Structural modifications to tetrahydropyridine-3-carboxylate esters en route to the discovery of M5-preferring muscarinic receptor orthosteric antagonists

Article abstract of DOI:10.1021/jm301774u

The M₅ muscarinic acetylcholine receptor is suggested to be a potential pharmacotherapeutic target for the treatment of drug abuse. We describe herein the discovery of a series of M₅-preferring orthosteric antagonists based on the scaffold of 1,2,5,6-tetrahydropyridine-3- carboxylic acid. Compound 56, the most selective compound in this series, possesses an 11-fold selectivity for the M₅ over M₁ receptor and shows little activity at M₂-M₄. This compound, although exhibiting modest affinity (K_i = 2.24 μM) for the [³H]N-methylscopolamine binding site on the M₅ receptor, is potent (IC₅₀ = 0.45 nM) in inhibiting oxotremorine-evoked [³H]DA release from rat striatal slices. Further, a homology model of human M₅ receptor based on the crystal structure of the rat M₃ receptor was constructed, and docking studies of compounds 28 and 56 were performed in an attempt to understand the possible binding mode of these novel analogues to the receptor.

Full text of DOI:10.1021/jm301774u

Article
pubs.acs.org/jmc
Structural Modifications to Tetrahydropyridine-3-carboxylate Esters
en Route to the Discovery of M5‑Preferring Muscarinic Receptor
Orthosteric Antagonists
Guangrong Zheng,*,† Andrew M. Smith,‡ Xiaoqin Huang,‡ Karunai L. Subramanian,‡
Kiran B. Siripurapu,‡ Agripina Deaciuc,‡ Chang-Guo Zhan,‡ and Linda P. Dwoskin‡
†Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock,
Arkansas 72205, United States
‡Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536, United States
S
* Supporting Information
ABSTRACT: The M5 muscarinic acetylcholine receptor is suggested to be a potential pharma-
cotherapeutic target for the treatment of drug abuse. We describe herein the discovery of a series
of M5-preferring orthosteric antagonists based on the scaffold of 1,2,5,6-tetrahydropyridine-3-
carboxylic acid. Compound 56, the most selective compound in this series, possesses an 11-fold
selectivity for the M5 over M1 receptor and shows little activity at M2−M4. This compound,
although exhibiting modest affinity (Ki = 2.24 μM) for the [3H]N-methylscopolamine binding site
on the M5 receptor, is potent (IC50 = 0.45 nM) in inhibiting oxotremorine-evoked [3H]DA release
from rat striatal slices. Further, a homology model of human M5 receptor based on the crystal structure of the rat M3 receptor
was constructed, and docking studies of compounds 28 and 56 were performed in an attempt to understand the possible binding
mode of these novel analogues to the receptor.
Furthermore, microinfusion into VTA of an antisense oligo-
nucleotide targeting M5 receptor mRNA inhibits brain stimula-
tion reward in rats15 and microinfusion into the VTA of the
nonselective mAcChR antagonist, scopolamine (1, Figure 1),
reduces cocaine-facilitated DA release in nucleus accumbens.17
Microinfusion of the nonselective mAcChR antagonist atropine
(2, Figure 1) into the VTA in rats dose-dependently inhibits
morphine-induced conditioned place preference.22 Taken
together, these studies suggest that discovery of subtype-
selective M5 mAcChR antagonists may provide novel treat-
ments for drug abuse. Importantly, mice lacking M5 receptors
exhibit no difference from their wild-type littermates in various
behavioral and pharmacologic tests,18,23 suggesting that
centrally active M5 receptor antagonists will be well tolerated.
Subtype-selective M5 receptor antagonists also will be useful
pharmacological tools in defining the physiological functions of
this receptor. Currently, little information is available on the
effects of selective activation or inhibition of M5 receptors.
Recent success in identifying M5-selective positive allosteric
modulators may provide new information in this regard.24−26
Nevertheless, no subtype-selective orthosteric M5 antagonists
are available to date. Unlike allosteric binding sites, which are
usually more divergent across subtypes, orthosteric ACh binding
sites across the five mAcChR subtypes have been shown to be
highly conserved at the amino acid sequence level (73−83%
identity).5 As such, selectivity among the mAcChR subtypes is
likely based on conformational dissimilarities rather than upon
INTRODUCTION
■
Muscarinic acetylcholine receptors (mAcChRs) are G-protein-
coupled receptors (GPCRs) activated by the endogenous neuro-
transmitter acetylcholine (ACh) and the natural alkaloid muscarine.
Upon ACh activation, these receptors regulate a variety of
central and peripheral physiological functions such as cognition,
movement, sleep, cardiovascular function, smooth-muscle con-
tractility, and glandular secretion.1−4 Thus, mAcChRs have
emerged as important therapeutic targets for many diseases,
such as Alzheimer’s disease, Parkinson’s disease, psychosis,
pain, asthma, diabetes, and smooth-muscle disorders.3,4 Five
mAcChR subtypes (M1−M5) have been identified.2 Each of the
five mAcChR subtypes has a defined distribution pattern within
specific brain regions and peripheral tissues and mediates distinct
physiological functions.1−4 To avoid side effects, selectivity at
specific mAcChR subtypes is often a major focus of discovery of
mAcChR agonists and antagonists as therapeutic agents.
The M5 mAcChR was the last subtype to be cloned.2,5
A
growing body of evidence suggests that this subtype is a poten-
tial target for the discovery of treatments for drug abuse.6 The
rewarding effects of abused drugs are believed to be mediated
by the mesolimbic dopamine (DA) pathway, which projects
from the midbrain ventral tegmental area (VTA) to the nucleus
accumbens.7−10 M5 mAcChRs are the only muscarinic subtype
localized to VTA and that modulate DA release from VTA
DA neurons11−17 Consistent with M5 mAcChR modulation of
mesolimbic DA transmission, behavioral studies using M5
knockout mice show a reduction in reward and withdrawal re-
sponses following repeated morphine or cocaine administration, as
well as a reduction in the rate of cocaine self-administration.18−21
Received: November 21, 2012
Published: February 4, 2013
© 2013 American Chemical Society
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Figure 1. Structures of selected mAcChR antagonists and their literature reported mAcChR affinity data.
single amino acid residues.27−29 In fact, small molecule ortho-
RESULTS AND DISCUSSION
■
steric antagonists with preference for an individual subtype have
been reported for M1−M4 but not M5 (e.g., compound 3 for
M1,30 5 for M2,31 6 for M3,32 and 7 for M4,29 Figure 1), indicat-
ing the existence of sufficient differences among the agonist
recognition sites on the five mAcChR subtypes allowing target-
ing of selective compounds to these sites.
Radioligand binding assays using membranes from recombi-
nant cells expressing human mAcChRs (hM1−hM5) show that
compound 8 (1-ethyl-4-phenyl-1,2,5,6-tetrahydropyridine-3-
carboxylic acid 4-methoxyphenethyl ester, Figure 1) preferen-
tially binds to the M5 receptor (subtype selectivity: M1/M5 =
2.2-fold, M2/M5 = 44-fold, M3/M5 = 60-fold, M4/M5 = 48-fold,
Figure 1).33 Thus, the current exploration of the structure−
activity relationship (SAR) regarding M5 selective orthosteric
ligands is based on compound 8. To our best knowledge, com-
pound 8 and its analogue 9 (Figure 1) are the only orthosteric
small molecules reported to exhibit preference for M5 receptors.33
The current structural modification strategy is provided in
Figure 2. The 1,2,5,6-tetrahydropyridine-3-caboxylate core structure
Synthesis. The general synthetic methods for analogues
17−36 (Table 1) with modifications on the p-methoxyphen-
ethyl moiety of compound 8 are depicted in Scheme 1.
Transformation of commercially available ethyl 1-benzyl-4-oxo-
3-piperidinecarboxylate (11) into compound 12 was achieved
by initial conversion of 11 to N-BOC protected compound via
a two-step process,34 followed by triflation under diisopropyl-
amine and trifluoromethanesulfonic anhydride.35 Suzuki
coupling of 12 with phenylboronic acid afforded compound
13. Removal of BOC in 13 by TFA afforded compound 14,
which was then N-ethylated to form compound 15. Compound
15 was subjected to ester hydrolysis under basic conditions to
afford carboxylic acid 16. Treatment of 16 under a Steglich
esterification condition with appropriate alcohols afforded 8
and its analogues 17−36. The synthetic route for analogues
with variations on the N-ethyl moiety of compound 8 was in-
itially devised to introduce the N-substituent at the last step.
Accordingly, compound 13 was converted to compound 38 in a
sequence similar to that for compound 15 to 8 (Scheme 1).
However, deprotection of 38 to form 39 using various de-BOC
methods (TFA/CH2Cl2, HCl/EtOAc, AcCl/MeOH, or TsOH/
CH2Cl2) resulted in complex mixtures. Attempts to purify
compound 39 by silica gel column chromatography failed. Impure
intermediate 39 (obtained from TFA treatment) was employed to
provide N-Me, N-n-Pr, N-n-Bu, or N-p-methoxybenzyl analogues
via reductive amination, but only N-p-methoxybenzyl analogue
(compound 40) was obtained in pure form after preparative TLC
purification. Alternatively, a similar route to that for analogues 17−
36 was applied to prepare analogues 42−59 from compound 14
(Scheme 1, Table 2).
Analogues 61−69 (Table 2) were synthesized in a similar
manner to analogues 42−59 from triflate 12. Phenyl ring sub-
stituted phenylboronic acids were used instead of phenyl-
boronic acid as the Suzuki coupling partners (Scheme 2).
Analogues 70−73 were prepared by N-alkylation of com-
pound 14 with appropriate alkyl halides (Scheme 3). Analogues
74−76 were prepared by reductive amination of compound 14
with an N-sulfonated or N-acylated 4-piperidinone using
sodium triacetoxyborohydride (Scheme 3).
The syntheses of analogues 84 and 85 were initially attempted
by applying a route similar to that used for the synthesis of their
regioisomers in Scheme 1 (analogues 56 and 45, respectively),
starting from ethyl 1-benzyl-3-oxo-4-piperidinecarboxylate hy-
drochloride (77) (Scheme 4). However, de-BOC reaction of
Figure 2. General structural modification strategy.
was retained while modifying each part of the “appending”
pharmacophores in a stepwise manner. These “appending”
pharmacophores were to be rearranged around the core struc-
ture in compound 8 (Figure 2). Herein, we report the synthesis,
pharmacological evaluation, and SAR analysis of this new series
of mAcChR antagonists. Additionally, a homology model of
hM5 mAcChR was constructed based on the crystal structure of
the rat M3 mAcChR. Preliminary docking experiments were
performed using this model in an attempt to understand the
structural basis of the interaction between these ligands and the
receptor.
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compound 81, which was treated with methyl iodide to yield
the corresponding quaternary pyridinium salt 82. Reduction of
82 with sodium borohydride gave 1,2,5,6-tetrahydropyridine
derivative 83, which underwent ester hydrolysis and subsequent
esterification with appropriate alcohols to afford analogues 84
and 85.
Table 1. Structures and Binding Affinity for Scopolamine
(1), 8, and 17−36 at the hM1 and hM5 mAcChRs
a
Synthesis of analogue 89 was achieved by initial Suzuki
coupling of 4-bromopyridine with 2-(ethoxycarbonyl)phenyl-
boronic acid, followed by a route similar to that for analogues
84 and 85 from compound 81 (Scheme 5).
Radioligand Binding Assay. Receptor affinities were
determined by measuring inhibition of the binding of [3H]N-
methylscopolamine (NMS), an orthosteric antagonist probe,
to membranes from Chinese hamster ovary (CHO) cells individ-
ually expressing human hM1−hM5 mAcChR receptors. Com-
pound 1 (scopolamine) and compound 8 were used as reference
compounds. Methods for these binding assays are described briefly
in the Experimental Section. All synthesized analogues were
evaluated first at hM1 and hM5 subtypes (Table 1 and 2, Scheme
3−5). Selectivity for M5 over M1 is important because antagonism
of CNS M1 receptors has been suggested to result in cognitive
deficits36 and to increase DA release in striatum.37 Binding affinity
for analogues that exhibited binding preference for M5 over M1
was determined at each of the five mAcChR subtypes (Table 3).
Identification of compound 8 was the starting point. Com-
pound 8 was an analogue of the 1-ethyl-4-phenyltetrahydropyr-
idine-3-carboxylic acid scaffold aimed at identifying M1 selective
antagonists. Compound 8 was reported to have a 2.2-fold pre-
ference for M5 over M1 and to be more than 40-fold selective
for M5 over M2−M4 mAcChR subtypes (Figure 1).33 However,
in the current study, compound 8 showed no selectivity for M5
over M1−M4 (Table 3). Preliminary SAR from the previous
report33 suggested that small changes on the p-methoxyphenyl
ring of 8 afforded remarkably different binding affinity and
selectivity profiles at mAcChR subtypes. For example, when the
methoxy group on the phenyl ring of 8 was replaced by a
methyl group, the resulting analogue 10 bound preferentially to
the M1 receptor, while binding affinity at the M5 receptor
increased 35-fold, when compared to compound 8 (Figure 1).33
Results in Table 1 summarize the modifications to the
p-methoxyphenethyl moiety of compound 8 evaluated in the
current study and the influence of these modifications on
binding affinity and selectivity for M1 and M5 receptors. Results
from para-, meta-, and ortho-methoxy analogues 8, 17, and 18,
respectively, indicate that the meta-substitution was favorable to
M5 selectivity. Compared to 8 (Ki = 420 nM) and 17 (Ki =
1250 nM), meta-analogue (18, Ki = 150 nM) exhibited a small
3- and 8-fold, respectively, increase in affinity at M5 over M1.
Replacement of the ethylene link in the p-methoxyphenethyl
moiety by a methylene (compound 19, Ki = 3610 nM) or a
methylethylene link (compound 21, Ki > 100 μM) significantly
decreased binding potency at M5 receptor, whereas replace-
ment with a propylene link (compound 20, Ki = 470 nM)
retained binding affinity at M5 and markedly increased sele-
ctivity (7-fold) over M1 receptors. Interestingly, there were no
marked differences in M5 affinities when the 4-methoxy was
replaced with another para-substitution group, including halo-
gens (compounds 23−25) and nitro group (compound 26),
indicating modification at this position is somewhat tolerated.
However, compound 27, in which a 4-methylsulfonyl group
was attached to the phenyl ring, exhibited little affinity for
either M1 or M5 receptors. Compounds 28 and 31, wherein
both 3- and 4-positions of the phenyl ring were substituted, not
a
At least three independent experiments with samples evaluated in
duplicate were performed to obtain the Ki value.
compound 78 failed to yield the desired intermediate 79. As
an alternative, analogues 84 and 85 were synthesized starting
from 3-bromoisonicotinic acid (80). Ethyl esterification of 80
followed by Suzuki coupling with phenylboronic acid afforded
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a
Scheme 1. Synthesis of Compounds 8, 17−36, 40, and 42−59
a
Reagents and conditions: (a) H2, 10% Pd/C (5 w/w%), EtOH; (b) (BOC)2O, Et3N, CH2Cl2; (c) Tf2O, i-Pr2NH, CH2Cl2, −78 °C; (d)
PhB(OH)2, Pd(PPh4)3, Na2CO3 (2.0 M), THF, 65 °C; (e) TFA/CH2Cl2 (1:1); (f) EtI, K2CO3, EtOH; (g) 10% KOH (aq)/EtOH (1:1); (h)
alcohols, EDCI, DMAP, CH2Cl2; (i) p-methoxybenzaldehyde, NaBH(OAc)3, HOAc, THF; (j) aldehydes, NaBH3CN, EtOH; (k) alcohols, 2,4,6-
trichlorobenzoyl chloride, DMAP, Et3N, THF.
only retained affinity at the M5 receptor (Ki of 230 and 2460 nM,
respectively) but also exhibited preference for this subtype
(6.0- and 4.8-fold M5/M1, respectively). Trisubstitution of the
phenyl ring in compounds 32 and 33 revealed that this modi-
fication was detrimental to binding. Finally, replacement of the
4-methoxyphenyl ring with a heterocyclic ring, including pyridyl
(compounds 34 and 35) and thiophenyl (compound 36) rings,
resulted in retention of binding affinity at both M1 and M5
receptors.
In parallel with the modification on the p-methoxyphenethyl
group, the N-ethyl moiety of compound 8 (Table 2) also was
altered. Replacement of the ethyl group by a methyl group
(compound 42) resulted in an 8-fold and 14-fold increase in
binding affinity at M1 and M5 receptors, respectively. Increasing
the size of the substituted group from ethyl to n-propyl (compound
43), n-butyl (compound 44), or 4-methoxybenzyl group
(compound 40) eliminated affinity for these two receptors. Similar
results were observed for N-methyl, N-n-propyl, and N-n-butyl
analogues (compounds 45, 46, and 47, respectively) of compound
28. Interestingly, the M1/M5 selectivity of two N-methyl containing
analogues, compound 42 (0.7-fold) and 45 (4.8-fold), were similar
to their corresponding N-ethyl analogues, 8 (0.4-fold) and 28 (6.0-
fold), respectively.
On the basis of the above SAR data, our focus changed to
analogues bearing an N-methyl group in lieu of N-ethyl group,
and the SAR was extended regarding the substitution group of
the carboxylic ester moiety (Table 2). Replacement of the ethyl
link in compound 45 by a methylene (compound 49) or pro-
pylene (compound 50) link resulted in a decrease in binding
affinity at both M1 and M5 receptors and also resulted in a
decrease in selectivity for M5 over M1 receptors. SAR among
compounds 45, 49, and 50 was inconsistent with previous SAR
among compounds 8, 19, and 20, indicating an unpredictable nature
of this component of the structure. On the other hand, analogues
containing substituents at the meta-position or both the meta- and
para-position of the ester phenyl ring (compounds 53−56)
consistently exhibited a binding preference at M5 over M1
receptor. Compound 56 was identified as the most selective
(11-fold for M1/M5) M5 compound.
On the basis of compounds 45 and 56, SAR was further
extended by introduction of an electron-withdrawing (fluoro)
or electron-donating (methoxy) group onto the phenyl ring on
the C4 of the tetrahydropyridine core (compounds 61−69,
Table 2). In general, these analogues exhibited decreased or
completely abolished activity at either M1 or M5 receptor when
compared to compounds 45 and 56, suggesting that this com-
ponent of the molecule is less tolerant to structural modification.
Further SAR exploration was focused on previous hypotheses
that spatial rearrangement or reorientation of the pharmaco-
phore elements in mAcChR ligands would alter affinity and
selectivity profiles.38 For example, rearrangement of the amino
group in M1 preferring antagonist 3 (pirenzepine) afforded M2
preferring antagonist 4 (Figure 1).39 To test this hypothesis
with respect to the current analogues, we conducted three types
of pharmacophoric “rearrangements”. First, transposition of the
substituted group on the ester functionality and the ethyl group
on the N atom in compound 8 and analogues afforded com-
pounds 70−73 (Scheme 3). An N-acylated or N-sulfonated
piperidine-4-yl group was also introduced to the N atom of the
tetrahydropyridine ring (compounds 74−76, Scheme 3). The
N-substituents were selected because they are present in potent
mAcChR orthosteric antagonists such as darifenacin (90),
zamifenacin (91), and compounds 92 and 9340 (Figure 3).
Surprisingly, none of these new analogues displayed any activity
at either the M1 or M5 receptor, indicating a different receptor
binding mode for the basic N atom in compound 8 and its
analogues compared with compounds 90−93.
Second, the ester group on C3 and the phenyl group on C4
of the 1,2,5,6-tetrahydropyridine ring in compounds 56 and 45
were transposed to produce compounds 84 and 85 (Scheme 4),
respectively. Both compounds 84 (M1/M5 = 2.4) and 85
(M1/M5 = 2.1) displayed reduced selectivity for M5 over M1
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a
Table 2. Structures and Binding Affinity for Analogues 40, 42−59, and 61−69 at the hM1 and hM5 mAcChRs
a
At least three independent experiments with samples evaluated in duplicate were performed to obtain the Ki value.
receptors when compared to their corresponding position isomers,
56 and 45, respectively. Last, the ester functionality in compound
45 was moved from C3 of the tetrahydropyridine ring to the
phenyl ring on C4 (compound 89, Scheme 5). This “parallel”
shift resulted in a complete loss of binding affinity at both M1
and M5 receptors.
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a
a
Scheme 2. Synthesis of Compounds 61−69
Scheme 5. Synthesis of Compound 89
a
Reagents and conditions: (a) substituted phenylboronic acids,
Pd(PPh4)3, Na2CO3 (2.0 M), THF, 65 °C; (b) same as from
compound 13 to 42 in Scheme 1.
a
Scheme 3. Synthesis of Compounds 70−76
a
Reagents and conditions: (a) 4-bromopyridine, Pd(PPh3)4, Na2CO3
(2.0 M), THF, 65 °C; (b) MeI, acetone; (c) NaBH4, EtOH; (d) 10%
KOH (H2O)/EtOH (1:1); (e) 2-(2,3-dihydrobenzofuran-5-yl)-
ethanol, EDCI, DMAP, CH2Cl2.
over M1 receptor), had no affinity (Ki > 100 μM) at the M2,
M3, and M4 mAcChR subtypes. Compound 28 exhibited 6-fold,
>435-fold, 135-fold, and 46-fold for M5 over M1, M2, M3, and
M4, respectively. Although slightly less selective for the M5 over
the M1 receptor, it exhibited higher affinity at M5 when com-
pared to compound 56.
Inhibition of Oxotremorine-Evoked Striatal [3H]DA
Release. In vitro functional assays for mAcChR antagonists
measure the ability of molecules to block mAcChR agonist-
induced receptor activation at recombinant mAcChR subtypes
expressed in cells.41 Pharmacological studies of M5 receptors
using mouse basilar artery have also been reported.42 However,
these recombinant and native M5 receptors functional assays are
far removed from a potential role for M5 receptors in cocaine and
opiate addiction. Studies have shown that oxotremorine, a non-
selective mAcChR agonist, concentration-dependently increases
[3H]DA release from striatal slices prepared from wild-type mice
and that oxotremorine-evoked striatal [3H]DA release was reduced
significantly in M5 receptor knockout mice.18,43 We hypothe-
sized that an M5 receptor selective antagonist would also reduce
oxotremorine-mediated rat striatal [3H]DA release. Current
results show that oxotremorine evokes [3H]DA release from
rat striatal slices and that scopolamine inhibits this effect in a
concentration-dependent manner (Figure 4). These results
support the contention that this functional assay probes native
M5 receptors. Furthermore, this functional assay is highly
relevant to the underlying dopaminergic mechanisms involved
in drug reward and abuse.
a
Reagents and conditions: (a) alkyl bromides, KI, K2CO3, CH3CN,
reflux; (b) NaBH(OAc)3, HOAc, THF, 65 °C for 74, rt for 75 and 76.
a
Scheme 4. Synthesis of Compounds 84 and 85
a
Reagents and conditions: (a) EtOH, H2SO4 (conc), reflux; (b)
Results revealed that compound 56 inhibited (IC50 = 0.45
nM) oxotremorine (100 μM) evoked [3H]DA release from rat
striatal slices (Figure 5). Unlike scopolamine (1 μM), which
completely inhibits oxotremorine-mediated [3H]DA release
from rat striatal slices (Figure 4), compound 56 produced maximal
inhibition (Imax) of only 48% of the oxotremorine-evoked [3H]DA
release (Figure 5). These current results are consistent with pre-
vious reports that ∼50% of oxotremorine-evoked [3H]DA release
from striatal slices was eliminated in M5 knockout mice compared
to wild-type mice,18 indicating that other mAcChR subtype(s) also
mediate oxotremorine-evoked striatal DA release. In agreement
with this hypothesis, studies using mAcChR knockout mice sug-
gested that M3 and M4 receptors were also involved in mediating
striatal DA release.43 The observations that both compound 56
and the deletion of the M5 receptor resulted in similar effects on
oxotremorine-evoked striatal [3H]DA release, together with the
PhB(OH)2, Pd(PPh4)3, Na2CO3 (2.0 M), THF, 65 °C; (c) MeI,
acetone; (d) NaBH4, EtOH; (e) 10% KOH (H2O)/EtOH (1:1); (f)
alcohols, EDCI, DMAP, CH2Cl2.
At saturation concentrations, all analogues except for those
with Ki > 100 000 nM exhibited complete inhibition (maximal
inhibition Imax = 100%, data not shown) of the binding of the
orthosteric antagonist [3H]NMS at mAcChRs, which is con-
sistent with an orthosteric mechanism of action. Selected com-
pounds, including 20, 28, 45, 56, 57, 63, and 66, that exhibited
binding preference for hM5 over hM1 mAcChRs were also
evaluated for affinity at M2, M3, and M4 mAcChR subtypes
(Table 3). Results showed that five of these seven compounds
exhibited good selectivity for M5 over M2, M3, and M4
receptors. The most selective compound, 56 (11-fold for M5
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a
Table 3. Binding Affinity and Selectivity for Selected Analogues at the hM1−hM5 mAcChRs
Ki, nM
b
b
b
b
compd
hM5
hM1 (M1/M5)
hM2 (M2/M5)
hM3 (M3/M5)
hM4 (M4/M5)
1
17.6
420
470
230
60
7.5 (0.4)
9.5 (0.5)
6.5 (0.4)
36.9 (2.1)
8
170 (0.4)
1100 (2.6)
2030 (4.3)
1370 (3.3)
4070 (8.7)
31100 (135)
2840 (47)
1730 (4.1)
20
28
45
56
57
63
66
3290 (7.0)
1390 (6.0)
290 (4.8)
11500 (24)
10500 (46)
1890 (32)
>100000 (>435)
1170 (20)
2240
5040
740
3340
25300 (11)
24400 (4.8)
5270 (7.1)
27000 (8.0)
>100000 (>45)
>100000 (>20)
540 (0.7)
>100000 (>45)
>100000 (>20)
4890 (6.6)
>100000 (>45)
>100000 (>20)
5740 (7.8)
>100000 (>30)
>100000 (>30)
>100000 (>30)
a
b
At least three independent experiments with samples evaluated in duplicate were performed to obtain the Ki value. Numbers in the parentheses are
the ratios of binding affinity between M5 and the respective subtype.
Figure 5. Compound 56 inhibits oxotremorine (100 μM) evoked
[3H]DA release from rat striatal slices (data are expressed as the mean
SEM, n = 4).
Figure 3. Structures of darifenacin (90), zamifenacin (91), and
compounds 92 and 93.
orthosteric binding pocket.44 Compound 56 could have a
similar allosteric interaction with the receptor, causing the in-
hibition of oxotremorine-evoked striatal [3H]DA release. How-
ever, further pharmacological studies are needed to elucidate
the mechanism.
Binding Mode for M5 mAcChRs for Lead Compounds
28 and 56. To study the interaction of our compounds with
M5 mAcChR in atomic detail, homology modeling and mol-
ecular docking operations were performed. Compounds 28 and
56 were selected for these studies, since both analogues
preferencially bind to M5 mAcChR and thus can be considered
current lead compounds. Importantly, compounds 28 and 56
have an order of magnitude difference in binding affinities at
M5 receptor (Ki of 230 nM vs 2240 nM, respectively). This
moderate difference in affinity between 28 and 56 is ideal for
testing the reliability of our homology models. The structural
model of the human M5 mAcChR was constructed based on
selective binding of 56 to M5 over M3 and M4 receptors,
strongly suggest that 56 interacts with M5 receptors to inhibit
muscarinic agonist-induced striatal DA release.
It is noteworthy that compound 56 (IC50 = 0.45 nM) ap-
pears more potent than scopolamine in inhibiting oxotremorine-
evoked [3H]DA release from rat striatal slices, although its
[3H]NMS binding affinity on the M5 receptor is 127-fold less
than scopolamine. One explanation of the lack of correlation
between binding and function is that the [3H]NMS binding
assay was performed on a single receptor in a recombinant
system but the [3H]DA release assay was on heterogeneous
brain slices. Compound 56 may potently act at other sites
that also inhibit [3H]DA release. Alternatively, recent studies
on the crystal structure of the rat M3 receptor with antagonist
tiotropium bound to the orthosteric binding site suggested that
tiotropium binds transiently to an allosteric site en route to the
Figure 4. Scopolamine (0.01−1 μM) inhibits oxotremorine (10 and 100 μM) evoked [3H]DA release from rat striatal slices (data are expressed as
the mean SEM, n = 3).
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the newly available X-ray crystal structure of the rat M3
mAcChR with antagonist tiotropium bound to the orthosteric
binding site.44 Compounds 28 and 56 were docked into
possible binding sites among the transmembrane (TM) helices
of the M5 mAcChR. Binding structures were selected from
the docking results and subjected to energy minimization. In
accordance with the minimized binding structures, the bind-
ing site for compounds 28 and 56 at the M5 mAcChR is the
orthosteric site located near the extracellular end of TM3, TM5,
TM6, and TM7. As shown in Figure 6, TM2 and TM4 are also
through electrostatic attraction and strong hydrogen bonding.
Meanwhile, residue D110 is also hydrogen-bonded with the
side chain of S83 from TM2 and with the side chains of Y481
and Y485 from TM7. The cationic head of compound 28 is in
close contact with residues W106 and L107 from TM3. The
phenyl group on C4 of the tetrahydropyridine ring is closely
packed with Y87, Y90, and I91 from TM2, with W106 from
TM3, with F187 from EL-2, and with H478 from TM7. The
ethyl group at the cationic head of compound 28 makes contact
with the aromatic side chain of residue F82 from TM2. The
carbonyl oxygen of compound 28 is weakly hydrogen-bonded
with the hydroxyl group at the side chain of Y458 from TM6.
The tail group (2,3-dihydrobenzofuran-5-ethyl) of compound
28 is packed in parallel with the underneath Y111 from TM3
and packed perpendicularly with the side chain of W162 from
TM4. The tail group of compound 28 is also surrounded by
T197 from TM5 and by V462 from TM6.
As depicted in Figure 6B, the binding mode of the M5
mAcChR with compound 56 is essentially the same as that with
compound 28. One difference between the binding of 56 and
28 to the M5 mAcChR is that residues I193 and T194 from
TM5 are both within 5 Å of compound 56 (Figure 6B), while
these two residues have no contacts with compound 28 (Figure 6A).
Another difference is the distance of hydrogen bonding formed by
the side chain of residue D110 with the cationic headgroup of the
antagonist. As shown in Figure 6, the distance between the hydrogen
bonding between residue D110 and the cationic head of com-
pound 28 is shorter than that of the hydrogen bonding between
residue D110 and the cationic head of compound 56 (1.93 Å vs
1.99 Å, respectively), indicating a stronger bond with 28 than
with 56 to the protein. These structural differences, especially
the difference in hydrogen bonding distance, may contribute to
the difference in binding affinity as represented by the experi-
mentally measured values of Ki (230 nM for 28 vs 2240 nM for
56) for these two lead compounds (Table 3). The modeled
binding structures helped to qualitatively understand the ob-
served SAR and provided clues to design a valuable virtual
library of new analogues for computational screening.
SUMMARY
■
Starting from compound 8 as a lead structure, we have identi-
fied the first M5-preferring antagonists through a systematic
structural modification strategy. The greatest mAcChR
selectivity and potency shifts came from the modification of
the substituents on the ester group. Replacing the N-Et group
on the tetrahydropyridine ring with an N-Me group generally
resulted in a significant increase in mAcChR binding affinity while
maintaining mAcChR subtype-selectivity profile. This preliminary
SAR study provides a basis for further discovery of potent and
selective M5 ligands. In addition, we have successfully established a
functional assay for M5 receptors using oxotremorine-evoked DA
release from superfused rat striatal slices. One of our lead com-
pounds, 56, demonstrated inhibition of oxotremorine-mediated
striatal [3H]DA release, with a maximal inhibition of ∼50%. This
result is similar to the effects of M5 knockout on striatal [3H]DA
release, providing validation for the current assay. Further, we have
constructed a homology model of human M5 based on the newly
available crystal structure of the rat M3 receptor. Docking studies
performed on compounds 28 and 56 revealed that both possibly
interact with the orthosteric binding site on the M5 receptor,
which is consistent with the current results from the [3H]NMS
binding assay, indicating an orthosteric mechanism of action for
these new analogues. We are building and validating homology
Figure 6. Homology model of human M5 mAcChR based on the X-ray
crystal structure of rat M3 mAcChR (PDB entry of 4DAJ at 3.4 Å
resolution, A chain). (A) Top view of the energy-minimized binding
structure of M5 mAcChR−28 complex. (B) Top view on the energy-
minimized binding structure of M5 mAcChR−56 complex. The
receptor proteins in (A) and (B) are represented as ribbons in rainbow
color, and compounds 28 and 56 are shown as spheres (left panel) or
ball-and-stick (right panel). Residues within 5 Å of compound 28 and
56 are labeled and shown as sticks. 28 and 56 have very similar
hydrogen bonding interactions with the protein, including interactions
between the D110 side chain and the cationic heads of the com-
pounds, interactions between the carbonyl oxygen atoms of the
compounds and the Y458 side chain, and the interactions among side
chains of D110, S83, Y481, and Y485. These hydrogen bonding
interactions are shown as dashed lines along with the labeled distances.
partially involved in the formation of the antagonist-binding
site. In addition, the antagonist-binding site is partially covered
by extracellular loop 2 (EL-2). As depicted in Figure 6A, com-
pound 28 is orientated horizontally inside the binding pocket.
The cationic head of compound 28 is anchored around the
negatively charged side chain of residue D110 of TM3, interacting
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Sample collection began after 60 min of superfusion, when the rate of
release was stable. Consecutive 4 min (2.4 mL) samples were collected
to determine basal [3H]outflow. Superfusion continued in the absence
or presence of a range of analogue concentrations (0.1 nM to 1 mM)
for 40 min, followed by 40 min with oxotremorine (100 μM) added to
the superfusion buffer. Radioactivity in slices and superfusate samples
were determined via liquid scintillation spectroscopy. Data were
analyzed by weighted least-squares regression analysis of the sigmoidal
concentration−effect curves to obtain IC50 values.
Homology Modeling and Molecular Docking. The homology
model of human M5 mAcChR was built based on the X-ray crystal
structure of rat M3 mAcChR (PDB entry of 4DAJ at 3.4 Å resolution,
A chain)44 by using the Protein Modeling module of Discovery Studio
(version 2.5.5, Accelrys, Inc., San Diego, CA). The model of human
M5 mAcChR structure was constructed and refined in a very similar
manner as in our previous study;48 i.e., the best sequence alignment
was selected based on both the alignment score and the reciprocal
positions of the conserved residues among all mAcChR subtypes. The
coordinates of the conserved regions were directly transformed from
the template structure, while the nonconserved residues were mutated
from the template to the corresponding ones in M5 mAcChR. Struc-
tural optimization and energy minimization for the M5 mAcChR structue
were performed using the Amber 11 program suite. The convergence
criterion for the energy minization was set to 0.001 kcal mol−1 Å−1. On
the basis of the optimized M5 mAcChR structure, the binding mode of
the receptor with two lead compounds 28 and 56 was explored through
molecular docking using the AutoDock 3.0.5 program.48 This molecular
docking approach was similar to that described in our previous study.49 A
reasonable binding structure of M5 mAcChR in complex with either
compound 28 or 56 was obtained after energy minimization on each
complex structure.
models for the other four mAcChR subtypes, and these will be
used for virtual library screening. The predictability of these
models regarding mAcChR subtype selectivity remains to be
tested.
EXPERIMENTAL SECTION
■
Chemistry. All purchased reagents and solvents were used without
further purification unless otherwise noted. All reactions sensitive to
air and/or moisture were carried out under argon atmosphere in oven-
dried glassware. Flash column chromatography was carried out using
́
32−63 μm, 60 Å (230−400 mesh) silica gel. Analytical thin layer
chromatography was carried out on glass plates precoated with 250 μm
silica gel 60 F254. NMR spectra were recorded in CDCl3 on a Varian
300 or 500 MHz spectrometer, and chemical shifts are reported in
ppm relative to tetramethylsilane as the internal standard. Coupling
constants are reported in hertz (Hz). Mass spectra were recorded on a
JEOL JMS-700T MStation. GC−mass spectra were recorded on an
Agilent 6890 GC incorporating an Agilent 7683 autosampler and
an Agilent 5973 MSD. Elemental analyses were carried out on a
COSTECH elemental combustion system and are within 0.4% of
theory. All final compounds for biological testing were prepared as
salts in ≥95% purity, in accord with results from combustion analysis.
A detailed description of synthetic methodologies as well as analytical
and spectroscopic data for all described compounds is included in the
Supporting Information.
Binding Assay. Analogue binding affinities for the five mAcChR
subtypes were determined in assays evaluating inhibition of [3H]NMS
binding to membranes from CHO-K1 cells expressing one of the
recombinant hM1−hM5 mAcChRs. The CHO-K1 cell lines expressing
the five subtypes of muscarinic receptors were obtained as a gift from
Dr. Tom Bonner of National Institute of Mental Health (NIMH).
Cells were grown at 37 °C with 5% CO2 in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum,
4 mM glutamine, 50 ng/mL Geneticin G418 and 1% Pen−Strep. Cells
were harvested at 70−90% confluency. To obtain cell membranes, cells
were scraped into ice cold 50 mM Tris-HCl (pH 7.4), sonicated for 30 s,
and centrifuged (48000g for 30 min). Pellets were resuspended in
1.5 mL of ice-cold 50 mM Tris-HCl buffer, sonicated for 30 s, and
stored at −80 °C until assay. Assays were performed using 96-well
plates. Membrane aliquots (100 μL) containing 10−40 μg of protein
were added to wells containing 1 nM to100 μM of test compound
(25 μL), 0.3 nM [3H]NMS (25 μL, scopolamine methyl chloride
[N-methyl-3H], specific activity 82 Ci/mmol; Perkin-Elmer/NEN,
Boston, MA), and buffer (50 mM Tris-HCl, pH 7.4, 125 μL) for a
total volume of 250 μL. Atropine (1 mM) was used to determine
nonspecific binding. Samples were incubated for 120 min at 25 °C
with constant agitation. Reactions were terminated by rapid filtration
onto GF/B filters using a Filtermate harvester (PerkinElmer Life and
Analytical Sciences, Boston, MA). Samples were washed three times
with 350 μL of ice-cold 50 mM Tris-HCl buffer and dried for 60 min
at 50 °C. Subsequently, 40 μL of MicroScint 20 (PerkinElmer Life and
Analytical Sciences, Waltham, MA) was added to each well and
radioactivity bound determined using liquid scintillation spectrometry.
IC50 values were obtained and Ki values calculated using the equation
of Cheng and Prusoff.45
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic procedures and analytical data of all compounds
prepared. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: 501-526-6787. Fax: 501-526-5945. E-mail: gzheng@
uams.edu.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported financially by a Pilot Project Grant
from Center for Drug Abuse Research Translation at the
University of Kentucky (Grant DA05312) and grants from
National Institute of Drug Abuse, National Institutes of Health
(Grants DA025948, DA030667, and TR000117).
ABBREVIATIONS USED
■
Inhibition of Oxotremorine-Evoked [3H]DA Release Assay.
Assays were performed according to previously published methods46,47
with minor modifications. Striata were dissected and coronal slices
(500 μm, 4−6 mg) obtained with a McIlwain chopper. Slices were
incubated for 30 min in Krebs’ buffer (in mM: 108 NaCl, 4.7 KCl,
1.2 MgCl2, 1 NaH2PO4, 1.3 CaCl2, 11.1 glucose, 25 NaHCO3, 0.11
L-ascorbic acid, and 0.004 disodium EDTA, pH 7.4, saturated with
95% O2/5% CO2) in a metabolic shaker at 34 °C. Slices were in-
cubated with 0.1 μM [3H]DA during the latter 30 min of a 60 min
incubation period. Each slice was transferred to a superfusion chamber
and superfused (0.6 mL/min at 34 °C) for 60 min with Krebs’ buffer
containing nomifensine (10 μM) and pargyline (10 μM) to inhibit
reuptake and prevent metabolism, respectively, ensuring that [3H]-
overflow primarily represents [3H]DA rather than [3H]metabolites.47
VTA, ventral tegmental area; NMS, N-methylscopolamine;
CHO, Chinese hamster ovary; TM, transmembrane; EL,
extracellular loop
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