Synthesis and Pharmacological Evaluation of Heterocyclic Carboxamides: Positive Allosteric Modulators of the M1 Muscarinic Acetylcholine Receptor with Weak Agonist Activity and Diverse Modulatory Profiles

Article abstract of DOI:10.1021/acs.jmedchem.7b01812

Targeting allosteric sites at M₁ muscarinic acetylcholine receptors is a promising strategy for the treatment of Alzheimer's disease. Positive allosteric modulators not only may potentiate binding and/or signaling of the endogenous agonist acetylcholine (ACh) but also may possess direct agonist activity (thus referred to as PAM-agonists). Recent studies suggest that PAM-agonists with robust intrinsic efficacy are more likely to produce adverse effects in vivo. Herein we present the synthesis and pharmacological evaluation of a series of pyrrole-3-carboxamides with a diverse range of allosteric profiles. We proposed structural modifications at top, core, or pendant moieties of a prototypical molecule. Although generally there was a correlation between the degree of agonist activity and the modulatory potency of the PAMs, some derivatives displayed weak intrinsic efficacy yet maintained strong allosteric modulation. We also identified molecules with the ability to potentiate mainly the affinity or both affinity and efficacy of ACh.

Full text of DOI:10.1021/acs.jmedchem.7b01812

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Synthesis and Pharmacological Evaluation of Heterocyclic Carboxamides:
Positive Allosteric Modulators of the M1 Muscarinic Acetylcholine
Receptor with Weak Agonist Activity and Diverse Modulatory Profiles
Juliana C. C. Dallagnol, Elham Khajehali, Emma T. van der Westhuizen, Manuela Jörg, Celine
Valant, Alan G. Goncalves, Ben Capuano, Arthur Christopoulos, and Peter J. Scammells
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01812 • Publication Date (Web): 15 Mar 2018
Downloaded from http://pubs.acs.org on March 16, 2018
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Synthesis and Pharmacological Evaluation of Heterocyclic
Carboxamides: Positive Allosteric Modulators of the M Muscarinic
1
Acetylcholine Receptor with Weak Agonist Activity and Diverse
Modulatory Profiles
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†,§,¶
‡,¶
‡
Juliana C. C. Dallagnol,
Elham Khajehali, Emma T. van der Westhuizen,
†
‡
§
†
Manuela Jörg, Celine Valant, Alan G. Gonçalves, Ben Capuano, Arthur
‡,*
†,*
Christopoulos, Peter J. Scammells
†
Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences; Monash University, 381
Royal Parade, Parkville, VIC 3052, Australia
‡
§
Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences & Department of
Pharmacology, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia
Department of Pharmacy, Federal University of Parana, Avenida Prefeito Lothario Meissner 632,
Jardim Botanico, Curitiba, Parana, Brazil
¶
These authors contributed equally to this manuscript
Keywords: Positive allosteric modulators, indole, azaindole, pyrrole, muscarinic acetylcholine
receptor, Alzheimer`s disease.
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ABSTRACT: Targeting allosteric sites at M muscarinic acetylcholine receptors is a promising
1
strategy for the treatment of Alzheimer’s disease. Positive allosteric modulators may not only
potentiate binding and/or signaling of the endogenous agonist acetylcholine (ACh), but may also
possess direct agonist activity (thus referred to as PAM–agonists). Recent studies suggest that
PAMꢀagonists with robust intrinsic efficacy are more likely to produce adverse effects in vivo.
Herein we present the synthesis and pharmacological evaluation of a series of pyrroleꢀ3ꢀ
carboxamides with a diverse range of allosteric profiles. We proposed structural modifications at
top, core or pendant moieties of a prototypical molecule. Although, generally, there was a
correlation between the degree of agonist activity and the modulatory potency of the PAMs, some
derivatives displayed weak intrinsic efficacy yet maintaining strong allosteric modulation. We also
identified molecules with the ability to potentiate mainly the affinity or both affinity and efficacy of
ACh.
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ꢀ
INTRODUCTION
Alzheimer's disease (AD) is a progressive neurodegenerative disorder manifested by the
decline of cognitive function including memory, attention and language. The most recent World
Alzheimer Report prevalence study estimated that 47 million people worldwide were suffering from
AD in 2015 and further projected that without advances in rational drug therapy, the number of
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symptomatic cases might reach 131 million by 2050. Dysfunction of cholinergic neurons and
deficient acetylcholine (ACh) levels are believed to be the earliest events that trigger the
2
–4
pathology.
Based on these findings, targeting cholinergic receptors is a promising strategy to
5
slow down the disease progression and ameliorate symptoms. In particular, the M
1
muscarinic
acetylcholine receptor (M
1
mAChR) subtype has been linked to the process of cognition, thus
6,7
considered an attractive therapeutic target.
Consequently, current pharmacotherapy for AD
includes acetylcholinesterase inhibitors (i.e. anticholinesterases), which are able to increase the
levels of the endogenous orthosteric ligand ACh. Even though these drugs provide some
symptomatic relief they are not well tolerated. The indiscriminate activation of all mAChR
1 5
subtypes (M ꢀM ) leads to significant cholinergic toxicity, typically causing gastrointestinal and
cardiovascular adverse effects, which are mainly mediated by activation of the peripheral M and
2
8
M
3
mAChRs. For many years researchers had sought to develop M
1
mAChR selective agonists,
9
and despite the numerous efforts in this area, little was achieved. The recent high resolution crystal
10–12
structures of the mAChRs
confirmed that this approach is unlikely to succeed due to the high
homology of the orthosteric binding pocket across all five mAChR subtypes.
A new therapeutic opportunity has emerged from targeting the topographically distinct lessꢀ
1
conserved allosteric site on M mAChRs which proved to be a fruitful strategy to overcome the lack
of receptor subtype selectivity of orthosteric ligands. BQCA (1, Figure 1) was the first selective
13
1
positive allosteric modulator (PAM) of the M mAChR to be described in the literature.
Subsequent to its disclosure a vast range of novel chemotypes was disclosed, and insights into the
1
2,14–17
molecular basis of receptorꢀligand interactions
as well as enriched structureꢀactivity
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relationship (SAR) studies
have been published. Allosteric molecules may also possess direct
agonist activity in their own right, and are referred to as ‘allosteric agonists’ if they activate the
receptor but do not modulate the orthosteric ligand, or ‘PAMꢀagonists’ (also ‘agoꢀPAMs’) if they
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1
possess both positive allosteric modulatory properties and agonism. Although several M PAMꢀ
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agonists with aligned physicochemical properties and efficacy in rat cognitive function models were
developed, recent studies reported evidence of cholinergic toxicity associated with highly selective
2
2–24,26
M
1
PAMs.
1
The authors suggested that overactivation of the M mAChR, as a result of the
direct agonist activity of PAMꢀagonists, could underlie the AEs. It was therefore hypothesized that
pure M
1
PAMs would have improved safety profiles.
O
O
O
O
N
OH
HN
F HN
N
OH
O
HN
N
OH
O
N
OH
O
N
N
OH
O
O
F
N
OMe
N
S
N
O
N
N
N
N
BQCA (1)
PF-06764427 (2)
PAM EC50 : 30 nM
Ago EC50 : 590 nM
PF-06767832 (3)
PF-06827443 (4)
VU6004256 (5)
PAM EC50 : 823 nM
Ago EC50 : NA
τB: 2.09
PAM EC50 : 60 nM
Ago EC50 : 3900 nM
τB : 10.5
PAM EC50 : 47 nM
Ago EC50 > 5000 nM
τB: NA
PAM EC50 : 210 nM
Ago EC50 : 4800 nM
τB : NA
τB : 13.2
13
21,22,24
Figure 1. Examples of M
1
mAChR PAMꢀagonists from Merck 1 , Pfizer 2,3,4
and
2
3
Vanderbilt 5 with the relevant pharmacological data. PAM EC50 concentrations were obtained
using calcium mobilization assays in the presence of a fixed EC20 concentration of ACh
(
methodologies may differ slightly). Allosteric agonist EC50 concentrations (Ago EC50) were
obtained from concentrationꢀresponse curves for the modulators in the absence of ACh; τ values
represent a measure of the efficacy of the modulators derived from operational modelꢀfitting, and
were estimated in the present work from IP accumulation assays. NA ꢀ not available.
B
1
Recently, Pfizer published the SAR associated with a series of potent azaindole M mAChR
1
2
1
PAMꢀagonists, with the lead compound being PFꢀ06764427 (2). Shortly after, the same group
disclosed a pyridine series (lead compound PFꢀ06767832 ‒ 3), also reporting in vivo adverse effects
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for this class of compounds. A subsequent study reported a series of γꢀ and δꢀlactam PAMs (lead
compound PFꢀ06827443 ‒ 4), structurally related to pyridine derivative 3, which, despite displaying
24
less agonist activity, presented adverse effects. An independent study by the Vanderbilt Center
for Neuroscience Drug Discovery investigated the adverse effect liability of compound 2 together
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with their fluorinated indole derivative, VU6004256 (5). They have reported that 5 acts as a M
1
mAChR PAMꢀagonist in vitro, and unlike compound 2 enhances recognition memory without
severe behavioral convulsions and peripheral cholinergic effects in rodents. Nonetheless, it was not
possible to draw a clear correlation between the subtle structural changes of the studied compounds
23
and their pharmacological and safety profiles. It has therefore remained unclear which structural
features drive allosteric agonism relative to allosteric modulation or contribute to adverse effect
liability.
Herein, we report the development of a pyrroleꢀ3ꢀcarboxamide series closely related to
compound 2 and detailed pharmacological characterization of selected PAMs in this series. We
performed systematic modifications on three different regions (top, core and pendant, Figure 2) of a
prototypical molecule 6a in order to gain molecular diversity. Representative building blocks were
selected to be incorporated in each of the regions of the molecule, which included revisiting some
21,22,24
23
of the moieties used in previous studies by Pfizer
obtained an enriched allosteric SAR profile by dissecting different pharmacological parameters (i.e.
binding affinity; K , binding and functional cooperativity with ACh; α and αβ, respectively, and
and Vanderbilt University.
We have
B
27,28
direct allosteric agonist efficacy; τ
B
),
thus providing new insights into the structural basis of
allosteric modulation.
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Structural modifications
took place at three diffrent regions
of the prototypical molecule
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Figure 2. Chemical structure of the representative M mAChR PAMꢀagonist 6a, labeling top, core
1
and pendant components of the molecule.
1
Our optimization efforts were focused towards the development of M mAChR PAMs with
distinct pharmacological properties, in particular PAMs with weak agonist activity. The full
pharmacological characterization of six selected PAMs in our series and three literature compounds,
in binding and functional assays, indicated that there was no correlation between the binding and
functional cooperativity with ACh, suggesting that these PAMs could modulate only the affinity or
both affinity and efficacy of ACh. However, there was a significant correlation between the
intrinsic efficacy of PAMs and their functional cooperativity with ACh, consistent with a twoꢀstate
28,29
model of action.
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ꢀ
RESULTS AND DISCUSSION
Chemistry. We first synthesized our prototypical compound 6a, which consists of a
hydroxycyclohexyl top, 1Hꢀpyrrolo[3,2ꢀb]pyridine core and a 4ꢀ(1ꢀmethylꢀ1Hꢀpyrazolꢀ4ꢀyl)benzyl
3
0
pendant. The 2ꢀhydroxycyclohexylcarbamoyl motif was first employed in Merck analogues and
has been present in many M PAMs ever since. It appears to be well tolerated and maintain
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cooperativity in a range of different scaffolds. The 1Hꢀpyrroloꢀ[3,2ꢀb]pyridine core (also referred
to as 4ꢀazaindole) was maintained in order to preserve the intramolecular hydrogen bond (IMHB)
between the fused pyridine nitrogen and the carboxamide hydrogen in some of our analogues. This
1
IMBH was observed in both H NMR and computational torsional energy experiments of 2 and it
was considered key to preꢀorganize the ligand into a putative bioactive conformation thus
21
diminishing interactionꢀenergy requirements to a favorable binding. Finally, the 4ꢀ(1ꢀmethylꢀ1Hꢀ
pyrazolꢀ4ꢀyl)benzyl moiety was chosen as it has been used in a range of M
1
mAChR allosteric
ligands (similar to the moiety in the PAMꢀagonist 5), and has shown to contribute to affinity
18–20
towards the receptor.
While studying structural modifications in certain regions of the molecule
the remaining two other key moieties were kept unaltered allowing a direct comparison with 6a.
We used a practical twoꢀstep synthetic route (Schemes 1ꢀ4), thereby taking advantage of two
key disconnection points of prototypical compound 6a, namely the CꢀN bonds of the carboxamide
group and the Nꢀalkylated nitrogen of the azaindole core, to obtain a variety of compounds. A
collection of 1Hꢀindoleꢀ3ꢀcarboxylic acids and related building blocks were commercially available,
thus allowing the installation of a variety of amines on the top part of the molecule via amide
coupling. NꢀAlkylation was achieved by nucleophilic substitution by the secondary amine of the
core structure with the corresponding benzyl halide pendant. The modifications to the top region
included Nꢀsubstituted homoꢀ or heterocyclic alcohols and their amine bioisosteres as well as
acyclic and aromatic amides (Scheme 1ꢀ2). The core modifications were largely based on bicyclic
scaffolds related to indole, halogenated bicyclic analogues and the truncated pyrrole, where the
peripheral fused aryl ring was absent (Scheme 3). Finally, for the pendant modifications, we
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deleted or replaced the Nꢀmethylpyrazole moiety at the 4ꢀposition of the benzyl group by
bioisosteric motifs (oxazole, thiazole) or more simple substituents (–OMe, –COOMe, –COOH or –
2
CONH ) (Scheme 4). These modifications allowed us to explore the structural determinants of the
different allosteric features (affinity, cooperativity with ACh and intrinsic efficacy of PAMs).
The synthesis of Nꢀsubstituted carboxamides from a variety of primary amines 7aꢀp is shown in
Scheme 1. The reaction of transꢀ2ꢀaminocyclohexanol (7a) and 4ꢀazaindoleꢀ3ꢀcarboxylic acid (8)
using HCTU (2ꢀ(6ꢀchloroꢀ1Hꢀbenzotriazoleꢀ1ꢀyl)ꢀ1,1,3,3ꢀtetramethylaminium hexafluorophosphate)
as the coupling agent afforded the amide 9a in 46% yield. Attempts with different coupling
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reagents,
including
HATU
and
(2ꢀ(7ꢀazaꢀ1Hꢀbenzotriazoleꢀ1ꢀyl)ꢀ1,1,3,3ꢀtetramethyluronium
EDC•HCl (1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide
hexafluoroꢀphosphate)
hydrochloride), produced significantly lower yields of the desired product (29% and 15% yield,
respectively). Amides 9bꢀp were obtained from their parent amines 7bꢀp by applying the same
methodology used for the synthesis of 9a, giving yields ranging from 21 to 45%. Some of the
amines were prepared from suitable starting materials or Bocꢀprotected prior to incorporation
(Scheme 1, bottom). The reaction of 3,7ꢀdioxabicyclo[4.1.0]heptane (11) and concentrated
ammonium hydroxide aqueous solution resulted in both positional isomers transꢀaminotetrahydroꢀ
2
Hꢀpyranol 7b and 7c in a 1:1 ratio, which were separable by column chromatography yielding 30%
of each isomer. Bocꢀprotected amine 7d was prepared using an excess of transꢀ1,2ꢀ
diaminocyclohexane 12 to avoid the doubly protected product. The desired amine 7d was obtained
in 15% yield after column chromatography.
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a,b
Scheme 1. Synthetic route to obtain a diverse range of top motifs
R
R
HN
HO
HN
O
N
N
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R
(a)
(b)
+
H N
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H
Cl
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a-p
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3
4
5
6
7
8
9
0
N
N
N
10
N
FGI (c) or protection (d)
when necessary
Deprotection (e) or FGI (f)
when necessary
Amine precursor
Final molecules
11 and 12
6q-s
6, 7 and 9
HO
HO
HO
R =
O
O
O
HO
HO
HO
HO
BocHN
OH
a
g
b
c
d
e
f
Boc
O
N
m = H
n = Me
o = Et
HO
p = CH CH OH
h
i
j
k
l
2
2
Amine precursor
O
O
O
(
c)
(d)
OH
NH
2
NH2
NHBoc
O
NH2
7b + 7c
OH
NH2
12
NH2
11
7d
Final molecules
R =
OH
O
Boc
H
N
N
(
e)
(e)
(f)
BocHN
H N
2
6
d
6q
6i
6r
6j
6s
a
Reagents and conditions: (a) HCTU, DIPEA, DMF; 21–46% yield, (b) 10, K CO , DMF; 58–94%
2
3
4 2
yield (c) NH OH(aq), MeOH; 38% and 33% yield for 7b and 7c respectively, (d) Boc O,
DCM/dioxane, 0 ˚C to rt; 15% yield, (e) TFA, DCM, 0 ˚C to rt; 67% and 86% yield for 6q and 6r
b
respectively (f) CrO , H SO , H O, acetone, 0 ˚C to rt; 13% yield. All reactions were conducted
3
2
4
2
overnight at rt, unless otherwise stated.
The second step was the Nꢀalkylation to attach the 4ꢀ(1ꢀmethylꢀ1Hꢀpyrazolꢀ4ꢀyl)benzyl moiety
to intermediates 9aꢀp. The benzyl chloride 10 was prepared inꢀhouse (see Scheme 4). We used a
convergent synthesis, preparing this moiety prior to the nucleophilic substitution in order to avoid
one additional step for the synthesis of each individual analogue. Alternatively, intermediates 9aꢀp
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
would have had to be Nꢀalkylated with bromobenzyl bromide followed by a Suzuki reaction to
introduce the pyrazole moiety. The Nꢀalkylations were performed in DMF using potassium
carbonate as base obtaining compounds 6aꢀp in good yields from 58ꢀ94% (except for compound 6l).
The synthesis of 6l afforded two byproducts due to Oꢀalkylation of the phenolic group. The more
complex mixture forced us to adopt a different purification strategy, which included the use of
preparative HPLC, resulting in a final yield of 18%. Bocꢀprotection was essential for the synthesis
of intermediates 6d and 6i to prevent alkylation of the free amino groups. These derivatives were
subsequently subjected to Bocꢀdeprotection (Scheme 1, bottom) under acidic conditions to give the
final compounds 6q and 6r in good yields. Ketone analogue 6s was obtained in 13% yield by
oxidation of the hydroxyl group of derivative 6j using Jones reagent. The reaction was stopped as
soon as byproducts were detected by TLC, even though total consumption of the starting material
was not observed at this point. Although alternative oxidizing agents could be considered, ample
material was obtained for pharmacological evaluation therefore optimization of this reaction was
not attempted.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
One of the most promising derivatives of our initial series was the primary amide 6m.
Therefore, we designed structurally related N,Nꢀdisubstituted amides (Scheme 2). Both acyclic
13aꢀd and cyclic 16aꢀe amines were installed using the same approach presented before.
Intermediates 14aꢀd (open carbon chains) were obtained in 17ꢀ25% yield and 17aꢀe (cyclic
analogues) in 30ꢀ51% yield. Final compounds 15aꢀd and 18aꢀe were obtained upon Nꢀalkylation of
the corresponding amide intermediates (14, 17) with the benzyl chloride pendant 10. The
unprotected piperazine derivative 18f was afforded after Bocꢀdeprotection of 18e under acid
conditions (Scheme 2). As a bioisostere of 6m, compound 21 was synthesized by functionalizing
the carboxylic acid group in 8 to the corresponding ester in 19 and then undergoing a
cyclocondensation to obtain the oxazole top motif in 20. The next step consisted of the introduction
of the pendant group to afford 21. We failed to obtain the mentioned oxazole compound by
3
1,32
reacting carboxamide 6m and 2ꢀchloroꢀ3ꢀbutanone following literature methodologies.
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7
8
9
a,b
Scheme 2. Synthesis of N,Nꢀdisubstituted amides and oxazole top motifs
R1
O
R1
R2
O
N
N
R2
R1
N
N
(b)
(a)
HN
+
8
R2
N
R
N
H
15a-d
1
3a-d
14a-d
O
O
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
N
A
N
N
A
(b)
(a)
H
N
A
+ 8
N
R
N
H
1
6a-e
17a-e
1
8a-e
15, 18 and 21
R =
O
N
N
O
O
N
O
N
(
d)
N
O
(e)
(b)
8
N
N
N
H
N
R
N
H
2
0
21
19
13, 14 and 15
16, 17 and 18
a: R = R2 = methyl
1
A =
N
NR3
1
b: R = R2 = ethyl
N
N
N
O
N
1
2
c: R = R = allyl
e: R3 = Boc
8f: R3 = H
a
b
c
d
d: R = methyl; R2 = cyclohexyl
1
(c)
1
a
Reagents and conditions: (a) HCTU, DIPEA, DMF; 17ꢀ25% for 14aꢀd and 30ꢀ51% yield for 17aꢀe
(
b) 10, K CO , DMF; 46ꢀ55% for 15aꢀd, 58ꢀ98% for 18aꢀe and 19% yield for 21 (c) TFA, DCM,
2 3
0
˚C to rt; 56% yield (d) 2ꢀhydroxyꢀ3ꢀbutanone, EDC•HCl, DMAP, DMF; 19 was not isolated (e)
b
NH
4
OAc, AcOH, 120 ˚C; 2% yield. All reactions were conducted overnight at rt, unless otherwise
stated.
A smaller series of analogues with modifications on the core region of 6a was achieved by
attaching the key moieties (7a and 10) to commercially available heterocyclic carboxylic acids
22aꢀe) (Scheme 3). Amide intermediates 23aꢀe were obtained from their corresponding parent
(
carboxylic acids 22aꢀe by applying the same amide coupling conditions used in the synthesis of 9a,
in yields ranging from 34ꢀ66%. The benzyl pendant 10 was installed as described previously. The
electronic withdrawing character of the core moiety had the opposite effect on the yields of the Nꢀ
alkylation since the nucleophile strength depends on the availability of the lone pair of electrons on
the arylꢀ1Hꢀpyrrole nitrogen. As a result, the lowest yields were observed for 24c and 24d (54%
and 47%, respectively), while 24b was synthesized in 82% yield.
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a,b
Scheme 3. Synthetic route to obtain a diverse range of core motifs
HN
R
OH
O
HN
OH
O
COOH
(
a)
(b)
R
R
N
N
N
H
H
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
22a-e
23a-e
24a-e
N
N
R =
F
Cl
N
N
N
N
N
N
a
b
c
d
e
a
Reagents and conditions: (a) HCTU, transꢀ2ꢀaminocyclohexanol 7a, DIPEA, DMF; 34ꢀ66% yield
b
(
b) 10, K CO , DMF; 47ꢀ82% yield. All reactions were conducted overnight at rt.
2
3
The final set of compounds shown in Scheme 4 possess different benzyl pendants. Benzyl
alcohols 27a and 27b were prepared inꢀhouse through Suzuki coupling between 4ꢀbromobenzyl
alcohol 25 and the corresponding 1ꢀmethylpyrazole boronic acid pinacol esters. Next, the alcohol
was activated using thionyl chloride to give the corresponding substituted benzyl chlorides 10 (from
2
7a) and 28b (from 27b). Attempts to produce the benzyl bromide analog of 10 were unsuccessful.
All classic methodologies of bromination (using hydrogen bromide, phosphorus tribromide and
NBS) failed to yield the expected product or resulted in a complex mixture of products. Benzyl
halides 28cꢀg were purchased from commercial suppliers rather than prepared inꢀhouse.
Compounds 29bꢀg were obtained after Nꢀalkylation of carboxamide 9a with the respective benzyl
halide 28bꢀg. Final derivatives 29h and 29i were afforded by hydrolysis or aminolysis of the
methyl ester 29g, respectively.
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9
a,b
Scheme 4. Synthetic route to obtain compounds with a range of pendant motifs
OH
X
OH
O
(
a)
(b)
(c)
+
O
B
HN
N
OH
Br
R
R
9a
N
O
R
2
5
26a-b
27a-b
10, 28b-g
X = Cl, Br)
(
R =
e = H
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
f = OMe
g = COOMe
9h = COOH
N
N
O
S
R
N
N
N
(d)
2
(e)
6a, 29b-g
N
29i = CONH2
a
b
c
d
Synthetic routes:
2
6a
6b
27a
27b
10
6a
2
28b
29b
29c-g
28c-g (purchased)
9g 29(h,i)
2
a
Reagents and conditions: (a) PdCl
2
(PPh
3
)
2
, THF/1 M Na
2
CO
3
, reflux; 70ꢀ72% yield (b) SOCl
2
,
DCM, 0 ᴼC to rt; 95% yield (c) 9a, K
2
CO
3
, DMF; 26ꢀ83% yield (d) EtOH/NaOH(aq), reflux; 45%
b
yield (e) MeOH/NH
4
OH(aq), reflux, 3 d; 32% yield. All reactions were conducted overnight at rt,
unless otherwise stated.
Pharmacology. We previously established a rational analysis methodology to describe
18
allosterism and provided enriched SAR of BQCA analogues as well as exploring different
19
20
M
1
ꢀ PAM chemotypes, 4ꢀphenylpyrphenylpyridinones and fused arylpyrimidinones.
strategy allowed us to correlate chemical modifications to the changes in parameters that describe
allosteric modulation and agonism at the M mAChR. Here, we adopted a similar approach to
evaluate the allosteric features of our selected heterocyclic carboxamide derivatives.
Our initial IP accumulation assays provided a robust method to assess the modulation of ACh
response by 41 novel putative M mAChR PAMs at 1 ꢁM and 10 ꢁM. The ability of the novel
This
1
1
1
PAMs to modulate the ACh concentrationꢀresponse curves was determined by calculating the
difference in pEC50 (ꢀpEC50) as a surrogate for the cooperativity (αβ), and the increase in baseline
(∆baseline) was used as a surrogate for the intrinsic efficacy (τ
shows the magnitude of the increase in potency of the ACh concentrationꢀresponse curve and the
elevation in the baseline in the presence of 10 ꢁM of the test compound compared to ACh alone
B
) of the test allosteric ligand. Figure
3
(
∆pEC50 vs. ∆baseline). In this manner, we could systematically track the chemical modifications
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
yielding distinct pharmacological properties, such as weak intrinsic efficacy (low ∆baseline) while
preserving cooperativity (large ∆pEC50).
IP1 accumulation
110
N
R
1
9
7
5
0
0
0
ACh (basal)
N
ACh + 10 ꢀM BQCA
ACh + 10 ꢀM 15b
1
ꢀ
pEC50
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
30
ꢀ
baseline
1
0
N
-
10
N
basal -10 -9 -8 -7 -6 -5 -4 -3
log[ACh](M)
Carboxamide variants in A
HO
HO
HO
Carboxamide variants in B
O
O
O
OH
O
OH
O
OH
O
NH
O
2
N
O
N
O
N
O
N
O
HN
HN
HN
HN
HN
O
6b
6c
6q
6r
6e
6f
15a
15b
15c
15d
O
OH
H
O
N
O
BocN
HN
O
HN
O
HN
O
HN
O
HN
O
HN
O
N
O
N
O
N
O
N
O
N
O
6
g
6h
6s
6j
6k
18a
18b
18c
18d
18e
OH
HN
OH
O
HN
H
2
N
O
HN
O
HN
O
O
N
O
O
N
HN
6l
6m
6n
6o
6p
18f
21
1
00
100
80
60
40
20
0
A
B
6
s
3
3
2
k
6a
2
6
a
6g
8
0
0
0
0
0
6b 6e
6
6
j
6r
6
h
6
4
2
6c
6
f
6
p
1
1
6q
15d
1
8f
18c
6
o
6
l
6
m
n
21 18a
18d
6
15c
15b
1
8b
15a
1
8e
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4
ꢀ pEC50
ꢀ pEC50
Figure 3. Plot of ∆pEC50 vs ∆baseline for carboxamide variants. Parameters were estimated from
ACh concentrationꢀresponse curves in the presence or absence of 10 µM of each compound in IP
accumulation assays in FlpIn CHO cells stably expressing the hM mAChR. Plotted values
represent the means ± SEM of at least three experiments performed in duplicate. ∆pEC50 = pEC50 of
ACh + 10 µM compound) – pEC50 of ACh (basal). ∆baseline = maximal stimulation of (ACh basal
10 µM compound) – maximal stimulation of ACh (basal). ∆pEC50 was used as a surrogate of
1
1
(
+
functional cooperativity for 10 µM of the modulator and ∆baseline as a surrogate of its direct
agonism at the same concentration. In all graphs 1, 2, 3 (in blue) and 6a (green) were included as
reference compounds. Graphs: A. Plot of Nꢀsubstituted carboxamides 6bꢀs – top variants. B. Plot of
N,Nꢀdisubstituted carboxamides 15aꢀd, 18aꢀf and 21 – top variants. The estimated ∆pEC50 and
∆baseline values are listed in Supplementary Table 1.
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Figure 3A shows the effect of the modifications to the top motif. Compounds 6a (2ꢀ
hydroxyclyclohexyl), 6b (2ꢀhydroxytetrahydopyranꢀ3ꢀyl), 6c (2ꢀhydroxytetrahydopyranꢀ4ꢀyl), 6e
(2ꢀhydroxycyclopentyl),
6f
(1ꢀdeoxyꢀ3,6ꢀanhydrogalactitol),
6g
(cyclohexyl),
6h
(tetrahydropyranyl), 6j (4ꢀhydroxyclohexyl), 6k (phenyl), 6r (piperidinyl) and 6s (4ꢀoxocyclohexyl)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
displayed varying degrees of cooperativity with ACh but stronger intrinsic efficacy than BQCA (1)
when tested at 10 ꢁM, suggesting that the presence of cyclic substituents at the top of the molecule
correlates with strong intrinsic efficacy of the allosteric modulator.
Compound 6l (2ꢀ
hydroxyphenyl), was an exception to this general phenomenon, displaying low intrinsic efficacy
1
and only weakly potentiating the AChꢀmediated IP
1
response. The H NMR spectrum of the
compound showed deshielding of the phenolic group, with the –OH signal moving downfield
10.02 ppm), supporting the presence of an additional IMHB. This may impair the activity of 6l by
(
increasing the energy required to adopt its bioactive conformation upon the receptorꢀligand
interaction. Deletion of –OH group as in 6k effectively rescued the intrinsic efficacy of the
molecule (∆baseline 6l ~14%; 6k ~73%). Interestingly, whereas compound 6q (2ꢀaminocyclohexyl
1
top motif) displayed a stronger potentiation of the AChꢀmediated IP response, its intrinsic efficacy
was significantly lower than that observed for compound 6a (2ꢀhydroxycyclohexyl), suggesting that
positively charged groups in the top part of the molecule may have an important influence on
intrinsic efficacy. The primary carboxamide 6m and acyclic Nꢀsubstituted carboxamide 6n and 6o
also reduced the intrinsic efficacy of the compounds relative to 1 while maintaining a similar
potentiation of the ACh response. However, by the simple addition of a hydroxyl group to the
carbon chain, as in 6p, the intrinsic efficacy of the molecule was returned to that in the range
observed with BQCA (1).
The presence of Hꢀbond donors (–OH, –NH) and acceptors (O / N) commonly has a substantial
impact on the receptorꢀligand interactions, possibly creating (or deleting) interactions within amino
33
acids in the binding site.
In our series, the deletion of the hydroxyl group in compound 6a
(
∆pEC : 0.44 ± 0.63) resulted in a significant increase in the pEC (compound 6g ‒ ∆pEC : 2.55
50
50
50
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
±
0.75). This top moiety has never been reported in previous SAR of indoles or azaindoles M1
2
1,23
mAChR PAMs.
However, the presence or absence of hydroxyl groups or –O– or HN–
heteroatoms on the cyclohexyl top motif did not have a significant impact on the agonist activity of
the derivatives (Figure 3A). This represents an opportunity to modify physicochemical properties
without losing activity. Polyhydroxylated compound 6f, for example, is unlikely to cross the BBB
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
34
35
(
predicted cLogP = 0.177 – while preferred range for brain penetration is cLogP 2ꢀ4).
activity as M mAChR PAMꢀagonist could therefore be repurposed to treat peripheral disorders in
which the M mAChR is involved (e.g. exocrine glands and gut smooth muscle conditions).
Its
1
1
Figure 3B shows that addition of an N,Nꢀdisubstituted amide to the top part of the molecule, as
seen in analogues 15aꢀd and 18aꢀf, decreased the intrinsic efficacy of the compounds when
compared to BQCA 1. Compounds 15a (N,Nꢀdimethyl), 15b (N,Nꢀdiethyl), 15d (NꢀmethylꢀNꢀ
cyclohexyl) and 18b (pyrrolidine) potentiated the ACh response to a similar extent as seen with
BQCA 1 (∆pEC50 for 1: 1.17 ± 0.34; 15a: 0.89 ± 0.09; 15b: 1.07 ± 0.12; 15d: 1.00 ± 0.17;
18b: 0.76 ± 0.12), while having lower intrinsic efficacy. Compound 15d also had lower intrinsic
efficacy than its monoꢀsubstituted counterparts, 6a and 6g. However, the piperidinyl and NꢀBocꢀ
piperazinyl top motifs (e.g. in 18a, 18e) resulted in a complete loss of ACh potentiation.
Interestingly, the piperazine top motif, as in compound 18f, increased the intrinsic efficacy relative
to the bioisostere 18d (morpholine), while the bioisostere 18a (piperidine) remained essentially
inactive. The more rigid incorporation of a dimethyloxazole (compound 21) was not well tolerated
and completely obliterated the intrinsic efficacy and cooperativity with ACh. Overall, the N,Nꢀ
disubstituted compounds (15 and 18) proved that the removal of the capacity to form an IMHB by
replacing the amide hydrogen with an alkyl chain, is not significantly detrimental to the modulation
of ACh response and that this modification may contribute to the diminished intrinsic efficacy of
these PAMs. However, compounds 6mꢀo which have the IMHB intact, also showed reduced
intrinsic efficacy compared to 1, and retained strong cooperativity with ACh.
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In Figure 4A, the effects of modifications to the core part of the molecule and the benzyl
pendant were explored. Compound 6a and other bicyclic scaffolds, 24aꢀd, showed essentially a
21
PAMꢀagonist profile, and were all more potent allosteric agonists than 1. Creating a new scaffold
by simplification of the azaindole core, as shown in compound 24e, caused a dramatic drop in
intrinsic efficacy, while still maintaining cooperativity with ACh (7ꢀfold potentiation of ACh
activity). Similar to the N,Nꢀdisubstituted carboxamide series, the absence of a IMHB did not result
in a critical decrease in the modulatory effect.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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8
9
0
Core variants in A
Pendant variants in B
F
N
O
S
N
N
N
N
HN
N
OH
O
N
N
N
HN
N
OH
O
29b
29c
29d
2
4a
24b
24c
H
OMe
COOMe
Cl
29e
29f
29g
R
N
N
N
N
COOH
CONH
2
29h
29i
24d
24e
1
00
100
80
60
40
20
0
A
B
3
3
6a
2
6a
2
2
9i
80
60
40
20
0
24c
2
4b
29b
2
9f
2
4d
4a
2
9d
2
1
1
29c
2
9e
2
9g
29h
24e
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4
ꢀ pEC50
ꢀ pEC50
Figure 4. Plot of ∆pEC50 vs ∆baseline for core and pendant variants. Parameters were estimated
from ACh concentrationꢀresponse curves in the presence or absence of 10 µM of each compound in
1 1
IP accumulation assays in FlpIn CHO cells stably expressing the hM mAChR. Plotted values
represent the means ± SEM of three experiments performed in duplicate. Plotted values represent
the means ± SEM of at least three experiments performed in duplicate. ∆pEC = pEC of (ACh +
50
50
1
0 µM compound) – pEC50 of ACh (basal). ∆baseline = maximal stimulation of (ACh basal + 10
µM compound) – maximal stimulation of ACh (basal). ∆pEC50 was used as a surrogate of
functional cooperativity for 10 µM of the modulator and ∆baseline as a surrogate of its direct
agonism at the same concentration. In all graphs 1, 2, 3 (in blue) and 6a (green) were included as
reference compounds. Graphs: A. Plot of compounds 24aꢀe – core variants. B. Plot of compounds
2
9bꢀi – pendant variants. The estimated ∆pEC and ∆baseline values are listed in Supplementary
50
Table 1.
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6
Figure 4B shows the effects of alterations to the pendant moiety on the intrinsic efficacy and
cooperativity of the novel PAMs. The positional isomers 4ꢀ and 3ꢀNꢀmethylpyrazole motif, as in
compounds 6a and 29b, had significantly different cooperativity values (∆pEC50: 0.45 ± 0.63 and
0
1
2
3
4
5
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7
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9
0
1
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0
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2
.35 ± 0.30, respectively), consistent with previously published data by Rook and colleagues. By
replacing the methylpyrazole motif in 6a with an –OMe group (29f), the cooperativity with ACh
was decreased while the strong intrinsic efficacy was maintained, in agreement with previously
22
published results by Davoren and colleagues.
Changing the pendant group to an oxazole
(compound 29c), thiazole (29d) or hydrogen (29e) had minimal overall effects on the cooperativity
or intrinsic efficacy of these compounds when compared to 1. However, changing the pendant
group to a –CONH group (29i) afforded a significant increase in intrinsic efficacy and improved
2
18–20
cooperativity with ACh, consistent with our previous findings on other scaffolds.
In contrast,
its bioisosteres 29h (ꢀCOOH) and 29g (ꢀCOOMe) had similar cooperativity but lower intrinsic
efficacy compared to 1.
Overall, in search of compounds with improved cooperativity with ACh and reduced intrinsic
efficacy compared to the prototypical allosteric modulator BQCA 1, compounds 6o, 15b, 15d (top
part modified), 24e (core modified) and 29h (pendant modified); as well as 6a and the literature
compounds 2 and 3 (for comparison); were selected for further pharmacological characterisation.
To determine the affinity and binding cooperativity of the allosteric modulators with the
3
radiolabeled antagonist, [ H]NMS, at the M
1
mAChR, whole cell equilibrium binding studies were
3
performed. Increasing concentrations of the endogenous agonist, ACh, competed for [ H]NMS
1 i
binding to the M mAChR (pK = 5.41 ± 0.08). Compounds 1, 2, 3 and 6a also almost completely
3
inhibited [ H]NMS binding (i.e. via high negative cooperativity), and their binding affinity (pK
B
)
values could therefore be estimated from these experiments (Table 1 and Supporting Information
Figure 1A). All the other PAMs tested had no or weak effects in their own right. To determine the
binding cooperativities (Logα) between ACh and PAMs, increasing concentrations of the
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modulators were titrated against an EC20 concentration of ACh (3 ꢁM) (Supporting Information,
Figure 1B), and the data were analyzed using an allosteric ternary complex model (Eq. 1,
36
Experimental section). The binding affinities for 6o, 15b, 15d, 24e and 29h were also obtained
from these analyses (Table 1).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 1. Binding and functional allosteric parameters for selected carboxamides and
literature compounds at the M mAChR.
1
3
radioligand binding ([ H]NMS)
IP accumulation
1
c
d
e
f
pK
5.48 ± 0.09
B
Logα′
Logα (α)
Logαβ (αβ)
B B
Logτ (τ )
a
1
2
3
ꢀ0.91 ± 0.12
ꢀ1.28 ± 0.05
ꢀ1.32 ± 0.12
ꢀ1.35 ± 0.28
ND
1.58 ± 0.09 (38.0)
1.52 ± 0.08 (33.1)
1.71 ± 0.08 (51.3)
1.66 ± 0.07 (45.7)
1.55 ± 0.09 (35.5)
1.28 ± 0.06 (19.0)
1.23 ± 0.07 (17.0)*
1.81 ± 0.08 (64.6)
1.76 ± 0.12 (57.5)
1.44 ± 0.10 (27.5)
1.94 ± 0.10 (87.1)*
1.97 ± 0.08 (93.3)*
2.66 ± 0.07 (457)*†
2.19 ± 0.05 (155)*†
1.34 ± 0.08 (21.9)
1.73 ± 0.07 (53.7)†
2.06 ± 0.11 (115)*
1.97 ± 0.06 (93.3)*
0.32 ± 0.05 (2.09)
1.12 ± 0.03 (13.2)*
1.02 ± 0.03 (10.5)*
1.36 ± 0.03 (22.9)*
0.56 ± 0.04 (3.63)
0
a
a
a
b
6.41 ± 0.05*
6.20 ± 0.04*
5.92 ± 0.09*
6a
6o 4.61 ± 0.02*
b
15b 4.43± 0.09*
ꢀ0.11 ± 0.04
ND
b
15d 4.91 ± 0.09*
ꢀ0.24 ± 0.11 (0.57)*
0.52 ± 0.09 (3.31)
0.46 ± 0.04 (2.88)
b
b
2
4e 4.19 ± 0.06*
ꢀ0.05 ± 0.01
ꢀ0.26 ± 0.15
2
9h 4.63 ± 0.20*
a
3
b
Values were estimated from one way competition with [ H]NMS. Values were estimated from
3
competition with [ H]NMS in the presence of an EC20 concentration of ACh. For compound 1, 2, 3
3
and 6a, the Logα′ with [ H]NMS was fixed to the value obtained in (a), for all other ligands was
c
3
d
e
fixed to 0. Binding cooperativity with [ H]NMS. Binding cooperativity with ACh. Functional
cooperativity with ACh. For compound 1, 2, 3 and 6a, pK
all other PAMs was fixed to the value obtained in b.
B
was fixed to the value obtained in a, for
f
Intrinsic efficacy of the modulator.
*Significant difference (p < 0.05) was determined using oneꢀway ANOVA with Dunnett’s post test
†
compared to 1 as the reference PAM. Significant difference (p < 0.05) between Logα and Logαβ
values for each compound was determined using unpaired tꢀtest. Data represent the mean ± SEM of
at least four individual experiments in duplicate.
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5
6
As shown in Table 1, the binding affinities were improved for 2, 3 and our prototypical PAM,
6a (2ꢀhydroxycyclohexyl top motif) compared to BQCA 1. However, modifications to the core, top
21
1
and pendant motif of 6a reduced affinities of PAMs at the M mAChR. Davoren and coworkers
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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have proposed that the π–π interactions between the azaindole core and Trp400 and Tyr179 residues
are important to the overall potency of this class of compounds. Similarly, interaction between the
heterocycle at the 4ꢀposition of the benzyl pendant is supposed to be key for eletrostatic and π
interactions within a tyrosine and aromatic pocket. However, no information about the relevance
of the hydroxycyclohexyl top moiety interactions within the allosteric pocket is available in the
literature. Interestingly, all PAMs tested in our work, except for 15d, displayed similar binding
cooperativities with ACh, as seen by BQCA 1, although having lower affinities.
To investigate the effects of selected PAMs on receptor function, concentrationꢀresponse
curves for AChꢀstimulated IP
increasing concentrations of the test compounds (Supporting Information Figure 2). An operational
model of allosterism and agonism was applied to estimate their intrinsic efficacy (τ ) and functional
cooperativity with ACh (αβ). The binding affinities were fixed to the values obtained in the binding
1
accumulation were generated in the absence or presence of
B
22,23
studies, shown in Table 1. Consistent with previously published data,
we observed
approximately 5ꢀfold increase in intrinsic efficacy of 2 and 3 compared to 1 (Table 1). However, in
2
3,24
contrast to previous studies,
where 3 displayed lower agonism than 2 (Ago EC50 = 590 nM for 2
values for these compounds. In our series, the
and 3900 nM for 3, Figure 1), we obtained similar τ
B
two N,Nꢀdisubstituted carboxamides (15b and 15d) displayed the weakest intrinsic efficacy,
whereas the agonist activity of only 6a was increased compared to 1.
Interestingly, unlike the binding cooperativities, the functional cooperativities of all the PAMs
tested, except for 15b and 15d, were significantly increased compared to 1 (Table 1). The Logαβ
values for compounds 6a, 6o and 15d were significantly higher than the Logα values, whereas for
all other PAMs tested there were no significant differences between Logα and Logαβ values,
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suggesting that the modulation of ACh signaling by these PAMs is mainly derived from their
binding cooperativity. Overall, there was no significant correlation between the Logα and Logαβ
values (Figure 5A), indicating that the differences are not solely due to the different experimental
1
conditions in binding and IP accumulation assays. Compounds 6a, 6o and 15d may therefore
1
1
1
1
1
1
1
1
1
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2
2
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9
0
modulate both the affinity and efficacy of ACh whereas compounds 1, 2, 3, 15b, 24e and 29h may
act as only affinity modulators.
A
B
Figure 5. Correlation analysis of the binding and functional parameters for the selected PAMs at
the M mAChR. A. No significant correlation between binding cooperativity (α) estimates from
1
whole cell radioligand binding assays and functional cooperativity (αβ) estimates from IP
1
accumulation assays. B. Significant correlation between functional cooperativity and intrinsic
efficacy (τ ) values estimated from IP accumulation assays. The solid line is the line of best fit.
B
1
26
As it was suggested by Alt and coꢀworkers,
1
M PAMs with weak agonist activity and
appreciable potentiation of ACh response might have better in vivo safety profiles by fineꢀtuning
native receptor signaling. Our previous studies demonstrate that generally the magnitude of ACh
potentiation by an allosteric modulator (αβ) is correlated to an increase in the intrinsic efficacy (τ
B
)
of the PAM. However, particular structural modifications can tune specifically one of the allosteric
1
8–20
features over the other.
The results of the present study also show a direct correlation between
Logτ and Logαβ (Figure 5B), consistent with a twoꢀstate model of action, where the functional
B
cooperativity of a PAM tracks with the degree of orthosteric agonist efficacy and stimulusꢀresponse
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coupling.
Although such a mechanism can result in higher degrees of potentiation for stronger
agonists or for more amplified (e.g. downstream) signaling pathways, it does not predict biased
24
1
modulation. Intriguingly a recent study by Davoren et al. suggested that even M PAMs with
weak agonist activity could exhibit cholinergic side effects (lead compound 4, Figure 1). However,
it remains unclear whether this was a chemotypeꢀspecific effect since the fused lactam ring in 4 is
solely a covalent formalization of the IMHB present in 3.
0
1
2
3
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0
Notably, a fluorinated indole derivative 5, which potentially lacks the IMHB, typically present
on structural related compounds (as seen in 2), was reported to display stronger modulatory effects
than direct agonist activity. Although the extirpation of the IMHB in our series also resulted in
compounds with lower τ
B
1
values, further studies are required to link structural features of M PAMs
to their in vivo profile.
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ꢀ
CONCLUSIONS
In this study, we reported the synthesis and optimization of the PAM activity of azaindole,
21
indole and pyrrole derivatives inspired by prototypical M
1
PAMꢀagonist, PFꢀ06764427 (2), and
provided a detailed in vitro pharmacological evaluation of selected compounds within our series
and three reference M PAMs from literature, BQCA (1), PFꢀ06764427 (2) and PFꢀ06767832 (3).
Although some of the indole and azaindole scaffolds were evaluated in recent studies by Pfizer
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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2
3
and Vanderbilt University, we were able to obtain a series of compounds bearing a wider range of
structural modifications. Typically, the 2ꢀhydroxyclyclohexyl (as in 6a) and 2ꢀ
hydroxytetrahydopyranꢀ3ꢀyl motifs (as in 6b) are present in the structure of several M mAChR
1
PAMꢀagonists; here we have demonstrated that many other cyclic motifs, such as the cyclohexyl as
in 6g, the bulky polyhydroxylated furanoside as in 6f and the aromatic phenyl group as in 6k, are
tolerated on the top part of the molecule without suffering significant changes to the allosteric
effects. This represents an exceptional opportunity to optimize physicochemical properties, and to
link structural features to pharmacological parameters, and ultimately to in vivo response profiles of
1
the M PAMs.
We showed that unprecedented modifications, such as the substitution of the amide hydrogen
by short alkyl chains (as in 15b, 15d) and the deletion of the core fused aryl ring (as in 24e), yielded
promising PAMs with weak agonist activities. Interestingly, the removal of the cyclic top part (6mꢀ
p, Figure 3A) and the elimination of the IMHB (in 15a, 15b, 15d, 18b and 18c, Figure 3B) were not
1
too detrimental to the modulatory effect of the M PAMs. Similarly, the deletion of the
heteroaromatic nitrogen in the fused pyridine ring, by either replacing it with a carbon (24aꢀd) or
removing the aromatic ring (compound 24e) did not affect the modulatory response (Figure 4A).
Modifications to the benzyl pendant in the original structure 6a (Figure 4B) provided compounds
with diverse pharmacological behaviors. The N,Nꢀdisubstituted series standout with compounds
presenting a modulatory effect similar to the reference PAM 1 but much lower inherent agonism.
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6
Compound 24e bears a pyrrole core and represents a new scaffold to M PAMs, thus offering a
1
promising lead for future structureꢀactivity studies of this class of compounds.
Our detailed analysis of the allosteric features of selected M
profiles (Table 1). Overall, when compared to the reference molecule 1, all the selected compounds
but our prototypical PAM 6a have lower affinities (pK ) at the M mAChR, however, except for
5d, they all display similar binding cooperativities with ACh (α). Interestingly, all modifications,
except N,Nꢀdisubstituted carboxamides, which display the weakest intrinsic efficacies (15b and
5d), result in higher functional cooperativities with ACh (αβ), when compared to 1. The
1
PAMs revealed diverse allosteric
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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0
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0
1
2
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0
1
2
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4
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6
7
8
9
0
B
1
1
1
significant difference between Logα and Logαβ values for 6a, 6o and 15d indicates that the
potentiation of ACh signaling by these PAMs is derived from both modulation of ACh affinity and
efficacy, whereas all the other PAMs are mainly affinity modulators. Within our series, only 6a has
higher intrinsic efficacy compared to 1, and generally, the degree of ACh potentiation correlates
with the intrinsic efficacy of PAMs, consistent with a twoꢀstate model of allosterism. Taken all
together, we suggest compound 15d as a promising PAM for future studies as it has appreciable
affinity at the M
possessing significantly lower intrinsic efficacy (Table 1). Our detailed pharmacological
evaluations provide novel insights into the SAR at the M mAChR, which may aid in predicting in
vivo efficacy and AEs liability of M PAMs. However, further studies on more physiologically
1
mAChR, functional cooperativity in the same range of the reference PAM 1 yet
1
1
relevant systems, such as mouse primary culture neurons, pharmacokinetic and toxicology studies
are needed prior to testing this compound in animal models.
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ꢀ
EXPERIMENTAL SECTION
Chemistry. Chemicals and solvents were purchased from standard suppliers and used without
further purification. Davisil silica gel (40−63 ꢁm), 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). PFꢀ06764427 and PFꢀ06767832 were purchased from SigmaꢀAldrich.
Reactions were monitored by thin layer chromatography on commercially available precoated
aluminiumꢀbacked plates (Merck Kieselgel 60 F254). Visualization was by examination under UV
light (254 and 366 nm). Appropriate staining was carried out using a solution of ninhydrin (in
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
13
ethanol) to visualize primary and secondary amines. H NMR and C 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) 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). Twoꢀdimensional (2D)
experiments including HSQC and HMBC where used to assign the compounds only when
necessary. LCꢀMS were run to verify reaction outcome and purity using the following system:
Agilent 6120 Series Single Quad coupled to an Agilent 1260 Series HPLC; Buffers: A, 0.1% formic
acid in H
2
O; B, 0.1% formic acid in MeCN; The gradient was: 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; the flow rate
was 0.5 mL/min and total run time was 5 min at a Poroshell 120 ECꢀC18 50 × 3.0 mm 2.7 ꢁm
R
column. Retention times (t ) are quoted, in minutes, for all final coumpounds. 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. HRMS analyses were carried out on an Agilent 6224 TOF LC/MS
spectrometer coupled to an Agilent 1290 Infinity (Agilent, Palo Alto, CA). Preparative HPLC was
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1
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1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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6
performed using an Agilent 1260 infinity coupled with a binary preparative pump and Agilent 1260
FCꢀPS fraction collector, using Agilent OpenLAB CDS software (Rev C.01.04), and an Agilent 7
ꢁm XDBꢀC8 21.2 × 250 mm column. The following buffers were used: buffer A, 0.1% TFA in
2
H O; buffer B, 0.1% TFA in MeCN, with sample being run at a gradient of 5% buffer B to 100%
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
buffer B over 10 min, at a flow rate of 20 mL/min. All screening compounds were of >95% purity.
General Procedure A. Amide Coupling. A mixture of the carboxylic acid (1.0 equiv) and
HCTU (1.2 equiv) in DMF (1 mL) was stirred for 5 min until a homogenous suspension was
observed. A solution of the respective amine (1.2 equiv) and DIPEA (2 equiv) in DMF (1 mL) was
added dropwise and the reaction was stirred overnight at rt DMF was coꢀevaporated with water and
ethanol under reduced pressure at 50 ˚C. The residue was diluted with 1 M Na
mL) and extracted with EtOAc (3 × 80 mL). The combined organic layers were washed with brine
80 mL), filtered over anhydrous Na SO and dried under reduced pressure. Unless stated
2 3
CO solution (80
(
2
4
otherwise stated, the crude products were purified by flash column chromatography.
General Procedure B. NꢀAlkylation. A mixture of the carboxamide (1 equiv), the respective
2 3
benzyl halide (1 equiv) and K CO (2 equiv) in DMF (1 mL/0.2 mmol of starting material) was
stirred at rt overnight. DMF was coꢀevaporated with water and ethanol under reduced pressure at
5
0 ˚C. Unless stated otherwise, crude products were purified by flash column chromatography.
ꢀ((1 ,2 )ꢀ2ꢀHydroxycyclohexyl)ꢀ1ꢀ(4ꢀ(1ꢀmethylꢀ1 ꢀpyrazolꢀ4ꢀyl)benzyl)ꢀ1 ꢀpyrrolo[3,2ꢀ
]pyridineꢀ3ꢀcarboxamide (6a). Compound 9a (50 mg, 0.19 mmol) and 10 (40 mg, 0.19 mmol)
N
S
S
H
H
b
were reacted according to general procedure B. The crude product was purified by FCC (eluent
1
CHCl /MeOH 100:1) to afford 6a as a white solid (73 mg, 89%). Η NMR (DMSOꢀd ) δ 8.77 (d, J
3
6
= 7.7 Hz, 1H), 8.49 (dd, J = 4.7/1.2 Hz, 1H), 8.39 (s, 1H), 8.09 (dd, J = 8.3/1.2 Hz, 1H), 8.08 (s,
1
5
2
H), 7.81 (s, 1H), 7.52 (d, J = 8.2 Hz, 2H), 7.30 (dd, J = 8.3/4.7 Hz, 1H), 7.27 (d, J = 8.2 Hz, 2H),
.50 (s, 2H), 4.79 (d, J = 5.4 Hz, 1H), 3.84 (s, 3H), 3.77‒3.69 (m, 1H), 3.46–3.38 (m, 1H), 2.08–
13
.00 (m, 1H), 1.92–1.85 (m, 1H), 1.68–1.60 (m, 2H), 1.37–1.20 (m, 4H). C NMR (DMSOꢀd
6
) δ
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65.2, 143.5, 143.3, 135.9, 135.4, 134.1, 132.4, 129.8, 127.8, 127.6, 125.4, 122.4, 118.8, 117.2,
+
109.5, 72.8, 54.4, 50.0, 37.5, 33.7, 31.2, 24.0, 23.6. HRMS m/z [M+H] (QꢀTOF/ESI) C25
H
28
N
5
O
2
R
calcd: 430.2243; found: 430.2247. LCꢀMS t = 2.99.
Nꢀ((3R
,4
S)ꢀ3ꢀHydroxytetrahydroꢀ2
Hꢀpyranꢀ4ꢀyl)ꢀ1ꢀ(4ꢀ(1ꢀmethylꢀ1Hꢀpyrazolꢀ4ꢀyl)benzyl)ꢀ
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
H
ꢀpyrrolo[3,2ꢀb
]pyridineꢀ3ꢀcarboxamide (6b). Compound 9b (25 mg, 0.10 mmol) and 10 (20
mg, 0.10 mmol) were reacted according to general procedure B. The crude product was purified by
1
FCC (eluent CHCl
3
/MeOH 100:1) to afford 6b as a white solid (26 mg, 62%). Η NMR (DMSOꢀd
6
)
δ 8.83 (d, J = 7.4 Hz, 1H), 8.50 (dd, J = 4.7/1.2 Hz, 1H), 8.42 (s, 1H), 8.08 (dd, J = 8.3/1.2 Hz, 1H),
.06 (s, 1H), 7.80 (s, 1H), 7.51 (d, J = 8.0 Hz, 2H), 7.30 (d, J = 8.0 Hz, 2H), 7.24 (dd, J = 8.3/4.7
Hz, 1H), 5.50 (s, 2H), 5.18 (d, J = 5.4 Hz, 1H), 3.94–3.87 (m, 1H), 3.87–3.78 (m, 5H), 3.52–3.45
8
13
(m, 1H), 3.45–3.40 (m, 1H), 3.16–3.10 (m, 1H), 2.12–2.05 (m, 1H), 1.58–1.46 (m, 1H). C NMR
(DMSOꢀd
6
) δ 163.0, 144.0, 143.1, 136.5, 136.3, 134.8, 132.7, 129.7, 128.5, 128.2, 125.7, 121.8,
+
119.8, 117.7, 110.3, 71.6, 69.3, 66.1, 52.2, 50.0, 39.1, 32.3. HRMS m/z [M+H] (QꢀTOF/ESI)
C
24
H
26
N
5
O
3
calcd: 432.2036; found: 432.2046. LCꢀMS t
)ꢀ4ꢀHydroxytetrahydroꢀ2 ꢀpyranꢀ3ꢀyl)ꢀ1ꢀ(4ꢀ(1ꢀmethylꢀ1
ꢀpyrrolo [3,2ꢀ ]pyridineꢀ3ꢀcarboxamide (6c). Compound 7c (25 mg, 0.10 mmol) and 10 (20
R
= 2.87.
Nꢀ((3
S
,4
S
H
Hꢀpyrazolꢀ4ꢀyl)benzyl)ꢀ
1
H
b
mg, 0.10 mmol) was reacted according to general procedure B. The crude product was purified by
1
FCC (eluent CHCl
3
/MeOH 100:1) to afford the 6c as a white solid (25 mg, 60%). Η NMR
(DMSOꢀd
6
) δ 8.85 (d, J = 7.7 Hz, 1H), 8.50 (dd, J = 4.4 Hz, 1H), 8.41 (s, 1H), 8.07 (m, 2H), 7.80
(s, 1H), 7.51 (d, J = 8.0 Hz, 2H), 7.30 (m, 3H), 5.50 (s, 2H), 5.12 (d, J = 5.4 Hz, 1H), 4.01–3.95 (m,
1
2
H), 3.83 (s, 3H) , 3.83–3.78 (m, 1H), 3.73–3.66 (m, 1H), 3.53–3.45 (m, 1H), 3.37–3.34 (m, 1H),
1
3
6
.27–2.17 (m, 1H), 1.97–1.90 (m, 1H), 1.57–1.49 (m, 1H). C NMR (DMSOꢀd ) δ 163.3, 144.1,
143.1, 136.5, 136.4, 134.8, 132.7, 129.7, 128.5, 128.3, 125.7, 121.9, 119.9, 117.8, 110.3, 68.3, 68.0,
+
65.0, 51.8, 50.0, 39.1, 33.4. HRMS m/z [M+H] (QꢀTOF/ESI) C24
H
26
N
5
O
3
calcd: 432.2036; found:
R
432.2048. LCꢀMS t = 2.88.
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1
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1
1
2
2
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2
2
2
2
2
2
2
3
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tertꢀButyl(2ꢀ(1ꢀ(4ꢀ(1ꢀmethylꢀ1Hꢀpyrazolꢀ4ꢀyl)benzyl)ꢀ1Hꢀpyrrolo[3,2ꢀb]pyridineꢀ3ꢀ
carboxamido)cyclohexyl)carbamate (6d). Compound 9d (89 mg, 0.25 mmol) and 10 (51 mg,
0.25 mmol) were reacted according to general procedure B. The crude product was purified by
1
FCC (eluent CHCl
3
/MeOH 100:1) to afford 6d as a colourless oil (97 mg, 74%). Η NMR (CDCl
3
)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
δ 8.94 (d, J = 8.2 Hz, 1H), 8.46 (dd, J = 4.7/1.2 Hz, 1H), 8.03 (s, 1H), 7.68 (s, 1H), 7.57 (dd, J =
8
5
1
.3/1.2 Hz, 1H), 7.54 (s, 1H), 7.37 (d, J = 8.2 Hz, 2H), 7.10–7.06 (m, 3H), 5.35 (d, J = 7.0, 1H),
.25 (s, 2H), 3.99–3.90 (m, 1H), 3.88 (s, 3H), 3.50–3.40 (m, 1H), 2.23–2.16 (m, 1H), 2.11–2.04 (m,
+
+
H), 1.78–1.67 (m, 2H), 1.57–1.30 (m, 4H), 1.21 (s, 9H). LRMS m/z [M+H] (TOF ES ) 529.2.
ꢀ((1 ,2 )ꢀ2ꢀHydroxycyclopentyl)ꢀ1ꢀ(4ꢀ(1ꢀmethylꢀ1 ꢀpyrazolꢀ4ꢀyl)benzyl)ꢀ1 ꢀpyrrolo[3,2ꢀ
]pyridineꢀ3ꢀcarboxamide (6e). Compound 9e (61 mg, 0.25 mmol) and 10 (51 mg, 0.25 mmol)
N
S
S
H
H
b
were reacted according to general procedure B. The crude product was purified by FCC (eluent
1
CHCl
3
/MeOH 100:1) to afford 6e as a white solid (84 mg, 80%). Η NMR (CD
3
OD) δ 8.43 (dd, J
=
=
4.6/0.8 Hz, 1H), 8.13 (s, 1H), 7.81 (dd, J = 8.3/0.8 Hz, 1H), 7.80 (s, 1H), 7.71 (s, 1H), 7.42 (d, J
8.2 Hz, 2H), 7.17 (dd, J = 8.3/4.6 Hz, 1H), 7.15 (d, J = 8.2 Hz, 2H), 5.33 (s, 2H), 4.22–4.11 (m,
2
H), 3.84 (s, 3H), 2.28–2.17 (m, 1H), 2.06–2.96 (m, 1H), 1.88–1.74 (m, 2H), 1.69–1.57 (m, 2H).
1
3
C NMR (CD OD) δ 165.0, 143.6, 143.2, 135.9, 135.2, 134.0, 132.4, 129.8, 127.8, 127.6, 125.4,
3
+
122.3, 118.8, 117.2, 109.2, 77.6, 58.0, 50.0, 37.6, 31.7, 29.7, 20.4. HRMS m/z [M+H]
(QꢀTOF/ESI) C24 calcd: 416.2087; found: 416.2087. LCꢀMS t = 3.00.
H
26
N
5
O
2
R
N
ꢀ((2
ꢀpyrazolꢀ4ꢀyl)benzyl)ꢀ1
.11 mmol) and 10 (24 mg, 0.11 mmol) were reacted according to general procedure B. The crude
R
)ꢀ2ꢀ((3
R
,4
S
)ꢀ3,4ꢀDihydroxytetrahydrofuranꢀ2ꢀyl)ꢀ2ꢀhydroxyethyl)ꢀ1ꢀ(4ꢀ(1ꢀmethylꢀ
1
H
H
ꢀpyrrolo[3,2ꢀ ]pyridineꢀ3ꢀcarboxamide (6f). Compound 9f (36 mg,
b
0
3
product was purified by FCC (eluent CHCl /MeOH 100:1) to afford 6f as a white oil (32 mg, 58%).
1
3
Η NMR (CD OD) δ 8.40 (d, J = 4.2 Hz, 1H), 8.11 (s, 1H), 7.79 (s, 1H), 7.73 (d, J = 8.0 Hz, 1H),
7
4
.70 (s, 1H), 7.37 (d, J = 8.2 Hz, 2H), 7.10 (m, 3H), 5.29 (s, 2H), 4.14 (br s, 1H), 4.07 (br s, 1H),
.00–3.94 (m, 2H), 3.85–3.81 (m, 4H), 3.79–3.75 (m, 1H), 3.74–3.72 (m, 1H), 3.62–3.55 (m, 1H).
1
3
C NMR (CD OD) δ 165.3, 143.6, 143.2, 136.0, 135.3, 134.0, 132.4, 129.7, 127.8, 127.7, 125.4,
3
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9
+
1
22.3, 118.7, 117.2, 109.4, 86.6, 78.8, 77.4, 73.3. 70.1, 50.0, 42.0, 37.6. HRMS m/z [M+H]
28 5 5 R
(QꢀTOF/ESI) C25H N O calcd: 478.2090; found: 478.2085. LCꢀMS t = 2.77.
NꢀCyclohexylꢀ1ꢀ(4ꢀ(1ꢀmethylꢀ1
Hꢀpyrazolꢀ4ꢀyl)benzyl)ꢀ1Hꢀpyrrolo[3,2ꢀb]pyridineꢀ3ꢀ
carboxamide (6g). Compound 9g (61 mg, 0.25 mmol) and 10 (51 mg, 0.25 mmol) were reacted
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
according to general procedure B. The crude product was purified by FCC (eluent CHCl /MeOH
1
1
8
7
2
1
00:1) to afford 6g as a white solid (68 mg, 66%). Η NMR (CDCl ) δ 8.85 (d, J = 4.9 Hz, 1H),
3
.48 (dd, J = 4.2/1.2 Hz, 1H), 8.08 (s, 1H), 7.71 (s, 1H), 7.59–7.53 (m, 2H), 7.39–7.35 (m, 2H),
.13–7.07 (m, 3H), 5.29 (s, 2H), 4.08 (br s, 1H), 3.91 (s, 3H), 2.07–1.95 (m, 2H), 1.82–1.73 (m,
1
3
3
H), 1.65–1.23 (m, 6H). C NMR (CDCl ) δ 162.9, 143.7, 143.6, 136.7, 135.2, 133.3, 132.2, 129.6,
27.6, 127.0, 126.3, 122.4, 118.0, 117.1, 111.4, 50.8, 47.4, 47.3, 39.1, 33.3, 33.2, 25.8, 24.8.
+
HRMS m/z [M+H] (QꢀTOF/ESI) C H N O calcd: 414.2294; found: 414.2289. LCꢀMS t = 3.00.
25
28
5
R
1
ꢀ(4ꢀ(1ꢀMethylꢀ1
H
ꢀpyrazolꢀ4ꢀyl)benzyl)ꢀ
N
ꢀ(tetrahydroꢀ2
H
ꢀpyranꢀ4ꢀyl)ꢀ1
H
ꢀpyrrolo[3,2ꢀ
b
]pyridineꢀ3ꢀcarboxamide (6h). Compound 9h (25 mg, 0.10 mmol) and 10 (20 mg, 0.10 mmol)
were reacted according to general procedure B. The crude product was purified by FCC (eluent
1
CHCl
3
/MeOH 100:1) to afford 6h as a white solid (26 mg, 63%). Η NMR (CDCl
3
) δ 8.96 (d, J =
7
7
3
.8 Hz, 1H), 8.48 (dd, J = 4.7/1.2 Hz, 1H), 8.08 (s, 1H), 7.71 (s, 1H), 7.60 (dd, J = 8.3/1.2 Hz, 1H),
.57 (s, 1H), 7.39 (d, J = 8.2 Hz, 2H), 7.20–7.10 (m, 3H), 5.30 (s, 2H), 4.33–4.25 (m, 1H), 4.03–
13
.97 (m, 2H), 3.91 (s, 3H), 3.61–3.54 (m, 2H), 2.07–2.00 (m, 2H), 1.78–1.67 (m, 2H). C NMR
(CDCl
3
) δ 163.2, 143.8, 143.6, 136.7, 135.2, 133.2, 132.9, 129.7, 127.7, 127.0, 126.0, 122.4, 118.2,
+
117.2, 111.0, 66.8, 66.7, 50.9, 44.8, 44.7, 39.1, 33.3. HRMS m/z [M+H] (QꢀTOF/ESI)
C H N O calcd: 416.2087; found: 416.2095. LCꢀMS t = 2.95.
24
26
5
2
R
tertꢀButyl
4ꢀ(1ꢀ(4ꢀ(1ꢀmethylꢀ1
H
ꢀpyrazolꢀ4ꢀyl)benzyl)ꢀ1Hꢀpyrrolo[3,2ꢀb]pyridineꢀ3ꢀ
carboxamido)piperidineꢀ1ꢀcarboxylate (6i). Compound 9i (34 mg, 0.10 mmol) and 10 (20 mg,
0
.10 mmol) were reacted according to general procedure B. The crude product was purified by
1
FCC (eluent CHCl
3
/MeOH 100:1) to afford 6i as a light yellow oil (30 mg, 60%). Η NMR (CDCl
3
)
ACS Paragon Plus Environment