Synthesis and pharmacological evaluation of analogues of benzyl quinolone carboxylic acid (BQCA) designed to bind irreversibly to an allosteric site of the M1 muscarinic acetylcholine receptor

Article abstract of DOI:10.1021/jm500556a

Activation of the M₁ muscarinic acetylcholine receptor (mAChR) is a prospective treatment for alleviating cognitive decline experienced in central nervous system (CNS) disorders. Current therapeutics indiscriminately enhance the activity of the endogenous neurotransmitter ACh, leading to side effects. BQCA is a positive allosteric modulator and allosteric agonist at the M₁ mAChR that has high subtype selectivity and is a promising template from which to generate higher affinity, more pharmacokinetically viable drug candidates. However, to efficiently guide rational drug design, the binding site of BQCA needs to be conclusively elucidated. We report the synthesis and pharmacological validation of BQCA analogues designed to bind irreversibly to the M₁ mAChR. One analogue in particular, 11, can serve as a useful structural probe to confirm the location of the BQCA binding site; ideally, by co-crystallization with the M₁ mAChR. Furthermore, this ligand may also be used as a pharmacological tool with a range of applications.

Full text of DOI:10.1021/jm500556a

Article
pubs.acs.org/jmc
Synthesis and Pharmacological Evaluation of Analogues of Benzyl
Quinolone Carboxylic Acid (BQCA) Designed to Bind Irreversibly to
an Allosteric Site of the M1 Muscarinic Acetylcholine Receptor
Briana J. Davie,†,‡ Celine Valant,‡ Jonathan M. White,§ Patrick M. Sexton,‡ Ben Capuano,†
Arthur Christopoulos,*,‡ and Peter J. Scammells*,†
‡
†Medicinal Chemistry and Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal
Parade, Parkville, Victoria 3052, Australia
§School of Chemistry and the Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria
3010, Australia
S
* Supporting Information
ABSTRACT: Activation of the M1 muscarinic acetylcholine receptor
(mAChR) is a prospective treatment for alleviating cognitive decline
experienced in central nervous system (CNS) disorders. Current
therapeutics indiscriminately enhance the activity of the endogenous
neurotransmitter ACh, leading to side effects. BQCA is a positive
allosteric modulator and allosteric agonist at the M1 mAChR that has
high subtype selectivity and is a promising template from which to
generate higher affinity, more pharmacokinetically viable drug
candidates. However, to efficiently guide rational drug design, the
binding site of BQCA needs to be conclusively elucidated. We report
the synthesis and pharmacological validation of BQCA analogues
designed to bind irreversibly to the M1 mAChR. One analogue in
particular, 11, can serve as a useful structural probe to confirm the
location of the BQCA binding site; ideally, by co-crystallization with
the M1 mAChR. Furthermore, this ligand may also be used as a pharmacological tool with a range of applications.
cobound orthosteric ligand as well as (3) activating the receptor
in their own right.5
INTRODUCTION
■
The M1 mAChR is a Family A G protein-coupled receptor
(GPCR) that is well-established as a target of therapeutic
interest for the treatment of cognitive deficits associated with
Alzheimer’s disease and schizophrenia.1 This receptor is
primarily expressed in regions of the hippocampus, striatum,
and prefrontal cortex,2 and the prospective therapeutic utility of
M1 mAChR activation for enhanced cognitive function has
been extensively validated in vitro and in vivo.1,3 The primary
challenge impeding the development of clinically viable M1
mAChR drug candidates is achieving high receptor subtype
selectivity so as to avoid side effects associated with
indiscriminate activity at other mAChRs. Achieving such
selectivity with ligands that target the orthosteric (endogenous
ligand binding) site has been difficult due to the high sequence
conservation across the orthosteric sites of the M1−M5 mAChR
family. Allosteric ligands are attractive alternatives to orthosteric
drugs that have the potential to elicit more fine-tuned, highly
selective responses than their orthosteric counterparts.4
Allosteric ligands are capable of binding to topographically
distinct sites and can elicit their effects by three different
mechanisms: (1) by modulating the affinity of the cobound
orthosteric ligand, (2) by modulating the efficacy of the
An array of M1 mAChR-selective allosteric ligands has been
characterized to date.6 An exemplar molecule is 1-(4-
methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid
(more commonly abbreviated to “benzyl quinolone carboxylic
acid” or BQCA), a subtype-selective, orally bioavailable M1
mAChR positive allosteric modulator (PAM) and allosteric
agonist with an extraordinary degree of positive cooperativity
with orthosteric agonists, including the endogenous neuro-
transmitter acetylcholine (ACh).7 BQCA has undergone the
most extensive pharmacological evaluation and synthetic
derivatization of all M1 mAChR allosteric ligands reported to
date.7−12 While this campaign has led to the discovery of
several higher potency, more pharmacokinetically viable
analogues of BQCA, the structure−activity relationships
(SAR) reported to date have been frequently described as
“flat” or “shallow”,13−17 which is not an uncommon observation
in the broader field of allosteric ligand development.18
Performing more detailed pharmacological analyses that
deconstruct the binding and functional profile of allosteric
Received: April 9, 2014
Published: May 23, 2014
© 2014 American Chemical Society
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ligands has proven fruitful in obtaining more detailed,
“enriched” SAR12,19 and in further understanding their
functional capabilities.20 However, to more efficiently guide
rational drug design, identification and characterization of the
BQCA allosteric binding site by co-crystallization with the M1
mAChR would offer unequivocal insight into the critical
interactions that dictate its unique mode of action, the
geometry of the binding pocket with which it interacts, and
its global influence on receptor conformation. The recent
publication of the M2 mAChR co-crystallized with both an
orthosteric and an allosteric ligand21 demonstrates that this
approach is achievable, however, being a low-affinity ligand
(pKB ∼ 4),9,12 BQCA itself may not be an optimal candidate for
a similar study at the M1 mAChR.
An alternative approach to elucidating the BQCA binding
site has recently been published.22 Comprehensive mutagenesis
in conjunction with molecular dynamics simulations and ligand
docking revealed the BQCA binding site to likely reside in the
extracellular (EC) vestibule common to all mAChRs, over-
lapping with what is often dubbed the “common” allosteric
binding site.23 The BQCA binding site itself appears to involve
residues within transmembrane (TM) 2, TM 7, and EC loop 2,
building on observations made in the original BQCA
publication where the positive modulation of ACh-mediated
calcium mobilization by BQCA was absent at the Y179A (EC
loop 2) and W400A (TM 7) mutants.7 Furthermore, subtype-
specific transmission of cooperativity was proposed as the
potential mechanism underlying BQCA’s exquisite selectivity.
To further advance our understanding of the interaction
between BQCA and the M1 mAChR, and to aid interrogation
of such new insights, the current study sought to design novel
analogues of BQCA that bind irreversibly to the M1 mAChR
and form a covalently modified receptor complex. Such ligands
may be utilized as pharmacological tools in a range of
preclinical applications, including co-crystallization.
obtaining structural knowledge about ligand binding mode and
receptor conformation.
While irreversible orthosteric mAChR ligands have been
previously developed,27−30 this is the first time that develop-
ment of an irreversible purely allosteric mAChR ligand has been
achieved. We began our investigation with the synthesis of four
BQCA analogues, each containing a different electrophilic
functionality capable of reacting covalently with a nucleophilic
amino acid residue. The 4-benzylic position of BQCA was
selected as an ideal starting point for introducing our reactive
functionalities as this region of the molecule is highly tolerant
of a range of synthetic modifications10 and appears to be an
important determinant of analogue affinity.12 Furthermore,
molecular dynamics simulations suggest that this moiety lies in
close proximity to EC loops 2 and 3 as well as upper segments
of TMs 6 and 7.22 Using competition radioligand binding and
ERK1/2 phosphorylation assays, pharmacological evaluation of
these BQCA analogues confirmed the preservation of allosteric
binding and functional activation at hM1 mAChR-expressing
Chinese hamster ovary (CHO) cells. Utilizing saturation
radioligand binding assays to evaluate potential irreversible
interaction, we found that 11 binds irreversibly to the M1
mAChR and is therefore the first known alkylating PAM of a
GPCR.
RESULTS AND DISCUSSION
Chemistry. The 4-oxo-1,4-dihydroquinoline core of BQCA
was synthesized in two steps (Scheme 1) by employing well-
■
Scheme 1. Synthesis of BQCA (4b) and Analogues 4a, 4c,
a
and 4d
Irreversible ligands (also known as covalent modifiers) are
molecules that contain a reactive functionality rendering them
capable of binding to a site on a protein target of interest (such
as an enzyme or GPCR) and forming a covalent bond with one
or more of the protein’s amino acid residues. More relevant to
our experimental rationale, irreversible ligands have also been
developed as biological investigative tools for a range of protein
targets including adrenergic, histaminergic, imidazoline, mus-
carinic, opioid, and adenosine receptors.24−30 As with the
BQCA analogues published herein, such ligands most
commonly comprise a reactive moiety incorporated into a
known reversible ligand for which the target selectivity and
pharmacological profile is well established. In the past, these
modified ligands have enabled researchers to identify and map
ligand binding sites,29,31 characterize ligand binding modes,30
determine functional roles,32 and investigate cell surface
expression, receptor occupancy, and cellular processing of
different receptor families, and specific subtypes within those
families.25,33 The advent of fluorescently labeled irreversible
ligands has facilitated imaging of membrane-bound and
intracellular proteins in vitro.34,35 Ultimately, valuable structural
and functional insight into the role of biological targets in
physiological and pathophysiological processes is gained,
potentially informing the selection of novel points of
therapeutic intervention and directing subsequent rational
drug design. Adding further support to our rationale, co-
crystallization of an irreversible agonist-β2 adrenergic GPCR
complex was achieved,31 validating this strategy as a means for
a
Reagents and conditions: (a) diethylethoxymethylene malonate, 120
°C, 83%; (b) diphenyl ether, 240 °C, 48% or Eaton’s reagent, 100 °C,
quantitative; (c) 1 M LiOH(aq), THF, 80 °C, 75%; (d) benzyl halide
KI, DIPEA, ACN, 80 °C, 87% (4a), 97% (4b), 21% (4c), 53% (4d);
(e) NBS, benzoyl peroxide, EtOAc, microwave 100 °C, 81%; (f)
maleic anhydride, DMF, 110 °C, 100%; (g) sodium acetate, acetic
anhydride, 120 °C, 51%; (h) NBS, benzoyl peroxide, EtOAc,
microwave 100 °C, 45%.
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established reaction conditions reported previously.12,36,37 In
line with observations noted in a recent publication by Mistry
et al.,12 while the neat reaction between aniline and
diethylethoxymethylene malonate afforded an easily isolatable,
crystalline 1 in good yield, cyclization with diphenyl ether to
form 2 required unfavorably high temperatures and typically
resulted in widely variable yields and purity. Eaton’s reagent
(1:10 w/w P2O5 in methanesulfonic acid) was found to be an
ideal alternative, requiring milder conditions and resulting in
quantitative yields. In the earliest reported synthesis of BQCA,
Yang et al.36 follow this cyclization step with N-benzylation
followed by ester saponification to yield the target compound.
In our case, subsequent syntheses introducing irreversible
functionalities in place of the 4-methoxy group of BQCA
necessitate incorporation of the N-benzyl moiety as the final
synthetic step so as to avoid any unwanted reactivity in
subsequent synthetic steps. Hence, for pre-emptive optimiza-
tion purposes, the order of the third and final steps of the
BQCA synthesis was reversed from that of the initial
publication.36 Based on a procedure reported by Juli et al.,38
ester saponification to form 3 was achieved in good yield.
It was noted that, as a result of the need to perform the N-
benzylation step last, O-benzylation of the free carboxylic acid
moiety was also a possibility. The position of alkylation was
confirmed through a model study involving the alkylation of 3
with benzyl bromide where an X-ray crystal structure of the
product 4a was obtained (Figure 1) and confirmed the
after the reaction of p-toluidine and maleic anhydride resulted
in the uncyclized intermediate 4-oxo-4-(p-tolylamino)but-2-
enoic acid (structure not shown). Stirring with sodium acetate
in acetic anhydride rapidly recyclized the molecule to reform
the maleimide functionality, then radical bromination was
performed as previously described to yield the precursor 6b,
following column chromatography. Finally, N-benzylation with
3 gave the target maleimide analogue 4d in low but sufficient
yield for chemical and pharmacological characterization.
The bromoacetamide (10) and isothiocyanate (11) ana-
logues of BQCA were both accessed via the intermediate 9.
This compound was synthesized from the 4-oxo-1,4-dihydro-
quinoline core in three steps (Scheme 2). N-Benzylation with
4-nitrobenzyl bromide (7) was achieved in good yield by
employing reaction conditions established in Scheme 1.
Reduction of the nitro functionality to the amine with 10%
Pd/C under a hydrogen atmosphere gave 8 in quantitative
yield. The ester saponification conditions outlined in Scheme 1
(1 M LiOH(aq), THF) were insufficient for ester bond cleavage
of 8, however, 1 M KOH(aq) in 3:1 MeOH:H2O afforded
conversion to the target carboxylic acid 9 in excellent yield.
Cooling of 9, triethylamine (TEA), and acetonitrile (ACN)
in a dry ice/acetone bath (approximately −80 °C) was required
prior to addition of bromoacetyl bromide. Allowing the
reaction mixture to warm to room temperature (RT) naturally
was critical for obtaining clean conversion to the target
bromoacetamide analogue 10, which precipitated from the
reaction mixture and required no further purification (Scheme
2).
The isothiocyanate analogue 11 was isolated following
reaction of 9 with 1,1′-thiocarbonyldiimidazole (TCDI) and
catalytic 4-dimethylaminopyridine (DMAP), requiring only a
simple organic extraction to attain >95% purity (Scheme 2).
Pharmacology. To assess the binding activity, [3H]N-
methylscopolamine (NMS) equilibrium whole cell binding
interactions between ACh and each of our test allosteric ligands
(BQCA, 4c, 4d, 10, and 11) were conducted in CHO cells
stably expressing the hM1 mAChR (Figure 2). Application of an
allosteric ternary complex model to these data allowed us to
estimate each ligand’s affinity for the free receptor (pKB),
binding cooperativity with [3H]NMS (log α[NMS]), and binding
cooperativity with ACh (log α[ACh]) (Table 1).
To assess functional activity, ERK1/2 phosphorylation assays
were performed in CHO cells stably expressing the hM1
mAChR. Time course assays were first performed to establish
the length of incubation time required to measure the peak
phosphorylated ERK1/2 response for each test compound
(data not shown). Concentration−response curves (CRCs)
were subsequently constructed using this peak time point to
estimate the pEC50 of each compound and to select a suitable
concentration range for interaction studies with ACh (Figure 3,
Table 1). Interaction studies were conducted between ACh and
each of our test allosteric ligands (BQCA, 4c, 4d, 10, and 11)
(Figure 3). Application of an operational model of allosterism
to these data allowed us to estimate each ligand’s signaling
efficacy (τB) and the combined binding/activation cooperativity
(αβ) (Table 1).
Figure 1. Thermal ellipsoid plot for 4a (ellipsoids are at the 20%
probability level).
regioselectivity of the reaction. The N-alkylation with 4-
methoxybenzyl chloride proceeded in excellent yield, with the
pure product 4b precipitating from the reaction mixture
unaided. A HMBC 2D NMR experiment confirmed formation
of the N-benzylated product; the spectrum showed a cross peak
between H2 of the quinolinone ring system at δ 9.32 ppm and
the methylene carbon of the benzyl group at δ 55.5 ppm that
would not be apparent had O-benzylation occurred.
The fluorosulfonyl (4c) and maleimide (4d) analogues of
BQCA both required preparation of the requisite substituted
benzyl bromide precursors (Scheme 1). Radical bromination of
p-toluenesulfonyl fluoride gave a mixture of mono- and
dibrominated species, with the former (5) easily isolated by
column chromatography prior to N-benzylation with 3 to yield
the target fluorosulfonyl analogue 4c. Synthesis of 1-(p-tolyl)-
1H-pyrrole-2,5-dione (6a) required a two-step methodology
As expected, BQCA exhibited a modest micromolar affinity
(KB = 36 μM), a moderate potency (EC50 = 170 nM), and
agonism in its own right (τB = 162) in this system. BQCA
demonstrated a very high degree of positive cooperativity with
ACh found to be driven predominantly by binding (α = 759),
rather than functional (β = 7) cooperativity. Importantly, the
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a
Scheme 2. Synthesis of BQCA Analogues 10 and 11
a
Reagents and conditions: (a) 4-nitrobenzyl bromide, DIPEA, ACN, 80 °C, 78%; (b) H2, 10% Pd/C, DMF, RT, 93%; (c) 1 M KOH(aq), 3:1
MeOH:water, 80 °C, 82%; (d) bromoacetyl bromide, TEA, ACN, −80 °C−RT, 74%; (e) 1,1′-thiocarbonyldiimidazole, DMAP, dry DCM, RT, 96%.
results of these binding and functional experiments confirmed
the preservation of allosteric activity in each of our four BQCA
analogues, although the impact on individual parameters varied
across the set. Our results suggest that interaction of the 4-
methoxy group of BQCA with the receptor is an important
factor in the ligand’s high degree of positive binding
cooperativity with ACh as well as its signaling efficacy.
Introduction of the fluorosulfonyl moiety (4c) appears to
preserve this critical interaction; log α[ACh] and log τB values
were not significantly different from BQCA. However, this
interaction is clearly less-favored by the maleimide (4d),
bromoacetamide (10), and isothiocyanate (11) moieties, as
evidenced by the significant reduction in the log α[ACh] values
(p < 0.0001) as well as the log τB values (p < 0.01). In contrast,
the affinity (pKB) values for the maleimide and isothiocyanate
analogues were significantly improved relative to BQCA (p <
0.001) while the potency values were unaffected.
Prior to assessing the ability of our BQCA analogues to bind
irreversibly, we conducted [3H]NMS saturation whole cell
binding assays in CHO cells stably expressing the hM1 mAChR
following preincubation with buffer, the reversible endogenous
agonist, ACh, or the known irreversible ligand, acetylcholine
mustard (AChM), a nonselective muscarinic orthosteric
agonist, for which protocols are well-established.30,39
density), due to a reduction in cell-surface receptor expression
or by competition between [3H]NMS and AChM at the
orthosteric site, respectively. A key assumption in this
experiment is that, if a given incubation time does not induce
receptor internalization by ACh, any effect observed following
the same incubation time with AChM is likely the result of
receptor alkylation. No internalization was observed following
30 min preincubation with ACh, and 60% receptor alkylation
was observed following 30 min preincubation with AChM.
Hence, this time was selected as a suitable starting point for
probing the irreversibility of our BQCA analogues. In applying
this protocol to our experiments with allosteric ligands, it
should be noted that, rather than direct blockade of the
radioligand binding site, we hypothesized that irreversible
binding of our allosteric ligands would stabilize a conformation
of the receptor that would be unfavorable for [3H]NMS
binding. Hence, we would expect to see a reduction in Bmax as a
result of high irreversible negative cooperativity (Table 1),
rather than direct competition.
No change in Bmax was observed following 30 min
preincubation with BQCA and subsequent washout (Table 3,
Figure 5A,C), indicating that the ligand does not cause receptor
internalization during this time period. Preincubation with
BQCA in the presence of ACh gave a small, nonsignificant
reduction in Bmax, suggesting that the positive cooperativity
between the two molecules potentially induced a small amount
of receptor internalization not observed with either compound
alone. This modest effect allowed us to hypothesize that any
substantial reduction in Bmax observed with our putative
irreversible ligands would be most likely due to receptor
Time course experiments were performed to determine the
preincubation time at 37 °C necessary to observe at least 50%
receptor alkylation (irreversible binding) by AChM but without
observing significant internalization by ACh (Table 2, Figure
4). Both receptor internalization and receptor alkylation would
be expected to manifest as a decrease in Bmax (total receptor
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Figure 2. Pharmacological characterization of BQCA and the putative irreversible analogues in radioligand competition whole cell binding assays.
(A−E) Radioligand competition binding experiments performed in hM1 mAChR-expressing CHO cells in the presence of a KD concentration of
radiolabeled orthosteric antagonist [3H]NMS (0.1 nM), increasing concentrations of ACh with or without increasing concentrations of BQCA (A),
4c (B), 4d (C), 10 (D), or 11 (E). Values represent the mean SEM from at least three to four experiments performed in duplicate.
Table 1. Ternary Complex Model Parameters for the Binding Interaction between [3H]NMS, ACh, and Each Allosteric Ligand,
and the Operational Model of Allosterism Functional Parameters for the Activity of Each Allosteric Ligand Alone and in the
a
Presence of Acetylcholine (ACh)
BQCA
4c
4d
10
11
b
pKB
log α[NMS]
4.44 0.07
−3.00*
2.88 0.11
3.97 0.05
−3.00*
2.66 0.09
5.09 0.04
−3.00*
1.46 0.11
4.72 0.14
−0.06 0.01
1.29 0.09
19
5.22 0.04
−3.00*
1.26 0.13
c
d
log α[ACh]
α[ACh]
759
457
29
18
e
pEC50
6.77 0.07
2.21 0.08
162
6.50 0.09
1.79 0.35
62
6.38 0.12
1.10 0.06
13
5.80 0.10
1.04 0.07
11
6.41 0.11
0.83 0.04
7
f
log τB
τB
g
log αβ
3.71 0.18
0.83 (7)
3.62 0.21
0.96 (9)
1.91 0.17
0.45 (3)
2.15 0.18
0.87 (7)
1.33 0.26
0.07 (1)
h
log β (β)
a
b
Estimated parameter values represent the mean SEM from at least three to four experiments performed in duplicate. Negative logarithm of the
c
equilibrium dissociation constant of the allosteric ligand. Logarithm of the binding cooperativity factor between [3H]NMS and the allosteric ligand;
* denotes when the parameter was constrained to an arbitrary low value consistent with very high negative cooperativity between the allosteric ligand
d
and the radioligand. Logarithm of the binding cooperativity factor between ACh and the allosteric ligand. pKB value was fixed to that estimated for
e
f
each ligand in whole cell binding experiments. Negative logarithm of the ligand concentration that produces half the maximal response. Logarithm
g
of the operational efficacy parameter of the ligand. Logarithm of the product of the binding and activation cooperativity factors between ACh and
the allosteric ligand. Logarithm (and antilogarithm) of the activation cooperativity factor between ACh and the allosteric ligand (derived by fixing
h
the binding cooperativity value to that estimated for each ligand in whole cell binding experiments (log α[ACh])).
alkylation and not receptor internalization. No significant
reductions in Bmax were observed following preincubation with
4c, 4d, and 10 (data not shown), however, we observed a
significant reduction in Bmax following preincubation with the
isothiocyanate analogue 11 (Table 3, Figure 5B,D). Thirty
minutes preincubation of 11 in the presence of ACh resulted in
a further reduction in Bmax. This is most likely due to the
positive cooperativity between the two molecules facilitating
additional binding and receptor alkylation by 11 than when
preincubated alone. However, given our observations with
BQCA and ACh, it is also possible that receptor internalization
may be contributing to this reduction in Bmax to some extent.
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Figure 3. Pharmacological characterization of BQCA and the putative irreversible analogues in ERK1/2 phosphorylation assays. (A−F) ERK1/2
phosphorylation experiments performed in hM1 mAChR-expressing CHO cells. (A) Increasing concentrations of ACh, BQCA, 4c, 4d, 10, and 11
alone. (B−F) Increasing concentrations of ACh with or without increasing concentrations of BQCA (B), 4c (C), 4d (D), 10 (E), or 11 (F). Values
represent the mean SEM from at least three to four experiments performed in duplicate.
Table 2. Bmax (% Vehicle) Values (Total Receptor Density)
Following Different Pre-incubation Times of M1 mAChR-
Expressing Cells with Vehicle, ACh, or AChM
highly selective positive allosteric modulator BQCA were
synthesized, incorporating different reactive functionalities
capable of forming a covalent bond with the receptor:
fluorosulfonyl (4c), maleimide (4d), bromoacetamide (10)
and isothiocyanate (11). The pharmacological profile of BQCA
was preserved, though to varying extents, in all four analogues;
they were able to bind to (pKB) and activate (τB) the M1
mAChR and showed positive binding (α) and functional (β)
cooperativity with the endogenous neurotransmitter and
orthosteric agonist ACh in CHO cells stably expressing the
receptor of interest.
Saturation radioligand binding experiments demonstrated
that 30 min incubation of 11 with M1 mAChR-expressing cell
membranes resulted in a 50% reduction in the total detectable
receptor density (Bmax). This is indicative of irreversible
noncompetitive blockade of previously available binding sites
by high negative cooperativity between 11 and the radioligand
[3H]NMS.
5 min
10 min
15 min
20 min
30 min
vehicle
ACh
99.87
98.61
84.81
99.87
100.43
79.20
99.87
98.30
58.49
99.87
88.35
50.32
99.87
95.43
38.25
AChM
[3H]NMS saturation radioligand binding experiments with
30 min preincubation of ACh, AChM, BQCA ( ACh) and 11
( ACh) were repeated in CHO cell membranes stably
expressing the hM1 mAChR to eliminate the possibility of
observing receptor internalization and, hence, ascertain whether
the reduction in Bmax observed in the whole cell binding
experiments was indeed the result of receptor alkylation.
Encouragingly, we still observed no change in Bmax following
preincubation with BQCA but a significant reduction in Bmax
following preincubation with 11 ( ACh), conclusively
confirming that 11 is binding irreversibly to the M1 mAChR
(Table 3, Figure 6).
Taken together, we confirm that 11 is likely binding
irreversibly to an allosteric site of the M1 mAChR, suggesting
the presence of a nucleophilic amino acid residue within this
allosteric domain. This ligand may be employed in crystallo-
graphic studies to gain further structural insight into the BQCA
binding site. Alternatively, this ligand may also be used as a
chemical probe in proteomics studies to identify the interacting
CONCLUSIONS
Herein we report the development of an irreversible allosteric
ligand for a GPCR, the M1 mAChR. Four analogues of the
■
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Figure 4. Pharmacological characterization of ACh and AChM in radioligand saturation whole cell binding assays. (A−D) Radioligand saturation
binding experiments performed in hM1 mAChR-expressing CHO cells in the presence of increasing concentrations of radiolabeled orthosteric
antagonist [3H]NMS. (A) Preincubation with vehicle or ACh for increasing lengths of time. (B). Preincubation with vehicle or AChM for increasing
lengths of time. (C−D) Bmax (% vehicle) bar graphs where values significantly different from Vehicle are indicated by an * (where * = p <0.05, ** =
p <0.01, *** = p <0.001, and **** = p <0.0001). Values represent the mean SEM from three experiments performed in duplicate.
Graphics workstation. Chemical shifts (δH) for all 1H NMR spectra are
Table 3. Bmax (% Vehicle) Values (Total Receptor Density)
reported in parts per million (ppm) using the center peak of the
Following 30 min Pre-incubation of Either M1 mAChR-
Expressing Whole Cells or M1 mAChR-Expressing Cell
deuterated solvent chemical shift as the reference: CDCl3 (7.26) and
DMSO-d6 (2.50).40 Each resonance was assigned according to the
Membranes with Vehicle, BQCA ( ACh), or 11 ( ACh)
following convention: chemical shift (δ) (multiplicity, coupling
constant(s) in Hz, and number of protons). Coupling constants (J)
are reported to the nearest 0.5 Hz. In reporting spectral data, the
following abbreviations have been used: s, singlet; d, doublet; t, triplet;
q, quartet; p, pentet; m, multiplet; br, broad; app, apparent; as well as
combinations of these where appropriate.
vehicle
BQCA BQCA + ACh
11
11 + ACh
whole cells
membranes
99.22
107.06
90.79
88.49
91.42
64.67
49.96
36.75
33.71
100.00
amino acid residue29 which may subsequently inform molecular
modeling of the binding site. This ligand may also serve as a
template for the design of novel fluorescent irreversible
allosteric ligands for the M1 mAChR. Furthermore, 11 may
aid classification of forthcoming orthosteric and allosteric
ligands at the M1 mAChR30 and may prove useful in examining
the effects of prolonged M1 mAChR allosteric site occupation.
Finally, we postulate that 11 may “lock” the M1 mAChR in a
distinct conformation, thereby allowing us to build a
pharmacological fingerprint or “allosteric phenotype” using
different classes of orthosteric ligand and different functional
assay pathways, pursuing conformation-specific pharmacology.
13C NMR spectra were routinely recorded at 100 MHz using a
Bruker Avance 400 Ultra Shield Plus spectrometer equipped with a
̈
Silicon Graphics workstation. Chemical shifts (δC) for all 13C NMR
spectra are reported in parts per million (ppm), using the center peak
of the deuterated solvent chemical shift as the reference: CDCl3
(77.16) and DMSO-d6 (39.52).40 Coupling to fluorine (19F) is
reported as JCF, JCF, JCF, and JCF to the nearest Hz.41
1
2
3
4
HSQC, HMBC, and COSY spectra were obtained using the
standard Bruker pulse sequence to assist with structural assignment of
̈
the compounds.
Low resolution mass spectrometry (LRMS) analyses were recorded
in the specified ion mode using a Micromass Platform II single
quadropole mass spectrometer in acetonitrile:water (1:1) at a cone
voltage of 20 V. The principle ions (m/z) are reported. High
resolution mass spectrometry (HRMS) analyses were recorded in the
specified ion mode using a LCT Premier XE TOF mass spectrometer
coupled to 2795 Alliance separations module at cone voltages of 45 V
(ESI+) and 60 V (ESI−).
Analytical reverse-phase high performance liquid chromatography
(HPLC) was performed on a Waters HPLC system using a
Phenomenex Luna C8 (2) 100 Å column (150 mm × 4.6 mm, 5
μm) and a binary solvent system; solvent A, 0.1% TFA/H2O; solvent
B, 0.1% TFA/80% MeOH/H2O. Isocratic elution was carried out
using the following protocol (time, % solvent A, % solvent B): 0 min,
100, 0; 10 min, 20, 80; 11 min, 20, 80; 12 min, 100, 0; 20 min, 100, 0;
at a flow rate of 1.0 mL/min monitored at 254 nm using a Waters 996
photodiode array detector.
EXPERIMENTAL SECTION
■
Chemistry. All materials were reagent grade and purchased
commercially from Sigma-Aldrich or Matrix Scientific. Anhydrous
solvents were obtained from a MBraun MB SPS-800 solvent
purification system. Analytical thin layer chromatography (TLC) was
performed on Silica Gel 60 F254 precoated plates (0.25 mm, Merck
ART 5554) and visualized by ultraviolet light, iodine or ninhydrin as
necessary. Silica gel 60 (Fluka) was used for silica gel flash
chromatography.
Melting points (mp) were determined on a Mettler Toledo MP50
melting point system.
1H NMR spectra were routinely recorded at 400 MHz using a
Bruker Avancell Ultrashield Plus spectrometer equipped with a Silicon
̈
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Figure 5. Pharmacological characterization of BQCA and 11 in radioligand saturation whole cell binding assays. (A−D) Radioligand saturation
binding experiments performed in hM1 mAChR-expressing CHO cells in the presence of increasing concentrations of radiolabeled orthosteric
antagonist [3H]NMS. (A) 30 min preincubation with vehicle, BQCA, or BQCA + ACh. (B) 30 min preincubation with vehicle, 11, or 11 + ACh.
(C−D) Bmax (% vehicle) bar graphs where values significantly different from Vehicle are indicated by an * (where * = p <0.05, ** = p <0.01, *** = p
<0.001 and **** = p <0.0001). Values represent the mean SEM from at least three to four experiments performed in duplicate.
Figure 6. Pharmacological characterization of BQCA and 11 in radioligand saturation membrane binding assays. (A−D) Radioligand saturation
binding experiments performed in hM1 mAChR-expressing CHO cell membranes in the presence of increasing concentrations of radiolabeled
orthosteric antagonist [3H]NMS. (A) 30 min preincubation with vehicle, BQCA, or BQCA + ACh. (B) 30 min preincubation with vehicle, 11, or 11
+ ACh. (C−D) Bmax (% vehicle) bar graphs where values significantly different from vehicle are indicated by an * (where * = p <0.05, ** = p <0.01,
*** = p <0.001, and **** = p <0.0001). Values represent the mean SEM from three experiments performed in duplicate.
Liquid chromatography−mass spectrometry (LCMS) was per-
Characterization requirements for final compounds were set as mp,
1H NMR, 13C NMR, LRMS, HRMS, and HPLC (254 and 214 nm)
formed on an Agilent 1200 series coupled to the 6120 quadrupole
purity >95%.
mass spectrometer. Elution was also monitored at 254 nm.
Diethyl 2-((Phenylamino)methylene)malonate (1). Aniline (9.80
mL, 107 mmol) and diethylethoxymethylene malonate (21.70 mL, 107
mmol) were syringed into an RBF and heated at 120 °C for 4 h. The
reaction mixture was removed from the heat, 20 mL of cold petroleum
Characterization requirements for intermediate compounds were
set as mp, 1H NMR, 13C NMR, LRMS (or LCMS if unattainable), and
HPLC (254 nm) or LCMS purity.
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spirits added and the flask cooled on an ice bath for 30 min. The
yellow crystals that formed were transferred to a conical flask and
sonicated (in small batches) in cold petroleum spirits for 2−3 min.
The target compound was obtained as pale yellow−white crystals
following vacuum filtration and additional washes with petroleum
spirits (23.5 g, 83%); mp 43−44 °C. δH (CDCl3) 11.02 (br d, J = 13.5
Hz, 1H), 8.55 (d, J = 13.5 Hz, 1H), 7.39 (app t, J = 8.0 Hz, 2H), 7.21−
7.12 (m, 3H), 4.33 (q, J = 7.0 Hz, 2H), 4.27 (q, J = 7.0 Hz, 2H), 1.40
(t, J = 7.0 Hz, 3H), 1.35 (t, J = 7.0 Hz, 3H); δC (CDCl3) 169.2, 165.7,
151.9, 139.4, 129.8, 125.0, 117.2, 93.6, 60.4, 60.1, 14.4, 14.3. m/z
LRMS (TOF ES+) [MH]+ 264.3. HPLC: tR = 10.67 min, purity =
97.3%
Ethyl 4-Oxo-1,4-dihydroquinoline-3-carboxylate (2). Procedure 1
(diphenyl ether): Diphenyl ether (8 mL) was heated to 220 °C in a
small beaker directly placed on the hot plate. Diethyl 2-
((phenylamino)methylene)malonate (1) (1.00 g, 3.80 mmol) was
added and the reaction mixture heated, uncovered, for 20 min. The
beaker was removed from the heat and allowed to cool to RT,
resulting in product formation and almost total solidification of the
reaction mixture. The target compound was isolated as a gray−white
powder by vacuum filtration and copious washing with cold petroleum
spirits (395 mg, 48%). Procedure 2 (Eaton’s reagent): Diethyl 2-
((phenylamino)methylene)malonate (1) (23.5 g, 89.1 mmol) and
Eaton’s reagent (1:10 w/w phosphorus pentoxide solution in
methanesulfonic acid, 80 mL) were stirred at 100 °C for 3.5 h. The
reaction mixture was cooled to RT before being slowly pouring into
saturated sodium bicarbonate. The precipitated product was collected
as an orange−yellow powder by vacuum filtration and washed
copiously with cold petroleum spirits (19.0 g, quantitative); mp 271−
273 °C. δH (DMSO-d6) 8.63 (s, 1H), 8.25 (dd, J = 8.0, 1.5 Hz, 1H),
7.79 (ddd, J = 8.5, 7.0, 1.5 Hz, 1H), 7.71 (dd, J = 8.5, 1.0 Hz, 1H), 7.50
(ddd, J = 8.0, 7.0, 1.0 Hz, 1H), 4.31 (q, J = 7.0 Hz, 2H), 1.37 (t, J = 7.0
Hz, 3H). δC (DMSO-d6) 173.4, 164.9, 144.8, 139.0, 132.3, 127.3,
125.6, 124.6, 118.7, 110.0, 59.5, 14.4. m/z LRMS (TOF ES+) [MH]+
218.2. HPLC: tR = 6.61 min, purity = 98.8%
4-Oxo-1,4-dihydro-quinoline-3-carboxylic Acid (3). Ethyl 4-oxo-
1,4-dihydroquinoline-3-carboxylate (2) (500 mg, 2.30 mmol) and
THF (10 mL) were brought to 0 °C in an ice bath. A solution of
aqueous 1 M LiOH(aq) (4.60 mL, 4.60 mmol) was added dropwise
while stirring, and then the reaction mixture was heated at 80 °C for
24 h. Following addition of excess aqueous 1 M HCl(aq), the target
molecule precipitated out and was isolated as an off-white powder by
vacuum filtration, washed with distilled water to remove the residual
acid, and then dried under vacuum overnight (327 mg, 75%); mp
273−275 °C. δH (DMSO-d6) 15.48 (br s, 1H), 8.88 (s, 1H), 8.29 (dd,
J = 8.0, 1.5 Hz, 1H), 7.89 (ddd, J = 8.5, 7.0, 1.5 Hz, 1H), 7.82 (dd, J =
8.0, 1.5 Hz, 1H), 7.60 (ddd, J = 8.0, 7.0, 1.5 Hz, 1H). δC (DMSO-d6)
178.3, 166.5, 145.1, 139.4, 133.9, 126.3, 125.0, 124.4, 119.6, 107.7. m/z
LRMS (TOF ES+) [MH]+ 190.2. HPLC: tR = 6.44 min, purity =
>99.0%
General Procedure for N-B of Either Ethyl 4-Oxo-1,4-dihydroqui-
noline-3-carboxylate (2) or 4-Oxo-1,4-dihydro-quinoline-3-carbox-
ylic Acid (3). The ethyl carboxylate (2) or carboxylic acid (3) (30−500
mg) and acetonitrile (7−15 mL) were placed in an RBF and cooled to
0 °C in an ice bath. The appropriately substituted benzyl halide (2
equiv) and N,N-diisopropylethylamine (DIPEA) (4 equiv) were added
and the reaction stirred at 80 °C for between 2 and 22 h. Note: 1.1
equiv of potassium iodide was added at the beginning of the reaction if
a substituted benzyl chloride was being used. If the product was
observed to precipitate from the reaction mixture, the resulting solid
was isolated by vacuum filtration and washed with diethyl ether or hot
ethanol. If no precipitate was observed, the reaction mixture was
partitioned between chloroform (10 mL) and 1 M HCl(aq) (10 mL)
and the aqueous layer extracted with chloroform (3 × 10 mL). The
combined organic fractions were dried over anhydrous MgSO4,
filtered, and the solvent evaporated in vacuo to yield solid product.
1-(4-Methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic
Acid (4b). Fine white crystals; 3 h; 790 mg, 97%; mp 250−251 °C. δH
(DMSO-d6) 15.17 (s, 1H), 9.26 (s, 1H), 8.39 (dd, J = 8.0, 1.5 Hz, 1H),
7.96 (d, J = 8.5 Hz, 1H), 7.88 (ddd, J = 8.5, 7.0, 1.5 Hz, 1H), 7.63
(ddd, J = 8.0, 7.0, 1.0 Hz, 1H), 7.28 (d, J = 9.0 Hz, 2H), 6.92 (d, J =
9.0 Hz, 2H), 5.80 (s, 2H), 3.78 (s, 3H, CH3). δC (DMSO-d6) 177.9,
166.0, 159.0, 149.8, 139.4, 134.1, 128.4, 127.0, 126.3, 125.9, 125.7,
118.7, 114.3, 107.8, 55.9, 55.1. m/z LRMS (TOF ES+) [MH]+ 310.4.
m/z HRMS (TOF ES+) C18H16NO4 [MH]+ calcd 310.1074; found
310.1086. HPLC: tR = 9.13 min, purity (254 nm) = 98.3%, purity (214
nm) = 99.0%
1-(4-(Fluorosulfonyl)benzyl)-4-oxo-1,4-dihydroquinoline-3-car-
boxylic Acid (4c). Clear glassy crystals; 22 h; 12.1 mg, 21%; mp 289−
290 °C. δH (DMSO-d6) 15.12 (br s, 1H), 9.35 (s, 1H), 8.42 (dd, J =
8.0, 1.5 Hz, 1H), 8.12 (dd, J = 8.0, 6.5 Hz, 2H), 7.86 (ddd, J = 8.5, 7.0,
1.5 Hz, 1H), 7.75 (d, J = 8.5 Hz, 1H), 7.68−7.59 (m, 3H), 6.08 (s,
2H). δC (DMSO-d6) 178.1, 165.9, 150.7, 144.8, 139.3, 134.4, 130.8,
129.0, 128.2, 126.5, 126.1, 125.7, 118.3, 108.2, 55.8. m/z LRMS (TOF
ES+) [MH]+ 362.3. m/z HRMS (TOF ES+) C17H13FNO5S [MH]+
calcd 362.0493; found 362.0485. HPLC: tR = 9.56 min, purity (254) =
98.8%, purity (214) = 98.9%
1-(4-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzyl)-4-oxo-1,4-di-
hydroquinoline-3-carboxylic Acid (4d). Pale purple-tinged white
solid; 2 h; 42 mg, 53%; mp 292−293 °C. δH (DMSO-d6) 15.18 (br s,
1H), 9.35 (s, 1H), 8.42 (d, J = 7.5 Hz, 1H), 7.95−7.84 (m, 2H), 7.64
(ddd, J = 8.0, 5.5, 2.5 Hz, 1H), 7.40 (d, J = 8.5 Hz, 2H), 7.32 (d, J =
8.5 Hz, 2H), 7.17 (s, 2H), 5.94 (s, 2H). δC (DMSO-d6) 178.0, 169.8,
166.0, 150.4, 139.4, 134.9, 134.7, 134.3, 131.2, 127.14, 127.07, 126.4,
126.0, 125.7, 118.6, 108.0, 56.0. m/z LCMS [MH]+ 375.0. m/z HRMS
(TOF ES+) C21H15N2O5 [MH]+ calcd 375.0981, found 375.0966.
HPLC: tR = 8.32 min, purity (254) = 97.8%, purity (214) = 97.9%
Ethyl 1-(4-Nitrobenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxy-
late (7). White crystals; 22 h; 253 mg, 78%; mp 282−283 °C. δH
(DMSO-d6) 8.98 (s, 1H), 8.27 (dd, J = 8.0, 1.5 Hz, 1H), 8.22 (d, J =
9.0 Hz, 2H), 7.67 (ddd, J = 8.5, 7.0, 1.5 Hz, 1H), 7.59−7.39 (m, 4H),
5.86 (s, 2H), 4.26 (q, J = 7.0 Hz, 2H), 1.31 (t, J = 7.0 Hz, 3H). δC
(DMSO-d6) 172.9, 164.6, 150.3, 147.0, 143.9, 138.9, 132.7, 128.4,
127.6, 126.5, 125.0, 124.0, 117.5, 110.6, 59.8, 54.9, 14.3. m/z LRMS
(TOF ES+) [MH]+ 353.4.HPLC: tR = 9.19 min, purity = 99.1%
General Procedure for Radical Bromination of Either p-
Toluenesulfonyl Fluoride or 1-(p-Tolyl)-1H-pyrrole-2,5-dione. p-
Toluenesulfonyl fluoride or 1-(p-tolyl)-1H-pyrrole-2,5-dione (50−
250 mg), N-bromosuccinimide (NBS) (1.1−1.8 equiv), and benzoyl
peroxide (0.05 equiv) were placed in a microwave vial with ethyl
acetate (4 mL), and the reaction mixture was stirred briefly to dissolve.
The vessel was sealed and reacted in the microwave for 30 min at 100
°C. The reaction mixture was filtered to remove any precipitated NBS,
then the solvent evaporated in vacuo. The target molecule was isolated
by column chromatography (stationary phase, silica; mobile phase,\
5:2 hexane:DCM or 2:1 chloroform:hexane). Percentage purity was
observed to be low due to difficulty in separating the desired
monobrominated product from the dibrominated side product.
p-Sulfonylfluorobenzyl Bromide (5). White crystalline powder; 293
mg, 81%; mp 65−66 °C. δH (DMSO-d6) 7.92 (d, J = 8.5 Hz, 2H), 7.58
(d, J = 8.0 Hz, 2H), 4.44 (s, 2H). δC (DMSO-d6) 145.9, 130.2, 129.0,
128.2 (2JCF = 48 Hz), 30.7. m/z LRMS (TOF ES−) [MBr]−: 331.2.
LCMS: tR = 6.04 min, purity = 87.4%.
1-(p-Tolyl)-1H-pyrrole-2,5-dione (6a). Step A: p-Toluidine (1.00 g,
9.33 mmol) was dissolved in DMF (15 mL) and maleic anhydride
added (1.1 g, 10.27 mmol). The reaction mixture was stirred at 110 °C
for 5 h, after which time the solvent evaporated in vacuo to yield the
intermediate (Z)-4-oxo-4-(p-tolylamino)but-2-enoic acid as a yellow
solid (1.92 g, 100%); mp 180−181 °C. δH (DMSO-d6) 10.37 (br s,
1H), 7.96 (s, 1H), 7.52 (d, J = 8.5 Hz, 2H), 7.14 (d, J = 8.0 Hz, 2H),
6.47 (d, J = 12.0 Hz, 1H), 6.30 (d, J = 12.0 Hz, 1H), 2.27 (s, 3H). δC
(DMSO-d6) 166.7, 163.0, 135.9, 133.0, 131.6, 130.7, 129.2, 119.6,
20.5. m/z LCMS [M − H]− 204.1. HPLC: tR = 7.45 min, purity =
97.3%. Step B: (Z)-4-Oxo-4-(p-tolylamino)but-2-enoic acid (2 g, 9.75
mmol) and sodium acetate (400 mg, 4.87 mmol) were refluxed in 10
mL of acetic anhydride at 120 °C for 2.5 h. The reaction mixture was
then cooled to RT and the solvent evaporated in vacuo. Addition of
methanol (30 mL) resulted in precipitation of the target compound as
yellow needle-like crystals that were collected by vacuum filtration.
The crude filtrate was concentrated and purified by column
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chromatography (stationary phase, silica; mobile phase, 3:1 chlor-
oform:hexane) to isolate additional product (925 mg, 51%); mp 151−
153 °C. δH (DMSO-d6) 7.29 (d, J = 8.0 Hz, 2H), 7.20 (d, 2H, J = 8.5
Hz), 7.17 (s, 2H), 2.35 (s, 3H). δC (DMSO-d6) 170.0, 137.2, 134.6,
129.3, 128.9, 126.7, 20.7. m/z LCMS [MH]+ 188.1. HPLC: tR = 7.36
min, purity = 96.5%
1-(4-(Bromomethyl)phenyl)-1H-pyrrole-2,5-dione (6b). Yellow
solid; 32 mg, 45%; mp 113−114 °C. δH (CDCl3) 7.43 (d, J = 8.5
Hz, 2H), 7.28 (d, 2H, J = 8.5 Hz), 6.79 (s, 2H), 4.44 (s, 2H); δC
(CDCl3) 169.3, 137.4, 134.3, 131.2, 129.9, 126.1, 32.5. m/z LCMS
[MH]+ 267.9. HPLC: tR = 8.52 min, purity = 81.9%.
organic fractions were dried over anhydrous MgSO4, filtered, and the
solvent evaporated in vacuo to yield the target molecule as a fine pearly
white powder (54.7 mg, 96%); mp 237−238 °C. δH (DMSO-d6) 15.14
(s, 1H), 9.30 (s, 1H), 8.41 (dd, J = 8.0, 1.5 Hz, 1H), 7.87 (ddd, J = 8.5,
7.0, 1.5 Hz, 1H), 7.81 (d, J = 8.5 Hz, 1H), 7.64 (ddd, J = 8.0, 7.0, 1.0
Hz, 1H), 7.42 (d, J = 8.5 Hz, 2H), 7.36 (d, J = 8.5 Hz, 2H), 5.90 (s,
2H). δC (DMSO-d6) 178.0, 165.9, 150.4, 139.3, 135.3, 134.2, 133.7,
129.6, 128.1, 126.45, 126.41, 126.0, 125.7, 118.5, 108.0, 55.8. m/z
LCMS [MH]+ 337.1. m/z HRMS (TOF ES+) C18H12N2O3 [M + H]+
calcd 337.0569, found 337.0639. HPLC: tR = 10.76 min, purity (254)
= 97.7%, purity (214) = 97.4%
Crystallography. Intensity data were collected with an Oxford
Diffraction SuperNova CCD diffractometer using Cu Kα radiation,
and the temperature during data collection was maintained at 100.0(1)
using an Oxford Cryosystems cooling device. The structure of
compound 4a was solved by direct methods and difference Fourier
synthesis.42 Thermal ellipsoid plots were generated using the program
ORTEP-343 integrated within the WINGX44 suite of programs.
Crystal Data for 4a. C17H13NO3, M = 279.28, T = 100.0 K, λ =
1.54180, orthorhombic, space group Pna21, a = 16.2869(6) b =
15.6800(6), c = 5.0797(2) Å, V 1297.25(9) Å3, Z = 4, Dc = 1.430 mg
M−3 μ(Cu Kα) 0.808 mm−1, F(000) = 584, crystal size 0.67 × 0.04 ×
0.02 mm3, 3118 reflections measured, 1714 independent reflections
[R(int) = 0.0367]; the final R was 0.0578, [I > 2σ(I)] and wR(F2) was
0.1601 (all data).
Pharmacology. Radioligand Competition Whole Cell Binding
Assays. Plate preparation: FlpIn-CHO cells stably expressing human
muscarinic M1 receptors (hM1 mAChR FlpIn-CHO) were cultured at
37 °C in 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM)
supplemented with 5% (v/v) fetal bovine serum (FBS) and 16 mM
HEPES. Cells were seeded into white opaque Isoplates at 8.0 × 103
cells per well and then grown at 37 °C for 20−24 h. Cells were then
washed twice with cold HEPES-buffered saline, 140 μL per well, cold
HEPES-buffered saline was added, and the plates kept on ice. Assay
protocol: A stock solution of ACh (10−2 M) was made up in cold
HEPES-buffered saline. Stock solutions of the interacting allosteric
ligands of choice (10−2 M) were made up in DMSO. Dilutions of all
ligands were made up in cold HEPES-buffered saline at 10× the
required concentration and added to stock plates on ice. Cells were
equilibrated at 4 °C for 4 h with 20 μL of ACh, 20 μL of a single
interacting allosteric ligand, and 20 μL of 0.1 nM [3H]NMS (total
volume 200 μL per well). Assay termination and data collection:
Assays were terminated by media removal, 2 × 200 μL per well washes
with 0.9% NaCl solution and addition of 100 μL per well Microscint-
20 scintillation liquid. The levels of remaining bound radioligand, and
therefore the degree of radioligand displacement, was measured in
disintegrations per minute (dpm) on the Microbeta2 LumiJET 2460
microplate counter (PerkinElmer).
AlphaScreen ERK1/2 Phosphorylation Assays. Plate preparation:
FlpIn-CHO cells stably expressing human muscarinic M1 receptors
(hM1 mAChR FlpIn-CHO) were cultured at 37 °C in 5% CO2 in
DMEM supplemented with 5% (v/v) FBS and 16 mM HEPES. Cells
were seeded into transparent 96-well plates at 5 × 104 cells per well
and grown for 6 h. Cells were then washed once with phosphate-
buffered saline (PBS) and incubated in serum-free DMEM (180 μL
per well) at 37 °C overnight (16−18 h) to allow FBS-stimulated
phosphorylated ERK1/2 levels to subside. Time course assays: A stock
solution of ACh (10−2 M) was made up in Milli-Q water. Stock
solutions of the test ligands (10−2 M) were made up in DMSO. All
ligands were diluted to 10−5 M in serum-free DMEM. Ligand additions
(20 μL) were made at predetermined time points over a 30 min period
(30, 25, 20, 15, 10, 8, 6, 5, 3, and 1 min) with the cells being incubated
at 37 °C following each addition. FBS, which triggers ERK1/2
phosphorylation via a tyrosine kinase receptor, was added at 6 min as a
measure of the maximal levels of pERK1/2 production. Concen-
tration−response curves (CRCs): A stock solution of ACh (10−2 M)
was made up in Milli-Q water. Stock solutions of the test ligands (10−2
M) were made up in DMSO. Dilutions of all ligands were made up in
FBS-free media at 10× the required concentration and added to stock
plates. Cells were incubated at 37 °C with 20 μL per well of each
Ethyl 1-(4-Aminobenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxy-
late (8). Ethyl 1-(4-nitrobenzyl)-4-oxo-1,4-dihydroquinoline-3-carbox-
ylate (7) (700 mg, 2 mmol) was dissolved in dimethylformamide
(DMF) (25 mL). Then 10% Pd/C (20 mg) was added under a
nitrogen atmosphere, following evacuation of nitrogen, the reaction
mixture was stirred for 5 h at RT under a hydrogen atmosphere. The
reaction mixture was then filtered through Celite, washed with
methanol, and the solvents evaporated in vacuo to yield a yellow solid
that required no further purification (594 mg, 93%); mp 186−187 °C.
δH (DMSO-d6) 8.88 (s, 1H), 8.29 (dd, J = 8.0, 1.0 Hz, 1H), 7.80 (d, J
= 8.5 Hz, 1H), 7.78−7.70 (m, 1H), 7.49 (app t, J = 7.0 Hz, 1H), 7.03
(d, J = 8.5 Hz, 2H), 6.58 (d, J = 8.5 Hz, 2H), 5.49 (s, 2H), 5.19 (s,
2H), 4.29 (q, J = 7.0 Hz, 2H), 1.34 (t, J = 7.0 Hz, 3H). δC (DMSO-d6)
172.9, 164.7, 149.5, 148.5, 139.0, 132.4, 128.4, 127.9, 126.3, 124.8,
121.90, 117.9, 113.9, 109.8, 59.7, 55.6, 14.3. m/z LRMS (TOF ES+)
[M + H]+ 323.5. HPLC: tR = 6.01 min, purity = 95.1%
1-(4-Aminobenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic Acid
(9). Ethyl 1-(4-aminobenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxy-
late (8) (700 mg, 2.17 mmol) was stirred in 3:1 methanol:distilled
H2O (12 mL). A solution of aqueous 1 M KOH(aq) (6.51 mL, 6.51
mmol) was added and the reaction stirred for at 80 °C for 6.5 h, at
which point the reaction was allowed to cool to room temperature.
Following addition of 1 M HCl(aq) (7.60 mL, 7.60 mmol), the target
molecule precipitated out and was isolated as a yellow powder by
vacuum filtration and dried under vacuum overnight. (524 mg, 82%);
mp 215−216 °C. δH (DMSO-d6) 15.24 (br s, 1H), 9.16 (s, 1H), 8.39
(dd, J = 8.0, 1.5 Hz, 1H), 8.02 (d, J = 8.5 Hz, 1H), 7.90 (ddd, J = 8.5,
7.0, 1.5 Hz, 1H), 7.64 (app t, J = 7.5 Hz, 1H), 7.02 (d, J = 8.5 Hz, 2H),
6.52 (d, J = 8.5 Hz, 2H), 5.63 (s, 2H), 5.19 (s, 2H). δC (DMSO-d6)
178.0, 166.0, 150.2, 139.4, 134.2, 133.8, 133.5, 128.0, 126.4, 125.9,
125.7, 122.7, 118.6, 107.9, 55.9. m/z LRMS (TOF ES+) [M + H]+
295.4. LCMS: tR = 4.55 min, purity = >99.0%
1-(4-(2-Bromoacetamido)benzyl)-4-oxo-1,4-dihydroquinoline-3-
carboxylic Acid (10). 1-(4-Aminobenzyl)-4-oxo-1,4-dihydroquinoline-
3-carboxylic acid (9) (200 mg, 680 μmol) was stirred in acetonitrile (8
mL) in a dry ice/acetone bath. TEA (231.7 μL, 1.70 mmol) and
bromoacetyl bromide (65.6 μL, 748 μmol) were added dropwise and
the reaction mixture allowed to slowly return to RT as the dry ice
evaporated. After 20 h stirring at RT, the precipitated target molecule
was isolated by vacuum filtration as a beige powder and washed with
0.1 M HCl(aq) to remove the residual base, followed by distilled water
to remove the residual acid, then dried under vacuum overnight (208
mg, 74%); mp 239−240 °C. δH (DMSO-d6) 15.23 (br s, 1H), 10.48 (s,
1H), 9.32 (s, 1H), 8.44 (d, J = 8.0 Hz, 1H), 7.96−7.88 (m, 2H), 7.67
(ddd, J = 8.0, 5.0, 3.0 Hz, 1H), 7.61 (d, J = 8.5 Hz, 2H), 7.33 (d, J =
8.5 Hz, 2H), 5.88 (s, 2H), 4.07 (s, 2H). δC (DMSO-d6) 178.0, 171.0,
165.4, 150.1, 138.9, 134.1, 130.5, 130.0, 127.3, 126.4, 125.9, 125.7,
119.8, 118.7, 107.8, 61.8, 56.1. m/z LRMS (TOF ES+) [M − Br]+
335.4. m/z HRMS (TOF ES−) C19H14BrN2O4 [M − H]− calcd
413.0142, found 413.0141. HPLC: tR = 8.60 min, purity (254) =
99.1%, purity (214) = 97.9%
1-(4-Isothiocyanatobenzyl)-4-oxo-1,4-dihydroquinoline-3-car-
boxylic Acid (11). 1-(4-Aminobenzyl)-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (9) (50 mg, 170 μmol) was stirred in dry DCM (4 mL)
under a N2 atmosphere. 1,1′-Thiocarbonyl diimidazole (41.5 mg, 233
μL) and DMAP (1.89 mg, 15.5 μmol) were added and the reaction
mixture stirred at RT for 5 h. The reaction mixture was partitioned
between chloroform (20 mL) and 1 M HCl(aq) (20 mL) and the
aqueous layer extracted with chloroform (3 × 20 mL). The combined
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concentration of all compounds at 5 min (as determined by time
course assays). Interaction studies: A stock solution of ACh (10−2 M)
was made up in Milli-Q water. Stock solutions of the allosteric test
ligands and atropine (10−2 M) were made up in DMSO. In the case of
the allosteric ligand interactions, dilutions of ACh and the interacting
ligand were made up in FBS-free media at 20× the ideal concentration
range (as determined by CRC assays). Equal volumes of each
concentration of ACh and each concentration of the interacting ligand
of choice were premixed in stock plates. Cells were incubated at 37 °C
with 20 μL per well of each ligand mixture at 5 min (as determined by
time course assays). In the case of atropine, dilutions of ACh and
atropine were made up in FBS-free media at 10× the ideal
concentration range as determined by laboratory colleagues. Atropine
was equilibrated with the receptor at 30 min before the addition of
ACh at 5 min. Assay termination and data collection: Agonist-
stimulated ERK1/2 phosphorylation was terminated by the removal of
drugs and the addition of 100 μL p/well of SureFire lysis buffer. The
cell lysates were agitated for 5−10 min. Following agitation, 5 μL of
cell lysates were transferred into a 384-well white opaque Optiplate,
followed by addition of 8.5 μL of a solution of reaction buffer/
activation buffer/acceptor beads/donor beads in a ratio of 6/1/0.3/0.3
(v/v/v/v) under green light conditions. The plates were then
incubated at 37 °C in the dark for 1 h, and fluorescence was
measured on a Fusion-α plate reader (PerkinElmer) using standard
AlphaScreen settings.
Radioligand Saturation Whole Cell Binding Assays. Plate
preparation: FlpIn-CHO cells stably expressing human muscarinic
M1 receptors (hM1 mAChR FlpIn-CHO) were cultured at 37 °C in
5% CO2 in DMEM supplemented with 5% (v/v) FBS and 16 mM
HEPES. Cells were seeded into white opaque Isoplates at 7.0 × 103
cells per well and then grown at 37 °C for 6 h. Cells were then washed
once with PBS and incubated in serum-free DMEM (160 μL per well)
at 37 °C overnight (16−18 h). Preincubation with controls and test
ligands: A stock solution of ACh (10−2 M) was made up in Milli-Q
water. A stock solution of AChM was made up in ethanol. Stock
solutions of other test ligands (10−2 M) were made up in DMSO. All
ligands were diluted to 10−3 M in HEPES-buffered saline, except
AChM, which was diluted to 10−3 M in 50 mM phosphate buffer and
incubated at 37 °C for 20 min to form the reactive aziridinium ion and
then stored on ice and used as quickly as possible. Buffer controls
(10% DMSO/HEPES-buffered saline and 10% ethanol/50 mM
phosphate buffer) were also made up. Ligand additions (20 and 20
μL buffer for single ligand additions), for a final volume of 200 μL,
were made at 30 min (or at 5, 10, 15, or 20 min during experimental
optimization) and the cells incubated at 37 °C, during which time any
irreversible binding of the test ligands would be expected to take place.
Cells were then washed twice with cold PBS and 160 μL per well of
cold HEPES-buffered saline was added and the plates kept on ice.
Assay protocol: Stock solutions of [3H]NMS (10−7.5 and 10−8 M)
were made up in HEPES-buffered saline. Dilutions of [3H]NMS were
made up in HEPES-buffered saline at 10× the required concentration
and added to a stock plate. A stock solution of atropine (10−4 M) was
made up at 10× the required concentration. Cells were equilibrated at
4 °C for 4 h with 20 μL of HEPES-buffered saline (specific wells) or
20 μL of atropine (nonspecific wells) and 20 μL per well of each
concentration of [3H]NMS (total volume 200 μL per well). Assay
termination and data collection: Assays were terminated by media
removal, 2 × 200 μL per well washes with 0.9% NaCl solution, and
addition of 100 μL per well of Microscint-20 scintillation liquid. The
levels of remaining bound radioligand, and therefore the degree of
radioligand displacement was measured in disintegrations per minute
(dpm) on the Microbeta2 LumiJET 2460 microplate counter
(PerkinElmer).
made up to 8 mL with cold HEPES-buffered saline, and the tubes kept
on ice. Preincubation with controls and test ligands: A stock solution
of ACh (10−2 M) was made up in Milli-Q water. A stock solution of
AChM was made up in ethanol. Stock solutions of other test ligands
(10−2 M) were made up in DMSO. All ligands were diluted to 10−3
M
in HEPES-buffered saline, except AChM which was diluted to 10−3 M
in 50 mM phosphate buffer and incubated at 37 °C for 20 min to form
the reactive aziridinium ion and then stored on ice and used as quickly
as possible. Buffer controls (10% DMSO/HEPES-buffered saline and
10% ethanol/50 mM phosphate buffer) were also made up. Then 1
mL ligand additions (and 1 mL buffer for single ligand additions), for a
final volume of 10 mL, were made at 30 min and the membrane tubes
incubated in a water bath at 37 °C, during which time any irreversible
binding of the test ligands would be expected to take place. Then 10
mL of ice-cold HEPES-buffered saline was added, and then additional
ice cold buffer was added to bring all tubes to equal mass. Tubes were
centrifuged on the Sorval Ultracentrifuge at 4 °C for 20 min at 40000
rpm. The supernatant was discarded, the membranes were
resuspended thoroughly in 1 mL of ice-cold HEPES-buffered saline,
and an additional 19 mL of ice-cold buffer added, and the tubes
brought to equal mass again. Tubes were centrifuged again on the
Sorval ultracentrifuge at 4 °C for 20 min at 40000 rpm. Two
centrifugation steps were used in place of the PBS washes in the whole
cell binding experiment described previously. The supernatant was
discarded and the membranes resuspended in 400 μL of cold HEPES-
buffered saline. A Bradford assay was performed to ascertain the
concentration of membrane in each treatment tube, and then the
required amount of cold HEPES-buffered saline was added to bring
the concentration of each to 30 μg/mL. Assay protocol: Stock
solutions of [3H]NMS (10−7.5 and 10−8 M) were made up in HEPES-
buffered saline. Dilutions of [3H]NMS were made up in HEPES-
buffered saline at 10× the required concentration. A stock solution of
atropine (10−4 M) was made up at 10× the required concentration.
Membranes (100 μL per tube) were equilibrated at 37 °C for 1 h with
100 μL of HEPES-buffered saline (specific wells) or 100 μL of
atropine (nonspecific wells) and 100 μL per well of each concentration
of [3H]NMS (in the required amount of HEPES-buffered saline to
make a total volume of 1 mL per tube). Assay termination and data
collection: Assays were terminated by harvesting the tubes in the
Brandel harvester and rinsing tubes twice with 2 mL of ice-cold 0.9%
NaCl solution. Membranes were dried under the heat lamp, picked,
and placed into 6 mL scintillation tubes. Then 4 mL per tube of
Ultima Gold scintillation liquid was added and the tubes capped,
vortexed, and left on the bench for 1 h at RT. The levels of bound
radioligand, and therefore the degree of radioligand blockade by any
irreversibly bound test ligands, was measured in disintegrations per
minute (dpm) on the TriCarb 2910 TR liquid scintillation analyzer
(PerkinElmer).
Data Analysis. All data analysis was managed using Prism 6
software (GraphPad Software, San Diego, CA). Experiments
measuring radioligand competition binding interactions were fitted
to the allosteric ternary complex model 1:
[A]
Y =
KAKB
[I]
KI
[B]
KB
α[I][B]
[A] +
1 +
+
+
KIKB
(
α ′ [B] + K )(
)
(1)
B
where Y is the percentage (vehicle control) binding, [A], [B], and [I]
are the concentrations of [3H]NMS, the allosteric ligand, and ACh,
respectively, KA and KB are the equilibrium dissociation constants of
[3H]NMS and the allosteric ligand, respectively, KI is the equilibrium
dissociation constant of ACh, and α and α′ are the cooperativities
between the allosteric ligand and [3H]NMS or ACh, respectively.
Values of α (or α′) > 1 denote positive cooperativity, values <1 (but
>0) denote negative cooperativity, and values = 1 denote neutral
cooperativity. Functional orthosteric and allosteric agonist concen-
tration−response curves were fitted via nonlinear regression to the
three-parameter logistic function 2:
Radioligand Saturation Membrane Binding Assays. Membrane
preparation: Preprepared FlpIn-CHO cell membranes stably express-
ing human muscarinic M1 receptors (hM1 mAChR FlpIn-CHO) were
thawed, diluted to a concentration of 1250 μg/mL, and resuspended
using the hand-held homogenizer (at slow speed to minimize
frothing). Ultracentrifuge tubes (one per buffer/ligand treatment)
were labeled, 500 μg of membrane pipetted into each, the volume
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concentration that gives a response halfway between Emax and basal.
Functional experiments measuring the interactions between ACh and
each allosteric ligand were fitted to the operational model of
allosterism and agonism 345,46 to derive functional estimates of
modulator affinity, cooperativity, and efficacy.
(Emax − basal)
E = basal +
50−log[A])
1 + 10−pEC
(2)
where E is response, Emax and basal are the top and bottom asymptotes
of the curve, respectively, log [A] is the logarithm of the agonist
concentration, and pEC50 is the negative logarithm of the agonist
Em(τA[A](KB + αβ[B]) + τB[B]KA)n
([A]KB + KAKB + [B]KA + α[A][B])n + (τA[A](KA + αβ[B]) + τB[B]KA)n
E =
(3)
where Em is the maximum attainable system response for the pathway
under investigation; [A] and [B] are the concentrations of orthosteric
and allosteric ligands, respectively; KA and KB are the equilibrium
dissociation constants of the orthosteric and allosteric ligands,
respectively; τA and τB are the operational measures of orthosteric
and allosteric ligand efficacy (which incorporate both signal efficiency
and receptor density), respectively; n is a transducer slope factor
linking occupancy to response; α is the binding cooperativity
parameter between the orthosteric and allosteric ligand; and β denotes
the magnitude of the allosteric effect of the modulator on the efficacy
of the orthosteric agonist.12,45 In all cases, the equilibrium dissociation
constant of each allosteric ligand was fixed to that determined from the
competition binding experiments.
ABBREVIATIONS USED
■
AChM, acetylcholine mustard; ACN, acetonitrile; BQCA,
benzyl quinolone carboxylic acid (1-(4-methoxybenzyl)-4-oxo-
1,4-dihydroquinoline-3-carboxylic acid); CHO, Chinese ham-
ster ovary; CNS, central nervous system; COSY, correlation
spectroscopy; CRC, concentration−response curve; DCM,
dichloromethane; DIPEA, N,N-diisopropylethylamine; DMAP,
4-dimethylaminopyridine; DMEM, Dulbecco’s Modified Eagle
Medium; DMF, dimethylformamide; DMSO, dimethyl sulf-
oxide; ERK, extracellular-regulated kinase; FBS, fetal bovine
serum; GPCR, G protein-coupled receptor; HEPES, 2-[4-(2-
hydroxyethyl)-1-piperazinyl]ethanesulfonic acid; HMBC, het-
eronuclear multiple-bond correlation; HPLC, high performance
liquid chromatography; HRMS, high resolution mass spec-
trometry; HSQC, heteronuclear single quantum coherence;
LCMS, liquid chromatography−mass spectrometry; LRMS, low
resolution mass spectrometry; mAChR, muscarinic acetylcho-
line receptor; MP, melting point; NBS, N-bromosuccinimide;
NMR, nuclear magnetic resonance; NMS, N-methylscopol-
amine; PAM, positive allosteric modulator; PBS, phosphate-
buffered saline; RBF, round-bottomed flask; RT, room
temperature; SAR, structure−activity relationship; TCDI,
thiocarbonyldiimidazole; TEA, triethylamine; TFA, trifluoro-
acetic acid; THF, tetrahydrofuran; TLC, thin layer chromatog-
raphy; TOF, time-of-flight; WBC, whole cell binding
Experiments measuring radioligand saturation binding were fitted to
the one site-specific binding model 4:
B
max[A]
Y =
+ NS[A]
Kd + [A]
(4)
where Y is the specific binding + nonspecific binding, Bmax is the
maximum receptor binding (in the same units as Y), [A] is the
concentration of [3H]NMS, and Kd is the equilibrium dissociation
constant of [3H]NMS in the same units as [A].
Statistical analyses were performed where appropriate using one-
way analysis of variance with Dunnett’s post test, and significance
indicated as follows: * = p < 0.05, ** = p <0.01, *** = p <0.001 and
**** = p <0.0001.
ASSOCIATED CONTENT
* Supporting Information
Assigned HMBC spectrum of BQCA, HPLC chromatograms
■
S
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■
Bmax (% vehicle) bar graphs for compounds 4c, 4d, and 10. This
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AUTHOR INFORMATION
Corresponding Authors
*For P.J.S.: phone, +61 (0)3 9903 9542; E-mail, Peter.
Scammells@monash.edu.
■
*For A.C.: phone, +61 (0)3 9903 9067; E-mail, Arthur.
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