Synthesis and evaluation of 4-substituted piperidines and piperazines as balanced affinity μ opioid receptor (MOR) agonist/δ opioid receptor (DOR) antagonist ligands

Article abstract of DOI:10.1016/j.bmcl.2013.12.021

In this letter, we describe a series of 4-substituted piperidine and piperazine compounds based on tetrahydroquinoline 1, a compound that shows balanced, low nanomolar binding affinity for the mu opioid receptor (MOR) and the delta opioid receptor (DOR).

Full text of DOI:10.1016/j.bmcl.2013.12.021

Accepted Manuscript
Synthesis and evaluation of 4-substituted piperidines and piperazines as bal-
anced affinity μ opioid receptor (MOR) agonist/ δ opioid receptor (DOR)
antagonist ligands
Aaron M. Bender, Mary J. Clark, Michael P. Agius, John R. Traynor, Henry I.
Mosberg
PII:
S0960-894X(13)01393-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2013.12.021
BMCL 21129
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Accepted Date:
6 November 2013
4 December 2013
Please cite this article as: Bender, A.M., Clark, M.J., Agius, M.P., Traynor, J.R., Mosberg, H.I., Synthesis and
evaluation of 4-substituted piperidines and piperazines as balanced affinity μ opioid receptor (MOR) agonist/ δ
opioid receptor (DOR) antagonist ligands, Bioorganic & Medicinal Chemistry Letters (2013), doi: http://dx.doi.org/
10.1016/j.bmcl.2013.12.021
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Synthesis and evaluation of 4-substituted piperidines and piperazines as balanced affinity μ
opioid receptor (MOR) agonist/ δ opioid receptor (DOR) antagonist ligands
Aaron M. Bender1,2, Mary J. Clark3, Michael P. Agius1,2, John R. Traynor3, and Henry I.
Mosberg1,2*
1 Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann
Arbor, MI 48109.
2 Interdepartmental Program in Medicinal Chemistry, University of Michigan, Ann Arbor, MI
48109.
3 Department of Pharmacology, Medical School, University of Michigan, Ann Arbor, MI 48109
* To whom correspondence should be addressed
him@med.umich.edu

Abstract
In this report, we describe a series of 4-substituted piperidine and piperazine compounds
based on tetrahydroquinoline 1, a compound that shows balanced, low nanomolar binding
affinity for the mu opioid receptor (MOR) and the delta opioid receptor (DOR). We have shown
that by changing the length and flexibility profile of the side chain in this position, binding
affinity is improved at both receptors by a significant degree. Furthermore, several of the
compounds described herein display good efficacy at MOR, while simultaneously displaying
DOR antagonism. The MOR agonist/DOR antagonist has shown promise in the reduction of
negative side effects displayed by selective MOR agonists, namely the development of
dependence and tolerance.
OH
OH
NH2
N
NH2
N
N
O
O
1

Although opioid analgesics represent the gold standard for the treatment of acute and
chronic pain, their usage is often accompanied by undesirable side effects such as the
development of dependence and tolerance. A considerable amount of research has thus been
done to find a potent analgesic that does not display these negative attributes. In general,
clinically used opioid analgesics such as morphine evoke both the desired and undesired effects
through activation of the mu opioid receptor (MOR). Numerous reports have indicated that the
undesired MOR-related side effects may be ameliorated by concomitant ligand interaction with
the delta opioid receptor (DOR). It has been shown that the co-administration of DOR-selective
agonists1 or antagonists2 with a MOR agonist can attenuate the dependence and tolerance
typically associated with the latter.
A ligand displaying good binding affinity for both MOR and DOR represents a
significant advantage over the co-administration of multiple drugs, due to both increased
pharmacokinetic simplicity as well as improved patient compliance. For these reasons, the
development of small molecule MOR/DOR bifunctional opioid ligands has attracted much
attention. MOR agonist/DOR antagonist compounds have been shown to be effective analgesics
with a diminished tolerance and dependence profile3,4 and have found use in other areas, such as
for the treatment of irritable bowel syndrome.5 Recently, we showed that a MOR agonist/DOR
antagonist compound was an effective analgesic after interperitoneal administration, with a
duration of action comparable to morphine.6
In an effort to further develop drug-like MOR/DOR bifunctional ligands, we turned our
attention to compound 1 (Figure 1) a compound previously synthesized by our lab that displays
equal binding affinity for both MOR and DOR, as well as for the kappa opioid receptor (KOR)
(Ki = 25.8 nM (MOR); 33.0 nM (DOR); 36.5 nM (KOR), unpublished observations). Given the

relative simplicity of the compound and its nonselective binding profile, we reasoned it would be
a good starting point for derivatization. Computational modeling suggested that position 4 would
be the optimal point for diversification, as an aromatic moiety at this position would be ideally
situated to interact with Asn125, Thr218, and Lys303 in the MOR active site, and the resulting
compound would thus function as a MOR agonist.7
Figure 1. Compound 1
1 was initially substituted with a benzyl group at the 4 position (2, Table 1). The
synthesis of 2 began by subjecting ketone 13 to a Wittig reaction to yield alkene 14, which was
subsequently hydrogenated and deprotected to give amine 15, to which was coupled Boc-
protected L-2,6-dimethyltyrosine (Boc-L-Dmt) and deprotected (Scheme 1). Binding affinity
(Ki) was obtained by competitive displacement of radiolabeled [3H]diprenorphine in C6 cells
stably expressing MOR or DOR or CHO cells stably expressing KOR. Efficacy was assessed by
agonist-stimulated [35S] GTPγS binding in the same cells.6,8,9
Compared to 1, the resulting compound 2 displayed no significant change in binding
affinity for MOR and DOR, but showed decreased affinity for KOR (Table 1).

Scheme 1. Synthesis of compound 2a
a. Reagents and conditions: (a) triphenylphosphinebenzyl bromide, n-BuLi, THF, reflux; (b) H2,
10% Pd/C, MeOH, 50 psi; (c) TFA, DCM; (d) Boc-L-Dmt, HATU, HOBt-Cl, DIEA, DMF, 4Å
molecular sieves, 40°C; (e) TFA, DCM.
Unfortunately, 2 also displayed no notable efficacy at MOR as determined by the [35S] GTPγS
assay. Because the synthesis of 2 proved somewhat laborious, and the resulting diastereomers
could not be resolved by RP-HPLC, we reasoned that synthesis of further analogues could be
simplified by the replacement of the tetrahydroquinoline (THQ) core of 2 with a piperidine,
effectively eliminating a stereocenter. The resulting compound 3 displayed roughly a tenfold
increase in binding affinity for MOR and DOR, but still lacked any efficacy at MOR. The
remainder of our SAR campaign was focused on changing the length and flexibility profile of the
side chain in an attempt to not only retain strong binding affinity for both MOR and DOR, but to
increase efficacy at MOR. For purposes of synthetic utility as well as increased solubility, the
piperidine core was also replaced with a piperazine for most of the analogues, the results of
which are summarized in Table 1.

Table 1. Binding affinity and efficacy data for analogues 2-12a
Structure
Ki (nM)
EC50 (nM)
% stimulation
MOR
DOR
KOR
MOR
DOR KOR
MOR
DOR KOR
29±9
14±2
310±50
dns
dns
dns
dns
dns
dns
dns
dns
dns
dns
dns
dns
dns
dns
dns
11±6
dns
dns
dns
dns
dns
dns
dns
dns
dns
dns
dns
dns
dns
dns
2
3
4
5
6
7
8
1.4±0.1
2.0±0.8
3.8±0.8
1.1±0.3
6.4±0.1
1.1±0.2
7.8±0.9
12±2
140±24
110±11
250±51
78±15
dns
119±39
150±49
85±10
dns
dns
26±2
23±2
17±3
dns
36±4
6.6±2
11±0.65
21±7
330±92
150±42
dns
dns
0.45±0.3
6.6±2
78±15
89±23
64±3
dns
dns
dns
43±6
36±4
dns
dns
12±1
16±3
9
0.29±0.07
23±8.9
20±3.6
1500±120
10
0.3±0.1
28±6.8
6.9±1.7
54±3.2
39±15
41±15
53±23
dns
dns
dns
49±5
42±1
dns
dns
11±7
12±5
11
12
0.42±0.15
660±80

a. All values are expressed as the mean ± SEM of three separate assays performed in duplicate.
dns: does not stimulate. Binding affinities (Ki) were obtained by competitive displacement of
radiolabeled [3H]diprenorphine in membrane preparations. Efficacy data were obtained using
agonist induced stimulation of [35S]GTPγS binding assay. Efficacy is represented as EC50 (nM)
and percent maximal stimulation relative to standard agonist DAMGO (MOR), DPDPE (DOR),
or U69,593 (KOR) at 10 μM.
Compounds 3-5, and 9 were synthesized by coupling a commercially available piperidine
or piperazine derivative with Boc-L-Dmt, followed by TFA-mediated deptrotection and HPLC
purification to yield the final compounds. In the case of 8 and 12, a commercially available
primary alcohol was first mesylated and refluxed with excess piperazine to give intermediates 16
and 17, which were then coupled with Boc-L-Dmt and deprotected under similar conditions
(Scheme 2). The remainder of the compounds were synthesized as shown in Scheme 3. The
appropriate commercially available aldehyde was subjected to a Horner-Wadsworth-Emmons
type olefination to give alkenes 18, 19 and 27, which were then reduced to the corresponding
alcohols using either DIBAL (for the formation of allylic alchols) or LAH (for the formation of
saturated alcohols). Before reduction, alkene 19 was first hydrogenated to give saturated ester 24.
All intermediates were then carried forward in a similar manner as in Scheme 2 to give finished
products.
Scheme 2. Synthesis of analogues 3-5, 8, 9, 12a

a. Reagents and conditions: (a) Boc-L-Dmt, PyBOP or HATU, HOBt-Cl, DIEA, DMF; (b) TFA,
DCM; (c) MsCl, Et3N, DCM, 0°C; (d) piperazine, THF, reflux.
Scheme 3. Synthesis of analogues 6, 7, 10, 11a
a. Reagents and conditions: (a) O=P(CH3CH2O)2CH2CO2CH3, NaH, THF, 0°C; (b)
O=P(CF3CH2O)2CH2CO2CH3, NaH, THF, 0°C; (c) DIBAL, DCM, -78°C; (d) H2, 10% Pd/C,
MeOH, 15 psi; (e) LAH, THF, 0°C; (f) MsCl, Et3N, DCM, 0°C; (g) piperazine, THF, reflux.
The synthesized analogues in Table 1 display a broad range of binding affinities for MOR
(29 nM to 0.29 nM), and to a lesser extent, DOR (150 nM to 6.6 nM). Extension of the side
chain of 3 from 1 to 3 methylene units did little to change binding at MOR or DOR, but
encouragingly, the resulting compound (4) behaved as a weak partial agonist at MOR.
Replacement of the piperidine core of 4 with a piperazine (5) proved inconsequential, and the
continued balanced MOR/DOR binding profile of this analogue led us to pursue other aromatic
moieties separated by three methylene units from the piperazine core. Analogue 6 in particular
showed an improved balanced MOR/DOR binding profile, and also displayed a partial agonist
profile at MOR. Interestingly, compound 7, in which the 1-naphthyl side chain of 6 is

constrained with an additional double bond, showed no efficacy in the [35S]GTPγS assay at all
three receptors, with an additional loss of binding affinity for KOR. The insertion of an extra
aromatic moiety as in the case of the diphenylmethyl analogue 8 did little to increase binding
affinity for either MOR or DOR. Further extension of the distance between the aromatic side
chain and the piperazine core (9) resulted in a boost in MOR binding, without drastically
affecting DOR. Although these 4 carbon analogues (9-11) suffered a slight loss of MOR/DOR
affinity balance, all displayed good efficacy at MOR, particularly the unsaturated analogues 10
and 11 (20 and 41 nM, respectively). Side chain extension to 5 methylene units (12) did little to
improve upon the profile of 10 or 11.
Structurally, these analogues exhibit some similarities to the class of trans-3,4-dimethyl-
4-(3-hydroxyphenyl)piperidine opioid antagonists originally described by Zimmerman10 and
explored by others.11 In our series, the 3-hydroxyphenyl moiety is replaced by 2,6-L-
dimethyltyrosine, and the piperidine (or piperazine) core is left unsubstituted. In both series,
receptor selectivity is modulated by the nature of the lipophilic side chain attached para to the
phenolic component of the molecule. The 11 piperidine and piperazine analogues of
tetrahydroquinoline 1 described here display a favorable balance between binding affinity at
MOR and DOR, and several (4-6, 9-12) display improved potency at MOR as compared to
morphine (Ki (MOR) = 6.3 nM, (DOR) = 171 nM; EC50 (MOR) = 194 nM).12,13 These
analogues are therefore promising leads for further derivatization and in vivo studies.
References and notes

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Tolerance than Morphine in Rats. J. Med. Chem. 1999, 42, 3520−3526.
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[3H]diprenorphine (50Ci/mmol) by the test compound from membrane preparations
containing opioid receptors. The assay mixture, containing membrane suspension (20 μg
protein/ tube) in 50 mM Tris-HCl buffer (pH 7.4), [3H]diprenorphine, and various
concentrations of test peptide, was incubated at room temperature for 1 h to allow
binding to reach equilibrium. The samples were rapidly filtered through Whatman GF/C
filters using a Brandel harvester (Brandel, Gaithersburg, MD, U.S.) and washed three
times with 50 mM Tris-HCl buffer. The radioactivity retained on dried filters was
determined by liquid scintillation counting after saturation with EcoLume liquid
scintillation cocktail in a Wallac 1450 MicroBeta (Perkin-Elmer, Waltham, MA, U.S.).
Nonspecific binding was determined using 10 μM naloxone. Ki values were calculated
using nonlinear regression analysis to fit a logistic equation to the competition data using
GraphPad Prism, version 5.01, for Windows (GraphPad Software Inc., La Jolla, CA). The
results presented are the mean ± standard error from at least three separate assays
performed in duplicate.
9. Membranes (10−20 μg of protein/tube) were incubated 1 h at room temperature in
GTPγS buffer (50 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl , pH 7.4) containing 0.1
nM [35S]GTPγS, 30 μM guanosine diphosphate (GDP), and varying concentrations of test
peptides. Peptide stimulation of [35S]GTPγS was compared with 10 μM standard

compounds [D-Ala2,N-MePhe4,Gly-ol]enkephalin (DAMGO) at MOR, D-Pen2,5-
enkephalin (DPDPE) at DOR, or U69,593 at KOR. The reaction was terminated by
rapidly filtering through GF/C filters and washing 10 times with GTPγS buffer, and
retained radioactivity was measured as described above. The results presented are the
mean ± standard error from at least three separate assays performed in duplicate;
maximal stimulation was determined using nonlinear regression analysis with GraphPad
Prism (GraphPad Software Inc., La Jolla, CA).
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for Narcotic Antagonists Defined in a 4-Phenylpiperidine Series. Nature. 1978, 275, 332-
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Acknowledgements

This work was supported by NIH grants DA003910 (H.I.M) and DA004087 (J.R.T). The
authors thank Lisa Rosenthal and Alisha Griffin for technical assistance.

OH
OH
NH2
N
NH2
N
N
O
O
1