Benzylideneoxymorphone: A new lead for development of bifunctional mu/delta opioid receptor ligands

Article abstract of DOI:10.1016/j.bmcl.2016.11.057

Opioid analgesic tolerance remains a considerable drawback to chronic pain management. The finding that concomitant administration of delta opioid receptor (DOR) antagonists attenuates the development of tolerance to mu opioid receptor (MOR) agonists has led to interest in producing bifunctional MOR agonist/DOR antagonist ligands. Herein, we present 7-benzylideneoxymorphone (6, UMB 246) displaying MOR partial agonist/DOR antagonist activity, representing a new lead for designing bifunctional MOR/DOR ligands.

Full text of DOI:10.1016/j.bmcl.2016.11.057

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Benzylideneoxymorphone: A new lead for development of bifunctional
mu/delta opioid receptor ligands
Jason R. Healy a,b, Padmavani Bezawada c, Nicholas W. Griggs d, Andrea L. Devereaux e,
Rae R. Matsumoto a,f, John R. Traynor d, Andrew Coop c, Christopher W. Cunningham e,
⇑
a Department of Basic Pharmaceutical Sciences, West Virginia University School of Pharmacy, 2036 Health Sciences North, Morgantown, WV 26506, USA
b Department of Pharmacology and Experimental Therapeutics, Thomas Jefferson University, 1170 Main Bldg., 132 S. 10th St., Philadelphia, PA 19107, USA
c Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, 20 Penn Street, Baltimore, MD 21201, USA
d Department of Pharmacology, University of Michigan Medical School, 1220A MSRB, Ann Arbor, MI 48109, USA
e Department of Pharmaceutical Sciences, Concordia University Wisconsin School of Pharmacy, 12800 N. Lake Shore Drive, Mequon, WI 53097, USA
f Department of Biological and Pharmaceutical Sciences, Touro University California College of Pharmacy, 1310 Club Drive, Vallejo, CA 94592, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Opioid analgesic tolerance remains a considerable drawback to chronic pain management. The finding
that concomitant administration of delta opioid receptor (DOR) antagonists attenuates the development
of tolerance to mu opioid receptor (MOR) agonists has led to interest in producing bifunctional MOR ago-
nist/DOR antagonist ligands. Herein, we present 7-benzylideneoxymorphone (6, UMB 246) displaying
MOR partial agonist/DOR antagonist activity, representing a new lead for designing bifunctional
MOR/DOR ligands.
Received 14 September 2016
Revised 20 November 2016
Accepted 21 November 2016
Available online xxxx
Keywords:
Ó 2016 Elsevier Ltd. All rights reserved.
Mu opioid receptor
Delta opioid receptor
Analgesic tolerance
Oxymorphone
Benzylideneoxymorphone
Chronic severe pain is a considerable burden facing public
health, costing the United States an estimated $560–635 billion
in health care costs and loss of productivity in 2011.1 Opioid anal-
gesics remain the standard treatment for severe pain, due to their
ability to inhibit nociceptive processing and maintain the patient
in a state of well-being.2 Despite their benefits and widespread
use, long-term use of opioids such as morphine and oxycodone is
hindered by the rapid development of analgesic tolerance.
Tolerance to intestinal motility does not develop as rapidly as anal-
gesic tolerance; this ‘‘differential tolerance” results in severe con-
stipation arising from the increased doses required to
compensate for the diminished analgesic effect.3 Analgesics that
produce a blunted tolerance effect would be expected to lower
healthcare costs and provide beneficial outcomes to patients.
Three opioid receptor (OR) types have been cloned and charac-
terized, namely mu (MOR), delta (DOR), and kappa (KOR), and are
members of the G protein-coupled receptor (GPCR) superfamily.
All approved opioid analgesics are MOR agonists, and produce
analgesic and euphoric effects. Bifunctional opioid receptor ligands
possessing dual profiles of OR type agonism and antagonism may
also offer certain pharmacologic advantages. For example,
buprenorphine (Suboxone; Subutex) is a MOR partial agonist/
KOR antagonist that is used as an alternative to methadone for
treating opioid dependence.4
There is considerable evidence that concomitant DOR antago-
nism attenuates the development of tolerance to MOR agonists.
Studies using DOR knockout mice5 and DOR-specific antibodies,6
as well as pharmacologic inhibition of DOR using naltrindole,7 sup-
port this hypothesis. These proof-of-concept studies have led to the
development of ligands that are bifunctional MOR agonists and
DOR antagonists. Building upon the success of DIPP-NH2[W], the
first bifunctional, tetrapeptide MOR agonist/DOR antagonist to
produce antinociception with a low propensity to produce toler-
ance and dependence,8 several small-molecule bifunctional
MOR/DOR probes have been produced (Fig. 1) and demonstrate
efficacy in vitro and in vivo.9–12 To overcome limitations of solubil-
ity, oral bioavailability, and receptor-type potency, we have set out
to identify new chemical leads for bifunctional MOR agonist/DOR
antagonist ligands.
The DOR antagonist benzylidenenaltrexone (BNTX, Fig. 2) was
first described as a DOR-selective antagonist.13 This compound
was rationalized using the ‘‘message-address concept,” whereby
the naltrexone ‘‘message” was selectively targeted to DOR by the
⇑
Corresponding author.
E-mail address: chris.cunningham@cuw.edu (C.W. Cunningham).
http://dx.doi.org/10.1016/j.bmcl.2016.11.057
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Healy J.R., et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.11.057

2
J.R. Healy et al. / Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Fig. 1. Three examples of small-molecule, bifunctional MOR agonist/DOR antagonist lead molecules.10–12
the appropriate alkyl bromide. Intermediates 2, 4, and 5 were
converted to C7-benzylidene analogues 6–8 under basic Claisen
conditions.13 Purified final products were converted to water-sol-
uble salts (HCl, oxalate) prior to pharmacologic evaluation.22
OR binding results are shown in Table 1. Compound 6 bound
preferentially to MOR and DOR over KOR. Affinity for MOR and
DOR generally decreased as a function of N-arylalkyl chain length,
whereas KOR affinity was improved for 7 and 8 compared to 6. This
caused a net decrease in MOR/DOR preference over KOR, similar to
BNTX.14 The N-methyl analogue (6) exhibited the highest affinity
for MOR and DOR of the series, approximately 3- and 20-fold
greater than N-phenylethyl (7) and N-phenylpropyl (8) analogues
for MOR, respectively. This was unexpected, as N-phenylethyl
and N-phenylpropyl 4,5-epoxymorphinans are generally higher
affinity MOR agonists than their parent N-methyl equivalents.23,24
Fig. 2. Schematic describing the ‘‘message-address” rationale for designing bifunc-
tional MOR agonist/DOR antagonist probes based on modification of oxymorphone.
CPM = cyclopropylmethyl.
7-benzylidene ‘‘address.” Subsequent reports14,15 demonstrate
BNTX binds with similar affinity to MOR and antagonizes the
effects of MOR agonists with similar potency as DOR agonists.16
Thus, we consider BNTX to be a MOR/DOR antagonist. The N-sub-
stituent frequently modulates efficacy in the 4,5-epoxymorphinan
series of MOR analgesics. According to a ligand-based, quantitative
conformationally-sampled pharmacophore describing DOR
ligands,17 N-alkyl substitutions do not alter predicted efficacy in
this same series. Applying the message-address concept,18,19 we
hypothesize that substitution of the N-cyclopropylmethyl
‘‘message” of BNTX with N-alkyl groups satisfying the ‘‘message”
(Fig. 2) would enhance MOR efficacy and maintain low efficacy at
DOR, resulting in MOR agonist/DOR antagonist bifunctional
ligands.
Table 2 shows the results of a [35S]GTP
cS assay to determine the
relative efficacy of compounds at MOR and DOR. Compounds 6 and
7 were found to be MOR partial agonists with 6 demonstrating
approximately 3-fold higher potency than 7. Efficacy data for 8
could not be determined due to low potency. Compounds 6 and
7 possess no significant agonist activity at DOR. Compound 6 was
tested further to determine DOR antagonist potency. In the GTPcS
functional assay, 6 caused a parallel rightward shift in the SNC80
concentration–response curve with Ke of 138 24 nM (Table 2).
Taken together, compound 6 (benzylideneoxymorphone, UMB
246) exhibited a profile of MOR/DOR preferential binding affinity,
partial agonist effects at MOR, and antagonist activity at DOR.
Compound 6 was therefore selected for further characterization
in vivo.
Acute antinociception assays in the mouse were performed fol-
lowing subcutaneous (s.c.) administration of increasing doses of
6.24,25 As shown in Fig. 3, 6 produced an increase in Emax to approx-
imately 40% MPE with Tmax of 50 min in the hot plate nociception
test. Peak antinociception effects for UMB2 46 were evident at
50 min post-administration for the 60 mg/kg treatment group.
Repeated measures ANOVA revealed a significant difference in
latency for hot plate nociception testing among treatment groups
(p < 0.01) and among time points (p < 0.001). Bonferroni post hoc
analysis revealed that the 50 mg/kg treatment group had signifi-
cantly greater antinociception, compared to saline control, at 30
(p < 0.05) minutes post-administration (Fig. 3). Bonferroni post
hoc analysis revealed that the 60 mg/kg treatment group had sig-
nificantly greater antinociception, compared to saline control, at
30 (p < 0.01), 50 (p < 0.001), and 70 (p < 0.05) minutes post-
administration.
Synthesis of compounds 6–8 was achieved from oxycodone as
shown in Scheme 1. Oxycodone (1) was converted to oxymorphone
(2) using BBr3 in the usual manner.20 To synthesize N-substituted
analogues 7 and 8, 2 was N-demethylated to noroxymorphone
(3) using
a
-chloroethyl chloroformate21 and N-alkylated using
Fig. 4 shows the results from the tail-flick nociceptive test. Peak
antinociception effects for 6 were evident at 30 min post-adminis-
tration for the 60 mg/kg treatment group. Repeated measures
ANOVA revealed a significant difference in latency for tail-flick
noticeptive testing among treatment groups (p < 0.01) and among
time points (p < 0.001). Bonferroni post hoc analysis revealed that
the 50 mg/kg treatment group had significantly greater
antinociception, compared to saline control, at 70 (p < 0.01) min-
utes post-administration (Fig. 4). Bonferroni post analysis revealed
Scheme 1. Synthesis of 6–8. Reagents and conditions: i) BBr3, CHCl3, 0 °C, 30 min;
NH4OH(aq), 0 °C, 30 min. ii) Ac2O, reflux, 24 h; 1-chloroethyl chloroformate, K2CO3,
Cl(CH2)2Cl, reflux, 24 h; NaOH, MeOH, reflux, 3 h. iii) R-Br, K2CO3, RT, 24 h. iv)
PhCHO, NaOH, MeOH, 0 °C, 18 h
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J.R. Healy et al. / Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
3
Table 1
Ki values of analogues 6–8 at MOR, DOR, KOR, and receptor selectivity ratios.
Compound
Ki (nM) SEMa
MOR
KOR/MOR
MOR/DOR
DOR
KOR
6
7
8
17.5 1.10
56.0 3.80
313 23
26
14.4 0.65
84.0 2.80
168 9.20
6.2
1067 39
602 60
793 58
48
61
1.2
0.7
1.9
4
10.8
2.5
2
BNTXb
a
Competition binding for compounds were performed in CHO cells stably transfected with and overexpressing the human opioid receptor types hMOR, hDOR and hKOR.
Assays were performed in triplicate of duplicates and reported as mean Ki values SEM. MOR was labeled using 1.3 nM [3H]DAMGO, DOR using 1.2 nM [3H]DPDPE, and KOR
using 1.7 nM [3H]U69,593. Ki values for standard compounds: DAMGO (MOR, 1.5 nM), DPDPE (DOR, 2.4 nM), U69,593 (KOR, 1.1 nM).
b
Data from Ref. 14.
that the 60 mg/kg treatment group had significantly greater
antinociception, compared to saline control at 30 (p < 0.001), 50
(p < 0.01), and 70 (p < 0.01) minutes post-administration.
Table 2
EC50 (nM) and %Emax of oxymorphone analogues 6–8 at MOR and DOR.a
Compound
MOR
DOR
The reduced opioid receptor binding affinity and potency for
compounds 7 and 8 was unexpected. Introduction of the C7-ben-
zylidene may introduce a steric effect that requires BOM to adopt
an altered binding pose within MOR and DOR active sites, thereby
precluding larger N-arylalkyl substituents.
EC50 (nM)
%Emax
EC50 (nM)
%Emax
6
7
8
72 11
253 70
N.D.
39 1.3
52 3.8
<10
N.D.
N.D.
N.D.
N.D.b
<10
N.D.
There was some discrepancy between the Ki value (14.4 nM)
and Ke value (138 nM) for 6. Ideally, for a completely neutral
antagonist these values should be the same. If taken at face value
the higher Ke value obtained in more physiologically relevant buf-
fer would suggest that 6 does have some agonist activity, albeit
a
Percent maximal stimulation relative to standard agonist (MOR, DAMGO; DOR,
DPDPE; KOR, U69,593).
b
Caused a parallel, rightward shift in SNC80 concentration–response curve using
GTP
c
S assay (Ke = 138 24 nM, n = 3). Tested as freebase.
insufficient to show up in the stringent [35S]GTP
cS assay. On the
other hand the values may be reflective of the different DORs used
in the assays (human versus rat) and/or the different environments
in the in different cell lines employed (CHO versus C6).
UMB 246 Timecourse (Hot Plate)
100
90
80
70
60
50
40
30
20
10
0
Saline
In summary, results presented here indicate 6 possesses a pro-
file of MOR partial agonism and DOR antagonism in vitro. 6 also
produces antinociception following s.c. administration, which sug-
gests an ability to cross the blood–brain barrier and activate MOR
in vivo. This initial structure–activity relationship (SAR) study
shows longer-chain N-alkyl substituents do not enhance MOR
potency as in the parent oxymorphone series. Further investigation
into the structural features that improve MOR efficacy and potency
in the 7-benzylideneoxymorphone series are underway and will be
reported in due course.
20 mg/kg
30 mg/kg
40 mg/kg
50 mg/kg
60 mg/kg
-10
-20
0
0
0
0
9
3
5
7
Time After UMB 246 Administration (min)
Acknowledgments
Fig. 3. Acute dose- and time-response curves for s.c. 6 treatment for the hot plate
assay. For details, see Ref. 25. Significance relative to saline control: *p < 0.05.
**p < 0.01. ***p < 0.001
The authors thank the National Institute on Drug Abuse,
National Institutes of Health (NIDA, NIH) (A.C., DA 13583; C.W.C.,
DA 021049; J.R.T., DA 039997). C.W.C. is grateful to Concordia
University Wisconsin School of Pharmacy for financial support. J.
R.H. is supported by the National Institutes of Health Postdoctoral
training grant no. T32GM008562. CHO cells stably transfected to
express each of the human opioid receptor types (hMOR-CHO,
hDOR-CHO, hKOR-CHO cells) were generously provided by Dr.
Larry Toll (Torrey Pines Institute for Molecular Studies, Port St.
Lucie, FL).
UMB 246 Timecourse (Tail-Flick)
100
Saline
90
80
70
60
50
40
30
20
10
0
20 mg/kg
30 mg/kg
40 mg/kg
50 mg/kg
60 mg/kg
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