Muscarinic acetylcholine receptor binding affinities of pethidine analogs

Article abstract of DOI:10.1016/j.bmcl.2015.10.029

A series of pethidine analogs were synthesized and their affinities for the [³H]N-methyl-scopolamine (NMS) binding site on muscarinic acetylcholine receptors (mAChRs) were determined using M₁, M₃ or M₅ human mAChRs expressed by Chinese hamster ovary (CHO) cell membranes. Compound 6b showed the highest binding affinities at M₁, M₃ and M₅ mAChRs (K_i = 0.67, 0.37, and 0.38 μM, respectively).

Full text of DOI:10.1016/j.bmcl.2015.10.029

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Muscarinic acetylcholine receptor binding affinities of pethidine
analogs
Na-Ra Lee a, Xuan Zhang b, Mahesh Darna a, Linda P. Dwoskin a, Guangrong Zheng b,
⇑
A Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536, United States
b Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205, United States
a r t i c l e i n f o
a b s t r a c t
A series of pethidine analogs were synthesized and their affinities for the [3H]N-methyl-scopolamine
(NMS) binding site on muscarinic acetylcholine receptors (mAChRs) were determined using M1, M3 or
M5 human mAChRs expressed by Chinese hamster ovary (CHO) cell membranes. Compound 6b showed
Article history:
Received 1 September 2015
Revised 7 October 2015
Accepted 12 October 2015
Available online xxxx
the highest binding affinities at M1, M3 and M5 mAChRs (Ki = 0.67, 0.37, and 0.38 lM, respectively).
Ó 2015 Elsevier Ltd. All rights reserved.
Keywords:
Pethidine
Muscarinic acetylcholine receptors
[3H]NMS binding affinity
Drug abuse
Many drugs of abuse including cocaine, amphetamine, metham-
phetamine, and morphine increase extracellular dopamine (DA) in
the nucleus accumbens (NAc) at doses that produce rewarding
effects.1 Indeed, bilateral microinjection of 6-hydroxydopamine
which produces dopaminergic neuron damage in NAc inhibited ini-
tiation of amphetamine self-administration in rats when 6-hydrox-
ydopamine was administered before self-administration training,
and disrupted responding during maintenance of amphetamine
self-administration.2 DA containing ventral tegmental area (VTA)
neurons project to NAc, and are important for drug seeking behav-
ior. Electrical stimulation of acetylcholine-containing laterodorsal
tegmental nucleus (LDT) neurons, which innervate VTA dopaminer-
gic neurons, results in an increase in extracellular DA concentra-
tions in NAc.3,4 The five subtypes of mAChRs (M1–M5) are
separated into two groups depending on their G protein coupling
a
functionality. M1, M3, and M5 mAChR subtypes preferentially acti-
vate G
proteins, leading to an increase in cytosolic calcium
aq/11
ion concentration. M2 and M4 mAChR subtypes preferentially cou-
ple with G protein, inhibiting the conversion of adenosine
a
i/o
triphosphate to cyclic adenosine monophosphate in the cytosol.5
Important to the current study, M5 mAChRs are highly expressed
on the postsynaptic DA neurons in VTA.6–9 In M5 mAChRs knockout
(KO) mice, LDT stimulation and morphine-induced DA release in
NAc is reduced relative to wild-type mice.10,11 M5 KO mice also have
decreased cocaine self-administration and cocaine- or morphine-
induced conditioned place preference when compared to wild-type
controls.11,12 Microinfusion into VTA of scopolamine, a mAChR
Figure 1. Structure of compounds 1 and 2, pethidine (3), and design of pethidine
analogs 4–10 as novel mAChR ligands.
antagonist, robustly decreased cocaine-seeking behavior during
withdrawal in rats.13 Together, these findings led us to hypothesize
that selective antagonism of M5 mAChRs represents a novel target
for the treatment of drug abuse.
⇑
Corresponding author.
http://dx.doi.org/10.1016/j.bmcl.2015.10.029
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Lee, N.-R.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.10.029

2
N.-R. Lee et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Table 1
Structures and binding affinity for analogs at M1, M3, and M5 mAChRsa
Compd
R
[3H]NMS binding Ki SEM (
lM)
M1
M3
M5
1b
2b
—
—
25.3
0.02 0.002
>100
NDc
2.24
0.03 0.005
4a
4b
10.8 0.67
1.20 0.11
5.26 0.33
6.95 0.47
0.64 0.037
0.87 0.053
5a
5b
>30
>10
>10
>30
3.29 0.80
6.97 0.77
6a
6b
7a
7b
3.63 0.22
0.67 0.078
>10
4.60 0.61
0.37 0.045
>30
2.14 0.22
0.38 0.011
>10
3.44 0.93
1.54 0.19
1.81 0.11
8
9
5.09 0.29
2.91 0.17
4.03 0.57
2.30 0.22
5.41 0.59
2.05 0.30
10a
10b
>10
>10
>10
5.40 1.22
3.55 0.11
4.30 0.29
a
b
c
Three independent experiments, each experiment included duplicate samples, were performed to obtain Ki values (mean SEM).
Data from Ref. 14.
Not determined.
We recently reported on a class of M5-preferring orthosteric
the piperidine ring. New analogs resulted from such rearrange-
ment resembling pethidine (3, Fig. 1), a once popular analgesic.
Interestingly, pethidine has been identified as an antagonist at
mAChRs in guinea-pig ileum assays.15 Thus, analogs based on the
pethidine scaffold may afford interesting SAR at mAChRs. In
addition to ester containing analogs (4 and 6), we also planned
to evaluate amides (5 and 7), carbamates (8 and 9), and carbamides
(10). Herein, we describe the synthesis of these novel analogs and
the evaluation of their binding affinity at mAChRs.
antagonists based on the scaffold of 1,2,5,6-tetrahydropyridine-
3-carboxylic acid.14 Compound 1 (Fig. 1, Table 1) was identified
as the most selective M5 mAChRs antagonist in this series. Interest-
ingly, removal of the meta-methoxy group in 1 (compound 2) sig-
nificantly increased binding affinities at both M1 and M5 receptors,
but resulted in a complete loss in selectivity for M5 over M1. To fur-
ther explore the structure–activity relationship (SAR), we planned
to reposition the carboxylate group in 1 and 2 from C-3 to C-4 of
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N.-R. Lee et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
Scheme 1. Reagents and conditions: (a) Pd(OH)2, H2, MeOH; (b) HCHO (37% aqueous), NaBH(OAc)3, THF; (c) 6 N HCl, reflux; (d) (1) SOCl2, DCM, reflux; (2) alcohol or amine,
TEA, DCM; (e) EtOH, SOCl2, reflux; (f) LAH, THF, 0 °C–rt; (g) 3-(3,4-dimethoxy or 4-methoxy)phenylpropanoyl chloride, TEA, DCM; (h) (1) 4-nitrophenyl chloroformate,
Na2CO3, THF; (2) 2-(3,4-dimethoxyphenyl)ethanamine Na2CO3, THF; (i) (1) LAH, THF, 0 °C–rt; (2) Boc2O, Na2CO3, THF/H2O; (3) TFA/DCM; (j) (1) 4-nitrophenyl chloroformate,
Na2CO3, THF; (2) alcohol or amine, Na2CO3, THF.
Compounds 4a, 4b, 5a, and 5b were synthesized by converting
1-methyl-4-phenylpiperidine-4-carboxylic acid (pethidinic acid,
14) to the corresponding carbonyl chloride in the presence of
SOCl2, followed by reacting with a phenyl ring substituted 2-phe-
nylethanol or 2-phenylethanamine (Scheme 1). Compound 14
was synthesized by initial catalytic hydrogenolysis of compound
11 under Pd(OH)2 to afford 12, followed by N-methylation to form
compound 13 and hydrolysis of the cyano group. Conversion of 14
to alcohol 16 was achieved by a standard two-step procedure.
Esterification between phenyl ring substituted 3-phenylpropanoyl
chloride and 16 afforded compounds 6a and 6b. Treatment of 16
with 4-nitrophenyl chloroformate followed by reaction with
2-(3,4-dimethoxyphenyl)ethanamine provided carbamate 8.
Similarly, amides 7a and 7b, carbamate 9, and carbamides 10a
and 10b were synthesized from amine intermediate 17. A three-
step process of an initial LAH reduction of the cyano group in 12
to amino group, followed by Boc protection to reduce polarity for
column purification and de-Boc afforded 17.
M3; 2 to 8-fold at M5) when compared to their corresponding
di-methoxy substituted analogs (4a, 5a, 6a, 7a, and 10a, respec-
tively). The corresponding carboxylate moiety repositioned in
molecule 4a exhibited 2- and 19-fold higher affinity at M1 and
M3 mAChRs, respectively, compared with compound 1. However,
the affinity of 4a at M5 mAChRs was decreased by 30%. Thus, com-
pound 4a was not subtype selective. It is unclear why reposition of
the carboxylate group in 1 resulted in a complete loss of subtype
selectivity.
In addition, replacement of the ester link in 4a/b or 6a/b with an
amide link (5a/b and 7a/b, respectively) resulted in a loss of affinity
at all three mAChR subtypes (4a vs 5a, 2 to 4-fold; 4b vs 5b, 5 to
8-fold; 6a vs 7a, 3 to 7-fold; 6b vs 7b, 4 to 5-fold). In general,
analogs with carbamate and carbamide linkers exhibited up to an
8-fold lower affinity compared to the esters. Furthermore, the
reverse ester of 4a (i.e., 6a) exhibited a moderate 1 to 3-fold
increase in affinity at all three mAChRs. A similar increase in affin-
ity was observed for the other reverse ester/amide series, that is,
4b versus 6b and 5b versus 7b. Analog 6b was identified as the
most potent compound at M5 in this series.
Subtype selectivity for M5 over M1 and M3 mAChRs is particu-
larly difficult to achieve because M5 exhibit the high amino acid
sequence identity with M3 and M1 mAChRs (85%, 79%, 73%, and
68% with M3, M1, M4, and M2 mAChRs, respectively).16 In addition,
In summary, a series of pethidine analogs was synthesized and
evaluated to determine binding affinity for the [3H]NMS binding
site on M1, M3, and M5 human mAChRs expressed by CHO cell
membranes. Compound 6b showed the highest binding affinity
all three subtypes prefer to bind G q/11. Thus, in this study, we
a
evaluated binding affinity of our analogs at M1, M3, and M5 mAChR
subtypes as a first approach, and then compared selectivity of ana-
logs at M5 over M1 and M3 mAChRs. Analog affinities for M1, M3
and M5 mAChRs were determined by measuring inhibition of
[3H]N-methylscopolamine (NMS) binding to Chinese hamster
ovary (CHO) cell membranes expressing M1, M3, or M5 recombi-
nant human mAChRs. CHO cells stably expressing each of the
human mAChRs were obtained from Dr. Tom Bonner of National
Institute of Mental Health (NIMH). Detailed materials and methods
for cell culture and cell membrane preparation were described
previously.14,17 IC50 values were obtained and Ki values were
calculated using the equation of Cheng and Prusoff.18 Results are
summarized in Table 1.
at M1, M3 and M5 mAChRs (Ki = 0.67, 0.37, and 0.38 lM,
respectively). However, this series of new analogs did not exhibit
selectivity for M5 mAChRs over M1 and M3 subtypes. Further SAR
and pharmacological evaluations are needed to identify potent
and selective M5 mAChR antagonists. Additionally, pethidine has
been reported to have weak
l
-opioid receptor agonist activity.19
Thus, in future studies, once analogs are demonstrated to have high
affinity and selectivity for M5 mAChRs, they will be evaluated also
for l-opioid receptor affinity to assure selectivity at the M5 mAChR
target.
Acknowledgment
Similar to the SAR generated for the parent compounds 1 and 2,
mono-methoxy substituted analogs (4b, 5b, 6b, 7b, and 10b) con-
sistently exhibited higher affinity (2 to 9-fold at M1; 3 to 19-fold at
This work was supported by funding from the National Insti-
tutes of Health (DA030667 and UL1 TR000117).
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4
N.-R. Lee et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
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