The effect of absolute configuration on activity, subtype selectivity (M3/M2) of 3α-acyloxy-6β-acetoxyltropane derivatives as muscarinic M3 receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2012.12.052

Both enantiomers of 3α-acyloxy-6β-acetoxyltropane derivatives 1-4 were prepared respectively and underwent functional studies and radioreceptor binding assays. 6S Enantiomers showed obvious muscarinic M3, M2 antagonistic activity, while the 6R ones elicit

Full text of DOI:10.1016/j.bmc.2012.12.052

Bioorganic & Medicinal Chemistry 21 (2013) 1234–1239
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Bioorganic & Medicinal Chemistry
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The effect of absolute configuration on activity, subtype selectivity
(M3/M2) of 3a-acyloxy-6b-acetoxyltropane derivatives as muscarinic
M3 receptor antagonists
a
Zhi-Peng Wang a, Hui-Zhong Liu a, Liang Zhu b, You-Min Hu c, Yong-Yao Cui b, Yin-Yao Niu a, , Yang Lu ,
⇑
Hong-Zhuan Chen b
a Division of Chemistry, Department of Pharmacy, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
b Division of Pharmacology, Department of Pharmacy, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
c Functional Training Laboratory, Department of Pharmacy, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Both enantiomers of 3a-acyloxy-6b-acetoxyltropane derivatives 1–4 were prepared respectively and
Received 19 November 2012
Revised 19 December 2012
Accepted 20 December 2012
Available online 9 January 2013
underwent functional studies and radioreceptor binding assays. 6S Enantiomers showed obvious musca-
rinic M3, M2 antagonistic activity, while the 6R ones elicited little muscarinic activity by functional stud-
ies. Besides, the affinity of 6S enantiomers to muscarinic M3 receptors of rat submandibulary gland, M2
receptors of rat left atria was much larger than that of corresponding 6R enantiomers. All these pharm-
alogical results indicated 6S configuration was favorable for 3a-acyloxy-6b-acetoxyltropane derivatives
Keywords:
to bind with muscarinic M3 or M2 receptors and elicited antagonistic activity. Furthermore, the musca-
rinic M3 activity and subtype selectivity (M3/M2) of 6S enantiomers could be improved by increasing the
electron density of carbonyl oxygen or introducing methylene group between the carbonyl and phenyl
Tropane derivative
6S Configuration
Muscarinic antagonist
Enantiomer
ring in C-3
(M3/M2) of 3
M3 antagonists with high activity and low side effects or toxicity.
Ó 2013 Elsevier Ltd. All rights reserved.
a
position. Understanding the effect of absolute configuration on activity, subtype selectivity
a-acyloxy-6b-acetoxyltropane derivatives will provide the clues for designing muscarinic
Subtype selectivity
1. Introduction
larger than that of corresponding 6R enantiomers, suggesting that
the absolute configuration of -acyloxy-6b-acetoxyltropane
3
a
Applying muscarinic M3 receptor antagonists has become one
of the most important pharmaceutical approaches in the treatment
of respiratory and urinary tract disorders such as chronic obstruc-
tive pulmonary disease and urinary incontinence.1–4 However,
muscarinic M3 receptor antagonists with lower selectivity towards
M2 receptors will exhibit undesirable side effects in clinical use.5
So, it is necessary to evaluate not only activity but also subtype
selectivity (M3/M2) for muscarinic M3 receptor antagonists. In
derivatives might play an important role in muscarinic activity, as
well as subtype selectivity (M3/M2). As in a chiral environment such
as in vivo, two enantiomers demonstrate different chemical, bio-
chemical, and pharmacologic behaviors, the investigation of single
isomers rather than racemates is more favorable for improving
activity and reducing side effects or toxicity.10–12 Therefore, it is
more significant to study the activity and subtype selectivity (M3/
M2) for both enantiomers of 3
tives, if compared with their racemates. Besides, up to date, the
examination of subtype selectivity (M3/M2) for chiral 3 -acyloxy-
a-acyloxy-6b-acetoxyltropane deriva-
our previous studies, many racemic 3a-acyloxy-6b-acetoxyltro-
pane derivatives elicited antagonistic activity to muscarinic M3
receptors and moderate subtype selectivity (M3/M2),6,7 and the
6b-acetoxyl was confirmed to be crucial for the muscarinic
activity,8,9 as well as subtype selectivity (M3/M2). Furthermore,
a
6b-acetoxyltropane derivatives through biological assays has not
been reported.
In this paper, we prepared both enantiomers of 1 (3
oxy-6b-acetoxytropane), (3 -orthochlorobenzoyloxy-6b-acet-
oxytropane), 3 (3 -orthonitrobenzoyloxy-6b-acetoxytropane) and
4 (3 -phenylacetoxy-6b-acetoxytropane) respectively and deter-
a-benzoyl-
the functional antagonistic activity of some (3S,6S)-3
a-acyloxy-
2
a
6b-acetoxyltropane derivatives1 to muscarinic M3 receptors was
a
a
mined each binding affinity of the eight enantiomers to muscarinic
M3 receptors of rat submandibulary gland, M2 receptors of rat left
atria. The in vitro functional activity of the four pairs of enantio-
mers on isolated guinea pig ilea (M3), left atria (M2) was also
investigated, as evaluation of antagonistic effect on these models
⇑
Corresponding author. Tel.: +86 21 63846590-776464; fax: +86 21 53065329.
E-mail address: niuyinyao@yahoo.com.cn (Y.-Y. Niu).
As the absolute configuration of C3, as well as C1 and C5 is in agreement with that
1
of C6 for discussed compounds in this article, the absolute configurations of their
enantiomers are simply expressed as 6S or 6R for convenience in the following
discussion.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.12.052

Z.-P. Wang et al. / Bioorg. Med. Chem. 21 (2013) 1234–1239
1235
may provide a better indication of their potential activity. The
structure–activity relationship (SAR) and structure–subtype selec-
tivity (M3/M2) relationship (SSSR) were preliminarily discussed.
Understanding the effect of absolute configuration on activity, sub-
type selectivity (M3/M2) will provide the clues for successive de-
sign of muscarinic M3 receptor antagonists with high activity
and low side effects or toxicity.
M3, M2 receptors are 6.45 0.22 and 6.31 0.24, respectively. Four
6S enantiomers could inhibit carbachol-induced contraction on
isolated ilea (M3) and carbachol-induced bradycardia on isolated
left atrial (M2). But their corresponding 6R enantiomers could
not do, indicating that 6R configuration was not favorable for 1–4
to elicit antagonistic activity to muscarinic M3 or M2 receptors.
Like (6S)-4 (Fig. 2), (6S)-1, (6S)-2 and (6S)-3 shifted the carba-
chol-induced response curves to the right with a parallel manner.
The pA2 values of four 6S enantiomers are 6.51 0.52,
6.56 0.34, 6.41 0.44 and 6.91 0.28 for ilea (M3), while
5.88 0.39, 6.20 0.35, 6.07 0.40 and 5.96 0.43 for left atria
(M2), respectively (Table 1). The pA2 value of each of 6S enantio-
mers for ilea (M3) is obviously larger than that for left atria
(M2), indicating that the 6S enantiomers act as selective functional
antagonists to the muscarinic M3 receptors.
2. Results and discussion
2.1. Chemistry
(ꢀ)-5 ((ꢀ)-3a-Hydroxy-6b-acetoxytropane) and (+)-5, two
enantiomers, were acquired by resoluting ( )-5 with (+) or (ꢀ)-
2,3-dibenzoyl tartaric acid in isopropanol. The absolute configuration
of (ꢀ)-5 and that of (+)-5 were determined as (6S)-5 and (6R)-5,
respectively according to the relationship between the absolute
configuration and optical activity of chiral tropane derivatives.13
The resoluted (6S)-5 and (6R)-5 were examined to be optically pure
by HPLC (Fig. 1). Carbonylating (6S)-5 and (6R)-5, respectively gave
four pairs of optically pure enantiomers of 1–4, which are (6S)-1,
(6S)-2, (6S)-3, (6S)-4, and (6R)-1, (6R)-2, (6R)-3, (6R)-4 (Chart 1).
2.2.2. [3H]NMS binding to rat submandibulary glands (M3), left
atria (M2)
[3H]NMS (3H-labelled N-methylscopolamine) binding studies
were performed with a crude membrane fraction prepared from
rat submandibulary glands (M3) or left atria (M2). Specific binding
of [3H]NMS to submandibulary glands (M3) or left atria (M2) was
saturable. The dissociation equilibrium constant (Kd) and receptor
density (Bmax) are 0.58 0.06 nM and 63.37 3.94 fmol/mg protein
(n = 3) for submandibulary glands (M3), while 0.30 0.06 nM and
27.09 2.29 fmol/mg protein (n = 3) for left atria (M2) respectively
(Fig. 3).
2.2. Pharmacology
2.2.1. Effects of carbachol on isolated guinea pig ilea (M3), left
atria (M2)
The affinities of both enantiomers of 1–4 to submandibulary
glands (M3), left atria (M2) were examined. The binding of
[3H]NMS to muscarinc M3 or M2 receptors decreased with increas-
ing concentrations of 6S or 6R enantiomer of 1–4 in a log concen-
tration-dependent manner (Fig. 4). As shown in Table 2, four 6R
enantiomers displayed very weak binding affinity, among which
Carbachol, a typical muscarinic agonist, could stimulate con-
traction of isolated ilea (M3) or induce bradycardia of isolated left
atria (M2). Cumulative addition of carbachol produced a log con-
centration-dependent contractile response to isolated ilea (M3),
while inhibiting-contractile response to isolated left atria (M2).
The pEC50 (negative logarithm of the concentration of agonist caus-
ing a half-maximal response) values of carbachol for muscarinic
the smallest Ki value is 66 lM for (6R)-3 to muscarinc M3
Figure 1. Dynamic chromatograms of (A) ( )-5, (B) (6S)-5,(C) (6R)-5 on Chiralpak AD (150 ꢁ 4.6 mm I.D.) under normal phase conditions. Eluent: n-hexane–2-propanol–
diethylamine 80:20:0.1 (v/v/v); flow rate: 0.7 mL/min; column temperature: 25 °C. The peak-time of (6S)-5, (6R)-5 is 9.71, 11.29 min, respectively.
CH
3
CH
3
N
N
carbonylate
carbonylate
AcO
AcO
6
3
3
6
R
HO
(6R)-(+)-5
R
O
C
1 Ph
2 o-ClPh
resolution
(6R)-1, 2, 3, 4
( )-5
O
3 o-NO Ph
CH
CH
2
3
3
N
N
4 PhCH
2
AcO
AcO
3
3
6
6
R
O
HO
(6S)-(-)-5
C
(6S)-1, 2, 3, 4
O
Chart 1. Synthesis of both enantiomers of 1–4.

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Z.-P. Wang et al. / Bioorg. Med. Chem. 21 (2013) 1234–1239
Figure 2. Effects of muscarinic agonist carbachol on ilea (M3), left atria (M2) with incubation of (6S)-4 (n = 3–5).
receptors. This finding could be used to explain that the antagonis-
tic effect of 6R enantiomers on isolated ilea (M3) or left atria (M2)
was not detected in above functional studies due to their weak
binding affinity. Besides, the Ki(M2)/Ki(M3) values of the four 6R
enantiomers are approximately equal to 1. So, it is less significant
to discuss the SAR or SSSR for 6R enantiomers.
Four 6S enantiomers displayed obvious inhibition to the binding
of [3H]NMS with muscarinc M3 or M2 receptors, if compared to
their corresponding 6R enantiomers. The binding affinity of 6S
enantiomers is at least 18-fold that of corresponding 6R enantio-
Table 1
Results of both enantiomers of 1–4 to dynamic test on isolated guinea pig ilea (M3),
left atria (M2) (n = 3–5)
Compd
Functional studies (pA2 SE)
Ilea (M3)a
Left atria (M2)b
(6S)-1
(6R)-1
(6S)-2
(6R)-2
(6S)-3
(6R)-3
(6S)-4
(6R)-4
6.51 0.52⁄
5.88 0.39⁄
—
—
6.56 0.34⁄
6.20 0.35⁄
—
—
6.41 0.44⁄
6.07 0.40⁄
—
—
mers for muscarinic M3 receptors ((6S)-3, Ki = 3.7
Ki = 66 M), while 7-fold that of 6R ones for M2 receptors ((6S)-
4, Ki = 12 M and (6R)-4, Ki = 86 M) (Table 2), indicating that 6S
configuration was favorable for 3 -acyloxy-6b-acetoxyltropane
lM and (6R)-3,
6.91 0.28⁄
—
5.96 0.43⁄
—
l
l
l
pA2: a logarithmic measure of the potency of the antagonist.
a
⁄P <0.05.
derivatives to bind with muscarinic M3 or M2 receptors. The re-
sults are in good agreement with that concluded from the func-
tional studies. Also, it can be found that for muscarinic M3
‘—’: Not detected.
a
The pEC50 value of carbachol for ilea (M3) is 6.45 0.22.
The pEC50 value of carbachol for left atria (M2), 6.31 0.24.
b
Figure 3. Saturation isotherms of [3H]NMS binding to submandibulary glands (M3), left atria (M2). [3H]NMS with increasing concentrations were incubated in rat
submandibulary glands (M3), left atria (M2) and specific binding (SB) was defined as the difference between total binding (TB) and nonspecific binding (NSB) observed in the
presence of 10
symbol.
lM atropine. Points represent the mean SEM of three experiments each performed in duplicate. In some points, the error deviation is hidden inside the
Figure 4. Specific binding of tropane derivatives as muscarinic antagonists competing against [3H]NMS. Glandular (M3), left atria (M2) protein was incubated with 0.29 nM
[3H]NMS and increasing concentrations of (6S)-4 as representative.

Z.-P. Wang et al. / Bioorg. Med. Chem. 21 (2013) 1234–1239
1237
Table 2
Binding affinity of both enantiomers of 1–4 to rat submandibulary glands (M3), left atria (M2)
Compd
Binding affinity (Ki SE,
Submandibulary glands (M3)a
l
M)
Subtype selectivity (M3/M2) (Ki(M2)/Ki(M3))
Left atria (M2)b
(6S)-1
(6R)-1
(6S)-2
(6R)-2
(6S)-3
(6R)-3
(6S)-4
(6R)-4
2.7 0.51
71 14
1.4 0.28
83 20
3.7 0.63
66 11
0.92 0.21
95 24
10.7 2.6
77 14
8.8 2.2
95 24
9.8 2.8
84 17
12 2.3
86 20
4.0
1.1
6.3
1.1
2.6
1.3
13.0
0.90
a
The Kd (dissociation constant), Bmax (maximal number of binding sites) and Hill coefficient of [3H]NMS are 0.58 nM, 63.37 (fmol/mg wet weight), 0.91 for submandibulary
glands (M3).
b
The Kd (dissociation constant), Bmax (maximal number of binding sites) and Hill coefficient of [3H]NMS are 0.30 nM, 27.09 (fmol/mg wet weight), 0.93 for left atria (M2).
receptors the stronger the binding affinity of 6S enantiomer is, the
weaker that of corresponding 6R enantiomer be.
with our former studies concerning the absolute configuration of
active tropane derivatives.14 Besides, the muscarinic M3 activity,
as well as subtype selectivity (M3/M2) of 6S enantiomers could
be improved by increasing the electron density of carbonyl oxygen
or introducing methylene group between the carbonyl and phenyl
Among the four 6S enantiomers, (6S)-4 elicited the strongest
binding affinity to muscarinic M3 receptors (Ki = 0.92
lM), while
the weakest binding affinity to M2 receptors (Ki = 12 M), resulting
l
in its largest subtype selectivity (M3/M2) (Ki(M2)/Ki(M3) = 13.0-
fold). This finding suggested that introducing methylene group be-
ring in C-3
the absolute configuration of
a
position. All these pharmalogical results implied that
-acyloxy-6b-acetoxyltropane
3a
tween the carbonyl and phenyl ring in C-3
a
position could improve
derivatives played an important role in the muscarinic M3 antago-
not only muscarinic M3 activity but also subtype selectivity (M3/
nistic activity and subtype selectivity (M3/M2).
M2). (6S)-2 displayed stronger inhibition to the binding of
[3H]NMS with muscarinc M3 receptors (Ki = 1.4
l
M), and subtype
selectivity (M3/M2) (Ki(M2)/Ki(M3) = 6.3-fold), if compared to
(6S)-1 (Ki = 2.7 M), and (Ki(M2)/Ki(M3) = 4.0-fold). Introduction
of 2-chloro substitution on the phenyl ring of C-3 increases the
electron density of carbonyl oxygen via p– conjugation, in which
the Cl atom is regarded as electron-donating group. The C-3 car-
4. Experimental
l
4.1. Instruments and materials
a
p
Melting points were recorded on a WRS-1A melting point appa-
ratus. NMR spectra were recorded in CDCl3 on a Bruck AM-400
NMR spectrophotometer operating. Mass spectra (MS) were deter-
mined on an HP-5988 GC/MS spectrophotometer and infrared (IR)
spectra on a Nicolet-Magna IR 750 spectrophotometer using KBr
discs. HPLC enantioseparations were performed by using Chiralpak
AD (150 ꢁ 4.6 mm I.D.) under normal phase conditions. Rotary
power measurements were performed on a Perkin-Elmer 241MC
polarimeter at 20 2 °C. Reactions were monitored by TLC using
silica gel type HSG (F254) of Qingdao Ocean Chemical Plant and
visualized with iodine vapours or Dragendorff reagent. Column
chromatography was performed on silica gel (Qingdao Ocean,
a
bonyl oxygen, an important pharmacophore atom and electron do-
nor, realizes the H-bond with certain polar residue in the
muscarinic receptors.8 Increasing the electron density of carbonyl
oxygen improved the binding affinity of (6S)-2 for muscarinic M3
receptors rather than M2 receptors. So, the subtype selectivity
(M3/M2) of (6S)-2 compared to (6S)-1 enhanced. (6S)-3 elicited
weaker inhibition to the binding of [3H]NMS with muscarinic M3
receptors (Ki = 3.7 lM), as well as subtype selectivity (M3/M2)
(Ki(M2)/Ki(M3) = 2.6-fold), if compared to (6S)-1. Nitro group is a
strong electron-withdrawing group. Introduction of 2-nitro substi-
tution on the phenyl ring of C-3a greatly reduces the electron den-
10–40 lM). Solvents for synthesis were redistilled. ( )-5 was ob-
sity of carbonyl oxygen. This decreased more binding affinity of
(6S)-3 for muscarinic M3 receptors. In that case, the subtype selec-
tivity (M3/M2) of (6S)-3 compared to (6S)-1 dropped.
tained from Hangzhou Minsheng Pharmaceutical Factory (China),
and (+),(ꢀ)-2,3-dibenzoyl tartaric acids were purchased from Al-
drich Chemical Inc. (USA). Tris-[hydroxymethyl] amino methane
(Tris) was purchased from Toronto Research Chemicals (Canada).
Carbachol and atropine were purchased from Sigma corporation
(USA), and [3H]NMS (spec. act. 43 Ci/mM) from Amersham Inc.
(England). (6S)-1, (6R)-1, (6S)-2, (6R)-2, (6S)-3, (6R)-3, (6S)-4 and
(6R)-4 were dissolved in distilled water as the test drugs and pre-
served under 4 °C for functional in vitro studies and radioreceptor
binding assays.
3. Conclusions
The synthesis from two chiral starting materials (6S)-5 and
(6R)-5 has afforded eight optically pure 3a-acyloxy-6b-acetoxyl-
tropane derivatives with definite absolute configuration, (6S)-1,
(6S)-2, (6S)-3, (6S)-4, and (6R)-1, (6R)-2, (6R)-3, (6R)-4. The eight
enantiomeric-pure compounds were applied to study the musca-
rinic M3, M2 antagonistic activity and subtype selectivity (M3/
M2) through functional studies in vitro and radioreceptor binding
assays. The functional studies showed that 6S enantiomers elicited
obvious antagonistic effects towards muscarinic M3 or M2 recep-
tors, while the corresponding 6R enantiomers had little biological
activities. The radioreceptor binding assays registered that the
affinity of 6S enantiomers was much larger than that of
corresponding 6R enantiomers. These findings indicated that 6S
4.2. Resolution of ( )-3a-hydroxy-6b-acetoxytropane (5)
7.524 g (37.8 mmol) ( )-5 and 12.969 g (36.2 mmol) (+)-2,3-
dibenzoyl- -tartaric acid were dissolved in 60 ml isopropanol,
D
placed at room temperature for 24 h. Precipitate was collected
and recrystallized in absolute alcohol. 8.214 g of white needle crys-
tals were obtained, mp 183–184 °C, ½a D20
ꢂ
+63.6° (c, 1.03, H2O). The
salt was treated in usual manner to give the base 3.224 g (43%)
configuration was favorable for 3
a-acyloxy-6b-acetoxyltropane
(6S)-5 as colourless oil, ½a D20
ꢀ20.5° (c, 1.10, CHCl3). The mother li-
ꢂ
derivatives to combine with muscarinic M3, as well as M2 recep-
tors and elicited antagonistic activities. It was in good agreement
quor was evaporated in vacuo. The residue was dissolved in water
and adjusted with concentrated ammonium hydroxide to pH 9–10.

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Z.-P. Wang et al. / Bioorg. Med. Chem. 21 (2013) 1234–1239
The solution was extracted with 30 ml CH2Cl2, and the organic
layer was dried over sodium carbonate and evaporated to dryness.
idly removed and gently cleaned of adhering connective tissue in a
pre-warmed (37 °C) and oxygenated (95% O2 + 5% CO2) medium of
the Kreb’s solution of the following composition: NaCl 6.6 g, CaCl2
0.28 g, KCl 0.35 g, MgSO4ꢃ7H2O 0.294 g, KH2PO4 0.162 g, NaHCO3
2.1 g, glucose 2.0 g in 1000 ml distilled water. Strips of ileal muscle
(1.5 cm) prepared were transferred to 10 ml organ baths with filled
Kreb’s solution and loaded with a tension of 1000 mg. The prepara-
tion was allowed to equilibrate for 30 min, changing the bath fluid
every 15 min. Contractions were recorded isotonically with an
electromechanical transducer connected to Bridge amplifier and
Powerlab system recorder. Cumulative concentration–response
curve was obtained for carbachol. The concentration of carbachol
in the organ bath was increased approximately 3-fold at each step,
with each addition being made only after the response to the pre-
vious addition had attained a maximal level and remained steady.
The contractile response of ileal muscle for each dose of carbachol
was expressed as a percentage of the maximal response in the con-
trol curve. The results were expressed in terms of EC50, the concen-
tration of agonist required to produce 50% of the maximum
contraction. After stable concentration–response curves for carba-
chol were obtained, (6S)-1, (6R)-1, (6S)-2, (6R)-2, (6S)-3, (6R)-3,
(6S)-4 or (6R)-4 was added and the tissue was stimulated
cumulatively with carbachol as before. For the antagonistic test,
three different concentrations of (6S)-1, (6R)-1, (6S)-2, (6R)-2,
(6S)-3, (6R)-3, (6S)-4 and (6R)-4 were investigated.
The residue 3.215 g with 6.530 g (18.2 mmol) (ꢀ)-2,3-dibenzoyl-
L-
tartaric acid in 25 ml isopropanol, treated as mentioned above,
9.269 g salt as white needle crystals were obtained, mp 180–
181 °C, ½a 2D0
ꢀ63.2° (c, 0.90, H2O), followed by getting the colour-
ꢂ
less oil 3.062 g (41%), (6R)-5, ½a D20
ꢂ
+19.7° (c, 0.94, CHCl3).
4.3. General procedure for the preparation of enantiomers of
1–4
Equimolar quantity of (6S)-5 or (6R)-5 was dissolved in 2 ml
CH2Cl2 with 0.1 ml pyridine. The 0.3–0.5 ml acid chloride was then
added dropwise while stirring at room temperature during 4–12 h.
The reaction liquor was evaporated in vacuo. The residue was dis-
solved in water and adjusted with concentrated ammonium
hydroxide to pH 9–10. The solution was extracted with CH2Cl2 (5
times ꢁ 6 ml). The organic phase was dried over anhydrous sodium
sulfate and evaporated to dryness. The crude product was purified
by column chromatography over silica gel. CH2Cl2/CH3OH (25:1)
eluted the pure ester.
4.3.1. (6S),(6R)-3a-benzoyloxy-6b-acetoxytropane (1)
Pale yellow oil, (6S)-1, (254 mg, 61%), ½a D20
ꢂ
+27.1° (c, 1.20,
CHCl3); (6R)-1 (232 mg, 76%), ½a D20
ꢀ26.7° (c, 1.13, CHCl3). IR
ꢂ
(KBr) cmꢀ1: 2950, 2809, 1718, 1602. EI-MS m/z: 303 (M+, 12.54),
182, 122, 94, 82, 43. 1H NMR (CDCl3, 400 MHz) d: 2.11 (s, 3H,
CH3CO), 2.73 (s, 3H, CH3N), 1.30–2.52 (m, 6H, 2, 4, 7-H), 3.41–
3.80 (m, 2H, 1, 5-H), 5.27–5.53 (m, 1H, 3-H), 5.70 (dd, J = 4.0,
8.0 Hz, 1H, 6-H),7.50–7.73 (m, 3H, Ph-H), 8.03–8.25 (m, 2H, Ph-H).
4.4.2. Electrically stimulated guinea pig left atria (M2)
The heart was rapidly removed, and the right and left atria were
separated. The left atria were amounted in Kreb’s solution and
loaded with a tension of 500 mg. After a period of stabilization of
45 min, tissues were electrically stimulated through platinum elec-
trode by square-wave submaximal pulse (2 Hz, 6 ms, 10 V) and
inotropic activity was recorded isometrically. The response of left
atria for each dose of carbachol was expressed as a percentage of
the contractile response before the agonist added. For the antago-
nistic test, two or three different concentrations of (6S)-1, (6R)-1,
(6S)-2, (6R)-2, (6S)-3, (6R)-3, (6S)-4 and (6R)-4 were studied.
4.3.2. (6S),(6R)-3a-orthochlorobenzoyloxy-6b-acetoxytropane
(2)
Pale yellow oil, (6S)-2, (195 mg, 65%), ½a D20
ꢂ
+10.1° (c, 0.85,
CHCl3); (6R)-2 (236 mg, 70%), ½a D20
ꢀ9.8° (c, 0.93, CHCl3). IR (KBr)
ꢂ
cmꢀ1: 2939, 2857, 1732, 1592. EI-MS m/z: 337 (M+, 11.67), 182,
139, 122, 94, 82, 43. 1H NMR (CDCl3, 400 MHz) d: 1.70, 1.87 (d,
J = 15.4 Hz, 2H, 2, 4-H), 2.00 (s, 3H, CH3CO), 2.05–2.30 (m, 3H, 2,
4, 7-H), 2.49 (s, 3H, CH3N), 2.56 (dd, J = 7.6, 15.0 Hz, 1H, 7-H), 3.18
(s, 1H, 5-H), 3.31 (m, 1H, 1-H), 5.27 (m, 1H, 3-H), 5.46 (dd, J = 3.0,
7.5 Hz, 1H, 6-H), 7.27–7.45 (m, 3H, Ph-H), 7.78–7.87 (m, 1H, Ph-H).
4.5. Radioreceptor binding assays
The binding of (6S)-1, (6R)-1, (6S)-2, (6R)-2, (6S)-3, (6R)-3, (6S)-
4 and (6R)-4 to muscarinic M3 or M2 receptors was determined
using rat submandibulary glands or left atria. A male SD rat
(220–250 g) was killed by cervical dislocation. The submandibu-
lary glands, left atria were removed, cleaned adhering tissue in
ice-cold 50 mM Tris buffer (pH 7.4). Homogenisation of the sub-
mandibulary glands, left atria was carried out in 1 g:20 ml (w:v)
volume ice-cold 0.32 M sucrose in Tris buffer using a Waring blen-
der and further disrupted with an Ultraturrax Tissuemizer. The
crude homogenate was centrifuged for 10 min at 1000g and the
resulting supernatant was centrifuged for 30 min at 20000g to
yield a membrane pellet. The pellet was resuspended in Tris buffer
as a crude membrane fraction. All the procedures were performed
at 4 °C. In the saturation binding assays, membranes (0.1 mg pro-
tein) were incubated vibrantly at 37 °C for 30 min in 0.080–
4.3.3. (6S),(6R)-3a-orthonitrobenzoyloxy-6b-acetoxytropane (3)
Pale yellow oil, (6S)-3, (228 mg, 68%), ½a D20
ꢂ
+42.8° (c, 1.15,
CHCl3); (6R)-3 (192 mg, 56%), ½a D20
ꢀ40.7° (c, 1.10, CHCl3). IR (KBr)
ꢂ
cmꢀ1: 2938, 2857, 1732, 1609. EI-MS m/z: 348 (M+, 9.27), 182,
122, 94, 82, 43. 1H NMR (CDCl3, 400 MHz) d: 1.72–2.30 (m, 5H, 2,
4, 7-H), 2.01 (s, 3H, CH3CO), 2.37 (dd, J = 7.7, 14.1 Hz, 1H, 7-H),
2.54 (s, 3H, CH3N), 3.18 (s, 1H, 5-H), 3.35 (s, 1H, 1-H), 5.26–5.36
(m, 2H, 3, 6-H), 7.60–7.76 (m, 3H, Ph-H), 7.90–7.98 (m, 1H, Ph-H).
4.3.4. (6S),(6R)-phenylacetoxy-6b-acetoxytropane (4)
Pale yellow oil, (6S)-4, (155 mg, 64%), ½a D20
ꢂ
+2.0° (c, 1.05, CHCl3);
(6R)-4, (139 mg, 57%), ½a D20
ꢂ
ꢀ2.3° (c, 1.22, CHCl3). IR (KBr) cmꢀ1
:
2947, 2858, 1732, 1604. EI-MS m/z: 317 (M+, 19.58), 182, 122,
94, 82, 43. 1H NMR (CDCl3, 400 MHz) d: 1.64–2.15 (m, 6H, 2,4,7-
H), 2.03 (s, 3H, CH3CO), 2.44 (s, 3H, CH3N), 3.08 (s, 1H, 5-H), 3.21
(m, 1H, 1-H), 3.61 (s, 2H, CH2), 4.95 (m, 1H, 3-H), 5.21 (dd,
J = 3.1, 7.6 Hz, 1H, 6-H), 7.20–7.36 (m, 5H, Ph-H).
1.52 nM [3H]NMS with or without 10
lM atropine sulfate in a total
volume of 0.4 ml. The reaction was terminated by rapid filtration
through glass fiber filters, washed three times with ice-cold Tris
buffer. The protein concentration was determined with the micro
BCA kit (Pierce, Rockford, IL), using bovine serum albumin as the
standard. For competition binding assays, membranes (0.1 mg pro-
tein) were incubated with 0.4 nM [3H]NMS at 37 °C for 60 min and
increasing concentrations of (6S)-1, (6R)-1, (6S)-2, (6R)-2, (6S)-3,
(6R)-3, (6S)-4 or (6R)-4 in total volume of 0.4 ml. All the dilutions
for the test compounds were made in Tris buffer. Assays were per-
formed in duplicate.15
4.4. Functional in vitro studies
4.4.1. Guinea pig ilea (M3)
Guinea pigs (220–260 g) of either sex provided by animal
experimental center of Shanghai Jiao Tong University were killed
by a blow to the head and exsanguinated. The ileal muscle was rap-

Z.-P. Wang et al. / Bioorg. Med. Chem. 21 (2013) 1234–1239
1239
4.6. Statistics and data analysis
References and notes
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For the ilea or atria contraction assay, EC50 values and the slopes
of the log concentration–response curves for carbachol were
calculated by means of nonlinear curve fitting of sigmoidal dose–
response logistic transformation using program GraphPad PRISM
4.0 (San Diego, CA, USA). The pA2 values for (6S)-1, (6S)-2, (6S)-3
and (6S)-4 were determined according to Arunlakshana and
Schild.16 In saturation binding tests, nonlinear curve fitting was
used to generate affinity (Kd) and capacity (Bmax) values for
[3H]NMS. The displacement data were analyzed using the commer-
cial software PRISM to obtain IC50 values for competing ligands.
The affinity, expressed as Ki was calculated from IC50 value accord-
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The statistically significant differences were determined by Stu-
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Acknowledgements
We wish to present our grateful thanks to Shanghai Municipal
Science Item (No. 10ZR1416900) and Doctor Innovation Fund of
School of Medicine in Shanghai Jiao Tong University (No. BXJ0707).