Structural determinants of opioid and NOP receptor activity in derivatives of buprenorphine

Article abstract of DOI:10.1021/jm2003238

The unique pharmacological profile of buprenorphine has led to its considerable success as an analgesic and as a treatment agent for drug abuse. Activation of nociceptin/orphanin FQ peptide (NOP) receptors has been postulated to account for certain aspects of buprenorphine's behavioral profile. In order to investigate the role of NOP activation further, a series of buprenorphine analogues has been synthesized with the aim of increasing affinity for the NOP receptor. Binding and functional assay data on these new compounds indicate that the area around C20 in the orvinols is key to NOP receptor activity, with several compounds displaying higher affinity than buprenorphine. One compound, 1b, was found to be a mu opioid receptor partial agonist of comparable efficacy to buprenorphine but with higher efficacy at NOP receptors.

Full text of DOI:10.1021/jm2003238

ARTICLE
pubs.acs.org/jmc
Structural Determinants of Opioid and NOP Receptor Activity in
Derivatives of Buprenorphine
Gerta Cami-Kobeci,† Willma E. Polgar,‡ Taline V Khroyan,‡ Lawrence Toll,‡ and Stephen M. Husbands*,†
†Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, U.K.
‡SRI International, Menlo Park, California 94025, United States
S Supporting Information
b
ABSTRACT: The unique pharmacological profile of buprenorphine has led to its considerable success as an
analgesic and as a treatment agent for drug abuse. Activation of nociceptin/orphanin FQ peptide (NOP)
receptors has been postulated to account for certain aspects of buprenorphine’s behavioral profile. In order
to investigate the role of NOP activation further, a series of buprenorphine analogues has been synthesized
with the aim of increasing affinity for the NOP receptor. Binding and functional assay data on these new
compounds indicate that the area around C20 in the orvinols is key to NOP receptor activity, with several
compounds displaying higher affinity than buprenorphine. One compound, 1b, was found to be a mu opioid receptor partial agonist
of comparable efficacy to buprenorphine but with higher efficacy at NOP receptors.
Chart 1. Structures
’ INTRODUCTION
The treatment of opiate abuse and dependence by detoxifica-
tion, substitution, and maintenance with methadone, LAAM and,
more recently, buprenorphine (1a)( Chart 1), is the most successful
pharmacotherapeutic program of substance abuse treatment.1
The pharmacological profile of buprenorphine has long been
recognized as having some unique features.2,3 Some of these
effects are explicable by the slow onset and even slower offset of
its interaction with mu opioid (MOP) receptors. These effects
include its long duration of action and the mildness of the
abstinence syndrome following its chronic administration and
the delay in the appearance of these effects.4 Of particular
significance to its use as a pharmacotherapy for heroin addiction
is its acute safety in overdosage, which clearly differentiates
buprenorphine from the abused opiates and the therapeutic
substitute, methadone. Though, as an MOP receptor partial
agonist, buprenorphine would be expected to show a ceiling to its
opiate effects, including respiratory depression, in fact, the level
of opiate effects from very high doses are lower than that from
intermediate doses, i.e., buprenorphine has bell-shaped do-
seꢀresponse curves for its opiate effects including respiratory
depression, thus, explaining its exceptional acute safety.5,6
The mechanism of the bell-shaped doseꢀresponse curves for
buprenorphine’s MOP receptor effects in vivo has been the
subject of considerable speculation. This has been addressed in a
review of buprenorphine’s basic pharmacology and the two most
favored explanations recognized as being (a) an interaction with
an opposing pronociceptive system and (b) an induction of
autoinhibition at higher doses, which can be attributed to
buprenorphine’s slow offset kinetics.6 The pronociceptive sys-
tem in question is the NOP receptor (nociceptin/orphanin FQ
receptor), a receptor to which most standard opioid ligands do
not bind with any significant affinity.7 Buprenorphine is therefore
somewhat unusual among opioids in having moderate affinity at
the NOP site.7 Although the affinity is 2 orders of magnitude
lower than the affinity for the MOP and KOP receptors, bupre-
norphine seems to have some agonist activity at NOP.8ꢀ10 Lutfy
and colleagues discovered that the bell-shaped antinociception
curve lost its downward component in the presence of an NOP
antagonist and in NOP knockout mice in the absence of an
antagonist.11 This finding led to the conclusion that NOP activa-
tion at high buprenorphine concentrations was the cause of the
downward part of the doseꢀresponse curve. Further support for
the influence of buprenorphine’s NOP activity was supplied by the
findings of Ciccocioppo and colleagues that low doses of bupre-
norphine enhanced alcohol consumption in rats but that high
doses of buprenorphine reduced the consumption of alcohol.12
Received: March 21, 2011
Published: August 25, 2011
r
2011 American Chemical Society
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Figure 1. Overlay of buprenorphine (colored by atom type) with Ro
64-6198 (gold, except for the NH, which is colored by atom type).
Figure 2. Overlay of buprenorphine (colored by atom type) and Ro 64-
6198 (gold, except for the NH, which is colored by atom type).
The reduced consumption produced by high buprenorphine
dosage was additive with the opiate antagonist naltrexone’s similar
effect but inhibited by a selective NOP antagonist.
Scheme 1a
Though the case for NOP involvement to explain buprenor-
phine’s bell-shaped doseꢀresponse curves in MOP receptor
mediated behavioral assays is compelling, it seems unlikely that
this is the whole story. This follows from the effect of the
competitive MOP receptor antagonist naloxone on buprenor-
phine’s antinociception bell-shaped doseꢀresponse curve.13
Naloxone induced a parallel displacement of the doseꢀresponse
curve to a higher dose range, without changing the bell-shape.
This is only possible when all components of the curve are sus-
ceptible to the competitive interaction with naloxone. The antag-
onist has no measurable affinity for NOP; therefore, it could not
affect the NOP component.14,15
The slow dissociation of buprenorphine from the MOP
receptor, though not involving any covalent interactions with
the receptor, corresponds to a pseudoirreversible binding. It can
be hypothesized that pseudoirreversible binding is preceded by
reversible binding during which time the ligand displays its
agonist effects.16 The prolonged occupation of the receptor
produces receptor blockade, which is manifested as an antagonist
effect of the ligand.6,17 The lower MOP receptor agonist effect
of higher doses of buprenorphine may be related to the observa-
tion by Bidlack and co-workers that the onset of the MOP
receptor antagonist effect in ligands related to methoclocinna-
mox (MC-CAM, 2: a ligand with a profile very similar to that of
buprenorphine) is dose-dependent with higher doses having a
very much faster onset.18,19
It is, therefore, unclear whether direct activation of NOP
receptors is the mechanism by which buprenorphine produces a
bell-shaped doseꢀresponse curve. This issue can be investigated
by identifying and characterizing novel ligands with differing
affinity and activity profiles at MOP and NOP receptors. Such
ligands might also allow for the development of substitution
therapies for opiate dependence with a duration of effect and
acute safety comparable to those of buprenorphine but with, for
example, a higher level of morphine-like effects to appeal to
addicts for whom methadone is presently prescribed. Alterna-
tively, it may allow for the identification of novel analgesics with
low or no abuse potential and low tolerance development.
a (i) N-Bromosuccinimide, 0.1 N H2SO4, 38% or N-chlorosuccinimide,
0.1 N HCl, 45%; (ii) PrSNa, HMPA, 120 °C, 56ꢀ89%.
A major feature of the pharmacophore is the presence of a basic
nitrogen to which is attached a large, lipophilic moiety. We have
previously demonstrated that replacement of the N-cyclopropyl-
methyl group of buprenorphine with the lipophilic cyclooctyl-
methyl group found in selective NOP receptor antagonists such as
J-113397,15 resulted in a significant decrease in affinity for the
NOP receptor7 suggesting that the N-17 substituent of buprenor-
phine does not play the same role as the N-substituent in high
affinity NOP receptor ligands and that an alternative group in the
buprenorphine structure interacts with the lipophilic site. Align-
ment (using the flexible alignment feature of MOE, v.2005.06) of
buprenorphine with known NOP receptor ligands (Figures 1 and
2 show alignments with Ro 64ꢀ6198 (3)21) indicates that either
the t-butyl group (Figure 1) or the aromatic A-ring (Figure 2) of
buprenorphine could be acting as the lipophilic group while still
allowing the basic nitrogen to occupy a position similar to that in 3.
These alignments suggest that either increasing the steric bulk of
the t-butyl substituent or adding a substituent to the aromatic ring
of buprenorphine could provide compounds with increased
affinity for NOP receptors.
’ DESIGN AND SYNTHESIS
To this end, 1-chloro- and 1-bromo-buprenorphine were
prepared using methods previously reported for the halogenation
of hydrocodone and oxycodone.22 Thus, buprenorphine 3-O-
The majority of NOP receptor ligands can be described by a
two-dimensional (2-D) pharmacophore as previously reported.20
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ARTICLE
Scheme 2a
a (i) RMgBr, THF/Toluene, 15ꢀ45%; (ii) PrSNa, HMPA, 120 °C, 52ꢀ80%.
methyl ether (4) was halogenated using the appropriate N-
halosuccinimide to give the 1-substituted thevinol (5) before
3-O-demethylation with propane thiolate (Scheme 1), while
bromination of buprenorphine itself with N-bromosuccinimide
gave 2-bromobuprenorphine (7) directly, analogous to a pre-
vious iodination of buprenorphine.23 Attempts to synthesize
2-chlorobuprenorphine using an identical procedure with N-
chlorosuccinimide failed to give the desired product due to,
among other things, competing 6-O-demethylation.
Table 1. Binding Affinities to Opioid and NOP Receptors
Ki (nM) ( SEMa
NOP
mu
delta
kappa
nociceptin
0.08 ( 0.03
>10,000
437 ( 13
2846 ( 512
300 ( 59
147 ( 3.4
306 ( 46
>10,000
DAMGO
1.59 ( 0.17
504 ( 10
DPDPE
>10,000
1.24 ( 0.09
>10,000
U69,593
1a
>10,000
>10,000
1.6 ( 0.26
2.5 ( 1.2
Access to analogues of buprenorphine in which the t-butyl
group is replaced by alternative bulky groups was by the standard
procedure of Grignard addition to a ketone (8). This is a reaction
that is low yielding (30ꢀ40%) for buprenorphine itself due to the
size of the Grignard reagent. Unsurprisingly, it proved difficult to
increase bulk without adversely affecting this addition step. p-(t-
Butyl)phenylmagnesium bromide, phenanthren-9-ylmagnesium
bromide, and t-pentylmagnesium chloride were prepared from
their corresponding halides, and each gave the expected addi-
tion product (9b,e, 9g) on reaction with (8) (Scheme 2).
The attempted addition of (3-methylbut-2-en-2-yl)magnesium
bromide or 1-methylcyclopentylmagnesium bromide failed to
yield any of the desired tertiary alcohol. In the case of the alkyl
and arylalkyl Grignard reagents, the addition of a methylene
group to separate the reacting and bulky centers resulted, as
expected, in a more facile Grignard addition (9c,d,f). The
generally low yields (15ꢀ45%) were the result of rearrangements
leading to C4-phenols and, in the reaction with t-pentylmagne-
sium chloride, the secondary alcohol product of Grignard
reduction.24 In each case, the thevinol (9) was then 3-O-
demethylated to the corresponding orvinol (1) using propane
thiolate. Despite numerous attempts, this step failed for 9g (no
reaction took place), and the corresponding orvinol could not be
obtained.
77.4 ( 16
8.46 ( 1.3
18.5 ( 0.68
22.0 ( 0.55
133 ( 8.2
16.0 ( 1.5
237 ( 64
67.8 ( 20
159 ( 1.4
1.5 ( 0.8
6.1 ( 0.4
1b
2.14 ( 0.79
2.95 ( 0.65
2.19 ( 0.65
5.56 ( 1.3
11.4 ( 0.17
14.3 ( 0.88
13.2 ( 0.97
10.5 ( 3.86
1.59 ( 0.28
1.27 ( 0.21
3.66 ( 0.92
3.03 ( 0.61
7.13 ( 1.3
6.79 ( 0.99
17.2 ( 0.26
8.67 ( 0.81
5.63 ( 1.3
1.59 ( 0.25
4.15 ( 1.3
8.92 ( 0.34
9.87 ( 1.3
9.93 ( 3.7
45.6 ( 3.4
35.5 ( 3.9
1c
1d
1e
1f
6a
6b
7
a Binding to cloned human opioid receptors transfected into CHO cells
(method in ref 25). Values are the average of two experiments each
carried out in duplicate. Tritiated ligands were [3H]DAMGO (MOP),
[3H]Cl-DPDPE (DOP), [3H]U69593 (KOP), and [3H] N/OFQ.
respectively. Buprenorphine (1a) was found to bind with nano-
molar affinity to MOP, KOP, and DOP receptors with around 50-
fold lower affinity for NOP receptors compared to that for MOP
receptors (Table 1). Halogenation at C-1 or C-2 (6a, 6b, 7) had
little effect on NOP and DOP receptor affinity but resulted in
somewhat lower affinity for MOP and KOP receptors. This might
imply that the aryl ring of buprenorphine does not occupy the
lipophilic site described within the NOP receptor pharmacophore.
Because of this result, no further aryl ring substituted analogues
were synthesized and evaluated and attention focused on the
C20 group.
Of the analogues of 1a with variation in the C20 substituent,
1b and 1c, having t-pentyl and neopentyl groups, are the closest
in structure to 1a, being homologues with one extra methylene
group. Compared to 1a, 1b retained affinity at opioid receptors
(all 1.6ꢀ5.6 nM) and was 1 order of magnitude higher in affinity
at NOP receptors (8.5 nM versus 77 nM). Therefore, 1b has
essentially equivalent affinities for each of the opioid and NOP
’ RESULTS AND DISCUSSION
Binding affinities of the new compounds, plus various stan-
dards, were determined in Chinese hamster ovary (CHO) cells
transfected with human receptor cDNA, as previously described;25
the displaced selective radioligands were [3H]DAMGO, [3H]Cl-
DPDPE, [3H]U69593, and [3H]N/OFQ for binding to the MOP,
delta opioid (DOP), kappa opioid (KOP), and NOP receptors,
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Table 2. Stimulation (EC50/nM and % Stimulation) of [35S]GTPγS Binding to Opioid and NOP Receptors
MOR
NOP
DOR
KOR
EC50
% stima
100
EC50
% stima
EC50
% stima
100
EC50
% stima
DAMGO
35.3 ( 0.53
6.86 ( 0.4
nociceptin
8.1 ( 1.4
116 ( 88
78.6 ( 49
100
78.5 ( 8.80
100
1a
1b
1c
1d
1e
1f
10.2 ( 2.2
6.0 ( 2.1
2.6 ( 1.2
28.7 ( 1.1
20.1 ( 8.7
26.5 ( 8.0
16.8 ( 1.3
0
21.0 ( 8.4
48 ( 13
5.4 ( 3.4
42.7 ( 21
9.3
>10,000
>10,000
10.8 ( 6.8
20.6 ( 0.6
42.7 ( 6.7
0
0
50.1 ( 7.1
16.9 ( 1.5
33.7 ( 3.4
0
2380 ( 156
111 ( 10
3411 ( 819
0.88 ( 0.58
5.33 ( 1.8
6.1 ( 2.7
67.7 ( 7.0
18.6 ( 19.0
13.8 ( 4.8
17.1 ( 1.1
58.2 ( 20
0
49.3 ( 4.6
61.3 ( 21
17.0 ( 4.8
0
52.4 ( 7.9
0
6a
6b
7
0
12.4
17.0 ( 1.9
12.9 ( 4.9
6.05 ( 3.6
a Percent maximal stimulation with respect to the standard agonists DAMGO (MOP), U69593 (KOP), DPDPE (DOP), and nociceptin (NOP). Values
are the means of 5 or 6 experiments. EC50 values were not calculated if the % stimulation was < 20%.
receptors. Compound 1c behaves similarly in binding, though
the effect at NOP receptors is not as profound, being 4- to 5-fold
higher in affinity than 1a. Once again, 1c has high, nonselective
affinity for the three opioid receptors comparable with that of
1a. Compound 1d, which is structurally related to 1c, having a
phenyl group in place of one of the methyls of the neopentyl
group, has an affinity similar to that of 1c for all four receptors
and is 10-fold selective for MOP over NOP receptors. Com-
pound 1f, in which the C20 substituent is a bulky tricyclic group
attached through a methylene spacer to C20, also shows
increased affinity for NOP receptors compared to that of 1a
but couples this with a reduced affinity for opioid receptors such
that it has near 10 nM affinity for each of the four receptors. The
final ligand, 1e, has the t-butyl group separated from C20 by a
phenyl ring; it displays no increase in affinity for NOP receptors
over 1a, while having similar or slightly lower opioid receptor
affinity. Overall, the binding data suggest that it is the C20
substituent of 1a and related orvinols that occupy the putative
lipophilic site in the NOP receptor, as required for high affinity
binding.
The ligands were assessed for functional activity in the
[35S]GTPγS-binding assay in human receptor transfected CHO
cells as described previously.25 The results from these assays are
shown in Table 2. Buprenorphine (1a) was confirmed as a low
efficacy partial agonist at the MOP receptor with 29% stimulation
relative to the standard MOP receptor agonist DAMGO and as a
low potency (116 nM) and low efficacy (21% relative to
nociceptin) partial agonist at NOP receptors. The data for 1a
is consistent with the suggestion that its in vivo profile is due to it
having MOP receptor mediated effects at low doses with NOP
receptor activation being noticeable at higher doses.12,26
of 15% at 30 mg/kg) and only reached a peak of 20% in the much
lower stimulus intensity PPQ-induced antiwrithing assay in
which even low efficacy partial agonists are active.27 Compound
6b did act as an antagonist versus morphine in the tail flick assay,
AD50 4.7 mg/kg, which is comparable to that obtained with 1a
(AD50 1 mg/kg). However, 1a was an agonist in both the tail flick
(ED50 0.14 mg/kg) and PPQ-induced writhing assays (ED50
0.016 mg/kg) confirming it as a partial agonist in vivo.
Of the C20 analogues of buprenorphine (1a), the one carbon
homologues 1b and 1c displayed efficacy similar to that of 1a at
MOP receptors (all 20ꢀ29% relative to DAMGO) and were of
very similar potency (2.6ꢀ10.2 nM). Quite substantial differ-
ences in efficacy were found between these three ligands at the
NOP receptor (5ꢀ48% relative to nociceptin). Compound 1a
was of similar efficacy at the NOP receptor as at the MOP
receptor, but an order of magnitude less potent at the former,
while the homologue having a methylene between C20 and the t-
butyl group (1c) had little or no efficacy at the NOP receptors. In
contrast, 1b was more efficacious, and of similar or higher
potency, at the NOP receptors than was 1a. As found with 1a,
1b was around 10-fold more potent at the MOP receptor com-
pared to the NOP receptor. The low efficacy at MOP and KOP
receptors displayed by 1b and 1c is notable due to their structural
relationship to the n-propyl orvinols 10a and 10b. In the original
orvinol and N-CPM nororvinol series, a C20 R-group three
carbon atoms in length was associated with a peak in analgesic
activity, through the activation of MOP or KOP receptors.28
Both 1b and 1c have a substituted three carbon chain and yet
have low efficacy at both MOP and KOP receptors indicating
that the effect of branching in lowering efficacy at opioid
receptors outweighs the higher efficacy associated with a three
carbon-long chain. Compound 1d, in which a phenyl ring
replaces a methyl group in 1c, had low efficacy at MOP receptors
and only very low potency at NOP receptors. The most notice-
able feature about 1d’s profile in this assay was the increase in
efficacy at KOP receptors, such that it was a potent low efficacy
partial agonist. This compares with the equivalent phenethyl
compound (1, R = (CH2)2Ph; unpublished) that demonstrates
negligible efficacy at any of the opioid receptors under these
conditions but was a partial agonist at NOP receptors (35%
stimulation). Similarly, 1f displayed KOP receptor partial agonist
activity, suggesting that the introduction of the methylene group
The halogenated analogues (6a, 6b, and 7) had little efficacy at
any receptor studied. Each had around half the efficacy of
buprenorphine at the MOP receptors with only the 2-bromo
analogue (7) demonstrating any efficacy at the NOP receptors.
1-Chlorobuprenorphine (6b) is interesting as a highly lipophilic,
universal opioid and NOP receptor antagonist. In vitro, compound
6b inhibited agonist stimulated [35S]GTPγSactivityat MOP, DOP,
KOP, and NOP receptors (see Figure S1 in the Supporting
Information). Its MOP receptor antagonist activity has been
confirmed in vivo in mice, where it was found to have little to no
efficacy in the tail flick assay up to a dose of 30 mg/kg (peak effect
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ARTICLE
between C20 and the bulky substituent leads to a rise in KOP
receptor activation, as would be predicted from earlier studies.29,30
Compound 1f is profiled as a partial agonist at each of the four
receptors, with highest potency at MOP and KOP receptors.
Compound 1e had little to no efficacy at any receptor, and like
the 1-chloro analogue (6b), it may prove to be a universal opioid
and NOP receptor antagonist.
Because 1b has an in vitro profile similar to that of 1a, but with
higher affinity and efficacy for the NOP receptor, it was evaluated
in an antinociceptive assay (tail flick) in the mouse and has been
found to be an agonist of higher efficacy than buprenorphine
and equivalent in efficacy to morphine.31 This may indicate that
the MOP receptor mediated effects of BU08028 predominate
in vivo.
(purple solution) and titrating with 1 M 2-butanol (anhydrous) in THF
(end point, pale yellow solution).
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 N-cyclopropylmethyl-6,14-endo-ethanonorthevinone (8) (500 mg,
1.18 mmol) in anhydrous toluene (12 mL). After stirring at room
temperature for 20 h, the reaction was quenched by the addition of a
saturated aqueous ammonium chloride solution (20 mL). The phases
were separated, and the aqueous phase was 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 solu-
tion/hexane.
General Procedure C: 3-O-Demethylation. A solution of the
appropriate methyl ether (0.1 mmol) in anhydrous HMPA (0.5 mL)
under an inert atmosphere was treated with sodium hydride (8.5 mg,
0.35 mmol) followed by 1-propanethiol (32 μL, 0.35 mmol). After the
addition was complete, the reaction mixture was heated to 120 °C and
stirred until completion (∼3 h). Upon cooling to room temperature,
NH4Cl (sat, aq) was added, and the mixture was extracted with diethyl
ether. The organic extracts were washed with water (3ꢁ) and brine.
The organic phase was dried over MgSO4, filtered, and evaporated to
dryness. The residue was purified by column chromatography over silica gel.
The HCl salts were prepared bythe addition of5 N HCl in isopropanol
(2 equiv)toa solutionofthe orvinol indiethyl ether. The whiteprecipitate
that formed was collected by filtration, washed with ether, and dried under
high vacuum.
’ CONCLUSIONS
The notable rise in affinity at NOP receptors for compounds
1b, 1c, 1d, and 1f, but not with 6a, 6b, and 7, indicates that it is
the area around the C20 carbon of buprenorphine (1a) and
analogues that fulfils the role of the large lipophilic group found
in NOP receptor ligands. A methylene group inserted between
the bulky lipophilic group and C20 (1c, 1d, 1f) led to an increase
in efficacy at KOP receptors compared to that of 1a with less
consistent effects on MOP receptor activity. One compound, 1b,
profiled as an MOP receptor partial agonist of comparable
efficacy to buprenorphine, but with higher efficacy at NOP
receptors, is the subject of more extensive pharmacological
evaluation in vivo.31
(20R,5α,6R,7R,14α)-20-(1-Bromo-17-cyclopropylmethyl-
7,8-dihydro-4,5-epoxy-6,14-ethano-3-hydroxy-6-methoxy-
morphinan-7-yl)-30,30-dimethylbutan-20-ol (6a). Compound
5a was treated as described in General Procedure C, and 6a was isolated
as a white solid (52 mg, 89%). Rf 0.20; 1H NMR (270 MHz, CDCl3) δ
0.10ꢀ0.14 (2H, d, J = 8.0 Hz), 0.48ꢀ0.50 (2H, t, J = 7.8, 8.9 Hz),
0.73ꢀ0.75 (1H, m), 1.22 (9H, s), 1.22ꢀ1.27 (1H, m), 1.34 (3H, s),
1.62ꢀ2.17 (6H, m), 2.19 ꢀ2.23 (3H, m), 2.30ꢀ2.32 (2H, d, J = 6.4 Hz),
2.58ꢀ2.60 (1H, m), 2.75ꢀ2.82 (1H, d, J = 18.1 Hz), 2.85ꢀ2.86 (1H,
m), 3.01ꢀ3.03 (1H, d, J = 5 Hz), 3.50 (3H, s), 4.46 (1H, s), 5.84 (1H, s),
’ EXPERIMENTAL SECTION
Full details are available in the Supporting Information. Reagents and
solvents were purchased from Aldrich or Lancaster and used as received.
M.p.: Gallenkamp MFB-595 melting point apparatus, uncorrected.
NMR Spectra: Jeol Delta-270-MHz instrument, 1H at 270 MHz; Varian
Mercury-400-MHz 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: Perkin-Elmer 240C analyzer. Ligands were
tested as their hydrochloride salts and prepared by adding one equiva-
lent of (5 N hydrochloric acid in isopropanol) to an ether solution of the
compound. All compounds were >95% pure by microanalysis.
General Procedure A: Halogenation. Buprenorphine or bu-
prenorphine 3-O-methyl ether (0.31 mmol), was dissolved in H2SO4
(0.1 N, 16 mL) for bromination or HCl (0.1 N, 4 mL) for chlorination,
and N-halosuccinimide (0.37 mmol) was added in one portion. The
reaction mixture was stirred until TLC indicated that the starting
material had been consumed (approximately 3 h), and the mixture
was then poured into a separatory funnel containing dichloromethane
(17 mL). Sufficient aqueous sodium hydroxide (10%) solution was
added to raise the pH of the aqueous layer to ca. 10, the organic layer was
separated, and the aqueous layer was extracted further with three
portions of 9:1 dichloromethane/methanol (10 mL). The organic
extracts were combined and dried over anhydrous sodium sulfate, and
the solvent was removed under reduced pressure. The residue was
purified by column chromatography over silica gel eluting with a
gradient from 5% to 10% ethyl acetate in hexane.
6.90 (1H, s). ESIMS m/z: 547 [M + 1]+. Anal. (C29H40BrNO4
0.75H2O) CHN.
3
(20R,5α,6R,7R,14α)-20-(1-Chloro-17-cyclopropylmethyl-
7,8-dihydro-4,5-epoxy-6,14-ethano-3-hydroxy-6-methoxy-
morphinan-7-yl)-30,30-dimethylbutan-20-ol (6b). Compound
5b was treated as described in General Procedure C, and 6b was isolated
as a white solid (8 mg, 56%). Rf 0.15; 1H NMR (270 MHz, CDCl3) δ
0.12ꢀ0.13 (2H, d, J = 8.0 Hz), 0.47ꢀ0.49 (2H, m), 0.49ꢀ0.51 (1H, m),
0.78ꢀ0.83 (4H, m), 1.03 (9H, s), 1.25ꢀ1.36 (4H, m), 1.56 (3H, brs),
1.68ꢀ1.97 (4H, m), 2.04ꢀ2.14 (3H, m), 2.32ꢀ2.34 (1H, d, J = 6.3 Hz),
2.61ꢀ2.65 (1H, m), 2.81ꢀ2.82 (2H, m), 3.02ꢀ3.03 (1H, d, J = 6.3 Hz),
3.51 (3H, s), 4.47 (1H, s), 5.77 (1H, s), 6.75 (1H, s). ESIMS m/z: 503
[M + 1]. Anal. (C29H40ClNO4 HCl 0.75H2O) CHN.
3
3
(20R,5α,6R,7R,14α)-20-(2-Bromo-17-cyclopropylmethyl-
7,8-dihydro-4,5-epoxy-6,14-ethano-3-hydroxy-6-methoxy-
morphinan-7-yl)-30,30-dimethylbutan-20-ol (7). From bupre-
norphine (1a) using the halogenation procedure in General Procedure
1
A, 7 was obtained as a white solid (70 mg, 60%). Rf 0.25; H NMR
(270 MHz, CDCl3) δ 0.08ꢀ0.14 (2H, d, J = 8.0 Hz), 0.47ꢀ0.50 (2H, t,
J = 7.8, 8.9 Hz), 0.99ꢀ1.07 (1H, m), 1.22 (9H, s), 1.23ꢀ1.27 (3H, m),
1.35 (3H, s), 1.61ꢀ2.12 (5H, m), 2.19 ꢀ2.30 (3H, m), 2.30ꢀ2.33 (2H,
d, J = 6.4 Hz), 2.58ꢀ2.60 (1H, m), 2.75ꢀ2.82 (1H, d, J = 18.1 Hz),
2.85ꢀ2.86 (1H, m), 3.00ꢀ3.03 (1H, d, J = 5 Hz), 3.53 (3H, s), 4.45 (1H,
s), 5.80 (1H, s), 6.90 (1H, s). ESIMS m/z: 547 [M + 1]+. Anal.
General Procedure B: Grignard Addition. The Grignard
reagents were prepared from the corresponding bromides (6 mmol)
by reaction with magnesium (218 mg, 9 mmol) in anhydrous THF
(6 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 1,10-phenanthroline (∼2 mg) in anhydrous THF (2 mL)
(C29H40BrNO4 0.45H2O) CHN.
3
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Journal of Medicinal Chemistry
ARTICLE
(10RS,5α,6R,7R,14α)-20-(17-Cyclopropylmethyl-7,8-dihy-
dro-3,6-dimethoxy-4,5-epoxy-6,14-ethano-morphinan-7-
yl)- 30,30-dimethylpentan-20-ol (9b). General Procedure B was
used to isolate 9b as a clear oil (428 mg, 15%). Rf 0.58; 1H NMR (270
MHz, CDCl3) δ 0.08ꢀ0.11 (2H, m), 0.44ꢀ0.53 (2H, m), 0.81ꢀ0.86
(3H, m), 0.83ꢀ0.86 (3H, m), 0.89 (6H, s), 1.24ꢀ1.31 (2H, m), 1.34
(1H, s), 1.41 (3H, s), 1.55ꢀ1.80 (3H, m), 1.95ꢀ2.03 (2H, m),
2.27ꢀ2.34 (5H, m), 0.84 (1H, m), 2.79ꢀ2.84 (1H, t, J = 9 Hz),
2.94ꢀ2.98 (1H, m), 3.53 (3H, s), 3.86 (3H, s), 4.41 (1H, s), 5.96 (1H,
s), 6.53 (1H, d, J = 8.0 Hz), 6.67 (1H, d, J = 8.0 Hz, m). ESIMS m/z: 496
[M + 1]+.
2.03ꢀ2.10 (1H, m), 2.13ꢀ2.20 (5H, m), 2.46ꢀ2.48 (1H, m), 2.77ꢀ278
(1H, m), 2.87ꢀ2.91 (1H, d, J = 18.3 Hz), 3.54 (3H, s), 4.43 (1H, s), 5.44
(1H, s), 6.44ꢀ6.46 (1H, d, J = 8.0 Hz), 6.61ꢀ6.63 (1H, d, J = 8.0 Hz),
7.32ꢀ7.33 (2H, d, J = 4.7 Hz), 7.34ꢀ7.35 (2H, d, J = 4.7 Hz); 13C NMR
(100.6 MHz, CDCl3) δ 3.4, 3.5, 9.1, 17.8, 23.0, 23.4, 29.8, 31.3, 32.4, 34.3,
35.5, 36.0, 43.2, 47.0, 48.3, 52.7, 58.5, 59.2, 80.7, 97.2, 116.3, 119.4, 124.6,
125.6, 128.1, 132.3, 137.2, 144.0, 145.4, 149.3. ESIMS m/z: 544 [M + 1]+.
Anal. (C35H45NO4) CHN.
(20R,5α,6R,7R,14α)-20-(17-Cyclopropylmethyl-7,8-dihy-
dro-4,5-epoxy-6,14-ethano-3-hydroxy-6-methoxy-morphi-
nan-7-yl)-10-(bicyclo[2.2.1]heptan-1-yl)-propan-20-ol (1f).
General Procedure C was used to prepare 1f, isolated as a white solid
(55 mg, 62%). Rf 0.23; 1H NMR (400 MHz, MeOD) δ 0.16ꢀ0.18 (2H,
m), 0.51ꢀ0.53 (2H, d, J = 8.1 Hz), 0.71ꢀ0.82 (3H, m), 1.14ꢀ1.35 (3H,
m), 1.37ꢀ1.38 (15 H, m), 1.54ꢀ1.86 (7H, m), 2.22ꢀ2.39 (4H, m), 2.69
(1H, m), 2.99ꢀ3.00 (1H, m), 3.03ꢀ3.05 (1H, d, J = 18.1 Hz), 3.58 (3H,
s), 4.36 (1H, s), 6.47ꢀ6.50 (1H, d, J = 8.0 Hz), 6.62ꢀ6.64 (1H, d, J =
8.0 Hz); 13C NMR (100.6 MHz, CDCl3) δ 4.0, 4.4, 10.2, 19.1, 23.8, 24.8,
29.6, 30.7, 32.9, 36.0, 37.2, 38.6, 39.8, 40.9, 43.5, 44.8, 46.9, 53.0, 60.8,
67.1, 69.2, 81.9, 97.9, 117.9, 120.4, 128.5, 133.6, 139.5, 147.3. ESIMS m/
z: 520 [M + 1]+. Anal. (C33H45NO4 HCl 0.5H2O) CHN.
(20R,5α,6R,7R,14α)-20-(17-Cyclopropylmethyl-7,8-dihy-
dro-4,5-epoxy-6,14-ethano-3-hydroxy-6-methoxy-morphi-
nan-7-yl)-30,30-dimethylpentan-20-ol (1b). General Procedure C
was used to isolate (1b) as a clear oil (182 mg, 80%). Rf 0.23; 1H NMR
(400 MHz, CDCl3) δ 0.08ꢀ0.10 (2H, d, J = 8.0 Hz), 0.44ꢀ0.49 (2H, t,
J = 7.8, 8.9 Hz), 0.65ꢀ0.69 (1H, m), 0.72ꢀ0.80 (1H, m), 0.86ꢀ0.90
(3H, m), 0.91 (6H, s), 1.03ꢀ1.06 (1H, m), 1.31ꢀ1.34 (2H, m), 1.35
(3H, s), 1.40ꢀ1.45 (1H, m), 1.60ꢀ1.75 (3H, m), 1.80ꢀ1.86 (1H, m),
1.93ꢀ2.00 (1H, m), 2.15ꢀ2.36 (5H, m), 2.57ꢀ2.62 (1H, dd, J = 5 Hz),
2.79ꢀ2.86 (1H, m), 2.93ꢀ2.96 (1H, d, J = 17.0 Hz), 2.96ꢀ2.98 (1H, d,
J = 7.0 Hz), 3.51 (3H, s), 4.43 (1H, s), 5.95 (1H, s), 6.49 (1H, d, J = 8.1
Hz), 6.67 (1H, d, J = 8.1 Hz); 13C NMR (100.6 MHz, CDCl3) δ 3.2, 4.1,
9.1, 9.4, 18.3, 20.6, 21.4, 21.7, 22.8, 28.5, 29.5, 33.3, 35.5, 35.8, 42.7, 43.0,
43.6, 46.3, 52.4, 58.2, 59.4, 80.4, 80.8, 96.9, 116.3, 119.5, 128.3, 132.6,
3
3
’ ASSOCIATED CONTENT
S
Supporting Information. Full experimental details (spectra
b
137.2, 145.3. ESIMS m/z: 482 [M + 1]+. Anal. (C30H43NO4 0.3
and microanalysis data) plus figures detailing antagonist activity of
6b in vitro. This material is available free of charge via the Internet
at http://pubs.acs.org.
3
H2O) CHN.
(20R,5α,6R,7R,14α)-20-(17-Cyclopropylmethyl-7,8-dihy-
dro-4,5-epoxy-6,14-ethano-3-hydroxy-6-methoxy-morphi-
nan-7-yl)-40,40-dimethylpentan-20-ol (1c). Isolated as a white
solid, by using General Procedure C to yield 1c (20 mg, 52%). Rf
’ AUTHOR INFORMATION
1
Corresponding Author
*Tel: 44 (0)1225 383103. Fax: 44 (0)1225 386114. E-mail: s.m.
husbands@bath.ac.uk.
0.46; H NMR (400 MHz, CDCl3) δ 0.10ꢀ0.11 (2H, d, J = 8.0 Hz),
0.47ꢀ0.50 (2H, m), 0.72ꢀ0.75 (1H, m), 0.81ꢀ0.88 (2H, m), 1.08 (9H,
s), 1.24 (2H, s), 1.44 (6H, m), 1.63ꢀ1.66 (1H, d, J = 3 Hz) 1.73ꢀ1.81
(2H, m), 1.84ꢀ2.04 (2H, m), 2.17ꢀ2.40 (4H, m), 2.61ꢀ2.65 (1H, m),
2.81ꢀ2.87 (1H, m), 2.94ꢀ2.99 (1H, d, J = 18.1 Hz), 2.99ꢀ3.00 (1H, d, J =
5 Hz), 3.50 (3H, s), 4.42 (1H, s), 5.14 (1H, s), 6.48ꢀ6.50 (1H, d, J = 8.0
Hz), 6.66ꢀ6.68 (1H, d, J = 8.0 Hz); 13C NMR (100.6 MHz, CDCl3) δ 3.4,
4.1, 9.4, 18.0, 22.6, 24.0, 29.8, 31.9, 32.0, 32.6, 35.5, 36.0, 47.1, 48.4, 52.0,
52.6, 58.2, 59.8, 78.2, 80.9, 97.5, 116.2, 119.4, 128.4, 132.4, 137.1, 145.4.
ESIMS m/z: 482 [M + 1]. Anal. (C30H43NO4 HCl 0.3H2O) CHN.
’ ACKNOWLEDGMENT
This work was funded by the National Institutes of Health
National Institute on Drug Abuse grant DA023281.
’ ABBREVIATIONS USED
3
3
(20R,5α,6R,7R,14α)-20-(17-Cyclopropylmethyl-7,8-dihy-
dro-4,5-epoxy-6,14-ethano-3-hydroxy-6-methoxy-morphi-
nan-7-yl)-40-methyl-40-phenylpentan-20-ol (1d). General Procedure
C was used to prepare 1d and was isolated as a clear oil (60 mg, 80%). Rf 0.34;
1H NMR (400 MHz, CDCl3) δ 0.13ꢀ0.14 (2H, d, J = 8.1 Hz), 0.52ꢀ0.53
(2H, m), 0.55ꢀ0.56 (1H, m), 0.85ꢀ0.96 (6H, m), 1.41 (3H, s), 1.52 (5H,
m), 1.62ꢀ1.64 (2H, t, J = 6.8 Hz), 1.73ꢀ1.85 (3H, m), 2.03ꢀ2.07 (1H, d,
J=14.5Hz), 2.16ꢀ2.35(4H, m), 2.58ꢀ2.71(2H, m), 2.92ꢀ2.96 (1H, d, J=
18.4 Hz), 2.95ꢀ2.96 (1H, m), 3.43 (3H, s), 4.27 (1H, s), 5.18 (1H, brs),
6.46ꢀ6.48 (1H, d, J = 8.1 Hz), 6.64ꢀ6.66 (1H, d, J = 8.1 Hz), 7.15ꢀ7.17
(1H, t, J = 7.1 Hz), 7.27ꢀ7.29 (2H, d, J = 7.1 Hz), 7.44ꢀ7.45 (2H, d, J = 7.2
Hz); 13C NMR (100.6 MHz, CDCl3) δ 3.5, 4.0, 9.4, 18.3, 22.6, 23.8, 29.6,
32.6, 33.4, 35.3, 35.9, 37.9, 43.5, 46.6, 46.8, 52.4, 52.7, 58.3, 59.9, 80.8, 96.9,
116.3, 119.4, 125.0, 126.1, 127.9, 128.1, 132.4, 137.1, 145.4, 150.5. ESIMS m/
z: 544 [M + 1]+. Anal. (C35H45NO4 HCl 1.5H2O) CHN.
(20R,5α,6R,7R,14α)-10-(400-t-Butyl-phenyl)-10-(17-cyclopro-
pylmethyl-7,8-dihydro-4,5-epoxy-6,14-ethano-3-hydroxy-
6-methoxy-morphinan-7-yl)-ethan-10-ol (1e). Compound 1e
was prepared using General Procedure C and was isolated as a white
solid (50 mg, 79%). Rf 0.33; 1H NMR (400 MHz, CDCl3) δ ꢀ0.07ꢀ0.00
(2H, m), 0.31ꢀ0.33 (2H, d, J = 8.0 Hz), 0.68ꢀ0.72 (1H, m), 0.73ꢀ0.86
(1H, m), 0.86ꢀ0.92 (1H, m), 0.99ꢀ1.03 (1H, m), 1.31 (9H, s),
1.52ꢀ1.55 (1H, d, J = 12.5 Hz), 1.77 (6H, m), 1.81ꢀ1.84 (1H, m),
MOP receptor, mu opioid receptor; NOP receptor, nociceptin/
orphanin FQ receptor; MC-CAM, methoclocinnamox; CHO,
Chinese hamster ovary; KOP receptor, kappa opioid receptor;
DOP, delta opioid receptor
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