Orvinols with mixed kappa/Mu opioid receptor agonist activity

Article abstract of DOI:10.1021/jm301543e

Dual-acting kappa opioid receptor (KOR) agonist and mu opioid receptor (MOR) partial agonist ligands have been put forward as potential treatment agents for cocaine and other psychostimulant abuse. Members of the orvinol series of ligands are known for their high binding affinity to both KOR and MOR, but efficacy at the individual receptors has not been thoroughly evaluated. In this study, it is shown that a predictive model for efficacy at KOR can be derived, with efficacy being controlled by the length of the group attached to C20 and by the introduction of branching into the side chain. In vivo evaluation of two ligands with the desired in vitro profile confirms both display KOR, and to a lesser extent MOR, activity in an analgesic assay suggesting that, in this series, in vitro measures of efficacy using the [³⁵S]GTPγS assay are predictive of the in vivo profile.

Full text of DOI:10.1021/jm301543e

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Article
Orvinols with Mixed Kappa/Mu Opioid Receptor Agonist Activity
Benjamin Greedy, Faye Bradbury, Mark P. Thomas, Konstantinos Grivas,
Gerta Cami-Kobeci, Ashley Aarchambeau, Kelly Bosse, Mary J Clark,
Mario Aceto, John W. Lewis, John R. Traynor, and Stephen M Husbands
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm301543e • Publication Date (Web): 25 Feb 2013
Downloaded from http://pubs.acs.org on March 13, 2013
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Orvinols with Mixed Kappa/Mu Opioid Receptor Agonist
Activity
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1Greedy, Benjamin M.; 2Bradbury, Faye.; 1Thomas, Mark P.; 3Grivas, Konstantinos; 1CamiꢀKobeci,
Gerta; 2Archambeau, Ashley.; 2Bosse, Kelly.; 2Clark, Mary J.; 4Aceto, Mario; 1Lewis, John W.;
2Traynor, John R.; 1Husbands, Stephen M.
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1Department of Pharmacy and Pharmacology, University of Bath, Bath, BA2 7AY, UK;
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2Department of Pharmacology, University of Michigan, Ann Arbor, Michigan, School of
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Chemistry, University of Bristol, Bristol, BS8 1TS, UK. Department of Pharmacology and
Toxicology, Virginia Commonwealth University, Richmond, Virginia.
Corresponding author:
Stephen M. Husbands
Department of Pharmacy and Pharmacology
University of Bath
Claverton Down
Bath, BA2 7AY,
UK
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Abstract
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Dualꢀacting kappa opioid receptor (KOR) agonist and mu opioid receptor (MOR) partial agonist
ligands have been put forward as potential treatment agents for cocaine and other psychostimulant
abuse. Members of the orvinol series of ligands are known for their high binding affinity to both
KOR and MOR but efficacy at the individual receptors has not been thoroughly evaluated. In this
study, it is shown that a predictive model for efficacy at KOR can be derived, with efficacy being
controlled by the length of the group attached to C20 and by the introduction of branching into the
side chain. In vivo evaluation of two ligands with the desired in vitro profile confirms both display
KOR, and to a lesser extent MOR, activity in an analgesic assay suggesting that, in this series, in
vitro measures of efficacy using the [35S]GTPγS assay are predictive of the in vivo profile.
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Introduction
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The continuing illicit use of psychoactive substances, with the resulting health and social
consequences, emphasizes the need for improved pharmacotherapies for drug abuse. Treatments
for opiate abuse, such as methadone and buprenorphine (1n), are available and have proven
successful against a range of addict populations. However there are no such approved
pharmacotherapies for cocaine abuse, though a wide range of possible treatment agents have been
evaluated in the laboratory and in clinical trials.1
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There has been interest in the use of kappa opioid receptor (KOR) agonists as potential
pharmacotherapies for cocaine and other psychostimulant abuse,2ꢀ7 particularly as repeated
administration of KOR agonists has been shown to prevent, or reduce many of cocaine’s
behavioural effects.4,8ꢀ10 Primarily due to the dysphoric effects produced by KORꢀagonists, the
development of a KORꢀagonist pharmacotherapy for human use is not straightforward. For
example, while enadoline (CIꢀ977), a high efficacy, selective KORꢀagonist, appeared to be better
tolerated in subjects with a history of drug use compared to naive individuals, it still caused some
dysphoria.11 Overall the evidence suggests that higher efficacy KORꢀagonists with some additional
mu opioid receptor (MOR)ꢀagonist activity, such as ethylketazocine (EKC), are more effective in
reducing cocaine selfꢀadministration, and display fewer side effects, than their more KORꢀselective
counterparts such as enadoline and spiradoline.4,12 Presumably, the presence of some MORꢀagonist
effects helps attenuate any dysphoria induced by the KORꢀagonism.
In the treatment of opiate abuse, a longꢀduration of action is of benefit for successful
treatment agents. The main pharmacotherapies methadone and buprenorphine (1n), but also
LAAM, are long acting and their success is at least partly as a result of this property.13 Together
with its reduced efficacy, the slow onset of effects displayed by buprenorphine (1n) appears to
reduce its abuse potential. EKC and the other KORꢀagonists effective in reducing cocaine selfꢀ
administration all have short duration of action.4 A strategy for the treatment of cocaine abuse
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could therefore be the utilization of mixed KORꢀagonists/MORꢀpartial agonists having extended
duration of action. Of interest in this regard are the orvinols, a series of opioids displaying a range
of pharmacological profiles including the longꢀacting MOR partial agonist 1n.14 The orvinols have
not previously been thoroughly evaluated as KORꢀagonists, but it has become clear that a number
of them do display substantial KORꢀefficacy in vivo and that their lack of absolute KORꢀselectivity,
in particular over MOR, makes them of particular interest to this project. An early example was the
isopentyl orvinol M320 (5) which was the subject of a detailed pharmacological study by Boura and
Fitzgerald.15 5 shows a KOR/MOR profile similar to that of EKC but it is more potent and very
much longer acting. In this manuscript we describe our initial work towards analogues of 5 and 1n
as potential therapies for cocaine abuse. There is a particular focus on orvinols having a branched
chain attached to C20 as these regions (above and away from C6/C7 and below C8) are associated
with efficacy at KOR.16,17
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Synthesis
The compounds were prepared using the standard techniques for orvinol synthesis.14,18,19 Only one
significant change has been made to the synthetic route and relates to how and when the Nꢀmethyl
group is replaced with cyclopropylmethyl in series 1. We have found that this process is most
reliably performed (simpler and higher yielding) with diisopropyl azodicarboxylate (DIAD) on
methyl ketone 6 (Scheme 1). Grignard addition to ketone 8 provided series 1 via 9. Series 2 – 4
were all synthesised from aldehyde 10, itself prepared from Nꢀcyclopropylnorthebaine,20 the initial
step being the addition of the appropriate Grignard reagent which gave a mixture of diastereomeric
secondary alcohols, with the major isomer (11a) as shown (Scheme 2). This major product in each
case was isolated in sufficient quantity to allow 3ꢀOꢀdemethylation to series 4. Swern oxidation of
11a alone or the mixture of diastereomeric alcohols gave ketones 12 that on treatment with
methylmagnesium bromide and subsequent 3ꢀOꢀdemethylation gave the diastereomeric 3oꢀalcohol
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series (2). Reduction of 12 gave the 2o alcohols 11b (major product) and 11a (minor product), with
3ꢀOꢀdemethylation of 11b giving access to series 3.
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Results
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Four series of orvinols were studied. These were tertiary alcohols having the same relative
stereochemistry as buprenorphine (1a -1n, Scheme 1), a more limited series of diastereomeric
tertiary alcohols (2a-2i), secondary alcohols with the same relative stereochemistry as
buprenorphine (3a-3i) and finally a series of diastereomeric secondary alcohols (4a-4k) (Scheme 2).
Binding affinities of the new compounds for MOR and KOR were determined by displacement of
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[ H]ꢀdiprenorphine binding from C6ꢀrat glioma cells expressing recombinant rat MOR and CHO
cells expressing recombinant human KOR. Details of the assay have been described previously.21
Delta opioid receptor (DOR) activity of the new ligands was only determined for the ligands
progressed to in vivo studies as in no case has DOR activity proved significant in the
pharmacological profile of orvinol based ligands. As expected, all of the ligands bound with high to
very high affinity to both KOR and MOR with little or no selectivity for one receptor over the other
(Table 1). If any selectivity was observed, it was for the KOR (e.g. 4b and 4d had 11ꢀfold higher
affinity for KOR than for MOR). Affinities for KOR were in the subnanomolar range (0.015 to
0.22 nM) for all the ligands, with affinities for the MOR in the range 0.029 ꢀ 1.0 nM. Binding
affinities for five of the ligands (1l, 1m, 2g, 2h, 2i), all synthesized at an earlier time, were
evaluated in guinea pig brain membranes using established methods.22 1a was evaluated in both
binding assays, providing a reference to enable comparison with the rest of the series. Affinities
were lower in the guinea pig brain membrane assay, but confirmed the lack of selectivity, with
equal affinity for KOR and MOR. Affinities for 1b and 1c at DOR were determined by
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displacement of [ H]ꢀdiprenorphine binding from C6ꢀrat glioma cells expressing recombinant
DOR. They bound with high affinity, with Ki values of 0.34 nM and 0.42 nM respectively.
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The primary in vitro assay used to determine opioid receptor functional activity was the [35S]GTPγS
assay which, like the binding assays, was performed on recombinant opioid receptors transfected
into C6ꢀrat glioma cells (for MOR) and CHO cells (for KOR). Assays were performed as
previously described by Traynor and Nahorski.23 Agonist efficacy at these opioid receptors was
determined in comparison to the standard selective agonists DAMGO (MOR), and U69593 (KOR)
(Table 1). In this assay buprenorphine (1n) displayed no efficacy at KOR while M320 (5) was a
very potent, full agonist (91% stimulation). The new compounds displayed a range of efficacies at
KOR, from low efficacy partial agonists (e.g. 1a, 1d, 1j, 3a had efficacies from 30 – 49%) to full
agonists of equivalent efficacy to the standard U69593 (e.g. most of series 4, plus 2c, 2d, 3i
amongst others).
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The majority of ligands displayed lower efficacy at MOR than at KOR; the most extreme examples
being 3b and 3i which are potent, full agonists at KOR, with no efficacy at MOR. They proved to
be potent MOR antagonists shifting the concentration effect curve for the standard agonist DAMGO
in a parallel fashion giving Ke(MOR) of 0.50 nM and 0.35 nM respectively. 4a was similarly
selective, but with partial agonist activity at the KOR. Substantial selectivity in efficacy for KOR
was seen in a number of other ligands including 1b, 2b, 2c, 2d, 2f, 3g, 4b and 4d. Within series 1,
2 and 3 highest MOR efficacy was typically found with the cyclohexylmethyl and closely related
benzyl side chains. 4h, having a cyclohexylmethyl side chain, also had high efficacy, although in
this series 4j (having a benzyl group) was only a partial agonist with moderate efficacy. The two
ligands, 1b and 1c, evaluated at DOR were both of very low efficacy (2% and 8% stimulation
respectively).
Five ligands (1l, 1m, 2g, 2h, 2i) were evaluated in the guinea pig ileum (GPI) and mouse vas
deferens (MVD) isolated tissue assays, instead of [35S]GTPγS assays (Table 2), using standard
procedures.22 1a and 1n were also tested in these isolated tissue assays to allow meaningful
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comparison between results from the different assays. The GPI has both MOR and KOR
populations and is sensitive to KOR agonist effects, while the MVD has all three types of opioid
receptor and is particularly sensitive to DOR agonists and least sensitive to KOR agonists. The nꢀ
propyl analogue (1l) and the diastereomeric nꢀpentyl analogue (2h) were partial agonists in the GPI
whereas the other ligands evaluated in this assay were all of higher efficacy. Thus 1l had lower
efficacy than its iꢀpropyl isomer 1a and also lower than the bulky tꢀbutyl containing 1n and also
1m, having the longer nꢀpentyl chain. 2g, the diastereomer of 1l, was also higher efficacy as was
the tꢀbutyl containing 2i. The effects of these ligands in the GPI could not readily be reversed by
the standard antagonists CTAP and norBNI. Each of the compounds was found to be an antagonist
in the MVD with 1a, 1l and 1m displaying some selectivity for MOR. Antagonist Ke’s at KOR
were not determined for 2g and 2i as they had demonstrated efficacy in the GPI.
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Comparative molecular field analysis (CoMFA) is a technique used to generate a description of a
3D structureꢀactivity relationship in a quantitative manner, i.e. it is a 3D variant of a quantitative
structureꢀactivity relationship (QSAR). The output of a CoMFA is a 3D model showing structural
features of the input data that are likely to affect, beneficially or adversely, the activity of the
compounds. A CoMFA was used to help identify the regions near C20 that are beneficial or
detrimental to KOR efficacy and to help confirm the conformation adopted by the different isomeric
series on binding to the KOR. All the compounds evaluated in the [35S]GTPγS assay, including 1n
and 5, were used to develop the model. With all compounds overlaid in their lowest energy, C6ꢀ
OMe to C20ꢀOH hydrogen bonded conformation (as depicted for 1n in Figure 1) and using the %
stimulation at KOR data, an R2 of 0.911 and crossꢀvalidated R2 of 0.447 were obtained. As can be
seen in Figure 1, the model predicts that for high KOR efficacy there are two areas near to C20 that
could beneficially be occupied by lipophilic groups, the first is away from C7 and the second is the
region below C8. The model also suggests that lipophilic groups closer to C20 are not well
tolerated, leading to lower KOR efficacy. Evaluation of alternative conformations about the C7ꢀ
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C20 bond (e.g. so that the bulky R group of series 1 and 3 occupies the region below C8) led to
substantial reductions in R2, and in particular the crossꢀvalidated R2.
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The docking of 1n to the recently published crystal structures of the MOR and KOR opioid
receptors was examined. The crystal structure24 of the KOR was determined in the presence of the
selective KOR antagonist JDTic and presumably represents the antagonist conformation of the
receptor. JDTic is enclosed in a largely hydrophobic pocket with only one direct hydrogen bond to
the protein. There is a network of waterꢀmediated hydrogen bonds between the ligand and the
protein. The docked 1n structure overlays the 7ꢀhydroxyꢀtetrahydroisoquinoline with the
buprenorphine phenolic hydroxy group making the same interactions as the equivalent group in
JDTic (Figure 2). There is a hydrogen bond between the protonated nitrogen and the side chain of
Asp138. The isopropyl group of JDTic fits into and fills a tight pocket formed by Val108, Asn141,
Trp287 and Tyr320: this interaction is replicated by the cyclopropyl moiety of 1n. The tꢀbutyl group
makes no specific interactions with the protein, but projects into the large pocket that the
phenylpiperidine motif of JDTic occupies.
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The MOR was crystallised with the irreversible antagonist βꢀFNA present in the binding site,25 a
ligand with significant structural similarity to buprenorphine. βꢀFNA is covalently bound to the side
chain nitrogen of Lys233 (Figure 3). The docked 1n structure approximately overlays βꢀFNA but,
without the covalent restraint, lies a little deeper in the binding site. There is a hydrogen bond
between the positively charged nitrogen and the sidechain of Asp147. The cyclopropyl moiety
overlays that of βꢀFNA occupying, but not filling, a small pocket formed by Asn150, Trp293,
Ile322 and Tyr326. The tꢀbutyl group makes no specific interactions with the protein, but projects
into the same large pocket occupied by the methyl ester of βꢀFNA.
Two ligands, the iꢀbutyl (1b) and iꢀpentyl (1c) analogues of buprenorphine (1n) were selected for
preliminary in vivo evaluation in the paraꢀphenylquinone (PPQ) abdominal stretch assay (Table
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3).26 In this assay PPQ acts as a fairly low intensity nociceptive stimulus such that even partial
opioid agonists are active as antinociceptives. These two ligands were chosen as they had the
desired lower efficacy partial agonist activity at MOR in the [35S]GTPγS assay coupled with higher
efficacy at KOR. As it is difficult to accurately predict the in vivo profile of mixedꢀaction ligands,
one ligand (1b) was chosen that was substantially biased towards KOR while the other (1c) had a
more balanced profile. In the PPQ assay 1b was a potent agonist (ED50 0.02 mg/kg s.c.) that could
be reversed by norBNI (AD50 6.5 mg/kg s.c.) but only partly reversed by βꢀFNA (a maximum
reversal of 27% at the highest βꢀFNA dose) (Table 3). 1c was equally potent (0.02 mg/kg s.c.) and
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again could be reversed by norBNI (AD50 2.45 mg/kg s.c.) and partially reversed by βꢀFNA (59%
reversed at 10 ꢀg/brain βꢀFNA).
Discussion
As predicted, based on earlier work by ourselves and others,16,17,27 the Nꢀ
cyclopropylmethylnororvinols synthesised and evaluated in this study had very high affinity, but
little to no MOR/KOR selectivity in binding assays. Also as predicted, in the [35S]GTPγS assay
used to determine the level of agonist activity at each receptor, the majority of the ligands were
KOR agonists with lower efficacy at the MOR. Importantly, the [35S]GTPγS data for the known
compounds buprenorphine (1n) and M320 (5) were in agreement with previously reported isolated
tissue data and findings from in vivo studies, with 1n displaying no efficacy at the KOR while 5
was a very potent, full agonist at this receptor and both displayed partial agonist activity at the
MOR, such that 1n profiled as a MOR partial agonist, KOR antagonist and 5 as a high efficacy
KOR agonist with some MOR activity.14,28 Similarly, evaluation of 1a in both [35S]GTPγS and
isolated tissue assays (Tables 1 and 2) gave results in good agreement, indicating low efficacy at
each receptor.
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We have previously proposed that KOR agonist activity can be achieved in orvinol and related
series through interaction with either a region above and away from C7 (relating to the site occupied
by the Rꢀgroup in series 1 and 3) or a region below C8 (relating to the site occupied by the Rꢀgroup
in series 2 and 4).16,17 The results of the CoMFA analysis performed on the compounds prepared
for this study are in agreement with this hypothesis. With all compounds held in their low energy
Hꢀbonding conformation, an excellent correlation was found between predicted and actual KOR
efficacy and two regions, corresponding to the two lipophilic sites just described, were highlighted
as being beneficial to KOR efficacy (Figure 1). Importantly, this appears to confirm that the
hydrogen bonded conformation of the orvinols is preserved on interaction with the KOR.
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In general the 2o and 3o alcohols having opposite relative stereochemistry to buprenorphine (series 2
and 4) display higher KOR efficacy than series 1 and 3, which have the same relative
stereochemistry to buprenorphine. Substantial differences in efficacy between series was also
found at the MOR, with series 4 > series 1 > series 2 ≈ series 3. Comparing the buprenorphineꢀlike
3o alcohols (1) with their diastereoisomers (2), the results from the [35S]GTPγS assays indicate that
those having the same relative stereochemistry as buprenorphine have equal or higher efficacy at
MOR than their diastereoisomers (2), but that 2 have higher efficacy at KOR, often substantially so.
This finding was replicated in the GPI assay where buprenorphine (1n) was found to be a MOR
partial agonist while its isomer (2i) was a potent KOR agonist. Within the 2o alcohols those with
the opposite relative stereochemistry (4) to buprenorphine mostly had higher efficacy for both MOR
and KOR than their diastereoisomers (3). The 2o alcohols (3, 4) were in general of higher efficacy
at both MOR and KOR than the 3o alcohols (1, 2).
Within tertiary alcohols (1), KOR efficacy was lowest in those ligands having branching at C21,
while insertion of one or two methylene units increased KOR efficacy. So, for example, 1a having
an isopropyl group was a low efficacy partial agonist (30% relative to U69,593) whereas 1b and 1c
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(isobutyl and isopentyl containing) were higher efficacy agonists (84% and 69% relative to
U69,593). A similar increase in efficacy on going from isopropyl to isobutyl and isopentyl was
seen in the diastereomeric series of tertiary alcohols (2a versus 2b and 2c) but efficacy at the KOR
was generally higher for this series than for their diastereomers. The isopropyl group also led to
lowest KOR efficacy in both series of secondary alcohols (3a and 4a) though in these series other
ligands having branching at C21, for example the cyclopentyl in 3d and 4d had significantly higher
efficacy. These results are consistent with previous studies in isolated tissue assays with 17ꢀNMe
orvinols (Series 1, but Nꢀcyclopropylmethyl replaced by NꢀMe) where it was found that branching
of the alkyl group at the point of attachment to the carbinol function (i.e. at C21) resulted in lower
potency and efficacy at the MOR, while in general, MOR and KOR potency and efficacy increased
with increasing chain length.17,29 The results of the CoMFA study provide further support for these
conclusions with the region adjacent to C21 found to be detrimental to efficacy at the KOR (Figure
1), appearing to confirm our hypothesis that the loss of KOR efficacy in 1n relative to closely
related orvinols is due to a methyl group of the tꢀbutyl moiety interacting favourably with the
antagonist conformation of KOR receptor and/or disfavouring the agonist conformation.17,27
Docking of 1n to the recently published crystal structures of the KOR confirms that the tꢀbutyl
group accesses a large lipophilic region when the receptor is in an antagonistꢀbound conformation,
but does not provide any more detailed information on why buprenorphine is not an agonist at
KOR. The modeling does shed some light on why the Nꢀcyclopropyl group, and related moieties
such as allyl, help confer substantial antagonist character to the orvinols and other series; groups of
this size appear to bind tightly to a pocket formed by Val108, Asn141, Trp287 and Tyr320 in the
KOR and Asn150, Trp293, Ile322 and Tyr326 in the MOR.
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Comparison of 5 and 1c suggests that reducing the 6,14ꢀbridge from etheno to ethano has little or no
effect on efficacy at MOR but substantially decreases efficacy at KOR. Previously it has been
suggested that reduction of bridge leads to some attenuation of MOR intrinsic activity,14 though this
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conclusion was based on in vivo antinociceptive activity and without the benefit of selective
antagonists or of data from isolated tissue assays.
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The in vivo evaluation of 1b and 1c gave results entirely in keeping with the in vitro analysis. Both
had substantial KOR agonism with 1c having a greater MOR component than 1b. While there is
not a clear dose dependency found in the experiments using βꢀFNA pretreatment, this may simply
be indicative of the low partial agonist character of these ligands at the MOR meaning we are
looking at inhibition in vivo of a group of receptors responsible for only part of the activity
(analgesia) that is the measured endꢀpoint. The current in vitro analysis also help rationalise the in
vivo data that has been reported for a limited number of these compounds previously.30 The in vivo
data was collected on compounds of type 1 with R groups from methyl to butyl (including branched
isomers) and suggested that compounds with R = nꢀpropyl or larger (up to tꢀbutyl) were MOR
partial agonists that in some cases had high efficacy KOR agonist activity.30 The in vitro data
generated for the compounds common to these previous studies and the current work (1a, 1b, 1l,
1n; R = iꢀpropyl, iꢀbutyl, nꢀpropyl and tꢀbutyl) is mostly consistent with these findings with low
MOR efficacy, but variable KOR efficacy that appears to initially increase with size of the R group
(e.g. iꢀpropyl → iꢀbutyl, 30 → 84%) before then decreasing again (iꢀbutyl → tꢀbutyl, 84 → 0%).
The nꢀpropyl analogue (1l) was only evaluated in isolated tissue assays where it was found to be a
low efficacy agonist in guinea pig ileum (GPI). The difficulty in reversing the agonist effects of the
ligands in the GPI by selective antagonists for MOR and KOR (CTAP and norBNI) makes
commenting on the receptor selectivity of this activity difficult, but is typical of the orvinol series
and results from their tight, longꢀlived binding to the receptors and can be indicative of an extended
duration of action.17,31 Differences between the GPI and the cell based [35S]GTPγS assays may be
related to the easier access to receptors in the cell homogenates. The activity of these compounds as
antagonists in the mouse vas deferens (MVD) suggested that all were partial, rather than full,
agonists. However, in vivo 1l, having a nꢀpropyl group, appeared to have higher efficacy than the
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C20 branched chain analogues 1n (tꢀbutyl) and 1a (iꢀpropyl),30 contrary to the GPI derived data just
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Conclusions
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The present study confirms the orvinols lack of selectivity in binding to opioid receptors and that
KOR/MOR agonists of varying efficacy can be obtained from this series. As predicted, efficacy
can be controlled by the chain length of the C20 R group and by the introduction of branching or
ring systems into the chain; a full range of profiles from EKCꢀlike KOR agonists/MOR partial
agonists (e.g. 1c, 1e), selective KOR agonists (e.g. 3b, 3i, 4a), nonꢀselective agonists (e.g. 1h, 4f)
and even a ligand with predominant MOR activity (1j) were seen. Preliminary in vivo evaluation of
1b and 1c, coupled with the known profiles of 1n and 5a suggests that the [35S]GTPγS data is
predictive of the in vivo activity of these ligands. Compounds have successfully been obtained with
the targeted profile of potent, moderate to high efficacy KOR agonism combined with potent partial
agonist activity at the MOR.
Experimental
Reagents and solvents were purchased from SigmaꢀAldrich or Alfa Aesar and used as received.
Buprenorphine (1n) was supplied by the National Institute on Drug Abuse, Bethesda, Maryland. 1H
and 13C NMR spectra were obtained with a Bruckerꢀ400ꢀMHz instrument (1H at 400 MHz, 13C at
100 MHz); δ in ppm, J in Hz with TMS as an internal standard. ESIMS: microTOF (BRUKER).
Microanalysis: PerkinꢀElmer 240C analyzer. Column Chromatography was performed using
RediSep preꢀpacked columns with a Teledyne Isco CombiFlash instrument. Ligands were tested as
their hydrochloride salts, prepared by adding 5 equivalents of HCl (1 N solution in diethyl ether) to
a solution of compound in anhydrous methanol. All reactions were carried out under an inert
atmosphere of nitrogen unless otherwise indicated. All compounds were > 95% pure as determined
by microanalysis. A representative synthesis for each series is reported here.
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General procedure A: Grignard addition
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The Grignard reagents were prepared form the corresponding bromides (5 mmol) by reaction with
magnesium (182 mg, 7.5 mmol) in anhydrous THF (5 ml) containing a crystal of iodine. The
Grignard reagents were titrated prior to use by adding 1 ml of the Grignard solution to a flask
containing 1,10ꢀphenanthroline (~2 mg) in anhydrous THF (2 ml) (purple solution) and titrating
with 1M 2ꢀbutanol (anhydrous) in THF (end point pale yellow solution).
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A solution of the appropriate Grignard reagent (1 M in THF, 1.2 ml, 1.2 mmol) was treated
dropwise at room temperature with a solution of Nꢀcyclopropylmethylꢀ6,14ꢀendoꢀ
ethanonorthevinone (8) (500 mg, 1.18 mmol) or Nꢀcyclopropylmethylꢀ6,14ꢀendoꢀethanonorthevinal
(10) (500 mg, 1.22 mmol) in anhydrous toluene (12 ml). After stirring at room temperature for 20 h,
the reaction was quenched by addition of saturated aqueous ammonium chloride solution (20 ml).
The phases were separated and the aqueous phase extracted with EtOAc. The combined organic
phases were washed with saturated aqueous sodium bicarbonate, dried over MgSO4, filtered and
evaporated in vacuo. The residue was purified by column chromatography over silica gel eluting
with a gradient from 10% to 30% ethyl acetate in hexane. Rf values are recorded from TLC eluted
with 30:1:69 ethyl acetate/ammonia solution/hexane.
General Procedure B: 3-O-demethylation with propane thiolate and HCl salt formation
A solution of the appropriate thevinol (0.25 mmol) in anhydrous HMPA (1 ml) under an inert
atmosphere was treated with sodium hydride (21 mg, 0.875 mmol) followed by 1ꢀpropanethiol (79
ꢁl, 0.875 mmol). After the addition was complete, the reaction mixture was heated to 120°C and
stirred for 3 h. On cooling to room temperature, NH4Cl (sat, aq) was added and the mixture
extracted with diethyl ether. The organic extracts were washed with water (3×) and brine. The
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organic phase was dried (MgSO4) filtered and evaporated to dryness. The residue was purified by
column chromatography over silica gel.
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The HCl salts were prepared by the addition of 2M HCl in diethyl ether (1.2 equiv.) to a solution of
the orvinol in diethyl ether. The white precipitate which formed was collected by filtration, washed
with ether and dried under high vacuum.
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General Procedure C: Reduction with LiAlH4
A solution of the ketone (0.215 mmol) in anhydrous THF (7 ml) was added dropwise to a
suspension of LiAlH4 (20.5 mg, 0.54 mmol) in THF at room temperature. The resulting mixture
was allowed to stir for 1 hour and then treated carefully with saturated aqueous sodium sulphate
sulphate solution until all aluminium salts had precipitated. The aluminium salts were removed by
filtration and washed with Et2O. The filtrate was dried over MgSO4, evaporated to dryness and the
residue purified by column chromatography over silica gel (30% ethyl acetate, 0.5% NH4OH in
hexane).
General Procedure D: Swern oxidation
A solution of DMSO (170 ꢂl, 2.4 mmol) in anhydrous DCM (5 ml) was added dropwise to a
solution of oxalyl chloride (93 ꢂl, 1.1 mmol) in DCM (2 ml) at ꢀ78°C. The resulting solution was
stirred at ꢀ78°C for 10 min and then treated dropwise with a solution of the alcohol (1 mmol) in
DCM (5 ml). The reaction was stirred for a further 15 min before triethylamine (0.7 ml, 5 mmol)
was added slowly and the resulting solution allowed to warm to room temperature. Water (10 ml)
was added and the resulting mixture stirred for 10 min. The phases were separated and the aqueous
phase extracted with DCM. The combined organic extracts were washed with saturated ammonium
chloride, dried over MgSO4, filtered and the solvent removed under reduced pressure. The residue
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was purified by column chromatography over silica gel (0.5% NH4OH, 30% ethyl acetate in
hexane) to afford the product.
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(2′R, 5α, 6R, 7R, 14α)-2′-(4,5-epoxy-7,8-dihydro-3,6-dimethoxy-17-cyclopropylmethyl-6,14-
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ethano-morphinan-7-yl)-5′-methyl-hexan-2′-ol (9c)
Using general procedure A on 8, with isopentyl magnesium bromide, 9c was isolated as a white
solid (216 mg, 37%). 1H NMR (400 MHz, CDCl3) δ 0.08ꢀ0.12 (2H, m), 0.44ꢀ0.52 (2H, m), 0.70ꢀ
0.83 (2H, m), 0.91 (3H, d), 0.93 (3H, d), 1.01ꢀ1.08 (2H, m), 1.19ꢀ1.56 (5H, m), 1.34 (3H, s), 1.66
(1H, dd), 1.74ꢀ1.85 (2H, m), 1.90ꢀ2.03 (2H, m), 2.19ꢀ2.38 (4H, m), 2.64 (1H, dd), 2.79ꢀ2.86 (1H,
m), 2.95ꢀ3.01 (2H, m), 3.54 (3H, s), 3.88 (3H, s), 4.42 (1H, s), 5.10 (1H, s), 6.54 (1H, d), 6.70 (1H,
d). HRMS (ESI+) calcd for C31H46NO4 (MH+), 496.3421; found 496.3414 (100%).
(2′R, 5α, 6R, 7R, 14α)-2′-(4,5-epoxy-7,8-dihydro-3-hydroxy-6-methoxy-17-cyclopropylmethyl-
6,14-ethano-morphinan-7-yl)-5′-methyl-hexan-2′-ol (1c)
Using the general procedure B with 9c (50 mg) gave 1c as a yellow solid (47 mg, 98%) 1H NMR
(400 MHz, CDCl3) δ 0.08ꢀ0.12 (2H, m), 0.46ꢀ0.50 (2H, m), 0.70ꢀ0.83 (2H, m), 0.91 (6H, d), 1.00ꢀ
1.08 (2H, m), 1.19ꢀ1.26 (1H, m), 1.34 (3H, s), 1.34ꢀ1.54 (4H, m), 1.65ꢀ1.69 (1H, m), 1.77ꢀ1.81 (2H,
m), 1.90ꢀ2.03 (2H, m), 2.18ꢀ2.38 (4H, m), 2.66 (1H, dd), 2.79ꢀ2.86 (1H, m), 2.95ꢀ3.00 (2H, m),
3.52 (3H, s), 4.42 (1H, s), 5.16 (1H br s), 6.50 (1H, d, J=8.0 Hz), 6.69 (1H, d). HRMS (ESI+) calcd
for C30H44NO4 (MH+), 482.3265; found 482.3241 (100%); Anal. (C30H43NO4ꢃHClꢃ1.5H2O) C, H, N.
(1′S, 5α, 6R, 7R, 14α)-1′-(4,5-epoxy-7,8-dihydro-3,6-dimethoxy-17-cyclopropylmethyl-6,14-
ethano-morphinan-7-yl)- 2′-cyclohexyl-ethan-1′-ol (11a: R = cyclohexylmethyl)
Using general procedure A on 10 with cyclohexylmethylmagnesium bromide gave 11a, isolated as
a white solid, 89%. 1H NMR (400 MHz, CDCl3) δ 0.09ꢀ0.10 (2H, m), 0.46ꢀ0.50 (2H, m), 0.68ꢀ0.72
(1H, m), 0.78ꢀ0.89 (3H, m), 0.95ꢀ0.98 (1H, m), 1.13ꢀ1.28 (5H, m), 1.47ꢀ1.53 (4H, m), 1.63ꢀ1.71
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(8H, m), 1.83ꢀ1.86 (1H, m), 2.00ꢀ2.02 (2H, m), 2.21ꢀ2.29 (3H, m), 2.32ꢀ2.42 (1H, m), 2.58ꢀ2.66
(2H, m), 2.95ꢀ3.00 (1H, d), 3.06ꢀ3.08 (1H, d), 3.41 (1H, s), 3.86 (3H, s), 4.41 (1H, s), 6.53 (1H, d),
6.68 (1H, d); HRMS (ESI+) calcd for C32H46NO4 (MH+), 508.34; found 508.34.
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(5α, 6R, 7R, 14α)-1’-(4,5-epoxy-7,8-dihydro-3,6-dimethoxy-17-cyclopropylmethyl-6,14-
ethano-morphinan-7-yl)-2′-cyclohexylethanone (12, R = cyclohexylmethyl)
Using general procedure D on 11a (R = cyclohexylmethyl) gave 12 (R = cyclohexylmethyl) as a
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white solid (80 mg, 32%). H NMR (400 MHz, CDCl3) δ 0.07ꢀ0.08 (2H, m), 0.45ꢀ0.49 (2H, m),
0.68ꢀ0.78 (2H, m), 0.84ꢀ0.95 (2H, m), 1.12ꢀ1.16 (1H, m), 1.22ꢀ1.35 (4H, m), 1.50ꢀ1.51 (1H, m),
1.54 (3H, s), 1.58ꢀ1.68 (5H, m), 1.83ꢀ1.85 (1H, m), 1.90ꢀ2.00 (1H, m), 2.26ꢀ2.35 (3H, m), 2.37ꢀ2.38
(1H, d), 2.47ꢀ2.53 (1H, dd), 2.59ꢀ2.63 (1H, m), 2.66ꢀ2.73 (1H, m), 2.94ꢀ2.99 (2H, m), 3.04ꢀ3.06
(1H, d), 3.39 (1H, s), 3.86 (3H, s), 4.44 (1H, s), 6.54 (1H, d), 6.69 (1H, d); HRMS (ESI+) calcd for
C32H44NO4 (MH+), 506.33; found 506.33.
(2′R, 5α, 6R, 7R, 14α)-2′-(4,5-epoxy-7,8-dihydro-3,6-dimethoxy-17-cyclopropylmethyl-6,14-
ethano-morphinan-7-yl)-3′-cyclohexyl-propan-2′-ol (13: R = cyclohexylmethyl)
Using procedure A for the addition of methylmagnesium bromide to 12 (R = cyclohexylmethyl), 13
(R = cyclohexylmethyl) was isolated as a white solid (34 mg, 89%), 1H NMR (400 MHz, CDCl3) δ
0.08ꢀ0.10 (2H, m), 0.47ꢀ0.50 (2H, m), 0.69ꢀ0.72 (2H, m), 0.90ꢀ0.99 (2H, m), 1.04ꢀ1.10 (2H, m),
1.15 (3H, s), 1.20ꢀ1.28 (3H, m), 1.56ꢀ1ꢀ68 (8H, m), 1.74ꢀ1.77 (2H, m), 1.85 (1H, m), 1.99ꢀ2.03
(2H, m), 2.18ꢀ2.24 (2H, m), 2.26ꢀ2.36 (1H, m), 2.58ꢀ2.63 (1H, m), 2.78ꢀ2.86 (1H, m), 2.96ꢀ2.99
(1H, d), 2.99ꢀ2.03 (1H, d), 3.51 (1H, s), 3.87 (3H, s), 4.38 (1H, s), 4.68 (1H, s), 6.53 (1H, d), 6.69
(1H, d); HRMS (ESI+) calcd for C33H48NO4 (MH+), 522.36; found 522.36.
(2′S, 5α, 6R, 7R, 14α)-2′-(4,5-epoxy-7,8-dihydro-3-hydroxy-6-methoxy-17-cyclopropylmethyl-
6,14-ethano-morphinan-7-yl)-3′-cyclohexyl-propan-2′-ol (2e)
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Using General Procedure B on 13 (R = cyclohexylmethyl) gave 2e as a white solid. 1H NMR, 270
MHz (CDCl3) δ 0.07ꢀ0.11 (2H, m), 0.46ꢀ0.50 (2H, m), 0.67ꢀ0.81 (2H, m), 0.86ꢀ1.30 (10H, m),
1.55ꢀ1.89 (12H, m), 1.94ꢀ2.08 (2H, m), 2.14ꢀ2.26 (3H, m), 2.34ꢀ2.41 (1H, m), 2.55 (1H, dd), 2.76ꢀ
2.86 (1H, m), 2.92 (1H, d), 2.99 (1H, d), 3.50 (3H, s), 4.40 (1H, s), 4.71 (1H, s), 6.48 (1H, d), 6.66
(1H, d); HRMS, m/z for (C32H46NO4) [MH]+, calcdꢀ 508.3427, foundꢀ 508.3424. Anal.
(C32H45NO4ꢃHClꢃ1.5H2O) C, H, N.
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(1′R, 5α, 6R, 7R, 14α)-1′-(4,5-epoxy-7,8-dihydro-3,6-dimethoxy-17-cyclopropylmethyl-6,14-
ethano-morphinan-7-yl)-3′-phenyl-propan-1′-ol (11a, R = phenethyl)
Phenethylmagnesium bromide addition to 10 using general procedure A gave 11a, isolated as a
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white solid (218 mg, 58%). Rf 0.48: H NMR (400 MHz, CDCl3) δ 0.08ꢀ0.10 (2H, m), 0.46ꢀ0.49
(2H, m), 0.72ꢀ0.96 (2H, m), 1.15ꢀ1.25 (3H, m), 1.51ꢀ1.66 (2H, m), 1.72ꢀ1.79 (4H, m), 1.90ꢀ2.03
(2H, m), 2.21ꢀ2.35 (4H, m), 2.62ꢀ2.69 (3H, m), 2.95ꢀ3.06 (2H ,dd), 3.38 (3H, s), 3.86 (3H, s), 4.18
(1H, m), 4.40 (1H, s), 6.53 (1H, d), 6.68 (1H, d), 7.16ꢀ7.30 (5H, m); HRMS (ESI+) calcd for
C33H42NO4 (MH+), 516.31; found 516.31.
(5α, 6R, 7R, 14α)-(4,5-epoxy-7,8-dihydro-3,6-dimethoxy-17-cyclopropylmethyl-6,14-ethano-
morphinan-7-yl)-3-phenyl-propanone (12: R = phenethyl)
11a, R = phenylethyl (0.36 mmmol) was treated as described in general procedure D. The residue
was purified by column chromatography using combi flash machine (30% ethyl acetate in hexane)
to afford 12 (R = phenethyl). Isolated as a white solid (100 mg, 54%) Rf 0.55: 1H NMR (400 MHz,
CDCl3) δ 0.07ꢀ0.09 (2H, d), 0.44ꢀ0.49 (2H, m), 0.73ꢀ0.76 (1H, m), 1.15ꢀ1.32 (2H, m), 1.53ꢀ1.57
(1H, m), 1.56ꢀ1.71 (2H, m), 1.99ꢀ2.03 (2H, m), 2.23ꢀ2.32 (4H, m), 2.59ꢀ2.60 (1H, m), 2.70ꢀ2.78
(2H, m), 2.89ꢀ3.08 (5H, m), 3.37 (3H, s), 3.86 (3H, s), 4.43 (1H, s), 6.54 (1H, d), 6.68 (1H, d), 7.14ꢀ
7.24 (5H, m);; HRMS (ESI+) calcd for C33H40NO4 (MH+), 514.30; found 514.30.
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(1′R, 5α, 6R, 7R, 14α)-1′-(4,5-epoxy-7,8-dihydro-3,6-dimethoxy-17-cyclopropylmethyl-6,14-
ethano-morphinan-7-yl)-3′-phenyl-propan-1′-ol (11b: R = phenethyl)
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give 11b (R = phenethyl). Isolated as a white solid (86 mg, 86%). Rf 0.51. H NMR (400 MHz,
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56
57
58
59
60
CDCl3) δ 0.07ꢀ0.09 (2H, m), 0.47ꢀ0.49 (2H, m), 0.72ꢀ0.80 (2H, m), 0.87ꢀ0.99 (2H, m), 1.59ꢀ1.82
(5H, m), 1.96ꢀ2.04 (2H, m), 2.18ꢀ2.34 (5H, m), 2.61ꢀ2.72 (2H, m), 2.74ꢀ2.82 (1H, m), 2.92ꢀ2.99
(3H, m), 3.54 (3H, s), 3.87 (3H, s), 4.47ꢀ4.48 (1H, d), 5.46 (1H, s), 6.54 (1H, d), 6.70 (1H, d), 7.15ꢀ
7.18 (1H, m), 7.24ꢀ7.28 (4H, m); HRMS (ESI+) calcd for C33H42NO4 (MH+), 516.31; found 516.31.
(1′R, 5α, 6R, 7R, 14α)-1′-(4,5-epoxy-7,8-dihydro-3-hydroxy-6-methoxy-17-cyclopropylmethyl-
6,14-ethano-morphinan-7-yl)-3′-phenyl-propan-1′-ol (3i)
11b (R = phenylethyl) (54 .0 mg, 0.10 mmol) was treated as described in general procedure B to
1
give 3i. Isolated as a white solid (48 mg, 96%). Rf 0.14; H NMR, 270 MHz (CDCl3) δ 0.06ꢀ0.09
(2H, m), 0.45ꢀ0.50 (2H, m), 0.64ꢀ0.80 (2H, m), 0.82ꢀ0.98 (2H, m), 1.58ꢀ1.86 (5H, m), 1.91ꢀ2.08
(2H, m), 2.13ꢀ2.32 (4H, m), 2.60ꢀ2.84 (3H, m), 2.91ꢀ3.03 (3H, m), 3.50 (3H, s), 3.81 (1H, t), 4.49
(1H, s), 5.45 (1H, s), 6.48 (1H, d), 6.67 (1H, d), 7.13ꢀ7.32 (5H, m); HRMS, m/z for (C32H40NO4)
[MH]+, calcdꢀ 502.2957, foundꢀ 502.2956. Anal. (C32H39NO4ꢃHClꢃ1.5H2O) C, H, N.
(1′S, 5α, 6R, 7R, 14α)-1′-(4,5-epoxy-7,8-dihydro-3-hydroxy-6-methoxy-17-cyclopropylmethyl-
6,14-ethano-morphinan-7-yl)-3′-phenyl-propan-1′-ol (4k)
1
11a (R = phenethyl) was treated as described in General Procedure B to give 4k. H NMR (400
MHz, CDCl3) δ 0.08ꢀ0.09 (2H, m), 0.45ꢀ0.50 (2H, m), 0.66 (1H, m), 079 (1H, m), 1.12ꢀ1.13 (1H,
m), 1.39ꢀ1.48 (1H, m), 1.52ꢀ1.57 (2H, m), 1.63ꢀ1.67 (1H, d), 1.70ꢀ1.80 (4H, m), 1.90ꢀ1.99 (3H, m),
2.19ꢀ2.38 (5H, m), 2.60ꢀ2.70 (3H, m), 2.85 (1H, m), 2.93ꢀ2.98 (1H, d), 3.04ꢀ3.05 (1H, d), 3.35 (3H,
s), 4.41 (1H, s), 6.49 (1H, d), 6.67 (1H, d), 7.16 (1H, m), 7.16ꢀ7.30 (5H, m); HRMS (ESI+) calcd for
C32H40NO4 (MH+), 502.2957; found 502.3002. Anal. (C32H39NO4.HCl.0.5H2O) C, H, N.
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Molecular Modeling. CoMFA - All the modelling was carried out using the SybylꢀX 1.0
molecular modelling environment from Tripos Inc. The molecules were built, assigned with
GasteigerꢀHuckel charges and minimised using the MMFF94s force field. The CoMFA analysis
was carried out as per the instructions in the SybylꢀX 1.0 documentation. Docking ꢀ The 4DJH
crystal structure of the KOR and the 4DKL crystal structure of the MOR were prepared for docking
work by running them through the Protein Preparation Wizard tool of the Schrödinger software. 1n
was built using the Schrödinger software. GOLD was used to dock 1n into both prepared protein
structures. The binding site was defined as a sphere of 5Å radius centred on the centroid of the
crystal structure ligand with the requirement that the centroid of the docked ligand lie within this
sphere. Water molecules in the crystal structure were left in place.
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Supporting Information. Full experimental details. This material is available free of charge via the
Internet at http://pubs.acs.org
Acknowledgement
This work was funded by the National Institutes of Health National Institute on Drug Abuse grant
DA07315 (SMH). MPT was supported by the Wellcome Trust (Programme Grant 082837 to BVL
Potter, University of Bath).
Abbreviations used
MOR, mu opioid receptor; DOR, delta opioid receptor; KOR, kappa opioid receptor; LAAM, levoꢀ
αꢀacetylmethadol (or (3S,6S)ꢀ(6ꢀdimethylaminoꢀ4,4ꢀdiphenylꢀheptanꢀ3ꢀyl)acetate)
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Table 1: Binding affinitiesa of ligands to opioid receptors and stimulation of [35S]GTPγS bindingb
3
4
5
to CHOꢀKOR and C6ꢀMOR membranes.
6
7
8
9
R
Ki/nM – MOR Ki/nM ꢀ KOR EC50/nM, % stim’
MOR
EC50/nM,
KOR
% stim’
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
iPr
iPr
ibutyl
ipentyl
cpentyl
cpentylmethyl 0.29±0.063
cpentylethyl
chexyl
chexylmethyl
chexylethyl
benzyl
phenethyl
nPropyl
nPentyl
tButyl
ipropyl
ibutyl
ipentyl
cpentyl
chexylmethyl
chexylethyl
nPropyl
nPentyl
tButyl
0.15±0.013
0.60±0.05
0.20±0.037
0.22±0.018
0.21±0.042
0.051±0.024
0.40±0.20
2.1±1.4,
12±4.4
14±1.0
0.083±0.048, 30±5.3
1a
1ac
1b
1c
1d
1e
1f
1g
1h
1i
0.092±0.025
0.060±0.013
0.074±0.024
0.069±0.019
0.12±0.019
0.10±0.033
0.051±0.016
0.18±0.022
0.082±0.025
0.065±0.022
1.2±0.10
1.2±0.56,
0.19±0.049, 33±7.6
2.8±1.7, 25±3.7
0.56±0.12, 38±0.35 0.011±0.0033, 93±1.2
2.9±0.51,
0.057±0.029, 84±4.1
0.071±0.027, 69±5.1
0.068±0.045, 46±4.9
0.62±0.17
0.21±0.069
0.34±0.12
1.0±0.14
0.15±0.036
0.32±0.067
0.90±0.20
2.4±1.0
0.19±0.018
0.091±0.011
0.19±0.036
0.21±0.099
0.18±0.048
0.37±0.12
0.47±0.21
1.3±0.0
56±4.7
0.027±0.0067, 82±5.3
0.031±0.0061, 53±0.22
0.017±0.0012, 75±1.6
0.041±0.035, 89±1.7
0.071±0.011, 44±6.6
0.016±0.011, 89±6.9
N.D.
0.43±0.10, 35±2.8
1.7±0.86,
4.8±1.5,
1.3±0.98,
4.9±3.0,
N.D.
63±5.7
43±4.6
75±8.6
47±8.6
1j
1k
1lc
1mc
1n
2a
2b
2c
2d
2e
2f
4.7±0.10
N.D.
N.D.
0.067±0.021
0.11±0.024
0.11±0.024
0.15±0.044
0.15±0.037
0.22±0.057
0.16±0.012
2.0±0.50
0.27±0.094, 18±0.99 ꢀ,
0
53±2.6
1.27±0.72, 11±1.1
0.85±0.59, 7.6±1.7
0.43±0.098, 25±1.3
1.0±0.66, 9.3±2.1
0.72±0.015, 46±1.8
2.50±0.98, 8.5±1.8
N.D.
0.46±0.13,
0.18±0.078, 89±4.0
0.16±0.084, 96±11
0.083±0.016, 95±5.2
0.31±0.17,
0.46±0.15,
N.D.
87±1.5
72±2.4
2gc
2hc
2ic
3a
3b
3c
3d
3e
3f
3g
3h
3i
4a
4b
4c
4d
4e
4f
2.7±0.85
5.5±0.90
0.50±0.10
N.D.
N.D.
N.D.
N.D.
0.40±0.05
0.33±0.16
0.054±0.013
0.031±0.0086
0.31±0.23
0.15±0.035
0.043±0.016
0.060±0.021
0.038±0.018
0.029±0.0035
0.88±0.19
0.68±0.20
0.12±0.0074
1.0±0.48
ipropyl
ibutyl
ipentyl
cpentyl
0.067±0.011
0.059±0.014
0.033±0.011
0.078±0.015
0.090±0.042
0.015±0.0043
0.019±0.0030
0.020±0.0066
0.025±0.0039
0.16±0.079
0.057±0.025
0.067±0.023
0.086±0.048
0.086±0.040
0.062±0.017
0.12±0.0082
0.12±0.028
0.086±0.016
0.041±0.0060
0.047±0.0054
0.082±0.019
0.39±0.11
1.0±0.41, 6.9±3.8
ꢀ,
0.63±0.22, 49±3.1
0.17±0.045, 89±3.9
0.042±0.0027, 89±2.0
0d
1.5±0.99, 39±4.8
0.60±0.057, 11±1.2
0.26±0.063, 48±3.8
0.56±0.098, 45±4.5
2.0±0.45, 23±1.6
0.32±0.11, 41±2.4
0.29±0.12,
71±5.4
chexyl
0.053±0.0088, 78±1.9
0.19±0.037, 81±4.6
0.24±0.027, 94±9.3
0.077±0.039, 87±8.7
0.052±0.020, 109±8.2
0.035±0.018, 59±7.2
0.013±0.0092, 107±7.3
0.0061±0.002, 113±3.3
0.020±0.080, 104±12
0.007±0.0007, 103±3.9
0.0077±0.0045, 95±8.1
chexylmethyl
chexylethyl
benzyl
phenethyl
ipropyl
ꢀ,
ꢀ,
0d
0
ibutyl
ipentyl
0.48±0.24, 20±2.0
0.16±0.059, 65±2.0
0.29±0.12, 35±35
0.16±0.060, 80±3.3
0.16±0.026, 83±7.8
0.12±0.0070, 73±8.1 0.0098±0.003, 102±6.2
0.094±0.029, 91±9.3 0.029±0.015, 108±1.9
0.20±0.10, 34±2.9
0.23±0.13, 45±4.0
21.0±17.5, 7.5±2.0
1.0±0.42, 34±3.2
cpentyl
cpentylmethyl 0.36±0.067
cpentylethyl
chexyl
chexylmethyl
chexylethyl
benzyl
0.49±0.058
0.41±0.14
0.27±0.12
4g
4h
4i
4j
4k
5
0.23±0.17
0.047±0.003, 83±6.7
0.076±0.043, 63±7.3
0.030±0.0041
0.029±0.0058
0.18±0.068
phenethyl
0.73±0.58,
58±4.9
iPentyle
0.051±0.022, 91±3.7
2.2±0.66, 93±1.3
EKC
4.4±2.5,
26±1.0
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aKi (nM) versus [3H]diprenorphine, values are an average ± SEM from three separate experiments.
bPercent maximal stimulation (% stim) with respect to the standard agonists DAMGO (MOR) and
U69,593 (KOR), values are an average ± SEM from three separate experiments, N.D. not
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c
determined. Binding to Hartley guinea pig membranes, Ki (nM) versus [3H]DAMGO (MOR) and
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[3H]U69,593 (KOR). d 3b Ke (versus DAMGO) 0.50±0.08 nM, 3i Ke (versus DAMGO) 0.35±0.11.
eC6ꢀC14 etheno bridged. F Affinities for 1b and 1c at DOR were determined by displacement of
3
[ H]ꢀdiprenorphine binding from C6ꢀrat glioma cells expressing recombinant DOR; Ki values were
0.34±0.07 nM and 0.42±0.18 nM respectively. Efficacy at DOR was 1b:2.3±1.3%, EC50 not
determined and 1c: 7.9±1.7%; EC50 values not determined
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Table 2: Agonist activity (IC50/nM) in the guinea pig ileum (GPI) and antagonist activity (Ke/nM)
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in the mouse vas deferens (MVD).
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GPI
Ke (nM)a
MVDe
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IC50 (nM)
0.52 ± 0.49
P.A.c
CTAP norBNI
N.R.b N.R.
N.D.d N.D.
Ke (MOR)
Ke (KOR)
Ke (DOR)
0.90±0.32
0.16±0.03
0.06±0.02
ꢀ
1a iPr
1l nPr
0.034±0.004 0.37±0.21
0.007±0.0002 0.20±0.12
0.004±0.001 0.11±0.04
1m nPent 12.3 ± 2.1
N.R.
N.R
N.R.
N.R
1n tBu
2g nPr
8.1 ± 3.6
ꢀ
ꢀ
ꢀ
0.49±0.31
N.R.
N.D.
N.R.
0.71±0.07 0.028±0.003
N.D. 0.02±0.006
1.8±1.33 0.043±0.009
0.31±0.08
2h nPent P.A.
2i tBu 2.0±1.4
0.01±0.002 0.35±0.06
15±5.9
ꢀ
aKe (nM) of the selective antagonists norBNI (KOR) and CTAP (MOR) versus test compound.
bN.R. – the antagonists did not reverse the activity of the test compound. cPartial agonist with
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maximum inhibition of twitch of 30 – 50%. N.D. Not determined. Antagonist Ke (nM) of the test
compound versus the standard agonists DAMGO (MOR), U69,593 (KOR) and DPDPE (DOR).
Values are from two experiments each carried out in triplicate
Table 3: Antinociceptive activity in the PPQ induced abdominal stretch assay
Reversal by selective opioid antagonists: AD50 or % reversal
ED50, mg/kg s.c.
norBNI, mg/kg s.c. (KOR)
6.5 (2.6 – 16)
βꢀFNA, ꢀg/brain i.c.v (MOR)
0.02 (0.01 – 0.03) 15% at 1, 5% at 10, 27% at 30
0.02 (0.01 – 0.04) 14% at 3, 59% at 10, 12% at 30
1b
1c
2.5 (0.53 – 11.4)
Methods as described previously (ref 26)
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Figure 1: Two views of the areas, predicted by COMFA analysis, where interaction with a
lipophilic group would be beneficial for KOR activation (shown in green) and where interaction
with a lipophilic group would be detrimental to efficacy (shown in yellow).
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