Synthetic Studies of Neoclerodane Diterpenes from Salvia divinorum: Identification of a Potent and Centrally Acting μ Opioid Analgesic with Reduced Abuse Liability

Article abstract of DOI:10.1021/acs.jmedchem.6b01235

Opioids are widely used to treat millions suffering from pain, but their analgesic utility is limited due to associated side effects. Herein we report the development and evaluation of a chemical probe exhibiting analgesia and reduced opioid-induced side effects. This compound, kurkinorin (5), is a potent and selective μ-opioid receptor (MOR) agonist (EC₅₀ = 1.2 nM, >8000 μ/κ selectivity). 5 is a biased activator of MOR-induced G-protein signaling over β-arrestin-2 recruitment. Metadynamics simulations of 5's binding to a MOR crystal structure suggest energetically preferred binding modes that differ from crystallographic ligands. In vivo studies with 5 demonstrate centrally mediated antinociception, significantly reduced rewarding effects, tolerance, and sedation. We propose that this novel MOR agonist may represent a valuable tool in distinguishing the pathways involved in MOR-induced analgesia from its side effects.

Full text of DOI:10.1021/acs.jmedchem.6b01235

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Article
Synthetic Studies of Neoclerodane Diterpenes from
Salvia divinorum: Identification of a Potent and Centrally
Acting µ Opioid Analgesic with Reduced Abuse Liability
Rachel Saylor Crowley, Andrew P. Riley, Alexander M. Sherwood, Chad E. Groer,
Nirajmohan Shivaperumal, Miguel Biscaia, Kelly Paton, Sebastian Schneider,
Davide Provasi, Bronwyn M. Kivell, Marta Filizola, and Thomas E. Prisinzano
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01235 • Publication Date (Web): 28 Nov 2016
Downloaded from http://pubs.acs.org on December 1, 2016
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Synthetic Studies of Neoclerodane Diterpenes
from Salvia divinorum: Identification of a Potent and
Centrally Acting  Opioid Analgesic with Reduced
Abuse Liability
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Rachel Saylor Crowley,1† Andrew P. Riley,2† Alexander M. Sherwood,1 Chad E. Groer,1 Nirajmohan
Shivaperumal,3Miguel Biscaia,3 Kelly Paton,3 Sebastian Schneider,4 Davide Provasi,4 Bronwyn M.
Kivell,3 Marta Filizola,4 and Thomas E. Prisinzano1*
1 Department of Medicinal Chemistry, School of Pharmacy, The University of Kansas, Lawrence, KS,
USA. 2 Department of Chemistry, The University of Kansas, Lawrence, KS, USA. 3School of Biological
Sciences, Centre for Biodiscovery, Victoria University of Wellington, Wellington, New Zealand.
4Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY,
USA. †These authors contributed equally to this work.
prisinza@ku.edu
RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required
according to the journal that you are submitting your paper to)
*To whom correspondence should be addressed. Thomas E. Prisinzano, Professor and Chair, 1251
Wescoe Hall Drive, 4070 Malott Hall, Lawrence, Kansas 66045-7572. Telephone: (785) 864-3267. Fax:
(785) 864-5326.
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Abstract. Opioids are widely used to treat millions suffering from pain, but their analgesic utility is
limited due to associated side effects. Herein we report the development and evaluation of a chemical
probe exhibiting analgesia and reduced opioid-induced side effects. This compound, kurkinorin (5), is a
potent and selective µ-opioid receptor (MOR) agonist (EC50 = 1.2 nM, >8,000 / selectivity). 5 is a
biased activator of MOR-induced G-protein signaling over β-arrestin-2 recruitment. Metadynamics
simulations of 5’s binding to a MOR crystal structure suggest energetically preferred binding modes that
differ from crystallographic ligands. In vivo studies with 5 demonstrate centrally-mediated
antinociception significantly reduced rewarding effects, tolerance, and sedation. We propose that this
novel MOR agonist may represent a valuable tool in distinguishing the pathways involved in MOR-
induced analgesia from its side effects.
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Introduction
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Pain affects nearly 115 million Americans, which is more than cancer, heart disease, and diabetes
combined. Unfortunately, the use of opioids, one of the most common drug classes used to treat pain, is
problematic due to their high abuse liability.1 In 2014, an estimated 54 million people over the age of 12
had used prescription drugs, such as opioids, non-medically in their lifetimes,2 and according to the
Centers for Disease Control and Prevention (CDC), 2014 had the highest rate of drug overdose deaths
than any year prior, with an estimated 40 Americans dying each day from overdosing on prescription
painkillers.3 Thus, a strong analgesic devoid of abuse liabilities is desperately needed in an attempt to
combat this trend and prevent these deaths.
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The prototypical opioid, morphine, has been used for decades for the management of pain, and its
powerful analgesic effects have stimulated innumerable synthetic and semi-synthetic investigations aimed
at optimizing its biological effects.4 While these studies have resulted in most of the clinically useful
treatments for pain, including codeine, oxycodone, meperidine, methadone, and fentanyl, these drugs have
not been able to overcome the drawbacks associated with opioid use. All of these morphine-derived
compounds are -opioid receptor (MOR) agonists and suffer from severe adverse effects such as sedation,
tolerance, dependence, constipation, and respiratory depression.4a Tolerance and dependence are
particularly significant effects because they can lead to opioid addiction, which is often the most
concerning effect for both patients and prescribers.5
Most clinically used MOR ligands are structurally similar to morphine, and they all have very similar
activity profiles. To date, it has been very difficult to differentiate opioid-induced analgesic activity from
abuse liability. The development of novel, non-morphine-derived scaffolds of MOR ligands may allow
for this differentiation. Using the natural product salvinorin A as our novel scaffold, we present herein the
design and synthesis of 5 (kurkinorin), a salvinorin A analogue that demonstrates remarkable potency and
selectivity for MORs. Results from in vitro, in silico, and in vivo evaluations indicate 5 is a useful probe
of MORs and represents a crucial step towards separating analgesic activity from the adverse effects of
current opioid ligands.
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Results and Discussion
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Of the known, naturally occurring opioid ligands, the most structurally diverse is the κ-opioid receptor
(KOR) agonist salvinorin A (1a) found in the leaves of the mint plant Salvia divinorum (Fig. 1). Most
notably, 1a lacks a basic nitrogen that was once believed to be necessary for binding to opioid receptors.6
The structural uniqueness of 1a has led to several semi-synthetic studies aimed at elucidating structure-
activity relationships (SAR).7 Among the information gained from these investigations was that the
orientation of the C2 substituent can have an effect on the affinity and activity at KORs.8 It was also
demonstrated that replacement of the C2 acetate with a benzoate resulted in a loss of activity at KORs but
an increase in MOR activity. This analogue, referred to as herkinorin (2), represents the first non-
nitrogenous MOR agonist.9 Further evaluation of 2’s interaction with MORs indicated that it was a
functionally selective compound, as it activates MORs without promoting the recruitment of β-arrestin-2
or internalization of the receptor, both of which have been indicated to play a critical role in development
of morphine-like side-effects.10 Although 2 has proven to be a useful tool for probing MORs in vitro10-11
and in vivo,12 it lacks sufficient potency (EC50 = 39.0  4.0 nM at MOR) and selectivity (4-fold selective
for MOR over KOR) for further exploration of more complex systems of pain and drug abuse. The utility
of 2 is also limited by the fact that it is peripherally restricted,12b and thus cannot be used to probe centrally
mediated processes.
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Interestingly, the structurally similar amide 3 is significantly more potent and selective for MORs than
2.11a On the basis of molecular modeling, it was suggested that differences in receptor binding and
regulation for 2 and 3 are the result of differing phenyl rings orientations.11a To support this hypothesis,
we have recently solved the first X-ray crystal structures for both 2 and 3 (Supplementary Fig. 1). A
comparison of the two structures indicates that indeed, the phenyl ring of 3 lies more in the plane with the
decalin core than the phenyl ring of 2. Although these structures represent only two possible
conformations, and not necessarily the conformations bound to the receptor, they suggested that
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modifying the configuration of the C2 position of 2 might result in improved selectivity and potency for
MORs.
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Based on the previously understood activities of 1a, 2, and 3 combined with this new understanding of
the X-ray crystal structures of 2 and 3, it was envisioned that locking the conformation of the C2
substituent would impart selectivity between the MOR and KORs. The selectivity of 3 could be attributed
to one of two different interactions occurring due to its conformational restriction, one being the
coplanarity of the phenyl and decalin ring systems and the other being an upward movement of the C2
substituent from the - to the -face of the decalin core. Previous studies have demonstrated that inversion
of the C2 substituent of 1a to 1c (Scheme 1) results in a loss of activity at the KOR.8, 13 Thus, reduced
KOR activity and increased MOR activity was expected if the substituent was locked in the plane of the
decalin core, as seen in the crystal structure of 3, but to determine if further movement to the -face was
tolerated, or even preferred, the C2 stereochemistry had to be inverted.
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Although the synthesis for analogues of 1a and 2 with inverted C2 centers has been described
previously, 1c and 4 (Scheme 1), respectively,13 the effects of this inversion on MOR and KOR activity
have not been fully reported. Furthermore, the introduction of an additional degree of unsaturation
between C2 and C3 resulting in 5 (Scheme 1), was envisioned to lock the C2 substituent in the plane of
the decalin core. We discuss herein the synthetic strategies employed to arrive at 5 and how these
modifications to the orientation of the C2 substituents affects potency and selectivity at MORs and KORs.
Chemical Synthesis
As mentioned above, the synthesis of compounds and 1c and 4 have been previously reported (Scheme
1).13 Access to 5 containing the additional degree of unsaturation was envisioned to arise from the
oxidation of salvinorin B (6) to a transient diketone with subsequent tautomerization to form diosphenol
7a. Initial attempts to prepare 7a via a Swern oxidation led to only trace amounts of product. However,
Cu(OAc)2 accomplished the transformation in more acceptable yields (Scheme 1).14 Although remaining
6 could be removed from the reaction mixture via silica gel chromatography, a rapid equilibration between
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the pseudosymmetric -diketone to the two isomeric enols 7a and 7b prevented further purification.
Following chromatography, immediate characterization of 7a by NMR spectroscopy revealed a 3:1
mixture of 7a and 7b. However upon standing, solutions containing 7a slowly equilibrated to
thermodynamically stable enol 7b. Pure samples of 7b were readily obtained.
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To further explore the nature of the oxidation and tautomerization of 6, it was determined that the action
of lead tetraacetate in MeOH/benzene on 6 produced similar mixtures of products corresponding to 7a-b
by TLC. Following isolation of the mixture, chromatographic fractions were combined and subjected to
slow evaporation resulting in crystalline 7b, which proved to be spectroscopically identical to the major
product obtained from the Cu(OAc)2 reaction and was unambiguously characterized by single crystal X-
ray diffraction. Surprisingly, acylation of either the mixture of 7a-b or pure 7b with benzoyl chloride
provided a single regioisomer (Scheme 1), confirmed to be the desired 5 by 2D NMR studies. Briefly, the
C3 proton correlated via COSY to the C4 proton and via HMBC to C1, C2, C4, and C5. The proton of
C10 lacked any observable COSY correlations. Additionally, HSQC confirmed that carbons C3 and C10
each bear a single proton, further validating the regiochemistry of 5 (see Supplementary Fig. 2).
To elucidate additional SAR for this modification, two parallel series of analogues, one with and one
without saturation at the C2-C3 bond, possessing substitutions at the phenyl ring were synthesized
(Scheme 2). Due to an observed variability in the purities of commercially available acid chlorides,
commercially available carboxylic acids were coupled to 6 and 7a through the aid of EDC and DMAP
(Scheme 1). Compounds 8a – 22a were synthesized using a MiniBlock XT parallel synthesis system and
purified using mass-directed reverse phase HPLC. Compounds 8b – 22b proved to be unstable to this
purification process and were synthesized in batch mode and purified using a combination of silica gel
chromatography and preparative reverse phase HPLC.
In Vitro Opioid Receptor Activity
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To determine how the configuration of the C2 position in 2 affects potency and selectivity at MORs,
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the C2-epimerized analogue 413 and the unsaturated analogue 5 were evaluated for activity at MORs and
KOR. Both compounds were tested for opioid activity using a functional assay that measures forskolin-
induced cAMP accumulation in CHO cells stably expressing either MORs KORs, or -opioid receptors
(DORs) (Table 1).15 The epimerization of 2 at the C2 position (e.g. 4) resulted in a complete loss of
activity at both MOR and KORs. Of note, previously published activity profiles of 2 at KOR indicate that
it is a full agonist, while in this assay it appears to only be a partial agonist. Differences in the functional
readouts can account for these changes, and are not surprising as many compounds activate different
pathways to different extents. The introduction of an additional degree of unsaturation to the core of 2,
however, resulted in a potent MOR agonist, 5, with an EC50 value of 1.2 ± 0.6 nM, possessing an improved
activity profile compared to that of 3. Additionally, 5 was extremely selective for MORs, possessing no
activity at KORs at 10 M. This impressive >8,000-fold selectivity for MORs is drastically greater than
that of both 2 (4.25-fold selective over KOR) and morphine (66-fold selective over KOR). Furthermore,
5 is of similar potency to DAMGO, a peptidic MOR ligand commonly employed for its high potency and
selectivity for MORs. 5 does have an EC50 values of 74 ± 10 nM, which was unanticipated as few
salvinorin-like compounds have DOR activity. Even so, 5’s MOR selectivity is still greater than 60-fold.
However, this result opens the possibility of identifying DOR selective neoclerodanes in the future.
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To confirm that this MOR activity and selectivity cannot be attributed to the product of ester hydrolysis
of 5, both 7a and 7b were tested for MOR and KOR activity. While 7a cannot be fully resolved, and thus
fully characterized, neither compound tested showed any activity at MORs (>10,000 nM). Interestingly,
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the 3:1 mixture of 7a:7b (as characterized by H NMR) was active at KORs, however pure 7b was
completely inactive (Table 1).
Although the MOR and KORs share a high degree of similarity, the selectivity of 5 for MORs over
KORs supported our hypothesis that altering the conformational restriction of the C2 substituent is able
to grant preferential binding for one receptor over the other and thus provides a tool for designing selective
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ligands. To further confirm the hypothesis that the KOR does not tolerate this conformational constraint,
the unsaturated and C2-epimerized analogues (1b and 1c, respectively) of 1a were synthesized and
evaluated at KOR. Compound 1b was synthesized from 6 as described above and 1c as described
previously.8 Neither of these changes improved potency at the KOR or resulted in MOR activity. In fact,
both the desaturation and the epimerization of 1a decreased potency at the KOR by 20-fold and >3,000-
fold, respectively (Table 1). These data indicate that stereochemical modifications at C2 are not tolerated
at the KOR, and may be a reason for the high selectivity of 5 at MORs.
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The improved potency and selectivity of 5 clearly warranted additional studies to establish SAR for
compounds containing this modified core, thus analogues of 2 and 5 were evaluated for their activity at
MORs (Supplementary Table 1). All of the analogues evaluated retained full efficacy at the MORs. In
all cases, the unsaturated variants were more potent than their saturated analogues, by a factor of 1.3 to
97-fold. A scatter plot comparison of the pEC50 of the two libraries shows a clear correlation of activity
between the two series, indicating that parallel changes in structure correspond to parallel changes in
activity (Fig. 2). This correlation suggests that both the saturated and unsaturated series of analogues bind
at a similar location in the MORs. Generally, substitutions at the 4-position were well tolerated, with 3-
substituted analogues being slightly less active, and 2-substituted analogues being considerably less active
than the 4-substituted analogues. Of these substituted phenyl analogues, the chloro- and nitro-substituted
analogues were the least potent, indicating that electron-poor aromatic rings may be detrimental to
activity. Furthermore, none of the substituted phenyl analogues were more potent than 5, suggesting that
the additional steric bulk of the substitutions is poorly tolerated and thus reduces the potency of the
compounds.
Compounds that displayed potencies below 100 nM at MORs (8b, 9b, 10b, 13b, 16b, 21b, and 22b)
were additionally tested at KORs and DORs to further assess their selectivity (Table 2). Compounds 8b
and 10b were the only analogues active at KOR with EC50 values of 160 ± 50 and 260 ± 120 nM,
respectively. All of these compounds possessed weaker activity at DORs relative to MORs. Furthermore,
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all analogues were less selective for MOR than 5 itself, with selectivity values ranging from 10 to 56-fold
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Compounds 8b, 9b, 10b, 13b, 16b, 21b, and 22b were further evaluated for their ability to recruit β-
arrestin-2 through MOR activation. The DiscoverX (Fremont, CA) β-arrestin PathHunterTM technology
was used for this analysis, which utilizes enzyme fragment complementation (EFC). Both the receptor
and β-arrestin-2 are tagged with a fragment of β-galactosidase that is only activated upon
complementation. The recruitment of β-arrestin-2 to the receptor results in the activated enzyme, and the
addition of substrate that is converted into a luminescent product allows for a dose-dependent increase in
luminescence. This luminescence is then used to determine the extent of β-arrestin-2 recruitment.
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DAMGO has previously been shown to highly recruit β-arrestin-2 through MOR activation,16 and was
thus used as the positive control to which all data were normalized. Morphine does not recruit to the same
extent as DAMGO, and in the EFC assay utilized herein, this trend holds true (Table 3). As mentioned
above, 2 has been shown to activate the MOR without recruiting β-arrestin-2,10-11 however previous assays
have employed fluorescent whole-cell imaging, and in this EFC assay, 2 does weakly recruit β-arrestin-2
with an EC50 > 3 µM and an efficacy of 72%. The previously published trend that 3 recruit β-arrestin-2
to a higher extent than 2 holds true in this assay, and as would be expected from its conformational
similarity to 3, 5 recruits β-arrestin-2 as well. All of the 5-like analogues tested had efficacies in the 70-
90% ranges, with the exception of 16b which was more efficacious than DAMGO at 110% efficacy, but
5 itself induced recruitment at the same efficacy as DAMGO.
The differing trends between 5 and its analogues in the two pathways evaluated suggest a signaling
bias. Much work has been done to develop models for quantifying this bias that accounts for the
differences between the assays and pathways,17 and a simplified equation is used herein that normalizes
data to the activity of DAMGO in both assays, similar to previous work17c, 17e (see Experimental section
for full equation). Bias factor values less than 1 indicate bias towards the G-protein activation pathway
and those greater than 1 indicate bias towards β-arrestin-2 recruitment. As the standard to which all data
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is normalized, DAMGO has a bias factor value of 1, indicating no signaling bias. The more sensitive β-
arrestin-2 assay used here establishes that 2 does recruit β-arrestin-2, and in fact is only slightly biased
towards the G-protein coupled pathway, with a bias factor of 0.95. Compounds 3 and 5 have similar
profiles, as originally predicted in the project rationale, having bias factor values of 0.32 and 0.57,
respectively, indicating a bias towards G-protein coupled pathway. The analogues of 5 are all biased
towards the β-arrestin-2 recruitment pathway, with bias factor values ranging from 3.1 to 9.4 Variations
u72in signaling bias within this small set of structurally similar compounds is intriguing, however a
rationale for these differences is not readily apparent and will require additional studies to more fully
elucidate the structure-functional selectivity relationships for this class of molecules.
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In an effort to determine if the changes in activity may be a result of changes in the physicochemical
properties between the compound series, the polar surface area (PSA) and cLogP values18 as well as ligand
efficiency (LE) and lipophilic ligand efficiency (LELP) values were calculated (Supplementary Tables 2
and 3). While there are slight variations between these values for the two series, no major trends or
correlations are seen. The modest set of analogues outlined in this work was designed to efficiently assess
the SAR around the phenyl ring, and with the SAR developed from this campaign, further analogues with
more diverse physicochemical properties can be designed and synthesized that optimize both activity and
properties.
Predicted Modes of Binding
Both 2 and 5 are MOR agonists and, although structurally similar, their potency and selectivity for the
MOR differ significantly. These activity differences incited exploration of their binding modes at the
MOR. As no X-ray crystal structure of a non-nitrogenous agonist bound to the MOR has been solved yet,
we resorted to well-tempered multiple-walker metadynamics simulations19 to predict the energetically
preferred modes of binding of 2 and 5 at a flexible MOR. We previously demonstrated the ability of this
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technique to predict accurate binding poses of opioid ligands20 and described its advantages and protocol
in the literature.21
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The analysis led to the identification of two different low-energy bound conformations for compound
2 with comparable free energies. A full description of the clusters and their relative stability is reported
in Supplementary Table 4. Although poses 1 and 2 of 2 partially overlap with the co-crystallized ligand
BU72 (23)22 in the recently published active structure of MOR23 (black sticks in Fig. 3), their orientation
in the binding pocket differs (see right panel of Fig. 3a and Supplementary Table 4 for the corresponding
ligand-receptor interactions). Specifically, while in pose 1 (blue in Fig. 3a), the ligand’s furan ring is
oriented towards helices TM5 and TM6, forming hydrophobic interactions with F2375.44 (superscripts
refer to the Ballesteros-Weinstein numbering scheme24) and edge-to-face interactions with H2976.52, in
pose 2 (salmon in Fig. 3a), the ligand’s furan ring points towards the receptor extracellular side, interacting
with I1443.29 and L219 in the ECL2.
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The phenyl ring of 2 interacts with W2936.48 and Y3267.43 in pose 1, but with the latter only in pose 2,
in addition to Y751.39 and Y1282.64. In both poses, 2 interacts via water molecules with aspartic acid
D1473.32, which usually forms a salt bridge with nitrogenous ligands. Additional interactions that are
occurring with high probability between the ligand and the receptor are listed in Supplementary Table 4.
Among them, water-mediated interactions with Y1483.33 and N1503.35 and edge-to-face interaction with
Y1282.64 are specific to pose 2, while interactions unique to pose 1 are the aforementioned interactions in
TM5 and 6, as well as V3006.55
.
Even larger structural variability was observed in the low-energy poses of ligand 5, for which three
different poses had similar free-energies. The first two poses of 5 do not overlap with the co-crystalized
ligands in the active and inactive crystal structures of MOR (e.g., black lines in Fig. 3b, right panel,
correspond to 23), as the ligand is located more towards the receptor extracellular side (see Supplementary
Table 4 for the ligand-receptor interactions formed by 5 in the three lowest-energy poses). In pose 1 (light
blue in Fig. 3b), 5 interacts with residues in TM1/2/6 and 7, and has the phenyl ring forming interactions
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with Y751.39, Y1282.64, and Y1217.43. In contrast, the ligand’s furan ring points towards TM6, forming
interactions with W3187.35. Pose 2 (red sticks in Fig. 3b) represents a different orientation of 5, with the
ligand’s furan ring interacting with D1473.32, Q1242.60, and V1433.28 and the ligand’s phenyl ring directed
towards Y1282.64. Pose 3 of 5 is in a similar location to pose 2 of 2, but it exhibits a different orientation
of the furan ring towards TM5, stabilized by a water-mediated interaction with K2335.40. In this pose, the
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ligand’s phenyl ring is located closer to TM2 but it also forms interactions with Y751.39 and L1212.57
.
Interactions that were found to be specific to compound 2 are with residues V2365.43, F2375.44, and
H2976.52 (pose 1), N1503.35 (pose 2), and Y3267.43 (pose 1 and 2). On the other hand, compound 5 formed
unique interactions with residues K3036.58 (pose 1); N1272.63, Q1242.60, V1433.28, and T3157.32 (pose 2);
K2335.40 and Y2996.54 (pose 3). The ligand’s phenyl ring is found to form hydrophobic interactions with
L1212.57 (a methionine in the -opioid receptor) during simulation of both compounds 2 (pose 2) and 5
(pose 1 and 3), albeit with different probabilities (~50% for pose 2 of 2 and ~65% or ~80% for pose 1 or
pose 3 of 5). As expected, the phenyl ring of 5 was observed to adopt a planar conformation with respect
to the decalin core, whereas it displayed a higher flexibility in the bound pose of 2. Mutation of these
residues will need to be performed next to help discriminate between the energetically indistinguishable
poses of each ligand, discriminate between the mode of binding and/or function of 2 and 5, and identify
the molecular determinants responsible for 2/5 subtype specificity.
We also used tools from the 2016-3 Schrödinger Suite (see Supporting Information for details) to
rationalize the observed activity variations among compound 2 derivatives (molecules 8a-22a) and
compound 5 derivatives (molecules 8b-22b). Specifically, we built a SAR model based on estimated
ligand-receptor interaction probabilities and the experimentally-derived EC50 values reported in Table 2.
For each derivative, an interaction probability was obtained from weighted averages of ligand-receptor
interaction fingerprints of the derivative in each of their parent compound’s (2 or 5) poses identified by
metadynamics (see discussion above). Specifically, the interaction fingerprints were weighted according
to estimated ligand binding affinities from approximated free-energy values (see details in the Supporting
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Journal of Medicinal Chemistry
Information). Linear regression models with no more than five regressors were ranked based on Akaike
information criteria (AIC), with the smallest AIC indicating the best fitting model for the measured
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activities. This model (AIC=116.9) identifies ligand interaction probabilities with residues I711.35
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S1252.62, K2335.40, V3006.55, and K3036.58 as best at discriminating between low- and high-potency
compounds (see Supplementary Fig. 7). Among these, ligand interactions with S1252.62 and K2335.40
exhibit the largest coefficients and are therefore believed to contribute more significantly to ligand
potency. This model also captures the increased potency of 5, as well as that of some of its derivatives
(specifically, 9b, 10b, 22b; see cluster at the bottom left of Supplementary Fig. 7). According to
approximated estimates of ligand binding affinity, the most stable poses of these compounds are involved
in simultaneous interaction with residues S1252.62 and K2335.40. Notably, the less potent 2 and all its
derivatives never engage in simultaneous interaction with both residues in either of the two most stable
poses (see Supplementary Fig. 8, top panels), as also seen with the less potent 5 derivatives (e.g., 15b,
17b, and 20b; see top right of Supplementary Fig. 7). Interestingly, all molecules based on 2 (8a-22a)
present similar interactions with these residues in both metadynamics-derived poses: all compounds
interact with K2335.40 but not with S1252.62 in pose 1, and with S1252.62 but not with K2335.40 in pose 2
(see panels on the top row of Supplementary Fig. 8). This suggests that different potencies within the
herkinorin series might be affected by modulation of the ligand-receptor interaction probability, and
therefore differences in binding affinity, more than by differences in allosteric coupling.
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In Vivo Activity
The increased potency and selectivity of 5 for the MOR relative not only to 2 but also morphine
prompted further investigation into how this novel compound behaves in vivo. Given the historic use of
MOR agonists as useful analgesics, the antinociceptive properties of 5 were investigated and compared
to those of morphine and 2.
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Previously, 2 had demonstrated efficacy in both the nociceptive and inflammatory phases of the rodent
formalin test.12b However, these effects appeared to be limited to the site of injections, suggesting 2 is
restricted to the periphery. To provide additional support of this hypothesis and determine if 5 was
similarly peripherally restricted, the antinociceptive effects of both 2 and 5 were assessed in the hot water
(50°C) tail-flick test in male B6-SJL mice (22-29 g), a method of testing nociceptive processes in the
spinal cord.25 Using this model, the centrally mediated antinociceptive effects of 2 and 5 were assessed at
1, 5, and 10 mg/kg doses alongside morphine (10 mg/kg, i.p.) with a 10 s time cutoff to prevent tissue
injury. Consistent with the previous studies, 2 had no significant antinociceptive effects in the tail-flick
assay (Fig. 4a). However, at 5 and 10 mg/kg doses, 5 produced a significant antinociceptive effect. Two-
way repeated measures ANOVA revealed a significant effects of time F(5,33)=64,p<0.0001, and drug
F(5,33)=99, p<0.0001. At the larger dose, 5 produced similar effects to that of morphine at the same dose
with maximal effects observed at 30 min (Fig. 4b). Pre-treatment with the opioid antagonist naloxone (10
mg/kg s.c. 45 min) blocked the antinociceptive effects of 5 (Fig. 4b), demonstrating that these effects
were opioid receptor mediated. Thus, it appears that in addition to increasing the potency and selectivity
of the compound, the conversion of 2 into 5 results in a compound with the antinociceptive efficacy of
morphine and the first non-nitrogenous opioid with centrally mediated antinociceptive activity. The
differing abilities of 2 and 5 to penetrate the CNS is particularly surprising given the similarities not only
in their structures but also their cLogP and PSA.
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Although interesting and potentially promising that such a small structural change might affect in vivo
activity so greatly, the penetration of 5 into the central nervous system may pose some additional risks.
For instance, the sedation and abuse potential observed with clinically used opioids, such as morphine,
are centrally mediated.26 Therefore, we sought to determine if 5 would also produce similar negative side
effects.
Tolerance to the analgesic effects of opioids is a major problem for pain management where increasing
doses of opioids are required to maintain analgesic effects.26 These escalating doses are also associated
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with increased side effects. With this in mind, we utilized the dose-response tail-flick assay in mice using
a within subject design to quantify the antinociceptive effects of 5 and morphine, 30 min following each
cumulative dose.27 Nonlinear regression analysis was used to calculate ED50 values. These data show that
the two compounds are equipotent, with no significant potency differences seen between morphine (ED50
5.3 mg/kg) and 5 (ED50 5.0 mg/kg) on day 1 (Fig. 5a, filled symbols). Differences in dose–response curves
between day 1 and day 9 were used to evaluate chronic tolerance following daily administration of either
morphine or 5, (10 mg/kg/s.c.) on days 2-8. One-way ANOVA of EC50 values followed by Bonferroni’s
multiple comparison test shows that both morphine and 5 show tolerance on day 9 compared to day 1,
however, morphine shows significantly more tolerance on day 9 (EC50 11.6 mg/kg) compared to 5 (7.9
mg/kg) (Fig. 5a).
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Another known side effect of morphine is its ability to produce sedation. To evaluate the sedative
properties of 5, the accelerating rotarod behavioral assay in mice was used. In this assay, compounds that
induce motor dysfunction or sedation, such as morphine, will reduce the amount of time a test animal
remains on an elevated rotating rod set to accelerate from 4 to 40 rpm over 300 s (Fig. 5b). At a dose that
elicited maximal antinociceptive effects in the tail-flick assay (10 mg/kg), 5 demonstrated slight sedation
only at the 15 min time point with significantly less effect than morphine upon motor coordination overall
(Two-way repeated measures ANOVA shows a significant time F(8,120)=17.18,p<0.0001 and drug
interaction F(2,15)=9.36,p<0.01).Thus it appears that while 5 clearly acts upon the central nervous
system, as evidenced by its efficacy in the tail-flick test, its impairment of motor coordination is
considerably reduced in comparison to that of morphine. Compound 2, which is restricted to the peripheral
nervous system, produced no observable effect on motor co-ordination (data not shown).
Finally, the rewarding effects of 5 were tested using the conditioned place preference (CPP) assay in
male Sprague Dawley rats (240-350 g). Following a 15 min free roaming pre-test on day 1, rats were
paired with either drug (morphine, 2 or 5) or vehicle in daily 60 min conditioning sessions for 6 days
using a counterbalanced design. During the post-conditioning test rats were allowed to freely roam the
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chambers for 15 min. The change in time spent in the drug-paired chamber pre- and post-conditioning is
shown as the CPP score (expressed as %). Using this paradigm, 5 resulted in a CPP score similar to the
vehicle control at both 5 mg/kg and 10 mg/kg and significantly less than the same doses of morphine (Fig.
6). These data indicate that 5 produces less rewarding effects than morphine, at both 5 and 10 mg/kg doses
suggesting it may possess a lower liability for abuse. Taken together, the effects of 5 in animal models of
antinociception, motor coordination, tolerance, and reward are very promising.
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Conclusions
Through SAR studies on the naturally-occurring KOR agonist salvinorin A, we have identified 5 as a
potent, selective, and centrally active probe for opioid receptors. 5’s activation of MORs potently induces
G-protein mediated effects and, to a lesser extent, recruits β-arrestin-2. Enhanced molecular dynamics
simulations suggest a different mode of binding of this compound than typical opioid ligands.
Interestingly, 5 has reduced tolerance, sedation, and rewarding properties compared to morphine, the
standard opioid analgesic.
The molecular nature of 5’s reduced side effect profile in vivo is unclear, as it does recruit β-arrestin-2
to a greater extent than morphine, but given its distinctive properties and desirable in vivo effects, 5 is an
attractive probe for potentially differentiating analgesia from abuse liability. Furthermore, our findings
provide additional evidence that moving away from morphine-based opioid ligands may serve as a viable
strategy for dissociating central antinociception from common opioid induced side effects.
Experimental
General experimental procedures. Salvinorin A was isolated from the leaves of Salvia divinorum and
converted to 1b, 4 and 6 as previously described.13 All other chemical reagents were purchased from
commercial suppliers and used without further purification. All solvents were obtained from a solvent
purification system in which solvent was passed through two columns of activated alumina under argon.
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Reactions performed in standard glassware were performed under an atmosphere of argon using glassware
dried overnight in an oven at 120 °C and cooled under a stream of argon. Reactions were monitored by
thin-layer chromatography (TLC) on 0.25 mm Analtech GHLF silica gel plates and visualized using a
UV Lamp (254 nm) and vanillin solution. Flash column chromatography was performed on silica gel (4-
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63 mm) from Sorbent Technologies. H and 13C NMR were recorded a 500 MHz Bruker AVIII
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spectrometer equipped with a cryogenically-cooled carbon observe probe or a 400 MHz Bruker AVIIIHD
spectrometer using tetramethyl silane as an internal standard. Chemical shifts (δ) are reported in ppm and
coupling constants (J) are reported in Hz. High-resolution mass spectrum (HRMS) was performed on a
LCT Premier (Micromass Ltd., Manchester UK) time of flight mass spectrometer with an electrospray
ion source in either positive or negative mode. Melting points were measured with a Thomas Capillary
Melting Point Apparatus and are uncorrected. HPLC was carried out on an Agilent 1100 series HPLC
system with diode array detection at 209 nm on an Agilent Eclipse XDB-C18 column (250 x 10 mm, 5
mm). Compounds were identified as ≥95% pure by HPLC before all in vitro and in vivo analyses.
Methyl
(2S,4aR,6aR,7R,10aR,10bR)-9-acetoxy-2-(furan-3-yl)-6a,10b-dimethyl-4,10-dioxo-
1,4,4a,5,6,6a,7,10,10a,10b-decahydro-2H-benzo[f]isochromene-7-carboxylate (1b). A solution of 7 (37
mg, 0.095 mmol) in CH2Cl2 (5 mL) was treated with acetic anhydride (18 μL, 0.189 mmol), DIPEA (33
μL, 0.189 mmol), and DMAP (1.2 mg, 0.0095 mmol). After stirring overnight the reaction mixture was
rinsed with HCl (1 M, 5 mL), saturated NaHCO3 (5 mL), and brine (5 mL). The organic layer was dried
over Na2SO4 and concentrated in vacuo. The resulting residue was purified by FCC 30%EtOAc/Pent. to yield 1b
(23 mg, 56%). 1H NMR (500 MHz, CDCl3) δ 7.47 – 7.34 (m, 2H), 6.52 (d, J = 2.2 Hz, 1H), 6.39 (dd, J = 1.9, 0.9
Hz, 1H), 5.62 – 5.50 (m, 1H), 3.78 (s, 3H), 3.52 (d, J = 2.3 Hz, 1H), 3.04 (dd, J = 13.7, 5.3 Hz, 1H), 2.40 (s, 1H),
2.24 (s, 3H), 2.17 (d, J = 5.6 Hz, 1H), 2.11 – 2.05 (m, 1H), 1.67 (ddd, J = 11.8, 9.7, 3.1 Hz, 3H), 1.36 (s, 3H), 1.30
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– 1.24 (m, 1H), 1.17 (s, 3H). C NMR (126 MHz, CDCl3) δ 191.00, 171.36, 170.32, 168.76, 145.21, 143.67,
139.37, 129.80, 125.41, 108.45, 72.01, 63.33, 56.35, 52.43, 51.31, 44.12, 43.72, 38.39, 35.80, 20.23, 17.91, 16.72,
14.82. HRMS calculated for C23H26O8: [M+Na]+: 453.1539 (found); 453.1525 (calc). Melting point: =
171-175 °C (dec).
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(2S,4aR,6aR,7R,10aR,10bR)-Methyl
9-((benzoyl)oxy)-2-(furan-3-yl)-6a,10b-dimethyl-4,10-dioxo-
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2,4,4a,5,6,6a,7,10,10a,10b-decahydro-1H-benzo[f]isochromene-7-carboxylate (5). A solution of 7 (40
mg, 0.103 mmol) in CH2Cl2 (8 mL) was treated with benzoyl chloride (24 μL, 0.206 mmol), DIPEA (36
μL, 0.206 mmol), and DMAP (1.3 mg, 0.206 mmol). After stirring overnight the reaction mixture was
rinsed with HCl (1 M, 8 mL), saturated NaHCO3 (8 mL), and brine (8 mL). The organic layer was dried
over Na2SO4 and concentrated in vacuo. The resulting residue was purified by FCC 30%EtOAc/Pent. to
yield 5 as a white solid (26.4 mg, 52%). 1H NMR (400 MHz, CDCl3)  8.11 (d, J = 7.14 Hz, 2H), 7.62 (t,
J = 7.45 Hz, 1H), 7.48 (t, J = 7.74 Hz, 2H), 7.41 (s, 1H), 7.39 (t, J = 1.66 Hz, 1H), 6.66 (d, J = 2.15 Hz,
1H), 6.39 (d, J = 0.95 Hz, 1H), 5.54 (dd, J = 5.28, 11.47 Hz, 1H), 3.80 (s, 3H), 3.60 (d, J = 2.13 Hz, 1H),
3.07 (dd, J = 5.34, 13.66 Hz, 1H), 2.47 (s, 1H), 2.20 (dd, J = 5.66, 8.50 Hz, 2H), 2.12 (m, 1H), 1.69 (m,
3H), 1.38 (s, 3H), 1.25 (s, 3H). 13C NMR (126 MHz, CDCl3)  190.91, 171.39, 170.41, 164.61, 145.47,
143.67, 139.37, 133.94, 130.28, 129.98, 128.63, 128.28, 125.43, 108.45, 72.07, 63.47, 56.51, 52.48,
51.39, 44.20, 43.81, 38.46, 35.85, 17.95, 16.83, 14.86. HRMS calculated for C28H28O8: [M-H]-: 491.1704
(found); 491.1711 (calc). Melting point 104-108 °C.
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Methyl-(2S,4aR,6aR/S,7R,10aR,10bR)-2-(furan-3-yl)-9-hydroxy-6a,10b-dimethyl-4,10-dioxo-
2,4,4a,5,6,6a,7,10,10a,10b-decahydro-1H-benzo[f]isochromene-7-carboxylate
(7a)
Methyl-
(2S,4aR,6aS,7R,10bS)-2-(furan-3-yl)-10-hydroxy-6a,10b-dimethyl-4,9-dioxo-1,4,4a,5,6,6a,7,8,9,10b-
decahydro-2H-benzo[f]isochromene-7-carboxylate (7b). A combination of CH2Cl2 (40 mL) and MeOH
(40 mL) was added to a flask containing 6 (250 mg, 0.640 mmol) and Cu(OAc)2 (349 mg, 1.92 mmol).
After stirring overnight at RT the reaction was concentrated in vacuo. The residue was re-dissolved in
CH2Cl2 (40 mL) and H2O (40 mL). The aqueous layer was re-extracted with CH2Cl2 (2 ×40 mL). The
combined organic layers were washed with saturated NH4Cl (50 mL) and brine (50 mL) then dried over
Na2SO4. The solvent was removed in vacuo and the residue purified FCC eluting with 12.5%
1
EtOAc/CH2Cl2 to yield 7a and 7b as a 3:1 mixture (139 mg, 52%) as a white solid. H NMR for 7a (500
MHz, CDCl3)  7.44 (m, 1H), 7.42 (m, 1H), 6.41 (dd, J = 0.80, 1.79 Hz, 1H), 6.02 (d, J = 2.50 Hz, 1H),
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Journal of Medicinal Chemistry
5.59 (dd, J = 5.25, 11.62 Hz, 1H), 3.76 (s, 3H), 3.41 (d, J = 2.49 Hz, 1H), 3.14 (dd, J = 5.13, 13.35 Hz,
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1H), 2.33 (s, 1H), 2.18 (m, 2H), 2.01 (m, 1H), 1.67 (m, 3H), 1.36 (s, 3H), 1.11 (s, 3H). C NMR (126
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MHz, CDCl3)  194.70, 171.32, 171.25, 146.69, 143.77, 139.38, 125.46, 112.18, 108.42, 71.92, 62.70,
55.98, 52.24, 51.27, 44.67, 43.88, 38.20, 35.78, 17.86, 16.64, 14.72. HRMS calculated for C21H24O7:
[M+Na]+: 411.1409 (found); 411.1420 (calc). Melting point: 165-170 °C (dec).
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To an ice cold stirring suspension of 6 (140 mg, 0.36 mmol) in methanol (3 mL) and benzene (12 mL),
lead tetraacetate (213 mg, 0.48 mmol) was added in four portions over 30 minutes producing a deep red
solution that slowly faded to transparent pale yellow as the reaction came to room temperature and stirred
overnight. The reaction was quenched by addition of 1M Na2S2O3 (15 mL) and sat. NaHCO3 (15 mL).
The resulting mixture stirred for 30 minutes and was then extracted with EtOAc (3 x 25 mL). The
combined organic extracts were washed with brine (50 mL), dried (Na2SO4) and concentrated. The
solvent was removed in vacuo and the residue purified FCC eluting with 12.5% EtOAc/CH2Cl2 to yield
7b (35 mg, 25 %) as a white crystalline solid. Rf = 0.6 (8 % EtOAc / CH2Cl2).
1H NMR for 7b (500 MHz, CDCl3)  7.49 - 7.42 (m, 2H), 6.79 (s, 1H), 6.45 (dd, J = 1.9, 0.9 Hz, 1H),
5.59 (ddd, J = 10.5, 6.4, 1.1 Hz, 1H), 3.75 (s, 3H), 3.75 - 3.73 (m, 1H), 3.19 - 3.08 (m, 1H), 3.11 - 3.01
(m, 1H), 2.74 (dd, J = 17.3, 3.0 Hz, 1H), 2.46 (dd, J = 12.4, 3.0 Hz, 1H), 2.22 (dq, J = 14.2, 3.4 Hz, 1H),
2.16 - 2.03 (m, 1H), 1.80 (tdd, J = 14.0, 12.3, 3.0 Hz, 1H), 1.71 (dt, J = 13.6, 3.2 Hz, 1H), 1.46 (s, 3H),
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1.43 (s, 3H), 1.27 (s, 1H). C NMR (126 MHz, CDCl3)  192.99, 171.84, 171.70, 143.82, 139.36, 139.25,
126.03, 108.62, 71.54, 52.00, 51.74, 50.57, 41.53, 38.64, 37.94, 37.24, 34.82, 29.72, 24.44, 19.02, 17.77.
HRMS calculated for C21H24O7: [M+H]+: 389.1606 (found); 389.1595 (calc). Melting point: = 215-216
°C (dec).
Synthesis of analogues. Analogues of 2 were prepared with a MiniBlox XT Parallel Synthesizer to
acylate 6 with various benzoyl chlorides using an analogous procedure described for the synthesis of 5.
Analogues of 5 were prepared by EDC and DMAP-mediated couplings of 7 with various benzoic acids.
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Full synthetic procedures and full characterization of compounds are provided in the Supplementary
Information.
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Calculated properties. Optimized structures of the compounds were generated in Maestro using the
OPLS_2005 force field at pH=7.4. Properties of the optimized structures were calculated using QikProp.18
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In vitro pharmacology. Cell lines and cell culture. The HitHunterTM Chinese hamster ovary cells
(CHO-K1) stably expressing the human µ-opioid receptor (OPRM1, catalog # 95-0107C2) the human κ-
opioid receptor (OPRK1, catalog # 95-0088C2), or the human -opioid receptor, (OPRD1, catalog # 95-
0108C2) and the PathHunterTM Chinese hamster ovary cells stably expressing the human µ-opioid
receptor β-arrestin-2 EFC cell line (catalog # 93-0213C2) were purchased from DiscoverX Corp.
(Fremont, CA) and maintained in F-12 media with 10% fetal bovine serum (Life Technologies, Grand
Island, NY), 1% penicillin/streptomycin/ ʟ-glutamine (Life Technologies), and 800 µg/mL Geneticin
(Mirus Bio, Madison, WI). The media of the PathHunterTM cells was supplemented with an additional
250 µg/mL Hygromycin B (Mirus Bio). All cells were grown at 37 °C and 5% CO2 in a humidified
incubator.
Forskolin-induced cAMP accumulation. Assays proceeded as previously described.15
β-arrestin-2 EFC recruitment assay. The MOR agonist-mediated β-arrestin-2 recruitment was
measured using the DiscoverX PathHunterTM technology. On day 1, ~80% confluent CHO-K1 OPRM1
β-arrestin-2 cells were detached from culture plates using nonenzymatic cell dissociation buffer (Life
Technologies) and counted using a hemocytometer. Cells were plated at 5 000 cells/well in 20 μL of Cell
Plating Reagent 2 (DiscoveRx) in 384-well tissue culture plates and incubated at 37 °C overnight. On day
2, stock solutions of test compounds were generated in 100% DMSO to 5 mM. The stock solutions were
used to make 11 serial dilutions and then diluted to yield 5x compound concentrations in assay buffer
(Hank’s Balanced Salt Solution [HBSS, Life Technologies] with 10 mM HEPES [Life Technologies]).
The cells were treated with 5 µL of the test compound solutions, final concentration of 1X compound and
1% DMSO. Cells were incubated for 90 minutes at 37oC. Cells were then treated with detection reagents,
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12.5 µL per well, according to manufacturer’s instructions and incubated at room temperature for at least
1 h protected from light. Luminescence was quantified using a Synergy 2 plate reader with Gen5 Software
(BioTek, Winooski, VT). Data were normalized to vehicle (no compound, 1% DMSO final concentration)
to remove any background luminescence. The highest dose(s) of DAMGO was used as 100% recruitment
and all data was converted to percentages based upon the DAMGO response.
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Data analysis. Data were analyzed using nonlinear regression analysis in GraphPad Prism 5.0 software
(GraphPad, La Jolla, CA) to generate sigmoidal dose-response curves for both the cAMP accumulation
assay and the β-arrestin-2 recruitment assay. cAMP accumulation data were normalized to vehicle and
forskolin only control values, and β-arrestin-2 recruitment data were normalized to vehicle and DAMGO
maximum response values. All compounds were run in parallel assays in triplicate in > 2 individual
experiments. EC50 and Emax values are reported as the means ± S.E.M. and represent the average of each
individual experiment following nonlinear regression analysis. Bias factors were calculated using relative
activity values, as shown in equation 1,
퐸푚푎푥푡푒푠푡 ∗ 퐸퐶50퐷퐴푀퐺푂
퐸푚푎푥푡푒푠푡 ∗ 퐸퐶50퐷퐴푀퐺푂
퐸퐶50푡푒푠푡 ∗ 퐸푚푎푥퐷퐴푀퐺푂
(
)
풍풐품 푩풊풂풔 푭풂풄풕풐풓 = (log (퐸퐶50
) )
− (log (
) )
(Eq. 1)
∗ 퐸푚푎푥퐷퐴푀퐺푂
푡푒푠푡
훽−푎푟푟푒푠푡푖푛
푐퐴푀푃
Metadynamics simulations. Simulations were performed using protocols that have been reported
previously.21 Full computational details are presented in the Supporting Information.
Animal studies. Adult male B6-SJL mice (22-29 g) and male Sprague Dawley rats (240-350 g) were
housed on a 12 h light cycle and experiments were conducted during the light cycle. All animals were
bred and housed at the Victoria University of Wellington (VUW) small animal facility, New Zealand. All
experimental procedures were approved by the VUW Animal Ethics Committee and carried out in
accordance to their guidelines for animal care. Food and water were provided ad libitum except during
experimental procedures.
Hot water tail-flick assay. The tail-flick assay was carried out in mice as previously described.28 Briefly,
mice were habituated in a plexi-glass restrainer (internal diameter 24 mm) for 15 min for 2 days prior to
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testing. Latencies were recorded by immersing one third of the tail in a water bath (50 ± 0.5 °C) and
measuring the time taken to show a withdrawal response. To avoid tissue damage, the maximum time for
the tail immersion was set at 10 s. Control latencies were measured in triplicate before testing and an
average calculated.
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To measure the duration of action of anti-nociceptive effects, mice were administered a single dose of
compound, morphine sulphate (Hospira New Zealand Limited) or vehicle (dimethyl sulfoxide
(DMSO):Tween-80:saline at a ratio of 2:1:7; i.p.) and the withdrawal latencies repeatedly measured at 1,
5, 10, 15, 30, 45, 60, 90 and 120 min. The maximum possible effect (MPE) was calculated using the
following formula: %MPE = 100 × (test latency–control latency)/(10−control latency).
Dose-dependent antinociceptive and tolerance evaluation: The cumulative dose-response tail-flick
assay was carried out in mice following repeated daily subcutaneous (s.c.) administration of either
Morphine or 5 (10 mg/kg) for 9 days as previously as previously described.27
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Accelerating rotarod performance assay. To evaluate motor incoordination, mice were assessed in an
accelerating rotarod protocol using previously described protocols,29 Full experimental description are
provided in the Supplementary Information.
Conditioned place preference assay. Methods were conducted as previously described,30 and a more
detailed description can be found in the Supplementary Information.
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Associated Content
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Supporting Information. Data includes H NMR, and C NMR spectra of 1b, 4, 7a-b, 8a-16a, and
8b-22b, 1H NMR, 13C NMR, COSY, HSQC, and HMBC spectra for 5, HPLC chromatograms for 1b, 2,
4, 5, 8a-17a, and 8b-22b, further experimental details, and supplementary figures and tables. This material
is available free of charge via the Internet at http://pubs.acs.org.
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Author Information
Corresponding Author. Phone (785) 864-3267. Fax: (785) 864-5326. E-mail: prisinza@ku.edu.
Notes. The University of Kansas has filed a provisional patent related to this work. Other than this, the
authors declare no competing financial interests.
Acknowledgements. This work was supported by DA018151 and GM111385 (to TEP), DA03049,
MH107053, and DA026434 (to MF), GM008545 (to APR and RSC), AFPE Pre-doctoral Fellowship in
Pharmaceutical Sciences (to RSC), and the Neurological Foundation and Health Research Council of New
Zealand and Victoria University of Wellington (to BMK). Support for the NMR instrumentation was
provided by NIH Shared Instrumentation Grant #S10RR024664 and NSF Major Research
Instrumentation Grant #0320648. 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 (to MF), which is supported by National Science Foundation grant
number ACI-1053575. The content is the sole responsibility of the authors and does not necessarily
represent the official views of the National Institute on Drug Abuse, National Institutes of Health, or the
National Science Foundation. The authors would like to thank Dr. Jeff Aubé, Dr. Sudeshna Roy, and Dr.
Edward Stahl for their assistance.
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Figure 1. Many opioids are derived structurally from morphine, including codeine, oxycodone,
meperidine, methadone, and fentanyl. The structure of traditional MOR agonists morphine and DAMGO
differ significantly from those of the KOR agonist salvinorin A (1) and the MOR agonists herkinorin (2)
and herkamide (3).
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