Structure-activity relationships and discovery of a g protein biased μ opioid receptor ligand, [(3-methoxythiophen-2-yl)methyl]({2-[(9 r)-9-(pyridin-2-yl)-6-oxaspiro-[4.5]decan-9-yl]ethyl})amine (TRV130), for the treatment of acute severe pain

Article abstract of DOI:10.1021/jm4010829

The concept of ligand bias at G protein coupled receptors has been introduced to describe ligands which preferentially stimulate one intracellular signaling pathway over another. There is growing interest in developing biased G protein coupled receptor ligands to yield safer, better tolerated, and more efficacious drugs. The classical μ opioid morphine elicited increased efficacy and duration of analgesic response with reduced side effects in β-arrestin-2 knockout mice compared to wild-type mice, suggesting that G protein biased μ opioid receptor agonists would be more efficacious with reduced adverse events. Here we describe our efforts to identify a potent, selective, and G protein biased μ opioid receptor agonist, TRV130 ((R)-30). This novel molecule demonstrated an improved therapeutic index (analgesia vs adverse effects) in rodent models and characteristics appropriate for clinical development. It is currently being evaluated in human clinical trials for the treatment of acute severe pain.

Full text of DOI:10.1021/jm4010829

Article
pubs.acs.org/jmc
Structure−Activity Relationships and Discovery of a G Protein Biased
μ Opioid Receptor Ligand, [(3-Methoxythiophen-2-yl)methyl]({2-
[(9R)‑9-(pyridin-2-yl)-6-oxaspiro-[4.5]decan-9-yl]ethyl})amine
(TRV130), for the Treatment of Acute Severe Pain
Xiao-Tao Chen,* Philip Pitis, Guodong Liu, Catherine Yuan, Dimitar Gotchev, Conrad L. Cowan,
David H. Rominger, Michael Koblish, Scott M. DeWire, Aimee L. Crombie, Jonathan D. Violin,
and Dennis S. Yamashita
Trevena, Inc., 1018 West 8th Avenue, Suite A, King of Prussia, Pennsylvania 19406, United States
S
* Supporting Information
ABSTRACT: The concept of “ligand bias” at G protein
coupled receptors has been introduced to describe ligands
which preferentially stimulate one intracellular signaling
pathway over another. There is growing interest in developing
biased G protein coupled receptor ligands to yield safer, better
tolerated, and more efficacious drugs. The classical μ opioid
morphine elicited increased efficacy and duration of analgesic
response with reduced side effects in β-arrestin-2 knockout
mice compared to wild-type mice, suggesting that G protein
biased μ opioid receptor agonists would be more efficacious
with reduced adverse events. Here we describe our efforts to identify a potent, selective, and G protein biased μ opioid receptor
agonist, TRV130 ((R)-30). This novel molecule demonstrated an improved therapeutic index (analgesia vs adverse effects) in
rodent models and characteristics appropriate for clinical development. It is currently being evaluated in human clinical trials for
the treatment of acute severe pain.
which explains the high proportion (>80%) of patients who
report inadequate treatment of their pain.7
INTRODUCTION
■
Morphine, an alkaloid natural product isolated from the poppy
plant Papaver somniferum, is the principal component of opium
and has been used as an analgesic since the Neolithic Age.1 The
pharmacological actions of morphine are mediated by agonism
of the μ opioid receptor (MOR),2 a G protein coupled receptor
(GPCR) that is highly expressed in the central nervous system
(CNS). A wide range of MOR agonists have been developed to
treat severe pain including the morphine derivatives oxycodone,
hydromorphone, oxymorphone, levorphanol, and buprenor-
phine and the fully synthetic opioids such as fentanyl,
sufentanil, meperidine, and butorphanol.3 However, despite
the effectiveness of these powerful analgesics, all of these agents
suffer from similar adverse effects including constipation,
nausea, vomiting, sedation, pruritus, and respiratory depression.
In addition, MOR agonists cause euphoria and physical
dependence which can lead to abuse and addiction. Chronic
use of MOR agonists can also result in analgesic tolerance,4
necessitating dose escalation to control pain. In the post-
operative setting, adverse effects from MOR agonists such as
nausea, vomiting, and constipation, especially in cases of open
abdominal surgery, can delay patient recovery and hospital
discharge.5 Moreover, the risk of severe respiratory depression
often limits the dose of MOR agonists used postoperatively,6
GPCRs are seven-transmembrane (7TM) receptors that bind
to agonists and couple to G proteins resulting in activation of
complex intracellular signaling pathways. In addition to G
protein signaling, GPCRs can activate parallel and sometimes
distinct signaling pathways. Principal among these is signaling
mediated by β-arrestins, which bind activated receptors to
desensitize G protein signaling, promote receptor internal-
ization, and can initiate distinct signal transduction cascades
independent of G protein pathways. GPCR “biased ligands” are
compounds that selectively bind a target GPCR and are capable
of activating select intracellular responses, including G protein
or β-arrestin specific responses.8 A G protein biased ligand may
have equivalent or better efficacy in activating a G protein
relative to a reference agonist compound, but has lowered
efficacy or even is capable of blocking or inhibiting β-arrestin
recruitment and activation.
Pharmacological studies conducted with morphine in
knockout (KO) mice suggested that ligand bias may confer
unique pharmacologic signals. In β-arrestin-2 KO mice, the
morphine analgesic response was prolonged with reduced
Received: July 17, 2013
© XXXX American Chemical Society
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Table 1. Identification of a Spirocyclic Core for G Protein Biased μ Opioid Receptor Ligand
MOR cAMP pEC50
MOR cAMP % efficacy MOR β-Arr-2 pEC50 MOR β-Arr-2 % efficacy
a
compd
R1
R2
R3, R4
R5
R6
SD
SD
SD
SD
morphine
7.4 0.1
8.7 0.1
6.3 0.1
5.0 0.1
7.4 0.1
5.8 0.1
6.6 0.1
7.1 0.1
7.5 0.1
6.3 0.1
6.4 0.1
6.3 0.1
6.0 0.1
5.5 0.1
100
55
1
1
1
1
3
2
3
2
1
4
2
2
2
4
6.3 0.1
100
NQ
32
NQ
3
b
buprenorphine
NQ
1
Me
Me
F
OH
OH
OH
H
Et, Me
H, H
H
H
74
5.7 0.4
NQ
5
2
H
H
41
3
Et, Me
Me, Me
Et, Et
H
H
108
79
5.8 0.2
NQ
236 19
NQ
4
F
H
H
5
F
H
H
H
96
5.2 0.2
6.2 0.4
6.4 0.3
5.1 0.1
5.1 0.2
NQ
55
27
7
4
2
6
F
H
(CH2)4
(CH2)4
(CH2)4
(CH2)5
(CH2)4
(CH2)4
(CH2)4
H
H
72
(R)-6
(S)-6
7
F
H
H
H
79
20
F
H
H
H
110
104
75
165
56
3
F
H
H
H
6
(R)-8
(R)-9
(R)-10
F
H
Me
H
H
NQ
F
H
Me
Me
65
NQ
NQ
NQ
F
H
Me
84
NQ
a
b
Data are means standard error, displayed as percentage of maximum morphine efficacy, for greater than three independent experiments. NQ:
the activity was too low to detect, and therefore, was not quantifiable.
tolerance compared to wild-type littermates.9 In addition, β-
arrestin-2 KO mice treated with morphine experienced reduced
constipation as measured by fecal boli accumulation and
reduced respiratory depression as measured by plethysmog-
raphy.10 We and others hypothesized that G protein biased
MOR agonists, compounds that selectively activate MOR G
protein signaling with minimal to no β-arrestin-2 signaling,
would retain powerful analgesic activity and possess a superior
therapeutic index with respect to constipation and respiratory
depression compared to morphine.11 Herein, we disclose a
structure−activity relationship (SAR) study of a novel class of
MOR ligands that led to the identification of [(3-
methoxythiophen-2-yl)methyl]({2-[(9R)-9-(pyridin-2-yl)-6-
oxaspiro[4.5]decan-9-yl]ethyl})amine, (R)-30 (TRV130), a
MOR G protein biased agonist currently being evaluated in
human clinical trials for the treatment of acute severe pain.12
MOR β-arrestin recruitment activity of compound 2 was too
low to detect and therefore was not quantifiable (NQ).
Several additional commercially available analogues of
compound 1 were obtained; the 4-fluorophenyl analogue (3)
had a 13-fold increase in G protein MOR potency compared to
the corresponding 4-methylphenyl analogue (1), but the β-
arrestin recruitment efficacy also increased to 236% relative to
morphine. Because potency increased with the 4-fluorophenyl
substitution, this was held fixed while the tetrahydropyran
substitution was examined to evaluate impact on potency and
efficacy. In addition, the phenol group on the side chain of
compound 3 (R2 = OH) was eliminated to remove a potential
site of glucuronidation. Therefore, the 2,2-dimethyl analogue
(4) and 2,2-diethyl analogue (5) were prepared with a simple
benzyl group on the ethylamine side chain. Compound 4 was
40 times less potent than compound 3, while compound 5 was
only 6-fold less potent in the cAMP accumulation assay. More
importantly, compound 5 had lower β-arrestin recruitment
efficacy than morphine and compound 3. Because 2,2-diethyl
substitution retained promising activities in the cAMP
accumulation assay, a set of spirocyclic ring analogues were
thus prepared. The spirocyclopentane analogue (6) was 3-fold
more potent than the 2,2-diethyl analogue (5) and 5-fold more
potent than the corresponding spirocyclohexane analogue (7)
with lower β-arrestin recruitment efficacy than morphine.
Because of the promising in vitro profile of the racemate (6),
the enantiomers of compound 6 were synthesized and tested
separately. (R)-6 was 16-fold more potent than its opposite
enantiomer ((S)-6) and also had much lower β-arrestin efficacy
(20% vs 165%). The higher β-arrestin efficacy of the (S)-
enantiomer is not evident in the racemate, likely because the
(R)-enantiomer has higher affinity for the receptor and is thus
responsible for the majority of receptor occupancy in the
racemic mixture. Tolerance of methyl substitution at the
benzylic position of (R)-6 was examined. Both monomethyla-
tions ((R)-8 and (R)-9) and gem-dimethyl substitution ((R)-
RESULTS AND DISCUSSION
■
We began a lead discovery effort by screening an internal
compound collection of small molecules and identified a novel
MOR agonist (1, Table 1). This hit was a 4-phenyl-4-(2-
benzylaminoethyl)-tetrahydropyran analogue described as
having anti-inflammatory activity in rats.13 The submicromolar
G protein MOR potency and low MOR β-arrestin recruitment
activity (32% compared to 100% for morphine) of compound 1
made it an interesting starting point for a further SAR
investigation with the aim of improving its MOR G protein
potency while maintaining its low β-arrestin recruitment
activity.
Because the 2-methyl-2-ethyl disubstitution on the tetrahy-
dropyran (THP) ring resulted in a chiral center, the effects of
no substitution at the 2-position of the THP ring were
explored. The unsubstituted analogue (2) was prepared and
was shown to be 20-fold less potent than the corresponding 2-
methyl-2-ethyl substituted analogue (1) and had reduced G
protein efficacy compared to morphine and compound 1. The
B
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Table 2. A Structure−Activity Relationship Study on the 2-Pyridyl Spirocyclic Core
a
compd
R1
MOR cAMP inh pEC50 SD
MOR cAMP inh % efficacy SD MOR β-Arr-2 pEC50 SD MOR β-Arr-2 % efficacy SD
11
Ph
Ph
Ph
7.3 0.1
7.9 0.1
6.2 0.1
8.0 0.2
8.6 0.1
6.8 0.1
6.7 0.1
5.9 0.1
6.2 0.1
6.1 0.1
7.8 0.1
7.9 0.1
6.8 0.1
7.5 0.1
74
82
109
30
91
34
53
31
35
84
95
89
87
69
1
2
2
1
1
1
1
1
1
2
1
1
4
2
6.7 0.4
6.8 0.3
4.8 0.1
16
17
79
2
2
5
(R)-11
(S)-11
(R)-12
(R)-13
(R)-14
(R)-15
(R)-16
(R)-17
(R)-18
(R)-19
(R)-20
(R)-21
(R)-22
b
2-Cl-Ph
NQ
NQ
59
NQ
3-Cl-Ph
7.3 0.3
NQ
6
2-pyridyl
3-pyridyl
NQ
NQ
NQ
NQ
4-pyridyl
NQ
2-pyrazyl
NQ
4-pyrimidyl
2-thiophenyl
3-thiophenyl
2-naphthyl
cyclopentyl
5.7 0.3
6.6 0.3
7.2 0.3
4.7 0.1
NQ
23
3
2
1
15
12
245 21
NQ
a
b
Data are means standard error, displayed as percentage of maximum morphine efficacy, for greater than three independent experiments. NQ:
the activity was too low to detect, and therefore, was not quantifiable.
10) resulted in a 6−40-fold loss in potency in the cAMP
accumulation assay; it was concluded that substitution at the
benzylic position was not preferred and no further chemistry
efforts were undertaken at this position.
potency and reduced β-arrestin recruitment efficacy, while the
4-pyrimidine ((R)-18) had decreased potency compared to the
phenyl analogue ((R)-11). Most interestingly, both regioiso-
meric thiophene analogues ((R)-19 and (R)-20) retained
similar in vitro activity profiles as (R)-11, i.e., potent MOR G
protein agonist activity with low β-arrestin recruitment efficacy.
Because of the promising in vitro activity profile of (R)-19, it
was selected for an evaluation of efficacy and adverse effects in
animal models.16 (R)-19 was more efficacious in a mouse hot-
plate assay (56 °C) than morphine, with an ED50 of 1.1 mg/kg
administered subcutaneously (sc). In the mouse hot-plate assay
(56 °C), 6 mg/kg sc morphine exhibited 69% of maximal
possible analgesic effect (MPE) at 30 min while 3 mg/kg of
(R)-19 showed 79% of MPE at 30 min. Therefore, the 6 mg/kg
dose of morphine was considered to be equianalgesic to the 3
mg/kg dose of (R)-19. In contrast to the analgesic effect, the 6
mg/kg dose of morphine exhibited a more severe constipation
effect than the 3 mg/kg dose of (R)-19 over a 4 h interval in a
mouse glass bead retention model of colonic motility (158 min
retention time for morphine vs 65 min retention time for (R)-
19). Because both morphine and (R)-19 have similar
pharmacokinetic profiles in the mouse after sc administration
(including similar half-life and Tmax, data not shown), the
differentiation in the in vivo pharmacology cannot be attributed
to pharmacokinetics. Similarly, in the mouse fecal boli
accumulation model, at equianalgesic doses, mice in the (R)-
19 treated group produced approximately 2-fold more fecal boli
(less constipation) than did mice in the morphine treated
group over a 4 h interval. This in vivo pharmacology suggested
that (R)-19 could be a potential candidate for further
preclinical development. Unfortunately, potent inhibition of
hERG channel current in vitro and cardiac conduction
abnormalities in arterially perfused rabbit ventricular wedge17
precluded further preclinical development of (R)-19 (see
detailed Discussion below).
Heterocyclic replacements of the phenyl group attached to
the tetrahydropyran core were explored. Among the heteroaryl
groups examined, the 2-pyridine group appeared to be the most
promising. A 2-pyridine analogue (11, Table 2) possessed good
potency in the cAMP accumulation assay and low efficacy in
the β-arrestin recruitment assay. Overall, the in vitro activity
profile of the racemate 11 was better than that of the racemate
6. Similar to the pair of enantiomers of 6, (R)-11 had a superior
in vitro profile compared to the (S)-enantiomer with increased
potency in the cAMP accumulation assay (pEC50 = 7.9 and 6.2,
respectively) and reduced efficacy in the β-arrestin recruitment
assay (17% and 79%, respectively). Because of that, the
remaining SAR studies were conducted on single (R)-
enantiomers. Keeping the 2-pyridine fixed, substituted benzyl
amine analogues and heterocyclic methylene amine analogues
were investigated. The 2-chlorophenyl analogue ((R)-12) was a
weak partial agonist in the MOR G protein assay, whereas the
3-chlorophenyl analogue ((R)-13) retained high MOR G
protein agonist activity. Also, all of the regioisomeric pyridine
analogues ((R)-14, (R)-15, and (R)-16) and the 2-pyrazine
analogue ((R)-17) were MOR G protein partial agonists. The
2-naphthyl analogue ((R)-21) retained MOR G protein agonist
activity but also had increased β-arrestin recruitment efficacy
(245% relative to morphine). The marked change in β-arrestin
efficacy without substantive differences in G protein coupling
efficacy (e.g., (R)-20 vs (R)-21) suggests distinct structure−
activity relationships for β-arrestin recruitment and G protein
coupling. This is consistent with the hypothesis that distinct
sets of receptor conformations are stabilized by biased and
unbiased ligands.14 However, it should be noted that comparing
efficacies in different assays is not a rigorous method of
quantifying ligand bias; other methods are required, and our
approach to quantify bias was described elsewhere.15,12 The
cyclopentyl analogue ((R)-22) retained MOR G protein
Because (R)-19 demonstrated a favorable in vivo pharmacol-
ogy profile, substitution of the 2-thiophene and 2-furan was
further examined (Table 3). All of the analogues retained good
C
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Table 3. A Structure−Activity Relationship Study Leading to Identification of (R)-30
MOR cAMP inh pEC50
MOR cAMP inh % efficacy
MOR β-Arr-2 pEC50
MOR β-Arr-2 % efficacy
a
compd
X
R1
R2
SD
SD
SD
SD
b
(R)-23
(R)-24
(R)-25
(R)-26
(R)-27
(R)-28
(R)-29
(R)-30
S
3-Me
4-Me
5-Me
5-Me
3-Me
4-Me
4-Me
3-OMe
H
8.2 0.1
8.3 0.1
7.8 0.1
7.3 0.1
7.6 0.1
8.3 0.1
7.5 0.1
8.1 0.1
75
101
96
1
1
2
1
2
2
1
1
NQ
NQ
S
H
6.4 0.2
NQ
95
NQ
8
S
H
O
S
H
84
6.7 0.5
6.2 0.2
6.3 0.2
6.5 0.2
7.3 0.4
13
18
2
1
5-Me
5-Me
5-Me
H
84
S
104
98
197 13
O
S
48
15
4
2
84
a
b
Data are means standard error, displayed as percentage of maximum morphine efficacy, for greater than three independent experiments. NQ:
the activity was too low to detect, and therefore, was not quantifiable.
potency and efficacy for G protein coupling and low β-arrestin
recruitment efficacy at human MOR, with the exception of
compounds (R)-24 and (R)-28, which had higher β-arrestin
recruitment efficacies (95 and 197%, respectively, relative to
morphine). During the SAR investigation, selectivity over κ
opioid receptor (KOR) and δ opioid receptor (DOR) was
closely monitored. In general, compounds within this series
were highly MOR selective. (R)-19 had potencies of 0.90 and
0.75 μM in human KOR and human DOR cAMP accumulation
assays, respectively, yielding 71-fold selectivity over KOR and
56-fold selectivity over DOR. Similarly, (R)-30 had potencies of
1.4 and 2.8 μM in human KOR and human DOR cAMP
accumulation assays, respectively, yielding 178-fold selectivity
over KOR and 355-fold selectivity over DOR.12
Several compounds were evaluated in mouse and rat
analgesia models and in mouse constipation models.
Ultimately, it was found that (R)-30 had a similar in vivo
pharmacology profile as that of (R)-19. It had high potency
(3−10 times more potent than morphine) in mouse and rat
hot-plate models as well as an improved therapeutic index with
respect to glass bead retention (colonic motility) and fecal boli
constipation measures.12 (R)-30 was also shown to have a
superior therapeutic index of analgesia vs respiratory
suppression (hot-plate vs CO2 blood gases) in the rat. On
the basis of its improved in vivo profile, we evaluated the
cardiovascular liability of (R)-30. A summary of these
assessments for (R)-30 and (R)-19 is listed in Table 4. The
compounds were evaluated for their ability to inhibit currents
of the hERG, Cav1.2, and Nav1.5 human cardiac ion channels
expressed in HEK293 cells, using an in vitro automated patch
clamp assay.18 (R)-30 and (R)-19 inhibited the hERG current
with IC50 values of 6.2 and 2.3 μM, respectively. (R)-30 and
(R)-19 demonstrated similar potencies at Nav1.5 channels. (R)-
19 did not inhibit Cav1.2 current up to the highest
concentration tested (100 μM), while (R)-30 inhibited the
current with an IC50 value of 36 μM. We sought at least a 30-
fold and preferably greater than 100-fold safety margin between
estimated target potency and hERG IC50. The margin for (R)-
30 between MOR agonism (functional EC50 = 8 nM,
radioligand binding Ki = 6 nM) and ion channel inhibition
was greater than 200-fold in all cases.
The potential CV liability of (R)-30 and (R)-19 was assessed
further in the isolated rabbit left ventricular wedge model.17
Briefly, a TdP score is calculated from drug-induced changes in
QT, Tp‑e/QT ratio, and arrhythmic events related to early after
depolarizations (EAD), which is utilized to semiquantitatively
describe the relative TdP risk of compounds. Validation
studies17 of this model show that compounds with scores of
2.5 or greater at concentrations less than 100 times their free
therapeutic plasma Cmax have arrhythmogenic potential. In
contrast, compounds which are not torsadogenic in humans,
elicited TdP scores of less than 2.5 (and, in some cases,
negative values).
(R)-19 increased QT and Tp−e intervals (Table 4) in
agreement with the hERG inhibition potency prolonging the
Tp−e interval to a greater extent than the QT interval.
Composite TdP scores of 2.3 and 4.0 were determined at
compound concentrations of 0.3 and 1.0 μM, respectively.
Therefore, (R)-19 poses a potential risk for arrhythmia. In
contrast, (R)-30 caused a very mild increase (3%) in QT
interval at 0.3 μM, but there was no significant effect at higher
doses. (R)-30 may have caused a very mild increase in Tp−e
interval at 0.3 and 1 μM, but caused a decreased Tp−e interval at
higher doses with only slight increase in QT interval over the
range of concentrations tested. It did not cause any
proarrhythmic events at tested concentrations. The estimated
TdP scores determined are either zero or negative up to 30 μM.
The results of both the ion channel inhibition and rabbit wedge
assays predict that (R)-30 will have little or no potential to
generate significant QT prolongation, leading to TdP or other
abnormal arrhythmias.
Table 4. A Summary of Cardiovascular Liability Assessment
of (R)-30 and (R)-19
(R)-30
IC50 (μM)
(R)-19
IC50 (μM)
ion channel
hERG
6.2
2.3
human Nav 1.5
phasic
4.7
16.5
36.0
9.0
20.0
tonic
human Cav 1.2
rabbit wedge
>100.0
(R)-30
(R)-19
highest TdP score
peak % QT interval
peak % Tp‑e interval
<0
2.3 at 0.3 μM, 4.0 at 1.0 μM
+40% at 1.0 μM
+3% at 0.3 μM
+7% at 1.0 μM
+65% at 1.0 μM
D
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Consistent with the results presented here, in studies
evaluating cardiovascular safety in male cynomolgus monkeys,
(R)-30 did not reveal any biologically significant effect on QRS
duration, PR interval, QT, or corrected QT (QTc) interval.
(R)-30 was thus progressed to IND-enabling studies and is now
being evaluated in human clinical trials for the treatment of
acute severe pain.
In summary, we describe here a lead optimization program
that established a structure−activity relationship for a novel
chemotype targeting the μ opioid receptor, culminating in the
identification of (R)-30, which is a potent and strong agonist of
G protein coupling at the μ opioid receptor but engages very
little β-arrestin-2 recruitment compared to standard strong
agonists like morphine. This biased ligand represents a new
mode of μ opioid receptor pharmacology, distinct from strong
agonists like morphine and fentanyl and from partial agonists
like buprenorphine. In addition, this work developed SAR for
distinct receptor functions, likely related to the different
receptor conformations stabilized by biased and unbiased
ligands. This demonstrates that biased ligands can be
discovered through empirical SAR-based lead optimization.
a mixture of regioisomers (36), which were not separated in
most cases and used directly in the next step. Either aryllithium
or aryl Grignard reagent was added to the unsaturated 2-
cyanopropenoate (36) in the presence of catalytic amount of
copper(I) iodide to form adduct (37). Decarboxylation of 37
was achieved under strong basic conditions with heating to
afford the nitrile (38). Reduction of the nitrile to its primary
amine (39) was accomplished with lithium aluminum hydride
(LAH) reduction in ether. Selective monoalkylation of the
primary amine (39) was realized via a two-step, one-pot
reductive amination with a variety of aldehydes. First, the
primary amine and an aldehyde formed an imine in
dichloromethane, and then the imine was reduced by NaBH4
after a solvent switch from dichloromethane to methanol to
afford compounds (2, 5−7, and 11).
The majority of cyclic ketones (35) used for our studies are
either commercially available or can be accessed from simple
commercially available starting materials (Scheme 3). For
example, a Prins reaction20 between 3-pentanone (40) and 3-
buten-1-ol (41) in 75% sulfuric acid afforded a carbinol (42),
which was further oxidized to the ketone (35b) by Ley TPAP
oxidation.21
CHEMISTRY
To access the single enantiomers, enantiomers of nitrile
intermediates (38c and 38e) were isolated. Supercritical fluid
chromatography (SFC) chiral separation was employed to
separate two enantiomers. AD-3 column was used to separate
the enantiomers of nitrile (38c) and AD-H column to separate
the enantiomers of nitrile (38e). The faster eluting enantiomer
((S)-38c) was converted to (S)-6 (Scheme 4) following the
chemistry outlined in Scheme 2, and the slower eluting
enantiomer (R)-38c was converted to (R)-6. The more potent
μ opioid agonist was derived from (R)-38c, and its absolute
(R)-configuration was conferred based on an X-ray crystallog-
raphy analysis on analogue 43. Similarly, (R)-38e was
converted to (R)-30. The single crystal X-ray structures of 43
and (R)-30 confirmed the absolute (R)-configuration (Figure
1). The crystal data of 43 and (R)-30 are listed in Tables 5 and
6. CCDC-949885 and CCDC-949886 contains the supple-
mentary crystallographic data for 43 bis-TFA and (R)-30
fumarate, respectively. These data can be obtained free of
charge from the Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
■
Compounds disclosed in the paper were synthesized via two
different strategies. The initial approach (Scheme 1) permitted
a quick access to analogues of the original screening hits.
a
Scheme 1. Synthesis of Compound 4
a
Because the (S)-enantiomers were less potent as μ opioid
agonists, the SAR focus was largely on the (R)-enantiomers.
Compounds (R)-12 to (R)-30 were prepared from (R)-38e,
following the same chemistry outlined in Scheme 2.
Reagents and conditions: (i) 4-fluorophenyl bromide, n-BuLi, THF;
(ii) allyltrimethylsilane, BF3·Et2O, DCM; (iii) O3, DCM, then PPh3;
(iv) amine, DCM, then NaBH4, MeOH.
Compounds (R)-8 to (R)-10 were synthesized through an
aldehyde intermediate ((R)-44) (Scheme 5). DIBAL reduction
of (R)-38c gave rise to the aldehyde ((R)-44), which was
reacted with three different amines under reductive amination
conditions to afford (R)-8 to (R)-10.
Commercially available starting material cyclic ketone (31)
reacted with substituted phenyl lithium reagent, generated from
the corresponding phenylbromide and n-butyl lithium to afford
a tertiary alcohol (32). In turn, treatment of the tertiary alcohol
(32) with allyltrimethylsilane and trifluoroboron etherate led to
formation of allylated product (33), which was contaminated
with a byproduct (dehydration product of the tertiary alcohol).
The byproduct was difficult to remove from the product and it
was carried onto the next step. After ozonolysis, the
corresponding acetaldehyde (34) was isolated and character-
ized. Reductive amination of the aldehyde (34) with an amine
generated compound 4.
Because of the difficulty to remove the byproduct in the
allylation step (Scheme 1), a copper(I) mediated conjugated
Michael addition approach to install the quaternary carbon
center was evaluated (Scheme 2).19 Knoevenagel condensation
between a cyclic ketone (35) and methyl cyanoacetate provided
EXPERIMENTAL SECTION
■
General. All reagents and solvents were obtained from commercial
suppliers and used without further purification. Silica gel chromatog-
raphy was performed using prepacked silica gel cartridges (Biotage).
1H NMR spectra were obtained on a Bruker Avance 1 400 MHz
spectrometer with 5 mm NMR Tube 7 N51A for 400 MHz using the
residual solvent peak as the reference. All new final compounds were
purified to >95% purity at 220 and 254 nm as determined by HPLC.
The HPLC analysis was performed on an Agilent 1200 system, using
(1) Phenomenex Luna C-18 column (50 mm × 4.6 mm, 5 μm) at 40
°C with 1.0 mL/min flow rate using a gradient of 5−40% (0.05% TFA
in acetonitrile) in (0.05% TFA in water) over 1 min, followed by a
E
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a
Scheme 2. Synthetic Approach to MOR Ligands via a 1,4- Michael Addition
a
Reagents and conditions: (i) methyl cyanoacetate, NH4OAc, HOAc, PhH, reflux; (ii) ArMgBr or ArLi, CuI, Et2O, iced-water bath; (iii) KOH,
ethylene glycol, 120 °C; (iv) LAH, Et2O, 0 °C; (v) aldehyde, DCM, then NaBH4.
acetonitrile) in (0.05% TFA in water) over 5 min. Electrospray mass
(API-ES) measurements were obtained on an Agilent 1200 series
system with an Agilent 6120 quadrupole LC/MS detector, using the
following separation method: Phenominex Luna C-18 column (50 mm
× 4.6 mm, 5 μm) at 40 °C with 1.0 mL/min flow rate using a gradient
of 5−40% (0.05% TFA in acetonitrile) in (0.05% TFA in water) over 1
min, followed by a gradient of 40−100% (0.05% TFA in acetonitrile)
in (0.05% TFA in water) over 5 min.
2-[({2-[4-(4-Methylphenyl)oxan-4-yl]ethyl}amino)methyl]phenol
(2) (Method A). To a solution of 2-[4-(4-methylphenyl)oxan-4-
yl]ethan-1-amine (39a, 200.0 mg, 0.91 mmol) in anhydrous
dichloromethane (5.0 mL) and Na2SO4 (129.0 mg, 0.914 mmol) at
room temperature was added salicaldehyde (0.14 mL, 1.37 mmol).
The reaction was stirred for 4 h. The reaction mixture was filtered and
concentrated. The residue was dissolved in 5.0 mL of MeOH at 0 °C
and NaBH4 (41 mg, 1.10 mmol) added in one portion. The reaction
was stirred at 0 °C for 1 h. The solution was then quenched with H2O
Scheme 3. Representative Synthesis of Ketone via Prins
Reaction
a
a
Reagents and conditions: (i) 75% H2SO4, 27% yield; (ii) TPAP,
NMO, 4Å MS, 73% yield.
gradient of 40−100% (0.05% TFA in acetonitrile) in (0.05% TFA in
water) over 5 min; (2) Phenomenex Luna Phenyl-Hexyl column (100
mm × 2.0 mm, 5 μm) at 40 °C with 1.0 mL/min flow rate using a
gradient of 5−40% (0.05% TFA in acetonitrile) in (0.05% TFA in
water) over 1 min, followed by a gradient of 40−100% (0.05% TFA in
a
Scheme 4. Supercritical Fluid Chromatography Chiral Separation of Nitriles
a
Reagents and conditions: (i) supercritical fluid chromatography chiral separation; AD-3 column for 38c using 15% MeOH with 0.05% diethylamine
(DEA) and AD-H column for 38e using 50% MeOH with 0.05% DEA.
F
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Figure 1. X-ray Structure of Compounds 43 and (R)-30.
a
Table 5. Crystal Data of 43
Scheme 5. Synthesis of Benzylic Substituted Analogues
empirical formula
formula weight
crystal system
space group
C29H34F7NO5
609.57
monoclinic
P21
a, b, c (Å)
8.9509(2), 11.1348(3),
14.9160(3)
α, β, γ (deg)
volume (Å3)
temperature (K)
Z
90.00, 97.4010(10), 90.00
1474.24(6)
100(2)
2
a
Reagents and conditions: (i) DIBAL, toluene; (ii) amine, DCM, then
NaBH4, MeOH
goodness-of-fit on F2
1.071
final R indexes [3669 data; I > 2σ (I)] R1 = 0.0494, wR2 = 0.1300
final R indexes [all data]
R1 = 0.0529, wR2 = 0.1334
1
less oil (2, 200.0 mg, 67.0% yield). H NMR (400 MHz, CDCl3) δ
7.15 (m, 5H), 6.88 (dd, J = 7.4, 1.3, 1H), 6.79 (dd, J = 8.1, 1.0, 1H),
6.73 (td, J = 7.4, 1.1, 1H), 5.83 (s, 1H), 3.76 (m, 4H), 3.54 (ddd, J =
11.7, 9.4, 2.5, 2H), 2.37 (m, 5H), 2.13 (m, 2H), 1.82 (m, 4H). LC-MS
(API-ES) m/z = 326.2 (M + H).
Table 6. Crystal Data of (R)-30
empirical formula
formula weight
crystal system
space group
C26H34N2O6S
502.61
2-[({2-[4-(4-Fluorophenyl)-2,2-dimethyloxan-4-yl]ethyl}amino)-
methyl]phenol (4). H NMR (400 MHz, CDCl3) δ 9.22 (m, 2H),
1
monoclinic
P21
7.21 (dd, J = 5.0, 1.8, 3H), 7.08 (m, 4H), 6.92 (t, J = 8.6, 2H), 3.65
(dd, J = 6.7, 2.7, 2H), 3.59 (s, 2H), 2.55 (s, 1H), 1.93 (s, 5H), 1.65 (td,
J = 12.6, 4.6, 1H), 1.52 (t, J = 8.5, 2H), 1.09 (s, 3H), 0.55 (s, 3H). LC-
MS (API-ES) m/z = 342.2 (M + H).
Benzyl {2-[2,2-Diethyl-4-(4-fluorophenyl)oxan-4-yl]ethyl}amine
(5). Using a procedure described in method A, 2-[2,2-diethyl-4-(4-
fluorophenyl)oxan-4-yl]ethan-1-amine (39b, 30.0 mg, 0.11 mmol) was
converted to benzyl {2-[2,2-diethyl-4-(4-fluorophenyl)oxan-4-yl]-
a, b, c (Å)
6.2203(4), 17.3233(9),
11.9123(6)
α, β, γ (deg)
volume (Å3)
temperature (K)
Z
90.00, 92.951(5), 90.00
1281.92(12)
100.0
2
goodness-of-fit on F2
1.068
1
ethyl}amine as a TFA salt (white solid, 5, 15 mg, 45% yield). H
final R indexes [3491 data; I > 2σ (I)] R1 = 0.0543, wR2 = 0.1535
final R indexes [all data] R1 = 0.0669, wR2 = 0.1715
NMR (400 MHz, CDCl3) δ 7.26−7.14 (m, 3H), 7.13−7.02 (m, 4H),
6.91 (t, J = 8.6, 2H), 3.69−3.47 (m, 4H), 2.51 (td, J = 12.2, 4.7, 1H),
2.14−1.94 (m, 3H), 1.83 (td, J = 12.7, 4.3, 1H), 1.64 (td, J = 12.6, 4.7,
1H), 1.56−1.35 (m, 3H), 1.27 (tt, J = 27.2, 13.7, 1H), 0.95 (dq, J =
14.7, 7.4, 1H), 0.84−0.58 (m, 4H), 0.43 (t, J = 7.4, 3H). LC-MS (API-
ES) m/z = 370.1 (M + H).
(10.0 mL), extracted with dichloromethane (3 × 20.0 mL), washed
with brine (10.0 mL), dried over Na2SO4, and concentrated. The
residue was purified by flash column chromatography on a prepacked
silica gel column (0−10% MeOH in dichloromethane) to give 2-[({2-
[4-(4-methylphenyl)oxan-4-yl]ethyl}amino)methyl]phenol as a color-
Benzyl {2-[9-(4-Fluorophenyl)-6-oxaspiro[4.5]decan-9-yl]ethyl}-
amine (6). Using a procedure described in method A, 2-[9-(4-
G
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fluorophenyl)-6-oxaspiro[4.5]decan-9-yl]ethan-1-amine (39c, 50.0 mg,
0.18 mmol) was converted to benzyl {2-[9-(4-fluorophenyl)-6-
oxaspiro[4.5]decan-9-yl]ethyl}amine as a TFA salt (white solid, 6,
(m, 3H), 1.91−1.72 (m, 3H), 1.70−1.57 (m, 2H), 1.54 (d, J = 6.8,
3H), 1.51−1.37 (m, 4H), 1.30−1.13 (m, 1H), 0.78 (dt, J = 13.6, 8.8,
1H). LC-MS (API-ES) m/z = 382.3 (M + H).
1
66.0 mg, 68% yield). H NMR (400 MHz, CDCl3) δ 8.82 (d, J =
{2-[(9R)-9-(4-Fluorophenyl)-6-oxaspiro[4.5]decan-9-yl]ethyl}(2-
phenylpropan-2-yl)amine ((R)-10). Using a procedure described in
method B, 2-[(9R)-9-(4-fluorophenyl)-6-oxaspiro[4.5]decan-9-yl]-
acetaldehyde ((R)-44, 30.0 mg, 0.11 mmol) reacted with 2-
phenylpropan-2-amine (18.0 μL, 0.14 mmol) to provide {2-[(9R)-9-
(4-fluorophenyl)-6-oxaspiro[4.5]decan-9-yl]ethyl}(2-phenylpropan-2-
134.2, 2H), 7.31 (m, 3H), 7.16 (m, 4H), 7.00 (dd, J = 10.7, 6.5, 2H),
3.72 (m, 4H), 2.70 (s, 1H), 2.28 (s, 1H), 2.05 (m, 2H), 1.94 (td, J =
12.6, 4.6, 1H), 1.82 (m, 3H), 1.62 (m, 2H), 1.46 (m, 4H), 1.23 (m,
1H), 0.77 (dt, J = 13.6, 8.8, 1H). LC-MS (API-ES) m/z = 368.3 (M +
H).
1
yl)amine as a TFA salt ((R)-10, 31.4 mg, 57% yield). H NMR (400
Benzyl ({2-[(9R)-9-(4-Fluorophenyl)-6-oxaspiro[4.5]decan-9-yl]-
ethyl})amine ((R)-6). Using a procedure described in method A, 2-
[(9R)-9-(4-fluorophenyl)-6-oxaspiro[4.5]decan-9-yl]ethan-1-amine
((R)-39c, 100.0 mg, 0.36 mmol) was converted to benzyl ({2-[(9R)-9-
(4-fluorophenyl)-6-oxaspiro[4.5]decan-9-yl]ethyl})amine as a TFA
MHz, CDCl3) δ 7.37 (s, 5H), 7.28 (s, 1H), 7.17−6.99 (m, 3H), 6.93
(t, J = 8.6, 2H), 3.81−3.57 (m, 2H), 2.45 (d, J = 9.0, 1H), 2.04−1.72
(m, 7H), 1.66 (t, J = 10.7, 6H), 1.62−1.53 (m, 2H), 1.52−1.34 (m,
4H), 1.23 (s, 1H), 0.78 (d, J = 13.8, 1H). LC-MS (API-ES) m/z =
396.3 (M + H).
1
salt ((R)-6, 121.0 mg, 92% yield). H NMR (400 MHz, CDCl3) δ
7.24−7.17 (m, 2H), 7.16−7.09 (m, 3H), 7.01 (d, J = 7.8, 2H), 6.89 (d,
J = 8.0, 2H), 3.68 (ddd, J = 11.8, 5.0, 1.3, 1H), 3.62−3.49 (m, 3H),
2.32 (t, J = 7.3, 2H), 2.25 (s, 3H), 2.22−2.13 (m, 1H), 1.93 (dtd, J =
15.7, 7.7, 3.8, 1H), 1.81−1.66 (m, 2H), 1.65−1.56 (m, 1H), 1.37 (d, J
= 20.2, 1H), 1.20−1.05 (m, 2H), 1.01−1.02 (m, 2H), 0.86 (t, J = 12.7,
1H). LC-MS (API-ES) m/z = 368.3 (M + H).
Benzyl {2-[(9S)-9-(4-Fluorophenyl)-6-oxaspiro[4.5]decan-9-yl]-
ethyl}amine ((S)-6). Using a procedure described in method A, 2-
[(9S)-9-(4-fluorophenyl)-6-oxaspiro[4.5]decan-9-yl]ethan-1-amine
((S)-39c, 100.0 mg, 0.36 mmol) was converted to benzyl {2-[(9S)-9-
(4-fluorophenyl)-6-oxaspiro[4.5]decan-9-yl]ethyl}amine as a TFA salt
(white solid, (S)-6, 120.0 mg, 91% yield). 1H NMR (400 MHz,
CDCl3) δ 8.92 (d, J = 139.1, 2H), 7.31 (m, 3H), 7.16 (m, 4H), 7.00 (t,
J = 8.6, 2H), 3.73 (m, 4H), 2.67 (s, 1H), 2.26 (s, 1H), 2.02 (s, 2H),
1.94 (td, J = 12.6, 4.7, 1H), 1.85 (d, J = 13.9, 3H), 1.62 (s, 2H), 1.46
(dd, J = 7.8, 4.0, 4H), 1.24 (d, J = 12.7, 1H), 0.77 (dt, J = 13.6, 8.7,
1H). LC-MS (API-ES) m/z = 368.3 (M + H).
Benzyl ({2-[9-(Pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]ethyl})-
amine (11). Using a procedure described in method A, 2-[9-
(pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]ethan-1-amine (39e, 21.0
mg, 0.08 mmol) was converted to benzyl ({2-[9-(pyridin-2-yl)-6-
oxaspiro[4.5]decan-9-yl]ethyl})amine as a TFA salt (11, 3.0 mg, 11%
yield). 1H NMR (400 MHz, CDCl3) δ 8.49 (s, 1H), 8.03 (s, 1H), 7.53
(d, J = 8.0, 2H), 7.18 (m, 5H), 3.82 (s, 2H), 3.63 (s, 1H), 3.53 (dd, J =
23.8, 13.7, 1H), 2.84 (s, 1H), 2.38 (s, 1H), 2.27 (d, J = 7.4, 1H), 2.13
(d, J = 14.1, 3H), 1.84 (d, J = 14.2, 1H), 1.67 (m, 2H), 1.52 (d, J = 5.0,
1H), 1.32 (m, 4H), 1.01 (s, 1H), 0.61 (dt, J = 13.0, 8.9, 1H). LC-MS
(API-ES) m/z = 351.2 (M + H).
Benzyl ({2-[(9R)-9-(Pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]-
ethyl})amine ((R)-11). Using a procedure described in method A, 2-
[(9R)-9-(pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]ethan-1-amine ((R)-
39e, 225.0 mg, 0.86 mmol) was converted to benzyl ({2-[(9R)-9-
(pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]ethyl})amine as a TFA salt
((R)-11, 189.0 mg, 47.0% yield). 1H NMR (400 MHz, CDCl3) δ 8.61
(s, 1H), 8.18 (t, J = 7.7, 1H), 7.79−7.58 (m, 2H), 7.43−7.10 (m, 5H),
6.90 (d, J = 26.0, 4H), 3.88 (s, 2H), 3.72 (d, J = 12.7, 1H), 3.60 (t, J =
10.0, 1H), 2.90 (s, 1H), 2.39 (d, J = 34.6, 2H), 2.20 (t, J = 13.3, 3H),
1.92 (d, J = 14.8, 2H), 1.86−1.67 (m, 2H), 1.59 (d, J = 4.9, 1H), 1.53−
1.27 (m, 4H), 1.08 (s, 1H), 0.68 (dt, J = 13.2, 9.0, 1H). LC-MS (API-
ES) m/z = 351.3 (M + H).
Benzyl {2-[4-(4-Fluorophenyl)-1-oxaspiro[5.5]undecan-4-yl]-
ethyl}amine (7). Using a procedure described in method A, 2-[4-(4-
fluorophenyl)-1-oxaspiro[5.5]undecan-4-yl]ethan-1-amine (39d, 30.0
mg, 0.11 mmol) was converted to benzyl {2-[4-(4-fluorophenyl)-1-
oxaspiro[5.5]undecan-4-yl]ethyl}amine as a TFA salt (solid, 7, 34.1
1
mg, 65.0% yield). H NMR (400 MHz, CDCl3) δ 7.23 (m, 4H), 7.10
(dd, J = 4.6, 2.6, 4H), 6.92 (s, 2H), 3.64 (s, 5H), 2.63 (m, 1H), 2.07 (t,
J = 13.9, 3H), 1.74 (s, 2H), 1.48 (d, J = 8.3, 3H), 1.40 (d, J = 14.0,
2H), 1.29 (m, 3H), 1.06 (m, 4H), 0.57 (m, 1H). LC-MS (API-ES) m/
z = 382.3 (M + H).
Benzyl ({2-[(9S)-9-(Pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]ethyl})-
amine ((S)-11). Using a procedure described in method A, 2-[(9S)-9-
(pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]ethan-1-amine ((S)-39e, 20.0
mg, 0.076 mmol) was converted to benzyl ({2-[(9S)-9-(pyridin-2-yl)-
6-oxaspiro[4.5]decan-9-yl]ethyl})amine as TFA salt ((S)-11, 16.0 mg,
46.0% yield). 1H NMR (400 MHz, CDCl3) δ 8.61 (s, 1H), 8.18 (t, J =
7.7, 1H), 7.65 (m, 2H), 7.26 (m, 5H), 6.90 (d, J = 26.0, 4H), 3.88 (s,
2H), 3.72 (d, J = 12.7, 1H), 3.60 (t, J = 10.0, 1H), 2.90 (s, 1H), 2.39
(d, J = 34.6, 2H), 2.20 (t, J = 13.3, 3H), 1.92 (d, J = 14.8, 2H), 1.75
(m, 2H), 1.59 (d, J = 4.9, 1H), 1.41 (m, 4H), 1.08 (s, 1H), 0.68 (dt, J
= 13.2, 9.0, 1H). LC-MS (API-ES) m/z = 351.2 (M + H).
{2-[(9R)-9-(4-Fluorophenyl)-6-oxaspiro[4.5]decan-9-yl]ethyl}[(1S)-
1-phenylethyl]amine ((R)-8) (Method B). Into a vial were placed 2-
[(9R)-9-(4-fluorophenyl)-6-oxaspiro[4.5]decan-9-yl]acetaldehyde
((R)-44, 30.0 mg, 0.11 mmol), dichloromethane (3.0 mL), and
sodium sulfate (78.0 mg, 0.55 mmol). (1S)-1-Phenylethan-1-amine
(18.0 μL, 0.14 mmol) was added, and the reaction mixture was stirred
overnight. NaBH4 (5.3 mg, 0.14 mmol) was added to the mixture,
stirred for 10 min, and then was added 1.0 mL of MeOH. After stirring
for 1h, it was quenched with water and the organic layer was separated
and concentrated. The residue was purified by reverse-phase HPLC to
give {2-[(9R)-9-(4-fluorophenyl)-6-oxaspiro[4.5]decan-9-yl]ethyl}-
[(1S)-1-phenylethyl]amine as a TFA salt ((R)-8, 33.7 mg, 62%
[(2-Chlorophenyl)methyl]({2-[(9R)-9-(pyridin-2-yl)-6-oxaspiro-
[4.5]decan-9-yl]ethyl})amine ((R)-12). Using a procedure described in
1
method A, (R)-39e was converted to (R)-12 as a TFA salt. H NMR
(400 MHz, CD3CN) δ 8.75 (dd, J = 5.4, 1.2, 1H), 8.52 (s, 3H), 8.22
(td, J = 8.0, 1.7, 1H), 7.77 (d, J = 8.2, 1H), 7.67 (ddd, J = 7.5, 5.4, 0.9,
1H), 7.42 (m, 4H), 4.20 (d, J = 14.0, 2H), 3.72 (m, 2H), 3.05 (td, J =
12.0, 5.1, 1H), 2.53 (td, J = 12.0, 4.4, 1H), 2.36 (m, 3H), 2.17 (m,
1H), 2.01 (d, J = 14.2, 1H), 1.79 (ddd, J = 9.3, 6.7, 3.4, 2H), 1.52 (m,
5H), 1.17 (m, 1H), 0.78 (dt, J = 12.9, 8.8, 1H). LC-MS (API-ES) m/z
= 385.0 (M + H).
1
yield). H NMR (400 MHz, CDCl3) δ 9.21 (s, 1H), 8.44 (s, 1H),
7.35−7.04 (m, 7H), 7.03−6.88 (m, 2H), 6.91−6.76 (m, 2H), 3.95 (s,
1H), 3.70−3.45 (m, 2H), 2.47 (s, 1H), 1.93 (t, J = 14.2, 3H), 1.85−
1.62 (m, 4H), 1.59−1.44 (m, 2H), 1.42 (t, J = 5.8, 4H), 1.38−1.23 (m,
3H), 1.22−1.04 (m, 1H), 0.66 (dt, J = 13.5, 8.8, 1H). LC-MS (API-
ES) m/z = 382.3 (M + H).
{2-[(9R)-9-(4-Fluorophenyl)-6-oxaspiro[4.5]decan-9-yl]ethyl}-
[(1R)-1-phenylethyl]amine ((R)-9). Using a procedure described in
method B, 2-[(9R)-9-(4-fluorophenyl)-6-oxaspiro[4.5]decan-9-yl]-
acetaldehyde ((R)-44, 30.0 mg, 0.11 mmol) reacted with (1R)-1-
phenylethan-1-amine (18.0 μL, 0.14 mmol) to provide {2-[(9R)-9-(4-
fluorophenyl)-6-oxaspiro[4.5]decan-9-yl]ethyl}[(1R)-1-phenylethyl]-
amine as a TFA salt ((R)-9, 36.8 mg, 66% yield). 1H NMR (400 MHz,
CDCl3) δ 9.15 (s, 1H), 8.83 (s, 1H), 7.47−7.30 (m, 3H), 7.32−7.19
(m, 2H), 7.11 (dd, J = 8.9, 5.2, 2H), 6.98 (t, J = 8.6, 2H), 4.03 (s, 1H),
3.77−3.47 (m, 2H), 2.51 (s, 1H), 2.19 (d, J = 14.5, 1H), 2.07−1.91
[(3-Chlorophenyl)methyl]({2-[(9R)-9-(pyridin-2-yl)-6-oxaspiro-
[4.5]decan-9-yl]ethyl})amine ((R)-13). Using a procedure described in
1
method A, (R)-39e was converted to (R)-13 as a TFA salt. H NMR
(400 MHz, CD3CN) δ 8.72 (dd, J = 5.4, 1.1, 1H), 8.57 (s, 1H), 8.19
(td, J = 8.0, 1.8, 1H), 7.74 (d, J = 8.2, 1H), 7.64 (ddd, J = 7.6, 5.4, 0.9,
1H), 7.37 (m, 5H), 3.99 (d, J = 2.3, 2H), 3.70 (m, 2H), 2.95 (m, 1H),
2.36 (m, 4H), 2.12 (td, J = 12.9, 5.1, 1H), 1.76 (ddd, J = 14.2, 9.3, 5.1,
2H), 1.50 (m, 5H), 0.77 (dt, J = 13.0, 8.9, 1H). LC-MS (API-ES) m/z
= 385.0 (M + H).
{2-[(9R)-9-(Pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]ethyl}(pyridin-
2-ylmethyl)amine ((R)-14). Using a procedure described in method A,
H
dx.doi.org/10.1021/jm4010829 | J. Med. Chem. XXXX, XXX, XXX−XXX

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(R)-39e was converted to (R)-14 as a TFA salt. 1H NMR (400 MHz,
CD3CN) δ 10.49 (s, 1H), 8.81 (dd, J = 5.5, 1.2, 1H), 8.55 (dd, J = 3.7,
0.8, 1H), 8.30 (td, J = 8.0, 1.7, 1H), 7.91 (td, J = 7.8, 1.7, 1H), 7.83 (d,
J = 8.2, 1H), 7.75 (ddd, J = 7.6, 5.5, 1.0, 1H), 7.45 (dd, J = 11.3, 6.5,
2H), 4.24 (m, 2H), 3.73 (m, 2H), 3.06 (td, J = 12.0, 5.2, 1H), 2.57 (td,
J = 12.1, 4.4, 1H), 2.39 (m, 3H), 2.24 (m, 1H), 2.04 (d, J = 14.0, 1H),
1.82 (m, 2H), 1.63 (m, 1H), 1.50 (m, 4H), 1.19 (m, 1H), 0.81 (dt, J =
12.9, 8.8, 1H). LC-MS (API-ES) m/z = 352.3 (M + H).
{2-[(9R)-9-(Pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]ethyl}(pyridin-
3-ylmethyl)amine ((R)-15). Using a procedure described in method A,
(R)-39e was converted to (R)-15 as a TFA salt. 1H NMR (400 MHz,
CD3CN) δ 8.81 (s, 1H), 8.74 (m, 2H), 8.32 (d, J = 8.1, 1H), 8.26 (td,
J = 8.0, 1.7, 1H), 7.80 (m, 2H), 7.70 (m, 1H), 4.18 (m, 2H), 3.73 (m,
2H), 3.02 (td, J = 12.0, 5.1, 1H), 2.51 (td, J = 12.1, 4.3, 1H), 2.36 (m,
3H), 2.15 (m, 1H), 2.01 (d, J = 14.1, 1H), 1.80 (ddd, J = 9.8, 8.2, 4.7,
2H), 1.62 (m, 1H), 1.48 (m, 4H), 1.19 (m, 1H), 0.80 (dt, J = 13.0, 8.8,
1H). LC-MS (API-ES) m/z = 352.3 (M + H).
{2-[(9R)-9-(Pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]ethyl}(pyridin-
4-ylmethyl)amine ((R)-16). Using a procedure described in method A,
(R)-39e was converted to (R)-16 as a TFA salt. 1H NMR (400 MHz,
CD3CN) δ 8.73 (m, 3H), 8.20 (td, J = 8.0, 1.7, 1H), 7.82 (d, J = 6.5,
2H), 7.76 (d, J = 8.2, 1H), 7.65 (m, 1H), 4.22 (m, 2H), 3.73 (m, 2H),
3.03 (td, J = 12.0, 5.1, 1H), 2.53 (td, J = 12.1, 4.4, 1H), 2.37 (m, 3H),
2.16 (m, 1H), 2.00 (d, J = 14.2, 1H), 1.79 (m, 2H), 1.63 (ddd, J =
12.2, 8.8, 4.0, 1H), 1.49 (m, 4H), 1.19 (m, 1H), 0.80 (dt, J = 13.1, 8.9,
1H). LC-MS (API-ES) m/z = 352.3 (M + H).
(m, 2H), 7.54 (d, J = 8.1, 1H), 7.48 (dd, J = 8.5, 1.7, 1H), 7.34 (m,
1H), 4.19 (s, 2H), 3.69 (dt, J = 8.9, 5.1, 3H), 3.48 (brs, 1H), 3.02 (s,
1H), 2.52 (s, 1H), 2.33 (m, 2H), 2.19 (m, 1H), 2.02 (m, 1H), 1.89 (t,
J = 9.4, 1H), 1.70 (dq, J = 9.2, 5.1, 2H), 1.59 (m, 1H), 1.44 (m, 4H),
1.10 (m, 1H), 0.69 (dt, J = 13.1, 8.8, 1H). LC-MS (API-ES) m/z =
401.3 (M + H).
(Cyclopentylmethyl)({2-[(9R)-9-(pyridin-2-yl)-6-oxaspiro[4.5]-
decan-9-yl]ethyl})amine ((R)-22). Using a procedure described in
1
method A, (R)-39e was converted to (R)-22 as a TFA salt. H NMR
(400 MHz, CDCl3) δ 8.77 (d, J = 4.6, 2H), 8.26 (t, J = 7.6, 1H), 7.89−
7.60 (m, 2H), 3.85 (dd, J = 8.5, 4.2, 1H), 3.73 (t, J = 10.1, 1H), 3.00
(s, 1H), 2.81 (s, 2H), 2.42 (dt, J = 23.0, 9.5, 4H), 2.25 (t, J = 10.8,
1H), 2.19−1.98 (m, 2H), 1.98−1.33 (m, 13H), 1.16 (s, 3H), 0.76 (dt,
J = 13.1, 8.9, 1H). LC-MS (API-ES) m/z = 343.3 (M + H).
[(3-Methylthiophen-2-yl)methyl]({2-[(9R)-9-(pyridin-2-yl)-6-
oxaspiro[4.5]decan-9-yl]ethyl}) amine ((R)-23). Using a procedure
described in method A, (R)-39e was converted to (R)-23 as a TFA
1
salt. H NMR (400 MHz, CDCl3) δ 9.68 (s, 1H), 8.75 (s, 1H), 8.16
(m, 1H), 7.74 (d, J = 27.0, 2H), 7.27 (d, J = 1.5, 1H), 6.85 (d, J = 5.1,
1H), 4.10 (m, 2H), 3.84 (d, J = 12.7, 1H), 3.66 (d, J = 10.3, 1H), 2.96
(m, 1H), 2.69 (m, 1H), 2.54 (m, 3H), 2.35 (m, 4H), 2.11 (d, J = 14.0,
1H), 1.87 (d, J = 10.3, 3H), 1.57 (m, 5H), 1.06 (m 1H), 0.78 (d, J =
12.8, 1H). LC-MS (API-ES) m/z = 371.1 (M + H).
[(4-Methylthiophen-2-yl)methyl]({2-[(9R)-9-(pyridin-2-yl)-6-
oxaspiro[4.5]decan-9-yl]ethyl}) amine ((R)-24). Using a procedure
described in method A, (R)-39e was converted to (R)-24 as a TFA
salt. 1H NMR (400 MHz, CDCl3) δ 9.63 (s, 1H), 8.61 (d, J = 4.1, 1H),
8.08 (t, J = 7.8, 1H), 7.61 (d, J = 8.1, 1H), 7.53 (dd, J = 7.0, 5.6, 1H),
6.91 (s, 1H), 6.88 (s, 1H), 4.14 (m, 2H), 3.75 (dt, J = 19.0, 11.1, 2H),
3.02 (m, 1H), 2.61 (m, 1H), 2.40 (brs, 1H), 2.27 (m, 4H), 2.19 (d, J =
0.8, 3H), 1.95 (d, J = 14.0, 1H), 1.79 (m, 2H), 1.66 (dd, J = 12.1, 5.9,
1H), 1.47 (m, 4H), 1.16 (m, 1H), 0.74 (dt, J = 13.1, 8.9, 1H). LC-MS
(API-ES) m/z = 371.3 (M + H).
[(5-Methylthiophen-2-yl)methyl]({2-[(9R)-9-(pyridin-2-yl)-6-
oxaspiro[4.5]decan-9-yl]ethyl}) amine ((R)-25). Using a procedure
described in method A, (R)-39e was converted to (R)-25 as a TFA
salt. 1H NMR (400 MHz, CDCl3) δ 8.71 (d, J = 4.7, 1H), 8.14 (t, J =
7.6, 1H), 7.78−7.48 (m, 2H), 6.86 (d, J = 3.4, 1H), 6.78−6.53 (m,
1H), 4.09 (s, 2H), 3.76 (ddd, J = 40.6, 14.3, 7.2, 2H), 3.17−2.85 (m,
1H), 2.64−2.23 (m, 6H), 2.16 (dd, J = 16.4, 8.6, 1H), 1.99 (d, J = 14.2,
1H), 1.89−1.75 (m, 2H), 1.75−1.61 (m, 1H), 1.61−1.35 (m, 4H),
1.24−1.05 (m, 1H), 0.74 (dt, J = 13.2, 8.9, 1H). LC-MS (API-ES) m/z
= 371.2 (M + H).
(Pyrazin-2-ylmethyl)({2-[(9R)-9-(pyridin-2-yl)-6-oxaspiro[4.5]-
decan-9-yl]ethyl})amine ((R)-17). Using a procedure described in
1
method A, (R)-39e was converted to (R)-17 as a TFA salt. H NMR
(400 MHz, CDCl3) δ 8.79 (dd, J = 5.6, 1.4, 1H), 8.68−8.54 (m, 2H),
8.51 (dd, J = 2.3, 1.6, 1H), 8.32 (td, J = 8.0, 1.6, 1H), 7.93−7.66 (m,
3H), 4.30 (s, 2H), 3.85 (dt, J = 12.3, 4.2, 1H), 3.72 (t, J = 9.9, 1H),
3.19 (td, J = 11.7, 5.2, 1H), 2.72 (td, J = 11.8, 4.0, 1H), 2.62−2.45 (m,
1H), 2.45−2.27 (m, 3H), 2.10 (d, J = 14.2, 1H), 2.00−1.79 (m, 2H),
1.69 (dt, J = 9.9, 6.6, 1H), 1.63−1.41 (m, 4H), 1.19 (dd, J = 12.6, 6.5,
1H), 0.78 (dt, J = 13.1, 8.9, 1H). LC-MS (API-ES) m/z = 353.1 (M +
H).
{2-[(9R)-9-(Pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]ethyl}-
(pyrimidin-4-ylmethyl)amine ((R)-18). Using a procedure described
1
in method A, (R)-39e was converted to (R)-18 as a TFA salt. H
NMR (400 MHz, CD3CN) δ 9.16 (s, 1H), 8.78 (s, 2H), 8.70 (dd, J =
5.3, 1.1, 1H), 8.16 (td, J = 8.0, 1.8, 1H), 7.74 (d, J = 8.2, 1H), 7.62
(ddd, J = 7.6, 5.4, 0.9, 1H), 4.27 (brs, 1H), 4.04 (t, J = 7.7, 2H), 3.73
(m, 2H), 3.01 (td, J = 12.0, 5.1, 1H), 2.50 (td, J = 12.0, 4.4, 1H), 2.33
(m, 3H), 2.12 (ddd, J = 19.0, 11.7, 5.2, 1H), 1.99 (d, J = 10.1, 1H),
1.78 (m, 2H), 1.61 (m, 1H), 1.48 (m, 4H), 1.17 (m, 1H), 0.78 (dt, J =
13.1, 8.9, 1H). LC-MS (API-ES) m/z = 353.2 (M + H).
[(5-Methylfuran-2-yl)methyl]({2-[(9R)-9-(pyridin-2-yl)-6-oxaspiro-
[4.5]decan-9-yl]ethyl}) amine ((R)-26). Using a procedure described
1
in method A, (R)-39e was converted to (R)-26 as a TFA salt. H
NMR (400 MHz, CDCl3) δ 8.73 (d, J = 4.2, 1H), 8.18 (td, J = 8.0, 1.5,
1H), 7.80−7.53 (m, 2H), 6.28 (s, 1H), 5.92 (s, 1H), 4.00 (d, J = 1.4,
2H), 3.83 (dt, J = 12.4, 4.3, 1H), 3.79−3.63 (m, 1H), 3.10−2.86 (m,
1H), 2.64−2.44 (m, 1H), 2.45−2.27 (m, 3H), 2.27−2.11 (m, 4H),
2.02 (d, J = 14.2, 1H), 1.95−1.77 (m, 2H), 1.68 (dd, J = 9.5, 4.1, 1H),
1.62−1.39 (m, 4H), 1.26−1.05 (m, 1H), 0.77 (dt, J = 13.3, 9.0, 1H).
LC-MS (API-ES) m/z = 355.1 (M + H).
{2-[(9R)-9-(Pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]ethyl}-
(thiophen-2-ylmethyl)amine ((R)-19). Using a procedure described in
1
method A, (R)-39e was converted to (R)-19 as a TFA salt. H NMR
(400 MHz, CDCl3) δ 8.67 (d, J = 4.3, 1H), 8.14 (s, 1H), 7.66 (d, J =
8.2, 1H), 7.59 (s, 1H), 7.33 (dd, J = 5.1, 1.1, 1H), 7.12 (d, J = 2.7, 1H),
7.00 (dd, J = 5.1, 3.5, 1H), 4.22 (s, 2H), 3.80 (s, 1H), 3.72 (t, J = 9.8,
1H), 3.33−2.70 (m, 1H), 2.70−2.50 (m, 1H), 2.30 (d, J = 14.0, 3H),
2.19 (dd, J = 18.0, 7.1, 1H), 1.98 (d, J = 14.1, 1H), 1.83 (d, J = 4.6,
2H), 1.76−1.62 (m, 1H), 1.50 (dd, J = 20.1, 13.3, 5H), 1.16 (s, 1H),
0.75 (dt, J = 13.1, 9.1, 1H). LC-MS (API-ES) m/z = 357.2 (M + H).
{2-[(9R)-9-(Pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]ethyl}-
(thiophen-3-ylmethyl)amine ((R)-20). Using a procedure described in
[(3,5-Dimethylthiophen-2-yl)methyl]({2-[(9R)-9-(pyridin-2-yl)-6-
oxaspiro[4.5]decan-9-yl] ethyl})amine ((R)-27). Using a procedure
described in method A, (R)-39e was converted to (R)-27 as a TFA
1
salt. H NMR (400 MHz, CDCl3) δ 9.45 (brs, 1H), 8.70 (d, J = 5.0,
1H), 8.26 (t, J = 7.7, 1H), 7.75 (d, J = 8.1, 1H), 7.70 (m, 1H), 6.46 (d,
J = 0.8, 1H), 4.07 (s, 2H), 3.76 (ddd, J = 44.9, 13.9, 7.2, 2H), 3.05 (m,
1H), 2.58 (m, 1H), 2.43 (t, J = 10.6, 1H), 2.36 (d, J = 0.7, 3H), 2.24
(dd, J = 31.9, 17.7, 3H), 2.03 (m, 4H), 1.85 (m, 2H), 1.66 (dd, J =
13.8, 8.8, 1H), 1.48 (m, 4H), 1.15 (d, J = 7.9, 1H), 0.75 (dt, J = 13.1,
8.9, 1H). LC-MS (API-ES) m/z = 385.1 (M + H).
[(4,5-Dimethylthiophen-2-yl)methyl]({2-[(9R)-9-(pyridin-2-yl)-6-
oxaspiro[4.5]decan-9-yl] ethyl})amine ((R)-28). Using a procedure
described in method A, (R)-39e was converted to (R)-28 as a TFA
salt. 1H NMR (400 MHz, CDCl3) δ 9.46 (s, 1H), 8.62 (d, J = 4.2, 1H),
8.07 (t, J = 7.3, 1H), 7.60 (d, J = 8.1, 1H), 7.52 (m, 1H), 6.76 (s, 1H),
4.06 (q, J = 13.9, 2H), 3.75 (m, 2H), 3.01 (m, 1H), 2.57 (s, 1H), 2.29
(m, 7H), 2.19 (m, 1H), 2.04 (s, 3H), 1.95 (d, J = 14.0, 1H), 1.81 (m,
1
method A, (R)-39e was converted to (R)-20 as a TFA salt. H NMR
(400 MHz, CDCl3) δ 8.73 (d, J = 5.0, 1H), 8.27 (t, J = 7.5, 2H), 7.88−
7.62 (m, 2H), 7.48−7.23 (m, 1H), 7.04 (dd, J = 4.9, 1.0, 1H), 4.02 (s,
2H), 3.90−3.76 (m, 1H), 3.69 (t, J = 10.0, 1H), 2.95 (s, 1H), 2.62−
2.12 (m, 4H), 2.13−1.95 (m, 1H), 1.95−1.76 (m, 2H), 1.68 (dt, J =
13.5, 7.9, 1H), 1.62−1.30 (m, 5H), 1.16 (dd, J = 13.2, 6.6, 1H), 0.76
(dt, J = 13.0, 8.9, 1H). LC-MS (API-ES) m/z = 357.2 (M + H).
(Naphthalen-2-ylmethyl)({2-[(9R)-9-(pyridin-2-yl)-6-oxaspiro-
[4.5]decan-9-yl]ethyl})amine ((R)-21). Using a procedure described in
1
method A, (R)-39e was converted to (R)-21 as a TFA salt. H NMR
(400 MHz, CD3CN) δ 8.57 (dd, J = 5.0, 1.0, 1H), 7.90 (m, 5H), 7.59
I
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2H), 1.67 (d, J = 8.2, 1H), 1.47 (m, 4H), 1.15 (m, 1H), 0.74 (dt, J =
13.1, 8.8, 1H). LC-MS (API-ES) m/z = 385.3 (M + H).
oxaspiro[4.5]decan-9-one (35d, 3.8 g, 22.6 mmol) was converted to
methyl 2-cyano-2-[(4Z)-1-oxaspiro[5.5]undecan-4-ylidene]acetate as a
colorless oil (36d, 4.93 g, 87.7% yield). H NMR (400 MHz, CDCl3)
1
[(4,5-Dimethylfuran-2-yl)methyl]({2-[(9R)-9-(pyridin-2-yl)-6-
oxaspiro[4.5]decan-9-yl] ethyl})amine ((R)-29). Using a procedure
described in method A, (R)-39e was converted to (R)-29 as a TFA
δ 3.88 (t, J = 5.7, 1H), 3.83 (s, 3H), 3.80 (d, J = 5.7, 1H), 3.13 (t, J =
5.7, 1H), 3.04 (s, 1H), 2.74 (t, J = 5.7, 1H), 2.64 (s, 1H), 1.70 (m,
2H), 1.50 (m, 8H). LC-MS (API-ES) m/z = 250.1 (M + H).
Methyl 2-Cyano-2-[4-(4-methylphenyl)oxan-4-yl]acetate (37a)
(Method D). Into a 100 mL round-bottom flask was charged a 4-
methylphenyl magnesium bromide (1.0 M in THF, 6.63 mL, 6.63
mmol) in dry diethyl ether (15.0 mL). CuI (105.0 mg, 0.55 mmol)
was added, followed by a dropwise addition of methyl 2-cyano-2-
(oxan-4-ylidene)acetate (36a, 1.0 g, 5.52 mmol) in diethyl ether (15.0
mL) over 30 min while cooling the reaction flask in an iced water bath.
The mixture was then stirred at room temperature until reaction
completion. The reaction mixture was poured into a mixture of 50.0 g
of ice and 40.0 mL of 1.0 N HCl. The product was extracted with Et2O
(3 × 50.0 mL), washed with brine (50.0 mL), dried over Na2SO4, and
concentrated. The residue was purified by normal phase flash column
chromatography on a prepacked BioTage silica gel column (7 to 60%
EtOAc in hexanes) to give methyl 2-cyano-2-[4-(4-methylphenyl)-
oxan-4-yl]acetate as a white solid (37a, 1.09 g, 71.0% yield). 1H NMR
(400 MHz, CDCl3) δ 7.21 (s, 4H), 3.83 (dt, J = 11.9, 3.7, 2H), 3.63 (s,
1H), 3.51 (d, J = 8.1, 3H), 3.47 (m, 2H), 2.50 (m, 2H), 2.35 (s, 3H),
2.16 (m, 2H). LC-MS (API-ES) m/z = 274.1 (M + H).
1
salt. H NMR (400 MHz, CDCl3) δ 10.28 (brs, 1H), 9.39 (brs, 1H),
8.70 (d, J = 4.6, 1H), 8.12 (t, J = 7.5, 1H), 7.65 (d, J = 8.1, 1H), 7.58
(m, 1H), 6.14 (s, 1H), 3.91 (q, J = 14.4, 2H), 3.75 (m, 2H), 2.95 (dd, J
= 10.9, 5.9, 1H), 2.51 (t, J = 9.7, 1H), 2.33 (m, 3H), 2.10 (s, 3H), 1.99
(d, J = 14.1, 1H), 1.82 (m, 5H), 1.68 (m, 1H), 1.48 (m, 4H), 1.15 (m,
1H), 0.74 (dt, J = 13.2, 8.9, 1H). LC-MS (API-ES) m/z = 369.1 (M +
H).
[(3-Methoxythiophen-2-yl)methyl]({2-[(9R)-9-(pyridin-2-yl)-6-
oxaspiro[4.5]decan-9-yl] ethyl})amine ((R)-30). Using a procedure
described in method A, (R)-39e was converted to (R)-30 as a TFA
salt. 1H NMR (400 MHz, CDCl3) δ 11.70 (brs, 1H), 9.14 (d, J = 66.6,
2H), 8.72 (d, J = 4.3, 1H), 8.19 (td, J = 8.0, 1.4, 1H), 7.70 (d, J = 8.1,
1H), 7.63 (dd, J = 7.0, 5.8, 1H), 7.22 (d, J = 5.5, 1H), 6.78 (d, J = 5.6,
1H), 4.08 (m, 2H), 3.80 (m, 4H), 3.69 (dd, J = 11.2, 8.7, 1H), 2.99 (d,
J = 4.8, 1H), 2.51 (t, J = 9.9, 1H), 2.35 (m, 3H), 2.18 (td, J = 13.5, 5.4,
1H), 1.99 (d, J = 14.2, 1H), 1.82 (m, 2H), 1.65 (m, 1H), 1.47 (m,
4H), 1.14 (m, 1H), 0.73 (dt, J = 13.2, 8.9, 1H). LC-MS (API-ES) m/z
= 387.0 (M + H).
2,2-Diethyloxan-4-one (35b). To a solution of 2,2-diethyloxan-4-ol
(42, 500.0 mg, 3.2 mmol) in dichloromethane (10.0 mL) were added
N-methylmorpholine N-oxide (NMO, 750.0 mg, 6.41 mmol) and 4 Å
MS (molecular sieves, 2.0 g). The solution was stirred for 30 min, and
then TPAP (34.0 mg, 0.096 mmol) was added in one portion. The
reaction was allowed to stir for 10 h. After TLC indicated completion
of the reaction, it was filtered through a short pad of SiO2. The filtrate
was concentrated and purified by flash column chromatography on
prepacked Biotage silica gel column (0−50% EtOAc/hexanes) to give
2,2-diethyloxan-4-one (35b, 365.0 mg, 73% yield). 1H NMR (400
MHz, CDCl3) δ 3.76 (m, 2H), 2.6 (m, 2H) 2.49 (m, 2H), 1.47 (t, J =
6.7, 4H), 0.77 (t, J = 6.7, 6H).
Methyl 2-Cyano-2-[2,2-diethyl-4-(4-fluorophenyl)oxan-4-yl]-
acetate (37b). Using a procedure described in method D, methyl 2-
cyano-2-[(4Z)-2,2-diethyloxan-4-ylidene]acetate (36b, 1.0 g, 4.2
mmol) was converted to methyl 2-cyano-2-[2,2-diethyl-4-(4-
fluorophenyl)oxan-4-yl]acetate as a white solid (37b, 1.18 g, 84%
yield). LC-MS (API-ES) m/z = 334.2 (M + H).
Methyl 2-Cyano-2-[9-(4-fluorophenyl)-6-oxaspiro[4.5]decan-9-
yl]acetate (37c). Using a procedure described in method D, methyl
2-cyano-2-[(9E/Z)-6-oxaspiro[4.5]decan-9-ylidene]acetate (36c, 2.5 g,
10.5 mmol) was converted to methyl 2-cyano-2-[9-(4-fluorophenyl)-6-
oxaspiro[4.5]decan-9-yl]acetate as a colorless oil (37c, 3.24 g, 93%
1
yield). H NMR (400 MHz, CDCl3) δ 7.39 (m, 1H), 7.31 (m, 1H),
Methyl 2-cyano-2-(oxan-4-ylidene)acetate (36a) (Method C). A
50 mL round-bottom flask equipped with a Dean−Stark distillation
setup and condenser was charged with tetrahydro-4H-pyran-4-one
(35a, 2.3 mL, 25.0 mmol), methyl cyanoacetate (2.63 mL, 30.0
mmol), ammonium acetate (0.5 g, 6.5 mmol), acetic acid (0.285 mL,
5.0 mmol), and benzene (15.0 mL). The mixture was refluxed until no
more water was collected in the Dean−Stark (2 h) and cooled. Then
more benzene (15.0 mL) was added and the organic washed with
water (25.0 mL). The aqueous layer was extracted with dichloro-
methane (3 × 50.0 mL). The combined organic phase was washed
with saturated NaHCO3 (50.0 mL) and brine (50.0 mL) and dried
over MgSO4, filtered, and concentrated. The residue was purified by
flash column chromatography on prepacked silica gel column (10−
60% EtOAc in hexanes) to give methyl 2-cyano-2-(oxan-4-ylidene)
7.07 (td, J = 8.7, 3.1, 2H), 3.78 (dq, J = 12.5, 4.1, 1H), 3.66 (m, 2H),
3.49 (d, J = 1.2, 3H), 2.59 (m, 1H), 2.48 (dd, J = 14.0, 2.3, 1H), 2.35
(s, 1H), 2.11 (m, 2H), 2.0 (m, 1H), 1.80 (t, J = 10.4, 1H), 1.57 (m,
4H), 1.26 (t, J = 7.1, 1H), 1.20 (dd, J = 11.1, 4.6, 1H), 0.86 (m, 1H).
LC-MS (API-ES) m/z = 332.1 (M + H).
Methyl 2-Cyano-2-[4-(4-fluorophenyl)-1-oxaspiro[5.5]undecan-
4-yl]acetate (37d). Using a procedure described in method D, methyl
2-cyano-2-[(4Z)-1-oxaspiro[5.5]undecan-4-ylidene]acetate (36d, 1.0
g, 4.0 mmol) was converted to methyl 2-cyano-2-[4-(4-fluorophenyl)-
1-oxaspiro[5.5]undecan-4-yl]acetate as a yellow oil (37d, 1.4 g,
1
quantitative yield). H NMR (400 MHz, CDCl3) δ 7.43 (dd, J = 9.0,
5.1, 1H), 7.34 (dd, J = 9.0, 5.1, 1H), 7.14−7.01 (m, 2H), 3.91−3.74
(m, 1H), 3.65 (tdd, J = 12.4, 7.0, 1.8, 1H), 3.55 (d, J = 3.1, 1H), 3.50
(d, J = 2.4, 3H), 2.78−2.64 (m, 1H), 2.64−2.43 (m, 1H), 2.14−2.09
(m, 1H), 1.99 (ddd, J = 14.5, 11.5, 4.4, 1H), 1.95−1.89 (m, 1H), 1.75
(d, J = 14.1, 1H), 1.61 (d, J = 12.3, 2H), 1.53−1.37 (m, 3H), 1.18 (dd,
J = 17.7, 6.4, 3H), 0.81−0.56 (m, 1H). LC-MS (API-ES) m/z = 346.2
(M + H).
1
acetate as a colorless oil (36a, 4.0 g, 89.0% yield). H NMR (400
MHz, CDCl3) δ 3.86 (dd, J = 7.0, 4.1, 2H), 3.83 (s, 3H), 3.78 (t, J =
5.6, 2H), 3.17 (t, J = 5.6, 2H), 2.78 (t, J = 5.5, 2H). LC-MS (API-ES)
m/z = 182.1 (M + H).
Methyl 2-cyano-2-[(4Z)-2,2-diethyloxan-4-ylidene]acetate (36b).
Using a procedure described in method C, 2,2-diethyloxan-4-one
(35b, 4.1 g, 26.2 mmol) was converted to methyl 2-cyano-2-[(4Z)-2,2-
diethyloxan-4-ylidene]acetate as a colorless oil (36b, 4.01 g, 61.7%
yield). 1H NMR (400 MHz, CDCl3) 1H NMR (400 MHz, CDCl3) δ,
3.89 (t, J = 6.1, 1H), 3.82−3.49 (m, 3H), 3.14−2.84 (m, 1H), 2.64
(dd, J = 21.3, 15.7, 1H), 2.43−2.08 (m, 2H), 1.79−1.27 (m, 4H), 0.79
(qd, J = 7.8, 4.8, 6H). LC-MS (API-ES) m/z = 238.1 (M + H).
Methyl 2-Cyano-2-[(9E/Z)-6-oxaspiro[4.5]decan-9-ylidene]-
acetate (36c). Using a procedure described in method C, 6-
oxaspiro[4.5]decan-9-one (35c, 6.0 g, 39.0 mmol) was converted to
a mixture of E/Z isomers: methyl 2-cyano-2-[(9E/Z)-6-oxaspiro[4.5]-
Methyl 2-Cyano-2-[9-(pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]-
acetate (37e). Using a procedure described in method D, methyl 2-
cyano-2-[(9E/Z)-6-oxaspiro[4.5]decan-9-ylidene]acetate (36c, 8.3 g,
35.3 mmol) was converted to methyl 2-cyano-2-[9-(pyridin-2-yl)-6-
1
oxaspiro[4.5]decan-9-yl]acetate (37e, 9.34 g. 84% yield). H NMR
(400 MHz, CDCl3) δ 8.54 (m, 1H), 7.66 (qd, J = 7.7, 1.8, 1H), 7.37
(dd, J = 44.2, 8.0, 1H), 7.16 (td, J = 7.0, 2.8, 1H), 3.68 (m, 2H), 3.56
(s, 1H), 2.64 (m, 1H), 2.55 (m, 1H), 2.21 (d, J = 13.4, 1H), 2.07 (m,
2H), 1.97 (s, 2H), 1.68 (m, 2H), 1.55 (m, 3H), 1.38 (dddd, J = 17.5,
13.7, 12.0, 6.5, 3H), 0.96 (m, 1H). LC-MS (API-ES) m/z = 315.2 (M
+ H).
2-[4-(4-Methylphenyl)oxan-4-yl]acetonitrile (38a) (Method E). To
a solution of KOH (183.0 mg, 3.26 mmol) in ethylene glycol (10.0
mL) was added 2-cyano-2-[4-(4-methylphenyl)oxan-4-yl]acetate (37a,
445.0 mg, 1.63 mmol). The mixture was heated to 120 °C for 3 h, then
cooled. H2O (25.0 mL) was added, and the product extracted with
1
decan-9-ylidene]acetate as a colorless oil (36c, 8.93 g, 97% yield). H
NMR (400 MHz, CDCl3) δ 3.84 (m, 5H), 3.15 (m, 2H), 2.74 (S, 2H),
1.68 (m, 8H). LC-MS (API-ES) m/z = 236.1 (M + H).
Methyl 2-Cyano-2-[(4Z)-1-oxaspiro[5.5]undecan-4-ylidene]-
acetate (36d). Using a procedure described in method C, 6-
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Et2O (3 × 25.0 mL), washed with H2O (25.0 mL), dried over Na2SO4,
filtered, and concentrated. The residue was purified by flash column
chromatography on a prepacked silica gel column (7−60% EtOAc in
hexanes) to give 2-[4-(4-methylphenyl)oxan-4-yl]acetonitrile as a
white solid (38a, 350.0 mg, >99.0% yield). 1H NMR (400 MHz,
CDCl3) δ 7.24 (m, 4H), 3.78 (m, 2H), 3.57 (ddd, J = 11.9, 9.4, 2.6,
2H), 2.57 (s, 2H), 2.36 (s, 3H), 2.29 (ddd, J = 11.9, 5.1, 2.6, 2H), 1.97
(m, 2H). LC-MS (API-ES) m/z = 216.1 (M + H).
2-[2,2-Diethyl-4-(4-fluorophenyl)oxan-4-yl]acetonitrile (38b).
Using a procedure described in method E, methyl 2-cyano-2-[2,2-
diethyl-4-(4-fluorophenyl)oxan-4-yl]acetate (37b, 250.0 mg, 0.8
mmol) was converted to 2-[2,2-diethyl-4-(4-fluorophenyl)oxan-4-
yl]acetonitrile (38b, 184 mg, 84% yield). LC-MS (API-ES) m/z =
276.2 (M + H).
2-[9-(4-Fluorophenyl)-6-oxaspiro[4.5]decan-9-yl]acetonitrile
(38c). Using a procedure described in method E, methyl 2-cyano-2-[9-
(4-fluorophenyl)-6-oxaspiro[4.5]decan-9-yl]acetate (37c, 3.24 g, 9.8
mmol) was converted to 2-[9-(4-fluorophenyl)-6-oxaspiro[4.5]decan-
9-yl]acetonitrile (38c, 1.96 g, 73% yield). 1H NMR (400 MHz,
CDCl3) δ 7.35 (m, 2H), 7.08 (m, 2H), 3.76 (qd, J = 7.6, 3.9, 2H), 2.54
(dd, J = 40.0, 16.6, 2H), 2.34 (ddd, J = 9.4, 5.5, 3.3, 1H), 2.22 (dd, J =
13.9, 1.8, 1H), 2.04 (m, 1H), 1.87 (m, 2H), 1.69 (m, 1H), 1.52 (m,
4H), 1.32 (ddd, J = 13.6, 7.2, 3.0, 1H), 0.92 (dt, J = 13.5, 8.9, 1H). LC-
MS (API-ES) m/z = 274.2 (M + H).
2-[(9R)-9-(4-Fluorophenyl)-6-oxaspiro[4.5]decan-9-yl]acetonitrile
((R)-38c) and 2-[(9S)-9-(4-fluorophenyl)-6-oxaspiro[4.5]decan-9-yl]-
acetonitrile ((S)-38c). Racemic 2-[9-(4-fluorophenyl)-6-oxaspiro[4.5]-
decan-9-yl]acetonitrile (38c) (1.96 g) was separated by supercritical
fluid chromatography (SFC) on an AD-3 column using 15% MeOH
(0.05% diethylamine as a modifier) to give 2-[(9S)-9-(4-fluorophen-
yl)-6-oxaspiro[4.5]decan-9-yl]acetonitrile as a colorless oil (faster
eluting enantiomer, (S)-38c, 635 mg, 32% yield) with LC-MS (API-
ES) m/z = 274.2 (M + H) and 2-[(9R)-9-(4-fluorophenyl)-6-
oxaspiro[4.5]decan-9-yl]acetonitrile as a colorless oil (slower eluting
enantiomer, (R)-38c, 703 mg, 36%) with LC-MS (API-ES) m/z =
274.2 (M + H).
decan-9-yl]acetonitrile ((R)-38e, 800.0 mg, 40% yield) with LC-MS
(API-ES) m/z = 257.2 (M + H).
The absolute (R)-configuration was determined by a single crystal
X-ray analysis on compound (R)-30 (see the X-ray crystallography
result, Figure 1).
2-[4-(4-Methylphenyl)oxan-4-yl]ethan-1-amine (39a) (Method
F). To a solution of 2-[4-(4-methylphenyl)oxan-4-yl]acetonitrile
(38a, 846.0 mg, 3.93 mmol) in anhydrous ether (20.0 mL) at 0 °C
was added dropwise a solution of lithium aluminum hydride (LAH, 1.0
M in Et2O, 7.87 mL, 7.87 mmol). Upon reaction completion, the
reaction was quenched with 2.0 mL of H2O, 0.25 mL of 15% NaOH,
and then 2.0 mL of H2O. The reaction mixture was extracted with
Et2O (3 × 50.0 mL), dried over Na2SO4, and concentrated to give 2-
[4-(4-methylphenyl)oxan-4-yl]ethan-1-amine as an yellow oil, which
used without further purification (39a, 750.0 mg, 87.0% yield). LC-MS
(API-ES) m/z = 220.1 (M + H).
2-[2,2-Diethyl-4-(4-fluorophenyl)oxan-4-yl]ethan-1-amine (39b).
Using a procedure described in method F, crude 2-[2,2-diethyl-4-(4-
fluorophenyl)oxan-4-yl]acetonitrile (38b, 534 mg, approximately 1.95
mmol) was converted to 2-[2,2-diethyl-4-(4-fluorophenyl)oxan-4-
yl]ethan-1-amine (39b, 430 mg, 79%). 1H NMR (400 MHz,
CDCl3) δ 7.17 (m, 2H), 6.94 (m, 3H), 3.62 (m, 2H), 2.40 (s, 1H),
2.23 (m, 1H), 2.05 (dt, J = 12.1, 6.0, 2H), 1.62 (dddd, J = 14.4, 10.2,
8.2, 4.6, 2H), 1.49 (m, 2H), 1.36 (m, 4H), 1.00 (dq, J = 14.8, 7.5, 1H),
0.76 (m, 5H), 0.46 (t, J = 7.4, 3H). LC-MS (API-ES) m/z = 280.2 (M
+ H).
2-[9-(4-Fluorophenyl)-6-oxaspiro[4.5]decan-9-yl]ethan-1-amine
(39c). Using a procedure described in method F, 2-[9-(4-
fluorophenyl)-6-oxaspiro[4.5]decan-9-yl]acetonitrile (38c, 1.09 g,
3.99 mmol) was converted to 2-[9-(4-fluorophenyl)-6-oxaspiro[4.5]-
decan-9-yl]ethan-1-amine as a yellow oil, which used without further
purification (39c, 900 mg, 82% yield). LC-MS (API-ES) m/z = 278.2
(M + H).
2-[(9R)-9-(4-Fluorophenyl)-6-oxaspiro[4.5]decan-9-yl]ethan-1-
amine ((R)-39c). Using a procedure described in method F, 2-[(9R)-9-
(4-fluorophenyl)-6-oxaspiro[4.5]decan-9-yl]acetonitrile ((R)-38c,
500.0 mg, 1.8 mmol) was converted to 2-[(9R)-9-(4-fluorophenyl)-
6-oxaspiro[4.5]decan-9-yl]ethan-1-amine as a yellow oil, which used
without further purification ((R)-39c, 500.0 mg, quantitative yield).
LC-MS (API-ES) m/z = 278.2 (M + H).
The absolute configuration was determined by an X-ray
crystallography study on a (R)-enantionmer (43) (see the single
crystal X-ray analysis result in Figure 1).
2-[4-(4-Fluorophenyl)-1-oxaspiro[5.5]undecan-4-yl]acetonitrile
(38d). Using a procedure described in method E, methyl 2-cyano-2-[4-
(4-fluorophenyl)-1-oxaspiro[5.5]undecan-4-yl]acetate (37d, 1.4 g, 4.0
mmol) was converted to 2-[4-(4-fluorophenyl)-1-oxaspiro[5.5]-
undecan-4-yl]acetonitrile (38d). The crude material was used directly
2-[(9S)-9-(4-Fluorophenyl)-6-oxaspiro[4.5]decan-9-yl]ethan-1-
amine ((S)-39c). Using a procedure described in method F, 2-[(9S)-9-
(4-fluorophenyl)-6-oxaspiro[4.5]decan-9-yl]acetonitrile ((S)-38c, 200
mg, 0.73 mmol) was converted to 2-[(9S)-9-(4-fluorophenyl)-6-
oxaspiro[4.5]decan-9-yl]ethan-1-amine as an yellow oil, which used
without further purification ((S)-39c, 203 mg, quantitative yield). LC-
MS (API-ES) m/z = 278.2 (M + H).
1
in next step. H NMR (400 MHz, CDCl3) δ 7.29 (m, 2H), 6.99 (m,
2H), 3.69 (m, 2H), 3.41 (q, J = 7.0, 1H), 2.39 (m, 3H), 2.24 (dd, J =
14.0, 2.4, 1H), 1.77 (ddd, J = 14.2, 11.1, 4.7, 1H), 1.58 (m, 1H), 1.36
(m, 3H), 1.15 (m, 5H), 0.68 (m, 1H). LC-MS (API-ES) m/z = 288.2
(M + H).
2-[4-(4-Fluorophenyl)-1-oxaspiro[5.5]undecan-4-yl]ethan-1-
amine (39d). Using a procedure described in method F, 2-[4-(4-
fluorophenyl)-1-oxaspiro[5.5]undecan-4-yl]acetonitrile (38d, 1.04 g,
3.8 mmol) was converted to 2-[4-(4-fluorophenyl)-1-oxaspiro[5.5]-
undecan-4-yl]ethan-1-amine as an oil (39d, 0.98 g, 89.0% yield), which
2-[9-(Pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]acetonitrile (38e).
Using a procedure described in method E, methyl 2-cyano-2-[9-
(pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]acetate (37e, 18.6 g, 59.2
mmol) was converted to 2-[9-(pyridin-2-yl)-6-oxaspiro[4.5]decan-9-
yl]acetonitrile (38e, 9.9 g. 66.0% yield). 1H NMR (400 MHz, CDCl3)
δ 8.63 (ddd, J = 4.8, 1.8, 0.9, 1H), 7.73 (td, J = 7.8, 1.9, 1H), 7.42 (d, J
= 8.0, 1H), 7.23 (ddd, J = 7.5, 4.8, 1.0, 1H), 3.81 (m, 2H), 3.50 (d, J =
4.4, 1H), 2.72 (dd, J = 74.6, 16.5, 2H), 2.51 (m, 2H), 2.00 (t, J = 11.1,
1H), 1.92 (m, 1H), 1.83 (m, 1H), 1.69 (m, 2H), 1.55 (m, 3H), 1.44
(m, 1H), 1.23 (dddd, J = 7.5, 6.0, 5.5, 4.5, 1H), 0.81 (dt, J = 13.5, 8.9,
1H). LC-MS (API-ES) m/z = 257.2 (M + H).
2-[(9R)-9-(Pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]acetonitrile
((R)-38e) and 2-[(9S)-9-(pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]-
acetonitrile ((S)-38e). 2-[9-(Pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]-
acetonitrile (38e, 2.0 g) was separated by supercritical fluid
chromatography on AD-H column using 50% MeOH with 0.05%
DEA to give a pair of enantiomers: faster eluting enantiomer, 2-[(9S)-
9-(pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]acetonitrile ((S)-38e, 838.7
mg, 42% yield) with LC-MS (API-ES) m/z = 257.2 (M + H), and the
slower eluting enantiomer, 2-[(9R)-9-(pyridin-2-yl)-6-oxaspiro[4.5]-
1
was used without further purification. H NMR (400 MHz, CDCl3) δ
7.20 (m, 2H), 6.93 (m, 2H), 3.66 (m, 2H), 2.42 (td, J = 11.6, 5.2, 1H),
2.24 (m, 1H), 2.07 (m, 2H), 1.62 (m, 2H), 1.48 (m, 3H), 1.31 (m,
5H), 1.11 (m, 5H), 0.64 (m, 1H). LC-MS (API-ES) m/z = 292.2 (M
+ H).
2-[9-(Pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]ethan-1-amine
(39e). Using a procedure described in method F, 2-[9-(pyridin-2-yl)-6-
oxaspiro[4.5]decan-9-yl]acetonitrile (38e, 300.0 mg, 1.2 mmol) was
converted to 2-[9-(pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]ethan-1-
amine (39e, 230.0 mg, 79.0% yield) and was used without further
1
purification. H NMR (400 MHz, CDCl3) δ 8.58 (ddd, J = 4.8, 1.9,
0.9, 1H), 7.63 (m, 1H), 7.30 (m, 1H), 7.12 (ddd, J = 7.4, 4.8, 1.0, 1H),
3.76 (m, 2H), 2.55 (td, J = 11.6, 5.1, 1H), 2.46 (ddd, J = 13.7, 5.1, 2.7,
1H), 2.37 (dd, J = 13.7, 2.1, 1H), 2.14 (td, J = 11.6, 5.0, 1H), 1.92 (m,
2H), 1.70 (m, 4H), 1.51 (m, 3H), 1.39 (m, 1H), 1.22 (s, 2H), 1.12 (m,
1H), 0.71 (dt, J = 13.4, 8.8, 1H). LC-MS (API-ES) m/z = 261.2 (M +
H).
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2-[(9R)-9-(Pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]ethan-1-amine
((R)-39e). Using a procedure described in method F, 2-[(9R)-9-
(pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]acetonitrile ((R)-38e, 800.0
mg, 3.1 mmol) was converted to 2-[(9R)-9-(pyridin-2-yl)-6-oxaspiro-
[4.5]decan-9-yl]ethan-1-amine ((R)-39e, 670.0 mg, 84.0% yield),
stimulated cAMP accumulation in the presence of 1.5 mM NKH-477
(water-soluble forskolin, Tocris catalogue no. 1603) and 500 μM 3-
isobutyl-1-methylxanthine (IBMX). cAMP accumulation assays were
run in parallel with β-arrestin-2 recruitment, using the same cells, drug
dilutions, and assay buffers (1% DMSO, F12 Ham’s buffer) to ensure
accurate assay-to-assay comparison of data. Plates were read using a
time-resolved fluorescence ratio (665 nm/620 nm) on a PheraStar
plate reader.
β-Arrestin-2 Recruitment Assay. The PathHunter enzyme
complementation assay (DiscoveRx Corporation, Fremont, California)
was performed according to the manufacturer’s protocol and read for
chemiluminescence on a PheraStar plate reader (BMG Labtech,
Durham, North Carolina). Briefly, when β-arrestin-2 translocates to
active receptor, the complementary β-galactosidase fragments fused to
receptor and β-arrestin interact to form a functional enzyme, which is
detected by chemiluminescence. For all in vitro assays, data were
normalized as a percentage of maximal assay responses, typically
defined by morphine stimulated activity in the MOR assays.
1
which was used directly without further purification. H NMR (400
MHz, CDCl3) δ 8.58 (ddd, J = 4.8, 1.9, 0.9, 1H), 7.63 (m, 1H), 7.30
(m, 1H), 7.12 (ddd, J = 7.4, 4.8, 1.0, 1H), 3.76 (m, 2H), 2.55 (td, J =
11.6, 5.1, 1H), 2.46 (ddd, J = 13.7, 5.1, 2.7, 1H), 2.37 (dd, J = 13.7, 2.1,
1H), 2.14 (td, J = 11.6, 5.0, 1H), 1.92 (m, 2H), 1.70 (m, 4H), 1.51 (m,
3H), 1.39 (m, 1H), 1.22 (s, 2H), 1.12 (m, 1H), 0.71 (dt, J = 13.4, 8.8,
1H). LC-MS (API-ES) m/z = 261.2 (M + H).
2-[(9S)-9-(Pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]ethan-1-amine
((S)-39e). Using a procedure described in method F, 2-[(9S)-9-
(pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]acetonitrile ((S)-38e, 838.0
mg, 3.3 mmol) was converted to 2-[(9S)-9-(pyridin-2-yl)-6-oxaspiro-
[4.5]decan-9-yl]ethan-1-amine ((S)-39e, 710.0 mg, 83% yield), which
1
was used next step without further purification. H NMR (400 MHz,
CDCl3) δ 8.67−8.52 (m, 1H), 7.81−7.57 (m, 1H), 7.37−7.24 (m,
1H), 7.24−7.06 (m, 1H), 3.85−3.63 (m, 2H), 2.68−2.58 (m, 1H),
2.38 (ddt, J = 23.4, 11.8, 6.0, 2H), 2.24−2.15 (m, 1H), 2.04−1.94 (m,
2H), 1.81−1.68 (m, 3H), 1.63 (ddd, J = 15.3, 8.9, 3.7, 2H), 1.57−1.42
(m, 4H), 1.41−1.30 (m, 2H), 1.10 (dd, J = 10.5, 4.8, 1H), 0.67 (dt, J =
13.3, 8.8, 1H). LC-MS (API-ES) m/z = 261.2 (M + H).
ASSOCIATED CONTENT
■
S
* Supporting Information
1H NMR spectra of all of the final compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
2,2-Diethyloxan-4-ol (42). To a mixture of 3-butene-1-ol (41, 19.8
mL, 233.0 mmol) and 3-pentenone (40, 12.3 mL, 116.0 mmol) was
added 75% sulfuric acid dropwise at 0 °C. The reaction mixture was
allowed to warm to room temperature and stirred overnight. Water
(70.0 mL) was added to the mixture, then neutralized with NaOH
(pellets) to pH 8.0 and extracted with diethyl ether (3 × 150.0 mL).
The combined ether extracts were washed with an aqueous sodium
bisulfite solution (40.0 mL), dried over K2CO3, and concentrated. The
residue was distilled under reduced pressure to give 2,2-diethyloxan-4-
ol (42, 4.89 g, 27%, bp = 65−70 °C at 1 mmHg). 1H NMR (400 MHz,
CDCl3) δ 4.04−3.86 (m, 1H), 3.84−3.66 (m, 1H), 3.65−3.38 (m,
1H), 2.06−1.95 (m, 1H), 1.92−1.76 (m, 2H), 1.78−1.63 (m, 1H),
1.63−1.50 (m, 1H), 1.51−1.31 (m, 3H), 1.28−1.10 (m, 1H), 0.92−
0.68 (m, 6H).
2-[(9R)-9-(4-Fluorophenyl)-6-oxaspiro[4.5]decan-9-yl]-
acetaldehyde (44). To a solution of 2-[(9R)-9-(4-fluorophenyl)-6-
oxaspiro[4.5]decan-9-yl]acetonitrile ((R)-38c, 250.0 mg, 0.91 mmol)
in toluene at −78 °C was added dropwise a solution of DIBAL in
toluene (1.0 M, 1.0 mL, 1.0 mmol). The resulting mixture was stirred
at −78 °C for 1.5 h, and two more equivalents of DIBAL were added
until completion of the reaction monitored by LC-MS. The reaction
was quenched with saturated NH4Cl (10.0 mL) and warmed to room
temperature. Water (10.0 mL) was added, and the mixture was stirred
for 10 min. EtOAc (40.0 mL) was added to the mixture, which was
filtered to remove salts and extracted with EtOAc (3 × 10.0 mL) and
dried over MgSO4. After filtration and concentration, the residue was
purified by flash column chromatography on a prepacked silica gel
column (0−20% EtOAc in hexanes) to give 2-[(9R)-9-(4-fluorophen-
yl)-6-oxaspiro[4.5]decan-9-yl]acetaldehyde ((R)-44, 90.0 mg, 36%
yield). LC-MS (API-ES) m/z = 277.2 (M + H).
In Vitro Cellular Assays. Cell Culture and Preparation. HEK-293
cells stably transfected to overexpress β-arrestin-2 fused to a β-
galactosidase fragment were purchased from DiscoveRx (PathHunter
β-arrestin assay, DiscoveRx Corporation, Fremont California) and the
human OPRM1 gene (NM_000914.3, encoding human MOR),
human OPRD1 gene (NM_000911.3, human DOR), and human
OPRK1 gene (NM_000912.3, human KOR) receptors were fused to a
complementary β-galactosidase fragment using the pCMV-ProLink
plasmid purchased from DiscoveRx. Cells were grown in MEM with
10% FBS, 1% penicillin/streptomycin, and 150 μg/L of neomycin and
150 μg/L of hygromycin.
cAMP Accumulation Assay. Receptor G protein mediated
responses were determined by measuring changes in cAMP using
the cAMP-HTRF kit (Cisbio, Codolet, France), using the same cell
lines used to measure β-arrestin-2 recruitment. MOR couples to Gαi,
so G protein coupling was measured as inhibition of forskolin-
Accession Codes
CCDC-949885 and CCDC-949886
AUTHOR INFORMATION
Corresponding Author
*Phone: 610-354-8840. Fax: 610-354-8850. E-mail: dchen@
trevenainc.com.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Amy A. Sarjeant, Northwestern University, for
the X-ray structure analyses of (R)-30 and 43, and Dr. Richard
Keenan for his critical reading of the manuscript.
ABBREVIATIONS USED
■
1H NMR, proton nuclear magnetic resonance; 7TM receptor,
seven-transmembrane receptor; CV, cardiovascular; DOR, δ
opioid receptor; EAD, early after depolarization; IND,
investigational new drug; KO, knockout; KOR, κ opioid
receptor; MOR, μ opioid receptor; MPE, maximal possible
analgesic effect; PR interval, duration from start of the P-wave
to the beginning of the QRS complex of the ECG. Also used as
a measure of conduction time from SA node to AV node (along
with QRS interval); QRS interval, duration from the start of the
Q-wave to the end of the S-wave, corresponding to ventricular
depolarization; QT interval, duration from the start of the Q-
wave to the end of the T-wave, when the ECG becomes
isoelectric; this corresponds to ventricular depolarization and
repolarization; QTc, the QT interval corrected for heart rate;
TdP, torsade de pointes; Tp−e, duration from the peak of the T-
wave to the end of the T-wave or isoelectric point; this is a
measure of transmural dispersion or repolarization
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