Discovery of M3Antagonist-PDE4 Inhibitor Dual Pharmacology Molecules for the Treatment of Chronic Obstructive Pulmonary Disease

Article abstract of DOI:10.1021/acs.jmedchem.1c00204

In this paper, we report the discovery of dual M3 antagonist-PDE4 inhibitor (MAPI) compounds for the inhaled treatment of pulmonary diseases. The identification of dual compounds was enabled by the intuition that the fusion of a PDE4 scaffold derived from our CHF-6001 series with a muscarinic scaffold through a common linking ring could generate compounds active versus both the transmembrane M3 receptor and the intracellular PDE4 enzyme. Two chemical series characterized by two different muscarinic scaffolds were investigated. SAR optimization was aimed at obtaining M3 nanomolar affinity coupled with nanomolar PDE4 inhibition, which translated into anti-bronchospastic efficacy ex vivo (inhibition of rat trachea contraction) and into anti-inflammatory efficacy in vitro (inhibition of TNFα release). Among the best compounds, compound 92a achieved the goal of demonstrating in vivo efficacy and duration of action in both the bronchoconstriction and inflammation assays in rat after intratracheal administration.

Full text of DOI:10.1021/acs.jmedchem.1c00204

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Discovery of M3 Antagonist-PDE4 Inhibitor Dual Pharmacology
Molecules for the Treatment of Chronic Obstructive Pulmonary
Disease
Elisabetta Armani,* Andrea Rizzi, Carmelida Capaldi, Renato De Fanti, Maurizio Delcanale, Gino Villetti,
Gessica Marchini, Anna Rita Pisano, Vanessa Pitozzi, Maria Gloria Pittelli, Marcello Trevisani,
Michela Salvadori, Valentina Cenacchi, Paola Puccini, Francesco Amadei, Alice Pappani, Maurizio Civelli,
Riccardo Patacchini, Charles A.G. Baker-Glenn, Hervé Van de Poël, Wesley P. Blackaby, Kevin Nash,
and Gabriele Amari
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ABSTRACT: In this paper, we report the discovery of dual M3
antagonist-PDE4 inhibitor (MAPI) compounds for the inhaled
treatment of pulmonary diseases. The identification of dual
compounds was enabled by the intuition that the fusion of a
PDE4 scaffold derived from our CHF-6001 series with a
muscarinic scaffold through a common linking ring could generate
compounds active versus both the transmembrane M3 receptor
and the intracellular PDE4 enzyme. Two chemical series
characterized by two different muscarinic scaffolds were inves-
tigated. SAR optimization was aimed at obtaining M3 nanomolar
affinity coupled with nanomolar PDE4 inhibition, which translated
into anti-bronchospastic efficacy ex vivo (inhibition of rat trachea contraction) and into anti-inflammatory efficacy in vitro (inhibition
of TNFα release). Among the best compounds, compound 92a achieved the goal of demonstrating in vivo efficacy and duration of
action in both the bronchoconstriction and inflammation assays in rat after intratracheal administration.
Inhalers containing a binary combination of drugs, for
example, Combivent (ipratropium bromide/salbutamol), can
simplify a COPD patient’s drug regimen by reducing the
number of inhalations per day. The concept of combining two
activities in a single molecule has also been investigated in the
recent years [i.e., dual-pharmacology inhaled muscarinic
antagonist-β2 agonist (MABA) molecules for COPD treat-
ment]2 and this approach may offer potential advantages over
the combination of two different drugs including the potential
benefit of an increase in affinity over the monofunctional and
monovalent parent compounds, a high molecular weight that
often translates into greater lung retention, low oral
bioavailability, and reduced systemic exposure. Moreover, the
development of a single compound with dual activity is
simplified in terms of a unique pharmacokinetic profile,
INTRODUCTION
■
Chronic obstructive pulmonary disease (COPD) describes a
group of lung conditions and is characterized by progressive
airflow limitation. These conditions include persistent
bronchitis, which is long-term inflammation of the airways,
and emphysema, which damages the air sacs in the lungs, and
both these conditions can occur together. COPD is a major
worldwide health problem, with an estimated 3 million deaths
caused globally by the disease in 2015, which is around 5% of
all deaths in that year.1
The current treatment guidelines for COPD involve the use
of bronchodilators, usually muscarinic (M3) antagonists or β-
adrenergic agonists. However, although these drugs may be
effective in improving symptoms, they fail to address the
underlying chronic inflammation. As such, corticosteroids are
also recommended for COPD patients at high risk of
exacerbations, but these drugs have shown limited efficacy as
anti-inflammatory agents in COPD. However, PDE4 inhib-
itors, which elevate cAMP levels, have shown promise in the
treatment of COPD by reducing the responses of inflammatory
cells, and the latest clinically validated COPD therapy is
roflumilast, a potent, long-lasting phosphodiesterase 4 (PDE4)
inhibitor.
Received: February 3, 2021
Published: June 18, 2021
https://doi.org/10.1021/acs.jmedchem.1c00204
© 2021 American Chemical Society
J. Med. Chem. 2021, 64, 9100−9119
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Figure 1. Known MABA (GSK-961081 and AZD-2115), BAPI (GS-5759), and MAPI (UCB 101333-3, Ex 111 in WO 2009100170) compounds.
Figure 2. PDE4 inhibitor CHF-6001 and muscarinic antagonist scaffolds.
formulation, and clinical development programs, so avoiding
the associated challenges of the clinical development and co-
formulation of two active pharmaceutical ingredients. On the
other hand, whereas the flexible dosage of each active
component is possible in the combination product, in the
single dual product, the dosage is the same for the different
components, meaning that the ratio of the two pharmaco-
logical activities cannot be adjusted as needed. Balancing of the
pharmacological activities should be an intrinsic property of
the dual molecule in order to avoid the overdosage of one
component and the associated risk of unwanted side effects.
The combination in a single molecule of a muscarinic
antagonist and a β-adrenergic agonist (MABA) has been
widely reported,2 with compounds from GSK (GSK-961081)
and AZ (AZD-2115) being progressed into the clinic.
Compounds combining either a β-adrenergic agonist with a
PDE4 inhibitor (BAPI), such as GS-5759,3 or combining
muscarinic antagonist activity with a PDE4 inhibiting activity
(MAPI), such as UCB 101333-34 or a series of pyrazolopyr-
idines from Glaxo5 (Figure 1), have also been reported.
In this paper, we report the discovery of a family of dual M3
antagonist-PDE4 inhibitor molecules derived from our
previously reported PDE4 inhibitor, CHF-6001.6
RESULTS AND DISCUSSION
■
Initial Strategy. Our aim was the identification of
compounds that demonstrated activity both at the trans-
membrane M3 receptor and at the intracellular PDE4 protein
to deliver a MAPI for inhaled administration that displayed a
balanced in vivo efficacy as a bronchodilator and anti-
inflammatory agent with limited systemic exposure to
minimize any potential side effects resulting from interaction
with peripheral drug targets. Our strategy was to combine the
structure of PDE4 inhibitor CHF-6001 (Figure 2) with that of
a muscarinic M3 antagonist: a number of muscarinic antagonist
scaffolds were investigated, but this paper will focus on work
derived from two related scaffolds, phenlyglycine scaffold 17
and phenylcarbamate scaffold 28 (Figure 2).
Unlike many of the MABA compounds reported in the
literature, and the GSK MAPI series, which contain a linker
between the two pharmacophoric fragments, we began our
investigation by designing compounds where the two frag-
ments are overlapped through a common linking ring. A series
of four analogues were synthesized, all containing a meta-linked
phenyl as the shared ring: three of these compounds contain a
muscarinic head group derived from scaffold 1, with different
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Figure 3. First MAPI compounds with muscarinic scaffold 1 (25, 87a, and 33) or scaffold 2 (47b).
Table 1. Biological Data and Calculated logD for Compounds 25, 33, 87a, 47b, 24, 32, 86a, and 46b
Cmp
−X−
bond
−SO2−
−CH2−
R1
R2
stereochem
M3 pKi
GPT pIC50
LARBS pIC50
clogD (7.4)
25
33
87a
47b
24
32
86a
46b
CH2cPr
CH2cPr
CH2cPr
CH2cPr
Me
Me
Me
Me
CHF2
CHF2
CHF2
CHF2
Me
Me
Me
Me
diast. mix
diast. mix
diast. mix
9.1
8.9
9.2
8.9
9.4
9.2
9.4
9.5
<6
9.0
10.0
9.5
9.9
9.8
9.6
9.5
9.0
4.3
3.9
4.6
4.6
3.4
2.9
3.7
3.8
7.3
7.6
6.5
7.7
8.2
8.2
9.2
bond
−SO2−
−CH2−
diast. mix
diast. mix
diast. mix
linkers between the phenylglycine amine and the shared ring,
and one is derived from carbamate scaffold 2 (Figure 3).
Pleasingly, compounds 25, 33, 87a, and 47b all demon-
strated good affinity for the M3 receptor coupled with good
inhibition of PDE4 in a LARBS assay. Based on this promising
initial result, the compounds were assessed in the isolated
guinea pig trachea (GPT) assay, measuring the inhibition of
the contraction of the isolated tissue caused by carbachol
(Cch) (Table 1). It was hypothesized that the poor translation
from M3 binding affinity to functional efficacy in GPT with at
least a 40-fold reduction may be due to the physical properties
of the molecules. The less lipophilic 3,4-dimethoxycatechol
analogues of the four MAPI compounds were synthesized to
investigate the impact of a reduced lipophilicity on GPT
functional efficacy.
The dimethoxy catechol-containing analogues 86a, 24, 32,
and 46b in Table 1 retained a good affinity for the M3 receptor
coupled with good inhibition of PDE4 and showed a reduced
drop-off between the M3 binding and GPT assays.
Optimization of Phenylcarbamate Series. Compounds
containing a carbamate M3 fragment showed a better chemical
stability in comparison with their phenylglycine analogues,
several of which displayed a significant reduction in chemical
purity when stored as solid over a 2 month time period, mainly
due to hydrolysis of the quinuclidine ester bond in the M3
fragment. The first round of optimization was therefore
undertaken using the carbamate M3 fragment, coupled with a
PDE4 fragment containing a dimethoxy catechol substituent.
At this point in the project, a technically simpler PDE4 assay
was also introduced: the cell-free activity of the compounds
was determined against human recombinant PDE4B2 protein
where, in general, MAPI compounds showed a lower level of
inhibition compared to the original LARBS assay. A PDE4 cell-
based assay was added to the cell-free assay: the best
compounds were progressed to the in vitro evaluation of
activity in peripheral blood mononuclear cells (PBMCs)
measuring the inhibition of (LPS)-induced tumor necrosis
factor-alpha (TNF-α) release.
We focused on the optimization of the linking ring of
phenylcarbamate series, synthesizing a set of derivatives that
retain the (R)-quinuclidinyl-phenylcarbamate and dimethox-
yphenyl-dichloropyridine oxide portions whilst varying the
substitution pattern around the phenyl linking ring and the
linking ring itself (Table 2).
Both meta- and para-substitution of the linking phenyl ring
in 46b and 46a was tolerated at the muscarinic receptor with
an improvement in PDE4 inhibitory activity for para-
substitution. Fluorine introduction on the phenyl linking ring
of 46a and 46b resulted in the retention of M3 activity and the
improvement of PDE4 cell-based activity for compounds 46d
and 46e. Introduction of a methylene between the linking ring
and the PDE4 ester fragment showed a good M3 affinity
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Table 2. Biological Data of Compounds 46a−e and 60
Table 3. Biological Data of Analogues of 60 with Variations
in the Non-linking M3 Ring
Cmp
Ar
M3 pKi GPT pIC50 PDE4B2 pIC50 PBMC pIC50
60
57
61
58
59
62
63
64
Ph
2-F-Ph
3-F-Ph
3-Py
10.0
9.9
9.7
8.1
9.3
9.1
8.5
9.5
8.4
9.1
8.1
9.0
9.3
9.0
9.1
8.9
9.2
2-Py
8.7
8.4
8.8
9.0
9.2
9.2
9.5
9.1
9.3
9.3
9.7
9.4
2-thiazolyl
3-F2CH-Ph
3-HO-Ph
analogue 62 did not show the same drop in muscarinic
affinity. The analogues of 57 with alternative substitution
patterns on the catechol fragment (69−72 in Table 4)
confirmed the earlier hypothesis around the impact of the
physical properties on the translation from binding affinity to
functional efficacy.
without any improvement in PDE4B2 inhibition for com-
pound 46c. When the phenyl ring was replaced with a series of
five- and six-membered heterocycles a significant instability of
the PDE4 ester fragment was observed (data not shown).
However, the introduction of a 2,5-disubstituted thiophene as
linking ring resulted in a stable compound: 60 showed a good
in vitro activity and retained efficacy in the GPT assay.
Table 4. Biological Data, Calculated logD, and Kinetic
Solubility of Compounds 57 and 69−72
Fluorophenyl 46d and thiophene 60 showed the best overall
in vitro profiles. Based on the observation that thiophene 60
demonstrated a better chemical stability over time than
fluorophenyl derivatives, the next stage of optimization was
undertaken using thiophene as the linking ring.
To investigate the importance of the non-linking M3 ring, a
series of carbamate derivatives with different M3 rings was
targeted (Table 3). Introduction of a fluorine onto the phenyl
ring of 60 was tolerated (57 and 61), whilst introduction of
larger substituents such as methoxy or cyano were not well
tolerated, resulting in a drop in affinity for the M3 receptor
(data not shown). It was hypothesized that introduction of a
hydroxy group at the ortho-position may enable a hydrogen-
bonding interaction with the M3 receptor, but both the 2-
substituted phenol and the corresponding benzyl alcohol
demonstrated a rapid degradation, with the hydroxyl group
cyclizing onto the carbamate to release (R)-quinuclidin-3-ol.
In contrast, the meta-substituted phenol 64 was stable and
demonstrated good affinity for the M3 receptor coupled with a
good inhibition of PDE4B2, which translated into a good
efficacy in the GPT and PBMC assays. The bioisosteric meta-
difluoromethyl analogue 63 showed a significant drop-off in
the affinity for the M3 receptor in comparison with 64,
although it regained efficacy in the GPT assay. Introduction of
a basic 3-pyridyl group as the non-linking M3 ring (58)
resulted in a significant drop in affinity for the M3 receptor. In
contrast, the 2-pyridyl analogue 59 and the 2-thiazolyl
M3
GPT
clogD
(7.4)
kinetic solubility
Cmp
R1/R2
Me/Me
Me/CHF2
Et/CHF2
iPr/CHF2
cPrCH2/CHF2
pKi
pIC50
(μM)
57
69
70
71
72
9.9
9.6
9.5
9.5
9.7
9.1
8.2
8.3
7.5
8.2
3.4
3.5
3.9
4.2
4.2
134
<5
<5
<5
<5
Replacement of the methoxy group at the 4-position of the
catechol in 57 with a difluoromethoxy group to give 69
resulted in an increase in the drop-off between the M3 binding
and the GPT assays. Increasing the lipophilicity of the
substituent at the 3-position of the catechol resulted in a
corresponding increase in the clogD, and the compounds (70−
72) showed negligible kinetic solubility in phosphate-buffered
saline solution (PBS) and a large drop-off in the GPT assay.
At this stage, a set of compounds were next evaluated in an
in vivo model of bronchoconstriction (Table 5). The
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Table 5. Bronchodilator Effect of Compounds 57, 59, and
60
Table 6. Biological Data for Compounds 89, 90, 91a−c, and
92a−c
bronchodilator effect bronchodilator effect
M3
pKi
GPT
PDE4B2
pIC50
PBMC
pIC50
M3
GPT in vivo ED50 (nmol/ @ 1/16 h (%) (dose
Cmp
R1/R2 isomer
pIC50
Cmp Ar
pKi
pIC50
kg) @ 1 h
in nmol/kg)
89
90
Fast
Slow
9.8
9.7
9.9
10.2
10.0
9.8
9.0
9.1
8.9
9.0
8.6
8.5
8.4
8.1
8.8
8.8
9.2
9.2
8.7
8.5
8.9
8.6
8.5
8.8
9.0
8.9
8.5
8.7
60 Ph
10.0
9.9
8.4
9.1
1.6
0.5
82/− (3)
97/38 (3)
57 2-F-
91a
92a
91b
92b
91c
92c
H/H
H/H
H/F
H/F
Me/H
Me/H
Fast
Slow
Fast
Slow
Fast
Slow
Ph
59 2-Py
9.3
8.7
78/− (10)
9.2
9.2
compounds were dosed by solution via intra-tracheal
administration to anesthetized guinea pigs and their impact
on bronchoconstriction induced by acetylcholine (ACh) was
tested.
When tested at 3 nmol/kg, both 60 and 57 showed good
and dose-dependent inhibition of bronchoconstriction at 1 h
timepoint, giving ED50 values of 1.6 and 0.5 nmol/kg,
respectively. The 2-pyridyl analogue 59 displayed only 78%
inhibition of bronchoconstriction at 1 h timepoint when dosed
at 10 nmol/kg. Based on this data, the duration of action of 57
was investigated; disappointingly, the compound showed only
modest levels of inhibition 16 h after dosing.
Our focus at this point returned to MAPI compounds
derived from a phenylglycine muscarinic fragment, in particular
to the benzylamine-linked compounds, such as 87a containing
a second, mildly basic, amine functionality in addition to
quinuclidine basic center. Our aim was to verify if the presence
of the second basic center could improve the duration of action
after inhalation.9
Optimization of Phenylglycine Series. The cross-over
analogues of the best carbamate derivatives, 60 and 57, were
synthesized as a racemic mixture and then separated by chiral
SFC into the single diastereomers, identified on the basis of the
elution order as a Fast isomer or Slow isomer.
All the four compounds 91a, 92a, 91b, 92b demonstrated
good affinity for the M3 receptor coupled with good inhibition
of PDE4B2, and these data translated into moderate to good
efficacy in both the GPT and PBMC assays (Table 6). Capping
of the phenylglycine nitrogen atom in 91a and 92a with a
methyl group resulted in compounds 91c and 92c showing a
conserved PDE4 activity but a clear drop in M3 affinity. For
this reason, other N-methyl derivatives were not investigated.
The diastereoisomers from each pair displayed similar in
vitro profiles, as was also observed for 89 and 90, the two single
diastereoisomers of the phenyl linked analogue 86a shown in
Table 1.
To allow for investigation of the balance of in vivo activity at
the two pharmacological targets in the same species, it was
decided to run both in vivo assays in rat at this stage. To
support this testing paradigm, 91a and 92a were tested in a rat
trachea (RT) assay measuring, as for the corresponding assay
in guinea pig, the inhibition of the contraction of the isolated
tracheal tissue caused by carbachol and a good correlation was
observed between the data in the two species. Indeed 91a
showed RT pIC50 = 8.7 (GPT IC50 = 8.9) and 92a showed RT
pIC50 = 9.0, the same value obtained in GPT assay. Based on
this good correlation, the RT assay replaced the GPT assay at
this step in the project cascade.
The importance of the (R)-quinuclidinol head group found
in all the compounds made up until this point in the program
was investigated, replacing this group by a number of
alternative amine-containing head groups (Table 7). The
diastereomeric compounds in each case showed the same or
only marginally different PDE4 profile both in cell-free and
cell-based assay, but a significant difference in the affinity at the
M3 receptor was observed in contrast with the previous pairs of
diastereomers, reported in Table 6, containing an (R)-
quinuclidinol head group. For all of the non-(R)-quinuclidinol
head groups, one diastereomer showed an M3 affinity that was
significantly higher than the other. This difference in affinity
was carried through into the RT assay, except for 91f, which
regained activity in the rat trachea compared with M3 affinity.
At this stage, compounds that demonstrated an pIC50 ≥ 8.5
in both the rat trachea and hPBMC assays were evaluated in an
in vivo model of bronchoconstriction in rat. Compounds 91a,
92a, 92e, and 92f were dosed by solution via intra-trachea (IT)
administration to an anesthetized rat, and their impact on
bronchoconstriction induced by carbachol was assessed at 1 h
after dosing (Table 8). All compounds demonstrated an ED50
below 10 nmol/kg in this assay and were progressed to
investigation of the bronchodilator effect at 16 h, each
compound dosed at a concentration where at least 70%
reduction of bronchoconstriction was observed at 1 h
The thiophene-linked phenylglycine series, showing an
improved chemical stability overtime in comparison with the
phenyl-linked phenylglycine series, was progressed to further
investigation.
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and inflammation assays, even if the 40:1 ratio of ED50s
between the OVA and bronchoconstriction assays raised
concerns around the ability of the compound to display a
clinical effect without effectively overdosing the compound at
one of the target proteins. Indeed, both pharmacological
activities are relevant for COPD, where maintaining airway
caliber and counteracting inflammation are primary therapeutic
objectives. For this reason, it is necessary that both
pharmacophores of the preferred bifunctional compound are
effective on their respective targets over similar concentrations
in vivo.
Table 7. Biological Data for Compounds 91a, 91d−j, 92a,
and 92d−j
The in vivo anti-inflammatory activity, 40-fold lower than
bronchodilating activity, cannot be attributed to a difficulty of
compound 92a entering the inflammatory cells; indeed, the
apical to basolateral apparent permeability (Papp A-B) value
measured in Caco2 cells (Table 11) can be classified as a value
of medium permeability. The ability of 92a compound to cross
the cells is also confirmed in a PBMC in vitro test, where the
pIC50 data is comparable to that observed in cell-free PDE4B2.
At this stage of the project, the key factors for a balanced in
vivo activity remained unexplained.
The ADME profile of 92a (Table 11) showing high levels of
clearance in microsomes and a reduced stability in plasma
proved to be in alignment with the aim of limiting the systemic
exposure in order to reduce the risk of potential systemic side
effects.
The emetic side effect, well known for most of the PDE4
inhibitors, was investigated for compound 92a after intra-
tracheal administration in ferret, considered a current gold
standard to study the nausea and emetic effect of a
compound,12 at two level doses, 1 and 10 μmol/kg. The 1
μmol/kg a dose produced a sub-maximal anti-inflammatory
activity in the rat ovalbumin model of lung inflammation (ED50
in OVA model = 30 nmol/kg) and was compared with the oral
compound roflumilast at 10 μmol/kg, a dose associated with a
sub-maximal efficacy in the rat ovalbumin model (data not
shown) (Figure 4).
The mean of nausea-like score for the reference compound
showed a significant difference in comparison with the vehicle
(72.50 18.55 for roflumilast vs 12.83 5.10 for the vehicle),
whereas compound 92a showed at both doses a mean nausea-
like score much lower than the reference compound and,
importantly, without statistically significant differences from
the vehicle and between the two tested doses (30.67 6.13 for
compound 92a 1 μmol/kg; 19.83 6.48 for compound 92a 10
timepoint. Only (R)-quinuclidin-3-ol 92a was observed to
show greater than 50% inhibition at 16 h timepoint.
The sustained duration of action of 92a in the animal model
was confirmed by in vitro investigation on association and
dissociation kinetics from the M3 receptor in comparison with
the muscarinic antagonists long-acting tiotropium10 and short-
acting ipratropium.11 Association and dissociation t1/2 values,
obtained from the association and dissociation rate constants,
were very similar for compound 92a and tiotropium with a
dissociation t1/2 much higher than ipratropium (Table 9), so
suggesting that the observed long duration of action can be
attributed to the off-rate at the receptor. Selectivity vs receptor
M2 was also investigated at this stage: the lack of selectivity for
compound 92a was not considered a critical point, being
typical of most of the M3 antagonists, tiotropium included.10
Compound 92a was next tested in a rat OVA-induced
airway inflammation model (Table 10), where it showed an
ED50 of 30 nmol/kg, with about 90% reduction of eosinophils
at 1 μmol/kg, and a good duration of action when dosed at 1
μmol/kg (Table 10).
μmol/kg and 12.83
5.10 for the vehicle). Notably,
compound 92a was devoid of gastrointestinal side effects at
both dose levels, therefore also at a dose that exceeds the
pharmacologically active doses by a factor 10, whereas
roflumilast induced emesis and nausea at a pharmacological
relevant dose.
The absolute configuration of compound 92a was
determined by single-crystal high-resolution X-ray diffraction.
The configuration of stereocenters in the PDE4 alcohol and
quinuclidinol portions were known, respectively, S and R.
Based on that information, the single-crystal structure (CSD
code: 2035364) allowed to attribute the configuration R to the
stereocenter in the phenylglycine portion.
A preliminary evaluation of the developability of 92a as a dry
powder inhaler (DPI) was also started, but significant
hydrolysis of the quinuclidine ester fragment was observed
for all the identified solid forms when stored in accelerated
conditions, suggesting a potential issue for further progression.
Compound 92a achieved the goal of demonstrating efficacy
in vivo and duration of action in both the bronchoconstriction
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Table 8. Bronchodilator Effect of Compounds 91a, 92a, 92e, and 92f
Table 9. M3/M2 Binding and M3 Binding Kinetics of
Compound 92a vs Tiotropium, Ipratropium
M3/M2
pKi
M3 association t1/2
M3 dissociation t1/2
Cmp
92a
tiotropium
(min)
(min)
10.2/9.7
9.9/9.8
9.1/9.1
822
618
9.4
1768
1350
20.1
ipratropium
Table 10. Anti-inflammatory Effect of Compound 92a
eosinophilia
reduction in OVA
eosinophilia reduction
PDE4B2 PBMC model ED50 (nmol/ in OVA model @ 24 h
Figure 4. Evaluation of nausea-like score of 92a compared to
roflumilast. Differences in the mean of nausea-like scores were
assessed with Kruskal−Wallis one-way analysis of variance on ranks
followed by Dunn’s test. A level of probability of 0.05 or less was
accepted as significant.
Cmp
pIC50
pIC50
kg)
(%) (dose)
92a
8.8
8.8
30
54 (1 μmol/kg)
Table 11. ADME Profile of Compound 92a
Kin.
Solub.
(μM)
PPB (%
bound)
h/r
Plasma stab.
t1/2 (min)
h/r
Mics. Clint
Caco2
(nm/s)
Papp A-B
(μL/min/
The synthetic pathway for the synthesis of aniline derivatives
24 and 25 is depicted in Scheme 3. Chiral alcohols 11 and 12
were condensed with 3-nitrobenzoic acid 17 and then
reduction of the nitro group with tin(II) chloride led to the
amine intermediates 20 and 21, which underwent a multi-
component Petasis reaction involving phenylboronic acid and
glyoxylic acid. The obtained α-amino acids 22 and 23 were
directly coupled with (R)-quinuclidin-3-ol using the standard
DCC/HOBt procedure to afford anilines 24 and 25.
The synthesis of sulfonamides 32 and 33 is shown in
Scheme 4. Sulfonylation of tert-butyl 2-amino-2-phenylacetate
27 with methyl 3-(chlorosulfonyl)benzoate gave sulfonamide
28. Sulfonamides 30 and 31 were prepared by hydrolysis of
methyl ester 28 under basic conditions followed by coupling
with the suitable enantiomerically pure alcohols 11 or 12 in the
presence of EDC and DMAP. Acidic removal of the tert-butyl
group followed by DCC/HOBt coupling with (R)-quinuclidin-
3-ol afforded the final compounds 32 and 33.
Cmp
mg) h/r
92a
145
93/91.40
11/9
213/126
16
Combined with the non-optimal balance at the two
pharmacological targets, this finding resulted in the progression
of 92a being halted.
CHEMISTRY
■
The synthetic pathway employed for the preparation of the
PDE4 catechol fragment is shown in Schemes 1 and 2. Alcohol
11 was prepared following a similar synthetic approach used
for the preparation of (S)-alcohol 12.6 The absolute
configuration (S) of alcohol 11 was confirmed by the X-ray
of the solid crystals of alcohol 10 (data not shown). Acidic
cleavage of the cyclopropylmethyl group in 12 followed by
alkylation of the catecholic hydroxyl group with K2CO3 and
the appropriate alkyl iodide reagent led to compounds 14, 15,
and 16 (Scheme 2).
Carbamates 46a−e and 47b synthesized to explore the
linking ring SAR were prepared following the synthetic
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Scheme 1. Preparation of (S)-Alcohol 11
a
Reagents and conditions: (a) 1 N LHMDS, THF, −78 °C to rt, 1 h; (b) DMAP, EDC, DMF, rt, 2 h; (c) crystallization in CHCl3, MeOH, 2 h;
(d) t-BuOK, MeOH, toluene, rt, 24 h; (e) m-CPBA, EtOAc, rt, 5 h.
Scheme 2. Preparation of (S)-Alcohols 14−16
a
Reagents and conditions: (a) 37% HCl, rt, 3 min; (b) K2CO3, DMF, R1I, rt, 4 h.
Scheme 3. General Route for the Preparation of MAPI Compounds with Aniline Saffold
a
Reagents and conditions: (a) 11 or 12, EDC, DMAP, DCM, rt, 16 h; (b) SnCl2·2H2O, THF, 75 °C, 16 h; (c) phenylboronic acid, glyoxylic acid,
DCM, rt, 16 h; (d) (R)-quinuclidin-3-ol, DCC, HOBt, THF, rt, 16 h.
approach outlined in Scheme 5. Reductive amination of
commercially available carboxaldehydes 34a−c with aniline led
to amines 36a−c. Amine 36d was prepared via bromine
displacement of 35d with N-Boc-aniline in the presence of
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Scheme 4. General Route for the Preparation of MAPI Compounds with Sulfonamide Scaffold
a
Reagents and conditions: (a) isobutylene, sulfuric acid, 1,4-dioxane, rt, 48 h; (b) methyl 3-(chlorosulfonyl)benzoate, Et3N, THF, rt, 30 min; (c) 1
N LiOH, THF, MeOH, rt, 18 h; (d) 11 or 12, EDC·HCl, DMAP, DMF, rt, 18 h; (e) TFA, DCM, 0 °C, 2.5 h to rt, 0.5 h; (f) (R)-quinuclidin-3-ol,
DCC, HOBt, THF, rt, 18 h.
Scheme 5. General Route for the Preparation of MAPI Compounds with Carbamate Scaffold
a
Reagents and conditions: (a) (L = a−c) Aniline, AcOH, rt, 64 h then NaBH(OAc)3, rt, 2 h; (b) (L = d) Aniline, K2CO3, DMF, 60 °C, 18 h; (c)
trichloromethyl chloroformate, CH3CN, 0 °C, 1 h then rt, 16 h; (d) for the preparation of 39a and 39b: 38, pyridine, 0 °C to rt, 48 h; for the
preparation 39c and 39d: 38, DMAP, pyridine, rt, 48 h; (e) LiOH·H2O, THF, H2O, rt, 18 h; (f) 11, EDC·HCl, DMAP, DMF, rt, 18 h; (g) 11 or
12, EDC·HCl, DMAP, DCM, rt, 18 h; (h) aniline, AcOH, DCM, rt, 6−18 h then NaBH(OAc)3, rt, 18 h; (i) 38, CH3CN, microwave 80 °C, 15
min.
potassium carbonate followed by cleavage of the Boc group
under acidic conditions. Final carbamates 46a−d were
prepared by reaction of amines 36a−d with key chloroformate
intermediate 38 followed by hydrolysis of the ester group and
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Scheme 6. General Route for the Preparation of MAPI Compounds with Carbamate Scaffold
a
Reagents and conditions: (a) 5-formyl-2-thiophenecarboxylic acid, EDC·HCl, DMAP, DCM, rt, 18 h; (b) CyNH2, AcOH, DCM, rt, 6−18 h then
NaBH(OAc)3, rt, 18 h; (c) 38, CH3CN, microwave 80 °C.
EDC/DMAP coupling with enantiomerically pure alcohol 11.
A partially modified synthetic route was followed for
compounds 46e and 47b in which enantiomerically pure
alcohols 11 or 12 were coupled with commercially available
acids 41b and 41e followed with reductive amination with
aniline and carbamate formation using chloroformate inter-
mediate 38. In Scheme 5, the intermediate 42b is described to
be used in Scheme 7.
Carbamate 57, with a thiophene as the linking ring, was
prepared following a similar approach from aldehyde 48 via
reductive amination with the appropriate cyclic amine followed
by carbamate formation (Scheme 6). The analogous
compounds 58−64 and 69−72, with the same linking ring,
synthesized to explore the M3 non-linking ring and the
cathecolic substitution, were analogously prepared (Scheme 6).
The synthetic pathway for the preparation of MAPI
compounds with a phenylglycine scaffold is outlined in
Scheme 7. Boc-protected amino acids 73−75 were coupled
with the suitable alcohol 76−83 in the presence of DCC and
HOBt, and the subsequent Boc cleavage followed by a
reductive amination reaction led to phenyl glycine analogues
86a, 87a, and 88a−j as a mixture of two diastereoisomers. 86a
and 88a−j were separated by chiral SFC to afford single
diastereoisomers 89, 90, 91a−j, and 92a−j (Scheme 8).
PDE4 activity in vitro and efficacy in vivo in both
bronchoconstriction and inflammation assays. Our strategy
differed from the MAPIs reported by UCB, which combined
both activities in a shared scaffold, and from the GSK MAPI
series, which contains a linker between M3 and PDE4 portions,
in that the two pharmacophoric fragments overlapped through
a common linking ring.
We showed that the introduction of a second basic center
into our MAPI molecules could improve the duration of action
in the bronchoconstriction assay, and optimization of the
benzylamine-linked series led to 92a. The in vitro pharmaco-
logical characterization of 92a demonstrated that this
compound has low nanomolar potency for both M3 muscarinic
receptor and PDE4B2 isoenzyme. Functional potency of 92a
evaluated in rat isolated tracheal strips and in human PBMCs
was in the low nanomolar range in both assays with no drop-off
in potency compared with cell-free IC50 values. A key factor for
bifunctional compounds such as MAPI molecules is that a
balanced pharmacological profile at the two targets in vitro
translates into a bronchodilator and anti-inflammatory activity
that can be achieved in a similar dose range in relevant in vivo
models. Experiments performed in the rat looked at
bronchodilator and anti-inflammatory activity in two separate
models to investigate potency and duration of action of 92a
using the same formulation and methodology of inhaled
administration. The compound displayed full efficacy and a
sustained duration of action in both the bronchoconstriction
and OVA assays; however, the ED50 for anti-inflammatory
activity was 40-fold higher than the one for the bronchodilator
CONCLUSIONS
■
We have demonstrated that the PDE4 scaffold structurally
related to CHF-6001 can be combined with a muscarinic
antagonist scaffold to yield compounds that display dual M3/
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Scheme 7. General Route for the Preparation of MAPI Compounds with Phenylglycine Scaffold
a
Reagents and conditions: (a) DCC, HOBt, THF, rt, 18 h; (b) for the preparation of 85a,b: 2 N HCl in diethylether, 1,4-dioxane, rt, 20 h; for the
preparation of 85c−j: 4 N HCl in 1,4-dioxane, 1,4-dioxane, rt, 20 h; (c) 42b or 43b or 48, CH3CN, AcOH, rt, 20 h then NaBH(OAc)3, rt, 18 h.
Scheme 8. Preparation of Single Diastereoisomers 89, 90, 91a−j and 92a−j
a
Reagents and conditions: (a) Chiral SFC or chiral HPLC.
effect, meaning that a significant and sustained anti-
inflammatory activity may not be achieved within the window
of doses for bronchodilation. These data raised concerns not
only for the effectiveness of the compound but also for its
safety profile since the need of overdosing to reach a significant
local PDE4 inhibition may determine the appearance of
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gradients were selected based on acetonitrile and methanol solvent
systems under either acidic or basic conditions. The modifiers used
under acidic/basic conditions were formic acid or trifluoroacetic acid
(0.1% v/v) and ammonium bicarbonate (10 mM), respectively. The
purification was controlled by Waters Fractionlynx software through
monitoring at 210−400 nm and triggered a threshold collection value
at 260 nm and, when using the Fractionlynx, the presence of target
molecular ion as observed under APi conditions. Collected fractions
were analyzed by LCMS (Waters Acquity systems with Waters SQD).
The diastereomeric separation of compounds was achieved either by
chiral high-performance liquid chromatography (HPLC) using a
Gilson Trilution preparative HPLC system (322 pump, 155 UV/VIS,
GX281 liquid handler and fraction collector) or by supercritical luid
chromatography (SFC) using a Waters Thar Prep100 preparative
SFC system (P200 CO2 pump, 2545 modifier pump, 2998 UV/VIS
detector, 2767 liquid handler with stacked injection module). The
Waters 2767 liquid handler acted as both an auto-sampler and fraction
collector. The column used for the preparative purification of the
compounds was a Diacel Chiralpak IA/IB or an YMC Amylose-C at 5
μm 250 × 20−21.2 mm ID. Appropriate isocratic methods were
selected based on methanol, ethanol, or isopropanol solvent systems
under un-modified or basic/acidic conditions. The standard SFC
method used was modifier, CO2, 100 mL/min, 120 bar backpressure,
and 40 °C column temperature. The standard HPLC method used
was modifier, heptane, 5 mL/min, and room temperature. The
modifier used under basic conditions was diethylamine (0.1% v/v).
The modifier used under acidic conditions was either formic acid
(0.1% v/v) or trifluoroacetic acid (0.1% v/v). Chiral analysis methods
1−10 are described in the Supporting Information.
unwanted systemic side effects due to muscarinic antagonism.
These considerations, coupled with the developability
evaluation, which introduced questions about the chemical
stability of the compound, caused that 92a was not progressed
any further, and the MAPI program was continued to address
the key issues of compound 92a; among them, the need of
balancing the bronchodilator/anti-inflammatory profile, while
minimizing side effects, proved to be particularly challenging to
govern and quite unpredictable because a number of factors,
including in vitro affinities on M3 and PDE4 targets and
pharmacokinetic and physicochemical properties, contribute to
in vivo efficacy. The knowledge gained during the investigation
described in the present paper was the basis for the
identification of MAPI compounds chemically stable and
pharmacologically balanced, which will be subject of forth-
coming publication.
EXPERIMENTAL SECTION
■
Animals. Male albino Dunkin-Hartley guinea pigs (450−550 g),
male CD Sprague Dawley rats (220−250 g), and male Brown-Norway
rats (150−200 g) were obtained from Charles River Laboratories
Italia (Calco, Italy), while male Putorious Furo ferrets (0.9−1.5 kg)
were obtained from Marshall Ferrets France. Animals housed in
plastic cages (Tecniplast Gazzada, Varese, Italy) in air-conditioned
rooms at 22 °C in a 12 h light/dark cycle. Food and water were
available at libitum. All animals were acclimatized for at least 5 days
before any experimental work began. All the experiments were carried
out in accordance with the national and European legislation and
approved by local ethical committees.
All compounds submitted for biological screening had a purity
>95%.
Chemistry. All reagents and starting materials were obtained from
The preparation of intermediates 11, 14, 15, 16, 29, 39a−d, and
85a−j, the analytical characterization of intermediates 19, 21, 23, 31,
42b, 42e, 43b, 44e, 45b, 65−68, and 50−55, LCMS and SFC-MS
traces for compounds 92e, 92f, 91a, and 92a, and the crystal structure
determination of 92a are reported in the Supporting Information.
Preparation of Final Compounds 24 and 25. [(1S)-2-(3,5-
Dichloro-1-oxido-pyridin-1-ium-4-yl)-1-(3,4-dimethoxyphenyl)-
ethyl] 3-Nitrobenzoate (18). To a stirred solution of (1S)-2-(3,5-
dichloro-1-oxido-pyridin-1-ium-4-yl)-1-(3,4-dimethoxyphenyl)-
ethanol 11 (0.500 g, 1.45 mmol) and 3-nitrobenzoic acid (0.242 g,
1.45 mmol) in DCM (15.0 mL) was added 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (0.560 g, 2.91 mmol) and N,N-
dimethylpyridin-4-amine (0.089 g, 0.73 mmol). The reaction was
stirred at room temperature for 16 h. The reaction was quenched by
addition of saturated aqueous sodium bicarbonate solution (25.0 mL)
and extracted with DCM (2 × 60 mL). The combined organic
extracts were dried on magnesium sulfate and filtered, and the solvent
was removed in vacuo to afford a yellow oil. The crude material was
purified by chromatography on silica gel, eluting sequentially with
isohexane, ethyl acetate, and 10% methanol in ethyl acetate to afford
the title compound as a yellow solid (0.642 g, 90% yield).
commercial suppliers and used without further purification unless
otherwise stated. H nuclear magnetic resonance (NMR) spectros-
1
copy was carried out using a Bruker instrument operating at 400 MHz
using the stated solvent at around room temperature unless otherwise
stated. In all cases, NMR data were consistent with the proposed
structures. Characteristic chemical shifts (δ) are given in parts per
million using conventional abbreviations for designation of major
peaks: s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet of
doublets; dt, doublet of triplets; m, multiplet; br, broad. UV purity
and [MH+] of compounds were assessed by LCMS analysis
performed on a Waters 2795 Alliance HT HPLC with Waters 2996
Diode Array Detector coupled to a Micromass ZQ, single quadrupole
mass spectrometer using a Phenomenex Luna C18 (2) column (5 μm,
100 × 4.6 mm plus guard cartridge) with a linear gradient of 5−95%
acetonitrile/water (with 0.1% formic acid in each mobile phase)
within 3.5 min and held at 95% for 2.0 min or using a Waters Xterra
MS C18 column (5 μm, 100 × 4.6 mm plus guard cartridge) initially
held at 5% acetonitrile/water (with 10 mM ammonium bicarbonate)
for 0.5 min followed by a linear gradient of 5−95% within 3.5 min and
then held at 95% for 1.5 min. Alternatively, UV purity and [MH+] of
compounds were assessed by UPLC-MS analysis performed on a
Waters UPLC H Class with a Diode Array Detector coupled to a
Waters XEVO-TQS, triple quadrupole mass spectrometer with an
electrospray ionization (ESI) source, using a Acquity UPLC CSH
C18 1.7 μm 50 × 2.10 with mobile phase A consisting of ammonium
formate buffer (25 mM at pH 3) and mobile phase B consisting of
0.1% formic acid in acetonitrile. The flow rate was 0.35 mL/min, and
the gradient started from 20% to 80% of mobile phase B in 5.5 min
followed by an isocratic step at 80% for 2 min. Preparative HPLC
purification was performed by reversed-phase HPLC using a Waters
Fraction Lynx preparative HPLC system (2525 pump, 2996/2998
UV/VIS detector, 2767 liquid handler) or an equivalent HPLC
system such as a Gilson Trilution UV directed system. The Waters
2767 liquid handler acted as both an auto-sampler and fraction
collector. The columns used for the preparative purification of the
compounds were a Waters Sunfire OBD Phenomenex Luna Phenyl
Hexyl or Waters Xbridge Phenyl at 10 μm 19 × 150 mm or Waters
CSH Phenyl Hexyl, 19 × 150, 5 μm column. Appropriate focused
1H NMR (400 MHz, CDCl3): δ 8.89 (d, J = 2.0 Hz, 1 H), 8.44−
8.41 (m, 1 H), 8.33−8.30 (m, 1 H), 8.14 (s, 2 H), 7.63 (t, J = 8.0 Hz,
1 H), 7.06−7.03 (m, 2 H), 6.88 (d, J = 8.4 Hz, 1 H), 6.30 (dd, J = 9.6,
4.4 Hz, 1 H), 3.92 (s, 3 H), 3.90 (s, 3 H), 3.78 (dd, J = 9.6, 14.0 Hz, 1
H), 3.38 (dd, J = 14.0, 4.4 Hz, 1 H). LCMS: [MH+] = 493.
[(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-ium-4-yl)-1-(3,4-
dimethoxyphenyl)ethyl] 3-Aminobenzoate (20). To a stirred
solution of [(1S)-2-(3,5-dichloro-1-oxido-pyridin-1-ium-4-yl)-1-(3,4-
dimethoxyphenyl)ethyl] 3-nitrobenzoate 18 (0.640 g, 1.30 mmol) in
anhydrous THF (30.0 mL) was added tin chloride dihydrate (1.18 g,
5.2 mmol). After heating at 75 °C for 16 h, the reaction was allowed
to cool to room temperature. The reaction was partitioned between
ethyl acetate and saturated aqueous sodium bicarbonate solution and
extracted with ethyl acetate (2 × 60 mL). The combined organic
extracts were dried on magnesium sulfate and filtered, and the solvent
was removed in vacuo to afford the title compound as a brown solid
(0.520 g, 87% yield).
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1H NMR (400 MHz, DMSO-d6): δ 8.58 (s, 2 H), 7.20−7.15 (m, 3
H), 7.04−6.98 (m, 3 H), 6.83−6.81 (m, 1 H), 6.22−6.19 (dd, J = 4.4,
9.6 Hz, 1 H), 5.39 (s, 2 H), 3.82 (s, 3 H), 3.78 (s, 3 H), 3.63−3.59
(dd, J = 9.6, 14.0 Hz, 1 H), 3.33−3.29 (m, 1 H). LCMS: [MH+] =
463.
hydrochloride (1.192 g, 6.22 mmol), and the reaction was stirred at
room temperature for 18 h. After this time, the reaction mixture was
concentrated under reduced pressure and water (30 mL) was added;
the resulting off-white precipitate was filtered and dried in air. The
crude material was purified by chromatography on silica gel, eluting
with 0−100% EtOAc in isohexane to give the title compound as a
white solid (1.34 g, 60%).
2-[3-[(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-ium-4-yl)-1-(3,4-
dimethoxyphenyl)ethoxy]carbonylanilino]-2-phenylacetic Acid
(22). To a stirred solution of [(1S)-2-(3,5-dichloro-1-oxido-pyridin-
1-ium-4-yl)-1-(3,4-dimethoxyphenyl)ethyl] 3-aminobenzoate 20
(0.850 g, 1.83 mmol) in DCM (20 mL) was added phenylboronic
acid (0.225 g, 1.83 mmol) and glyoxylic acid (205 μL, 1.83 mmol).
The reaction was stirred at room temperature for 16 h. The reaction
was quenched by addition of water and extracted with DCM (2 × 60
mL). The combined organic extracts were dried on magnesium sulfate
and filtered, and the solvent was removed in vacuo. A small sample of
crude residue (90 mg) was purified by preparative HPLC to provide
the title compound as a white solid (33.6 mg). The remaining amount
of crude residue (0.940 g) was taken on to the next step without
further purification.
1H NMR (400 MHz, CDCl3): δ 8.33 (dt, J = 7.9, 1.6 Hz, 1 H),
8.18 (s, 1 H), 8.16 (s, 1 H), 8.19−8.05 (m, 1 H), 7.90−7.82 (m, 1 H),
7.43 (q, J = 7.6 Hz, 1 H), 7.21−7.08 (m, 5 H), 7.07−6.98 (m, 2 H),
6.88 (d, J = 8.3 Hz, 1 H), 6.26 (dd, J = 9.5, 4.8 Hz, 1 H), 6.07 (dd, J =
33.2, 7.9 Hz, 1 H), 4.96 (t, J = 7.8 Hz, 1 H), 3.93 (s, 2H), 3.92 (s, 1
H), 3.89 (s, 3 H), 3.8−3.69 (m, 1 H), 3.39 (dt, J = 14.0, 4.2 Hz, 1 H),
1.22 (s, 6 H), 1.21 (s, 3 H). LCMS: [MH+] = 717.
[(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-ium-4-yl)-1-(3,4-
dimethoxyphenyl)ethyl] 3-[[2-Oxo-1-phenyl-2-[(3R)-quinuclidin-3-
yl]oxyethyl]sulfamoyl]benzoate (32). Trifluoroacetic acid (5.0 mL)
was added to a stirred solution of [(1S)-2-(3,5-dichloro-1-oxido-
pyridin-1-ium-4-yl)-1-(3,4-dimethoxyphenyl) ethyl] 3-[(2-tert-bu-
toxy-2-oxo-1-phenyl-ethyl)sulfamoyl]benzoate 30 (1.0 g, 1.39
mmol) in DCM (5.0 mL) at 0 °C, and stirring was maintained at 0
°C for 2.5 h. The reaction mixture was then stirred at ambient
temperature for 30 min, after which time toluene (50 mL) was added
and the solvent removed in vacuo to yield a yellow solid. The crude
intermediate 2-[[3-[(1S)-2-(3,5-dichloro-1-oxido-pyridin-1-ium-4-yl)-
1-(3,4-dimethoxyphenyl)ethoxy]-carbonylphenyl]sulfonylamino]-2-
phenylacetic acid was dissolved in THF (20 mL), and N,N′-
dicyclohexylcarbodiimide (431 mg, 2.09 mmol), 1-hydroxybenzo-
triazole hydrate (282 mg, 2.09 mmol), and (R)-quinuclidin-3-ol (355
mg, 2.79 mmol) were sequentially added. The resulting reaction
mixture was stirred at ambient temperature for 18 h, after which time
the reaction mixture was filtered through a pad of Celite, washing with
THF (10 mL). The filtrate was concentrated in vacuo, the resulting
crude was partitioned between ethyl acetate (20 mL) and water (10
mL), the organic phases were dried over magnesium sulfate, filtered,
and concentrated in vacuo. The resulting crude was triturated in
diethyl ether and purified by preparative HPLC to afford the title
compound as a pale yellow solid (17.9 mg, 2%).
1H NMR (400 MHz, DMSO-d6): δ 8.54*or† (s, 2 H), 8.53*or† (s,
2H), 7.51−7.49 (m, 2 H), 7.39−7.35 (m, 2 H), 7.32−7.30 (m, 1 H),
7.26−7.24 (m, 1 H), 7.18−7.16 (m, 2 H), 7.01−6.92 (m, 4 H), 6.68−
6.51 (brs, 1 H), 6.18−6.14 (m, 1 H), 5.08 (s, 1H), 3.59 (s, 3 H), 3.58
(s, 3 H), 3.56−3.51 (m, 1 H), 3.32−3.27 (m , 1 H). OH not
observed. † and * refer to different isomers. LCMS: [MH+] = 597.
[(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-ium-4-yl)-1-(3,4-
dimethoxyphenyl)ethyl] 3-[[2-Oxo-1-phenyl-2-[(3R)-quinuclidin-3-
yl]oxyethyl]amino]benzoate (24). To a stirred solution of previously
obtained crude 22 (0.940 g, 1.57 mmol) in anhydrous THF (30.0
mL) was added (R)-quinuclidin-3-ol (0.300 g, 2.35 mmol), N,N′-
dicyclohexylcarbodiimide (0.375 g, 1.80 mmol), and 1-hydroxybenzo-
triazole (0.245 g, 1.80 mmol). The reaction was stirred at room
temperature under nitrogen for 16 h. The reaction was filtered
through fine sintered glass, and the filtrate was removed in vacuo. The
residue was partitioned between ethyl acetate and saturated aqueous
sodium bicarbonate solution and extracted with ethyl acetate (2 × 50
mL). The combined organic layers were dried on magnesium sulfate
and filtered, and the solvent was removed in vacuo. The residue was
purified by preparative HPLC to afford the title compound 24 as a
white solid (118 mg, 9% yield over two steps).
1H NMR (400 MHz, CDCl3): δ 8.30 (s, 1 H), 8.16 (s, 2 H), 8.06
(d, J = 7.4 Hz, 1 H), 7.82 (d, J = 8.0 Hz, 1 H), 7.42 (t, J = 8.0 Hz, 1
H), 7.20−7.08 (m, 5 H), 7.07−6.98 (m, 2 H), 6.88 (d, J = 8.5 Hz, 1
H), 6.26 (dd, J = 9.6, 4.8 Hz, 1 H), 5.10 (s, 1 H), 4.72−4.62 (m, 1
H), 3.94 (s, 3H), 3.89 (s, 3 H), 3.73 (dd, J = 14.0, 9.6 Hz, 1 H), 3.39
(dd, J = 14.0, 4.7 Hz, 1 H), 2.99 (ddd, J = 13.0, 8.1, 2.0 Hz, 1 H),
2.74−2.53 (m, 3 H), 2.48−2.35 (m, 1 H), 2.23 (d, J = 14.8 Hz, 1 H),
1.90−1.81 (m, 1 H) 1.76−1.2 (m, 4 H). LCMS: [MH+] = 770.
[(1S)-1-[3-(Cyclopropylmethoxy)-4-(difluoromethoxy)phenyl]-2-
(3,5-dichloro-1-oxido-pyridin-1-ium-4-yl)ethyl] 3-[[2-Oxo-1-phe-
nyl-2-[(3R)-quinuclidin-3-yl]oxyethyl]sulfamoyl]benzoate (33).
Compound 33 was synthesized following the same procedure
described for the preparation of 32 using enantiomerically pure
alcohol 12 instead of 11 via intermediate 31.
1H NMR (400 MHz, DMSO-d6): δ 8.61*or† (s, 2 H), 8.57*or† (s, 2
H), 7.57 (d, J = 7.5 Hz, 2 H), 7.47−7.31 (m, 4 H), 7.28−7.20 (m, 2
H), 7.06−6.97 (m, 4 H), 6.78−6.71 (m, 1 H), 6.21 (dd, J = 9.4, 4.7
Hz, 1 H), 5.37−5.29 (m, 1 H), 4.80−4.71 (m, 1 H), 3.83−3.76 (m, 6
H), 3.65−3.56 (m, 1 H), 3.18−3.10 (m, 1 H), 3.07−2.96 (m, 1 H),
2.68−2.57 (m, 3 H), 2.32−2.22 (m, 1 H), 2.11 (d, J = 14.5 Hz, 1 H),
1.93 (s, 1 H), 1.68−1.60 (m, 1 H), 1.60−1.54 (m, 1 H), 1.53−1.43
(m, 1 H), 1.34−1.17 (m, 1 H). † and * refer to different isomers.
LCMS: [MH+] = 706.
[(1S)-1-[3-(Cyclopropylmethoxy)-4-(difluoromethoxy)phenyl]-2-
(3,5-dichloro-1-oxido-pyridin-1-ium-4-yl)ethyl] 3-[[2-Oxo-1-phe-
nyl-2-[(3R)-quinuclidin-3-yl]oxyethyl]amino]benzoate Trifluoroa-
cetate Salt (25). Compound 25 was synthesized following the same
procedure described for the preparation of 24 using enantiomerically
pure alcohol 12 instead of 11 via intermediates 19, 21, and 23.
1H NMR (300 MHz, DMSO-d6): δ 9.36 and 9.50 (br. s, 1 H), 8.54
(s, 2 H), 7.49−7.68 (m, 3 H), 7.12−7.49 (m, 6 H), 6.90−7.12 (m, 3
H), 7.07 (t, 1 H), 6.75 (m, 1 H), 6.19 (dd, 1 H), 5.35 and 5.40 (d, 1
H), 4.95−5.13 (m, 1 H), 3.92 and 3.93 (d, 2H), 3.48−3.77 (m, 2 H),
3.01−3.29 (m, 4 H), 2.67−2.80 (m, 1 H), 2.54−2.61 (m, 1 H), 1.94−
2.08 and 2.20−2.26 (m, 1 H), 1.61−1.93 (m, 3 H), 1.36−1.57 (m, 1
H), 1.08−1.36 (m, 1 H), 0.45−0.70 (m, 2 H), 0.25.0.45 (m, 2 H).
LCMS [MH+] = 782.
lH NMR (300 MHz, DMSO-d6) δ ppm 9.52 and 9.59 (br. s., 1 H),
9.14 (d, 1 H), 8.55 and 8.57 (s, 2 H), 8.23 (t,1 H), 8.07 and 8.14 (dt,
1 H), 7.93 and 8.01 (dt, 1 H), 7.58 and 7.66 (t, 1 H), 7.09−7.32 (m, 8
H), 7.08 (t, 1 H), 6.10−6.30 (m, 1 H), 5.15 and 5.19 (d, 1 H), 4.81−
4.99 (m, 1 H), 3.95 (d, 2 H), 3.46−3.75 (m, 2 H), 3.30−3.45 (m, 1
H), 2.74−3.27 (m, 5 H), 1.89−1.98 and 1.98−2.10 (m, 1 H), 0.99−
1.87 (m, 5 H), 0.47−0.66 (m, 2 H), 0.30−0.42 (m, 2 H). MS/ESI+
[MH+] = 846.
Preparation of Final Compounds 46a−d. [(1S)-2-(3,5-
Dichloro-1-oxido-pyridin-1-ium-4-yl)-1-(3,4-dimethoxyphenyl)-
ethyl] 4-[(N-[(3R)-Quinuclidin-3-yl]oxycarbonylanilino)methyl]-
benzoate (46a). To a stirred solution of methyl 4-[(N-[(3R)-
quinuclidin-3-yl]oxycarbonylanilino)methyl]benzoate 39a (0.451 g,
1.144 mmol) in THF (5.8 mL) and methanol (5.8 mL) was added a
solution of lithium hydroxide monohydrate (0.096 g, 2.29 mmol) in
water (2.3 mL). The reaction was stirred rapidly at room temperature
for 18 h. The mixture was cooled using an ice bath and acidified by
dropwise addition of concentrated hydrochloric acid (0.46 mL, 5.52
Preparation of Final Compounds 32 and 33. [(1S)-2-(3,5-
Dichloro-1-oxido-pyridin-1-ium-4-yl)-1-(3,4-dimethoxyphenyl)-
ethyl] 3-[(2-tert-Butoxy-2-oxo-1-phenyl-ethyl)sulfamoyl]benzoate
(30). 3-[(2-tert-Butoxy-2-oxo-1-phenyl-ethyl)sulfamoyl]benzoic acid
29 (1.216 g, 3.11 mmol) was added to a stirred suspension of (1S)-2-
(3,5-dichloro-1-oxido-pyridin-1-ium-4-yl)-1-(3,4-dimethoxyphenyl)-
ethanol 11 (1.07 g, 3.11 mmol) in DMF (12 mL). To the resultant
solution was added DMAP (0.189 g, 1.55 mmol) followed by EDC
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mmol). The mixture was allowed to warm to room temperature, and
then the solvent was removed in vacuo (3 × toluene azeotrope
followed by 2 × acetonitrile azeotrope), and the residue was dried in
the vacuum oven at 40 °C to afford a pale yellow solid. The crude 4-
[(N-[(3R)-quinuclidin-3-yl]oxycarbonylanilino)methyl]benzoic acid
40a was dissolved in DMF (5 mL) and added to a stirred suspension
of (1S)-2-(3,5-dichloro-1-oxido-pyridin-1-ium-4-yl)-1-(3,4-
dimethoxyphenyl)ethanol 11 (0.394 g, 1.144 mmol) in DMF (12
mL). To the resultant solution was added DMAP (0.070 g, 0.572
mmol) followed by EDC hydrochloride (0.439 g, 2.288 mmol), and
the reaction was stirred at room temperature for 18 h. The majority of
DMF was removed in vacuo, and the residue was partitioned between
ethyl acetate and saturated aqueous sodium bicarbonate solution. The
organic layer was washed with brine, and the solvent was removed in
vacuo to afford an off-white solid. The crude material was partially
purified using an SCX-2 cartridge eluting sequentially with 1:1:1
methanol:acetonitrile:water and then methanol and 2.3 M methanolic
ammonia. Final purification was achieved by preparative HPLC to
afford the title compound as a pale yellow solid (0.240 g, 30%).
1H NMR (400 MHz, CDCl3): δ 8.13 (s, 2 H), 7.97 (d, J = 8.0 Hz,
2 H), 7.35−7.21 (m, 5 H), 7.10 (br s, 2 H), 7.04−6.95 (m, 2 H), 6.85
(d, J = 8.2 Hz, 1 H), 6.30 (dd, J = 9.7, 4.6 Hz, 1 H), 4.95−4.89 (m, 1
H), 4.89 (s, 2 H), 3.90 (s, 3 H), 3.88 (s, 3 H), 3.71 (dd, J = 14.0, 9.7
Hz, 1 H), 3.40−3.28 (m, 2 H), 2.90 (t, J = 9.1 Hz, 2 H), 2.81 (d, J =
18.1 Hz, 2 H), 2.73−2.59 (m, 1 H), 2.16−2.10 (m, 1 H), 1.84−1.73
(m, 1 H), 1.72−1.60 (m, 1 H), 1.55−1.45 (m, 1 H), 1.44−1.33 (m, 1
H). LCMS: [MH+] = 706.
between DCM and saturated aqueous sodium bicarbonate solution.
The organic layer was washed with brine and passed through a
hydrophobic frit, and the solvent was removed in vacuo. The crude
material was purified by chromatography on silica gel, eluting with 0−
100% EtOAc in DCM, to afford the title compound (488 mg, 39%) as
a yellow oil.
1H NMR (400 MHz, CDCl3): δ 9.97 (s, 1 H), 8.15 (s, 2 H), 7.81
(d, J = 3.6 Hz, 1 H), 7.72 (d, J = 3.6 Hz, 1 H), 7.03−6.99 (m, 2 H),
6.87 (d, J = 8.7 Hz, 1 H), 6.26 (dd, J = 4.4, 10.0 Hz, 1 H), 3.91 (s, 3
H), 3.88 (s, 3 H), 3.72 (dd, J = 10.0, 14.0 Hz, 1 H), 3.33 (dd, J = 4.4,
14.0 Hz, 1 H). LCMS: [MH+] = 482.
Intermediates 42b, 42e, and 43b were synthesized following the
same procedure of 48 using the suitable formyl acid.
Preparation of Intermediates 44e, 45b, 49, 50−55, and 65−
68. [(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-ium-4-yl)-1-(3,4-
dimethoxyphenyl)ethyl] 5-[(2-Fluoroanilino)methyl]thiophene-2-
carboxylate (49). To a stirred solution of [(1S)-2-(3,5-dichloro-1-
oxido-pyridin-1-ium-4-yl)-1-(3,4-dimethoxyphenyl)ethyl] 5-formylth-
iophene-2-carboxylate 48 (128 mg, 0.265 mmol) in DCM (5 mL) was
added 2-fluoroaniline (29 mg, 0.265 mmol) followed by glacial acetic
acid (0.015 mL, 0.265 mmol). The reaction was stirred at room
temperature for 6 h. Sodium triacetoxyborohydride (140 mg, 0.662
mmol) was added, and the reaction was stirred at room temperature
for 18 h. Water was added to quench the reaction, and the organic
layer was washed with brine and passed through a hydrophobic frit,
and the solvent was removed in vacuo. The crude material was purified
by chromatography on silica gel, eluting with 0−100% EtOAc in
DCM, to afford the title compound (50 mg, 32%) as a yellow oil.
1H NMR (400 MHz, CDCl3): δ 8.11 (s, 2 H), 7.65 (d, J = 4.0 Hz,
1 H), 7.02−6.95 (m, 5 H), 6.84 (d, J = 8.0 Hz, 1 H), 6.71−6.65 (m, 2
H), 6.18 (dd, J = 4.4, 9.6 Hz, 1 H), 4.57−4.56 (m, 2 H), 4.51−4.48
(m, 1 H), 3.87 (s, 3 H), 3.86 (s, 3 H), 3.64 (dd, J = 9.6, 14.0 Hz, 1
H), 3.29 (dd, J = 4.4, 14.0 Hz, 1 H). LCMS: [MH+] = 577.
Intermediates 44e, 45b, and 50−55 were synthesized following the
same procedure of intermediate 49 from intermediate 48 using
suitable anilines.
Intermediates 65−68 were synthesized in two steps reacting
alcohols 12, 14, 16, 18, and 5-formyl-2-thiophenecarboxylic acid, as
described for intermediate 48 and then the formyl derivatives
obtained with 2-fluoroaniline, as described for intermediate 49.
Preparation of Final Compounds 46e, 47b, 57, 69−72, 58−
63, and 64. [(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-ium-4-yl)-1-
(3,4-dimethoxyphenyl)ethyl] 5-[(2-Fluoro-N-[(3R)-quinuclidin-3-yl]-
oxycarbonylanilino)methyl]thiophene-2-carboxylate Formate Salt
(57). A microwave tube was charged with [(1S)-2-(3,5-dichloro-1-
oxido-pyridin-1-ium-4-yl)-1-(3,4-dimethoxyphenyl)ethyl] 5-[(2-
fluoroanilino)methyl]thiophene-2-carboxylate 49 (50 mg, 0.09
mmol), [(3R)-quinuclidin-3-yl] carbonochloridate hydrochloride
(81 mg, 0.36 mmol), and anhydrous acetonitrile (0.8 mL). The
mixture was heated at 80 °C for 3 min under microwave irradiation.
The reaction was evaporated to dryness. The residue was dissolved in
DMSO (1.5 mL) and purified by preparative HPLC to afford the tittle
compound as a white solid (26 mg, 41%).
Compounds 46b−d were synthesized following the same
procedure described for the preparation of compound 46a, using
intermediates 39b−d.
[(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-ium-4-yl)-1-(3,4-
dimethoxyphenyl)ethyl] 3-[(N-[(3R)-Quinuclidin-3-yl]-
1
oxycarbonylanilino)methyl]benzoate (46b). H NMR (400 MHz,
CDCl3): δ 8.11 (s, 2 H), 7.93−7.91 (m, 2 H), 7.48−7.46 (m, 1 H),
7.40−7.36 (m, 1 H), 7.32−7.22 (m, 3 H), 7.14−7.12 (m, 2 H), 7.00−
6.96 (m, 2 H), 6.86−6.84 (m, 1 H), 6.27 (dd, J = 9.6, 4.4 Hz, 1 H),
4.89 (s, 2 H), 4.82−4.81 (m, 1 H), 3.89 (s, 3 H), 3.87 (s, 3 H), 3.68
(dd, J = 14.0, 9.6 Hz, 1 H), 3.33 (dd, J = 14.0, 4.4 Hz, 1 H), 3.22−
3.18 (m, 1H), 2.76−2.62 (m, 5 H), 1.97−1.96 (m, 1 H), 1.65−1.62
(m, 1 H), 1.55−1.54 (m, 1 H), 1.40−1.39 (m, 1 H), 1.25−1.21 (m, 1
H). LCMS: [MH+] = 706
[(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-ium-4-yl)-1-(3,4-
dimethoxyphenyl)ethyl] 2-[4-[(N-[(3R)-Quinuclidin-3-yl]-
oxycarbonylanilino)methyl]phenyl]acetate (46c). 1H NMR (400
MHz, DMSO-d6): δ 8.54 (s, 2 H), 8.31 (s, 1 H), 7.34−7.30 (m, 2 H),
7.26−7.21 (m, 2 H), 7.20−7.12 (m, 4 H), 6.93−6.90 (m, 1 H), 6.83−
6.81 (m, 2 H), 5.95 (dd, J = 9.6, 4.8 Hz, 1 H), 4.87 (s, 2 H), 4.67−
4.64 (m, 1 H), 3.74 (s, 3 H), 3.70 (s, 3 H), 3.64−3.54 (m, 2 H),3.49−
3.35 (m, 2 H), 3.11−3.05 (m, 1 H), 2.67−2.47 (m, 5 H), 1.88−1.83
(m, 1 H), 1.58−1.52 (m, 1 H), 1.50−1.39 (m, 1 H), 1.37−1.22 (m, 1
H), 1.19−1.12 (m, 1 H). LCMS: [MH+] = 720
[(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-ium-4-yl)-1-(3,4-
dimethoxyphenyl)ethyl] 2-Fluoro-4-[(N-[(3R)-quinuclidin-3-yl]-
1H NMR (400 MHz, CDCl3): δ 8.40 (s, 1 H), 8.15 (s, 2 H), 7.59
(d, J = 3.6 Hz, 1 H), 7.32−7.28 (m, 1 H), 7.15−7.11 (m, 3 H), 6.99−
6.96 (m, 2 H), 6.86−6.84 (m, 2 H), 6.20 (dd, J = 4.8, 10.0 Hz, 1 H),
4.95−4.93 (m, 3 H), 3.90 (s, 3 H), 3.88 (s, 3 H), 3.66 (dd, J = 10.0,
14.0 Hz, 1 H), 3.38−3.28 (m, 2 H), 2.97−2.91 (m, 3 H), 2.83−2.61
(m, 2 H), 2.18−2.16 (m, 1 H), 1.84−1.79 (m, 1 H), 1.78−1.70 (m, 1
H), 1.50−1.37 (m, 2 H). LCMS: [MH+] = 730.
1
oxycarbonylanilino)methyl]benzoate (46d). H NMR (400 MHz,
CDCl3): δ 8.13 (s, 2 H), 7.85 (t, J = 7.7 Hz, 1 H), 7.33 (t, J = 7.6 Hz,
2 H), 7.28−6.93 (m, 6 H), 6.84 (d, J = 8.3 Hz, 1 H), 6.33 (dd, J = 9.1,
5.1 Hz, 1 H), 4.94−4.88 (m, 1 H), 4.86 (d, J = 6.8 Hz, 2 H), 3.91 (s, 3
H), 3.88 (s, 3 H), 3.67 (dd, J = 13.9, 9.1 Hz, 1 H), 3.35 (dd, J = 13.9,
5.0 Hz, 1 H), 3.30 (ddd, J = 14.8, 8.2, 2.2 Hz, 1 H), 2.87 (t, J = 7.8
Hz, 3 H), 2.78 (d, J = 14.8 Hz, 1 H), 2.71−2.62 (m, 1 H), 2.13−2.08
(m, 1 H), 2.05−1.71 (m, 2 H), 1.69−1.58 (m, 1 H), 1.54−1.44 (m, 1
H), 1.44−1.33 (m, 1 H). LCMS: [MH+] = 724.
Preparation of Intermediates 42b, 42e, 43b, and 48. [(1S)-2-
( 3 , 5 - D i c h l o r o - 1 - o x i d o - p y r i d i n - 1 - i u m - 4 - y l ) - 1 - ( 3 , 4 -
dimethoxyphenyl)ethyl] 5-Formylthiophene-2-carboxylate (48). To
a stirred solution of 5-formyl-2-thiophenecarboxylic acid (400 mg,
2.56 mmol) in DCM (20 mL) was added (1S)-2-(3,5-dichloro-1-
oxido-pyridin-1-ium-4-yl)-1-(3,4-dimethoxyphenyl)ethanol 11 (881
mg, 2.56 mmol) followed by DMAP (156 mg, 1.28 mmol) and
EDC hydrochloride (983 mg, 5.12 mmol). The resulting mixture was
stirred at room temperature for 18 h. The reaction was partitioned
Compounds 46e, 47b, 69−72, and 58−63 were synthesized
following the same procedure using the intermediates 44e, 45b, 65−
68, and 50−55.
[(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-ium-4-yl)-1-(3,4-
dimethoxyphenyl)ethyl] 4-Fluoro-3-[(N-[(3R)-quinuclidin-3-yl]-
1
oxycarbonylanilino)methyl]benzoate (46e). H NMR (400 MHz,
CDCl3): δ 8.10−8.06 (m, 3 H), 7.95−7.91 (m, 1 H), 7.33−7.29 (m, 2
H), 7.24−7.18 (m, 3 H), 7.05 (t, J = 8.9 Hz, 1 H), 6.99−6.95 (m, 2
H), 6.85 (d, J = 8.0 Hz, 1 H), 6.25 (dd, J = 4.4, 9.6 Hz, 1 H), 4.97 (s,
2 H), 4.79−4.77 (m, 1 H), 3.88 (s, 3 H), 3.87 (s, 3 H), 3.66 (dd, J =
9.6, 14.0 Hz, 1 H), 3.33 (dd, J = 4.4, 14.0 Hz, 1 H), 3.17−3.15 (m, 1
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H), 2.72−2.59 (m, 5 H), 2.00−1.92 (m, 1 H), 1.60−1.57 (m, 1 H),
1.52−1.47 (m, 1 H), 1.42−1.31 (m, 1 H), 1.24−1.17 (m, 1 H).
LCMS: [MH+] = 724
(m, 1 H), 7.86−7.82 (m, 1 H), 7.75−7.73 (m, 1 H), 7.66 (d, J = 3.7
Hz, 1 H), 7.23−7.20 (m, 1 H), 7.10 (d, J = 3.7 Hz, 1 H), 6.99−6.96
(m, 3 H), 6.11 (dd, J = 4.1, 9.7 Hz, 1 H), 5.37−5.28 (m, 2 H), 4.78−
4.76 (m, 1 H), 3.75 (s, 3 H), 3.74 (s, 3 H), 3.56 (dd, J = 9.7, 14.2 Hz,
1 H), 3.33−3.28 (m, 1 H), 3.18−3.13 (m, 1 H), 2.80−2.63 (m, 5 H),
1.96−1.95 (m, 1 H), 1.67−1.61 (m, 1 H), 1.59−1.51 (m, 2 H), 1.30−
1.29 (m, 1 H). LCMS: [MH+] = 713.
[(1S)-1-[3-(Cyclopropylmethoxy)-4-(difluoromethoxy)phenyl]-2-
(3,5-dichloro-1-oxido-pyridin-1-ium-4-yl)ethyl] 3-[(N-[(3R)-Quinu-
clidin-3-yl]oxycarbonylanilino)methyl]benzoate (47b). 1H NMR
(300 MHz, DMSO-d6) δ 8.51 (s, 2 H), 7.81−7.94 (m, 2 H), 7.54
(dt, 1 H), 7.48 (t, 1 H), 7.30−7.44 (m, 2 H), 7.17−7.30 (m, 5 H),
7.05 (dd, 1 H), 7.06 (t, 1 H), 6.21 (dd, 1 H), 4.88−5.04 (m, 2 H),
4.63−4.82 (m, 1 H), 3.92 (d, 2 H), 3.59 (dd, 1 H), 3.31−3.40 (m, 1
H), 3.08−3.26 (m, 1 H), 2.56−2.85 (m, 5 H), 1.77−1.99 (m, 1 H),
1.42−1.75 (m, 2 H), 1.10−1.42 (m, 3 H), 0.44−0.68 (m, 2 H), 0.22−
0.44 (m, 2 H). MS/ESI+ [MH+] = 782.
[(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-ium-4-yl)-1-(3,4-
dimethoxyphenyl)ethyl] 5-[(N-[(3R)-Quinuclidin-3-yl]-
oxycarbonylanilino)methyl]thiophene-2-carboxylate Formate Salt
1
(60). H NMR (400 MHz, CDCl3): δ 8.42 (s, 1 H), 8.15 (s, 2 H),
7.60 (d, J = 3.8 Hz, 1 H), 7.39−7.32 (m, 2 H), 7.29 (d, J = 7.2 Hz, 1
H), 7.17−7.06 (m, 2 H), 7.00−6.95 (m, 2 H), 6.85 (dd, J = 5.9, 2.2
Hz, 2 H), 6.21 (dd, J = 9.8, 4.5 Hz, 1 H), 5.02−4.93 (m, 2 H), 4.94−
4.87 (m, 1 H), 3.90 (s, 3 H), 3.88 (s, 3 H), 3.67 (dd, J = 14.0, 9.8 Hz,
1 H), 3.37−3.27 (m, 1 H), 3.31 (dd, J = 13.9, 4.9 Hz, 1 H), 2.92−
2.84 (m, 3 H), 2.79 (d, J = 15.5 Hz, 1 H), 2.74−2.62 (m, 1H), 2.17−
2.07 (m, 1 H), 1.82−1.72 (m, 1 H), 1.71−1.58 (m, 1 H), 1.56−1.31
(m, 2 H). LCMS: [MH+] = 712.
[(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-ium-4-yl)-1-(3,4-
dimethoxyphenyl)ethyl] 5-[(3-Fluoro-N-[(3R)-quinuclidin-3-yl]-
oxycarbonylanilino)methyl]thiophene-2-carboxylate (61). 1H
NMR (400 MHz, CDCl3): δ 8.15 (s, 2 H), 7.61 (d, J = 3.8 Hz, 1
H), 7.34 (td, J = 8.2, 6.2 Hz, 1 H), 7.05−6.94 (m, 3 H), 6.95−6.88
(m, 1 H), 6.91−6.80 (m, 3 H), 6.21 (dd, J = 9.8, 4.5 Hz, 1 H), 5.04−
4.97 (m, 1 H), 4.98−4.91 (m, 2 H), 3.91 (s, 3 H), 3.88 (s, 3 H), 3.68
(dd, J = 14.0, 9.8 Hz, 1 H), 3.45−3.38 (m, 1 H), 3.32 (dd, J = 14.0,
4.6 Hz, 1 H), 3.08−2.88 (m, 3 H), 2.90−2.76 (m, 2 H), 2.29−2.21
(m, 1 H), 1.91−1.86 (m, 1 H), 1.82−1.69 (m, 1 H), 1.68−1.46 (m, 2
H). LCMS: [MH+] = 730.
[(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-ium-4-yl)-1-(3,4-
dimethoxyphenyl)ethyl] 5-[[[(3R)-Quinuclidin-3-yl]oxycarbonyl-
thiazol-2-yl-amino]methyl]thiophene-2-carboxylate (62). 1H
NMR (400 MHz, CDCl3): δ 8.13 (s, 2 H), 7.61 (d, J = 3.8 Hz, 1
H), 7.49 (d, J = 3.6 Hz, 1 H), 7.09 (d, J = 3.7 Hz, 1 H), 7.01 (d, J =
3.6 Hz, 1 H), 6.98−6.93 (m, 2 H), 6.84 (d, J = 8.1 Hz, 1 H), 6.18 (dd,
J = 9.8, 4.5 Hz, 1 H), 5.52 (s, 2 H), 4.97 (s, 1 H), 3.88 (s, 3 H), 3.87
(s, 3 H), 3.64 (dd, J = 14.0, 9.8 Hz, 1 H), 3.33 (dd, J = 14.9, 8.6 Hz, 1
H), 3.29 (dd, J = 14.0, 4.3 Hz, 1 H), 2.92−2.73 (m, 5 H), 2.19−2.14
(m, 1 H), 1.80−1.69 (m, 1 H), 1.66−1.53 (m, 1 H), 1.52−1.40 (m, 1
H), 1.31−1.23 (m, 1 H). LCMS: [MH+] = 719.
[(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-ium-4-yl)-1-(3,4-
dimethoxyphenyl)ethyl] 5-[[3-(Difluoromethyl)-N-[(3R)-quinucli-
din-3-yl]oxycarbonylanilino]methyl]thiophene-2-carboxylate For-
mate Salt (63). 1H NMR (400 MHz, CDCl3): δ 8.41 (s, 1 H),
8.15 (s, 2 H), 7.61 (d, J = 3.8 Hz, 1 H), 7.48−7.39 (m, 2 H), 7.33 (s,
1 H), 7.26 (s, 1 H), 7.01−6.95 (m, 2 H), 6.88−6.81 (m, 2 H), 6.64 (t,
J = 56.3 Hz, 1 H), 6.21 (dd, J = 9.8, 4.5 Hz, 1 H), 5.02−4.90 (m, 3
H), 3.90 (s, 3 H), 3.88 (s, 3 H), 3.67 (dd, J = 14.5, 9.7 Hz, 1 H),
3.39−3.26 (m, 2 H), 2.99−2.71 (m, 5 H), 2.14 (s, 1 H), 1.82−1.76
(m, 1 H), 1.72−1.60 (m, 1 H), 1.63−1.34 (m, 2 H). LCMS: [MH+]
= 762
[(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-ium-4-yl)-1-(3,4-
dimethoxyphenyl)ethyl] 5-[(3-Hydroxy-N-[(3R)-quinuclidin-3-yl]-
oxycarbonylanilino)methyl]thiophene-2-carboxylate (64). To a
solution of 3-aminophenol (0.67 g, 6.15 mmol) in hexamethyldisi-
lazane (10 mL) was added a catalytic amount of concentrated sulfuric
acid (0.05 mL), and the mixture was heated at reflux for 18 h. The
mixture was cooled to room temperature, and the excess solvent was
removed in vacuo. Trituration with diethyl ether gave a precipitate,
which was filtered, and the filtrate was evaporated in vacuo. The crude
was purified by chromatography on silica gel, eluting with 0−100%
EtOAc in isohexane to give 3-((trimethylsilyl)oxy)aniline as a red-
brown oil (782 mg, 70%, LCMS: [MH+] = 182). A mixture of 3-
((trimethylsilyl)oxy)aniline (0.14 g, 0.77 mmol) and [(1S)-2-(3,5-
dichloro-1-oxido-pyridin-1-ium-4-yl)-1-(3,4-dimethoxyphenyl)ethyl]
5-formylthiophene-2-carboxylate 48 (0.186 g, 0.39 mmol) in DCM (5
mL) was stirred at room temperature for 18 h. NaBH(OAc)3 (0.206
g, 0.97 mmol) and acetic acid (0.043 mL, 0.75 mmol) were added,
and the reaction mixture was stirred at room temperature for a further
3 h. The reaction mixture was diluted with DCM (10 mL) and
[(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-ium-4-yl)-1-[4-(difluoro-
methoxy)-3-methoxyphenyl]ethyl] 5-[(2-Fluoro-N-[(3R)-quinucli-
din-3-yl]oxycarbonylanilino)methyl]thiophene-2-carboxylate For-
1
mate Salt (69). H NMR (400 MHz, DMSO-d6): δ 8.55 (s, 2 H),
8.20 (s, 1 H), 7.66 (d, J = 3.6 Hz, 1 H), 7.39−7.30 (m, 2 H), 7.28−
7.20 (m, 4 H), 7.07−6.88 (m, 3 H), 6.15 (dd, J = 4.0, 9.2 Hz, 1 H),
5.05−4.96 (m, 2 H), 4.71−4.67 (m, 1 H), 3.85 (s, 3 H), 3.60−3.54
(m, 1 H), 3.35−3.30 (m, 1 H), 3.16−3.13 (m, 1 H), 2.81−2.61 (m, 4
H), 2.50−2.35 (m, 1 H), 1.98−1.86 (m, 1 H), 1.67−1.40 (m, 2 H),
1.24−1.15 (m, 2 H). LCMS: [MH+] = 766.
[(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-ium-4-yl)-1-[4-(difluoro-
methoxy)-3-ethoxyphenyl]ethyl] 5-[(2-Fluoro-N-[(3R)-quinuclidin-
3-yl]oxycarbonylanilino)methyl]thiophene-2-carboxylate Formate
Salt (70). 1H NMR (400 MHz, DMSO-d6): δ 8.53 (s, 2 H), 8.20 (s, 1
H), 7.66 (d, J = 3.6 Hz, 1 H), 7.38−7.33 (m, 2 H), 7.27−7.18 (m, 4
H), 7.05−6.98 (m, 3 H), 6.13 (dd, J = 4.0, 9.2 Hz, 1 H), 5.04−4.97
(m, 2 H), 4.72−4.68 (m, 1 H), 4.15−4.06 (m, 2 H), 3.59−3.52 (m, 1
H), 3.32 (dd, J = 4.0, 14.2 Hz, 1 H), 3.15−3.12 (m, 1 H), 2.76−2.63
(m, 4 H), 2.49−2.42 (m, 1 H), 1.89−1.73 (m, 1 H), 1.65−1.42 (m, 2
H), 1.32 (t, J = 7.0 Hz, 3 H), 1.24−1.09 (m, 2 H). LCMS: [MH+] =
780
[(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-ium-4-yl)-1-[4-(difluoro-
methoxy)-3-isopropoxyphenyl]ethyl] 5-[(2-Fluoro-N-[(3R)-quinucli-
din-3-yl]oxycarbonylanilino)methyl]thiophene-2-carboxylate Tri-
1
fluoroacetate Salt (71). H NMR (400 MHz, CDCl3): δ 8.22 (s, 2
H), 7.60 (d, J = 3.8 Hz, 1 H), 7.38−7.30 (m, 1 H), 7.19−7.10 (m, 4
H), 7.05−6.98 (m, 2 H), 6.88 (d, J = 3.8 Hz, 1 H), 6.56 (t, J = 75.3
Hz, 1 H), 6.19 (dd, J = 9.9, 4.4 Hz, 1 H), 5.14−5.06 (m, 1 H), 4.97−
4.89 (m, 2 H), 4.57 (h, J = 5.96 Hz, 1 H), 3.66 (dd, J = 14.1, 10.0 Hz,
1 H), 3.62−3.54 (m, 1 H), 3.32 (dd, J = 14.5, 4.6 Hz, 1 H), 3.28−
3.14 (m, 3 H), 3.10 (d, J = 15.2 Hz, 1 H), 2.96−2.89 (m, 1 H), 2.39−
2.31 (m, 1 H), 2.02−1.93 (m, 1 H), 1.91−1.81 (m, 1 H), 1.71−1.58
(m, 2 H), 1.36 (dd, J = 11.4, 6.0 Hz, 6 H). LCMS: [MH+] = 794
[(1S)-1-[3-(Cyclopropylmethoxy)-4-(difluoromethoxy)phenyl]-2-
(3,5-dichloro-1-oxido-pyridin-1-ium-4-yl)ethyl] 5-[(2-Fluoro-N-
[(3R)-quinuclidin-3-yl]oxycarbonylanilino)methyl]thiophene-2-car-
boxylate Formate Salt (72). 1H NMR (400 MHz, CDCl3): δ 8.43 (s,
1 H), 8.16 (s, 2 H), 7.60 (d, J = 3.8 Hz, 1 H), 7.34−7.27 (m, 1 H),
7.21−7.09 (m, 4 H), 7.06−7.00 (m, 2 H), 6.87 (d, J = 3.8 Hz, 1 H),
6.62 (t, J = 75.3 Hz, 1 H), 6.18 (dd, J = 10.0, 4.2 Hz, 1 H), 4.94 (m, 3
H), 3.89 (d, J = 6.9 Hz, 2 H), 3.64 (dd, J = 14.1, 10.1 Hz, 1 H), 3.36−
3.23 (m, 3 H), 2.87 (m, 3 H), 2.76 (m, 1 H), 2.09 (s, 1 H), 1.82−1.55
(m, 2 H) 1.32−1.24 (m, 3 H), 0.68−0.62 (m, 2 H), 0.40−0.35 (m, 2
H). LCMS: [MH+] = 806
[(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-ium-4-yl)-1-(3,4-
dimethoxyphenyl)ethyl] 5-[[3-Pyridyl-[(3R)-quinuclidin-3-yl]-
oxycarbonylamino]methyl]thiophene-2-carboxylate (58). 1H
NMR (400 MHz, DMSO-d6): δ 8.56 (s, 2 H), 8.50 (brs, 1 H),
8.44 (d, J = 4.4 Hz, 1 H), 7.72−7.70 (m, 1 H), 7.66−7.65 (m, 1 H),
7.44−7.41 (m, 1 H), 7.04−6.97 (m, 4 H), 6.12 (dd, J = 4.0, 9.6 Hz, 1
H), 5.11 (brs, 2 H), 4.707−4.688 (m, 1 H), 3.77 (s, 3 H), 3.75 (s, 3
H), 3.60−3.54 (dd, J = 9.6, 14.0 Hz, 1 H), 3.31−3.26 (m, 1 H), 3.13−
3.07 (m, 1 H), 2.67−2.54 (m, 6 H), 1.81−1.79 (m, 1 H), 1.58−1.56
(m, 1 H), 1.55−1.50 (m, 1 H), 1.24−1.22 (m, 1 H). LCMS: [MH+]
= 713
[(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-ium-4-yl)-1-(3,4-
dimethoxyphenyl)ethyl] 5-[[2-Pyridyl-[(3R)-quinuclidin-3-yl]-
oxycarbonylamino]methyl]thiophene-2-carboxylate Formate Salt
1
(59). H NMR (400 MHz, DMSO-d6): δ 8.54 (s, 2 H), 8.45−8.44
9114
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washed with saturated aqueous NaHCO3 solution (25 mL). The
aqueous phase was further extracted with DCM (10 mL), and the
combined organic phases were washed with brine (20 mL) and
filtered through a phase separator cartridge, and the solvent was
removed in vacuo, co-evaporated with MeCN to give the crude [(1S)-
2-(3, 5-dichloro-1-oxido-pyridin-1-ium-4-yl)-1-(3, 4-
dimethoxyphenyl)ethyl] 5-[(3-trimethylsilyloxyanilino)methyl]-
thiophene-2-carboxylate (56) as a light brown gum. A mixture of
the crude 56 (0.185 g, 0.29 mmol) and [(3R)-quinuclidin-3-yl]
carbonochloridate (0.162 g, 0.72 mmol) in MeCN (5 mL) was heated
in a microwave at 80o C for 6 min. Additional [(3R)-quinuclidin-3-yl]
carbonochloridate (0.08 g, 0.35 mmol) was added and heated in the
microwave at 80o C for a further 6 min. The solvent was removed in
vacuo, and the residue was partitioned between EtOAc (20 mL) and
water (20 mL). The separated aqueous phase was basified by the
addition of saturated aqueous NaHCO3 solution and extracted with
EtOAc (2 × 20 mL). The organic extracts were combined and filtered
through a phase separator, and the solvent was removed in vacuo.
Purification by preparative HPLC gave the title compound as a white
solid (0.02 g, 9.6%).
7.73 (m, 8 H), 7.19−7.27 (m, 2 H), 7.10 (dd, 1 H), 7.07 (t, 1 H),
6.16−6.31 (m, 1 H), 4.98−5.19 (m, 1 H), 4.03 (br. s., 2 H), 3.93 (d, 2
H), 3.57−3.77 (m, 1 H), 3.63 (dd, 1 H), 2.78−3.30 (m, 6 H), 2.00−
2.14 and 2.32−2.45 (m, 1 H), 1.42−1.95 (m, 4 H), 1.06−1.28 (m, 1
H), 0.48−0.64 (m, 2 H), 0.23−0.45 (m, 2 H). LCMS: [MH+] = 796
[(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-ium-4-yl)-1-(3,4-
dimethoxyphenyl)ethyl] 5-[[[2-Oxo-1-phenyl-2-[(3R)-quinuclidin-3-
yl]oxyethyl]amino]methyl]thiophene-2-carboxylate (88a). 1H
NMR (400 MHz, DMSO-d6): δ 8.56 (s, 2 H), 7.69−7.68 (d, J =
3.8, 1 H), 7.45−7.28 (m, 5 H), 7.04−6.94 (m, 4 H), 6.14 (dd, J = 9.7,
4.3 Hz, 1 H), 4.73−4.65 (m, 1 H), 4.43 (d, J = 9.3, Hz, 1 H), 3.92−
3.82 (m, 2 H), 3.80−3.74 (m, 6 H), 3.66−3.54 (m, 2 H), 3.28−3.26
(m, 1 H), 3.10−3.03*or† (m, 1 H), 3.02−2.95*or† (m, 1 H), 2.63−
2.50 (m, 3 H), 2.46−2.29 (m, 1 H), 2.16 (m, 1 H), 1.89−1.84*or† (m,
1 H), 1.75−1.71*or† (m, 1 H), 1.61−1.38 (m, 3 H), 1.24−1.18 (m,1
H). † and * refer to different isomers (arbitrarily assigned). LCMS:
[MH+] = 726.
[(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-ium-4-yl)-1-(3,4-
dimethoxyphenyl)ethyl] 5-[[[1-(2-Fluorophenyl)-2-oxo-2-quinucli-
din-3-yloxyethyl]amino]methyl]thiophene-2-carboxylate (88b).
1H NMR (400 MHz, CDCl3): δ 8.14 (s, 2 H), 7.64 (dd, J = 3.8,
1.5 Hz, 1 H), 7.42−7.35 (m, 1 H), 7.35−7.29 (m, 1 H), 7.21−7.13
(m, 1 H), 7.13−7.05 (m, 1 H), 7.02−6.96 (m, 2 H), 6.92−6.88 (m, 1
H), 6.85 (d, J = 8.1 Hz, 1 H), 6.22 (dd, J = 9.7, 4.5 Hz, 1 H), 4.90−
4.79 (m, 1 H), 4.76 (d, J = 4.2 Hz, 1 H), 3.97 (s, 2 H), 3.90 (s, 3 H),
3.88 (d, J = 1.3 Hz, 3 H), 3.70−3.62 (m, 1 H), 3.35−3.28 (m, 1 H),
3.24−3.09 (m, 1 H), 2.80−2.62 (m, 5 H), 2.54−2.34 (m, 1 H), 2.01−
1.85 (m, 1 H), 1.57−1.43 (m, 2 H), 1.36−1.13 (m, 2 H). LCMS:
[MH+] = 744
1H NMR (400 MHz, DMSO-d6): δ 9.53 (s, 1 H), 8.55 (s, 2 H),
7.66 (d, J = 3.78 Hz, 1 H), 7.15 (t, J = 8.00 Hz, 1 H), 7.03−6.96 (m, 4
H), 6.70−6.61 (m, 3 H), 6.14 (dd, J = 9.71, 4.26 Hz, 1 H), 5.00 (s, 2
H), 4.68−4.63 (m, 1 H), 3.76 (d, J = 6.84 Hz, 6 H), 3.58 (dd, J =
14.23, 9.79 Hz, 1 H), 3.54 (dd, J = 14.19, 9.83 Hz, 1 H), 3.25 (dd, J =
14.20, 4.22 Hz, 1 H), 3.10 (dd, J = 14.57, 7.97 Hz, 1 H), 2.69−2.55
(m, 4 H), 1.87 (s, 1 H), 1.62−1.42 (m, 3 H), 1.21 (s, 1 H). LCMS:
[MH+] = 728.
Preparation of Final Compounds 86a, 87a, and 88a−j. [(1S)-
2-(3,5-Dichloro-1-oxido-pyridin-1-ium-4-yl)-1-(3,4-dimethoxy-
phenyl)-ethyl] 3-[[[2-Oxo-1-phenyl-2-[(3R)-quinuclidin-3-yl]-
oxyethyl]amino]methyl]benzoate (86a). To a suspension of (3R)-
quinuclidin-3-yl 2-amino-2-phenylacetate hydrochloride 85a (0.200 g,
0.600 mmol) in EtOAc (5 mL) was added Et3N (0.176 mL, 1.26
mmol). The reaction mixture was stirred at room temperature for 2 h.
The precipitate obtained was filtered and washed with EtOAc (∼5
mL), and the solvent was removed in vacuo. This residue was
dissolved in CH3CN (4 mL), and to the solution was added [(1S)-2-
(3,5-dichloro-1-oxido-pyridin-1-ium-4-yl)-1-(3,4-dimethoxyphenyl)-
ethyl] 3-formylbenzoate 42b (0.286 g, 0.6 mmol) followed by acetic
acid (0.034 mL, 0.6 mmol). The reaction mixture was stirred at room
temperature for 20 h. NaBH(OAc)3 (0.318 g, 1.5 mmol) was added,
and the reaction mixture was stirred at room temperature for a further
24 h. The excess solvent was removed in vacuo, and the residue was
partitioned between EtOAc (70 mL) and saturated aqueous NaHCO3
solution (15 mL). The organic layer was washed with saturated brine
(2 × 15 mL), dried on magnesium sulfate, and filtered, and the
solvent was removed in vacuo. Purification by preparative HPLC gave
a yellow gum (0.158 g), which was dissolved in EtOAc (25 mL),
washed with saturated aqueous NaHCO3 solution (5 mL), and
saturated brine (5 mL), dried on magnesium sulfate, and filtered. The
solvent was removed in vacuo, and trituration with diethyl ether gave
the title compound (1:1 mixture of diastereoisomers) as a yellow solid
(126.9 mg, 29%).
[(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-ium-4-yl)-1-(3,4-
dimethoxyphenyl)ethyl] 5-[[Methyl-[2-oxo-1-phenyl-2-[(3R)-quinu-
clidin-3-yl]oxyethyl]amino]methyl]thiophene-2-carboxylate (88c).
1H NMR (400 MHz, DMSO-d6): δ 8.56 (s, 2 H), 7.68 (d, J = 3.8 Hz,
1 H), 7.45−7.36 (m, 5 H), 7.04−7.03 (m, 2 H), 7.01−6.96 (m, 2 H),
6.14 (dd, J = 4.4, 9.7 Hz, 1 H), 4.83−4.76 (m, 1 H), 4.51 (d, J = 6.8
Hz, 1 H), 3.89−3.83 (m, 1 H), 3.78 (s, 3 H), 3.76 (s, 3 H), 3.75−3.72
(m, 1 H), 3.60 (dd, J = 9.9, 14.1 Hz, 1 H), 3.32−3.27 (m, 1 H), 3.16−
3.03 (m, 1 H), 2.68−2.58 (m, 4 H), 2.37−2.31 (m, 1 H), 2.22 (s, 3
H), 1.92−1.84 (m, 1 H), 1.62−1.39 (m, 3 H), 1.29−1.21 (m, 1 H).
LCMS: [MH+] =740
[(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-ium-4-yl)-1-(3,4-
dimethoxyphenyl)ethyl] 5-[[[2-[[(1R,5R)-8-Methyl-8-azabicyclo-
[3.2.1]octan-3-yl]oxy]-2-oxo-1-phenylethyl]amino]methyl]-
thiophene-2-carboxylate (88d). 1H NMR (400 MHz, CDCl3): δ
8.13 (s, 2 H), 7.64 (d, J = 3.8 Hz, 1 H), 7.37−7.32 (m, 5 H), 7.01−
6.96 (m, 2 H), 6.88−6.83 (m, 2 H), 6.26−6.20 (m, 1 H), 5.01 (s, 1
H), 4.37 (s, 1 H), 3.95−3.85 (m, 8 H), 3.70−3.63 (m, 1 H), 3.34−
3.28 (m, 1 H), 3.01 (s, 1 H), 2.88 (s, 1 H), 2.70 (s, 1 H), 2.19 (s, 3
H), 2.10 (d, J = 15.7 Hz, 1 H), 2.01 (s, 1 H), 1.90−1.80 (m, 1 H),
1.70−1.57 (m, 3 H), 1.41 (d, J = 15.2 Hz, 1 H), 1.04 (d, J = 9.7 Hz, 1
H). LCMS: [MH+] = 740
[(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-ium-4-yl)-1-(3,4-
dimethoxyphenyl)ethyl] 5-[[[2-[(1-Methyl-4-piperidyl)oxy]-2-oxo-1-
phenylethyl]amino]methyl]thiophene-2-carboxylate (88e). 1H
NMR (400 MHz, CDCl3): δ 8.14*or† (s, 2 H), 8.13*or† (s, 2 H),
7.64 (d, J = 3.8 Hz, 1 H), 7.37−7.30 (m, 5 H), 7.02−6.96 (m, 2 H),
6.91−6.83 (m, 2 H), 6.25−6.19 (m, 1 H), 4.88−4.77 (m, 1 H), 4.42
(d, J = 3.7 Hz, 1 H), 3.97−3.85 (m, 8 H), 3.67 (dd, J = 14.1, 9.7 Hz, 1
H), 3.34−3.27 (m, 1 H), 2.60−2.40 (m, 2 H), 2.30−2.15 (m, 2 H),
2.21 (s, 3 H), 1.92−1.80 (m, 2 H), 1.75−1.45 (m, 3 H), * and † refer
to different isomers. LCMS: [MH+] = 714
1H NMR (400 MHz, CDCl3): δ 8.12*or† (s, 2 H), 8.11*or† (s, 2 H),
8.00−7.97 (m, 1 H), 7.92 (d, J = 7.8 Hz, 1 H), 7.54−7.52 (m, 1 H),
7.42−7.30 (m, 6 H), 7.03−6.98 (m, 2 H), 6.85 (d, J = 8.2 Hz, 1 H),
6.30−6.28 (m, 1 H), 4.88−4.78 (m, 1 H), 4.40*or† (s, 1 H), 4.38*or†
(s, 1 H), 3.89 (s, 3 H), 3.88 (s, 3 H), 3.78−3.71 (m, 3 H), 3.38−3.34
(m, 1 H), 3.25−3.10 (m, 1 H), 2.81−2.65 (m, 4 H), 2.60−2.35 (m, 1
H), 2.00−1.88 (m, 1 H), 1.75−1.45 (m, 3 H), 1.40−1.10 (m, 2 H). *
and † refer to different isomers. LCMS: [MH+] =720.
[(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-ium-4-yl)-1-(3,4-
dimethoxyphenyl)ethyl] 5-[[[2-[(3R)-1-Methylpyrrolidin-3-yl]oxy-2-
oxo-1-phenylethyl]amino]methyl]thiophene-2-carboxylate (88f).
1H NMR (400 MHz, CDCl3): δ 8.14*or† (s, 2 H), 8.13*or† (s, 2
H), 7.63 (d, J = 3.5 Hz, 1 H), 7.38−7.33 (m, 5 H), 7.01−6.96 (m, 2
H), 6.89−6.83 (m, 2 H), 6.25−6.19 (m, 1 H), 5.26−5.17 (m, 1 H),
4.43 (s, 1 H), 3.95−3.91 (m, 2 H), 3.91*or† (s, 3 H), 3.90*or† (s, 3 H),
3.88 (s, 3 H) 3.66 (dd, J = 9.9, 13.9 Hz, 1 H), 3.35−3.27 (m, 1 H),
2.73−2.58 (m, 3 H), 2.50−2.43 (m, 1 H), 2.29−2.19 (m, 5 H), 1.88−
Compounds 87a and 88a−j were synthesized following the same
procedure described for the preparation of 86a using intermediates
85a−j and 43b or 48.
[(1S)-1-[3-(Cyclopropylmethoxy)-4-(difluoromethoxy)phenyl]-2-
(3,5-dichloro-1-oxido-pyridin-1-ium-4-yl)ethyl] 3-[[[2-Oxo-1-phe-
nyl-2-[(3R)-quinuclidin-3-yl]oxyethyl]amino]methyl]benzoate Hy-
drobromide Salt (87a). 1H NMR (300 MHz, DMSO-d6) δ ppm
9.44 and 9.55 (brs, 1 H), 8.54 (s, 2 H), 7.89−8.11 (m, 2 H), 7.37−
9115
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1.79 (m, 1 H). * and † refer to different isomers. NH not visible.
LCMS: [MH+] = 700.
[(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-ium-4-yl)-1-(3,4-
dimethoxyphenyl)ethyl] 5-[[[2-Oxo-1-phenyl-2-[[(3R)-quinuclidin-
3-yl]methoxy]ethyl]amino]methyl]thiophene-2-carboxylate (88g).
1H NMR (400 MHz, CD3CN): δ 8.19*or† (s, 2 H), 8.18*or† (s, 2 H),
7.69 (d, J = 3.8 Hz, 1 H), 7.40−7.38 (m, 5 H), 7.07−7.01 (m, 2 H),
6.97−6.93 (m, 2 H), 6.18 (dd, J = 4.5, 9.6 Hz, 1 H), 4.43 (s, 1 H),
4.15−4.02 (m, 2 H), 3.99−3.89 (m, 2 H), 3.84*or† (s, 3 H), 3.83*or†
(s, 3 H), 3.82 (s, 3 H), 3.67 (dd, J = 9.5, 14.0 Hz, 1 H), 3.34 (dd, J =
4.5, 14.1 Hz, 1 H), 2.86−2.80 (m, 2 H), 2.75−2.63 (m, 4 H), 2.29−
2.16 (m, 1 H), 1.89−1.86 (m, 1 H), 1.58−1.49 (m, 3 H), 1.44−1.39
(m, 1 H), 1.34−1.23 (m, 1 H). † and * refer to different isomers
(arbitrarily assigned). LCMS: [MH+] = 740
[(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-ium-4-yl)-1-(3,4-
dimethoxyphenyl)ethyl] 5-[[[2-[(1-Methyl-4-piperidyl)methoxy]-2-
oxo-1-phenylethyl]amino]methyl]thiophene-2-carboxylate (88h).
1H NMR (400 MHz, CDCl3 + D2O): δ 8.15*or† (s, 2 H), 8.14*or†
(s, 2 H), 7.64 (d, J = 3.8 Hz, 1 H), 7.39−7.35 (m, 5 H), 7.01−6.96
(m, 2 H), 6.89−6.84 (m, 2 H), 6.24−6.20 (m, 1 H), 4.42 (d, J = 3.0
Hz, 1 H), 3.93−3.87 (m, 10 H), 3.67 (dd, J = 9.5, 13.3 Hz, 1 H),
3.34−3.28 (m, 1 H), 2.84−2.81 (m, 2 H), 2.26 (s, 3 H), 1.91−1.83
(m, 2 H), 1.54−1.54 (m, 3 H), 1.31−1.24 (m, 2 H). † and * refer to
different isomers (arbitrarily assigned) LCMS: [MH+] = 728
[(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-ium-4-yl)-1-(3,4-
dimethoxyphenyl)ethyl] 5-[[[2-[[(2R)-1-Methylazetidin-2-yl]-
methoxy]-2-oxo-1-phenylethyl]amino]methyl]thiophene-2-carbox-
ylate (88i). 1H NMR (400 MHz, CD3CN): δ 8.19*or† (s, 2 H),
8.18*or† (s, 2 H), 7.69 (d, J = 3.8 Hz, 1 H), 7.43−7.39 (m, 5 H),
7.07−7.01 (m, 2 H), 6.97−6.94 (m, 2 H), 6.18 (dd, J = 4.5, 9.6 Hz, 1
H), 4.45 (s, 1 H), 4.22−4.12 (m, 1 H), 4.00−3.93 (m, 3 H), 3.84*or†
(s, 3 H), 3.83*or† (s, 3 H), 3.82 (s, 3 H), 3.71−3.64 (m, 1 H), 3.34
(dd, J = 4.5, 14.1 Hz, 1 H), 3.24−3.16 (m, 1 H), 3.11−3.02 (m, 1 H),
2.87 (s, 1 H), 2.73−2.64 (m, 1 H), 2.09*or† (s, 3 H), 2.09*or† (s, 3 H),
1.92−1.76 (m, 2 H). † and * refer to different isomers (arbitrarily
assigned). LCMS: [MH+] = 700.
[(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-ium-4-yl)-1-(3,4-
dimethoxyphenyl)ethyl] 5-[[[2-[2-(Dimethylamino)ethoxy]-2-oxo-
1-phenylethyl]amino]methyl]thiophene-2-carboxylate (88j). 1H
NMR (400 MHz, CDCl3): δ 8.14*or† (s, 2 H), 8.13*or† (s, 2 H),
7.64 (d, J = 3.8 Hz, 1 H), 7.38−7.35 (m, 5 H), 7.00−6.96 (m, 2 H),
6.89−6.83 (m, 2 H), 6.24−6.20 (m, 1 H), 4.42 (d, J = 2.8 Hz, 1 H),
4.23−4.09 (m, 2 H), 3.93 (d, J = 5.4 Hz, 2 H), 3.90*or† (s, 3 H),
3.90*or† (s, 3 H), 3.88 (s, 3 H), 3.66 (dd, J = 10.0, 13.8 Hz, 1 H), 3.31
(ddd, J = 2.7, 4.4, 13.9 Hz, 1 H), 2.18−2.15 (m, 2 H), 2.14 (s, 6 H),
1.75−1.69 (m, 2 H). † and * refer to different isomers (arbitrarily
assigned), NH not observed. LCMS: [MH+] = 688.
13.8, 9.6 Hz, 1 H), 3.35 (dd, J = 14.0, 4.6 Hz, 1 H), 3.19 (dd, J = 14.7,
8.4 Hz, 1 H), 2.80−2.65 (m, 4 H), 2.27−2.05 (m, 2 H), 1.89−1.83
(m, 1 H), 1.66−1.55 (m, 1 H), 1.54−1.44 (m, 1 H), 1.30−1.21 (m, 1
H), 1.21−1.10 (m, 1 H). LCMS: [MH+] = 720. Chiral analysis
(Method 3) at 6.59 min.
Single Diastereoisomer of [(1S)-2-(3,5-Dichloro-1-oxido-pyridin-
1-ium-4-yl)-1-(3,4-dimethoxyphenyl)ethyl] 5-[[(2-Oxo-1-phenyl-2-
[(3R)-quinuclidin-3-yl]oxyethyl)amino]methyl]thiophene-2-carbox-
ylate (91a, Fast Diastereoisomer). 1H NMR (400 MHz, DMSO-d6):
δ 8.56 (s, 2 H), 7.69 (d, J = 3.78 Hz, 1 H), 7.44−7.27 (m, 5 H),
7.04−6.96 (m, 4 H), 6.14 (dd, J = 9.62, 4.40 Hz, 1 H), 4.72−4.67 (m,
1 H), 4.43 (d, J = 9.25 Hz, 1 H), 3.88 (d, J = 5.70 Hz, 2 H), 3.76 (d, J
= 5.87 Hz, 6 H), 3.63−3.53 (m, 2 H), 3.06 (dd, J = 14.61, 8.26 Hz, 1
H), 2.59 (t, J = 10.90 Hz, 5 H), 2.45 (s, 1 H), 1.73 (s, 1 H), 1.61−
1.13 (m, 4 H). LCMS: [MH+] = 726. Chiral analysis (Method 4) at
12.97 min and (Method 10) at 2.57 min.
Single Diastereoisomer of [(1S)-2-(3,5-Dichloro-1-oxido-pyridin-
1-ium-4-yl)-1-(3,4-dimethoxyphenyl)ethyl] 5-[[(2-Oxo-1-phenyl-2-
quinuclidin-3-yloxyethyl)amino]methyl]thiophene-2-carboxylate
1
(92a, Slow Diastereoisomer). H NMR (400 MHz, DMSO-d6): δ
8.55 (s, 2 H), 7.68 (d, J = 3.78 Hz, 1 H), 7.45−7.29 (m, 5 H), 7.05−
6.95 (m, 4 H), 6.14 (dd, J = 9.59, 4.29 Hz, 1 H), 4.67 (d, J = 7.13 Hz,
1 H), 4.43 (d, J = 9.21 Hz, 1 H), 3.89 (t, J = 5.31 Hz, 2 H), 3.77 (d, J
= 8.16 Hz, 6 H), 3.59 (t, J = 11.05 Hz, 2 H), 3.00−2.93 (m, 1 H),
2.57 (s, 4 H), 2.36 (d, J = 20.05 Hz, 1 H), 2.15 (d, J = 14.66 Hz, 1 H),
1.86 (s, 1 H), 1.60−1.50 (m, 2 H), 1.44 (s, 1 H), 1.24 (s, 1 H).
LCMS: [MH+] = 726. Chiral analysis (Method 4) at 15.02 min and
(Method 10) at 3.29 min.
Single Diastereoisomer of [(1S)-2-(3,5-Dichloro-1-oxido-pyridin-
1-ium-4-yl)-1-(3,4-dimethoxyphenyl)ethyl] 5-[[[1-(2-Fluorophenyl)-
2-oxo-2-[(3R)-quinuclidin-3-yl]oxyethyl]amino]methyl]thiophene-
1
2-carboxylate (91b, Fast Diastereoisomer). H NMR (400 MHz,
DMSO-d6): δ 8.55 (s, 2 H), 7.68 (d, J = 3.8 Hz, 1 H), 7.57−7.51 (m,
1 H), 7.42−7.35 (m, 1 H), 7.28−7.19 (m, 2 H), 7.04−6.96 (m, 4 H),
6.14 (dd, J = 9.6, 4.3 Hz, 1 H), 4.72−4.66 (m, 2 H), 3.94 (d, J = 5.6
Hz, 2 H), 3.77 (s, 3 H), 3.76 (s, 3 H), 3.67−3.53 (m, 2 H), 3.30 (dd,
J = 14.3, 4.5 Hz, 1 H), 3.01 (dd, J = 14.8, 8.1 Hz, 1 H), 2.69−2.44 (m,
3 H), 2.38−2.27 (m, 1 H), 2.20−2.12 (m, 1 H), 1.89−1.82 (m, 1 H),
1.60−1.39 (m, 3 H), 1.28−1.14 (m, 1 H). LCMS: [MH+] = 744.
Chiral analysis (Method 9) at 15.61 min.
Single Diastereoisomer of [(1S)-2-(3,5-Dichloro-1-oxido-pyridin-
1-ium-4-yl)-1-(3,4-dimethoxyphenyl)ethyl] 5-[[[1-(2-Fluorophenyl)-
2-oxo-2-[(3R)-quinuclidin-3-yl]oxyethyl]amino]methyl]thiophene-
1
2-carboxylate (92b, Slow Diastereoisomer). H NMR (400 MHz,
DMSO-d6) δ 8.55 (s, 2 H), 7.69 (d, J = 3.8 Hz, 1 H), 7.56−7.50 (m, 1
H), 7.41−7.34 (m, 1 H), 7.26−7.18 (m, 2 H), 7.04−6.96 (m, 4 H),
6.14 (dd, J = 9.6, 4.4 Hz, 1 H), 4.77−4.67 (m, 2 H), 3.94 (d, J = 5.1
Hz, 2 H), 3.77 (s, 3 H), 3.76 (s, 3 H), 3.67−3.53 (m, 2 H), 3.30 (dd,
J = 14.1, 4.4 Hz, 1 H), 3.09 (dd, J = 14.8, 8.4 Hz, 1 H), 2.70−2.44 (m,
5 H), 1.77−1.71 (m, 1 H), 1.59−1.38 (m, 2 H), 1.31−1.09 (m, 2 H).
LCMS: [MH+] = 744. Chiral analysis (Method 9) at 17.65 min.
Single Diastereoisomer of [(1S)-2-(3,5-Dichloro-1-oxido-pyridin-
1-ium-4-yl)-1-(3,4-dimethoxyphenyl)ethyl] 5-[[Methyl-[2-oxo-1-
phenyl-2-[(3R)-quinuclidin-3-yl]oxyethyl]amino]methyl]thiophene-
2-carboxylate (91c, Fast Diastereoisomer). 1H NMR (400
MHz,CD3CN): δ 8.19 (s, 2 H), 7.67 (d, J = 3.8 Hz, 1 H), 7.51 (d,
J = 7.8 Hz, 2 H), 7.46−7.38 (m, 3 H), 7.08−7.01 (m, 2 H), 6.97−
6.93 (m, 2 H), 6.18 (dd, J = 4.5, 9.6 Hz, 1 H), 4.86−4.81 (m, 1 H),
4.44 (s, 1 H), 3.90−3.85 (m, 4 H), 3.83−3.77 (m, 4 H), 3.68 (dd, J =
9.9, 14.1 Hz, 1 H), 3.35 (dd, J = 4.5, 13.9 Hz, 1 H), 3.12 (ddd, J = 2.1,
8.2, 14.5 Hz, 1 H), 2.75−2.60 (m, 4 H), 2.46 (d, J = 14.4 Hz, 1 H),
2.30 (s, 3 H), 1.94−1.88 (m, 1 H), 1.76−1.50 (m, 3 H), 1.41−1.29
(m, 1 H). LCMS: [MH+] = 740. Chiral analysis (Method 7) at 2.27
min.
Purification of the 1:1 mixture of diastereoisomers 86a and 88a−j
by chiral preparative SFC or chiral preparative HPLC afforded the
single diastereoisomers 89, 90, 91a−j, and 92a−j.
Single Diastereoisomers of [(1S)-2-(3,5-Dichloro-1-oxido-pyri-
din-1-ium-4-yl)-1-(3,4-dimethoxyphenyl)ethyl] 3-[[[2-Oxo-1-phe-
nyl-2-[(3R)-quinuclidin-3-yl]oxyethyl]amino]methyl]benzoate (89,
1
Fast Diastereoisomer). H NMR (400 MHz, CDCl3): δ 8.12 (s, 2
H), 7.99 (s, 1 H), 7.92 (d, J = 7.8 Hz, 1 H), 7.55 (d, J = 7.7 Hz, 1 H),
7.42−7.29 (m, 6 H), 7.03−6.97 (m, 2 H), 6.85 (d, J = 8.2 Hz, 1 H),
6.29 (dd, J = 9.6, 4.7 Hz, 1 H), 4.82−4.76 (m, 1 H), 4.40 (s, 1 H),
3.89 (s, 3 H), 3.87 (s, 3 H), 3.78 (s, 2 H), 3.71 (dd, J = 13.9, 9.7 Hz, 1
H), 3.35 (dd, J = 13.9, 4.7 Hz, 1 H), 3.10 (ddd, J = 14.8, 8.2, 2.3 Hz, 1
H), 2.78−2.61 (m, 2 H), 2.56−2.47 (m, 1 H), 2.34 (d, J = 15.0 Hz, 1
H), 2.04−1.95 (m, 2 H), 1.69−1.59 (m, 2 H), 1.55−1.45 (m, 1 H),
1.35−1.25 (m, 2 H). LCMS: [MH+] = 720. Chiral analysis (Method
3) at 5.41 min.
Single Diastereoisomers of [(1S)-2-(3,5-Dichloro-1-oxido-pyri-
din-1-ium-4-yl)-1-(3,4-dimethoxyphenyl)ethyl] 3-[[[2-Oxo-1-phe-
nyl-2-[(3R)-quinuclidin-3-yl]oxyethyl]amino]methyl]benzoate (90,
Single Diastereoisomer of [(1S)-2-(3,5-Dichloro-1-oxido-pyridin-
1-ium-4-yl)-1-(3,4-dimethoxyphenyl)ethyl] 5-[[Methyl-[2-oxo-1-
phenyl-2-[(3R)-quinuclidin-3-yl]oxyethyl]amino]methyl]thiophene-
1
1
Slow Diastereoisomer). H NMR (400 MHz, CDCl3): δ 8.12 (s, 2
2-carboxylate (92c, Slow Diastereoisomer). H NMR (400 MHz,
H), 8.00 (s, 1 H), 7.92 (d, J = 7.8 Hz, 1 H), 7.53 (d, J = 7.7 Hz, 1 H),
7.42−7.27 (m, 6 H), 7.04−6.97 (m, 2 H), 6.85 (d, J = 8.2 Hz, 1 H),
6.28 (dd, J = 9.7, 4.6 Hz, 1 H), 4.87−4.82 (m, 1 H), 4.38 (s, 1 H),
3.89 (s, 3 H), 3.88 (s, 3 H), 3.78 (d, J = 3.6 Hz, 2 H), 3.71 (dd, J =
CD3CN): δ 8.19 (s, 2 H), 7.68 (d, J = 3.8 Hz, 1 H), 7.50 (d, J = 6.8
Hz, 2 H), 7.46−7.37 (m, 3 H), 7.08−7.02 (m, 2 H), 6.97−6.94 (m, 2
H), 6.18 (dd, J = 4.5, 9.6 Hz, 1 H), 4.88−4.83 (m, 1 H), 4.46 (s, 1
H), 3.91 (d, J = 15.2 Hz, 1 H), 3.84 (s, 3 H), 3.84−3.78 (m, 4 H),
9116
https://doi.org/10.1021/acs.jmedchem.1c00204
J. Med. Chem. 2021, 64, 9100−9119

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
3.68 (dd, J = 9.7, 14.0 Hz, 1 H), 3.35 (dd, J = 4.5, 14.1 Hz, 1 H), 3.19
(dd, J = 8.3, 14.7 Hz, 1 H), 2.79−2.64 (m, 4 H), 2.64−2.55 (m, 1 H),
2.30 (s, 3 H), 1.94−1.88 (m, 1 H), 1.70−1.50 (m, 3 H), 1.37−1.29
(m, 1 H). LCMS: [MH+] = 740. Chiral analysis (Method 7) at 3.08
min.
3.89 (s, 3 H), 3.87 (s, 3 H), 3.67 (dd, J = 9.9, 13.9 Hz, 1 H), 3.31 (dd,
J = 4.5, 13.9 Hz, 1 H), 2.77−2.70 (m, 1 H), 2.64−2.62 (m, 1 H), 2.48
(dd, J = 2.1, 11.0 Hz, 1 H), 2.34−2.29 (m, 2 H), 2.28 (s, 3 H), 1.86−
1.80 (m, 2 H). LCMS: [MH+] =700. Chiral analysis (Method 1) at
13.61 min.
Single Diastereomer of [(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-
ium-4-yl)-1-(3,4-dimethoxyphenyl)ethyl] 5-[[[2-Oxo-1-phenyl-2-
[[(3R)-quinuclidin-3-yl]methoxy]ethyl]amino]methyl]thiophene-2-
carboxylate (91g, Fast Diastereoisomer). 1H NMR (400 MHz,
CD3CN): δ 8.19 (s, 2 H), 7.69 (d, J = 3.8 Hz, 1 H), 7.41−7.32 (m, 5
H), 7.08−7.02 (m, 2 H), 6.97−6.94 (m, 2 H), 6.19 (dd, J = 4.5, 9.6
Hz, 1 H), 4.44 (s, 1 H), 4.11−4.08 (m, 2 H), 4.00−3.90 (m, 2 H),
3.84 (s, 3 H), 3.83 (s, 3 H), 3.71−3.64 (m, 1 H), 3.35 (dd, J = 4.5,
14.1 Hz, 1 H), 2.84 (dd, J = 9.9, 13.6 Hz, 2 H), 2.76−2.64 (m, 4 H),
2.26−2.19 (m, 1 H), 1.90−1.81 (m, 1 H), 1.61−1.50 (m, 3 H), 1.44−
1.25 (m, 2 H). LCMS: [MH+] = 740. Chiral analysis (Method 6) at
6.31 min.
Single Diastereomer of [(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-
ium-4-yl)-1-(3,4-dimethoxyphenyl)ethyl] 5-[[[2-Oxo-1-phenyl-2-
[[(3R)-quinuclidin-3-yl]methoxy]ethyl]amino]methyl]thiophene-2-
carboxylate (92g, Slow Diastereoisomer). 1H NMR (400 MHz,
CD3CN): δ 8.18 (s, 2 H), 7.69 (d, J = 3.8 Hz, 1 H), 7.43−7.34 (m, 5
H), 7.09−7.01 (m, 2 H), 6.98−6.94 (m, 2 H), 6.19 (dd, J = 4.4, 9.7
Hz, 1 H), 4.45 (s, 1 H), 4.15−4.04 (m, 2 H), 4.00−3.89 (m, 2 H),
3.85 (s, 3 H), 3.82 (s, 3 H), 3.71−3.64 (m, 1 H), 3.35 (dd, J = 4.4,
14.0 Hz, 1 H), 2.89−2.65 (m, 5 H), 2.31−2.23 (m, 1 H), 1.90 (dd, J
= 8.4, 20.4 Hz, 1 H), 1.61−1.44 (m, 3 H), 1.34−1.26 (m, 2 H), NH
not observed. LCMS: [MH+] = 740. Chiral analysis (Method 6) at
7.51 min.
Single Diastereoisomer of [(1S)-2-(3,5-Dichloro-1-oxido-pyridin-
1-ium-4-yl)-1-(3,4-dimethoxyphenyl)ethyl] 5-[[[2-[[(1R,5R)-8-Meth-
yl-8-azabicyclo[3.2.1]octan-3-yl]oxy]-2-oxo-1-phenylethyl]amino]-
1
methyl]thiophene-2-carboxylate (91d, Fast Diastereoisomer). H
NMR (400 MHz, CDCl3): δ 8.13 (s, 2 H), 7.64 (d, J = 3.8 Hz, 1 H),
7.38−7.36 (m, 5 H), 7.02−6.96 (m, 2 H), 6.88−6.84 (m, 2 H), 6.22
(dd, J = 4.3, 9.9 Hz, 1 H), 5.00 (dd, J = 5.4, 5.4 Hz, 1 H), 4.36 (s, 1
H), 3.93−3.87 (m, 8 H), 3.67 (dd, J = 10.0, 14.0 Hz, 1 H), 3.31 (dd, J
= 4.5, 13.9 Hz, 1 H), 3.03−3.00 (m, 1 H), 2.90−2.88 (m, 1 H), 2.62
(d, J = 70.9 Hz, 1 H), 2.19 (s, 3 H), 2.14−1.98 (m, 2 H), 1.86−1.81
(m, 1 H), 1.60−1.70 (m, 3 H), 1.42 (d, J = 15.2 Hz, 1 H), 1.1−1.0
(m, 1 H). LCMS: [MH+] = 740. Chiral analysis (Method 2) at 9.50
min.
Single Diastereoisomer of [(1S)-2-(3,5-Dichloro-1-oxido-pyridin-
1-ium-4-yl)-1-(3,4-dimethoxyphenyl)ethyl] 5-[[[2-[[(1R,5R)-8-Meth-
yl-8-azabicyclo[3.2.1]octan-3-yl]oxy]-2-oxo-1-phenylethyl]amino]-
1
methyl]thiophene-2-carboxylate (92d, Slow Diastereoisomer). H
NMR (400 MHz, CDCl3): δ 8.14 (s, 2 H), 7.65 (d, J = 3.8 Hz, 1 H),
7.39−7.35 (m, 5 H), 6.99 (d, J = 8.3 Hz, 2 H), 6.88−6.83 (m, 2 H),
6.23 (dd, J = 4.7, 9.7 Hz, 1 H), 5.01 (dd, J = 5.2, 5.2 Hz, 1 H), 4.38 (s,
1 H), 3.93−3.88 (m, 8 H), 3.67 (dd, J = 9.9, 13.9 Hz, 1 H), 3.31 (dd,
J = 4.7, 14.0 Hz, 1 H), 3.07−3.05 (m, 1 H), 2.92 (d, J = 3.0 Hz, 1 H),
2.21 (s, 3 H), 2.20−2.00 (m, 2 H), 1.86−1.70 (m, 4 H), 1.44 (d, J =
14.9 Hz, 1 H), 1.06−1.04 (m, 1H), NH not visible. LCMS: [MH+] =
740. Chiral analysis (Method 2) at 11.81 min.
Single Diastereomer of [(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-
ium-4-yl)-1-(3,4-dimethoxyphenyl)ethyl] 5-[[[2-[(1-Methyl-4-
piperidyl)oxy]-2-oxo-1-phenylethyl]amino]methyl]thiophene-2-
carboxylate (91e, Fast Diastereoisomer). 1H NMR (400 MHz,
CD3CN): δ 8.19 (s, 2 H), 7.69 (d, J = 3.8 Hz, 1 H), 7.43−7.32 (m, 5
H), 7.08−7.01 (m, 2 H), 6.97−6.92 (m, 2 H), 6.18 (dd, J = 4.5, 9.6
Hz, 1 H), 4.81−4.73 (m, 1 H), 4.42 (s, 1 H), 3.95 (dd, J = 15.0, 22.4
Hz, 2 H), 3.83 (s, 3 H), 3.82 (s, 3 H), 3.67 (dd, J = 9.6, 14.1 Hz, 1
H), 3.34 (dd, J = 4.5, 14.1 Hz, 1 H), 2.53−2.08 (m, 4 H), 2.15 (s, 3
H), 1.90−1.81 (m, 1 H), 1.77−1.59 (m, 2 H), 1.55−1.44 (m, 1 H),
NH not observed. LCMS: [MH+] = 714. Chiral analysis (Method 7)
at 1.68 min.
Single Diastereomer of [(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-
ium-4-yl)-1-(3,4-dimethoxyphenyl)ethyl] 5-[[[2-[(1-Methyl-4-
piperidyl)oxy]-2-oxo-1-phenylethyl]amino]methyl]thiophene-2-
carboxylate (92e, Slow Diastereoisomer). 1H NMR (400 MHz,
CD3CN): δ 8.18 (s, 2 H), 7.69 (d, J = 4.3 Hz, 1 H), 7.44−7.32 (m, 5
H), 7.07−7.01 (m, 2 H), 6.97−6.91 (m, 2 H), 6.18 (dd, J = 4.4, 9.7
Hz, 1 H), 4.81−4.73 (m, 1 H), 4.41 (s, 1 H), 3.95 (dd, J = 14.9, 25.1
Hz, 2 H), 3.83 (s, 3 H), 3.82 (s, 3 H), 3.67 (dd, J = 9.6, 14.1 Hz, 1
H), 3.33 (dd, J = 4.5, 14.1 Hz, 1 H), 2.56−2.11 (m, 4 H), 2.16 (s, 3
H), 1.89−1.81 (m, 1 H), 1.77−1.59 (m, 2 H), 1.55−1.45 (m, 1 H),
NH not observed. LCMS: [MH+] = 714. Chiral analysis (Method 7)
at 2.50 min.
Single Diastereoisomer of [(1S)-2-(3,5-Dichloro-1-oxido-pyridin-
1-ium-4-yl)-1-(3,4-dimethoxyphenyl)ethyl] 5-[[[2-[(3R)-1-Methyl-
pyrrolidin-3-yl]oxy-2-oxo-1-phenylethyl]amino]methyl]thiophene-
2-carboxylate (91f, Fast Diastereoisomer). 1H NMR (400 MHz,
CDCl3): δ 8.14 (s, 2 H), 7.64 (d, J = 3.8 Hz, 1 H), 7.37−7.29 (m, 5
H), 7.00−6.97 (m, 2 H), 6.87−6.84 (m, 2 H), 6.22 (dd, J = 4.4, 10.0
Hz, 1 H), 5.24−5.21 (m, 1 H), 4.43 (s, 1 H), 3.91−3.90 (m, 2 H),
3.89 (s, 3 H), 3.87 (s, 3 H), 3.66 (dd, J = 10.0, 14.0 Hz, 1 H), 3.31
(dd, J = 4.4, 14.0 Hz, 1 H), 2.69−2.67 (m, 3 H), 2.29 (s, 3 H), 2.27−
2.25 (m, 1 H), 2.19−2.14 (m, 1 H), 1.69−1.58 (m, 2 H). LCMS:
[MH+] =700. Chiral analysis (Method 1) at 11.66 min.
Single Diastereomer of [(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-
ium-4-yl)-1-(3,4-dimethoxyphenyl)ethyl] 5-[[[2-[(1-Methyl-4-
piperidyl)methoxy]-2-oxo-1-phenylethyl]amino]methyl]thiophene-
1
2-carboxylate (91h, Fast Diastereoisomer). H NMR (400 MHz,
CDCl3): δ 8.14 (s, 2 H), 7.64 (d, J = 3.8 Hz, 1 H), 7.37−7.35 (m, 5
H), 7.01−6.97 (m, 2 H), 6.89−6.84 (m, 2 H), 6.22 (dd, J = 4.5, 9.9
Hz, 1 H), 4.43 (s, 1 H), 3.98−3.91 (m, 4 H), 3.90 (s, 3 H), 3.88 (s, 3
H), 3.67 (dd, J = 9.7, 14.0 Hz, 1 H), 3.31 (dd, J = 4.5, 13.9 Hz, 1 H),
2.81 (d, J = 10.6 Hz, 2 H), 2.25 (s, 3 H), 1.89−1.82 (m, 2 H), 1.53−
1.52 (m, 3 H), 1.29−1.20 (m, 2 H). LCMS: [MH+] = 728. Chiral
analysis (Method 5) at 6.14 min.
Single Diastereomer of [(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-
ium-4-yl)-1-(3,4-dimethoxyphenyl)ethyl] 5-[[[2-[(1-Methyl-4-
piperidyl)methoxy]-2-oxo-1-phenylethyl]amino]methyl]thiophene-
1
2-carboxylate (92h, Slow Diastereoisomer). H NMR (400 MHz,
CDCl3): δ 8.13 (s, 2 H), 7.64 (d, J = 3.8 Hz, 1 H), 7.38−7.36 (m, 5
H), 7.02−6.96 (m, 2 H), 6.88−6.84 (m, 2 H), 6.22 (dd, J = 4.5, 9.9
Hz, 1 H), 4.42 (s, 1 H), 3.99−3.92 (m, 4 H), 3.91 (s, 3 H), 3.88 (s, 3
H), 3.67 (dd, J = 9.9, 13.9 Hz, 1 H), 3.31 (dd, J = 4.4, 14.0 Hz, 1 H),
2.86 (d, J = 8.8 Hz, 2 H), 2.29 (s, 3 H), 1.92−1.87 (m, 2 H), 1.54−
1.54 (m, 3 H), 1.35−1.20 (m, 2 H). LCMS: [MH+] = 728. Chiral
analysis (Method 5) at 7.21 min.
Single Diastereomer of [(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-
ium-4-yl)-1-(3,4-dimethoxyphenyl)ethyl] 5-[[[2-[[(2R)-1-Methylaze-
tidin-2-yl]methoxy]-2-oxo-1-phenylethyl]amino]methyl]-
thiophene-2-carboxylate (91i, Fast Diastereoisomer). 1H NMR
(400 MHz, CD3CN): δ 8.19 (s, 2 H), 7.69 (d, J = 3.8 Hz, 1 H), 7.44−
7.34 (m, 5 H), 7.07−7.01 (m, 2 H), 6.97−6.93 (m, 2 H), 6.19 (dd, J
= 4.5, 9.6 Hz, 1 H), 4.46 (s, 1 H), 4.20 (dd, J = 3.8, 11.4 Hz, 1 H),
4.02−3.90 (m, 3 H), 3.83 (s, 3 H), 3.82 (s, 3 H), 3.67 (dd, J = 9.6,
14.1 Hz, 1 H), 3.35 (dd, J = 4.5, 14.1 Hz, 1 H), 3.22−3.16 (m, 1 H),
3.09−3.01 (m, 1 H), 2.69 (ddd, J = 6.6, 8.0, 9.3 Hz, 1 H), 2.09 (s, 3
H), 1.91−1.76 (m, 2 H). Note: NH not visible LCMS: [MH+] = 700.
Chiral analysis (Method 6) at 3.44 min.
Single Diastereomer of [(1S)-2-(3,5-Dichloro-1-oxido-pyridin-1-
ium-4-yl)-1-(3,4-dimethoxyphenyl)ethyl] 5-[[[2-[[(2R)-1-Methylaze-
tidin-2-yl]methoxy]-2-oxo-1-phenylethyl]amino]methyl]-
thiophene-2-carboxylate (92i, Slow Diastereoisomer). 1H NMR
(400 MHz, CD3CN): δ 8.18 (s, 2 H), 7.69 (d, J = 3.8 Hz, 1 H), 7.43−
7.40 (m, 5 H), 7.07−7.02 (m, 2 H), 6.97−6.94 (m, 2 H), 6.19 (dd, J
= 4.5, 9.9 Hz, 1 H), 4.46 (s, 1 H), 4.19−4.12 (m, 1 H), 4.01−3.89 (m,
3 H), 3.83 (s, 3 H), 3.82 (s, 3 H), 3.71−3.63 (m, 1 H), 3.34 (dd, J =
4.5, 14.1 Hz, 1 H), 3.25−3.20 (m, 1 H), 3.15−3.07 (m, 1 H), 2.74−
Single Diastereoisomer of [(1S)-2-(3,5-Dichloro-1-oxido-pyridin-
1-ium-4-yl)-1-(3,4-dimethoxyphenyl)ethyl] 5-[[[2-[(3R)-1-Methyl-
pyrrolidin-3-yl]oxy-2-oxo-1-phenylethyl]amino]methyl]thiophene-
1
2-carboxylate (92f, Slow Diastereoisomer). H NMR (400 MHz,
CDCl3): δ 8.13 (s, 2 H), 7.64 (d, J = 3.8 Hz, 1 H), 7.37−7.29 (m, 5
H), 7.01−6.96 (m, 2 H), 6.86−6.84 (m, 2 H), 6.22 (dd, J = 4.4, 9.7
Hz, 1 H), 5.23−5.17 (m, 1 H), 4.43 (s, 1 H), 3.91−3.90 (m, 2 H),
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Article
2.67 (m, 1 H), 2.10 (s, 3 H), 1.91−1.76 (m, 2 H).NH not visible
LCMS: [MH+] = 700. Chiral analysis (Method 6) at 4.30 min.
Single Diastereoisomer of [(1S)-2-(3,5-Dichloro-1-oxido-pyridin-
1-ium-4-yl)-1-(3,4-dimethoxyphenyl)ethyl] 5-[[[2-[2-
(Dimethylamino)ethoxy]-2-oxo-1-phenylethyl]amino]methyl]-
thiophene-2-carboxylate (91j, Fast Diastereoisomer). 1H NMR
(400 MHz, CD3CN): δ 8.19 (s, 2 H), 7.70 (d, J = 3.5 Hz, 1 H), 7.42−
7.36 (m, 5 H), 7.08−7.01 (m, 2 H), 6.98−6.93 (m, 2 H), 6.18 (dd, J
= 4.5, 9.6 Hz, 1 H), 4.44 (s, 1 H), 4.23−4.14 (m, 2 H), 4.01−3.90 (m,
2 H), 3.84 (s, 3 H), 3.82 (s, 3 H), 3.67 (dd, J = 9.7, 14.0 Hz, 1 H),
3.35 (dd, J = 4.5, 14.1 Hz, 1 H), 2.47 (t, J = 5.7 Hz, 2 H), 2.15 (s, 6
H), NH not visible. LCMS: [MH+] = 688. Chiral analysis (Method
8) at 10.27 min.
Single Diastereoisomer of [(1S)-2-(3,5-Dichloro-1-oxido-pyridin-
1-ium-4-yl)-1-(3,4-dimethoxyphenyl)ethyl] 5-[[[2-[2-
(Dimethylamino)ethoxy]-2-oxo-1-phenylethyl]amino]methyl]-
thiophene-2-carboxylate (92j, Slow Diastereoisomer). 1H NMR
(400 MHz, CD3CN): δ 8.18 (s, 2 H), 7.69 (d, J = 3.7 Hz, 1 H), 7.42−
7.39 (m, 5 H), 7.07−7.01 (m, 2 H), 6.97−6.93 (m, 2 H), 6.21−6.15
(m, 1 H), 4.43 (s, 1 H), 4.23−4.14 (m, 2 H), 3.96 (dd, J = 15.2, 29.6
Hz, 2 H), 3.83 (s, 3 H), 3.82 (s, 3 H), 3.72−3.63 (m, 1 H), 3.34 (d, J
= 26.1 Hz, 1 H), 2.47 (t, J = 5.6 Hz, 2 H), 2.15 (s, 6 H), NH not
observed. LCMS: [MH+] = 688. Chiral analysis (Method 8) at 12.21
min.
Gino Villetti − Nuovo Centro Ricerche, Chiesi Farmaceutici
S.p.A., 43122 Parma, Italy
Gessica Marchini − Nuovo Centro Ricerche, Chiesi
Farmaceutici S.p.A., 43122 Parma, Italy
Anna Rita Pisano − Nuovo Centro Ricerche, Chiesi
Farmaceutici S.p.A., 43122 Parma, Italy
Vanessa Pitozzi − Nuovo Centro Ricerche, Chiesi Farmaceutici
S.p.A., 43122 Parma, Italy
Maria Gloria Pittelli − Nuovo Centro Ricerche, Chiesi
Farmaceutici S.p.A., 43122 Parma, Italy
Marcello Trevisani − Nuovo Centro Ricerche, Chiesi
Farmaceutici S.p.A., 43122 Parma, Italy
Michela Salvadori − Nuovo Centro Ricerche, Chiesi
Farmaceutici S.p.A., 43122 Parma, Italy
Valentina Cenacchi − Nuovo Centro Ricerche, Chiesi
Farmaceutici S.p.A., 43122 Parma, Italy
Paola Puccini − Nuovo Centro Ricerche, Chiesi Farmaceutici
S.p.A., 43122 Parma, Italy
Francesco Amadei − Nuovo Centro Ricerche, Chiesi
Farmaceutici S.p.A., 43122 Parma, Italy
Alice Pappani − Nuovo Centro Ricerche, Chiesi Farmaceutici
S.p.A., 43122 Parma, Italy
Maurizio Civelli − Nuovo Centro Ricerche, Chiesi
ASSOCIATED CONTENT
■
Farmaceutici S.p.A., 43122 Parma, Italy
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00204.
Riccardo Patacchini − Nuovo Centro Ricerche, Chiesi
Farmaceutici S.p.A., 43122 Parma, Italy
Charles A.G. Baker-Glenn − Chesterford Research Park,
Charles River Discovery Research Services UK Ltd, Saffron
Walden CB10 1XL, United Kingdom
Hervé Van de Poël − Chesterford Research Park, Charles
River Discovery Research Services UK Ltd, Saffron Walden
CB10 1XL, United Kingdom
Wesley P. Blackaby − Chesterford Research Park, Charles
River Discovery Research Services UK Ltd, Saffron Walden
CB10 1XL, United Kingdom; Present Address: Servier, 125
Chemin de Ronde, 78290 Croissy sur Seine, France.
Kevin Nash − Chesterford Research Park, Charles River
Discovery Research Services UK Ltd, Saffron Walden CB10
1XL, United Kingdom
Preparation of intermediates 11, 14, 15, 16, 29, 39a−d,
and 85a-j; analytical characterization of intermediates
19, 21, 23, 31, 42b, 42e, 43b, 44e, 45b, 65−68, and
50−55; chiral analytical methods, LCMS and SFC-MS
traces for compounds 92e, 92f, 91a, and 92a; crystal
structure determination of 92a; and methods for clogD
determination, kinetic solubility measure, assays:
PDE4B2 HTRF, PDE4 LARBS, M3 receptor radioligand
binding, THFa release in hPBMC, guinea pig and rat
isolated trachea contraction, bronchoconstriction in
anaesthetized guinea pigs and rats, M3 binding kinetics,
ovalbumin-induced eosinophilia in the sensitized Brown
Norway rat, nausea and emetic effect in ferret, in vitro
microsomial Stability, in vitro plasma protein binding, in
vitro plasma stability, and cellular permeability (PDF)
Gabriele Amari − Nuovo Centro Ricerche, Chiesi Farmaceutici
S.p.A., 43122 Parma, Italy
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00204
Molecular formula strings of final compounds with
biological data (CSV)
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
AUTHOR INFORMATION
■
Corresponding Author
Elisabetta Armani − Nuovo Centro Ricerche, Chiesi
Farmaceutici S.p.A., 43122 Parma, Italy; orcid.org/0000-
0002-2835-2652; Phone: +39 0521 279477;
Email: e.armani@chiesi.com; Fax: +39 0521 279880
Notes
The authors declare no competing financial interest.
All the experiments with animals were carried out in
accordance with national and European legislation and
approved by local ethical committees.
Authors
Andrea Rizzi − Nuovo Centro Ricerche, Chiesi Farmaceutici
ABBREVIATIONS
S.p.A., 43122 Parma, Italy
■
Carmelida Capaldi − Nuovo Centro Ricerche, Chiesi
Farmaceutici S.p.A., 43122 Parma, Italy
Renato De Fanti − Nuovo Centro Ricerche, Chiesi
Farmaceutici S.p.A., 43122 Parma, Italy; Present
Address: rdf0510noah@gmail.com, actually in retirement.
Maurizio Delcanale − Nuovo Centro Ricerche, Chiesi
Farmaceutici S.p.A., 43122 Parma, Italy
Ach, acetylcholine; ADME, absorption, distribution, metabo-
lism, excretion; Cch, carbachol; Clint, intrinsic clearance;
COPD, chronic obstructive pulmonary disease; CSD, crystal
structure databank; ED, effective dose; GPT, guinea pig
trachea; IC, inhibitory concentration; Ki, inhibition constant;
LARBS, low affinity rolipram binding site; LogD, distribution
coefficient logarithm; LPS, lipopolysaccharides; MABA,
9118
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muscarinic antagonist beta adrenergic agonist; MAPI, muscar-
inic antagonist, phosphodiesterase 4 inhibitor; M3, muscarinic
receptor 3; Mics, microsomes; OVA, ovalbumin; PBMC,
peripheral blood mononuclear cells; PDE4, phosphodiesterase
4; PDE4B2, isoform B2 of phosphodiesterase 4; PPB, plasma
protein binding; RT, rat trachea; TNFα, tumor necrosis factor
alpha
REFERENCES
■
(1) GBD 2015 Mortality and Causes of Death Collaborators. Global,
regional, and national life expectancy, all-cause mortality, and cause-
specific mortality for 249 causes of death, 1980−2015: a systematic
analysis for the Global Burden of Disease Study 2015. Lancet 2016,
388, 1459−1544.
(2) Cazzola, M.; Lopez-Campos, J.; Puente-Maestu, L. The MABA
approach: a new option to improve bronchodilator therapy. Eur.
Respir. J. 2013, 42, 885−887.
(3) Joshi, T.; Yan, D.; Hamed, O.; Tannheimer, S. L.; Phillips, G. B.;
Wright, C. D.; Kim, M.; Salmon, M.; Newton, R.; Giembycz, M. A.
GS-5759, a bifunctional β2-adrenoceptor agonist and phosphodiester-
ase 4 inhibitor for chronic obstructive pulmonary disease with a
unique mode of action: effects on gene expression in human airway
epithelial cells. J. Pharmacol. Exp. Ther. 2017, 360, 324−340.
(4) Provins, L.; Christophe, B.; Danhaive, P.; Dulieu, J.; Durieu, V.;
Gillard, M.; Lebon, F.; Lengelé, S.; Quéré, L.; van Keulen, B. First
dual M3 antagonists-PDE4 inhibitors: Synthesis and SAR of 4,6-
diaminopyrimidine derivatives. Bioorg. Med. Chem. Lett. 2006, 16,
1834−1839.
(5) Callahan, J. F.; Wan, Z.; Yan, H. Dual pharmacophores - PDE4-
muscarinic antagonistics. WO2009100170, Aug 13, 2009.
(6) Armani, E.; Amari, G.; Rizzi, A.; De Fanti, R.; Ghidini, E.;
Capaldi, C.; Carzaniga, L.; Caruso, P.; Guala, M.; Peretto, I.; La Porta,
E.; Bolzoni, P. T.; Facchinetti, F.; Carnini, C.; Moretto, N.;
Patacchini, R.; Bassani, F.; Cenacchi, V.; Volta, R.; Amadei, F.;
Capacchi, S.; Delcanale, M.; Puccini, P.; Catinella, S.; Civelli, M.;
Villetti, G. Novel class of benzoic acid ester derivatives as potent
PDE4 inhibitors for inhaled administration in the treatment of
respiratory diseases. J. Med. Chem. 2014, 57, 793−816.
(7) Amari, G.; Rizzi, A.; Patacchini, R.; Cenacchi, V.; Villetti, G.;
Catena Ruiz, J. L.; Masip Masip, I. Quinuclidine derivatives as M3
antagonists. WO2008012290, Jan 31, 2008.
(8) Caligiuri, A.; Riccaboni, M.; Amari, G. Alkaloid aminoester
derivatives and medicinal composition thereof. WO2010072338A1,
Jul 14, 2010.
(9) Alcaraz, L.; Bailey, A.; Cadogan, E.; Connolly, S.; Jewell, R.;
Jordan, S.; Kindon, N.; Lister, A.; Lawson, M.; Mullen, A.; Dainty, I.;
Nicholls, D.; Paine, S.; Pairaudeau, G.; Stocks, M. J.; Thorne, P.;
Young, A. From libraries to candidate: the discovery of new ultra
long-acting dibasic β2-adrenoceptor agonists. Bioorg. Med. Chem. Lett.
2012, 22, 689−695.
(10) Barnes, P. J. The pharmacological properties of tiotropium.
Chest 2000, 117, 63S−66S.
(11) Gross, N. J. Ipratropium bromide. N. Engl. J. Med. 1988, 319,
486−494.
(12) Holmes, A. M.; Rudd, J. A.; Tattersall, F. D.; Aziz, Q.; Andrews,
P. L. R. Opportunities for the replacement of animals in the study of
nausea and vomiting. Br. J. Pharmacol. 2009, 157, 865−880.
9119
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J. Med. Chem. 2021, 64, 9100−9119