Synthetic and Receptor Signaling Explorations of the Mitragyna Alkaloids: Mitragynine as an Atypical Molecular Framework for Opioid Receptor Modulators

Article abstract of DOI:10.1021/jacs.6b00360

Mu-opioid receptor agonists represent mainstays of pain management. However, the therapeutic use of these agents is associated with serious side effects, including potentially lethal respiratory depression. Accordingly, there is a longstanding interest in the development of new opioid analgesics with improved therapeutic profiles. The alkaloids of the Southeast Asian plant Mitragyna speciosa, represented by the prototypical member mitragynine, are an unusual class of opioid receptor modulators with distinct pharmacological properties. Here we describe the first receptor-level functional characterization of mitragynine and related natural alkaloids at the human mu-, kappa-, and delta-opioid receptors. These results show that mitragynine and the oxidized analogue 7-hydroxymitragynine, are partial agonists of the human mu-opioid receptor and competitive antagonists at the kappa- and delta-opioid receptors. We also show that mitragynine and 7-hydroxymitragynine are G-protein-biased agonists of the mu-opioid receptor, which do not recruit β-arrestin following receptor activation. Therefore, the Mitragyna alkaloid scaffold represents a novel framework for the development of functionally biased opioid modulators, which may exhibit improved therapeutic profiles. Also presented is an enantioselective total synthesis of both (-)-mitragynine and its unnatural enantiomer, (+)-mitragynine, employing a proline-catalyzed Mannich-Michael reaction sequence as the key transformation. Pharmacological evaluation of (+)-mitragynine revealed its much weaker opioid activity. Likewise, the intermediates and chemical transformations developed in the total synthesis allowed the elucidation of previously unexplored structure-activity relationships (SAR) within the Mitragyna scaffold. Molecular docking studies, in combination with the observed chemical SAR, suggest that Mitragyna alkaloids adopt a binding pose at the mu-opioid receptor that is distinct from that of classical opioids.

Full text of DOI:10.1021/jacs.6b00360

Article
pubs.acs.org/JACS
Synthetic and Receptor Signaling Explorations of the Mitragyna
Alkaloids: Mitragynine as an Atypical Molecular Framework for
Opioid Receptor Modulators
Andrew C. Kruegel,†,# Madalee M. Gassaway,†,# Abhijeet Kapoor,∥ Andras Varadi,⊥ Susruta Majumdar,⊥
́
́
Marta Filizola,∥ Jonathan A. Javitch,‡,§,∇ and Dalibor Sames*,†
‡
§
†Department of Chemistry, Department of Psychiatry, and Department of Pharmacology, Columbia University, New York, New
York 10027, United States
∥Department of Structural and Chemical Biology, Icahn School of Medicine at Mount Sinai, New York, New York 10029, United
States
⊥Department of Neurology, Memorial Sloan Kettering Cancer Center, New York, New York 10065, United States
∇Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, New York 10032, United States
S
* Supporting Information
ABSTRACT: Mu-opioid receptor agonists represent mainstays of pain management. However, the therapeutic use of these
agents is associated with serious side effects, including potentially lethal respiratory depression. Accordingly, there is a
longstanding interest in the development of new opioid analgesics with improved therapeutic profiles. The alkaloids of the
Southeast Asian plant Mitragyna speciosa, represented by the prototypical member mitragynine, are an unusual class of opioid
receptor modulators with distinct pharmacological properties. Here we describe the first receptor-level functional characterization
of mitragynine and related natural alkaloids at the human mu-, kappa-, and delta-opioid receptors. These results show that
mitragynine and the oxidized analogue 7-hydroxymitragynine, are partial agonists of the human mu-opioid receptor and
competitive antagonists at the kappa- and delta-opioid receptors. We also show that mitragynine and 7-hydroxymitragynine are
G-protein-biased agonists of the mu-opioid receptor, which do not recruit β-arrestin following receptor activation. Therefore, the
Mitragyna alkaloid scaffold represents a novel framework for the development of functionally biased opioid modulators, which
may exhibit improved therapeutic profiles. Also presented is an enantioselective total synthesis of both (−)-mitragynine and its
unnatural enantiomer, (+)-mitragynine, employing a proline-catalyzed Mannich−Michael reaction sequence as the key
transformation. Pharmacological evaluation of (+)-mitragynine revealed its much weaker opioid activity. Likewise, the
intermediates and chemical transformations developed in the total synthesis allowed the elucidation of previously unexplored
structure−activity relationships (SAR) within the Mitragyna scaffold. Molecular docking studies, in combination with the
observed chemical SAR, suggest that Mitragyna alkaloids adopt a binding pose at the mu-opioid receptor that is distinct from that
of classical opioids.
Indeed, MOR agonists, including not only morphine itself, but
also a vast number of synthetic and semisynthetic opioids,
remain the gold standard of pain therapy. Unfortunately, acute
MOR activation is also associated with serious side effects,
INTRODUCTION
■
The opioid receptors, and in particular, the mu-opioid receptor
(MOR), are among the longest and most intensely studied
molecular signaling systems in the central nervous system.1
Likewise, the prototypical small molecule agonist of these
receptors, morphine, has been used by humans as an important
analgesic and recreational euphoriant since ancient times.
Received: January 11, 2016
© XXXX American Chemical Society
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including respiratory depression, constipation, sedation, nausea,
and itching.1,2 At sufficiently high doses, the evoked respiratory
depression may be fatal. Further, the pronounced euphoria
produced by MOR agonists makes them major drugs of abuse.
These properties have made overdose from prescription opioid
analgesics a leading cause of accidental death in the United
States, killing more than 18 000 people in 2014.3,4 Another
shortcoming of MOR agonists is the rapid development of
tolerance to their analgesic effects. Thus, continuing escalation
of dose is required to maintain an equivalent level of pain
management. Similarly, when they are abused, tolerance to the
euphoric effects of opioids is also rapidly developed. Thus, in
either case, chronic use often results in severe physical
dependence on MOR agonists due to cellular- and circuit-
level adaptations to continuous receptor stimulation. Accord-
ingly, much effort has been dedicated to the development of
new MOR agonists retaining potent analgesic effects, while
mitigating or eliminating the deleterious side effects of the
agents currently in use.1−8
Historically, MOR agonists have also been applied in the
treatment of mood disorders, notably including major
depressive disorder (MDD). Indeed, until the mid-20th
century, low doses of opium itself were used to treat
depression, and the so-called “opium cure” was purportedly
quite effective.9 With the advent of tricyclic antidepressants
(TCAs) in the 1950s, however, the psychiatric use of opioids
rapidly fell out of favor and has been largely dormant since,
likely due to negative medical and societal perceptions
stemming from their abuse potential. However, there have
been scattered clinical reports (both case studies and small
controlled trials) since the 1970s indicating the effectiveness of
MOR agonists in treating depression. The endogenous opioid
peptide β-endorphin, as well as a number of small molecules,
have all been reported to rapidly and robustly improve the
symptoms of MDD and/or anxiety disorders in the clinical
setting, even in treatment-resistant patients.10−17 These results
have been recapitulated in rodent models, where a variety of
MOR agonists show antidepressant effects.18−21 Most recently,
we have found that the atypical antidepressant tianeptine,
which has been used clinically for several decades and
extensively studied in rodents and other mammalian species,
is an MOR agonist, suggesting that this agent exerts its
antidepressant effects via direct MOR activation.22 When taken
together, this body of work establishes a clear precedent for the
effectiveness of MOR agonists in the treatment of depression
and anxiety. Unfortunately, the treatment of mood disorders
with conventional MOR agonists is expected to suffer from the
same liabilities as their use in the context of pain management.
Accordingly, our laboratories have been concerned with the
exploration of structurally and pharmacologically distinct
classes of MOR agonists, with the aim of developing new
opioid-based treatments for mood disorders and pain, which
lack the classical side effects of these agents.
Figure 1. Leaves and fruiting bodies of Mitragyna speciosa and
chemical structures of notable alkaloids.
also known, including use as a treatment for fever, cough,
diarrhea, and depression. There is also a precedent for
recreational use of the plant, a fact that has contributed to
legal control of Mitragyna speciosa in both Thailand and
Malaysia, but the plant remains uncontrolled outside its
endemic regions.23−27
In light of its well documented medicinal properties, the
molecular constituents of Mitragyna speciosa responsible for its
psychoactive effects have been studied, with more than 20
unique indole alkaloids having been identified in the
plant.23−25,28 The indole alkaloid mitragynine (Figure 1) has
been universally cited as the primary alkaloid constituent of
Mitragyna speciosa, accounting for up to 66% by mass of crude
alkaloid extracts.23 The other major alkaloids in the plant have
been found to include paynantheine, speciogynine, and
speciociliatine (Figure 1).23 The quantities of these major
alkaloids, along with a wide variety of minor alkaloids, are
considerably varied among different regional varieties of the
plant and also depend on plant age, facts that considerably
complicate the interpretation of reported psychoactive effects
from the raw plant material.23−25,28 Among the minor alkaloids,
the oxidized derivative 7-hydroxymitragynine (7-OH)29 (Figure
1) is of particular interest, as it has been reported to induce
analgesic effects mediated through agonist activity at the MOR,
exceeding in potency those of the prototypical opioid agonist
morphine.27,30
The mechanism of action of Mitragyna alkaloids has also
been studied both in vitro and in vivo. In particular, Takayama
and co-workers have accumulated a large body of evidence
implicating the opioid receptor system as the primary mediator
of the psychoactive effects of these alkaloids. Specifically, both
mitragynine and 7-OH exhibit nanomolar binding affinities for
the MOR and possess functional activity in tissue assays.27,30
Likewise, the antinociceptive effects of mitragynine and 7-OH
It was in this context that we became interested in the
psychoactive plant Mitragyna speciosa (Figure 1), known as
“kratom” in Thailand, or “biak biak” in Malaysia, a substance
that has been used by humans in Southeast Asia for centuries to
treat a variety of ailments. The plant material is typically
consumed as a tea or chewed directly. At low doses, kratom is
primarily used for its stimulating effects. At higher doses,
opioid-like effects predominate, and the plant has been used as
a general analgesic and as a substitute for opium or to treat
opium withdrawal symptoms. Other medicinal applications are
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in several rodent models are also inhibited by naloxone.27,30,31
Despite this evidence for the involvement of opioid receptor
systems, and specifically MOR, in mediating the analgesic
effects of Mitragyna alkaloids, there are conflicting reports in
the literature. Most notably, an early study with mitragynine
indicated that the behavioral effects in cats, and analgesic effects
in rats, were not reversed by treatment with nalorphine, an
opioid antagonist, while at the same time, mitragynine was
found to produce markedly less respiratory depression than
codeine.32 Mitragynine has been shown to bind in some degree
to several non-opioid CNS receptors, including alpha-2
adrenergic receptor (α2R), adenosine A2a, dopamine D2, and
the serotonin receptors 5-HT2C and 5-HT7, but the strength of
these interactions has not been reported.33 Mitragynine
analgesia has also been shown to be inhibited by the α2R
antagonist idazoxan and by the nonspecific serotonin antagonist
cyproheptadine.34
Considering the many possible confounding factors present
at the tissue or system level, such in vivo and ex vivo tissue
assays are not ideal for confirmation of a functional effect at a
particular receptor. Although receptor-level functional activity
studies ([35S]GTPγS binding) using cloned opioid receptors
have recently been reported for several synthetic oxidized
analogues,35 no similar functional studies have been reported
for mitragynine itself, or for other naturally occurring alkaloids
in Mitragyna speciosa.36 Given the unique and still unclear
molecular pharmacology surrounding Mitragyna alkaloids, we
undertook a thorough examination of mitragynine and a
number of natural and novel analogues at the opioid receptors,
measuring receptor activation and intracellular signaling.
RESULTS AND DISCUSSION
■
Mitragynine is a Partial Agonist of Human MOR. We
isolated the four major alkaloids (Figure 1) from the Thai strain
of Mitragyna speciosa and prepared 7-OH by photochemical
oxidation of mitragynine (Scheme S1). Interestingly, only trace
quantities of 7-OH were observed (by mass spectrometry) in
our extractions of the raw plant material, and it was not possible
to isolate any measurable quantity of this derivative. Therefore,
it is doubtful that this alkaloid is a universal constituent of all
Mitragyna speciosa preparations and is unlikely to generally
account for the psychoactive properties of this plant.
Figure 2. Activity of mitragynine and 7-hydroxymitragynine (7-OH) at
the human mu-opioid receptor (hMOR). (A) Conceptual representa-
tion of the G protein BRET assay employing atomistic cartoons
derived from available X-ray crystal structures. To measure G protein
38
activation, hMOR37 (brown) was coexpressed with GαoB (red)
fused to RLuc839 (teal), β138 (blue), and mVenus40 (yellow-green)
fused to γ238 (yellow). (B) Agonist activity at hMOR; positive control
= [D-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin (DAMGO); curves
represent the average of n ≥ 3, with error bars representing SEM.
We then set out to evaluate the activity of these compounds
in HEK cells expressing the MOR, delta-opioid receptor
(DOR), or kappa-opioid receptor (KOR) using biolumines-
cence resonance energy transfer (BRET) functional assays. In
these G protein-dependent assays, the Gα subunit is fused with
a luciferase (RLuc8) donor, and the Gγ subunit is fused with a
fluorescent protein (mVenus) acceptor. Thus, on receptor
activation, the G protein subunits move apart and the observed
BRET signal decreases (Figure 2A).
At the human opioid receptors (hMOR, hKOR, and hDOR),
mitragynine acted as a partial agonist at hMOR (Table 1 and
Figure 2B, EC50 = 339 178 nM; maximal efficacy (Emax) =
34%). This result represents the first demonstration of
functional opioid agonist activity at human receptors with a
natural Mitragyna alkaloid, all prior functional studies having
been conducted in vivo in rodents or ex vivo in rodent tissue. In
contrast, at hKOR, mitragynine was a competitive antagonist
(Table 1, IC50 = 8.5 7.6 μM; pA2 = 1.4 0.40 μM, and
Figures S2 and S4), fully inhibiting the activity of the reference
agonist U-50,488. Similarly, mitragynine acted as an antagonist
at hDOR, but with very low potency (Figure S3). The other
Table 1. Functional Activity of Mitragyna Alkaloids at
Human Opioid Receptors as Determined in G Protein
BRET Assays
a
b
EC50 SEM (Emax
) or [IC50 SEM (pA2)] (μM)
compound
hMOR
hKOR
hDOR
mitragynine
paynantheine
speciogynine
speciociliatine
7-OH
0.339 0.178 (34%)
[2.2 10]
[8.5 7.6 (1.4)]
[>10]
[>10]
[>10]
[>10]
[>10]
[>10]
[5.7 2.8]
[>10]
[4.2 1.6]
[>10]
0.0345 0.0045 (47%)
[7.9 3.7 (0.49)]
a
Agonist activity indicated by EC50 values, maximal efficacy (Emax
)
b
relative to DAMGO in parentheses. Antagonist activity indicated by
IC50 values for inhibition of a reference agonist, pA2 determined from
Schild analysis in parentheses; All data points represent mean SEM
(μM) of n ≥ 3.
major natural alkaloids paynantheine, speciogynine, and
speciociliatine, showed no measurable agonist activity at any
of the human opioid receptors at concentrations up to 100 μM,
and only weak antagonist effects were observed (Table 1 and
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Figures S1, S2, and S3). The oxidized analogue, 7-OH, was also
characterized and found to be a potent, partial agonist at
significant challenges in the development of compounds in
this class as novel therapeutics, as activity in rodent models may
not be easily translatable to humans. In this regard, it is
fortunate that the more potent derivative, 7-OH, retains its
agonist activity at mMOR, as the oxidized scaffold represents a
more promising starting point for the development of
analgesics in this class. With respect to mitragynine, it is also
conceivable that the in vitro assays used in our study do not
exactly replicate the receptor activation and signaling in native
murine brain tissue. For example, a low efficacy partial agonist
in the brain may appear as an antagonist in the cell-based
assays. Thus, consideration of both interspecies and interassay
differences is advised for the future development of these
compounds.
hMOR (Table 1 and Figure 2B, EC50 = 34.5 4.5 nM; Emax
=
47%). Further, it acted as a competitive antagonist at both
hKOR (Table 1, IC50 = 7.9 3.7 μM; pA2 = 490 131 nM,
and Figures S2 and S5) and hDOR (Table 1, IC50 > 10 μM,
and Figure S3). The partial agonist activity of mitragynine and
7-OH at the human receptors was further confirmed in
antagonist experiments, as both compounds were able to
partially inhibit the response elicited by the full agonist [D-Ala2,
N-Me-Phe4, Gly-ol5]-enkephalin (DAMGO) (Figure S1).
In order to further confirm the opioid activity of the
Mitragyna alkaloids, we performed radioligand binding studies
to assess the affinity of the natural alkaloids and 7-OH for the
opioid receptors (Table 2). As expected from their activity in
Mitragyna Alkaloids Bias Intracellular Signaling
Toward G Proteins. Functionally selective (biased) agonists
of the opioid receptors, which preferentially activate only
certain downstream signaling pathways, hold promise as
analgesics or antidepressants with reduced side effects. Given
this potential to separate the well established therapeutic
benefits of MOR activation from negative side effects, the study
of functionally selective MOR ligands is a very active area of
research at present.41−44 In particular, some evidence indicates
that MOR agonists biased toward G protein signaling over β-
arrestin signaling display less respiratory depression, tolerance
development, and constipation, while remaining potent
analgesics.45−48 Therefore, we were interested in exploring
the potential for mitragynine’s unique molecular scaffold to
serve as a starting point for the development of biased MOR
agonists. To assess the level of β-arrestin recruitment induced
by Mitragyna alkaloids, we employed a recently described
BRET assay in transfected HEK cells, which was adapted for
use with the opioid receptors.49 In this assay, the unlabeled
receptor (in this case hMOR) is transfected alongside β-
arrestin-2 (arrestin-3), fused with luciferase (RLuc8) and an
SH3 binding domain (Sp1), and the membrane protein
GAP43, fused with a fluorescent protein (citrine) and an
SH3 domain. Accordingly, when an agonist induces β-arrestin
recruitment to MOR, the Sp1 domain on β-arrestin is brought
into proximity with the membrane-localized SH3 domain and
an interaction occurs. This results in concomitant association of
the luciferase donor and fluorescent protein acceptor, and a
BRET signal is observed (Figure 3A). An advantage of this
format is that native receptor can be used, avoiding potential
confounds related to C-terminal tagging on expression or
function.
As measured in this cell signaling assay, DAMGO induced
robust recruitment of β-arrestin. Interestingly, both mitragynine
and 7-OH were found to elicit no measurable β-arrestin
binding, even in the presence of G protein-coupled receptor
kinase 2 (GRK2), which typically enhances coupling to β-
arrestin (Figure 3B). Because of this extremely weak signal, we
were not able to quantitate the magnitude of functional
selectivity for mitragynine and 7-OH. However, since both
compounds exhibit significant efficacy for activation in the G
protein dissociation assay (Figure 2), there is a strong
qualitative bias in favor of G protein signaling in these cells.
Such selectivity for G protein-dependent signaling may explain
the reduced respiratory depression previously reported for
mitragynine compared to codeine,32 although any such
connection between upstream receptor signaling and physio-
logical properties must be considered speculative. In any case,
both mitragynine and 7-OH trigger downstream signaling
Table 2. Binding Affinities of Mitragyna Alkaloids at Human
Opioid Receptors
a
Ki SEM (μM)
compound
hMOR
hKOR
hDOR
mitragynine
paynantheine
speciogynine
speciociliatine
7-OH
0.233 0.048
0.410 0.152
0.728 0.061
0.560 0.168
0.047 0.018
0.772 0.207
2.56 0.37
>10
>10
>10
>10
3.20 0.36
0.329 0.112
0.188 0.038
0.219 0.041
a
All data points represent mean SEM (μM) of n ≥ 3.
our functional assays, both mitragynine and 7-OH exhibited
significant binding affinities for hMOR. Similarly, the other
natural alkaloids also bound to hMOR, consistent with their
antagonist activity in the functional assays. Binding was also
observed for mitragynine and 7-OH at hKOR and hDOR, again
in agreement with the results of the functional assays. Further,
the Ki values determined in the binding experiments were in
accord with the EC50 and IC50 (after correction by Cheng−
Prusoff equation) values determined in the functional experi-
ments, or with the pA2 values (an estimate of Kd) determined
by Schild analysis. Similar binding results were also obtained at
the mouse opioid receptors (Table S2). Further, our binding
data with 7-OH was consistent with previous literature reports
(using guinea pig brain homogenates), but the affinities
determined for mitragynine at MOR and DOR were much
weaker than previously described.27,30 Given the better
agreement between our in vitro data and the relative potencies
of mitragynine and 7-OH in vivo, this new data may be more
reliable. Taken together, the functional and binding results
provide a rigorous and internally consistent assessment of the
in vitro activity of the Mitragyna alkaloids at all three human
opioid receptors.
Surprisingly, analogous assays using rodent receptors (mouse
or rat) revealed that mitragynine exhibited no agonist activity,
and it was instead found to act as a competitive antagonist at
mouse MOR (mMOR) (Figure S6 and Table S1, IC50 = 1.1
μM; pA2 = 807
573 nM), fully inhibiting the activation
induced by the reference agonist DAMGO. The other isolated
natural products, paynantheine, speciogynine, and speciocilia-
tine, showed no notable agonist or antagonist activity at any of
the rodent opioid receptors. In contrast, the activity of 7-OH
was similar at the rodent receptors (Figure S6 and Table S1).
These observations complicate the interpretation of prior
reports of analgesic effects elicited by mitragynine in rodent
models. Further, this interspecies variation may present
D
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Figure 4. Key SAR locants of Mitragyna alkaloids (highlighted in
green) and select analogues designed to interrogate each position.
many known MOR ligands is proposed to be due to formation
of a water-mediated hydrogen bonding network with H2976.52
(superscript numbers refer to Ballesteros and Weinstein’s
generic numbering scheme52), as shown in two recent X-ray
crystal structures of MOR.37,53 Accordingly, it was hypothesized
that if mitragynine binds in a similar pose, then the
corresponding desmethyl analogue 1 (Figure 4) would be
expected to exhibit significantly increased potency. We also
envisioned simplification of the D ring substituents by selective
deletion of the enol ether (2) or pendant ethyl group (3)
(Figure 4), analogues which would probe previously unex-
plored SAR in this region.
Enantioselective Total Synthesis of Mitragynine and
Analogues. Although 7-OH and the desmethyl derivative 1
were prepared from the isolated natural product (Scheme S1),
we also desired a total synthesis of mitragynine that would
allow for the construction of more extensively modified
analogues. Accordingly, we envisioned a synthesis starting
from 4-methoxyindole, where ring C could be installed via a
Bischler-Napieralski reaction to give a 3,4-dihydro-β-carboline.
This intermediate would then be subjected to an enantiose-
lective, proline-catalyzed Mannich−Michael-type cyclization
with the appropriate enone to install ring D and set the
stereocenter at position 3. This transformation was developed
by Itoh and co-workers, and has been successfully employed in
the total synthesis of several structurally similar indole
alkaloids.54−56 The resulting tetracyclic ketone would serve as
a versatile intermediate for the synthesis of a variety of
analogues, including mitragynine itself (Figure 5).
The required 3,4-dihydro-β-carboline (4) was successfully
prepared in a six step sequence starting from commercially
available 4-methoxyindole (21% overall yield, Scheme S2A).
The required enone (5) was synthesized from methyl 2-ethyl-3-
oxobutanoate according to previously described procedures
(Scheme S2B).57
With the necessary building blocks in hand, we attempted the
key proline-mediated step for formation of ring D. Initial
experiments with crude β-carboline 4 treated with an excess of
enone 5 in the presence of proline, afforded the desired ketones
in moderate yield as a mixture of epimers 6 and 7. Importantly,
we found that the major diastereomer 6 was obtained in high
enantiomeric excess (ee), while the minor diastereomer 7 was
obtained in much lower ee. After optimization of the reaction
Figure 3. Mitragynine and 7-OH do not recruit β-arrestin. (A)
Conceptual representation of the β-arrestin BRET assay employing
atomistic cartoons derived from available X-ray crystal structures. To
measure β-arrestin recruitment, hMOR37 (brown) was coexpressed
with β-arrestin-250 (purple) fused to RLuc839 (teal) and Sp1, GAP43
fused to citrine40 (yellow-green) and SH3, and G protein-coupled
receptor kinase 2 (GRK2). On β-arrestin recruitment, association of
the Sp1 and SH3 domains results in an increase in the BRET signal
between RLuc8 and citrine. (B) β-arrestin recruitment at hMOR,
positive control = DAMGO; curves represent the average of n ≥ 3,
with error bars representing SEM.
significantly different from that induced by DAMGO. These
results provide a good rationale for future experiments aimed at
examining cellular signaling in relevant neuronal cells and
mapping the effects of structural modifications on the level of
functional selectivity.
SAR Exploration: Strategy. Having confirmed the MOR
agonist activity of both mitragynine and 7-OH, we chose to
explore several key structure−activity relationships (SAR)
within this scaffold. In particular, we were interested in the
effects of modifications at three positions: the methoxy group at
position 9, the ethyl group on ring D, and the β-
methoxyacrylate moiety (Figure 4). The aryl methoxy group
at position 9 was of particular interest, as in the classical
morphinan and benzomorphan opioid scaffolds, the change
from aryl methoxy to phenol at key position 3 (e.g., codeine to
morphine), typically results in a dramatic increase in potency.
Likewise, unsubstituted phenyl analogues in these scaffolds also
exhibit weak activity.51 The importance of a phenolic moiety in
E
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Figure 5. Retrosynthesis of mitragynine.
Scheme 1. Stereoselective Formation of D-Ring
conditions (it proved crucial to purify the β-carboline by
chromatography), the desired ketone isomer 6S could be
obtained in good yield and excellent enantiomeric excess
(Scheme 1A). To rationalize the stereoselectivity of this
transformation, a two step mechanism (Scheme 1B, D-proline
shown) can be imagined, in which a Mannich-like reaction
between the proline enamine of enone 5 and the β-carboline 4,
first sets the stereocenter at position 3. This is followed by
intramolecular Michael addition to close the ring. In the last
step, proline controls the face of protonation during hydrolysis,
thereby also influencing the stereocenter at position 19. Proline
is believed to be involved in control of the stereochemistry at
the 19-position, as the diastereomeric ratio obtained in the
reaction varies, and is different from that observed when either
ketone product (6 or 7) is epimerized under basic conditions.
Thus, the orientation of this stereocenter is not due simply to
thermodynamic equilibration of the ketone product.
less favorable axial configuration. As an alternative strategy, we
envisioned that pseudoallylic strain in the ene-ester product of a
Horner−Wadsworth−Emmons (HWE) reaction, or in the
corresponding transition state, might be used to perturb the
equilibrium away from the equatorial alignment.58 Therefore,
ketone 6S was treated with the carbanion formed from methyl
diethylphosphonoacetate, and partial epimerization was indeed
observed (Scheme 2A). The ene-ester with the desired axial
ethyl group was obtained as a mixture of E and Z isomers, 8E
and 8Z (55 mol % of total product by NMR), while the ene-
ester with retention of configuration at the ethyl group, 9, was
obtained exclusively as the E isomer (45 mol % of total product
by NMR). Unfortunately, the low reactivity of ketone 6S
necessitated the use of large excesses of phosphonoaceate, a
requirement that led to partial transesterification of the methyl
ester (products 8 and 9 contained ∼25 mol % of the
corresponding ethyl ester).
With key intermediate ketone 6S in hand, the next challenge
of the synthesis was to effect epimerization of the pendant ethyl
group into the appropriate β stereochemical configuration (as
in mitragynine). This could not be accomplished by
thermodynamic equilibration of the ketone with base or acid,
as the desired stereochemistry positions the ethyl group in the
With the ethyl group successfully epimerized to the
appropriate position, the mixed ene-esters 8E/Z were
converted to (−)-mitragynine by a sequence of several key
steps, including the simultaneous reduction of the ene-ester and
detosylation with magnesium, transesterification, Claisen
condensation (formylation), and O-methylation59 of the enol-
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Scheme 2. Total Synthesis of Mitragynine in Both Enantiomeric Forms
ester intermediate 10 (Scheme 2A). The final O-methylation
step provided both (−)-mitragynine and the isomeric analogue
(Z)-mitragynine (11) in modest yields. Starting from ketone
6R and employing the same synthetic sequence, the unnatural
enantiomer, (+)-mitragynine, was also obtained (Scheme 2B).
Comparison of this material to the natural product by chiral
HPLC confirmed their opposite stereochemistry, and thus, the
absolute stereochemistry of the ketone products 6 and 7
obtained in the proline-catalyzed cyclization.
Synthesis of Desethylmitragynine. Considering that
speciogynine, which is epimeric with mitragynine at the
pendant ethyl group on ring D, was inactive as an opioid
agonist in vitro, we reasoned that the nature and orientation of
the substituent at this position was important for activity, and
thus decided to further define the tolerance of the scaffold in
this region via synthesis of the desethylmitragynine analogue 3
(Figure 4). This dramatically modified analogue was prepared
by a synthetic sequence analogous to that applied for the
synthesis of mitragynine, starting with the treatment of β-
carboline 4 with methyl vinyl ketone in the presence of D-
proline (Scheme S3).
Opioid Activity of Synthetic Analogues. With a variety
of semi- and fully synthetic mitragynine derivatives in hand, key
SAR of this molecular scaffold at the opioid receptors could be
further explored. In particular, the synthesized analogues
allowed investigation of the key moieties highlighted above,
namely the aryl methoxy group at position 9, the β-
methoxyacrylate moiety, and the ethyl group on ring D (see
above, SAR Exploration: Strategy), as well as the importance of
the absolute stereochemistry. As expected, the unnatural
enantiomer (+)-mitragynine was found to be a much weaker
agonist at hMOR, in terms of both potency and maximal
efficacy (Table 3). Surprisingly, however, in contrast to
naturally occurring (−)-mitragynine, it was found to be a low
potency partial agonist at hKOR (EC50 = 9.1 4.3 μM; Emax
=
31%). Apparently, inversion of the stereochemistry in this
scaffold is sufficient to switch from antagonist to agonist activity
at hKOR.
In examining the specific functional groups identified above
(Figure 4), we observed several important trends. For one, the
switch from a methoxy group to a hydroxy group at position 9
had very little effect on activity at hMOR, with the phenolic
analogue 1 being of similar potency and efficacy to mitragynine
itself (Table 3). This result suggests that the mitragynine
scaffold is likely to adopt a unique binding pose compared to
classical morphinan- and benzomorphan-derived opioids, which
display a dramatic relationship between their MOR activity and
an exposed phenol group. Moving to the β-methoxyacrylate
moiety, removal of the enol ether, as in the total synthesis
intermediate 2, completely eliminated activity at hMOR (both
agonist and antagonist). In contrast, the very close analogue
(Z)-mitragynine (11), was found to possess very similar activity
to the natural product, indicating that stereochemical inversion
of the β-methoxyacrylate moiety is tolerated (Table 3). Lastly,
we turned our attention to the ethyl group on ring D. Results
with speciogynine (Table 1) had already demonstrated that
epimerization of this group was sufficient to switch from
agonist to antagonist activity at hMOR, while also reducing
binding affinity (see above, Tables 1 and 2, mitragynine vs
speciogynine). Examining the desethylmitragynine analogue 3,
we found that this derivative retained agonist activity at hMOR,
but with much lower potency (Table 3). Thus, the substituent
at position 19 is important for controlling both efficacy (agonist
vs antagonist) and potency. The synthetic derivatives described
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bonding network with H2976.52, an interaction that is common
to opioid ligands in the morphinan class (see above for
discussion, SAR Exploration: Strategy).37,51,53 In contrast, our
docking studies suggest that the methoxyindole domain of
(−)-mitragynine is preferentially directed toward an alternative
hydrophobic pocket formed by residues of TM2 and TM3
(Figure 6A), which appears to lack a means for forming an
analogous hydrogen bonding network. This result is in
agreement with the minimal activity difference observed in
vitro between (−)-mitragynine and phenolic derivative 1
(Table 3). Instead, the enol ether domain (part of the β-
methoxyacrylate) of mitragynine appears to be directed into the
same region as the phenol of BU72 (toward TM5 and TM6).
Thus, it can be speculated that this functional group
participates in an analogous hydrogen bonding network with
H2976.52, consistent with the complete loss of activity when this
group was removed (see compound 2, Table 3).
Table 3. Agonist Activity of Synthetic Analogues at hMOR as
Determined in G Protein BRET Assays
The importance of the ethyl group on ring D is also
explained by the predicted lowest-energy binding pose of
(−)-mitragynine. In comparing the orientation of this ligand
with that of BU72 (Figure 6A and Figure S7, respectively), it
can be seen that the ethyl group occupies a nearly identical
region as the N-methyl group of BU72, being in the vicinity of
several hydrophobic residues (M1513.36, Y1483.33, and Y3267.43).
Accordingly, loss of this group, as in compound 3, would be
expected to diminish hydrophobic ligand−receptor interactions
and reduce activity, as was observed (Table 3).
Docking of other mitragynine analogues described above also
produced results that were in agreement with the in vitro
activity data. For example, in accord with its similar in vitro
activity to (−)-mitragynine, (Z)-mitragynine (11) adopted a
nearly identical binding pose (Figure 6B). Likewise, the
predicted low-energy binding pose of 7-OH was also similar,
with all key functionalities (the acrylate, ethyl group, tertiary
amine, and indole) occupying very similar positions (Figure
6C). Interestingly, the 7-position hydroxy group does not
appear to make a close contact with the receptor surface, and
thus, potential hydrogen bonding interactions do not seem to
be involved in the significant increase in potency afforded by
oxidation at position 7. Instead, the slight bend introduced to
the core structure by this modification appears to be more
important. Notably, while (−)-mitragynine interacts with
W2936.48 through its acrylate group, 7-OH interacts with this
residue through both ethyl and acrylate groups, according to
the chosen cutoff for apolar interactions (4.5 Å between heavy
atoms; see Table S3). In contrast, docking of the unnatural
enantiomer, (+)-mitragynine, revealed a dramatically different
binding pose, reversed in its overall orientation in the pocket
(i.e., with the acrylate projecting toward TM2 and TM3, see
Figure S8A and compare with Figure 6A), in agreement with its
weak activity in the functional assays.
a
Agonist activity at hMOR indicated by EC50 values, maximal efficacy
(Emax) relative to DAMGO in parentheses; All data points represent
mean SEM (μM) of n ≥ 3.
here ((+)-mitragynine, 1, 2, 3, and 11) were also found to be
without activity at hKOR and hDOR (agonist and antagonist
mode, EC50 and IC50 both >30 μM).
Molecular Docking Reveals a Unique Binding Pose. In
order to rationalize our empirical SAR observations in the
mitragynine scaffold and inform future rational design, we
conducted preliminary molecular docking studies. Specifically,
AutoDock60 was used to dock mitragynine and several of the
analogues described above in the binding pocket of the agonist-
bound X-ray crystal structure of mMOR (PDBID = 5C1M)37
(Figure 6).
The top-scored binding pose of (−)-mitragynine in the
orthosteric site of the receptor partially overlapped with the
binding pose of the cocrystallized morphinan-derived agonist,
BU72, but exhibited some dramatic differences (compare
Figure 6A to Figure S7). Although (−)-mitragynine and BU72
share a polar interaction between their protonated amines and
D1473.32, which is well-known to be critical for the binding of
classical opioid agonists and antagonists,37,53 other important
ligand−receptor contacts are significantly different. In BU72
(Figure S7), the phenol occupies a hydrophobic pocket formed
by TM5 and TM6 and forms a water-mediated hydrogen
We also docked paynantheine, speciogynine, and specioci-
liatine, compounds which bind to hMOR, but exhibit no
agonist activity, instead serving as competitive antagonists.
Considering paynantheine and speciogynine, the inverted
stereochemistry at position 19 (vinyl or ethyl group) induces
a significant shift of the indole moiety toward TM1 and away
from TM3 (Figure 6D), which results in the formation of
additional contacts with TM1 (Y751.39) and TM6 (H3197.36
)
that are not seen in the docking of the active compounds.
Moreover, the hydrophobic interactions between the indole
methoxy group and V1433.28 and I1443.29 observed with
(−)mitragynine are lost in these analogues, potentially resulting
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Figure 6. Docking of (−)-mitragynine and other analogues to the active μ-opioid receptor crystal structure. Top-scoring binding poses of (A)
(−)-mitragynine, (B) (Z)-mitragynine, (C) 7-hydroxymitragynine, and (D) antagonists paynantheine (pink) and speciogynine (cyan). Only residue
side chains within 4 Å of the ligand are reported. Polar interactions are shown as dotted lines. TM helices are shown in cartoon representation (in
gray). ECL2 and part of TM5 have been omitted for clarity. Residues are labeled using one-letter amino acid code and Ballesteros and Weinstein’s
generic numbering scheme.
in reduced affinity for the active conformation of the receptor
and corresponding antagonist activity. Consistent with the
inverted stereochemistry of the core structure at position 3,
docking of speciociliatine revealed a completely different
binding pose with its indole group extending toward TM3
and forming a polar interaction with Y1483.33 (Figure S8B and
Table S3). Notably, unlike all other compounds reported here,
the acrylate moiety in speciociliatine is in axial rather than
equatorial position, pointing toward TM5 and TM6. All in all, a
number of different stereochemical configurations in the
Mitragyna alkaloid family appear to be sufficient to retain
binding at hMOR, while the absolute stereochemistry found in
mitragynine is required for activation of the receptor.
of competing agonist and antagonist effects at the opioid
receptors. It was also found that the potent oxidized analogue
7-OH is not necessarily present in all extracts of plant material
(as previously suggested), so the potential contribution of this
alkaloid to the effects of Mitragyna speciosa is not general, unless
7-OH is formed as a metabolite.
Exploration with several synthetic mitragynine analogues
allowed us to define basic SAR at each of the existing
substituents, including in regions (the D ring) that were only
accessible to derivatization via total synthesis. This work
revealed that opioid activity in the mitragynine scaffold is quite
sensitive to structural modification and suggested that the
Mitragyna alkaloids adopt a unique binding pose at MOR,
distinct from that of classical opioids. This possibility was
further supported by molecular docking studies at MOR. The
first total synthesis of the unnatural enantiomer, (+)-mitragy-
nine, likewise allowed us to confirm that the naturally occurring
isomer is the more active opioid agonist.
We also found that mitragynine and 7-OH display biased
signaling at MOR, being selective for the G protein pathway.
Thus, it is hoped that this scaffold may represent a starting
point for the development of novel opioid analgesics with
reduced side effects. Relatedly, the finding that, in addition to
their activity at MOR, both mitragynine and 7-OH are KOR
antagonists, suggests that Mitragyna alkaloids or their synthetic
derivatives may also serve as leads for novel dual-action
antidepressants. In this regard, mitragynine itself is of particular
interest, as it acts as both a low efficacy MOR partial agonist,
CONCLUSION
■
In this work, we have presented the first thorough, in vitro
characterization of the opioid receptor pharmacology and
signaling of the natural Mitragyna alkaloids in receptor-level
functional assays. The partial agonist activity of (−)-mitragy-
nine at MOR is likely to account for many of the effects of
Mitragyna speciosa extracts in humans. However, the other
major alkaloids isolated from the plant, paynanthiene,
speciogynine, and speciociliatine, all exhibited competitive
antagonist activity at this receptor. Considering that cumu-
latively, these secondary alkaloids accounted for an approx-
imately equal percentage of the total alkaloid content compared
to mitragynine in our extracts, the gross psychoactive effects of
crude plant material are likely to represent a complex interplay
I
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(3) National Institute on Drug Abuse. Overdose Death Rates.
https://www.drugabuse.gov/related-topics/trends-statistics/overdose-
death-rates (Accessed September 1, 2015).
(4) Quinones, S. Dreamland: The True Tale of America’s Opiate
Epidemic; Bloomsbury Press: New York, 2015.
(5) Pasternak, G. W. Clin. J. Pain 2010, 26 (Supplement 10), S3−S9.
(6) Grinnell, S. G.; Majumdar, S.; Narayan, A.; Le Rouzic, V.;
Ansonoff, M.; Pintar, J. E.; Pasternak, G. W. J. Pharmacol. Exp. Ther.
2014, 350, 710−718.
(7) Largent-Milnes, T.; Yamamoto, T.; Nair, P.; Moulton, J.; Hruby,
V.; Lai, J.; Porreca, F.; Vanderah, T. Br. J. Pharmacol. 2010, 161, 986−
1001.
and a KOR antagonist. Both of these mechanisms have been
shown to elicit antidepressant effects in animals, and in the case
of MOR, humans as well (see above). However, modulators of
the KOR have received considerably more attention in the
psychiatry community in recent years, likely due to their
apparent lack of abuse potential. Antagonists of this receptor
are antidepressant in animal models and oppose a variety of
stress-related responses that are believed to be mediated
through the endogenous KOR agonists, dynorphins.61−63
Further, several agents in this class have entered clinical trials
for MDD, but none have yet reached the market.64 Given the
ability of mitragynine to perturb both of these opioid signaling
systems in the direction consistent with mood enhancement, it
is unsurprising that it has been shown to exhibit antidepressant
activity in the forced swim test (a rodent model of
depression).65 Therefore, Mitragyna alkaloids also hold promise
in the treatment of mood disorders.
(8) Stevenson, G. W.; Luginbuhl, A.; Dunbar, C.; LaVigne, J.; Dutra,
J.; Atherton, P.; Bell, B.; Cone, K.; Giuvelis, D.; Polt, R.; Streicher, J.
M.; Bilsky, E. J. Pharmacol., Biochem. Behav. 2015, 132, 49−55.
(9) Kraepelin, E. Einfuhrung in die psychiatrische Klinik: Zweiund-
̈
dreissig Vorlesungen; Barth: Leipzig, 1905.
(10) Gerner, R. H.; Catlin, D. H.; Gorelick, D. A.; Hui, K. K.; Li, C.
H. Arch. Gen. Psychiatry 1980, 37, 642−647.
(11) Stoll, A. L.; Rueter, S. Am. J. Psychiatry 1999, 156, 2017.
(12) Dean, A. J.; Bell, J.; Christie, M. J.; Mattick, R. P. Eur. Psychiatry
2004, 19, 510−513.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.6b00360.
(13) Shapira, N. A.; Verduin, M. L.; DeGraw, J. D. J. Clin. Psychiatry
2001, 62, 205−206.
(14) Shapira, N. A.; Keck, P. E.; Goldsmith, T. D.; McConville, B. J.;
Eis, M.; McElroy, S. L. Depression Anxiety 1997, 6, 170−173.
(15) Emrich, H. M.; Vogt, P.; Herz, A. Ann. N. Y. Acad. Sci. 1982,
398, 108−112.
Copies of NMR spectra. (PDF)
Synthetic procedures and spectral data for all com-
pounds, additional binding data at rodent receptors,
additional functional data at rodent and human
receptors, and additional docking results (PDF)
(16) Karp, J. F.; Butters, M. A.; Begley, A. E.; Miller, M. D.; Lenze, E.
J.; Blumberger, D. M.; Mulsant, B. H.; Reynolds, C. F. J. Clin.
Psychiatry 2014, 75, e785−e793.
AUTHOR INFORMATION
Corresponding Author
*ds584@columbia.edu
(17) Bodkin, J. A.; Zornberg, G. L.; Lukas, S. E.; Cole, J. O. J. Clin.
Psychopharmacol. 1995, 15, 49−57.
■
(18) Besson, A.; Privat, A. M.; Eschalier, A.; Fialip, J.
Psychopharmacology 1996, 123, 71−78.
Author Contributions
́
(19) Rojas-Corrales, M. O.; Berrocoso, E.; Gibert-Rahola, J.; Mico, J.
#A.C.K. and M.M.G. contributed equally to this work.
A. Life Sci. 2002, 72, 143−152.
Notes
(20) Fichna, J.; Janecka, A.; Piestrzeniewicz, M.; Costentin, J.; do
The authors declare no competing financial interest.
Rego, J.-C. Neuropsychopharmacology 2007, 32, 813−821.
́
(21) Rojas-Corrales, M. O.; Gibert-Rahola, J.; Mico, J. A. Life Sci.
ACKNOWLEDGMENTS
1998, 63, PL175−PL180.
(22) Gassaway, M. M.; Rives, M.-L.; Kruegel, A. C.; Javitch, J. A.;
Sames, D. Transl. Psychiatry 2014, 4, e411.
(23) Takayama, H. Chem. Pharm. Bull. 2004, 52, 916−928.
(24) Adkins, J. E.; Boyer, E. W.; McCurdy, C. R. Curr. Top. Med.
Chem. 2011, 11, 1165−1175.
(25) Raffa, R. B.; Beckett, J. R.; Brahmbhatt, V. N.; Ebinger, T. M.;
Fabian, C. A.; Nixon, J. R.; Orlando, S. T.; Rana, C. A.; Tejani, A. H.;
Tomazic, R. J. J. Med. Chem. 2013, 56, 4840−4848.
(26) Matsumoto, K. Pharmacological Studies on 7-Hydroxymitragy-
nine, Isolated from the Thai Herbal Medicine Mitragyna speciosa:
Discovery of an Orally Active Opioid Analgesic. Ph.D. Dissertation,
Chiba University, Chiba, Japan, 2006.
■
The authors would like to thank Kevin Wulf for illustration of
Figure 1, and Professor Bryan L. Roth and the National
Institute of Mental Health’s Psychoactive Drug Screening
Program for their assistance in determining binding affinities at
the human opioid receptors. Work in the Sames laboratory was
supported by the Interdisciplinary Research Initiatives Seed
(IRIS) Fund Program of Columbia University College of
Physicians & Surgeons. Work in the Javitch laboratory was
supported by National Institutes of Health (NIH) grants
MH054137 and DA022413 (to J.J.). Work in the Filizola
laboratory was supported by NIH grants DA026434 and
DA034049 (to M.F.). Computations were run on resources
available through the Scientific Computing Facility at Mount
Sinai and the Extreme Science and Engineering Discovery
Environment (XSEDE) under MCB080109N, which is
supported by National Science Foundation grant number
OCI-1053575. Work in the Majumdar laboratory was
supported by NIH grant DA034106 (to S.M.) and in part
through the NIH/NCI Cancer Center Support Grant P30
CA008748.
(27) Takayama, H.; Ishikawa, H.; Kurihara, M.; Kitajima, M.; Aimi,
N.; Ponglux, D.; Koyama, F.; Matsumoto, K.; Moriyama, T.;
Yamamoto, L. T.; Watanabe, K.; Murayama, T.; Horie, S. J. Med.
Chem. 2002, 45, 1949−1956.
́
(28) Leon, F.; Habib, E.; Adkins, J. E.; Furr, E. B.; McCurdy, C. R.;
Cutler, S. J. Nat. Prod. Commun. 2009, 4, 907−910.
(29) Ponglux, D.; Wongseripipatana, S.; Takayama, H.; Kikuchi, M.;
Kurihara, M.; Kitajima, M.; Aimi, N.; Sakai, S. Planta Med. 1994, 60,
580−581.
(30) Matsumoto, K.; Horie, S.; Ishikawa, H.; Takayama, H.; Aimi, N.;
Ponglux, D.; Watanabe, K. Life Sci. 2004, 74, 2143−2155.
(31) Matsumoto, K.; Mizowaki, M.; Suchitra, T.; Takayama, H.;
Sakai, S.; Aimi, N.; Watanabe, H. Life Sci. 1996, 59, 1149−1155.
(32) Macko, E.; Weisbach, J. A.; Douglas, B. Arch. Int. Pharmacodyn.
Ther. 1972, 198, 145−161.
REFERENCES
■
(1) Pasternak, G. W.; Pan, Y.-X. Pharmacol. Rev. 2013, 65, 1257−
1317.
(2) Inturrisi, C. E. Clin. J. Pain 2002, 18 (4 Suppl), S3−S13.
J
DOI: 10.1021/jacs.6b00360
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
(33) Boyer, E. W.; Babu, K. M.; Adkins, J. E.; McCurdy, C. R.;
Halpern, J. H. Addiction 2008, 103, 1048−1050.
(34) Matsumoto, K.; Mizowaki, M.; Suchitra, T.; Murakami, Y.;
Takayama, H.; Sakai, S.; Aimi, N.; Watanabe, H. Eur. J. Pharmacol.
1996, 317, 75−81.
(35) Matsumoto, K.; Narita, M.; Muramatsu, N.; Nakayama, T.;
Misawa, K.; Kitajima, M.; Tashima, K.; Devi, L. A.; Suzuki, T.;
Takayama, H.; Horie, S. J. Pharmacol. Exp. Ther. 2014, 348, 383−392.
(36) Dale, O.; Ma, G.; Gemelli, C.; Husni, A.; McCurdy, C.; Avery,
B.; Leon, J.; Furr, E.; Manly, S.; Cutler, S. Planta Med. 2012, 78, 91.
(37) Huang, W.; Manglik, A.; Venkatakrishnan, A. J.; Laeremans, T.;
Feinberg, E. N.; Sanborn, A. L.; Kato, H. E.; Livingston, K. E.;
Thorsen, T. S.; Kling, R. C.; Granier, S.; Gmeiner, P.; Husbands, S. M.;
Traynor, J. R.; Weis, W. I.; Steyaert, J.; Dror, R. O.; Kobilka, B. K.
Nature 2015, 524, 315−321.
(61) Mague, S. D.; Pliakas, A. M.; Todtenkopf, M. S.; Tomasiewicz,
H. C.; Zhang, Y.; Stevens, W. C.; Jones, R. M.; Portoghese, P. S.;
Carlezon, W. A. J. Pharmacol. Exp. Ther. 2003, 305 (1), 323−330.
(62) Land, B. B.; Bruchas, M. R.; Lemos, J. C.; Xu, M.; Melief, E. J.;
Chavkin, C. J. Neurosci. 2008, 28 (2), 407−414.
(63) Bruijnzeel, A. W. Brain Res. Rev. 2009, 62 (1), 127−146.
(64) Urbano, M.; Guerrero, M.; Rosen, H.; Roberts, E. Bioorg. Med.
Chem. Lett. 2014, 24 (9), 2021−2032.
(65) Idayu, N. F.; Hidayat, M. T.; Moklas, M. A. M.; Sharida, F.;
Raudzah, A. R. N.; Shamima, A. R.; Apryani, E. Phytomedicine 2011,
18, 402−407.
(38) Rasmussen, S. G. F.; DeVree, B. T.; Zou, Y.; Kruse, A. C.;
Chung, K. Y.; Kobilka, T. S.; Thian, F. S.; Chae, P. S.; Pardon, E.;
Calinski, D.; Mathiesen, J. M.; Shah, S. T. A.; Lyons, J. A.; Caffrey, M.;
Gellman, S. H.; Steyaert, J.; Skiniotis, G.; Weis, W. I.; Sunahara, R. K.;
Kobilka, B. K. Nature 2011, 477, 549−555.
(39) Loening, A. M.; Fenn, T. D.; Gambhir, S. S. J. Mol. Biol. 2007,
374, 1017−1028.
(40) Rekas, A.; Alattia, J.-R.; Nagai, T.; Miyawaki, A.; Ikura, M. J. Biol.
Chem. 2002, 277, 50573−50578.
(41) Luttrell, L. M.; Maudsley, S.; Bohn, L. M. Mol. Pharmacol. 2015,
88, 579−588.
(42) Raehal, K. M.; Schmid, C. L.; Groer, C. E.; Bohn, L. M.
Pharmacol. Rev. 2011, 63, 1001−1019.
(43) Kyle, D. J. Research and Development of Opioid-Related Ligands;
Ko, M.-C., Husbands, S. M., Eds.; ACS Symposium Series; American
Chemical Society: Washington, D.C., 2013; Vol. 1131, pp 177−197.
(44) Pradhan, A. A.; Smith, M. L.; Kieffer, B. L.; Evans, C. J. Br. J.
Pharmacol. 2012, 167, 960−969.
(45) Groer, C. E.; Tidgewell, K.; Moyer, R. A.; Harding, W. W.;
Rothman, R. B.; Prisinzano, T. E.; Bohn, L. M. Mol. Pharmacol. 2007,
71, 549−557.
(46) Lamb, K.; Tidgewell, K.; Simpson, D. S.; Bohn, L. M.;
Prisinzano, T. E. Drug Alcohol Depend. 2012, 121, 181−188.
(47) DeWire, S. M.; Yamashita, D. S.; Rominger, D. H.; Liu, G.;
Cowan, C. L.; Graczyk, T. M.; Chen, X.-T.; Pitis, P. M.; Gotchev, D.;
Yuan, C.; Koblish, M.; Lark, M. W.; Violin, J. D. J. Pharmacol. Exp.
Ther. 2013, 344, 708−717.
(48) Soergel, D. G.; Subach, R. A.; Burnham, N.; Lark, M. W.; James,
I. E.; Sadler, B. M.; Skobieranda, F.; Violin, J. D.; Webster, L. R. Pain
2014, 155, 1829−1835.
(49) Clayton, C. C.; Donthamsetti, P.; Lambert, N. A.; Javitch, J. A.;
Neve, K. A. J. Biol. Chem. 2014, 289, 33663−33675.
(50) Zhan, X.; Gimenez, L. E.; Gurevich, W.; Spiller, B. W. J. Mol.
Biol. 2011, 406, 467−478.
(51) Hedberg, M. H.; Johansson, A. M.; Fowler, C. J.; Terenius, L.;
Hacksell, U. Bioorg. Med. Chem. Lett. 1994, 4, 2527−2532.
(52) Ballesteros, J. A.; Weinstein, H. Receptor Molecular Biology;
Methods in Neurosciences; Elsevier, 1995; Vol. 25, pp 366−428.
(53) Manglik, A.; Kruse, A. C.; Kobilka, T. S.; Thian, F. S.;
Mathiesen, J. M.; Sunahara, R. K.; Pardo, L.; Weis, W. I.; Kobilka, B.
K.; Granier, S. Nature 2012, 485, 321−326.
(54) Itoh, T.; Yokoya, M.; Miyauchi, K.; Nagata, K.; Ohsawa, A. Org.
Lett. 2003, 5, 4301−4304.
(55) Itoh, T.; Yokoya, M.; Miyauchi, K.; Nagata, K.; Ohsawa, A. Org.
Lett. 2006, 8, 1533−1535.
(56) Nagata, K.; Ishikawa, H.; Tanaka, A.; Miyazaki, M.; Kanemitsu,
T.; Itoh, T. Heterocycles 2010, 81, 1791−1798.
(57) Surber, W.; Schinz, H. Helv. Chim. Acta 1954, 37, 1239−1251.
(58) Openshaw, H. T.; Whittaker, N. J. Chem. Soc. 1963, 1461−1471.
(59) Bar
́ ́ ́ ́
czai-Beke, M.; Dornyei, G.; Toth, G.; Tamas, J.; Szantay, C.
̈
Tetrahedron 1976, 32, 1153−1159.
(60) Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew,
R. K.; Goodsell, D. S.; Olson, A. J. J. Comput. Chem. 2009, 30, 2785−
2791.
K
DOI: 10.1021/jacs.6b00360
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX