Novel Fused Arylpyrimidinone Based Allosteric Modulators of the M1 Muscarinic Acetylcholine Receptor

Article abstract of DOI:10.1021/acschemneuro.6b00018

Benzoquinazolinone 1 is a positive allosteric modulator (PAM) of the M₁ muscarinic acetylcholine receptor (mAChR), which is significantly more potent than the prototypical PAM, 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (BQCA). In this study, we explored the structural determinants that underlie the activity of 1 as a PAM of the M₁ mAChR. We paid particular attention to the importance of the tricyclic scaffold of compound 1, for the activity of the molecule. Complete deletion of the peripheral fused benzene ring caused a significant decrease in affinity and binding cooperativity with acetylcholine (ACh). This loss of affinity was rescued with the addition of either one or two methyl groups in the 7- and/or 8-position of the quinazolin-4(3H)-one core. These results demonstrate that the tricyclic benzo[h]quinazolin-4(3H)-one core could be replaced with a quinazolin-4(3H)-one core and maintain functional affinity. As such, the quinazolin-4(3H)-one core represents a novel scaffold to further explore M₁ mAChR PAMs with improved physicochemical properties.

Full text of DOI:10.1021/acschemneuro.6b00018

Research Article
pubs.acs.org/chemneuro
Novel Fused Arylpyrimidinone Based Allosteric Modulators of the M1
Muscarinic Acetylcholine Receptor
Shailesh N. Mistry,†,§ Herman Lim,‡,§ Manuela Jorg,† Ben Capuano,† Arthur Christopoulos,‡
̈
J. Robert Lane,*,‡ and Peter J. Scammells*,†
†Medicinal Chemistry and ‡Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052,
Victoria Australia
ABSTRACT: Benzoquinazolinone 1 is a positive allosteric modulator (PAM) of the M1 muscarinic acetylcholine receptor
(mAChR), which is significantly more potent than the prototypical PAM, 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (BQCA). In this study, we explored the structural determinants that underlie the activity of 1 as a PAM of the M1
mAChR. We paid particular attention to the importance of the tricyclic scaffold of compound 1, for the activity of the molecule.
Complete deletion of the peripheral fused benzene ring caused a significant decrease in affinity and binding cooperativity with
acetylcholine (ACh). This loss of affinity was rescued with the addition of either one or two methyl groups in the 7- and/or 8-
position of the quinazolin-4(3H)-one core. These results demonstrate that the tricyclic benzo[h]quinazolin-4(3H)-one core
could be replaced with a quinazolin-4(3H)-one core and maintain functional affinity. As such, the quinazolin-4(3H)-one core
represents a novel scaffold to further explore M1 mAChR PAMs with improved physicochemical properties.
KEYWORDS: M1 muscarinic acetylcholine receptor, positive allosteric modulator
lzheimer’s disease is a progressive and irreversible
Aneurodegenerative disorder affecting around 25 million
people worldwide.1 The disorder is primarily observed in the
aging population, and characteristic symptoms of the disease
include memory loss, confusion, and dementia.2,3 Currently,
pharmacological interventions for Alzheimer’s disease remain
limited, and provide only symptomatic relief to patients.4,5
The reduction of cholinergic neurons in the basal nuclear
complex is associated with the cognitive deficits observed in
patients with Alzheimer’s disease.6,7 A link between mAChR
function and disease pathology has been suggested, with the M1
mAChR subtype particularly highlighted for a role in
cognition.7,8
Acetylcholinesterase inhibitors are currently used to treat the
cognitive deficits associated with Alzheimer’s disease, but this
approach is limited by the moderate improvement in the
cognitive function of patients, as well as debilitating side effects
including nausea, diarrhea, hypotension and vomiting.9
However, they exert a nonselective effect at all muscarinic
receptor subtypes, because acetylcholine esterase inhibitors act
to inhibit acetylcholine breakdown. It is likely that such side-
effects are due activation of M2 and M3 mAChRs expressed in
the periphery.7
However, the design of selective orthosteric agonists for the M1
mAChR has proven difficult, due to the highly conserved
orthosteric pocket of all the mAChRs (M1−M5).10 However,
efforts to target the less-conserved, and topographically distinct
allosteric site of the receptor have proven more fruitful.11−14
Ligands that target such allosteric sites may act to potentiate
the binding and signaling activity of an orthosteric receptor
agonist (positive allosteric modulators, PAM) and/or activate
the receptor themselves (allosteric agonists). Allosteric ligands
of the M1 mAChR have been recognized as a potentially
promising novel drug class for the treatment of Alzheimer’s
disease.14
BQCA was reported as the first highly selective PAM for the
M1 mAChR.15 Structure−activity relationship (SAR) studies
around BQCA have revealed structurally related compounds
with higher affinity and potency.16−18 Furthermore, our group
has reported an enriched SAR study that used analytical
modeling of pharmacological data, to relate structural
modification to BQCA analogues, with variations in binding
affinity (pKB), binding (α) and functional (αβ) cooperativity,
Received: January 21, 2016
Accepted: February 18, 2016
Accordingly, there has been considerable focus upon the
design of ligands that selectively activate the M1 mAChR.
© XXXX American Chemical Society
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Figure 1. Top: Chemical structure of lead compounds 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (BQCA) and 3-((1S,2S)-
2-hydroxycyclohexyl)-6-((6-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)methyl)benzo[h]quinazolin-4(3H)-one (1). Bottom: Overview of the structures
and design strategy behind the novel analogues derived from compound 1. The numbering of the atoms in the quinazolinone ring bearing
substituents is shown in blue.
a
Scheme 1. Synthesis of Compound 1
a
Reagents and conditions: (a) (i) cat. Pd(P(tBu)3)2, degassed anhydrous THF, 0 °C; (ii) 0.5 M (2-chloro-5-pyridyl)methylzinc chloride/THF, 0 °C
to rt, 79%; (b) 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole, cat. PdCl2(PPh3)2, 1 M Na2CO3(aq)/THF 1:3 degassed, 100
°C, 64% (59% 1: 5% 12).
and intrinsic efficacy (τB).19 3-((1S,2S)-2-Hydroxycyclohexyl)-
6-((6-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)methyl)benzo-
[h]quinazolin-4(3H)-one (1), is a significantly more potent M1
mAChR PAM with a structural ancestry originating from
BQCA and related compounds (Figure 1).17,20 We recently
used a combination of site-directed mutagenesis, modeling of
pharmacological data and molecular dynamics simulations to
propose a binding mode for 1 at the M1 mAChR, similar to that
predicted for BQCA. In particular, residues Y852.64 and Y822.61
in transmembrane (TM) bundle 2, Y179 in extracellular loop
(ECL) 2, and W4007.35 in TM 7 were shown to be important
for the binding and function of both BQCA and compound 1.21
This approach also revealed that the higher potency of 1 was
predominantly driven by an increase in affinity, rather than
cooperativity with ACh, for the M1 mAChR allosteric site. The
current study aimed to explore the structural determinants that
underlie the activity of 1 as a PAM of the M1 mAChR and, in
particular, those that are responsible for its superior potency.
Furthermore, we aimed to move away from the tricyclic scaffold
of compound 1 and determine the importance of this moiety
for the activity of the molecule (Figure 1), using approaches
such as core trimming (compounds 2−9). An enriched SAR
profile was compiled to explore the important features of these
novel compounds to maintain high affinity, cooperativity, and
intrinsic efficacy.
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a
Scheme 2. Synthesis of Analogues 2−7
a
Reagents and conditions: (a) Br2, AcOH, DCM, rt, 95%; (b) cat. conc. H2SO4(aq), EtOH, reflux, 75%; (c) acetyl chloride, TEA, DCM, 0 °C to rt,
100%; (d) Br2, AcOH, rt, 23% (brsm); (e) 5 M NaOH(aq), EtOH, 90 °C, 81%; (f) cat. H2SO4(aq), MeOH, reflux, 83%; (g) (i) 1,4-dioxane/CCl4 1:1,
0 °C; (ii) Br2, 1,4-dioxane/CCl4 1:1, dropwise, 0 °C, 93%; (h) LiOH·H2O, THF, water, rt, 94%; (i) HCTU, (1S,2S)-2-aminocyclohexanol
hydrochloride, DIPEA, DMF, rt, 77−95%; (j) DMF-DMA, 85 or 115 °C, 63−95%; (k) formamide, 150 or 180 °C, 44−69%; (l)
triethylorthoformate, 100−150 °C, 65%; (m) (i) cat. Pd(P(tBu)3)2, dry THF, 0 °C; (ii) 0.5 M (2-chloro-5-pyridylmethyl)zinc chloride/THF, 0 °C
to RT or 55 °C, 14−90%; (n) 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole, cat. PdCl2(PPh3)2, 1 M Na2CO3(aq)/THF 1:3
degassed, 100 °C, Suzuki product 15−96%, dehalogenation product 0−14%.
Aside from pharmacological characterization of compounds
10, 12, and 1, which represent a stepwise buildup of the
(pyridin-3-yl)methyl pendant group, we also sought to
investigate the nature of the polyaromatic core. Given the
precedence for polyaromatic heterocycles potentially imparting
toxic DNA-chelation behavior to a scaffold,22 investigation of
related heteroaromatics seemed a prudent avenue of inves-
tigation. Initially we envisaged generating comparable ana-
logues of 1, incorporating gradual deletion of the benzo[h]-
quinazolin-4(3H)-one core toward a quinazolin-4(3H)-one
core. The deletion of the fused benzene ring was also
anticipated to make these analogues more “druglike”, through
reductions in lipophilicity (assessed through calculated log P),
topological polar surface area (tPSA), and molecular weight.23
With this strategy in mind, we synthesized analogues with
RESULTS AND DISCUSSION
■
Chemistry. The synthesis of lead compound 1, though
previously reported in patent literature,20 has only recently
been fully optimized and characterized in our hands.21 Seeking
to understand the basis for the PAM activity of compound 1,
we initially decided to pharmacologically characterize key
synthetic intermediates. Compound 10 was synthesized as
previously described, with subsequent Negishi coupling carried
out according to established methodology,20,21 affording
intermediate 11 in good yield. After the final Suzuki coupling
of 11 with commercially available 1-methyl-4-(4,4,5,5-tetra-
methyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole, we were able to
isolate and characterize both the desired Suzuki product 1, and
corresponding dehalogenated product 12 (Scheme 1).21
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a
Scheme 3. Synthesis of Compound 8 Containing a Pyrido[2,3-d]pyrimidinone Core
a
Reagents and conditions: (a) HCTU, (1S,2S)-2-aminocyclohexanol hydrochloride, DIPEA, DMF, rt, 94%; (b) formamide, 150 or 180 °C, 69%; (c)
(i) cat. Pd(P(tBu)3)2, dry THF, 0 °C; (ii) 0.5 M (2-chloro-5-pyridylmethyl)zinc chloride/THF, 0 °C to RT or 55 °C, 90%; (d) 1-methyl-4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole, cat. PdCl2(PPh3)2, 1 M Na2CO3(aq)/THF 1:3 degassed, 100 °C, 24%.
either complete deletion of the peripheral fused benzene ring
(2), or the presence of one or two methyl groups in the 7/8
positions of the quinazolin-4(3H)-one core, to give 4, 5 and 6.
Evaluation of these compounds was anticipated to address
whether the peripheral fused benzene ring of literature
compound 1, could be replaced by the steric presence of
either one or two methyl groups.
Our recent work determining the structural nature of the
interaction of compound 1 with the allosteric binding site at the
M1 mAChR, highlighted the importance of Y179 in ECL2,
making aromatic edge-face interactions with both the benzylic
pendant group and benzo[h]quinazolin-4(3H)-one core.21 In
addition, the proximity of the phenol moiety of Y179 could
facilitate additional polar interactions with an appropriately
positioned heteroatom incorporated into the ligand. With this
in mind, we also synthesized 8, the pyrido[2,3-d]pyrimidin-
4(3H)-one analogue of 2.
Synthesis of these analogues was carried out in a similar
manner to that of lead compound 1. In the case of 2,
commercially available 2-amino-5-bromobenzoic acid (21) was
employed, while the remaining substituted 2-amino-5-bromo-
benzoic acid intermediates (22−24), required synthesis from
unbrominated starting materials.
2-Amino-3,4-dimethylbenzoic acid (14) underwent initial
Fischer esterification to give the corresponding methyl ester 15,
followed by selective bromination of the 5-position to give
intermediate 16 as the hydrobromide salt, in excellent yield.
Subsequent basic hydrolysis afforded the desired 2-amino-5-
bromo-3,4-dimethylbenzoic acid (24). In the case of 2-amino-
3-methylbenzoic acid (13), we were able to directly brominate
in acetic acid at room temperature, to give 22, without the need
for esterification of the carboxylic acid moiety (Scheme 2).
Finally, in the case of 2-amino-4-methylbenzoic acid (23), our
attempts at direct bromination in the same manner as for 22,
gave a mixture of mono- and dibrominated products, in
addition to unreacted starting material, which proved difficult
to separate. However, selective bromination in the 5-postion
was achieved through esterification of the carboxylic acid
moiety, and acetylation of the aniline group. The acetanilide
derivative 19 facilitated selective monobromination of the 5-
position, allowing the isolation of 23 following the
saponification of the ester in the modest yield of 19% over
these two steps.
Subsequent synthetic steps proceeded in accordance with our
reported synthesis of lead compound 1.21 Briefly, the 2-amino-
5-bromobenzoic acid (21−24) intermediates first underwent
O-(1H-6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HCTU)-mediated coupling with
(1S,2S)-2-aminocyclohexanol hydrochloride in the presence
of DIPEA, in DMF at room temperature, furnishing the
corresponding amides (25−28) in good to excellent yield.
Cyclisation of these 2-amino arylcarboxamide derivatives was
achieved through heating in N,N-dimethylformamide-dimethy-
lacetal (DMF-DMA), formamide or triethylorthoformate,
affording 29-32. The more forcing conditions (150 or 180
°C in formamide) were found to prevent formation of stable 2-
(dimethylamino)-2,3-dihydroquinazolin-4(1H)-one-type inter-
mediates, which occurred in the presence of DMF-DMA. In
some cases, these intermediates failed to undergo elimination of
N,N-dimethylamine to furnish the desired product, necessitat-
ing the use of more forcing conditions.
Installation of the (pyridin-3-yl)methyl pendant group was
achieved as described for lead compound 1, through a sequence
of Negishi and Suzuki reactions, to give the desired compounds
2 and 4-6, bearing the (6-(1-methyl-1H-pyrazol-4-yl)pyridin-3-
yl)methyl group. During Suzuki coupling of final compounds 2
and 6, appreciable amounts of the dehalogenation side-product,
3 and 7 were also isolated, and deemed of interest for
pharmacological evaluation.
As part of the strategy to develop fused heteroaromatic
analogues of lead compound 1, the investigation of alternative
cores was also of interest. The pyrido[2,3-d]pyrimidinone
analogue of 2 (compound 8) was prepared using an analogous
D
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a
Scheme 4. Synthesis of Analogue 9 Containing a Thienopyrimidinone Core
a
Reagents and conditions: (a) (i) cat. PPh3, cat. PdCl2(PPh3)2, degassed DME/2 M Na2CO3(aq), 70 °C; (ii) 100 °C; (iii) 85 °C, 84% (E/Z 4:1); (b)
H2, wet 10% Pd/C, EtOAc, rt, 99%; (c) (i) anhydrous toluene, −78 °C; (ii) 1 M DIBAL-H in toluene, dropwise, −78 °C; (iii) MeOH quench, −78
°C; 88%; (d) (1S,2S)-2-aminocyclohexanol hydrochloride, HCTU, DIPEA, DMF, rt, 71%; (e) sulfur, TEA, EtOH, 60 °C, 57%; (f) formamide, 180
°C, 56%.
approach starting from 2-amino-5-bromonicotinic acid (37)
(Scheme 3).
Reduction of 45 in the presence of DIBAL-H at −78 °C,
afforded the desired aldehyde 46 in excellent yield.
The combination of 46, 48, and sulfur in the presence of
TEA and ethanol at 60 °C (one-pot Gewald conditions) gave
the substituted 2-aminothiophene-3-carboxamide 49 in mod-
erate yield. Finally cyclization of 49 was achieved by heating in
formamide at 180 °C, affording the desired thieno[2,3-
d]pyrimidin-4(3H)-one derivative 9. We elected to synthesize
9 bearing the biphenylmethyl pendant group, for initial ease of
access and to establish synthetic methodology. Furthermore,
this moiety has been shown to impart good affinity and
cooperativity on the BQCA scaffold (an early precursor of lead
compound 1 and related structures).7
Having synthesized the pyrido[2,3-d]pyrimidin-4(3H)-one
analogue 8, we turned our attention to 5−6 fused heterocyclic
scaffolds. The thieno[2,3-d]pyrimidin-4(3H)-one core was of
interest, since spatially isosteric replacements for the
quinazolin-4(3H)-one core have been reported in a number
of medicinal chemistry lead optimization campaigns, possessing
“drug-like” properties and biological activity.24−26
The thieno[2,3-d]pyrimidin-4(3H)-one core was accessible
through synthesis of the appropriately substituted 2-amino-
thiophene-3-carboxamide, which in turn was assembled in a
one-pot Gewald synthesis based on literature precedent.5
Components for the Gewald reaction were readily synthesized
from commercially available reagents (Scheme 4). Cyanoace-
tamide 48, was obtained through HCTU-mediated coupling of
cyanoacetic acid and (1S,2S)-2-aminocyclohexanol hydro-
chloride in the presence of DIPEA and DMF at room
temperature. A one-pot Wittig-Suzuki reaction was employed
to construct ester 44, from phenylboronic acid (41), 4-
bromobenzaldehyde (42), and (ethoxycarbonylmethylene)-
triphenylphosphorane (43), according to previously described
methodology.6 Though intermediate 44 was isolated as both
the E- and Z-isomers, these were combined before hydro-
genation of the double bond, to give saturated ester 45.
PHARMACOLOGY
■
We recently published an SAR study of the M1 mAChR PAM,
BQCA.19 By incorporating modeling into our pharmacological
analysis, we were able to relate modifications of the structural
features of BQCA, to changes in parameters that describe
allosteric ligand action. These comprise the affinity of the
modulator for the free receptor (KB), its modulatory effects on
the binding and efficacy of acetylcholine (α and β, respectively),
and its intrinsic efficacy (direct allosteric agonism) in the
system (τB). In particular, alternative substitution of the
quinolone ring in the 5- and 8-positions modulated intrinsic
efficacy; isosteric replacement of the carboxylic acid moiety or
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Table 1. Binding and Functional Parameters of 4-Phenylpyridin-2-one Analogues 2−12 at the M1 mAChR
a
Binding cooperativity with [3H]NMS; for instances where a complete inhibition of [3H]NMS binding by the allosteric modulator was observed
b
c
(consistent with a high level of negative cooperativity), log α′ was fixed to −3. Binding cooperativity with ACh. Functional cooperativity with ACh.
d
*
Intrinsic efficacy of the modulator; for instances where no intrinsic efficacy was observed, log τB was fixed to −3. Significant difference (p < 0.05)
relative to same parameter determined for 1, one-way ANOVA with Tukeys’s post-test. Values represent the mean SEM from at least three
experiments performed in duplicate.
Figure 2. (a−d) Pharmacological characterization of 1 and 5 in binding and function at the M1 mAChR. (a,b) Radioligand binding experiments were
performed using FlpIn-CHO cells expressing the M1 mAChR, 0.1 nM of the radiolabeled antagonist [3H]NMS, increasing concentrations of ACh,
with or without increasing concentrations of either 1 (a) or 5 (b). (c,d) IP1 accumulation experiments were performed using FlpIn-CHO cells
expressing the M1 mAChR and increasing concentrations of ACh with or without increasing concentrations of either compound 1 (c) or 5 (d). 100%
represents the maximal stimulation of ACh in the absence of test compound.
F
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amide derivatives of the acid function was important in
determining cooperativity, and replacement of the N-alkyl
group modulated ligand affinity.19 More recently, we focused
on the binding mode of 3-((1S,2S)-2-hydroxycyclohexyl)-6-
((6-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)methyl)benzo[h]-
quinazolin-4(3H)-one (1), a significantly more potent M1
mAChR PAM with a structural ancestry originating from
BQCA.21 To understand the structural determinants that
underlie the superior PAM activity of 1, we applied the same
approach as described above for BQCA for all ligands described
in this study. Competition binding studies between ACh and
the radiolabeled antagonist [3H]NMS at the M1 mAChR
expressed in FlpIN CHO cells were performed in the absence
and presence of increasing concentrations of each test
compound. Data were analyzed with an allosteric ternary
complex,27 to determine the KB of the test compound for the
allosteric site on the unoccupied M1 mAChR, and its binding
cooperativity (α) with ACh. To assess the ability of our test
compounds to modulate ACh function, we used myoinositol-1-
phosphate (IP1) accumulation as a measure of M1 mAChR
activation. Concentration curves of ACh were generated in the
presence of increasing concentrations of test compound, and an
operational model of allostery was applied to the data with the
KB fixed to that determined in the binding studies, thus
allowing an overall estimate of both functional cooperativity
with ACh (αβ) and the intrinsic efficacy of the allosteric ligand.
Values of α or β > 1 describe a positive modulatory effect upon
ACh, whereas values between 0 and 1 describe a negative
modulatory effect. It should be noted that because the
logarithms of affinity and cooperativity values are normally
distributed, whereas the corresponding absolute (antilogar-
ithms) are not,28 all statistical comparisons were performed on
the logarithmic values (Table 1).
As described before, 1 displays a significant 13-fold higher
affinity (KB = 1.3 μM) for the M1 mAChR as compared to
BQCA. In addition, in comparison to BQCA, 1 displays a 12-
fold increase in binding cooperativity (α = 692) and a 5-fold
increase in functional cooperativity (αβ = 370) with ACh
(Figure 2, Table 1). Finally, 1 displays superior intrinsic efficacy
compared to BQCA with a 15-fold increase in τB (τB = 3).
Complete deletion of the peripheral fused benzene ring, as in 2,
caused a 40-fold decrease in affinity (KB = 52) and an 8-fold
decrease in binding cooperativity with ACh (α = 91). However,
no change in intrinsic efficacy was observed (τB = 4). Further
deletion of the 4-(1-methylpyrazole-4-yl) substitution of the
(pyridin-3-yl)methyl pendant group to give 3, caused no
change in affinity relative to 2, but caused a significant 9-fold
loss of binding cooperativity, a significant 11-fold loss of
functional cooperativity, and a complete loss of observed
intrinsic efficacy. The addition of a methyl group at the 8- or 7-
positions of the quinazolin-4(3H)-one core (compounds 4 and
5, respectively) caused a significant 5- to 7-fold increase in
affinity compared to 2, but with no significant change in
cooperativity with ACh. While methyl substitution at the 8-
position (4) resulted in similar intrinsic activity relative to 2,
the addition of methyl at the 7-position (5) caused a significant
4-fold decrease in intrinsic efficacy (Figure 2). The addition of a
methyl group at both the 7- and 8-positions in compound 6
caused a 7-fold increase in affinity, and no significant change in
cooperativity with ACh or intrinsic efficacy relative to 2. Indeed
this analogue displayed a 2-fold higher intrinsic efficacy than 1.
Together these data reveal that the benzo[h]quinazolin-4(3H)-
one core is an important determinant of the affinity of 1.
Deletion of the fused benzene ring of this core (2) was
associated with a significant loss of affinity that was partly
rescued with the addition of methyl groups in the 7 and/or 8
position of a quinazolin-4(3H)-one core (5 and 6). Indeed,
compound 5 displayed no significant difference in affinity
compared to 1 (p > 0.05, one-way ANOVA with Tukey’s post-
test). In our recent SAR study,19 we observed that the
replacement of the N-alkyl group of the quinolone core of
BQCA modulated ligand affinity. In particular, changing the N-
(4-methoxy)benzyl group to N-(4-phenyl)benzyl tended to
improve affinity for the receptor without improving cooperative
binding with ACh. In contrast, the absence of a 4-(1-
methylpyrazole-4-yl) substituent from the (pyridin-3-yl)methyl
pendant group of 2 (with a quinazolin-4(3H)-one core) had no
effect upon affinity but instead caused a decrease in intrinsic
activity (compare compounds 2 and 3). However, when two
methyl groups were present on the quinazolin-4(3H)-one core,
the absence of the 4-(1-methylpyrazole-4-yl) moiety caused no
change in affinity, cooperativity or intrinsic activity (6
compared to 7, Table 1).
To further explore the role of the (pyridin-3-yl)methyl
pendant group in the determination of the activity of 1, we
characterized the synthetic intermediates 10 and 11 and
synthetic byproduct 12. Replacement of the (6-(1-methyl-1H-
pyrazol-4-yl)pyridin-3-yl)methyl group with a bromo-substitu-
ent (10) caused an 8-fold loss of affinity, a 170-fold and 70-fold
loss in binding and functional cooperativity with ACh,
respectively. In addition, a complete loss of intrinsic efficacy
was observed. Compound 12, which possessed an unsub-
stituted (pyridin-3-yl)methyl group, displayed an affinity for the
M1 mAChR that was not significantly different from 1 and had
similar binding and functional cooperativity with ACh (α = 223,
αβ = 436). Furthermore, 12 displayed 5-fold higher intrinsic
efficacy than 1 (τB = 14). In contrast, the (6-chloropyridin-3-
yl)methyl derivative 11 displayed 66-fold lower affinity than 1,
140-fold and 30-fold lower binding and functional cooperativity
with ACh and 8-fold lower intrinsic efficacy. Together these
data illustrate the importance of the benzylic pendant group for
the activity of 1. In contrast to our findings with BQCA, we
found that this moiety was not only important for affinity, but
also for the cooperativity with ACh and intrinsic efficacy
displayed by 1. However, removal of the 4-(1-methylpyrazole-
4-yl) substitution of the (pyridin-3-yl)methyl pendant group of
1 was tolerated both in terms of affinity, cooperativity with ACh
and intrinsic efficacy.
The pyrido[2,3-d]pyrimidin-4(3H)-one analogue (8: α = 69,
αβ = 66) displayed attenuated binding and functional
cooperativity with ACh relative to compound 2 (2: α = 91,
αβ = 195). Finally, the thieno[2,3-d]pyrimidin-4(3H)-one 9
showed negligible binding cooperativity with ACh.
CONCLUSIONS
■
We have recently reported that 3-((1S,2S)-2-hydroxycyclohex-
yl)-6-((6-(1-methyl-1H-pyrazol-4-yl)pyridin-3yl)methyl)benzo-
[h]quinazolin-4(3H)-one (1), while structurally related to
BQCA, is a significantly more potent PAM of the M1
mAChR, driven both by an increased affinity for the M1
mAChR and an increased level of positive cooperativity with
ACh.21 In addition, 1 displays higher intrinsic efficacy than
BQCA. Furthermore, we recently proposed a binding mode of
1 within the M1 mAChR that is similar to that predicted for
BQCA.21 In this study, we wanted to explore the structural
determinants that underlie the activity of 1 as a PAM of the M1
G
DOI: 10.1021/acschemneuro.6b00018
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ACS Chemical Neuroscience
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following gradient was used with a Poroshell 120 EC-C18 50 × 3.0
mm 2.7 μm column, and a flow rate of 0.5 mL/min and total run time
of 5 min; 0−1 min 95% buffer A and 5% buffer B, from 1 to 2.5 min up
to 0% buffer A and 100% buffer B, held at this composition until 3.8
min, 3.8−4 min 95% buffer A and 5% buffer B, held until 5 min at this
composition. Mass spectra were acquired in positive and negative ion
mode with a scan range of 100−1000 m/z. UV detection was carried
out at 214 and 254 nm. All retention times (tR) are quoted in minutes.
High resolution mass spectra (HRMS) were obtained from a Waters
LCT Premier XE (TOF) mass spectrometer fitted with an ESI ion
source, coupled to a 2795 Alliance Separations Module.
Preparative HPLC was performed using an Agilent 1260 infinity
coupled with a binary preparative pump and Agilent 1260 FC-PS
fraction collector, using Agilent OpenLAB CDS software (Rev
C.01.04), and an Altima 5 μM C8 22 × 250 mm column. The
following buffers were used: buffer A, H2O; buffer B, MeCN, with
sample being run at a gradient of 5% buffer B to 100% buffer B over 20
min, at a flow rate of 20 mL/min All screening compounds were of
>95% purity unless stated otherwise.
General Procedure A: HCTU-Mediated Amide Bond Formation.
Carboxylic acid (1 equiv), HCTU (1.1 equiv), and amine or amine salt
(1.1 equiv) were dispersed or dissolved in DMF (∼2 mL/mmol) at
RT. To this was added DIPEA (2.5 eq, for amine salts, an additional
1.0 equiv per salt form was also added), and the mixture allowed to stir
at RT overnight. LC-MS analysis was used to confirm reaction
completion. The mixture was diluted with water/sat. NaHCO3(aq) (1:1,
∼20 mL/mmol) and stirred for 30 min at RT. Where a solid
precipitate formed, this was collected by filtration (vacuum) and
washed with water. Where no solid could be isolated in this manner,
the aqueous slurry was extracted with EtOAc (3 times) and the
combined organic layers washed with brine, then concentrated under
reduced pressure. Where necessary, further purification was carried out
by FCC.
General Procedure B: Negishi Coupling of Aryl Bromides with (2-
Chloro-5-pyridylmethyl)zinc Chloride. Aryl bromide (1.0 equiv) was
dissolved in dry THF (2 mL/mmol), under an atmosphere of
nitrogen, and degassed for 5 min under a stream of nitrogen.
Pd(P(tBu)3)2 (0.03 equiv) was added and then vessel was evacuated
and refilled with nitrogen, before cooling to 0 °C over an ice bath. A
solution of 2-chloro-5-pyridylmethyl)zinc chloride (0.5 M in THF,
1.25 equiv) was added in a dropwise fashion, and stirring continued
over the ice bath for a further 5 min, before allowing the mixture to
warm to RT. Reactions were monitored by LC-MS analysis and
generally left to stir overnight. To quench, the mixture was cooled to 0
°C over an ice bath and a small amount of water added with care. The
quenched mixture was diluted with water, then washed three times
with equal volumes of EtOAc. The combined organic layers were
washed with brine, before drying over MgSO4, and concentrating
under reduced pressure. The crude product was further purified by
FCC (eluent EtOAc/PE 50:50 to 100:0).
mAChR. We have previously demonstrated that changing the
N-alkyl group of the quinolone core of BQCA modulated
ligand affinity but not cooperativity with ACh.19 In this study,
we demonstrate that the (pyrid-3-yl)methyl pendant group of 1
is not only important for affinity, but has an additional role in
determining cooperativity with ACh. In addition, we found that
removal of the 1-methyl-1H-pyrazol-4-yl moiety of 1 was well
tolerated in terms of both affinity and cooperativity with ACh
and generated a PAM (7) with superior intrinsic efficacy. We
paid particular attention to the importance of the tricyclic
scaffold of compound 1 for the activity of the molecule.
Complete deletion of the peripheral fused benzene ring caused
a significant decrease in affinity and binding cooperativity with
ACh, but no change in intrinsic activity. However, this loss of
affinity was partially rescued with the addition of methyl groups
in the 7- and/or 8-position of the quinazolin-4(3H)-one core
(compounds 4, 5 and 6). Indeed, compound 5 with the
addition of a methyl group in the 8-position displayed no
significant difference in affinity for the M1 mAChR compared to
1, but lower intrinsic activity (Figure 2). The addition of methyl
groups at the 7- and 8-positions (6) maintained this affinity and
rescued intrinsic efficacy. These results demonstrate that the
tricyclic benzo[h]quinazolin-4(3H)-one core could be replaced
with a quinazolin-4(3H)-one core. This may be important,
given the precedence of polyaromatic heterocycles as DNA
chelators. In addition, the quinazolin-4(3H)-one core repre-
sents a novel scaffold to explore further M1 mAChR PAMs with
improved physicochemical properties.
METHODS
■
Synthesis of Compounds. Chemicals and solvents were
purchased from standard suppliers and used without further
purification. Davisil silica gel (40−63 μm), for flash column
chromatography (FCC), was supplied by Grace Davison Discovery
Sciences (Victoria, Australia), and deuterated solvents were purchased
from Cambridge Isotope Laboratories, Inc. (distributed by Novachem
PTY. Ltd., Victoria, Australia).
Unless otherwise stated, reactions were carried out at ambient
temperature. Reactions were monitored by thin layer chromatography
on commercially available precoated aluminum-backed plates (Merck
Kieselgel 60 F254). Visualization was by examination under UV light
(254 and 366 nm). General staining was carried out with KMnO4 or
phosphomolybdic acid. A solution of Ninhydrin (in ethanol) was used
to visualize primary and secondary amines. All organic extracts
collected after aqueous workup procedures were dried over anhydrous
MgSO4 or Na2SO4 before gravity filtering and evaporation to dryness.
Organic solvents were evaporated in vacuo at ≤40 °C (water bath
temperature). Purification using preparative layer chromatography
(PLC) was carried out on Analtech preparative TLC plates (200 mm
× 200 mm × 2 mm).
General Procedure C: Suzuki Coupling of Substituted 2-
Chloropyridines with 1-Methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxa-
borolan-2-yl)-1H-pyrazole. Substituted 2-chloropyridine (1.0 equiv)
and 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyra-
zole (1.5 equiv) were dispersed in 1 M Na2CO3(aq)/THF (1:3, ∼ 10
mL/mmol) in a 10 mL microwave tube. The mixture was sonicated for
5 min, then degassed under a stream of nitrogen. PdCl2(PPh3)2 (0.1
equiv) was added, and the tube sealed, before heating (hot plate) at
100 °C for 2 h. The mixture was cooled to RT, then diluted with water
(20 mL), before extracting with EtOAc (3 × 20 mL). The combined
organic extracts were washed with brine (20 mL), then concentrated
under reduce pressure. The crude product was purified by normal
phase silica chromatography as specified under each monologue.
3-((1S,2S)-2-Hydroxycyclohexyl)-6-((6-(1-methyl-1H-pyrazol-4-
yl)pyridin-3-yl)methyl)quinazolin-4(3H)-one (2) and 3-((1S,2S)-2-
Hydroxycyclohexyl)-6-(pyridin-3-ylmethyl)quinazolin-4(3H)-one (3).
6-((6-Chloropyridin-3-yl)methyl)-3-((1S,2S)-2-hydroxycyclohexyl)-
quinazolin-4(3H)-one (33) (70 mg, 0.19 mmol) underwent Suzuki
coupling according to General Procedure C. The crude product was
purified by PLC (MeOH/EtOAc 6:94, plate run three times). The
1H NMR and 13C NMR spectra were recorded on a Bruker Avance
Nanobay III 400 MHz Ultrashield Plus spectrometer at 400.13 and
100.62 MHz, respectively. Chemical shifts (δ) were recorded in parts
per million (ppm) with reference to the chemical shift of the
deuterated solvent. Coupling constants (J) and carbon−fluorine
coupling constants (JCF) were recorded in Hz, and the significant
multiplicities described by singlet (s), doublet (d), triplet (t),
quadruplet (q), broad (br), multiplet (m), doublet of doublets (dd),
and doublet of triplets (dt). Spectra were assigned using appropriate
COSY, distortionless enhanced polarization transfer (DEPT), HSQC
and HMBC sequences. Specific optical rotation was determined using
a Jasco P-2000 polarimeter.
LC-MS were run to verify reaction outcome and purity using
following system: Agilent 6120 Series Single Quad coupled to an
Agilent 1260 Series HPLC. The following buffers were used; buffer A,
0.1% formic acid in H2O; buffer B, 0.1% formic acid in MeCN. The
H
DOI: 10.1021/acschemneuro.6b00018
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higher running band was found to be dehalogenated starting material:
(s, 1H), 3.82−2.86 (m, 1H), 2.52 (s, 3H), 2.33−2.13 (m, 4H), 2.03−
1.69 (m, 4H), 1.61−1.33 (m, 3H); 13C NMR (CDCl3) δ 162.3, 148.3,
146.1, 145.1, 143.3, 142.1, 137.7, 136.6, 134.9, 129.0, 125.3, 124.2,
119.7, 72.0, 37.7, 35.7, 31.0, 25.5, 24.6, 17.0, 13.7; m/z MS (TOF ES+)
364.2 [MH]+; HRMS - C22H26N3O2 [MH]+ calcd 364.2025; found
364.2031; LC-MS tR: 2.90 min; [α]2D7 = +13.87° (0.18, DMSO).
The lower running band was found to be 3-((1S,2S)-2-
hydroxycyclohexyl)-7,8-dimethyl-6-((6-(1-methyl-1H-pyrazol-4-yl)-
pyridin-3-yl)methyl)quinazolin-4(3H)-one (6), isolated as 10 mg
3-((1S,2S)-2-Hydroxycyclohexyl)-6-(pyridin-3-ylmethyl)quinazolin-
1
4(3H)-one (3), isolated as 9 mg (14%) of a white solid. H NMR
(CDCl3) δ 8.62−8.41 (m, 2H), 8.09 (s, 1H), 8.04 (d, J = 1.8 Hz, 1H),
7.68 (d, J = 7.9 Hz, 1H), 7.59 (d, J = 8.3 Hz, 1H), 7.49 (dd, J = 8.4/2.0
Hz, 1H), 7.45−7.32 (m, 1H), 4.74−4.40 (m, 1H), 4.13 (s, 2H), 4.08−
3.85 (m, 1H), 2.30−2.17 (m, 1H), 2.03−1.77 (m, 4H), 1.58−1.35 (m,
3H); 13C NMR (CDCl3) δ 161.8, 147.2, 146.4, 145.2, 144.9, 139.4,
138.2, 137.6, 135.1, 128.1, 126.7, 124.7, 122.2, 70.4, 38.8, 35.7, 31.0,
25.4, 24.5; m/z MS (TOF ES+) 336.2 [MH]+; HRMS - C20H22N3O2
1
(15%) of white solid. H NMR (CDCl3) δ 8.20 (s, 1H), 8.08−7.93
[MH]+ calcd 336.1712; found 336.1716; LC-MS tR: 2.76 min; [α]2D7
+5.10° (0.11, DMSO).
=
(m, 2H), 7.83 (s, 1H), 7.81 (s, 1H), 7.48−7.32 (m, 2H), 4.35 (s, 1H),
4.05 (s, 2H), 4.01−3.73 (m, 4H), 2.43 (s, 3H), 2.17 (s, 3H), 2.07 (d, J
= 10.1 Hz, 1H), 1.97−1.58 (m, 4H), 1.48−1.21 (m, 3H); 13C NMR
(CDCl3) δ 162.1, 151.6, 146.8, 144.9, 143.5, 142.2, 139.5, 137.6, 136.6,
134.6, 133.9, 129.9, 124.9, 120.6, 119.5, 112.1, 70.6, 38.9, 37.1, 35.1,
31.0, 25.2, 24.3, 16.7, 13.4; m/z MS (TOF ES+) 444.3 [MH]+; HRMS
- C26H30N5O2 [MH]+ calcd 444.2400; found 444.2411; LC-MS tR:
3.01 min; [α]2D7 = +6.44° (0.16, DMSO).
The lower running band was found to be 3-((1S,2S)-2-
hydroxycyclohexyl)-6-((6-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)-
methyl)quinazolin-4(3H)-one (2, isolated as 42 mg (53%) of a glassy
solid. 1H NMR (CDCl3) δ 8.39 (d, J = 1.7 Hz, 1H), 8.08 (s, 1H), 8.05
(s, 1H), 8.00 (d, J = 1.6 Hz, 1H), 7.88 (s, 1H), 7.53 (d, J = 8.3 Hz,
1H), 7.51−7.42 (m, 2H), 7.39 (d, J = 8.2 Hz, 1H), 4.51 (s, 1H), 4.06−
3.85 (m, 6H), 2.29−2.15 (m, 1H), 2.01−1.73 (m, 4H), 1.58−1.33 (m,
3H); 13C NMR (CDCl3) δ 161.8, 149.5, 148.1, 146.2, 144.6, 139.0,
138.6, 137.7, 135.1, 133.6, 129.6, 127.8, 126.5, 122.1, 120.0, 113.6,
71.6, 39.4, 38.5, 35.6, 31.0, 25.4, 24.6; m/z MS (TOF ES+) 416.3
[MH]+; HRMS - C24H26N5O2 [M-Na]+ calcd 438.1906; found
438.1872; LC-MS tR: 2.85 min; [α]2D7 = −1.04° (0.26, DMSO).
3-((1S,2S)-2-Hydroxycyclohexyl)-8-methyl-6-((6-(1-methyl-1H-
pyrazol-4-yl)pyridin-3-yl)methyl)quinazolin-4(3H)-one (4). 6-((6-
Chloropyridin-3-yl)methyl)-3-((1S,2S)-2-hydroxycyclohexyl)-8-meth-
ylquinazolin-4(3H)-one (34) (200 mg, 0.52 mmol) underwent Suzuki
coupling according to General Procedure C. The crude product was
purified by FCC (eluent MeOH/DCM 0:100 to 10:90) to give 214
mg (96%) of pale yellow glassy solid. 1H NMR (DMSO-d6) δ 8.46 (d,
J = 1.6 Hz, 1H), 8.38 (s, 1H), 8.22 (s, 1H), 7.93 (d, J = 0.6 Hz, 1H),
7.84 (d, J = 1.6 Hz, 1H), 7.61 (dd, J = 8.1/2.3 Hz, 1H), 7.58 (dd, J =
1.9/0.7 Hz, 1H), 7.55 (dd, J = 8.1/0.7 Hz, 1H), 4.90 (d, J = 5.4 Hz,
1H), 4.39 (s, 1H), 4.05 (s, 2H), 3.93 (s, 1H), 3.86 (s, 3H), 2.50 (s,
3H), 2.14−1.50 (m, 5H), 1.50−1.21 (m, 3H); 13C NMR (DMSO-d6)
δ 160.6, 149.8, 149.4, 144.9, 144.4, 139.1, 137.0, 136.9, 135.4, 135.2,
133.5, 129.6, 123.2, 122.7, 121.6, 119.1, 69.0, 38.7, 37.4, 35.2, 30.3,
25.0, 24.0, 17.0; m/z MS (TOF ES+) 430.3 [MH]+; HRMS -
C25H28N5O2 [MH]+ calcd 430.2243; found 430.2248; LC-MS tR: 3.29
min; [α]2D7 = +6.93° (0.29, DMSO).
3-((1S,2S)-2-Hydroxycyclohexyl)-6-((6-(1-methyl-1H-pyrazol-4-
yl)pyridin-3-yl)methyl)pyrido[2,3-d]pyrimidin-4(3H)-one (8). 6-((6-
Chloropyridin-3-yl)methyl)-3-((1S,2S)-2-hydroxycyclohexyl)pyrido-
[2,3-d]pyrimidin-4(3H)-one (40) (33 mg, 0.09 mmol) underwent
Suzuki coupling according to General Procedure C. The crude product
was purified by PLC (MeOH/EtOAc 7:93, plate run six times) to give
9 mg (24%) of white solid. 1H NMR (CD3OD) δ 8.88 (d, J = 2.4 Hz,
1H), 8.52 (s, 1H), 8.49−8.40 (m, 2H), 8.12 (s, 1H), 7.99 (d, J = 0.5
Hz, 1H), 7.70 (dd, J = 8.2/2.3 Hz, 1H), 7.62 (d, J = 8.2 Hz, 1H), 4.44
(s, 1H), 4.23 (s, 2H), 4.18−3.98 (m, 1H), 3.94 (s, 3H), 2.29−1.69 (m,
5H), 1.56−1.37 (m, 3H); 13C NMR (CD3OD) δ 163.1, 157.4, 157.2,
151.6, 150.4, 150.3, 139.3, 138.5, 137.3, 137.1, 134.6, 130.9, 123.9,
121.5, 118.0, 71.1, 39.1, 36.2, 36.0, 30.8, 26.4, 25.4; m/z MS (TOF
ES+) 417.3 [MH]+; HRMS- C23H25N6O2 [MH]+ calcd 417.2039;
found 417.2025; LC-MS tR: 3.16 min; [α]2D7 = −4.86° (0.20, DMSO).
6-([1,1′-Biphenyl]-4-ylmethyl)-3-((1S,2S)-2-hydroxycyclohexyl)-
thieno[2,3-d]pyrimidin-4(3H)-one (9). 5-([1,1′-Biphenyl]-4-ylmeth-
yl)-2-amino-N-((1S,2S)-2-hydroxycyclohexyl)thiophene-3-carboxa-
mide (49) (91 mg, 0.22 mmol) was dispersed in formamide (20 mL),
before heating at 180 °C for 4 h. LC-MS analysis indicated conversion
was complete. TLC analysis (EtOAc/PE 1:1, plate run twice)
indicated the starting material and product both had an Rf ∼ 0.4,
with only the starting material staining positive with ninhydrin. The
mixture was cooled, then poured onto ice/water, and the resulting
precipitate collected by filtration (vacuum), and further washed with
water. The crude product was purified by FCC (eluent EtOAc/PE
0:100 to 60:40, wet load in DCM), to give 52 mg (56%) of an off-
white solid. 1H NMR (DMSO-d6) δ 8.39 (s, 1H), 7.75−7.56 (m, 4H),
7.50−7.42 (m, 2H), 7.42−7.31 (m, 3H), 7.21 (s, 1H), 4.92 (s, 1H),
4.41 (s, 1H), 4.25 (s, 2H), 3.91 (s, 1H), 2.15−1.50 (m, 4H), 1.48−
1.11 (m, 4H); 13C NMR (DMSO-d6) δ 162.0, 156.8, 145.9, 142.1,
139.8, 138.9, 138.6, 129.2, 128.9, 127.4, 127.0, 126.6, 123.7, 119.6,
69.1, 35.3, 35.2, 30.6, 25.1, 23.9; m/z MS (TOF ES+) 417.3 [MH]+;
HRMS - C25H25N2O2S [MH]+ calcd 417.1637; found 417.1613; LC-
MS tR: 3.98 min; [α]D27 = +17.77° (0.35, DMSO).
3-((1S,2S)-2-Hydroxycyclohexyl)-7-methyl-6-((6-(1-methyl-1H-
pyrazol-4-yl)pyridin-3-yl)methyl)quinazolin-4(3H)-one (5). 6-((6-
Chloropyridin-3-yl)methyl)-3-((1S,2S)-2-hydroxycyclohexyl)-7-meth-
ylquinazolin-4(3H)-one (35) (328 mg, 0.97 mmol) underwent Suzuki
coupling according to General Procedure C. The crude product was
purified by FCC (eluent EtOAc/PE 50:50 to 100:0) to give 334 mg
1
(90%) of an off-white solid. H NMR (DMSO-d6) δ 8.38 (d, J = 1.6
Hz, 1H), 8.34 (s, 1H), 8.23 (s, 1H), 7.94 (d, J = 0.7 Hz, 1H), 7.84 (s,
1H), 7.56 (dd, J = 8.1/0.6 Hz, 1H), 7.53−7.42 (m, 2H), 4.90 (d, J =
5.4 Hz, 1H), 4.37 (s, 1H), 4.11 (s, 2H), 3.94 (s, 1H), 3.87 (s, 3H),
2.39 (s, 3H), 2.11−1.49 (m, 5H), 1.46−1.13 (m, 3H); 13C NMR
(DMSO-d6) δ 160.2, 149.8, 149.6, 146.2, 146.0, 143.7, 138.3, 137.0,
136.9, 132.3, 129.3, 127.8, 126.2, 122.6, 119.1, 119.0, 69.0, 38.7, 35.2,
35.0, 29.9, 25.1, 24.0, 19.7; m/z MS (TOF ES+) 430.3 [MH]+; HRMS
- C25H28N5O2 [MH]+ calcd 430.2243; found 430.2244; LC-MS tR:
3.24 min; [α]2D7 = +6.11° (0.67, DMSO).
Methyl 2-Amino-3,4-dimethylbenzoate (15).12 2-Amino-3,4-dime-
thylbenzoic acid (14) (4.92 g, 29.8 mmol) and conc. H2SO4 (3 mL)
were dissolved in MeOH (50 mL) and boiled under reflux for 48 h.
LC-MS analysis indicated approximately half of the starting material
had been converted. Additional H2SO4 (1 mL) was added, and heating
continued. After a total of 6 days, the mixture was cooled to RT, and
concentrated under reduced pressure. The residue was neutralized
with sat. NaHCO3(aq), and the resulting precipitate collected by
filtration (vacuum), and washed with water. After drying, 4.452 g
(83%) of an off-white solid was obtained. 1H NMR (CDCl3) δ 7.69 (d,
J = 8.2 Hz, 1H), 6.58 (d, J = 8.2 Hz, 1H), 6.18 (s, 2H), 3.86 (s, 3H),
2.30 (s, 3H), 2.13 (s, 3H); 13C NMR (CDCl3) δ 169.2, 147.8, 142.8,
128.4, 122.0, 119.4, 109.5, 51.7, 21.3, 13.0; m/z MS (TOF ES+) 180.2
[MH]+; LC-MS tR: 3.48 min.
3-((1S,2S)-2-Hydroxycyclohexyl)-7,8-dimethyl-6-((6-(1-methyl-
1H-pyrazol-4-yl)pyridin-3-yl)methyl)quinazolin-4(3H)-one (6) and
3-((1S,2S)-2-Hydroxycyclohexyl)-7,8-dimethyl-6-(pyridin-3-
ylmethyl)quinazolin-4(3H)-one (7). 6-((6-Chloropyridin-3-yl)-
methyl)-3-((1S,2S)-2-hydroxycyclohexyl)-7,8-dimethylquinazolin-
4(3H)-one (36) (61 mg, 0.15 mmol) underwent Suzuki coupling
according to General Procedure C. The crude product was purified by
PLC (MeOH/EtOAc 5:95, plate run three times). The higher running
band was found to be dehalogenated starting material: 3-((1S,2S)-2-
Hydroxycyclohexyl)-7,8-dimethyl-6-(pyridin-3-ylmethyl)quinazolin-
1
4(3H)-one (7), isolated as 5 mg (9%) of an off-white solid. H NMR
Methyl 2-Amino-5-bromo-3,4-dimethylbenzoate hydrobromide
(16). Methyl 2-amino-3,4-dimethylbenzoate (15) (3.40 g, 19.0 mmol)
was dissolved in 1,4-dioxane/CCl4 (1:1, 100 mL), and cooled to 0 °C
(CDCl3) δ 8.45 (s, 2H), 8.10 (s, 1H), 7.90 (s, 1H), 7.46 (d, J = 7.9 Hz,
1H), 7.33−7.19 (m, 1H), 4.53 (s, 1H), 4.13 (s, J = 7.5 Hz, 2H), 3.97
I
DOI: 10.1021/acschemneuro.6b00018
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ACS Chemical Neuroscience
Research Article
over an ice bath, after wrapping the flask in aluminum foil to exclude
light. To the cooled mixture, was added a solution of bromine (3.03 g,
18.97 mmol, 1.0 equiv) in 1,4-dioxane/CCl4 (1:1, 20 mL) in a
dropwise fashion. The mixture was stirred for 2 h at 0 °C, before
addition of Et2O, and collection of the resulting precipitate by filtration
(vacuum). After washing with further Et2O and drying, 5.96 g (93%)
The mixture was stirred at RT for 1 h, then diluted with Et2O. The
resulting precipitate was collected by filtration (vacuum) and washed
with Et2O. After drying, 4.07 g (95%) of a pale beige solid was
1
obtained as the hydrobromide salt. H NMR (DMSO-d6) δ 9.19 (s,
3H), 7.68 (dd, J = 2.5/0.4 Hz, 1H), 7.33 (dd, J = 2.5/0.8 Hz, 1H), 2.10
(s, 3H); 13C NMR (DMSO-d6) δ 168.9, 148.8, 136.4, 130.6, 126.4,
111.0, 104.8, 17.3; m/z MS (TOF ES+) 230.0 [MH]+; LC-MS tR: 3.62
min.
1
of an off-white solid was obtained. H NMR (DMSO-d6) δ 7.79 (s,
1H), 4.40 (s, 3H), 3.79 (s, 3H), 2.33 (s, 3H), 2.10 (s, 3H); 13C NMR
(DMSO-d6) δ 167.2, 148.7, 140.7, 130.7, 123.8, 109.7, 108.3, 51.7,
20.5, 14.4; m/z MS (TOF ES+) 258.0 [MH]+; LC-MS tR: 3.71 min.
Ethyl 2-Amino-4-methylbenzoate (18).10 2-Amino-4-methylben-
zoic acid (17) (2.10 g, 13.9 mmol) was dissolved in EtOH (50 mL)
with conc. H2SO4 (1 mL), before boiling under reflux for 2 h. LC-MS
analysis indicated little progression, so further conc. H2SO4 (1 mL)
was added, and heating continued for 65 h. The mixture was cooled,
then concentrated under reduced pressure. The resulting residue was
neutralized with sat. NaHCO3(aq), then extracted with DCM (3 × 30
mL). The combined organic layers were concentrated to give a brown
oil, which was further purified by FCC (eluent EtOA/PE 0:100 to
2-Amino-5-bromo-4-methylbenzoic Acid (23).30 Ethyl 2-acetami-
do-5-bromo-4-methylbenzoate (20) (672 mg, 2.24 mmol) was
dissolved in 5 M NaOH(aq) (20 mL) and EtOH (30 mL). The
resulting mixture was heated at 90 °C under a reflux condenser for 20
h. LC-MS analysis at this indicated hydrolysis was complete. The
mixture was cooled to RT, then concentrated under reduced pressure
to remove EtOH. The aqueous slurry was acidified with 2 M HCl(aq)
,
to pH 2 resulting in formation of a precipitate. This was collected by
filtration (vacuum), then washed with water and dried, to give 415 mg
1
(81%) of a beige solid. H NMR (DMSO-d6) δ 7.77 (s, 1H), 11.41−
6.62 (m, 2H), 6.72 (d, J = 0.6 Hz, 1H), 2.22 (s, 3H); 13C NMR
(DMSO-d6) δ 168.3, 150.7, 142.8, 133.7, 118.3, 109.4, 107.8, 22.7; m/
z MS (TOF ES+) 230.1 [MH]+; LC-MS tR: 3.56 min.
1
10:90), to give 1.87 g (75%) of pale yellow oil. H NMR (CDCl3) δ
7.85 (d, J = 8.1 Hz, 1H), 6.96 (s, 1H), 6.77 (d, J = 8.1 Hz, 1H), 4.36
(q, J = 7.2 Hz, 2H), 2.34 (s, 3H), 1.39 (t, J = 7.1 Hz, 3H); 13C NMR
(101 MHz, CDCl3) δ 168.2, 147.7, 145.2, 131.3, 119.8, 118.5, 110.5,
2-Amino-5-bromo-3,4-dimethylbenzoic Acid (24).13 Methyl 2-
amino-5-bromo-3,4-dimethylbenzoate hydrobromide (16) (5.93 g,
17.5 mmol) was dispersed in THF/water (1:1, 100 mL), and the flask
atmosphere purged with nitrogen. To this was added LiOH·H2O (3.67
g, 87.4 mmol, 5.0 equiv), and the mixture stirred at RT for 24 h. LC-
MS analysis after this time indicated complete hydrolysis had occurred.
The mixture was concentrated under reduced pressure to remove THF
and MeOH, and then acidified with excess 2 M HCl(aq). The resultant
precipitate was collected by filtration (vacuum), and washed with
water, before allowing to dry on the filter bed overnight. This was then
taken up in EtOAc (300 mL) and washed with brine (80 mL), before
drying over MgSO4. Concentration of dried organic layer under
1
60.6, 21.8, 14.5; H NMR; m/z MS (TOF ES+) C10H14NO2 180.1
[MH]+; LC-MS tR: 3.82 min.
Ethyl 2-Acetamido-4-methybenzoate (19)..10,11 Ethyl 2-amino-4-
methylbenzoate (18) (1.87 g, 10.4 mmol) and TEA (1.74 mL, 12.5
mmol, 1.2 equiv) were dissolved in DCM (50 mL) and cooled to 0 °C
under an atmosphere of nitrogen. Acetyl chloride (0.81 mL, 11.5
mmol, 1.1 equiv) was added and then the mixture was allowed to
warm to RT, before stirring overnight. LC-MS analysis indicated
incomplete conversion after this time, so further TEA (1.74 mL, 12.5
mmol, 1.2 equiv) and acetyl chloride (0.81 mL, 11.5 mmol, 1.1 equiv)
were added and stirring continued for a further 4 h. The mixture was
washed with water (50 mL), then sat. NaHCO3(aq) (50 mL), then
concentrated under reduced pressure, to give 2.56 g of yellow solid
1
reduced pressure gave 4.00 g (94%) of a pale brown solid. H NMR
(DMSO-d6) δ 7.78 (br s, 2H), 7.78 (s, 1H), 2.32 (s, 3H), 2.09 (s, 3H);
13C NMR (DMSO-d6) δ 168.9, 149.0, 140.3, 131.2, 123.5, 109.3,
1
109.1, 20.4, 14.3; m/z MS (TOF ES+) 244.1 [MH]+; LC-MS tR: 3.33
min.
(quantitative). H NMR (CDCl3) δ 11.10 (s, 1H), 8.53 (s, 1H), 7.92
(d, J = 8.2 Hz, 1H), 6.88 (ddd, J = 8.2/1.7/0.6 Hz, 1H), 4.35 (q, J =
7.1 Hz, 2H), 2.39 (s, 3H), 2.22 (s, 3H), 1.40 (t, J = 7.1 Hz, 3H); 13C
NMR (CDCl3) δ 169.2, 168.5, 145.9, 141.7, 130.8, 123.5, 120.7, 112.6,
61.3, 25.7, 22.3, 14.4; m/z MS (TOF ES+) 176.2 [M-OEt]+; LC-MS
tR: 3.79 min.
2-Amino-5-bromo-N-((1S,2S)-2-hydroxycyclohexyl)benzamide
(25). 2-Amino-5-bromobenzoic acid (21) (1.08 g, 5.00 mmol) and
(1S,2S)-2-aminocyclohexanol hydrochloride (834 mg, 1.1 equiv) were
coupled according to General Procedure A, to give 1.21 g (77%) of
precipitate as a yellow solid, requiring no further purification. 1H NMR
(DMSO-d6) δ 8.02 (d, J = 8.1 Hz, 1H), 7.70 (d, J = 2.4 Hz, 1H), 7.24
(dd, J = 8.8/2.4 Hz, 1H), 6.64 (d, J = 8.8 Hz, 1H), 6.47 (s, 2H), 4.65
(d, J = 5.2 Hz, 1H), 3.63−3.49 (m, 1H), 3.46−3.36 (m, 1H), 2.02−
1.72 (m, 2H), 1.71−1.43 (m, 2H), 1.19 (d, J = 6.6 Hz, 4H); 13C NMR
(DMSO-d6) δ 167.3, 148.6, 133.8, 130.5, 118.2, 117.2, 105.0, 71.0,
54.9, 34.6, 31.3, 24.6, 24.2; m/z MS (TOF ES+) 313.1 [MH]+; LC-MS
tR: 3.17 min.
Ethyl 2-Acetamido-5-bromo-4-methylbenzoate (20). Ethyl 2-
acetamido-4-methybenzoate (19) (2.55 g, 11.5 mmol) was dissolved
in acetic acid (8 mL) at RT, with stirring. A solution of bromine (1.84
g, 11.5 mmol, 1.0 equiv) in acetic acid (2 mL) was added in a dropwise
fashion. After 48 h of stirring, LC-MS indicated partial progression,
therefore another portion of bromine (0.30 mL, 921 mg, 5.76 mmol,
0.5 equiv) was added. After a further 72 h of stirring, further
progression was evident, with the appearance of a minor peak
indicating formation of the dibromo product. The mixture was stirred
with 5% Na2S2O3(aq) (20 mL) for 30 min, then sat. NaHCO3 (150
mL) added. This was extracted with DCM (3 × 50 mL), and the
combined organic extracts washed further with sat. NaHCO3(aq) (50
mL), before concentration under reduced pressure. Purification of the
crude material was attempted by FCC (eluent DCM), however, both
starting material and product were found to coelute. FCC was
reattempted (eluent DCM/PE 50:50 to 70:30, then 100:0) to give 687
mg of yellow solid (23%, brsm) and a further 995 mg of mixture
containing starting material and desired product. 1H NMR (CDCl3) δ
10.99 (s, 1H), 8.65 (s, 1H), 8.16 (s, 1H), 4.37 (q, J = 7.1 Hz, 2H), 2.43
(s, 3H), 2.22 (s, 3H), 1.42 (t, J = 7.1 Hz, 3H); 13C NMR (101 MHz,
CDCl3) δ 169.2, 167.4, 145.4, 140.7, 134.1, 122.3, 117.6, 114.5, 61.8,
25.7, 23.8, 14.3; m/z MS (TOF ES+) 300.2 [MH]+; LC-MS tR: 4.11
min.
2-Amino-5-bromo-N-((1S,2S)-2-hydroxycyclohexyl)-3-methylben-
zamide (26). 2-Amino-5-bromo-3-methylbenzoic acid hydrobromide
(22) (2.00 g, 6.43 mmol) and (1S,2S)-2-aminocyclohexanol hydro-
chloride (1.07 g, 1.1 equiv) were coupled according to General
Procedure A, to give 2.00 g (95%) of precipitate as a pale yellow solid,
1
requiring no further purification. H NMR (DMSO-d6) δ 8.03 (d, J =
8.1 Hz, 1H), 7.58 (d, J = 2.3 Hz, 1H), 7.22 (d, J = 1.7 Hz, 1H), 6.24 (s,
2H), 4.64 (d, J = 5.2 Hz, 1H), 3.68−3.47 (m, 1H), 3.46−3.34 (m,
1H), 2.07 (s, 3H), 1.98−1.71 (m, 2H), 1.72−1.51 (m, 2H), 1.34−1.06
(m, 4H); 13C NMR (DMSO-d6) δ 167.7, 146.6, 134.1, 128.2, 125.7,
117.2, 105.1, 70.9, 54.9, 34.6, 31.3, 24.5, 24.2, 17.3; m/z MS (TOF
ES+) 327.1 [MH]+; LC-MS tR: 3.62 min.
2-Amino-5-bromo-N-((1S,2S)-2-hydroxycyclohexyl)-4-methylben-
zamide (27). 2-Amino-5-bromo-4-methylbenzoic acid (23) (386 mg,
1.68 mmol) and (1S,2S)-2-aminocyclohexanol hydrochloride (281 mg,
1.1 equiv) were coupled according to General Procedure A, to give
508 mg (92%) of precipitate as a beige solid, requiring no further
purification. 1H NMR (DMSO-d6) δ 7.95 (d, J = 8.0 Hz, 1H), 7.74 (s,
1H), 6.64 (d, J = 0.5 Hz, 1H), 6.43 (s, 2H), 4.62 (d, J = 5.2 Hz, 1H),
3.68−3.45 (m, 1H), 3.45−3.34 (m, 1H), 2.20 (s, 3H), 1.97−1.71 (m,
2-Amino-5-bromo-3-methylbenzoic Acid Hydrobromide (22).29
2-Amino-3-methylbenzoic acid (13) (2.08 g, 13.8 mmol) was
dissolved in acetic acid (8 mL) and stirred at RT. A solution of
bromine (2.20 g, 13.8 mmol, 1.0 equiv) in acetic acid (2 mL) was
added in a dropwise fashion, followed by washings of DCM (5 mL).
J
DOI: 10.1021/acschemneuro.6b00018
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
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2H), 1.70−1.49 (m, 2H), 1.36−1.06 (m, 4H); 13C NMR (DMSO-d6)
δ 167.2, 148.9, 140.0, 131.2, 118.1, 115.0, 108.0, 70.9, 54.83, 34.6, 31.3,
24.5, 24.2, 22.4; m/z MS (TOF ES+) 327.1 [MH]+; LC-MS tR: 3.57
min.
NMR (CDCl3) δ 160.4, 145.6, 145.5, 145.1, 130.3, 128.3, 123.8, 121.1,
72.1, 35.7, 31.0, 25.4, 24.5, 23.8; m/z MS (TOF ES+) 337.1 [MH]+;
LC-MS tR: 3.64 min.
6-Bromo-3-((1S,2S)-2-hydroxycyclohexyl)-7,8-dimethylquinazo-
lin-4(3H)-one (32). 2-Amino-5-bromo-N-((1S,2S)-2-hydroxycyclohex-
yl)-3,4-dimethylbenzamide (28) (1.28 g, 3.75 mmol) was dispersed in
DMF-DMA (12 mL) under an atmosphere of nitrogen, and heated at
85 °C for 6 h, before LC-MS analysis indicated conversion was
complete. The mixture was cooled to RT, and carefully quenched with
water. The resulting precipitate was collected by filtration (vacuum),
washed further with water, and dried to give 1.25 g (95%) of an off-
2-Amino-5-bromo-N-((1S,2S)-2-hydroxycyclohexyl)-3,4-dimethyl-
benzamide (28). 2-Amino-5-bromo-3,4-dimethylbenzoic acid (24)
(1.00 g, 4.10 mmol) and (1S,2S)-2-aminocyclohexanol hydrochloride
(684 mg, 1.1 equiv) were coupled according to General Procedure A,
to give 1.32 g (94%) of precipitate as an off-white solid, requiring no
further purification. 1H NMR (DMSO-d6) δ 7.99 (d, J = 8.0 Hz, 1H),
7.66 (s, 1H), 6.27 (s, 2H), 4.62 (d, J = 5.2 Hz, 1H), 3.66−3.49 (m,
1H), 3.46−3.34 (m, 1H), 2.31 (s, 3H), 2.06 (s, 3H), 1.97−1.73 (m,
2H), 1.73−1.50 (m, 2H), 1.34−1.08 (m, 4H); 13C NMR (DMSO-d6)
δ 167.8, 146.7, 137.5, 128.7, 123.1, 115.4, 109.7, 70.9, 54.9, 34.6, 31.3,
24.5, 24.2, 20.1, 14.3; m/z MS (TOF ES+) 341.1 [MH]+; LC-MS tR:
3.31 min.
6-Bromo-3-((1S,2S)-2-hydroxycyclohexyl)quinazolin-4(3H)-one
(29). 2-Amino-5-bromo-N-((1S,2S)-2-hydroxycyclohexyl)benzamide
(25) (1.18 g, 3.8 mmol) was dispersed in DMF-DMA (12 mL) and
heated, with stirring at 85 °C for 3 h. LC-MS analysis indicated that
the dimethylamine addition product was the major component of the
reaction mixture, with elimination to the desired product progressing
slowly. Further DMF-DMA (5 mL) was added, and the reaction
temperature was increased to 115 °C, with heating continued for 72 h
(progress monitored by LC-MS). The mixture was cooled to RT and
carefully quenched with water (very exothermic), and the resulting
precipitate collected by filtration (vacuum) before washing with water.
After drying under air, the crude precipitate was recrystallized from
EtOH to 767 mg (63%) of a yellow solid. 1H NMR (CDCl3) δ 8.69 (s,
1H), 8.38 (d, J = 2.2 Hz, 1H), 7.83 (dd, J = 8.7/2.3 Hz, 1H), 7.70 (d, J
= 8.7 Hz, 1H), 4.81−4.43 (m, 1H), 4.14−3.91 (m, 1H), 2.30−2.16 (m,
1H), 2.11−1.97 (m, 1H), 1.96−1.65 (m, 3H), 1.63−1.32 (m, 3H); 13C
NMR (CDCl3) δ 160.6, 148.9, 144.7, 137.8, 129.7, 128.7, 123.4, 121.3,
61.5, 35.7, 31.1, 25.4, 24.5; m/z MS (TOF ES+) 323.1 [MH]+; LC-MS
tR: 3.21 min.
1
white solid. H NMR (DMSO-d6) δ 8.46 (s, 1H), 8.14 (s, 1H), 4.93
(d, J = 5.4 Hz, 1H), 4.36 (s, 1H), 3.94 (s, 1H), 2.59 (s, 3H), 2.49 (s,
3H), 2.17−1.54 (m, 5H), 1.52−1.06 (m, 3H); 13C NMR (DMSO-d6)
δ 159.6, 147.9, 144.7, 141.5, 136.0, 126.3, 122.9, 120.8, 68.7, 35.1, 26.8,
25.0, 23.9, 20.4, 14.1; m/z MS (TOF ES+) 351.1 [MH]+; LC-MS tR:
3.51 min.
6-((6-Chloropyridin-3-yl)methyl)-3-((1S,2S)-2-hydroxycyclohexyl)-
quinazolin-4(3H)-one (33). 6-Bromo-3-((1S,2S)-2-
hydroxycyclohexyl)quinazolin-4(3H)-one (29) (170 mg, 0.53 mmol)
underwent Negishi coupling according to General Procedure B, to give
129 mg (66%) of a pale yellow solid. 1H NMR (DMSO-d6) δ 8.40 (d,
J = 2.1 Hz, 1H), 8.37 (s, 1H), 8.01 (d, J = 1.8 Hz, 1H), 7.74 (dd, J =
8.2/2.5 Hz, 1H), 7.70 (dd, J = 8.4/2.1 Hz, 1H), 7.61 (d, J = 8.3 Hz,
1H), 7.44 (dd, J = 8.2/0.5 Hz, 1H), 4.91 (d, J = 5.3 Hz, 1H), 4.39 (s,
1H), 4.15 (s, 2H), 3.94 (s, 1H), 2.12−1.57 (m, 5H), 1.48−1.20 (m,
3H); 13C NMR (DMSO-d6) δ 160.3, 149.9, 148.8, 148.3, 146.0, 140.1,
139.0, 136.1, 134.9, 127.4, 125.7, 124.2, 121.7, 68.9, 36.6, 35.2, 27.6,
25.0, 23.9; m/z MS (TOF ES+) 370.2 [MH]+; LC-MS tR: 3.18 min;
[α]2D7 = +7.78° (0.43, DMSO).
6-((6-Chloropyridin-3-yl)methyl)-3-((1S,2S)-2-hydroxycyclohexyl)-
8-methylquinazolin-4(3H)-one (34). 6-Bromo-3-((1S,2S)-2-hydroxy-
cyclohexyl)-8-methylquinazolin-4(3H)-one (30) (462 mg, 1.37 mmol)
underwent Negishi coupling according to General Procedure B, to give
390 mg (74%) of pale yellow solid. 1H NMR (DMSO-d6) δ 8.40 (d, J
= 2.2 Hz, 1H), 8.39 (s, 1H), 7.85 (d, J = 1.4 Hz, 1H), 7.73 (dd, J =
8.2/2.5 Hz, 1H), 7.58 (s, 1H), 7.44 (d, J = 8.2 Hz, 1H), 4.91 (d, J = 5.4
Hz, 1H), 4.38 (s, 1H), 4.10 (s, 2H), 3.93 (s, 1H), 2.50 (s, 3H), 2.21−
1.53 (m, 5H), 1.51−1.12 (m, 3H); 13C NMR (DMSO-d6) δ 160.5,
149.9, 148.2, 144.9, 144.5, 140.1, 138.4, 136.2, 135.6, 135.2, 124.2,
123.4, 121.6, 68.6, 36.6, 35.2, 30.5, 25.0, 24.0, 17.0; m/z MS (TOF
ES+) 384.2 [MH]+; LC-MS tR: 3.63 min.
6-Bromo-3-((1S,2S)-2-hydroxycyclohexyl)-8-methylquinazolin-
4(3H)-one (30). 2-Amino-5-bromo-N-((1S,2S)-2-hydroxycyclohexyl)-
3-methylbenzamide (26) (1.50 g, 4.58 mmol) was dispersed in
triethylorthoformate (30 mL) under an atmosphere of nitrogen. The
mixture was heated under a reflux condenser, under nitrogen at 100 °C
for 19 h. LC-MS analysis indicated partial conversion had occurred, so
the temperature was increased to 150 °C for 96 h and monitored by
LC-MS. The mixture was cooled to RT before quenching with a small
amount of water (with care). On addition of water, a biphasic mixture
was formed, so EtOAc was added, and the water layer decanted. The
organic layer was then dried over MgSO4, before concentration under
reduced pressure to dryness. The crude residue was purified by FCC
(eluent EtOAc/PE 0:100 to 100:0), to give 996 mg (65%) of a pale
6-((6-Chloropyridin-3-yl)methyl)-3-((1S,2S)-2-hydroxycyclohexyl)-
7-methylquinazolin-4(3H)-one (35). 6-Bromo-3-((1S,2S)-2-hydroxy-
cyclohexyl)-7-methylquinazolin-4(3H)-one (31) (328 mg, 0.97 mmol)
underwent Negishi coupling according to General Procedure B, to give
1
334 mg (90%) of an off-white solid. H NMR (DMSO-d6) δ 8.35 (s,
1H), 8.31 (d, J = 2.1 Hz, 1H), 7.84 (s, 1H), 7.59 (dd, J = 8.2/2.5 Hz,
1H), 7.50 (s, 1H), 7.48−7.38 (m, 1H), 4.90 (d, J = 5.4 Hz, 1H), 4.37
(s, 1H), 4.15 (s, 2H), 3.93 (s, 1H), 2.35 (s, 3H), 2.07−1.48 (m, 5H),
1.47−1.14 (m, 3H); 13C NMR (DMSO-d6) δ 160.2, 150.0, 148.2,
146.4, 146.3, 143.6, 140.0, 137.6, 135.1, 128.0, 126.4, 124.2, 119.7,
68.9, 35.2, 34.4, 30.4, 25.1, 24.0, 19.7; m/z MS (TOF ES+) 384.2
[MH]+; LC-MS tR: 3.55 min.
1
yellow solid. H NMR (DMSO-d6) δ 8.48 (s, 1H), 8.06 (dd, J = 2.4/
0.5 Hz, 1H), 7.87 (dd, J = 2.3/0.9 Hz, 1H), 4.95 (d, J = 5.3 Hz, 1H),
4.38 (s, 1H), 3.94 (s, 1H), 2.53 (s, 3H), 2.14−1.56 (m, 5H), 1.47−
1.20 (m, 3H); 13C NMR (DMSO-d6) δ 159.6, 148.3, 145.0, 138.4,
136.8, 125.9, 121.8, 118.8, 67.9, 35.1, 29.7, 25.0, 23.9, 16.7; m/z MS
(TOF ES+) 337.1 [MH]+; LC-MS tR: 3.77 min.
6-((6-Chloropyridin-3-yl)methyl)-3-((1S,2S)-2-hydroxycyclohexyl)-
7,8-dimethylquinazolin-4(3H)-one (36). 6-Bromo-3-((1S,2S)-2-hy-
droxycyclohexyl)-7,8-dimethylquinazolin-4(3H)-one (32) (250 mg,
0.71 mmol) underwent Negishi coupling according to General
Procedure B. After stirring for 24 h, LC-MS analysis indicated only
partial conversion had occurred. Further Pd(P(tBu)3)2 (11 mg, 0.02
mmol, 0.03 equiv) were added and stirring continued for a further 24
h. After this time the mixture was heated to 55 °C for an additional 24
h. After this time, the reaction had not progressed any further, so the
mixture was cooled over an ice bath and quenched with a small
amount of water, before dilution with water (20 mL). The aqueous
slurry was extracted with EtOAc (3 × 20 mL), and the combined
organic layers washed with brine (20 mL) before concentration under
reduced pressure. The crude product was purified by FCC (eluent
EtOAc/PE 60:40 to 100:0), to give 164 mg of the starting material
(36) and 73 mg (75% brsm) of the desired product as an off-white
6-Bromo-3-((1S,2S)-2-hydroxycyclohexyl)-7-methylquinazolin-
4(3H)-one (31). 2-Amino-5-bromo-N-((1S,2S)-2-hydroxycyclohexyl)-
4-methylbenzamide (27) (481 mg, 1.46 mmol) was dispersed in
formamide (4 mL) in a 10 mL microwave vial, before sealing and
heating at 120 °C with stirring. LC-MS analysis after this time
indicated no reaction progression, so the temperature was increased to
150 °C, and stirring continued for 4 h. LC-MS analysis indicated the
reaction was complete, so the mixture was cooled to RT overnight,
before diluting with water (30 mL), then extracting with EtOAc (3 ×
30 mL). The combined organic layers were washed with water (30
mL) and brine (30 mL), before concentration under reduced pressure.
The crude residue was recrystallized from EtOH, to give 339 mg
(69%) of pale brown solid. Concentration of the mother liquor gave
141 mg of impure product. 1H NMR (CDCl3) δ 8.50 (s, 1H), 8.32 (s,
1H), 7.60−7.51 (m, 1H), 4.61 (s, 1H), 4.04 (s, 1H), 2.50 (s, 3H),
2.33−2.17 (m, 1H), 2.10−1.68 (m, 4H), 1.67−1.30 (m, 3H); 13C
K
DOI: 10.1021/acschemneuro.6b00018
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ACS Chemical Neuroscience
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1
glassy solid. H NMR (CDCl3) δ 8.19 (d, J = 2.1 Hz, 1H), 8.11 (s,
°C for 22.5 h, then at 100 °C for 3 h, followed by 85 °C overnight.
LC-MS analysis over this time indicated the Wittig reaction progressed
at a faster rate than the Suzuki coupling. The mixture was cooled, then
dilute with water (200 mL), before extracting with Et2O (3 × 100
mL). The combined organic layers were washed with brine (100 mL),
before concentration under reduced pressure. The residue was diluted
with Et2O/PE to effect precipitation of triphenylphosphine oxide, the
majority of which was removed by filtration (vacuum). The resulting
filtrated was reconcentrated under reduced pressure, and purified by
FCC (eluent Et2O/PE 0:100, followed by 8:92, then 10:90) to give
ethyl (Z)-3-([1,1′-biphenyl]-4-yl)acrylate as 677 mg (17%) of a clear
colorless oil and ethyl (E)-3-([1,1′-biphenyl]-4-yl)acrylate as 2.78 g
(68%) of a white solid. Total yield 3.46 g (84%, E/Z 4:1).
1H), 7.81 (s, 1H), 7.27 (dd, J = 8.6/2.8 Hz, 1H), 7.17 (d, J = 8.2 Hz,
1H), 4.49 (s, 1H), 4.16−3.85 (m, 3H), 3.10 (s, 1H), 2.47 (s, 3H), 2.21
(d, J = 15.6 Hz, 4H), 2.00−1.70 (m, 4H), 1.61−1.29 (m, 3H); 13C
NMR (CDCl3) δ 162.1, 149.7, 149.5, 144.5, 143.6, 142.1, 139.0, 136.8,
134.6, 134.3, 125.1, 124.2, 119.5, 71.6, 37.0, 35.5, 31.0, 25.4, 24.5, 16.9,
13.7; m/z MS (TOF ES+) 398.2 [MH]+; LC-MS tR: 3.41 min.
2-Amino-5-bromo-N-((1S,2S)-2-hydroxycyclohexyl)nicotinamide
(38). 2-Amino-5-bromopyridine-3-carboxylic acid (37) (1.09 g, 5.00
mmol) and (1S,2S)-2-aminocyclohexanol hydrochloride (834 mg, 1.1
equiv) were coupled according to General Procedure A, to give 1.47 g
(94%) of precipitate as an off-white solid, requiring no further
purification. 1H NMR (DMSO-d6) δ 8.22 (d, J = 8.2 Hz, 1H), 8.14 (d,
J = 2.4 Hz, 1H), 8.12 (d, J = 2.4 Hz, 1H), 7.17 (s, 2H), 4.70 (d, J = 5.0
Hz, 1H), 3.71−3.46 (m, 1H), 3.48−3.21 (m, 1H), 2.01−1.72 (m, 2H),
1.72−1.46 (m, 2H), 1.41−0.99 (m, 4H); 13C NMR (DMSO-d6) δ
166.0, 157.5, 150.9, 138.4, 111.8, 104.0, 71.0, 55.1, 34.4, 31.2, 24.5,
24.2; m/z MS (TOF ES+) 314.1 [MH]+; LC-MS tR: 3.32 min.
6-Bromo-3-((1S,2S)-2-hydroxycyclohexyl)pyrido[2,3-d]pyrimidin-
4(3H)-one (39). 2-Amino-5-bromo-N-((1S,2S)-2-hydroxycyclohexyl)-
nicotinamide (38) (515 mg, 1.64 mmol) was suspended in formamide
(4.5 mL) in a 10 mL microwave vial, before sealing the tube. The
mixture was heated to 180 °C for 1.5 h, then allowed to stir at RT
overnight, before a further period of heating at 180 °C for 2 h. The
mixture was cooled to RT, then quenched with water. The resulting
precipitate was collected by filtration (vacuum) to give 168 mg of
brown solid. The aqueous filtrate was extracted with EtOAc (3 × 30
mL), and the combined organic extracts washed with brine (30 mL).
TLC analysis (EtOAc) indicated that product was still trapped in the
aqueous layer. The aqueous layer was saturated with NaCl, before re-
extraction with EtOAc (2 × 30 mL) then MeOH/EtOAc (1:9, 30
mL). The combined organic layers were washed with brine (30 mL),
then dried over Na2SO4 before concentrating under reduced pressure,
to give an additional 366 mg of yellow solid. The crude solids were
combined, and purified by FCC (eluent MeOH/DCM 0:100 to 6:94),
E-isomer.14 1H NMR (CDCl3) δ 7.73 (d, J = 16.0 Hz, 1H), 7.67−
7.56 (m, 6H), 7.52−7.42 (m, 2H), 7.42−7.34 (m, 1H), 6.48 (d, J =
16.0 Hz, 1H), 4.28 (q, J = 7.1 Hz, 2H), 1.36 (t, J = 7.1 Hz, 3H); 13C
NMR (CDCl3) δ 167.2, 144.3, 143.1, 140.3, 133.6, 129.0, 128.7, 128.0,
127.7, 127.2, 118.3, 60.7, 14.5; m/z MS (TOF ES+) 253.2 [MH]+; LC-
MS tR: 3.83 min.
Z-isomer.15 1H NMR (CDCl3) δ 7.71 (d, J = 8.3 Hz, 2H), 7.65−
7.55 (m, 4H), 7.49−7.40 (m, 2H), 7.40−7.31 (m, 1H), 6.97 (d, J =
12.7 Hz, 1H), 5.97 (d, J = 12.7 Hz, 1H), 4.21 (q, J = 7.1 Hz, 2H), 1.28
(t, J = 7.1 Hz, 3H); 13C NMR (CDCl3) δ 166.4, 142.9, 141.9, 140.7,
133.9, 130.6, 129.0, 127.7, 127.2, 126.8, 119.8, 60.5, 14.3; m/z MS
(TOF ES+) 253.1 [MH]+; LC-MS tR: 3.83 min.
Ethyl 3-([1,1′-Biphenyl]-4-yl)propanoate (45).14,16 Ethyl 3-([1,1′-
biphenyl]-4-yl)acrylate (44) (3.39 g, 13.4 mmol, E/Z isomers
recombined) was dissolved in EtOAC (150 mL). Pd/C (10%, 300
mg, 0.1 wt eq) as a slurry in water (0.5 mL) was added and the mixture
degassed by sonication. The vessel was evacuated and filled with
hydrogen three times, then stirred under an atmosphere of hydrogen
(balloon) for 4 h at RT. LC-MS analysis indicated complete
consumption of starting material. The reaction mixture was filtered
through a bed of Celite, with washings of EtOAc, before concentrating
the filtrate under reduced pressure, to give 3.40 g (99%) of clear
colorless oil. 1H NMR (CDCl3) δ 7.60−7.55 (m, 2H), 7.52 (d, J = 8.1
Hz, 2H), 7.43 (dd, J = 7.5 Hz, 2H), 7.37−7.30 (m, 1H), 7.28 (d, J =
8.0 Hz, 2H), 4.15 (q, J = 7.1 Hz, 2H), 3.00 (t, J = 7.8 Hz, 2H), 2.75−
2.54 (m, 2H), 1.25 (t, J = 7.1 Hz, 3H); 13C NMR (CDCl3) δ 173.1,
141.1, 139.8, 139.4, 128.9, 127.4, 127.3, 127.2, 60.6, 36.0, 30.8, 14.4;
m/z MS (TOF ES+) 255.2 [MH]+; LC-MS tR: 3.78 min.
1
to give 233 mg (44%) of pale yellow solid. H NMR (DMSO-d6) δ
9.05 (d, J = 2.6 Hz, 1H), 8.68 (s, 1H), 8.65 (d, J = 2.6 Hz, 1H), 5.01
(d, J = 5.0 Hz, 1H), 4.35 (s, 1H), 3.96 (s, 1H), 2.12−1.50 (m, 5H),
1.49−1.10 (m, 3H); 13C NMR (DMSO-d6) δ 160.2, 156.4, 156.0,
149.3, 137.5, 118.1, 117.3, 68.3, 34.9, 30.1, 25.0, 23.9; m/z MS (TOF
ES+) 324.1 [MH]+; LC-MS tR: 3.35 min.
3-([1,1′-Biphenyl]-4-yl)propanal (46).16 Ethyl 3-([1,1′-biphenyl]-4-
yl)propanoate (45) (2.18 g, 8.57 mmol) was dissolved in dry toluene
(35 mL) under an atmosphere of nitrogen. The solution was degassed
under a stream of nitrogen, before cooling to −78 °C in a dry ice/
acetone bath. A solution of 1 M DIBALH in toluene (17 mL, 17
mmol, 2.0 equiv) was added in dropwise, and the mixture stirred at
−78 °C for 1.25 h. TLC analysis (DCM) indicated disappearance of
the starting material, so the mixture was quenched with care, with
dropwise addition of MeOH, while maintaining the temperature at
−78 °C. Once quenched, the mixture was allowed to warm to RT, and
stirred for 15 min, before addition of sat. Rochelle’s solution (50 mL)
and stirring for 30 min. The resulting mixture was then extracted with
Et2O (3 × 50 mL), and the combined organic layers washed with brine
(50 mL). On concentration under reduced pressure, 2.01 g of milky
white oil was obtained with an odor reminiscent of cinnamaldehyde.
The crude product was purified by FCC (eluent EtOAc/PE 0:100 to
30:70), to give 1.58 g (88%) of a white solid. 1H NMR (CDCl3) δ 9.86
(t, J = 1.3 Hz, 1H), 7.61−7.55 (m, 2H), 7.55−7.50 (m, 2H), 7.47−
7.40 (m, 2H), 7.38−7.31 (m, 1H), 7.28 (d, J = 8.4 Hz, 2H), 3.01 (t, J =
7.5 Hz, 2H), 2.89−2.78 (m, 2H); 13C NMR (CDCl3) δ 201.7, 141.0,
139.6, 139.5, 128.9, 128.9, 127.5, 127.3, 127.2, 45.4, 27.9; m/z MS
(TOF ES+) no mass peaks observed; LC-MS tR: 3.95 min.
6-((6-Chloropyridin-3-yl)methyl)-3-((1S,2S)-2-hydroxycyclohexyl)-
pyrido[2,3-d]pyrimidin-4(3H)-one (40). 6-Bromo-3-((1S,2S)-2-
hydroxycyclohexyl)pyrido[2,3-d]pyrimidin-4(3H)-one (39) under-
went Negishi coupling according to General Procedure B. After
quenching, the reaction mixture was concentrated under reduced
pressure (prior LC-MS analysis indicated extractive workup was not
suitable, due to solubility of the product in the aqueous layer). The
crude product was purified by FCC (eluent MeOH/DCM 0:100 to
6:94, slow gradient over 20 column volumes) to give 33 mg (14%) of
an off-white glassy solid. An additional 107 mg (37%) of yellow glassy
solid was also isolated form the column, eluting before the desired
product, and found to be 6-bromo-7-((6-chloropyridin-3-yl)methyl)-
3-((1S,2S)-2-hydroxycyclohexyl)-7,8-dihydropyrido[2,3-d]pyrimidin-
4(3H)-one. This was the major product of the reaction, resulting from
nucleophilic attack by the zincate 7-position of the pyrido[2,3-
1
d]pyrimidin-4(3H)-one ring system. H NMR (CD3OD) δ 8.90 (s,
1H), 8.58 (s, 1H), 8.49 (d, J = 2.4 Hz, 1H), 8.36 (d, J = 2.2 Hz, 1H),
7.74 (dd, J = 8.3/2.5 Hz, 1H), 7.42 (d, J = 8.2 Hz, 1H), 4.46 (s, 1H),
4.25 (s, 2H), 4.08 (s, 1H), 3.35 (s, 3H), 2.27−1.66 (m, 5H), 1.63−
1.31 (m, 3H); 13C NMR (CD3OD) δ 162.7, 157.2, 156.7, 151.0, 150.8,
150.8, 141.5, 137.9, 137.0, 136.3, 125.8, 118.5, 70.6, 36.2, 35.4, 30.9,
26.4, 25.3; m/z MS (TOF ES+) 371.2 [MH]+; LC-MS tR: 3.36 min.
Ethyl 3-([1,1′-Biphenyl]-4-yl)acrylate (44).6 Phenylboronic acid
(41) (2.47 g, 20.3 mmol, 1.25 equiv), 4-bromobenzaldehyde (42)
(3.00 g, 16.2 mmol), (ethoxycarbonylmethylene)-
triphenylphosphorane (43) (8.47 g, 24.3 mmol, 1.5 equiv),
PdCl2(PPh3)2 (398 mg 0.57 mmol, 0.035 equiv), and PPh3 (298
mg, 1.13 mmol, 0.07 equiv) were dispersed in degassed DME (64 mL)
and degassed 2 M Na2CO3(aq) (32 mL). The mixture was heated at 70
2-Cyano-N-((1S,2S)-2-hydroxycyclohexyl)acetamide (48). Cyano-
acetic acid (47) (419 mg, 4.92 mmol) and (1S,2S)-2-amino-
cyclohexanol hydrochloride (821 mg, 5.41 mmol) were coupled
according to General Procedure A. After stirring in water/sat.
NaHCO3(aq), no precipitate was evident, and the organic extracts of
this aqueous slurry contained mainly 1,1,3,3-tetramethylurea by-
product. The aqueous layer was concentrated to dryness under
L
DOI: 10.1021/acschemneuro.6b00018
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
Research Article
reduced pressure, and the resulting residue taken up in MeCN, and
stirred at RT for 30 min. The inorganic solid mass was removed by
filtration (vacuum) and the resulting filtrate concentrated under
reduced pressure to give 1.60 g of yellow solid. This was recrystallized
from EtOAc to give 400 mg of a yellow crystalline solid. The mother
liquor was reconcentrated and purified by FCC (eluent EtOAc/PE
50:50 to 100:0) to give a further 237 mg of an off-white solid. Total
yield 637 mg (71%). 1H NMR (DMSO-d6) δ 8.07 (d, J = 7.9 Hz, 1H),
3.57 (d, J = 0.7 Hz, 2H), 3.45−3.28 (m, 1H), 3.26−3.13 (m, 1H),
1.93−1.70 (m, 2H), 1.69−1.46 (m, 2H), 1.35−0.94 (m, 4H); 13C
NMR (DMSO-d6) δ 161.6, 116.4, 71.0, 54.9, 33.9, 30.8, 25.5, 24.1,
23.7; m/z MS (TOF ES−) 181.1 [M − H]−; LC-MS tR: 2.40 min.
5-([1,1′-Biphenyl]-4-ylmethyl)-2-amino-N-((1S,2S)-2-
hydroxycyclohexyl)thiophene-3-carboxamide (49). 3-([1,1′-Biphen-
yl]-4-yl)propanal (46) (334 mg, 1.59 mmol), 2-cyano-N-((1S,2S)-2-
hydroxycyclohexyl)acetamide (48) (289 mg, 1.59 mmol, 1.0 equiv),
sulfur (51 mg, 1.59 mmol, 1.0 equiv) and TEA (0.22 mL, 1.59 mmol,
1.0 equiv) were dispersed in EtOH (1.6 mL) in a 10 mL microwave
vial. The mixture was sonicated at RT for 5 min, before flushing the
atmosphere with nitrogen and sealing the vial. The mixture was heated
at 60 °C for 6 h, at which time LC-MS analysis indicated the reaction
was complete. The mixture was cooled to RT, before pouring onto
ice/water. The resulting brown solid was collected by filtration
(vacuum) and washed with water, then air-dried. The crude product
was purified by FCC (eluent EtOAc/PE 10:90 to 100:0) to give 367
Data Analysis. All data were analyzed using Prism 6.01 (GraphPad
Software, San Diego, CA). Binding-interaction studies with allosteric
ligands were fitted to the following allosteric ternary complex model
(eq 1):31
B
max[A]
Y =
KAKB
α ′ [B] + KB
[I]
[B]
KB
α[I][B]
[A] +
1 +
+
+
KIKB
(
)(
)
KI
(1)
where Y is percentage (vehicle control) binding, Bmax is the total
number of receptors, [A], [B], and [I] are the concentrations of
radioligand, allosteric modulator, and the orthosteric ligand,
respectively, KA, KB, and KI are the equilibrium dissociation constants
of the radioligand, allosteric modulator, and orthosteric ligand,
respectively. α′ and α are the binding cooperativities between the
allosteric ligand and [3H]NMS and the allosteric modulator and the
agonist acetylcholine, respectively. Saturation binding experiments
were used to determine the value of pKA for [3H]NMS (pKA = 9.70
0.01, KA = 0.2 nM). Values of α (or α′) > 1 denote positive
cooperativity; values < 1 (but > 0) denote negative cooperativity, and
value = 1 denotes neutral cooperativity. For the majority of
compounds, a complete inhibition of [3H]NMS binding by the
allosteric modulator was observed, consistent with a very high level of
negative cooperativity. In these cases to allow fitting of the data, logα′
was fixed to −3 to reflect this high negative cooperativity. The
dissociation constant of ACh (KI) was not fixed in these analyses but
rather determined for each separate experiment. No difference was
observed in the value of KI between experiments.
1
mg (57%) of red solid. H NMR (DMSO-d6) δ 7.80−7.56 (m, 4H),
7.53−7.40 (m, 2H), 7.40−7.28 (m, 3H), 7.24 (d, J = 7.9 Hz, 1H), 7.05
(s, 2H), 6.97 (s, 1H), 4.54 (s, 1H), 3.92 (s, 2H), 3.67−3.43 (m, 1H),
3.33 (s, 1H), 1.96−1.71 (m, 2H), 1.70−1.48 (m, 2H), 1.45−0.97 (m,
4H); 13C NMR (DMSO-d6) δ 165.4, 160.2, 134.0, 139.9, 138.2, 128.9,
128.9, 127.3, 126.8, 126.6, 122.4, 122.0, 106.9, 71.4, 54.3, 34.9, 34.6,
31.6, 24.6, 24.2; m/z MS (TOF ES+) 407.2 [MH]+; HRMS
C24H27N2O2S [MH]+ calcd 407.1793; found 407.1797; LC-MS tR:
3.92 min; [α]2D7 = +20.58° (0.14, DMSO).
Concentration−response curves for the interaction between the
allosteric ligand and the orthosteric ligand in the IP-One accumulation
assays were globally fitted to the following operational model of
allosterism and agonism (eq 2):32
E = E (τ [A](K + αβ[B]) + τ [B]K )n
{
}
A
m
A
B
B
/ ([A]K + K K + [B]K + α[A][B])n
{
B
A
B
A
Intact Cell Radioligand Binding Assays. Flp-In Chinese
hamster ovary (CHO) cells expressing the human muscarinic
acetylcholine M1 (hM1 mAChR) were grown in Dulbecco’s modified
Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented
with fetal bovine serum (FBS) (ThermoTrace (Melbourne, Australia)
and 0.2 mg/mL hygromycin-B (Roche, Mannheim, Germany). The
cells were plated at 104 cells per well in 96-well Isoplates
(PerkinElmer). Prior to assay the growth medium was removed and
the attached cells were used to perform radioligand binding studies in
the presence of 0.2 nM [3H]NMS and varying concentrations of
acetylcholine (Sigma, St. Louis, MO) and PAMs in a total volume of
200 μL of binding buffer (10 mM HEPES, 145 mM NaCl, 1 mM
MgSO4·7H2O, 10 mM glucose, 5 mM KCl, 2 mM CaCl2, 1.5 mM
NaHCO3, pH 7.4). The binding reaction mixtures were incubated for
1 h at 37 °C, in a humidified incubator and terminated by rapid
removal of radioligand followed by two 100 μL washes with ice-cold
0.9% NaCl buffer. Radioactivity was determined by addition of 100 μL
Microscint scintillation liquid (PerkinElmer Life Sciences) to each well
and counting in a MicroBeta plate reader (PerkinElmer Life Sciences).
IP-One Accumulation Assays. The IP-One assay kit (Cisbio,
France) was used for the direct quantitative measurement of
myoinositol 1 phosphate (IP1) in FlpIn CHO cells stably expressing
the hM1 mAChR. The cells were detached and resuspended in IP1
stimulation buffer (10 mM Hepes, 1 mM CaCl2, 0.5 mM MgCl2, 4.2
mM KCl, 146 mM NaCl, 5.5 mM glucose, 50 mM LiCl, pH 7.4). The
stimulations were performed in 384-well Proxy-plates (PerkinElmer)
in a total volume of 14 μL, in the absence or presence of increasing
concentrations of ACh and the PAMs, at cell density of 106 million
cells/ml for 1 h at 37 °C, 5% CO2. The reactions were terminated by
addition of 6 μL of lysis buffer containing HTRF reagents (the anti-
IP1 Tb cryptate conjugate and the IP1-D2 conjugate), followed by
incubation for 1 h at RT. The emission signals were measured at 590
and 665 nm after excitation at 340 nm using an Envision multilabel
plate reader (PerkinElmer), and the signal was expressed as the HTRF
ratio: F = ((fluorescence665nm/fluorescence590nm) × 104).
+ (τ [A](K + αβ[B]) + τ [B]K )n
(2)
}
A
A
B
B
where Em is the maximum possible cellular response, [A] and [B] are
the concentrations of orthosteric and allosteric ligands, respectively, KA
and KB are the equilibrium dissociation constant of the orthosteric and
allosteric ligands, respectively, τA and τB are operational measures of
orthosteric and allosteric ligand efficacy, respectively, α is the binding
cooperativity parameter between the orthosteric and allosteric ligand,
β denotes the magnitude of the allosteric effect of the modulator on
the efficacy of the orthosteric agonist, and n denotes the transducer
slope that describes the underlying stimulus-response coupling of the
ligand-occupied receptor to the signal pathway. This parameter was
constrained to be shared between all curves within a fitted data set for
each interaction study, and in all instances was not significantly
different from unity. In many instances, the individual model
parameters of eq 2 could not be directly estimated via the nonlinear
regression algorithm by analysis of the functional data alone due to
parameter redundancy. To facilitate model convergence, therefore, we
fixed the equilibrium dissociation constant of each ligand to that
determined from the whole cell binding assays. For compounds where
no agonism was observed, logτB was fixed to −3.
All affinity, potency, and cooperativity values were estimated as
logarithms, and statistical comparisons between values were by one-
way analysis of variance using a Tukey’s multiple comparison post test
to determine significant differences between mutant receptors and the
WT M1 mAChR. A value of p < 0.05 was considered statistically
significant.
AUTHOR INFORMATION
Corresponding Authors
*(P.J.S.) Phone: +61 (0)3 9903 9542. E-mail: Peter.
Scammells@monash.edu.
■
*(J.R.L.) Phone: +61 (0)3 9903 9095. E-Mail: Rob.Lane@
monash.edu.
M
DOI: 10.1021/acschemneuro.6b00018
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
Research Article
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Present Address
(S.N.M.) School of Pharmacy, Centre for Biomolecular
Sciences, University of Nottingham, University Park, Notting-
ham, NG7 2RD, UK.
Author Contributions
§S.N.M. and H.L. These authors contributed equally to this
work. The manuscript was written through contributions of all
authors, and all authors have given approval to the final version
of the manuscript.
Funding
This research was supported by Discovery Grant DP110100687
of the Australian Research Council as well as Program Grant
APP1055134 and Project Grant APP1049564 of the National
Health and Medicinal Research Council (NHMRC) of
Australia. J.R.L. is a R.D. Wright Biomedical Career Develop-
ment Fellow and a Larkin’s Fellow (Monash University,
Australia), while A.C. is a Senior Principal Research Fellow of
the NHMRC.
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Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
brsm, based on recovery of starting material; DCM, dichloro-
methane; DIPEA, diisopropylethylamine; DMF-DMA, N,N-
dimethylformamide, dimethylacetal; FCC, flash column
chromatography; HCTU, O-(1H-6-chlorobenzotriazole-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate; min, mi-
nutes; PE, petroleum spirits 40−60; TEA, triethylamine
REFERENCES
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