the
corresponding alkyl acrylate were added to a solution of the piperidine intermediate (6aꢀ6c) (9.67
mmol) dissolved in MeOH (50 mL). The crude mixture was refluxed overnight. The reaction
mixture was cooled to rt and the solvent removed under vacuum. The resulting crude product was
purified by flash chromatography.
16
ACS Paragon Plus Environment

Page 17 of 45
Journal of Medicinal Chemistry
1
2
3
tert-Butyl
3-(4',5'-dihydro-1H-spiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-yl)
4
5
6
propanoate (7a). The title compound was obtained from 6a (870 mg, 4.16 mmol) and tertꢀbutyl
7
8
acrylate (984 mg, 7.68 mmol) according to the general Michael addition procedure. The crude
9
product was purified by a plug of silica gel (CH2Cl2) to furnish 7a (1.3 g, 93%) as a colorless solid.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
LCꢀMS (ESI): m/z 338 (M+H)+. H NMR (300 MHz, CDCl3)
δ 7.17 (d, J=5.1Hz, 1H), 6.75 (d,
J=5.1Hz, 1H), 3.91 (t, J=5.7Hz, 2H), 2.74ꢀ2.66 (m, 6H), 2.48ꢀ2.39 (m, 4H), 2.04ꢀ1.93 (m, 4H), 1.45
(s, 9H).
tert-Butyl
3-(2'-chloro-4',5'-dihydro-1H-spiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-
yl)propanoate (7c). The title compound was obtained from 6c (2.35 g, 9.67 mmol) and tert-butyl
acrylate (2.44 g, 24.18 mmol) according to the general Michael addition procedure. The crude
product was purified by flash chromatography (CH2Cl2/MeOH=40:1) to furnish 7c (3.2 g, 89%) as
a light yellow oil. LCꢀMS (ESI): m/z 372 (M+H)+.
3-(2'-Fluoro-4',5'-dihydro-1H-spiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-yl)-2-(1H-
pyrazol-1-ylmethyl)propionic acid. Step 1: Ethyl 2ꢀ((1H)ꢀpyrazolꢀ1ꢀylmethyl)acrylate28 (150 mg,
0.83 mmol) was added to a solution of 6b14 (173 mg, 0.76 mmol) according to the general Michael
addition procedure. The crude product was purified by preparative thinꢀlayer chromatography
(TLC) (petroleum ether/ethyl acetate=1:1) to yield ethyl 3ꢀ(2'ꢀfluoroꢀ4',5'ꢀdihydroꢀ1Hꢀ
spiro[piperidineꢀ4,7'ꢀthieno[2,3ꢀc]pyran]ꢀ1ꢀyl)ꢀ2ꢀ(1H-pyrazolꢀ1ꢀylmethyl) propanoate (57 mg,
18%) as a yellow oil. LCꢀMS (ESI): m/z 408 (M+H)+. H NMR (300 MHz, CDCl3)
δ 7.50 (d,
1
J=1.5Hz, 1H), 7.38 (d, J=1.8Hz, 1H), 6.20 (t, J=1.8Hz, 1H), 6.11 (d, J=1.5Hz, 1H), 4.40 (d,
J=7.2Hz, 2H), 4.05ꢀ4.19 (m, 2H), 3.89 (t, J=5.7Hz, 2H), 3.24ꢀ3.35 (m, 1H), 2.35ꢀ2.76 (m, 7H),
1.94ꢀ2.02 (m, 2H), 1.62ꢀ1.85 (m, 3H), 1.20 (t, J=7.2Hz, 3H). Step 2: LiOH monohydrate (18 mg,
0.43 mmol) in THF (1 mL) and H2O (2 mL) was added to a solution of the compound obtained in
step 1 (55 mg, 0.14 mmol) in TFH (1 mL). The reaction mixture was stirred at rt for 16 h, acidified
to pH 2 with HCl solution (1N) and extracted with ethyl acetate. The combined organic extracts
17
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 45
1
2
3
were dried over Na2SO4, filtered, and concentrated under vacuum to afford the title compound (41
4
5
6
mg, 80%) as a yellow oil. LCꢀMS (ESI): m/z 380 (M+H)+. 1H NMR (300 MHz, CDCl3)
δ 7.49ꢀ7.59
7
8
9
(m, 2H), 6.55 (br, 1H), 6.25 (s, 1H), 6.14 (s, 1H), 4.61 4.65 (m, 1H), 4.44ꢀ4.50 (m, 1H), 3.88 (t,
J=5.4Hz, 2H), 3.43ꢀ3.47 (m, 1H), 3.22ꢀ3.33 (m, 3H), 3.06ꢀ3.11 (m, 1H), 2.82ꢀ3.00 (m, 2H), 2.55 (t,
J=4.8 Hz, 2H), 2.00ꢀ2.60 (m, 4H).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2-(Methylenepyrazole)-3-[spiro(benzothiophene-1(3H),4''-piperidine)propionic acid.
Step 1: Ethyl 2ꢀ((1H)ꢀpyrazolꢀ1ꢀylmethyl) acrylate28 (150 mg, 0.86 mmol) was added to a solution
of 6a14 (160 mg, 0.78 mmol) according to the general Michael addition procedure. The crude
product was purified by preparative TLC (petroleum ether/ethyl acetate=2:3) to yield ethyl 2ꢀ
(methylenepyrazole)ꢀ3ꢀ[spiro(benzothiopheneꢀ1(3H),4''ꢀpiperidin] propanoate (200 mg, 67%) as a
1
yellow oil. LCꢀMS (ESI): m/z 390(MH)+. H NMR (300 MHz, CDCl3): 7.50 (dd, J= 0.5, 1.8 Hz,
1H), 7.39 (dd, J= 0.5, 2.4 Hz, 1H), 7.13 (d, J= 5.1 Hz, 1H), 6.76 (d, J= 5.1 Hz, 1H), 6.20 (t, J= 2.1
Hz, 1H), 4.42ꢀ4.39 (m, 2H), 4.15ꢀ4.08 (m, 2H), 3.90 (t, J= 5.5 Hz, 2H), 3.27 (q, 1H), 2.72ꢀ2.52 (m,
8H), 2.03ꢀ1.90 (m, 4H), 1.19 (t, J= 7.1 Hz, 3H). Step 2: The compound obtained in step 1 was
hydrolyzed with LiOH according to the same procedure used for the preparation of compound 3ꢀ(2'ꢀ
fluoroꢀ4',5'ꢀdihydroꢀ1Hꢀspiro[piperidineꢀ4,7'ꢀthieno[2,3ꢀc]pyran]ꢀ1ꢀyl)2ꢀ(1H-pyrazolꢀ1ꢀ
ylmethyl)propionic acid. The title compound (85 mg, 75%) was obtained as a colorless oil. LCꢀMS
1
(ESI): m/z 362(MH)+; H NMR (300 MHz, CDCl3): 7.49 (d, J= 2.0 Hz, 1H), 7.42 (d, J= 1.6 Hz,
1H), 7.11 (d, J= 5.0 Hz, 1H), 6.73 (d, J= 5.0 Hz, 1H), 6.16 (t, J= 2.1 Hz, 1H), 4.56ꢀ4.34 (m, 2H),
3.84 (t, J= 5.4 Hz, 2H), 3.11ꢀ2.90 (m, 4H), 2.72ꢀ2.49 (m, 5H), 2.14ꢀ1.95 (m, 4H).
General Condensation Procedure: A solution of LiN(TMS)2 (1M) in dry THF (7.48 mL,
7.48 mmol) was added dropwise to a stirred solution of 7a-7c (3.74 mmol) in dry THF (12 mL) at ꢀ
78 oC. After 1 h of stirring, 1,3ꢀdimethylꢀtetrahydropyrimidinꢀ2(1H)ꢀone (0.45 mL, 3.74 mmol) was
o
added at ꢀ78 C and, the resulting mixture stirred for 30 min. Then the corresponding benzyl
bromide (4.13 mmol) in dry THF (10 mL) was added. Stirring was continued for 1 h, and the
o
temperature was allowed to rise to 0 C, where it was kept for 1h. Saturated aqueous NH4Cl
18
ACS Paragon Plus Environment

Page 19 of 45
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
solution was added. The mixture was extracted twice with ethyl acetate and the combined organic
extracts dried over Na2SO4, and evaporated under vacuum. The resulting crude product was purified
by chromatography.
9
Amide Formation General Procedure: TFA (2 mL) was added to a stirred solution of the
ester (0.37 mmol) in dichloromethane (2 mL), and stirred at rt for 18 h. The reaction mixture was
evaporated to dryness to afford the trifluoroacetate salt of the corresponding propionic acid which
was used without further purification. 1ꢀHydroxybenzotriazole (HOBt) (61 mg, 0.45 mmol) and 1ꢀ
ethylꢀ3(dimethylaminopropyl)ꢀcarbodiimide (EDCI) (86 mg, 0.45 mmol) in dry dichloromethane
(20 mL) were added to a stirred solution of the trifluoroacetate salt (0.37 mmol) and the
corresponding amine hydrochloride (0.37 mmol). Then diꢀisopropylethylamine (DIPEA) (173.8 ꢀL,
1.0 mmol) was added, and the mixture was stirred at rt for 18 h. The reaction mixture was washed
with HCl (1N) and brine, dried over Na2SO4, and concentrated under vacuum. The resulting crude
product was purified by chromatography.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
L-Tartaric Salt Formation General Procedure: LꢀTartaric acid (1 equiv) was added to a
stirred solution of the free base (1 equiv) in methanol. Evaporation of the solvent followed by
trituration with diethyl ether gave rise to the corresponding salt.
2-(Pyrazol-1-ylmethyl)-3-spiro[4,5-dihydrothieno[2,3-c]pyran-7,4'-piperidine]-1'-yl- N,
N-dimethylpropanamide ((±)-8). The racemic title compound was prepared from 2ꢀ
(methylenepyrazole)ꢀ 3ꢀ[spiro(benzothiopheneꢀ1(3H),4''ꢀpiperidine)propionic acid (560 mg, 1.56
mmol) and dimethylamine hydrochloride (127 mg, 1.56 mmol) according to the general amide
formation procedure. The crude product was purified by preparative HPLC (hexane/ethyl
acetate=8:1) to afford 8 (385 mg, 64%) as a yellow oil. LCꢀMS (ESI): m/z 389(MH)+, tR= 1.98 min,
purity 96%, method 1. 1HNMR (300 MHz, CDCl3): 7.47 (s, 1H), 7.34 (s, 1H), 7.10 (dd, J= 1.3, 4.9
Hz, 1H), 6.74ꢀ6.72 (m, 1H), 6.17ꢀ6.14 (m, 1H), 4.44ꢀ4.30 (m, 2H), 3.87 (t, J= 5.4 Hz, 2H), 3.69ꢀ
3.60 (m, 1H), 2.84 (d, J= 1.3 Hz, 3H), 2.79 (d, J= 1.3 Hz, 3H), 2.74ꢀ2.64 (m, 5H), 2.56ꢀ2.46 (m,
3H), 2.01ꢀ1.85 (m, 4H).
19
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 45
1
2
3
4
5
6
7
8
9
Resolution of (±)-8 by chiral HPLC method 1 (at 150 mL/min flow rate) gave (-)-8 (tR= 5.39
min) and (+)-8 (tR= 6.87 min). (-)-8: LCꢀMS (ESI): m/z 389 (M+H) +, tR= 1.75 min, purity 100%,
method 1. The Lꢀtartaric salt was prepared according to the general Lꢀtartaric salt formation
procedure. [α]D ꢀ16.2º (c 0.5, MeOH). HRMS (ESI): calcd for C20H28N4O2S , 388.1933, found
388.1944. (+)-8: LCꢀMS (ESI): m/z 389 (M+H) +, tR= 1.75 min, purity 100%, method 1. The Lꢀ
tartaric salt was prepared. [α]D +16.6º (c 0.5, MeOH). HRMS (ESI): calcd for C20H28N4O2S,
388.1933; found, 388.1934.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3-(2'-Fluoro-4',5'-dihydro-1H-spiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-yl)
2-(1H-
pyrazol-1-ylmethyl)-N, N-dimethylpropanamide ((±)-19). The racemic title compound was
prepared from 3ꢀ(2'ꢀfluoroꢀ4',5'ꢀdihydroꢀ1Hꢀspiro[piperidineꢀ4,7'ꢀthieno[2,3ꢀc]pyran]ꢀ1ꢀyl) 2ꢀ(1H-
pyrazolꢀ1ꢀylmethyl)propionic acid (41 mg, 0.11 mmol) and dimethylamine hydrochloride (8.91 mg,
0.11 mmol) according to the general amide formation procedure. The crude product was purified by
preparative HPLC (hexane/ethyl acetate=8:1) to afford (±)-19 (16 mg, 36%) as a colorless oil. LCꢀ
1
MS (ESI): m/z 407 (M+H)+, tR= 3.05 min, purity 93.7%, method 1. H NMR (300 MHz, CDCl3)
δ
7.49 (d, J=1.8Hz, 1H), 7.35 (d, J=2.7Hz, 1H), 6.17 (t, J=2.1Hz, 1H), 6.11 (d, J=1.8Hz, 1H), 4.28ꢀ
4.44 (m, 2H), 3.89 (t, J=5.1Hz, 2H), 3.60ꢀ3.70 (m, 1H), 2.05(s, 3H), 2.79 (s, 3H), 2.62ꢀ2.78 (m,
3H), 2.32ꢀ2.56 (m, 5H), 1.93ꢀ2.01 (m, 2H), 1.70ꢀ1,83 (m, 2H).
Resolution of (±)-19 by chiral HPLC method 1 gave (+)-19 (tR= 9.82 min) and (-)-19 (tR=
12.27 min). (+)-19: LCꢀMS (ESI): m/z 407 (M+H) +, tR= 1.88 min, purity 100%, method 1. The Lꢀ
tartaric salt was prepared. [α]D +15.3º (c 0.5, MeOH). HRMS (ESI): calcd for C20H27FN4O2S,
406.1839, found 406.1848.
2-Chlorophenylmethyl-3-(2-chlorospiro[4,5-dihydrothieno[2,3-c]pyran-7,4'-
piperidine]-1'-yl)-N, N-dimethylpropanamide ((±)-20). The racemic title compound was prepared
from tertꢀbutyl 2ꢀ[(2ꢀchlorophenyl)methyl]ꢀ3ꢀ(2ꢀchlorospiro[4,5ꢀdihydrothieno[2,3ꢀc]pyranꢀ7,4'ꢀ
piperidine]ꢀ1'ꢀyl)propanoate (241 mg, 0.51 mmol) and dimethylamine hydrochloride (42 mg, 0.51
20
ACS Paragon Plus Environment

Page 21 of 45
Journal of Medicinal Chemistry
1
2
3
mmol) according to the general amide formation procedure. The crude product was purified by
4
5
6
preparative HPLC (hexane/ethyl acetate=8:1) to afford (±)-20 (50 mg, 22%) as a colorless oil.
7
8
9
Resolution of (±)-20 by chiral HPLC method 1 gave (-)-20 (tR= 6.17 min) and (+)-20 (tR=
9.01 min). (-)-20: The Lꢀtartaric salt was prepared. LCꢀMS (ESI): m/z 467 (M+H)+, tR= 4.79 min,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
purity 100%, method 1. 1H NMR (300 MHz, CDCl3)
δ 7.34ꢀ7.31(m, 1H), 7.21ꢀ7.13 (m, 3H), 6.57
(s, 1H), 3.87 (t, J=5.7Hz, 2H), 3.48ꢀ3.36 (m, 1H), 3.15ꢀ3.06(m, 1H), 2.93ꢀ2.78 (m, 5H), 2.75ꢀ2.62
(m, 5H), 2.59ꢀ2.37(m, 5H), 1.95 (d, J=13.8Hz, 2H), 1.82ꢀ1.78 (m, 2H). [α]D ꢀ30.4º (c 0.52,
MeOH).
2-Chlorophenylmethyl-3-(2-chlorospiro[4,5-dihydrothieno[2,3-c]pyran-7,4'-
piperidine]-1'-yl)-N-methylpropanamide ((±)-21). The racemic title compound was prepared
from tertꢀbutyl 2ꢀ[(2ꢀchlorophenyl)methyl]ꢀ3ꢀ(2ꢀchlorospiro[4,5ꢀdihydrothieno[2,3ꢀc]pyranꢀ7,4'ꢀ
piperidine]ꢀ1'ꢀyl)propanoate (330 mg, 0.66 mmol) and methyl amine hydrochloride (45 mg, 0.66
mmol) according to the general amide formation procedure. The crude product was purified by
preparative TLC (CH2Cl2/MeOH=25:1) to afford (±)-21 (220 mg, 73%) as a white solid.
Resolution of (±)-21 by chiral HPLC method 1 (at 250 mL/min flow rate) gave (-)-21 (tR=
6.08 min) and (+)-21 (tR= 8.08 min). (-)-21: LCꢀMS (ESI): m/z 453 (M+H)+, tR= 4.31 min, purity
100%, method 1. 1H NMR (300 MHz, CDCl3) δ 7.34ꢀ7.25 (m, 2H), 7.19ꢀ7.14 (m, 2H), 6.57 (s, 1H),
3.87 (t, 2H, J=5.4 Hz), 3.27ꢀ3.21 (m, 1H), 2.86ꢀ2.65 (m, 5H), 2.76 (d, 3H, J=4.8 Hz), 2.59 (t, 2H,
J=5.4 Hz), 2.53ꢀ2.39 (m, 2H), 2.29ꢀ2.20 (m, 1H), 2.00ꢀ1.96 (m, 2H), 1.83ꢀ1.75 (m, 2H). [α]D ꢀ24.4º
(c 5.0, MeOH). HRMS (ESI): calcd for C22H26Cl2N2O2S, 452.1092; found, 452.1096.
2-Chlorophenylmethyl-3-(2-chlorospiro[4,5-dihydrothieno[2,3-c]pyran-7,4'-
piperidine]-1'-yl)propanamide ((±)-22). The racemic title compound was prepared from tertꢀbutyl
2ꢀ[(2ꢀchlorophenyl)methyl]ꢀ3ꢀ(2ꢀchlorospiro[4,5ꢀdihydrothieno[2,3ꢀc]pyranꢀ7,4'ꢀpiperidine]ꢀ1'ꢀ
yl)propanoate (119 mg, 0.24 mmol) and ammonium chloride (12.8 mg, 0.24 mmol) according to the
21
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 45
1
2
3
general amide formation procedure. The crude product was purified by preparative TLC (CH2Cl2/
4
5
6
MeOH=20:1) to afford (±)-22 (75 mg, 71%) as a as a white solid.
7
8
9
Resolution of (±)-22 by chiral HPLC method 2 gave (-)-22 (tR= 10.17 min) and (+)-22 (tR=
12.50 min). (+)-22: LCꢀMS (ESI): 439 (M+H)+, tR= 4.17 min, purity 95%, method 1. 1H NMR (300
MHz, CDCl3) δ 7.66 (brs, 1H), 7.36ꢀ7.27 (m, 2H), 7.22ꢀ7.14 (m, 2H), 7.58 (s, 1H), 5.41 (brs, 1H),
3.87 (t, 2H, J=5.4 Hz), 3.34ꢀ3.28 (q, 1H, J=5.1 Hz), 2.89ꢀ2.64 (m, 5H), 2.59 (t, 2H, J=5.4 Hz), 2.52ꢀ
2.36 (m, 2H), 2.24ꢀ2.15 (m, 1H), 2.05ꢀ1.70 (m, 4H). [α]D +6.4º (c 1.0, EtOH). HRMS (ESI): calcd
for C21H25Cl2N2O2S + H+, 439.1008; found, 439.1021.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2-Fluorophenylmethyl-3-(2-chlorospiro[4,5-dihydrothieno[2,3-c]pyran-7,4'-
piperidine]-1'-yl)-N, N-dimethylpropanamide ((±)-23). The racemic title compound was prepared
from tertꢀbutyl 2ꢀ[(2ꢀfluorophenyl)methyl]ꢀ3ꢀ(2ꢀchlorospiro[4,5ꢀdihydrothieno[2,3ꢀc]pyranꢀ7,4'ꢀ
piperidine]ꢀ1'ꢀyl)propanoate (136 mg, 0.28 mmol) and dimethylamine hydrochloride (23 mg, 0.28
mmol) according to the general amide formation procedure. The crude product was purified by
preparative TLC (CH2Cl2/MeOH=20:1) to afford (±)-23 (60 mg, 47%) as a light yellow oil.
Resolution of (±)-23 by chiral HPLC method 1 gave (+)-23 (tR= 6.44 min) and (-)-23 (tR=
8.78 min). (+)-23: LCꢀMS (ESI) 451 (M+H)+, tR= 4.45 min, purity 97.5%, method 1. 1H NMR (300
MHz, CDCl3) δ 7.13ꢀ7.22 (m, 2H), 6.95ꢀ7.11 (m, 2H), 6.56 (s, 1H), 3.86 (t, J=5.1Hz, 2H), 3.25ꢀ
3.35 (m, 1H), 2.75ꢀ3.02 (m, 9H), 2.30ꢀ2.70 (m, 7H), 1.90ꢀ2.00 (m, 2H), 1.70ꢀ1.85 (m, 2H). The Lꢀ
tartaric salt was prepared. [α]D + 6.4º (c 1.0, EtOH). HRMS (ESI): calcd for C23H29ClFN2O2S + H+,
451.1617; found, 451.1624.
2-Fluorophenylmethyl-3-(2-chlorospiro[4,5-dihydrothieno[2,3-c]pyran-7,4'-
piperidine]-1'-yl)-N-methylpropanamide ((±)-24). The racemic title compound was prepared
from tertꢀbutyl 2ꢀ[(2ꢀfluorophenyl)methyl]ꢀ3ꢀ(2ꢀchlorospiro[4,5ꢀdihydrothieno[2,3ꢀc]pyranꢀ7,4'ꢀ
piperidine]ꢀ1'ꢀyl)propanoate (742 mg, 1.54 mmol) and methyl amine hydrochloride (104 mg, 1.54
22
ACS Paragon Plus Environment

Page 23 of 45
Journal of Medicinal Chemistry
1
2
3
mmol) according to the general amide formation procedure. The crude product was purified by
4
5
6
preparative TLC (CH2Cl2/MeOH=25:1) to afford (±)-24 (392 mg, 58%) as a white solid.
7
8
Resolution of (±)-24 by chiral HPLC method 2 gave (-)-24 (tR= 6.43 min) and (+)-24 (tR=
9
1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
8.50 min). (-)-24: LCꢀMS (ESI) 437 (M+H)+, tR= 4.74 min, purity 100%, method 3. H NMR (300
MHz, CDCl3) δ 7.46 (br, 1H), 7.15ꢀ7.25 (m, 2H), 6.96ꢀ7.07 (m, 2H), 6.57 (s, 1H), 3.85 (t, J=5.4Hz,
2H), 3.15ꢀ3.21 (m, 1H), 2.52ꢀ2.79 (m, 10H), 2.37ꢀ2.52 (m, 2H), 2.22 (td, J=2.4Hz, 1H), 1.95ꢀ2.
[α]D ꢀ7.0º (c 1.0, EtOH). HRMS (ESI): calcd for C22H26ClFN2O2S, 436.1387; found, 436.1388.
3-(2'-Fluoro-4',5'-dihydro-1H-spiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-yl)
2-(2-
chlorobenzyl)-N, N-dimethylpropanamide (( )-25). The racemic title compound was prepared
±
from tertꢀbutyl 2ꢀ[(2ꢀchlorophenyl)methyl]ꢀ3ꢀ(2ꢀfluororospiro[4,5ꢀdihydrothieno[2,3ꢀc]pyranꢀ7,4'ꢀ
piperidine]ꢀ1'ꢀyl)propanoate14 (188 mg, 0.39 mmol) and dimethylamine hydrochloride (32 mg, 0.39
mmol) according to the general amide formation procedure. The crude product was purified by
column chromatography in silica gel (CH2Cl2/MeOH=30:1) to afford (±)-25 (120 mg, 68%) as a
pale yellow solid.
Resolution of (±)-25 by chiral HPLC method 1 gave (+)-25 (tR= 5.67 min) and (-)-25 (tR=
7.85 min). (+)-25: LCꢀMS (ESI) 451 (M+H)+, tR= 4.45 min, purity 97.8%, method 1. 1H NMR (300
MHz, CDCl3) δ 7.31ꢀ7.34 (m, 1H), 7.13ꢀ7.21 (m, 3H), 6.11 (d, J=1.5Hz, 1H), 3.89 (t, J=5.4Hz, 2H),
3.35ꢀ3.45 (m, 1H), 3.10ꢀ3.16 (m, 1H), 2.77ꢀ2.87 (m, 5H), 2.81ꢀ2.75 (m, 5H), 2.37ꢀ2.55 (m, 3H),
2.32ꢀ2.45 (m, 2H), 1.93ꢀ1.98 (m, 2H), 1.67ꢀ1.81 (m, 2H). [α]D +42.6º (c 1.0, EtOH). HRMS (ESI):
calcd for C23H29ClFN2O2S + H+, 451.1617; found, 451.1624.
3-(2'-Fluoro-4',5'-dihydro-1H-spiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-yl)
2-(2-
fluorobenzyl)-N, N-dimethylpropanamide (( )-27). The racemic title compound was prepared
±
from tertꢀbutyl 2ꢀ[(2ꢀfluorophenyl)methyl]ꢀ3ꢀ(2ꢀfluorospiro[4,5ꢀdihydrothieno[2,3ꢀc]pyranꢀ7,4'ꢀ
piperidine]ꢀ1'ꢀyl)propanoate14 (1.46 g, 3.15 mmol) and dimethylamine hydrochloride (257 mg, 3.15
mmol) according to the general amide formation procedure. The crude product was purified by
23
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 45
1
2
3
preparative TLC (CH2Cl2/MeOH=20:1) to afford (±)-27 (550 mg, 40%) as a light yellow oil. LCꢀ
4
5
6
MS (ESI) 435 (M+H)+, tR= 4.15 min, purity 98.0%, method 1. 1H NMR (300 MHz, CDCl3)
δ 7.14ꢀ
7
8
9
7.19 (m, 2H), 6.96ꢀ7.04 (m, 2H), 6.10 (d, J=1.2Hz, 1H), 3.88 (t, J=5.4Hz, 2H), 3.22ꢀ3.35 (m, 1H),
2.92ꢀ3.01 (m, 1H), 2.75ꢀ2.87 (m, 8H), 2.59ꢀ2.70 (m, 2H), 2.45ꢀ2.56 (m, 3H), 2.30ꢀ2.44 (m, 2H),
1.90ꢀ2.00 (m, 2H), 1.66ꢀ1.76 (m, 2H).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Resolution of (±)-27 by chiral HPLC method 1 gave (-)-27 (tR= 6.02 min) and (+)-27 (tR=
7.99 min). (-)-27: LCꢀMS (ESI) 435 (M+H)+, tR= 4.15 min, purity 98.0%, method 1. The Lꢀtartaric
salt was prepared. [α] Na = ꢀ15.7º (c 1.0, EtOH). HRMS (ESI): calcd for C23H28F2N2O2S:
434.1839, found: 434.1842. (+)-27: LCꢀMS (ESI) 435 (M+H)+, tR= 4.15 min, purity 97.0%, method
1. The Lꢀtartaric salt was prepared. [α]D +15.4º (c 1.0, EtOH). HRMS (ESI): calcd for
C23H28F2N2O2S, 434.1839; found, 434.1839.
3-(2'-Fluoro-4',5'-dihydro-1H-spiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-yl)
2-(2-
fluorobenzyl)-N-methyl-N-cyclopropylpropanamide (( )-28). The racemic title compound was
±
prepared from tertꢀbutyl 2ꢀ[(2ꢀfluorophenyl)methyl]ꢀ3ꢀ(2ꢀfluorospiro[4,5ꢀdihydrothieno[2,3ꢀ
c]pyranꢀ7,4'ꢀpiperidine]ꢀ1'ꢀyl)propanoate14 (74 mg, 0.15 mmol) and cyclopropyl methylamine
hydrochloride (16 mg, 0.15 mmol) according to the general amide formation procedure. The crude
product was purified by preparative TLC (CH2Cl2/MeOH=20:1) to afford (±)-28 (55 mg, 75%) as a
as a white solid.
Resolution of (±)-28 by chiral HPLC method 2 (with 0.2% DMEA in hexane/isopropyl
alcohol (90:10 v/v) and at 30 mL/min flow rate) gave (-)-28 (tR= 6.31 min) and (+)-28 (tR= 7.69
min). (-)-28: LCꢀMS (ESI) 461 (M+H)+, 96.58% (UV 214nm), tR= 4.62 min, purity 99.4%, method
1
1. H NMR (300 MHz, CDCl3)
δ 7.12ꢀ7.17 (m, 2H), 6.94ꢀ7.02 (m, 2H), 6.09 (d, J=1.8Hz, 1H),
3.79ꢀ3.89 (m, 3H), 3.03 (dd, J=12.9Hz, 5.1Hz, 1H), 2.68ꢀ2.79 (m, 7H), 2.45ꢀ2.53 (m, 3H), 2.31ꢀ
2.41 (m, 2H), 2.18ꢀ2.21 (m, 1H), 1.91ꢀ1.96 (m, 2H), 1.68ꢀ1.77 (m, 2H), 0.66ꢀ0.80 (m, 3H), 0.33ꢀ
24
ACS Paragon Plus Environment

Page 25 of 45
Journal of Medicinal Chemistry
1
2
3
4
0.44 (m, 1H). [α]D ꢀ14.3º (c 0.3, MeOH). HRMS (ESI): calcd for C25H31F2N2O2S + H+, 461.2069;
5
6
found, 461.2078.
7
8
9
3-(2'-Fluoro-4',5'-dihydro-1H-spiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-yl)
2-(2-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
chlorobenzyl)-N-methyl-N-cyclopropylpropanamide (( )-29). The racemic title compound was
±
prepared from tertꢀbutyl 2ꢀ[(2ꢀchlorophenyl)methyl]ꢀ3ꢀ(2ꢀfluorospiro[4,5ꢀdihydrothieno[2,3ꢀ
c]pyranꢀ7,4'ꢀpiperidine]ꢀ1'ꢀyl)propanoate14 (120 mg, 0.25 mmol) and cyclopropyl methylamine
hydrochloride (27 mg, 0.25 mmol) according to the general amide formation procedure. The crude
product was purified by preparative TLC (CH2Cl2/MeOH=20:1) to afford (±)-29 (85 mg, 71%) as a
as a white solid.
Resolution of (±)-29 by chiral HPLC method 2 (with 0.2% DMEA in hexane/EtOH (90:10
v/v)) gave (-)-29 (tR= 5.92 min) and (+)-29 (tR= 7.61 min). (-)-29: LCꢀMS (ESI) 477 (M+H)+ tR=
4.95 min, purity 99.4%, method 1. 1H NMR (300 MHz, CDCl3)
δ 7.33ꢀ7.30 (m, 1H), 7.21ꢀ7.12 (m,
3H), 6.12 (d, 1H, J=1.2 Hz), 3.99ꢀ3.93 (m, 1H), 3.92 (t, 2H, J=5.4 Hz), 3.25 (q, 1H, J=4.2 Hz),
2.85ꢀ2.68 (m, 4H), 2.76 (s, 3H), 2.57ꢀ2.50 (m, 3H), 2.46ꢀ2.32 (m, 2H), 2.05ꢀ1.94 (m, 3H),1.80ꢀ1.74
(m, 2H), 0.80ꢀ0.61 (m, 3H), 0.28ꢀ0.24 (m, 1H). [α]D ꢀ43.0o (c 0.5, MeOH). HRMS (ESI): calcd for
C25H31ClFN2O2S + H+, 477.1773; found, 477.1785.
In Vitro NOP Receptor Binding. A filtrationꢀbased [3H]ꢀnociceptin binding assay was
used to determine the affinity (Ki) of synthesized NOP compounds based on previous assay
formats,30 with minor modifications. Assay incubations were performed in deepꢀwell 96ꢀwell plates
with [3H]ꢀnociceptin (final assay concentration 0.2 nM) and 5−10 ꢁg of membrane protein (isolated
from CHO cells expressing cloned human NOP receptors) in a final volume of 0.5 mL of HEPES
buffer (20 mM; pH 7.4) containing, 5 mM MgCl2, 1 mM EDTA, 100 mM NaCl, and 0.1% BSA.
Incubations were performed for 60 min at 25 °C and terminated by filtration on glass fiber
filtermats (GF/C Filtermat A; pretreated with 0.3% polyethyleneimine for 1 h) on a Tometc cell
harvester. The filters were washed three times with 5 mL of iceꢀcold TrisꢂHCl buffer (50 mM, pH
25
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 45
1
2
3
4
5
6
7
8
9
7.4). Filtermats were then dried and embedded with Meltilux scintillant A and the radioactivity
counted in a Microbeta scintillation counter (Perkin Elmer Life and Analytical Sciences). Specific
binding was determined by displacement with 100 nM unlabeled nociceptin. Curves were plotted as
the percent of specific inhibition versus NOP compound concentration and IC50 values were
determined using fourꢀparameter nonlinear regression routines (XLFit version 4.0, IDBS). Ki values
were calculated from the IC50 by the equation of Cheng and Prusoff,31 where Ki = IC50 × (1 + D ×
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Kd−1)−1. Reported values for K are shown as geometric means ± the standard error of the mean
i
(SEM), with the number of replicate determinations indicated by n. Geometric means are calculated
by the equation GeoMean = 10^(Average (log K + log K +…log K )/ square root of the
i1 i2 in
number of replicates, n).
In Vitro Opioid Receptor Binding. To determine receptor selectivity to classical opioid
receptors, the binding affinity (Ki) of NOP compounds was determined in Chinese hamster ovary
(CHO) or human embryonic kidney (HEK) 293 cell membranes expressing either cloned human
mu, kappa or delta receptors, as previously described.32 Eleven point ligand displacement curves
were determined using [3H]ꢀdiprenorphine. Briefly, membranes (4ꢀ6 ꢀg protein per reaction) were
added to buffer containing 50 mM TrisꢀHCl, 100 mM NaCl, 1 ꢁM GDP, 5 mM MgCl2 and 1 mM
EDTA (pH 7.4). The reactions were initiated by the addition of 0.2 nM [3H]ꢀdiprenorphine to yield
a 500 ꢀl assay volume. The reaction was incubated for 120 min at rt Specific binding was
determined by displacement with 10 ꢀM naltrexone. Reactions were terminated by rapid filtration
through glass fiber filters, radioactivity measured and data analyzed using procedures identical to
those used for NOP receptor binding.
In Vitro Functional Blockade of NOP Receptor Agonist-mediated G-Protein Activation
– GTPγ-[35S] Binding. Receptorꢀmediated Gꢀprotein activation can be measured using the nonꢀ
hydrolyzable radiolabeled analog of GTP, GTPγꢀ[35S]. Thus, the NOP receptor antagonist affinity
(Kb) of test ligands was measured in membranes expressing cloned human NOP receptors with a
26
ACS Paragon Plus Environment

Page 27 of 45
Journal of Medicinal Chemistry
1
2
3
4
5
GTPγꢀ[35S] binding assay, according to previously described protocols with minor modifications.33,
34
6
7
8
9
Assays were conducted in deepꢀwell 96 well plates in a 200ꢀꢃL volume with the following
buffer composition: 100 mM NaCl, 20 mM HEPES, 5 mM MgCl2, 1 mM EDTA, 0.1% BSA, 3 ꢃM
GDP, 0.5 nM GTPγ[35S]. NOP receptor membrane suspension was added at a concentration of 20
ꢃg protein per well and receptor stimulation was achieved with 300 nM nociceptin. Wheat germ
agglutininꢀcoated scintillation proximity assay (SPA) beads (Perkin Elmer Life and Analytical
Sciences) were added at 1 mg per well to detect membraneꢀbound GTPγ[35S]. Plates were sealed
and incubated for 2 h at rt and then placed at 4 °C overnight to allow the SPA beads to settle. Plates
were then counted for radioactivity in a Microbeta Trilux instrument. Specific GTPγ[35S] binding
was determined as the difference in cpm observed in the absence and presence of 10 ꢃM unlabeled
GTPγS. Data were plotted as the percent of specific GTPγ[35S] bound from which IC50 values were
determined using fourꢀparameter nonlinear regression routines (XLFit version 4.0, IDBS).
Antagonist affinity (Kb) was estimated according to Delapp et al,33 using a modification of the
equation of Cheng and Prusoff31 where Kb = IC50 × (1 + D × EC50−1)−1. Reported values for Kb are
shown as geometric mean ± the standard error of the mean (SEM), with the number of replicate
determinations indicated by n.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Agonist functional GTPγ[35S] binding assays were similar to those described for antagonist
measurement, except that the GTPγ[35S] mixture did not have added nociceptin. Basal activity was
determined in the absence of added nociceptin. A control nociceptin curve (geometric mean EC50
for nociceptin was 2.25 nM, SEM=0.41, n=18) was included in each assay. The concentration range
tested for NOP compounds was 0.17 nM to 10 ꢀM (final assay DMSO concentration in all wells
was 1%). The percent efficacy is calculated, relative to nociceptin at 100%.
In Vivo Characterization of Potential tracers in Rat. Doses of potential tracers to be
intravenouly administered to rats were selected to be low (<10 ꢀg) but still allow accurate detection
27
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 45
1
2
3
4
5
6
7
8
9
and quantitation by LCꢀMS/MS. Thus, each test ligand was dissolved in 25% βꢀcyclodextrin
solution (1 mg/ mL) and then diluted to a final ligand concentration of 6 ꢁg/mL Each of these
solutions was administered intravenously to each of a separate group of three or four rats, injection
volume 0.5 mL/kg, via the lateral tail vein. In each group, rats were sacrificed at 10, 20, 40, or 60
min after ligand injection. Samples of hypothalamic, thalamic, and striatal tissue were weighed and
placed in conical centrifuge tubes on ice. Four volumes (w/v) of acetonitrile (ACN) containing
0.1% formic acid were added to each tube. These samples were then homogenized using an
ultrasonic probe and centrifuged at 14,000 rpm for 16 min Supernatant liquid was diluted by adding
sterile water (100−900 ꢁL) in HPLC injection vials for subsequent LCꢀMS/MS analysis.
Ligands were analyzed with a model 1200 HPLC apparatus (Agilent Technologies, Palo
Alto, CA) linked to an API 4000 mass spectrometer (Applied Biosystems, Foster City, CA, USA).
A C18 column (2.1 × 50 mm; Agilent; part # 971700ꢀ907) was used for the HPLC. The compound
specific isocratic methods consisted of mobile phase of various ratios of water (A) and acetonitrile
(B) with 0.1% formic acid. The ratios A/B used were the following: 72/28 ((-)-8), 80/20 ((+)-19),
65/35 ((-)-20), 65/35 ((-)-21), 65/35 ((+)-22), 60/40 ((+)-23), 65/35 ((-)-24), 60/40 ((+)-25), 60/40
((-)-26), 65/35 ((-)-27), 60/40 ((-)-28), 60/40 ((-)-29), 70/30 ((-)-30) and 60/40 ((-)-33). The ligands
were detected by monitoring the precursor to product ion transition. The following are the ion
transitions for the compounds listed in Table 5: 389.4→222.0 ((-)-8), 407.3→239.9 ((+)-19),
468.5→256.0 ((-)-20), 454.3→256.1 ((-)-21), 440.5→255.9 ((+)-22), 452.2→257.9 ((+)-23),
438.6→257.8 ((-)-24), 452.2→239.9 ((+)-25), 438.3→240.0 ((-)-26), 435.1→240.0 ((-)-27),
461.8→240.3 ((-)-28), 478.3→240.3 ((-)-29), 421.1→240.0 ((-)-30) and 463.8→240.0 ((-)-33).
Standards were prepared by adding known quantities of analyte to samples of brain tissue from
nonꢀtreated rats and processed as described above. The proportion of the injected ligand dose
reaching brain region tissue, normalized for animal weight, was expressed as %SUV (standardized
uptake value), which was calculated as follows:
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
%SUV = (ligand amount measured in tissue; (ng/mL)/(injected ligand dose; ꢁg/kg) ×100
28
ACS Paragon Plus Environment

Page 29 of 45
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Levels of ligand in the hypothalamus,16, 17 a region with high NOPꢀ1 receptor expression,
were taken to represent total ligand binding in a tissue. Thalamus was an intermediate NOP
expressing brain region. Striatal levels were used to represent nonspecific binding. It should be
noted, however, that striatum16, 17 does have low NOPꢀ1 receptor expression. Thus, striatum is a
pseudo receptorꢀnull region rather than a true null region. As such, the difference between the
ligand concentration measured in the total binding region, the hypothalamus, and that measured in
the pseudoꢀnull region, the striatum, is an underꢀestimation of the true NOP receptorꢀspecific
binding.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
To generate the dose occupancy curve, animals were administered an oral dose of 3 or
vehicle. One hour following oral pretreatment, animals received an i.v. dose of unlabeled tracer (-)-
27. Forty minutes after the tracer was injected, animals were sacrificed and hypothalamic and
striatal tissue were harvested and placed on ice. The procedures outlined above were then applied to
the tissue samples collected. Receptor occupancy calculations were made for each animal
employing the widely used ratio method.12, 13
Computation and Measurement of Lipophilicity. The cLogD values for ligands at pH 7.4
were computed with Pallas 3.7 software (CompuDrug). LogD values were experimentally
determined by the potentiometric method. LogD values at pH 7.4 were extrapolated from LogP
values determined on a Sirius Analytical GLpKa instrument and Refinement Pro software.
Titrations were performed over a defined pH range, with 1ꢀoctanol used as the partition solvent.
Acknowledgements. We would like to thank Jiapo Wang, Hao Yao, Heyang Shao, Jinting
Tang, Xi Liang, Zhongzhen Zhang for the preparation of many important compounds. We also
would like to acknowledge Anju Lewis, Keyue Chen and Lorrel Burison for the generation of the
in vitro binding and functional NOP data and the binding opioid receptor data.
Supporting Information: Experimental and spectroscopic data for compounds 9ꢀ18 and
HPLC analysis of enantiomerically pure final materials 8, 10, 19-25 and 27-29 are available.
29
ACS Paragon Plus Environment