Synthesis and biological comparison of enantiomers of mepenzolate bromide, a muscarinic receptor antagonist with bronchodilatory and anti-inflammatory activities

Article abstract of DOI:10.1016/j.bmc.2014.04.029

Chronic obstructive pulmonary disease (COPD) is characterized by abnormal inflammatory responses and airflow limitations. We recently proposed that the muscarinic antagonist mepenzolate bromide (mepenzolate) would be therapeutically effective against COPD due to its muscarinic receptor-dependent bronchodilatory activity as well as anti-inflammatory properties. Mepenzolate has an asymmetric carbon atom, thus providing us with the opportunity to synthesize both of its enantiomers ((R)- and (S)-mepenzolate) and to examine their biochemical and pharmacological activities. (R)- or (S)-mepenzolate was synthesized by condensation of benzilic acid with (R)- or (S)-alcohol, respectively, followed by quaternization of the tertiary amine. As predicted by computational simulation, a filter-binding assay in vitro revealed that (R)-mepenzolate showed a higher affinity for the muscarinic M3 receptor than (S)-mepenzolate. In vivo, the bronchodilatory activity of (R)-mepenzolate was superior to that of (S)-mepenzolate, whereas anti-inflammatory activity was indistinguishable between the two enantiomers. We confirmed that each mepenzolate maintained its original stereochemistry in the lung when administered intratracheally. These results suggest that (R)-mepenzolate may have superior properties to (S)-mepenzolate as a drug to treat COPD patients given that the former has more potent bronchodilatory activity than the latter.

Full text of DOI:10.1016/j.bmc.2014.04.029

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological comparison of enantiomers of mepenzolate
bromide, a muscarinic receptor antagonist with bronchodilatory and
anti-inflammatory activities
Yasunobu Yamashita a, Ken-Ichiro Tanaka a, Teita Asano a, Naoki Yamakawa a, Daisuke kobayashi a,
Tomoaki Ishihara a, Kengo Hanaya a, Mitsuru Shoji a, Takeshi Sugai a, Mitsuhito Wada b,c
,
Tadaaki Mashimo b,d, Yoshifumi Fukunishi e, Tohru Mizushima a,
⇑
a Faculty of Pharmacy, Keio University, Tokyo 105-8512, Japan
b Technology Research Association for Next Generation Natural Products Chemistry, 2-3-26, Aomi, Koto-ku, Tokyo 135-0064, Japan
c Biochemical Information Project, Fujitsu Limited, 1-9-3, Nakase, Mihama-ku, Chiba 261-8588, Japan
d Information and Mathematical Science and Bioinformatics Co., Ltd, Owl Tower, 4-21-1, Higashi-Ikebukuro, Toshima-ku, Tokyo 170-0013, Japan
e Molecular Profiling Research Center for Drug Discovery (molprof), National Institute of Advanced Industrial Science and Technology (AIST), Tokyo 135-0064, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Chronic obstructive pulmonary disease (COPD) is characterized by abnormal inflammatory responses and
airflow limitations. We recently proposed that the muscarinic antagonist mepenzolate bromide (mepen-
zolate) would be therapeutically effective against COPD due to its muscarinic receptor-dependent bron-
chodilatory activity as well as anti-inflammatory properties. Mepenzolate has an asymmetric carbon
atom, thus providing us with the opportunity to synthesize both of its enantiomers ((R)- and (S)-mepen-
zolate) and to examine their biochemical and pharmacological activities. (R)- or (S)-mepenzolate was
synthesized by condensation of benzilic acid with (R)- or (S)-alcohol, respectively, followed by quatern-
ization of the tertiary amine. As predicted by computational simulation, a filter-binding assay in vitro
revealed that (R)-mepenzolate showed a higher affinity for the muscarinic M3 receptor than (S)-mepen-
zolate. In vivo, the bronchodilatory activity of (R)-mepenzolate was superior to that of (S)-mepenzolate,
whereas anti-inflammatory activity was indistinguishable between the two enantiomers. We confirmed
that each mepenzolate maintained its original stereochemistry in the lung when administered intratrach-
eally. These results suggest that (R)-mepenzolate may have superior properties to (S)-mepenzolate as a
drug to treat COPD patients given that the former has more potent bronchodilatory activity than the
latter.
Received 5 March 2014
Revised 15 April 2014
Accepted 15 April 2014
Available online xxxx
Keywords:
COPD
Muscarinic receptor
Mepenzolate bromide
Enantiomers
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
press disease progression by controlling inflammation via
decreased reactive oxygen species.
Chronic obstructive pulmonary disease (COPD) is currently the
fourth leading cause of death in the world and its prevalence and
mortality rates are steadily increasing.1 The most important etio-
logic factor for COPD is cigarette smoke, with this disease defined
by a progressive and not fully reversible airflow limitation associ-
ated with abnormal inflammation and emphysema.1,2 Reactive
oxygen species, such as superoxide anion, are believed to play a
major role in this abnormal inflammation. Thus, for the clinical
treatment of COPD patients, it is important not only to improve
the airflow limitation by inducing bronchodilation, but also to sup-
Bronchodilators (such as muscarinic antagonists) are currently
used for the treatment of COPD owing to their ameliorative effect
on airflow limitation.1,2 On the other hand, steroids are used to
suppress inflammation in COPD patients; however recent clinical
studies revealed that steroids do not significantly modulate disease
progression or mortality,3,4 because the inflammation associated
with COPD tends to be resistant to steroid treatment.5 This insen-
sitivity can be explained in part by the notion that steroids sup-
press the expression of pro-inflammatory genes via their action
on histone deacetylase (HDAC) 2.6,7 Importantly, it was reported
that cigarette smoke inhibits the activity and expression of this
protein.6 Thus, the development of new types of anti-inflammatory
drugs to treat COPD patients is highly desirable.
⇑
Corresponding author. Tel.: +81 3 5400 2628.
E-mail address: mizushima-th@pha.keio.ac.jp (T. Mizushima).
http://dx.doi.org/10.1016/j.bmc.2014.04.029
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
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Y. Yamashita et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
The number of drugs reaching the marketplace each year is
decreasing, mainly due to the unexpected adverse effects of poten-
tial drugs being revealed at advanced clinical trial stages. For this
reason, we proposed a new strategy for drug discovery and devel-
opment (drug re-positioning).8 In this strategy, compounds with
therapeutically beneficial activity are screened from a library of
approved medicines with a view of developing them for new indi-
cations. The advantage of this approach is that there is a decreased
risk for unexpected adverse effects in humans because the safety
aspects of these drugs have already been well characterized.8 From
a library of approved medicines, we screened compounds that
could prevent elastase-induced pulmonary inflammation and
emphysema in mice, and selected mepenzolate bromide (mepen-
zolate).9 Mepenzolate is an orally administered muscarinic recep-
tor antagonist used to suppress the gastrointestinal hypermotility
associated with irritable bowel syndrome.10–12 We showed that
mepenzolate not only exerts an anti-inflammatory effect via a
muscarinic receptor-independent mechanism, but also a bron-
chodilatory effect via a muscarinic receptor-dependent mecha-
nism.9 This independence of the anti-inflammatory effect is
based on observations that other muscarinic receptor antagonists
such as ipratropium bromide (ipratropium) and tiotropium bro-
mide (tiotropium) could not exert ameliorative effects against elas-
tase-induced pulmonary emphysema.9 Although this animal
model (elastase-induced pulmonary inflammation and emphy-
sema) does not reflect some of the pathological features of COPD,
it has served as a convenient animal model for studying COPD
and we reported that mepenzolate could prevent cigarette
smoke-induced pulmonary inflammation and emphysema.9
As for the mechanism governing the anti-inflammatory activity
of mepenzolate, after confirmation of absence of direct inhibitory
effect of mepenzolate on elastase, we found that this drug can
restore HDAC activity under inflammatory conditions. We also
found that mepenzolate, but not steroids, decreased the pulmonary
level of superoxide anions. These results may explain why mepen-
zolate showed superior anti-inflammatory activity compared with
steroids in our animal model of COPD.9,13 Based on these findings,
we proposed that mepenzolate could serve as a candidate drug for
the treatment of COPD patients, given that it has both anti-inflam-
matory and bronchodilatory activities. Anti-inflammatory effect of
other muscarinic receptor antagonists was also reported
recently.14,15
Among the five types of muscarinic receptors (M1–5R), the mus-
carinic M3 receptor (M3R) expressed in airway and intestinal
smooth muscle positively regulates bronchoconstriction and intes-
tinal motility, respectively.16 Mepenzolate is a subtype-non-spe-
cific muscarinic antagonist12 whose bronchodilatory effect and
inhibitory effect on intestinal motility can be explained by its
antagonistic action on M3R. On the other hand, the muscarinic
M2 receptor (M2R) expressed in the sinoatrial node of the heart
negatively regulates heart rate,17 and we recently confirmed that
mepenzolate’s inhibitory action on this receptor leads to an
increased heart rate in mice (Tanaka et al., unpublished results).
Mepenzolate has one asymmetric carbon atom (Fig. 1) enabling
it to exist in the form of two enantiomers; a racemic mixture (( )-
mepenzolate) of these two enantiomers has been used in a clinical
setting. As the synthesis of one or other of these enantiomers of
mepenzolate has not been established, it has thus remained
unclear which of them is responsible for the drug’s anti-inflamma-
tory and anticholinergic activities. In various types of medicines,
including drugs used as muscarinic receptor antagonists, differ-
ences in stereochemistry can affect the biochemical and pharmaco-
logical activities of these compounds, meaning that the isolation of
distinct isomers can lead to the clinical development of more effec-
tive or safer medicines,18–20 however, there are still some advanta-
ges of racemates (such as cost). In the present study, we have
Figure 1. Structures of racemic mepenzolate and its enantiomers.
established a protocol for the synthesis of both (R)-mepenzolate
and (S)-mepenzolate (Fig. 1) and examined their biochemical and
pharmacological activities. Results showed that although anti-
inflammatory activity was indistinguishable between these enanti-
omers, the binding activity of (R)-mepenzolate to human M3R
(hM3R) in vitro and its bronchodilatory activity in vivo were supe-
rior to that of (S)-mepenzolate. These findings suggest that (R)-
mepenzolate may be preferable to (S)-mepenzolate as a candidate
drug to treat COPD patients.
2. Chemistry
The synthetic route for target compounds is outlined in
Scheme 1. The enantiomers of mepenzolate, (R)- and (S)-mepenzo-
late ((R)-1 and (S)-1), were synthesized in two steps from commer-
cially available benzilic acid (2) based on a procedure similar to
that previously described21 as outlined in Scheme 1. Condensation
of 2 with (R)- or (S)-3-hydroxy-1-methylpiperidine ((R)-3 or (S)-3)
in the presence of carbonyl diimidazol (CDI) afforded the corre-
sponding enantiomericaly pure tertiary amine ((R)- or (S)-1-
methyl-3-piperidyl benzilate ((R)-4 or (S)-4)), respectively. Quat-
ernization of intermediate (R)-4 or (S)-4 with methyl bromide in
acetonitrile provided desired compound (R)-1 or (S)-1,
respectively.
The final compounds were characterized by nuclear magnetic
resonance (NMR), infrared spectroscopy (IR) and high-resolution
mass spectra (HR-MS). The enantiomeric purity of each enantiomer
of 1 was determined by high performance liquid chromatography
(HPLC) with a chiral stationary phase.
3. Results and discussion
3.1. Binding of mepenzolate enantiomers to hM3R in silico and
in vitro
The interaction between hM3R and (R)-mepenzolate (or (S)-
mepenzolate) was predicted by molecular modelling and docking
studies. We constructed the structure of the complex between
hM3R and (R)-mepenzolate (or (S)-mepenzolate) based on the
recent reporting of the crystal structure of the complex between
rat M3R and tiotropium (another muscarinic antagonist22) (see
Materials and Methods). As for other cases of monoamine recep-
tors,23 hM3R has an aspartic acid residue in the third
a-helix, iden-
tified as Asp3.32 (Asp148). This residue of these monoamine
receptors strongly interacts with charged nitrogen atoms in the
agonists and antagonists,24 and subsequently we focused on this
residue in hM3R (Asp148).
As shown in Figure 2A, the nitrogen atom (N) in mepenzolate
interacts ionically with Asp148; we consider that this interaction
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Y. Yamashita et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
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Scheme 1. Synthesis of mepenzolate enantiomers.
Figure 2. Binding mode of (R)-mepenzolate and (S)-mepenzolate to hM3R in silico. (A) Initial coordinates of mepenzolate in hM3R. Results from MD simulations are shown for
(R)-mepenzolate ((R)-Mep) and (S)-mepenzolate ((S)-Mep). (B) Interatomic distances between the nitrogen atom (N) of (R)-mepenzolate and the two equivalent carboxylate
oxygen atoms (OD1 and OD2) of Asp148 of hM3R during the MD simulation production runs are indicated with red and orange lines, respectively. Those of (S)-mepenzolate
are indicated with purple and blue lines, respectively.
is important for the association between hM3R and mepenzolate.
We calculated the distance between the nitrogen atom and the
two equivalent carboxylate oxygen atoms (OD1 and OD2 in
Fig. 2A) of Asp148 during the molecular dynamics (MD) simulation
production run. As shown in Figure 2B, the distance was closer for
(R)-mepenzolate than for (S)-mepenzolate (the average distances
between OD1 or OD2 of Asp148 and the nitrogen atom of (R)-
mepenzolate were 4.596 Å or 4.104 Å, respectively, while those
for (S)-mepenzolate were 5.443 Å or 4.277 Å, respectively). These
results suggest that (R)-mepenzolate has a higher affinity for
hM3R than (S)-mepenzolate.
Other amino acid residues of hM3R seem to interact with both
(R)-mepenzolate and (S)-mepenzolate in an equivalent manner,
as mentioned in the following. Asn5086.52 is conserved among all
five types of muscarinic receptors and seems to be crucial for
ligand recognition.22,25 Hydrogen bonds between OD1 of
Asn5086.52 and the hydroxyl oxygen (O5 in Fig. 2A) of (R)- or (S)-
mepenzolate, and between ND2 of Asn5086.52 and the carbonyl
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Y. Yamashita et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Table 1
oxygen (O3 in Fig. 2A) of (R)- or (S)-mepenzolate, were stably
retained throughout the MD simulations. With regards to these
hydrogen bonds, there were no significant differences in the mea-
sured distances for the two enantiomers; the average distance
between OD1 of Asn5086.52 and the hydroxyl oxygen of (R)-mepen-
zolate or (S)-mepenzolate was 2.869 Å or 2.846 Å, respectively,
while between ND2 of Asn5086.52 and the carbonyl oxygen of
(R)-mepenzolate or (S)-mepenzolate the average distance was
2.849 Å or 2.977 Å, respectively. Further to this, no significant dif-
Affinity of (R)-mepenzolate, (S)-mepenzolate and ( )-mepenzolate for hM3R and
hM2R
Compound
Ki (nM)
hM2R
hM3R
(R)-Mepenzolate
(S)-Mepenzolate
(R)-Mepenzolate
0.45 0.13
2.52 0.64
0.68 0.01
2.11 0.23
28.0 1.70
2.60 0.22
Ki values were calculated based on data shown in Figure 3. Values shown are
mean SEM (n = 3).
ferences in the interactions of other residues (Ser1523.36
,
Tyr1493.33 and Tyr 5307.39) which surround (R)- or (S)-mepenzolate
were found (data not shown). Based on these data, we suggest that
the ionic interaction between the charged nitrogen atom and its
counter ion Asp1483.32 is important for the observed differences
in affinity of (R)- and (S)-mepenzolate for hM3R.
zolate was less than one-tenth of that of (S)-mepenzolate, showing
that the former has the much higher affinity for hM3R than the lat-
ter. The Ki value of ( )-mepenzolate was between the values of (R)-
mepenzolate and (S)-mepenzolate (Table 1). We also examined the
affinity of each mepenzolate to human M2R (hM2R) using a similar
approach. As shown in Figure 3B and Table 1, (R)-mepenzolate also
exhibited a higher affinity for hM2R than (S)-mepenzolate. Further
to this, the data in Table 1 demonstrate that each form of mepen-
zolate has a higher affinity for hM2R than for hM3R.
Our next step was to compare the binding affinities of (R)-
mepenzolate, (S)-mepenzolate and ( )-mepenzolate to hM3R by
carrying out radiolabelled [N-methyl-3H]scopolamine methyl bro-
mide ([3H]NMS) displacement studies on this receptor. As shown
in Figure 3A, all three forms of mepenzolate inhibit NMS-binding
to hM3R in a dose-dependent manner, showing that they are able
to bind to this receptor. The binding affinity of each mepenzolate
to hM3R was compared according to their antagonist dissociation
constant Ki value. As shown in Table 1, the Ki value of (R)-mepen-
3.2. Bronchodilatory and anti-inflammatory activities of
mepenzolate enantiomers in vivo
We tested for the possibility that enantiomerization of the
mepenzolate took place after the administration of (R)-mepenzo-
late or (S)-mepenzolate in the lung. Either (R)-mepenzolate or
(S)-mepenzolate was administered intratracheally, and then lung
homogenates were prepared and amounts of both enantiomers
were assessed by HPLC analysis. As shown in Table 2, (S)-mepenzo-
late or (R)-mepenzolate could not be detected after the administra-
tion of (R)-mepenzolate or (S)-mepenzolate, respectively, showing
that enantiomerization was not taking place in the lung. The
results in Table 2 also show that intratracheally administered
mepenzolate rapidly disappears from the lung and that the rate
of disappearance is indistinguishable between the two
enantiomers.
We then compared the bronchodilatory activities of (R)-mepen-
zolate and (S)-mepenzolate based on their capacity to inhibit the
increase in airway resistance induced by methacholine.9 As shown
in Figure 4A, at a dose of 38 lg/kg (one twentieth of clinical dose,
orally), the intratracheal administration of either (R)-mepenzolate
or ( )-mepenzolate completely suppressed the methacholine-
induced increase in airway resistance; in contrast the suppression
by (S)-mepenzolate was partial. On the other hand, at a dose of
3.8 lg/kg, (R)-mepenzolate and ( )-mepenzolate, but not (S)-
mepenzolate, showed an inhibitory effect on the methacholine-
induced increase in airway resistance (Fig. 4B). These results show
that (R)-mepenzolate has a more potent bronchodilatory activity
than (S)-mepenzolate. Considering that the bronchodilatory activ-
ity of mepenzolate is mediated via its antagonistic activity on M3R,
and the fact that the M3R amino acid sequence homology between
human and mouse is relatively high (97.4%), the results in Figure 4
are consistent with those shown in Figure 3.
We next compared the anti-inflammatory activities of (R)-
mepenzolate and (S)-mepenzolate. Porcine pancreatic elastase
(PPE)-induced pulmonary inflammatory responses were moni-
tored as a function of the number of leucocytes in bronchoalveolar
lavage fluid (BALF). As shown in Figure 5A, the total number of leu-
cocytes and the individual number of neutrophils in BALF
increased after the PPE treatment; this increase was partially sup-
pressed by the simultaneous intratracheal administration of each
mepenzolate. The extent of suppression was indistinguishable
between (R)-mepenzolate, (S)-mepenzolate and ( )-mepenzolate
Figure 3. Binding of (R)-mepenzolate, (S)-mepenzolate and ( )-mepenzolate to
hM3R and hM2R in vitro. Membrane fractions prepared from cells expressing hM3R
(A) or hM2R (B) were incubated with radiolabelled NMS (2 nM) in the presence of
indicated concentrations of (R)-mepenzolate ((R)-Mep), (S)-mepenzolate ((S)-Mep)
or ( )-mepenzolate (( )-Mep) for 2 h and NMS binding was determined by the
filter-binding assay. Values shown are mean SEM (n = 3).
at doses of 7.5 and 38 lg/kg (Fig. 5A). We also monitored
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Y. Yamashita et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
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Table 2
Pulmonary levels of (R)-mepenzolate and (S)-mepenzolate after intratracheal administration of (R)-mepenzolate or (S)-mepenzolate. Mice were intratracheally administered (R)-
mepenzolate or (S)-mepenzolate (20 mg/kg), and lung homogenates were prepared after indicated periods
Compound
Tissue concentration (
l
g/lung)
(R)-mepenzolate
(S)-mepenzolate
5 min
15 min
30 min
5 min
15 min
30 min
(R)-Mepenzolate
(S)-Mepenzolate
44.5 11.5
n.d.
6.3 4.2
n.d.
5.1 1.3
n.d.
n.d.
36.4 9.5
n.d.
7.6 0.5
n.d.
6.2 0.4
Levels of (R)-mepenzolate and (S)-mepenzolate were determined as described in the Materials and Methods. Values are mean SEM (n = 3). n.d., not detected.
suppression indistinguishable between the three forms (Fig. 5B).
The results in Figure 5 suggest that the anti-inflammatory effect
afforded by (R)-mepenzolate and (S)-mepenzolate is indistinguish-
able. Although the primary target molecule mediating the anti-
inflammatory activity of mepenzolate is not known as yet, it would
seem that, in contrast to the case of M3R, both enantiomers have
similar affinity or intrinsic efficacy for this target molecule.
3.3. Adverse effects of mepenzolate enantiomers in vivo
In relation to the clinical application of mepenzolate to treat
COPD patients, constipation and arrhythmia (heart palpitations)
have been noted as adverse side effects of this drug as a conse-
quence of its inhibitory effect on muscarinic receptors.26,27 To this
end, we compared the effects of (R)-mepenzolate and (S)-mepen-
zolate on defecation and heart rates in mice. Since the efficiency
of absorption of intratracheally administered mepenzolate into
the circulation is low (Tanaka et al., unpublished results), much
higher dose of mepenzolate was predicted to be required to affect
defecation and heart rates, compared to bronchodilatory and anti-
inflammatory activities.
Mice were subjected to restraint stress as a means to increase
fecal pellet output. As shown in Figure 6, the administration of
either (R)-mepenzolate or ( )-mepenzolate (4.7 mg/kg) suppressed
fecal pellet output with respect to control (vehicle) mice. (S)-
mepenzolate (4.7 mg/kg) also showed a tendency to suppress fecal
pellet output, but the suppression was not statistically significant
(Fig. 6). These findings suggest that (R)-mepenzolate has a more
potent inhibitory effect on fecal pellet output than (S)-mepenzo-
late. Since M3R expressed in the intestinal smooth muscle regulate
intestinal motility, the results in Figure 6 can be explained in terms
of the higher relative affinity for M3R of (R)-mepenzolate than (S)-
mepenzolate (Fig. 3A and Table 1).
The effect of mepenzolate on heart rate was measured by infra-
red sensor. As shown in Figure 7, the separate administration of
(R)-mepenzolate, (S)-mepenzolate and ( )-mepenzolate increased
heart rate to a similar extent. Since the stimulation of M2R
expressed in the heart mediates a reduction of heart rate, the
results in Figure 7 are not consistent with (R)-mepenzolate having
a higher relative affinity for M2R than (S)-mepenzolate (Fig. 3B and
Table 1). This contradiction may be explained by differences in the
sensitivities of the assays used, where the in vitro filter-binding
assay is more sensitive than the in vivo heart rate assay. Further-
more, the difference in binding affinity between (R)-mepenzolate
and (S)-mepenzolate is larger for M3R than for M2R (Fig. 3 and
Table 1), which may explain why the difference between (R)-
mepenzolate and (S)-mepenzolate for fecal pellet output is clearer
than that for heart rate.
Figure 4. Effect of intratracheal administration of (R)-mepenzolate, (S)-mepenzo-
late and ( )-mepenzolate on methacholine-induced airway constriction. (R)-
mepenzolate ((R)-Mep), (S)-mepenzolate ((S)-Mep) or ( )-mepenzolate (( )-Mep)
at 38 (A) or 3.8 (B)
lg/kg was administered intratracheally. After 1 h, mice were
exposed to nebulized methacholine 5 times for 20 s with a 40 s interval between
exposures and airway resistance was determined after each methacholine chal-
lenge as described in the Experimental section. Values shown are mean SEM. ⁄⁄P
<0.01 ((R)-mepenzolate versus (S)-mepenzolate).
PPE-induced pulmonary inflammatory responses based on the lev-
els of pro-inflammatory cytokines (tumor necrosis factor (TNF)-a)
Since COPD is characterized by airflow limitation and abnormal
and chemokines (macrophage inflammatory protein (MIP)-2,
monocyte chemotactic protein (MCP)-1 and keratinocyte-derived
chemokine (KC)) in BALF. These levels were increased by the PPE
administration, with this increase partially suppressed by the
simultaneous intratracheal administration of either (R)-mepenzo-
late, (S)-mepenzolate or ( )-mepenzolate, with the extent of
inflammatory responses,
a combination of anti-inflammatory
drugs (such as steroids) and bronchodilators is the standard treat-
ment regime.28,29 Since mepenzolate has both anti-inflammatory
and bronchodilatory activities, this drug may be beneficial for
treating COPD without the concomitant use of other medications.
In this study, we compared the bronchodilatory and anti-inflam-
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Y. Yamashita et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Figure 5. Effect of intratracheal administration of (R)-mepenzolate, (S)-mepenzolate and ( )-mepenzolate on PPE-induced pulmonary inflammatory responses. The indicated
dose of 7.5 or 38 g/kg of (R)-mepenzolate ((R)-Mep), (S)-mepenzolate ((S)-Mep) or ( )-mepenzolate (( )-Mep) was administered intratracheally, and 1 h later mice were
treated with or without (vehicle) PPE (20 U/kg). Six hours after the PPE administration, BALF was prepared. The total cell number and the number of neutrophils were
l
determined as described in the Experimental section (A). The amounts of TNF-
a, MIP-2, MCP-1 and KC in BALF were determined by ELISA (B). Values shown are mean SEM.
⁄P <0.05; ⁄⁄P <0.01.
matory activities of (R)-mepenzolate and (S)-mepenzolate and
found that although the anti-inflammatory activity was indistin-
guishable between the two enantiomers, the bronchodilatory
activity of (R)-mepenzolate was superior to that of (S)-mepenzo-
late. These results suggest that (R)-mepenzolate is likely to be
more appropriate than (S)-mepenzolate as a drug to treat COPD
patients. However, we also found that (R)-mepenzolate has a more
potent inhibitory effect on fecal pellet output than (S)-mepenzo-
late, suggesting that (R)-mepenzolate may cause more severe con-
stipation than (S)-mepenzolate as an adverse side effect associated
with its use. However, it should be noted that the intratracheally
administered mepenzolate showed both anti-inflammatory and
bronchodilatory effects in mice at a much lower dose than that
which affected defecation and heart rates. For this reason we con-
sider that intratracheally administered mepenzolate may achieve
both its anti-inflammatory and bronchodilatory effects without
affecting intestinal motility and heart rate in a clinical setting.
The relationship between the stereochemistry and potency of
activity for various muscarinic receptor antagonists has been
studied, and it was suggested that the former greatly affects the lat-
ter.30,31 In particular, similar to mepenzolate, 3-quinuclidinyl benzi-
late has an asymmetric carbon at the C3 position of the piperidine
skeleton containing a tertiary amine; it was reported that (R)-3-qui-
nuclidinyl benzilate has a higher affinity for muscarinic receptors
than (S)-3-quinuclidinyl benzilate.30 Furthermore, aclidinium bro-
mide and glycopyrronium bromide (glycopyrronium), both of which
are muscarinic receptor antagonists and used clinically for COPD
patients, also have an asymmetric carbon at this position and it
was suggested that the (R)-isomer of each of these has a higher affin-
ity for M3R than the corresponding (S)-isomer.32 Thus, it is interest-
ing that mepenzolate shares similar stereochemistry and
pharmacological properties with these other M3R antagonists.
It is difficult to determine which of (R)-mepenzolate and ( )-
mepenzolate should be developed as a drug to treat COPD patients,
as anti-inflammatory activity, bronchodilatory activity, inhibitory
effectonfecal pelletoutput, andstimulatoryeffect on heart ratewere
indistinguishable between the two forms. Because (R)-mepenzolate
has a higher affinity for hM3R than (S)-mepenzolate, (R)-mepenzo-
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Y. Yamashita et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
7
and we previously reported that the dose–response profile of
mepenzolate for bronchodilation was similar to that of ipratropi-
um.9 These results suggest that mepenzolate may be inferior to tiot-
ropium and glycopyrronium as a bronchodilator. On the other hand,
we recently reported that steroids do not provide protective or
therapeutic benefits against PPE-induced pulmonary emphysema,
alterations of lung mechanics or respiratory dysfunction,33 whereas
we showed that mepenzolate is effective against these disorders
under the same experimental conditions.9 Furthermore, we
reported that glycopyrronium suppressed the PPE-induced pul-
monary emphysema and alterations of lung mechanics, however,
the extent of suppression of emphysema was not as apparent as
that seen with mepenzolate, and glycopyrronium did not signifi-
cantly suppress the PPE-induced respiratory dysfunction.9 Tiotropi-
um did not suppress the PPE-induced pulmonary emphysema,
alterations of lung mechanics, or respiratory dysfunction.9 These
results suggest that mepenzolate may be superior to steroids, tiot-
ropium and glycopyrronium as an anti-inflammatory drug. Thus,
we consider that chemical modification of (R)-mepenzolate, which
is aimed both to change its bronchodilatory effect from short-acting
to long-acting and to maintain its anti-inflammatory activity,
would be important for identification more therapeutically benefi-
cial drugs for COPD.
Figure 6. Effect of intratracheal administration of (R)-mepenzolate, (S)-mepenzo-
late and ( )-mepenzolate on fecal pellet output. (R)-mepenzolate ((R)-Mep), (S)-
mepenzolate ((S)-Mep) or ( )-mepenzolate (( )-Mep) (4.7 mg/kg) was administered
intratracheally and one hour later mice were exposed to restraint stress. The
number of fecal pellets excreted during the restraint stress period (1 h) was
determined. Values shown are mean SEM. ⁄⁄P <0.01.
4. Conclusion
Results in this study suggest that (R)-mepenzolate may have
superior properties to (S)-mepenzolate as a drug to treat COPD
patients given that the former has more potent bronchodilatory
activity than the latter.
5. Experimental section
5.1. Chemicals and animals
All organic solvents and reagents used for the synthesis were
purchased from commercial sources and used without further
purification. (R)- and (S)-3-hydroxy-1-methylpiperidine were from
Pharma Block (Nanjing, China). Mepenzolate, PPE and HPLC-grade
acetonitrile were obtained from Sigma–Aldrich (St. Louis, MO).
Novo-Heparin for injection was from Mochida Pharmaceutical Co.
(Tokyo, Japan). Chloral hydrate was from Nacalai Tesque (Kyoto,
Japan). Diff-Quik was from Sysmex Co (Kobe, Japan). Isoflurane
was from Pfizer (New York, NY). The Amicon ultra-0.5 centrifugal
filter that we used was purchased from Merck Millipore (Billerica,
Figure 7. Effect of intratracheal administration of (R)-mepenzolate, (S)-mepenzo-
late and ( )-mepenzolate on heart rate. The indicated doses of (R)-mepenzolate
((R)-Mep), (S)-mepenzolate ((S)-Mep) or ( )-mepenzolate (( )-Mep) were admin-
istered intratracheally. The alteration of heart rate (beats per minute) was
monitored as described in the Experimental section, with the mepenzolate-
dependent alteration of heart rate (percentage change from the baseline to peak)
calculated. Values shown are mean SEM. ⁄P <0.05.
MA). ELISA kits for TNF-a, MIP-2, MCP-1 and KC were from R&D
Systems (Minneapolis, MN). Other solvents and reagents were pur-
chased from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan) or
Wako Pure Chemical Industries (Tokyo, Japan). ICR mice (4–
6 weeks old, male) were purchased from Charles River (Yokohama,
Japan). The experiments and procedures described here were car-
ried out in accordance with the Guide for the Care and Use of Lab-
oratory Animals as adopted and promulgated by the National
Institutes of Health, and were approved by the Animal Care Com-
mittee of Keio University.
late may be more effective and safer for clinical use than ( )-mepen-
zolate. On the other hand, a significantadvantage of ( )-mepenzolate
is that it already has regulatory approval, and some pre-clinical tests
(suchas genotoxicitytests) could beomitted fromthebatteryof tests
required for the approval. Therefore, it would perhaps be better to
initially consider ( )-mepenzolate for the treatment of COPD, fol-
lowed by the development and subsequent introduction of (R)-
mepenzolate.
Muscarinic antagonists used for COPD are usually categorised as
being long-acting (such as tiotropium and glycopyrronium) or
short-acting (such as ipratropium) and we previously reported that
mepenzolate belongs to short-acting ones.9 Furthermore, the clini-
5.2. Chemistry
Fourier transform IR spectra were recorded on a Jeol FT-IR
SPX60 spectrometer as attenuated total reflection for a solid. 1H
NMR and 13C NMR spectra were recorded on a VARIAN 500-MR
spectrometer (Agilent Technologies Japan, Tokyo, Japan) operating
at 500 MHz, in a ca. 2% solution of dimethyl sulfoxide (DMSO)-d6.
Coupling constant (J) values below are estimated in hertz (Hz)
and spin multiples are given as s (singlet), d (doublet), and m (mul-
cal dose of tiotropium (18
for COPD is much lower than that of ipratropium (160
l
g/day) or glycopyrronium (50
l
l
g/day)
g/day)
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8
Y. Yamashita et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
tiplet). HR-MS were measured on a Jeol JMS-700 MStation. The
progress of all reactions was monitored by thin-layer chromatogra-
phy with silica gel glass plates (60 F254) (Merck Ltd, Tokyo, Japan),
and spots were visualized with ultraviolet light (254 nm) and
stained with 5% ethanolic phosphomolybdic acid. Column chroma-
tography was performed using silica gel 60N (Kanto Chemical Co.,
Tokyo, Japan).
HPLC-UV chromatograms were acquired in a Waters Alliance
2695 chromatographer equipped with a Waters 2996 photodiode
array detector (Waters, Milford, MA). HPLC analysis was conduced
according to method A (see below) with the retention time
expressed in min detected at 220 nm. For HPLC method A, chroma-
(10À10 to 3 Â 10À5 M) of each mepenzolate were tested in triplicate
to generate competition curves. Non-specific binding was deter-
mined in the presence of atropine (2.5 lM). The samples were
passed through a GF/C filter (Filtermat A, PerkinElmer Life and
Analytical Sciences, Boston, MA) that was pre-incubated for 1 h
with wash buffer (50 mM Tris/HCl (pH 7.4), 100 mM NaCl) contain-
ing 1.0% polyethylenimine, and washed four times with ice-cold
wash buffer. Filters were then dried for 30 min before attachment
to MeltiLex A (melt-on scintillation sheet; PerkinElmer Life and
Analytical Sciences, Boston, MA). The radioactivity remaining on
the filter was monitored with MicroBeta Trilux microplate scintil-
lation counter (PerkinElmer Life and Analytical Sciences, Boston,
MA). Affinities at equilibrium were determined as equilibrium
antagonist dissociation constant (Ki) values after correcting the
experimentally determined IC50 values with the experimentally
determined Kd value of NMS for hM2R or hM3R and the concentra-
tion of NMS, as described previously.35 The Ki value was obtained
from three independent curves. All adjustments were performed
using Prism (GraphPad Software, Inc., San Diego, CA).
tography was performed on
a
Daicel Chiralpak IA
(250 mm  46 mm). The mobile phase, at a flow of 1.0 mL/min,
was a binary gradient of water (containing 0.1 M potassium hexa-
fluorophosphate) and acetonitrile, 30:70.
5.2.1. (R)-Mepenzolate ((R)-1)
To a solution of benzilic acid (830 mg, 3.6 mmol) in N,N-dimeth-
ylformamide (DMF) (8 mL), CDI (883 mg, 5.4 mmol) was added and
the mixture was stirred for 15 min at room temperature. To this
5.4. Homology modelling
mixture, a solution of (R)-3 (500 lL, 4.3 mmol) in DMF (4 mL)
was added dropwise at 80 °C and the resulting mixture was stirred
for 18 h at the same temperature. After cooling to room tempera-
ture, the reaction was quenched with water and organic materials
were extracted with ethyl acetate. The combined extract was
washed with brine, dried over sodium sulfate and concentrated
in vacuo. The residue was then applied to a short silica gel column,
eluted with ethyl acetate and concentrated in vacuo to give (R)-4 as
a yellow oil, which was used for the next step without further
purification.
To a solution of (R)-4 (860 mg, 2.6 mmol) in acetonitrile
(10 mL), methyl bromide (2.0 M in tetrahydrofuran, 6.2 mL,
12.4 mmol) was added and stirred for 5 h at room temperature.
The precipitates were filtered off and re-crystallized from dichloro-
methane and methanol to give (R)-1 as colorless fine needles
The structure of rat M3R bound to its antagonist, tiotropium,
was recently solved by X-ray crystallography (PDB code 4DAJ.22
Although a T4-lysozyme was fused to intracellular loop 3 (ICL3)
for enhancing crystallization, there were no residue gaps and few
residue mismatches between the crystal structure and hM3R
amino acid sequence except for both terminus regions and the
ICL3. Therefore, we constructed an hM3R model by fixing the miss-
ing atoms in the crystal and replacing the mismatched amino acid
residues with human ones. The fused T4-lysozyme is replaced with
a ten amino acid linker sequence (GGGGSGGGGS), because this
region has no reliable template for modelling. These modelling
procedures were performed using Modeller v9.4.36
5.5. Molecular dynamics
(980 mg, 64% 2 steps). IR
mmax: 3432, 3315, 1735, 1216, 1093,
1068, 923, 705 cmÀ1 1H NMR (DMSO-d6): d = 7.28–7.37 (m,
;
MD simulations were carried out in an explicit membrane and
water system by using myPresto/cosgene.37 The whole structure
consists of the hM3R model, (R)-1 or (S)-1, four cholesterols, 146
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 10,212
water molecules (TIP3P model) including 21 Na+ and 35 ClÀ parti-
cles.38 NaCl was added to achieve the saline condition and exces-
sive ClÀ was added to neutralize the total charge of the system.
The AMBER99 force field parameters39 were used for hM3R. Force
field parameters for (R)- or (S)-mepenzolate were obtained accord-
ing to the generalized AMBER force field procedure40 with partial
charges derived from quantum chemical calculations by GAUSSIAN
03 at the HF/6-31G⁄ level of theory. Periodic boundary conditions
were applied and Berendsen’s method for temperature and pres-
sure coupling was adopted (300 K and 1 atm, respectively). After
performing 400 steps of energy minimization by the steepest des-
cent method and the conjugate gradient method, the NPT (constant
particle number, constant pressure, and constant temperature)41
for 200 ps was performed to obtain an equilibrated system with
the following conditions: periodic boundary, 300 K, 1 atm, cut-
10H), 6.82 (s, 1H), 5.28 (m, 1H), 3.63 (dd, J = 13.5, 3.5 Hz, 1H),
3.49 (dd, J = 13.5, 4.7 Hz, 1H), 3.37 (m, 1H), 3.33 (m, 1H), 3.10 (s,
3H), 2.82 (s, 3H), 1.63–1.85 (m, 4H); 13C NMR (DMSO-d6):
d = 171.9, 143.0, 142.7, 128.0, 127.9, 127.7, 127.6, 127.0, 80.7,
26
66.8, 61.6, 60.8, 25.2, 16.2; [
a]
À8.12 (c 1.00, methanol); mp
D
224.2–224.9 °C; HR-MS (FAB): calcd for
C
21H26O3N: [M+1]+:
340.1913; found: m/z = 340.1905. HPLC analysis was done accord-
ing to method A (retention time, 16.8 min; single peak).
5.2.2. (S)-Mepenzolate ((S)-1)
The title compound ((S)-1) was synthesized using the same pro-
cedure described for the preparation of (R)-mepenzolate, except
for the use of (S)-3-hydroxy-1-methylpiperidine instead of (R)-iso-
mer. The final sample was a colorless needle (934 mg, 61% 2 steps).
25
[a
]
+8.33 (c 1.00, methanol). Other physical and spectral data of
D
this enantiomer were in good accordance with those of (R)-1. HPLC
analysis was done according to method
15.0 min; single peak).
A (retention time,
off = 12 Å, and 1.0 fs/step. The ZD method42 = 0.0 ÅÀ1) was
(a
5.3. Filter-binding assay
applied for the calculation of electrostatic force terms. Further
200 ps equilibrium calculations were performed under NVT (con-
stant particle number, constant volume, and constant tempera-
ture) conditions with the cell size maintained (300 K, 1.0 fs/step
for 50 ps, 0.75 fs/step for 60 ps, 1.0 fs/step for 40 ps and 2.0 fs/step
for 50 ps). After these preliminary calculations, a production run at
300 K was performed with the same cell size and the time step was
set to 2 fs. The SHAKE algorithm was applied to fix all the
bonds involving hydrogen during the NVT simulations. Snapshot
The filter-binding assay was done as described previously34
with some modifications. Membrane fractions prepared from
CHO-K1 cells expressing hM2R or hM3R (Membrane Target Sys-
tems, Perkin–Elmer Life and Analytical Sciences, Boston, MA; pro-
tein concentration, 10
[3H]NMS (85.5 Ci/mmol) at room temperature for 2 h in 200
PBS in the presence of each mepenzolate. A range of concentrations
lg/well) were incubated with 2 nM
lL
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Y. Yamashita et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
9
structures were obtained at every 10 ps as the target structure was
extracted from a trajectory of 100 ns.
fuged to obtain the final sample. An aliquot (300
l
l) of each sample
was ultrafiltered with an Amicon ultra-0.5 centrifugal filter to
extract the mepenzolate. The filtrate was analysed by analytical
HPLC using method A (see above).
5.6. Treatment of mice with PPE and mepenzolate
Mice maintained under anaesthesia with isoflurane were intrat-
racheally administered PPE (20 U/kg) or each mepenzolate (various
doses) in sterile saline (1 ml/kg) via micropipette. For control mice,
sterile saline alone was administered by the same procedure. The
administration of mepenzolate was performed 1 h prior to the
PPE administration.
5.12. Statistical analysis
All values are expressed as the mean SEM Tukey’s test was
used to evaluate differences between three or more groups. Differ-
ences were considered to be significant for values of P <0.05.
Acknowledgments
5.7. Measurement of airway resistance
This work was supported by Grants-in-Aid of Scientific
Research from the Ministry of Health, Labour, and Welfare of Japan,
Grants-in-Aid for Scientific Research from the Ministry of Educa-
tion, Culture, Sports, Science and Technology of Japan, and
Grants-in-Aid of the Japan Science and Technology Agency.
Airway resistance was monitored with a computer-controlled
small-animal ventilator (FlexiVent, SCIREQ, Montreal, Canada), as
described previously.13 Mice were anesthetized with chloral
hydrate (500 mg/kg), after 20 min a tracheotomy was performed,
and an 8 mm-long section of metallic tube (outer or inner diame-
ter, 1.27 mm or 0.84 mm, respectively) was inserted into the tra-
chea. Mice were mechanically ventilated at a rate of 150 breaths/
min, using a tidal volume of 8.7 ml/kg and a positive end-expira-
tory pressure of 2–3 cmH2O. For measurement of methacholine-
induced increases in airway resistance, 1 h after the mepenzolate
administration, mice were exposed to nebulised methacholine
(5 mg/ml) five times for 20 s with a 40 s interval between expo-
sures, and airway resistance was measured after each methacho-
line challenge by the snapshot technique. All data were analysed
using FlexiVent software.
Supplementary data
Supplementary data (1H NMR and 13C NMR spectra of final com-
pounds for (R)-mepenzolate (1) and (S)-mepenzolate (1)) associ-
ated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmc.2014.04.029.
References and notes
1. Rabe, K. F.; Hurd, S.; Anzueto, A.; Barnes, P. J.; Buist, S. A.; Calverley, P.; Fukuchi,
Y.; Jenkins, C.; Rodriguez-Roisin, R.; van Weel, C.; Zielinski, J. Am. J. Respir. Crit.
Care Med. 2007, 176, 532.
2. Barnes, P. J.; Stockley, R. A. Eur. Respir. J. 2005, 25, 1084.
3. Alsaeedi, A.; Sin, D. D.; McAlister, F. A. Am. J. Med. 2002, 113, 59.
4. Calverley, P. M.; Anderson, J. A.; Celli, B.; Ferguson, G. T.; Jenkins, C.; Jones, P.
W.; Yates, J. C.; Vestbo, J. N. Engl. J. Med. 2007, 356, 775.
5. Barnes, P. J.; Ito, K.; Adcock, I. M. Lancet 2004, 363, 731.
6. Rajendrasozhan, S.; Yang, S. R.; Edirisinghe, I.; Yao, H.; Adenuga, D.; Rahman, I.
Antioxid. Redox Signal. 2008, 10, 799.
7. Ito, K.; Yamamura, S.; Essilfie-Quaye, S.; Cosio, B.; Ito, M.; Barnes, P. J.; Adcock, I.
M. J. Exp. Med. 2006, 203, 7.
5.8. Analysis of PPE-induced inflammatory responses
Six hours after the PPE administration, BALF was collected by
cannulating the trachea and lavaging the lung with 1 ml of sterile
saline containing 50 U/ml heparin (2 times). About 1.8 ml of BALF
was routinely recovered from each animal. The total cell number
was counted using a hemocytometer. Cells were stained with
Diff-Quik reagents after centrifugation with CytospinÒ 4 (Thermo
Electron Corporation, Waltham, MA), and the ratio of number of
neutrophils to total cell number was calculated. Cytokine and che-
mokine levels were measured by ELISA, according to the manufac-
turer’s protocol.
8. Mizushima, T. J. Biochem. 2011, 149, 499.
9. Tanaka, K.; Ishihara, T.; Sugizaki, T.; Kobayashi, D.; Yamashita, Y.; Tahara, K.;
Yamakawa, N.; Iijima, K.; Mogushi, K.; Tanaka, H.; Sato, K.; Suzuki, H.;
Mizushima, T. Nat. Commun. 2013, 4, 1.
10. Chen, J. Y. Arch. Int. Pharmacodyn. Ther. 1959, 121, 78.
11. Buckley, J. P.; De, F. J.; Reif, E. C. J. Am. Pharm. Assoc. Am. Pharm. Assoc. (Baltim)
1957, 46, 592.
12. Long, J. P.; Keasling, H. H. J. Am. Pharm. Assoc. Am. Pharm. Assoc. (Baltim) 1954,
43, 616.
5.9. Analysis of fecal pellet output
13. Tanaka, K.; Sato, K.; Aoshiba, K.; Azuma, A.; Mizushima, T. Am. J. Physiol. Lung
Cell. Mol. Physiol. 2012, 302, L1250.
14. Bosnjak, B.; Tilp, C.; Tomsic, C.; Dekan, G.; Pieper, M. P.; Erb, K. J.; Epstein, M. M.
Pulm. Pharmacol. Ther. 2014, 27, 44.
One hour after the mepenzolate administration, mice were sub-
jected to restraint stress by being placed individually into a 50 ml
tube (Becton Dickinson, Franklin Lakes, NJ) for 1 h, as described
previously.43 These tubes are small enough to restrain a mouse
so that it is able to breathe but unable to move freely. The number
of fecal pellets excreted during the restraint stress period (1 h) was
measured.
15. Shen, L. L.; Liu, Y. N.; Shen, H. J.; Wen, C.; Jia, Y. L.; Dong, X. W.; Jin, F.; Chen, X.
P.; Sun, Y.; Xie, Q. M. Int. Immunopharmacol. 2014, 18, 358.
16. Eglen, R. M.; Hegde, S. S.; Watson, N. Pharmacol. Rev. 1996, 48, 531.
17. Eglen, R. M. Prog. Med. Chem. 2005, 43, 105.
18. Richter, J. E.; Kahrilas, P. J.; Johanson, J.; Maton, P.; Breiter, J. R.; Hwang, C.;
Marino, V.; Hamelin, B.; Levine, J. G. Am. J. Gastroenterol. 2001, 96, 656.
19. Bauernfeind, A. J. Antimicrob. Chemother. 1999, 43, 77.
20. Bartlett, J. B.; Dredge, K.; Dalgleish, A. G. Nat. Rev. Cancer 2004, 4, 314.
21. Dolle, F.; Hinnen, F.; Vaufrey, F.; Demphel, S.; Bramoulle, Y.; Fournier, D.;
Ponchant, M.; Valette, H.; Crouzel, C. J. Labelled Compd. Rad. 2001, 44, 337.
22. Kruse, A. C.; Hu, J.; Pan, A. C.; Arlow, D. H.; Rosenbaum, D. M.; Rosemond, E.;
Green, H. F.; Liu, T.; Chae, P. S.; Dror, R. O.; Shaw, D. E.; Weis, W. I.; Wess, J.;
Kobilka, B. K. Nature 2012, 482, 552.
23. Venkatakrishnan, A. J.; Deupi, X.; Lebon, G.; Tate, C. G.; Schertler, G. F.; Babu, M.
M. Nature 2013, 494, 185.
24. Suga, H.; Ehlert, F. J. Biochemistry 2013, 52, 4914.
25. Haga, K.; Kruse, A. C.; Asada, H.; Yurugi-Kobayashi, T.; Shiroishi, M.; Zhang, C.;
Weis, W. I.; Okada, T.; Kobilka, B. K.; Haga, T.; Kobayashi, T. Nature 2012, 482,
547.
5.10. Measurement of heart rate
Heart rate was measured with a MouseOx system (STARR Life
Sciences Corp., Allison Park, PA), as described previously.44 Mice
were anesthetized with chloral hydrate (500 mg/kg) and after
20 min the sensor was attached to the thigh. Heart rate was deter-
mined immediately after the mepenzolate administration, using
MouseOx software (STARR Life Sciences Corp., Allison Park, PA).
26. Agarwal, R.; Jindal, S. K. Eur. J. Intern. Med. 2008, 19, 227.
27. Verhamme, K. M.; Afonso, A. S.; van Noord, C.; Haag, M. D.; Koudstaal, P. J.;
Brusselle, G. G.; Sturkenboom, M. C. Pulm. Pharmacol. Ther. 2012, 25, 19.
28. Gross, N. J.; Giembycz, M. A.; Rennard, S. I. COPD 2010, 7, 141.
29. Miravitlles, M.; Anzueto, A. Int. J. Chron. Obstruct. Pulmon. Dis. 2009, 4, 185.
5.11. Determination of the level of mepenzolate in the lung
Whole lungs were taken from mepenzolate-treated mice,
homogenised in sterile PBS containing 50 U/ml heparin, and centri-
Please cite this article in press as: Yamashita, Y.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.029

10
Y. Yamashita et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
30. Kiesewetter, D. O.; Silverton, J. V.; Eckelman, W. C. J. Med. Chem. 1995, 38, 1711.
31. Rzeszotarski, W. J.; Mcpherson, D. W.; Ferkany, J. W.; Kinnier, W. J.;
Noronhablob, L.; Kirkienrzeszotarski, A. J. Med. Chem. 1988, 31, 1463.
32. Guerra, A.; Campillo, N.; Paez, J. A. Expert Opin. Ther. Pat. 2006, 16, 723.
33. Tanaka, K. I.; Sato, K.; Aoshiba, K.; Azuma, A.; Mizushima, T. Am. J. Physiol. Lung
Cell. Mol. Physiol. 2012.
34. Gavalda, A.; Miralpeix, M.; Ramos, I.; Otal, R.; Carreno, C.; Vinals, M.;
Domenech, T.; Carcasona, C.; Reyes, B.; Vilella, D.; Gras, J.; Cortijo, J.;
Morcillo, E.; Llenas, J.; Ryder, H.; Beleta, J. J. Pharmacol. Exp. Ther. 2009, 331,
740.
38. Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L. J.
Chem. Phys. 1983, 79, 926.
39. Wang, J. M.; Cieplak, P.; Kollman, P. A. J. Comput. Chem. 2000, 21, 1049.
40. Wang, J. M.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A.; Case, D. A. J. Comput.
Chem. 2004, 25, 1157.
41. Berendsen, H. J. C.; Postma, J. P. M.; Vangunsteren, W. F.; Dinola, A.; Haak, J. R. J.
Chem. Phys. 1984, 81, 3684.
42. Fukuda, I.; Kamiya, N.; Yonezawa, Y.; Nakamura, H. J. Chem. Phys. 2012, 137.
43. Asano, T.; Tanaka, K.; Suemasu, S.; Ishihara, T.; Tahara, K.; Suzuki, T.; Suzuki,
H.; Fukudo, S.; Mizushima, T. Biochem. Biophys. Res. Commun. 2012, 420, 444.
44. McLaughlin, B.; Buendia, M. A.; Saborido, T. P.; Palubinsky, A. M.; Stankowski, J.
N.; Stanwood, G. D. PLoS ONE 2012, 7, e36340.
35. Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099.
36. Sali, A.; Blundell, T. L. J. Mol. Biol. 1993, 234, 779.
37. Fukunishi, Y.; Mikami, Y.; Nakamura, H. J. Phys. Chem. B 2003, 107, 13201.
Please cite this article in press as: Yamashita, Y.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.029