The absolute configuration plays an important role in muscarinic activity of BGT-A and its analogs

Article abstract of DOI:10.1016/j.bmc.2008.10.047

Both enantiomers of 2, 3, and 4, three bioactive analogs of muscarinic agonist BGT-A were prepared respectively and underwent functional studies and radioreceptor binding assays. 6S enantiomers of 2, 3, and 4 showed obvious muscarinic activity, while 6R ones elicited little muscarinic activity by functional studies. Besides, the affinity of 6S enantiomers of 2, 3, and 4 was greatly larger than that of their 6R enantiomers respectively. All these pharmalogical results indicated the 6S configuration was beneficial for the active BGT-A analogs to bind with the muscarinic receptors. The finding was in good agreement with our previous SAR study to BGT-A and its active analogs by computational approach. The understanding to the relationship between muscarinic activity and absolute configuration will provide the basis for successive screening of BGT-A analogs as effective muscarinic agonists or antagonists in clinical use.

Full text of DOI:10.1016/j.bmc.2008.10.047

Bioorganic & Medicinal Chemistry 16 (2008) 10251–10256
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
The absolute configuration plays an important role in muscarinic activity
of BGT-A and its analogs
a,
Yin-Yao Niu a, , Liang Zhu b, , Yong-Yao Cui b, Hui-Zhong Liu a, Hong-Zhuan Chen b, , Yang Lu
*
*
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
a r t i c l e i n f o
a b s t r a c t
Article history:
Both enantiomers of 2, 3, and 4, three bioactive analogs of muscarinic agonist BGT-A were prepared
respectively and underwent functional studies and radioreceptor binding assays. 6S enantiomers of 2,
3, and 4 showed obvious muscarinic activity, while 6R ones elicited little muscarinic activity by func-
tional studies. Besides, the affinity of 6S enantiomers of 2, 3, and 4 was greatly larger than that of their
6R enantiomers respectively. All these pharmalogical results indicated the 6S configuration was beneficial
for the active BGT-A analogs to bind with the muscarinic receptors. The finding was in good agreement
with our previous SAR study to BGT-A and its active analogs by computational approach. The understand-
ing to the relationship between muscarinic activity and absolute configuration will provide the basis for
successive screening of BGT-A analogs as effective muscarinic agonists or antagonists in clinical use.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 6 September 2008
Revised 18 October 2008
Accepted 21 October 2008
Available online 1 November 2008
Keywords:
Analogs of Baogongteng A
Absolute configuration
Muscarinic agonist and antagonist
Mydriatic and myotic
Enantiomers
1. Introduction
configuration of carbon 6 of these analogs were crucial for the mAChR
activity, due to the fact that 6b-hydroxy analog of BGT-A was inac-
Muscarinic drugs have potential for therapeutic use in several
pathological states.1,2 As a consequence, the muscarinic compounds
have been studied widely in the past decades and the study of the
relationship between muscarinic activity and absolute configura-
tion of them has been interested by medicinal chemists.3,4 Bao-
tive11,12 and the value of the agonistic activity of synthetic ( )-BGT-
A was nearly half as that of the natural (6S)-BGT-A,13,14 and the ana-
lyzing result acquired by calculating the structural parameters of
some active analogs revealing that their mAChR activities being con-
nected with the 6S configuration.15 The understanding of the relation-
ship between mAChR activity and absolute configuration of BGT-A
analogs will provide the basis for successive screening of effective
hypotoxic mAChR agonists or antagonists in clinical use. However,
the knowledge of the relationship between the absolute configuration
and mAChR activity has not been further identified through biological
assay to the enantiomers of bioactive BGT-A analogs. In this paper, we
prepared both enantiomers of2, 3 and 4 respectively, and determined
each binding affinity of the six enantiomers to mAChR of the rat sub-
mandibulary gland. The in vitro functional activity of the three pairs
of enantiomers on isolated iris muscle was also investigated, as eval-
uation of the activity on this model may provide a better indication of
their potential activity. Both enantiomers of 2, 3 were tested for the
muscarinic activity as potential antagonists, while, both enantiomers
of 4 as agonists. Besides, the mydriatic or myotic effect of the enanti-
omers of 2, 3, 4 was tested in vivo.
gongteng A ([2S,6S]-2a-hydroxy-6b-acetoxynortropane, (6S)-BGT-
A)à (1) (Fig. 1) was the first naturally occurring tropane alkaloid as
muscarinic acetycholine receptor (mAChR) agonist isolated from the
stem of Chinese medicinal plant Baogongteng (Erycibe obtusifolia
Benth) and was used as a myotic agent to treat glaucoma in clinics.5
However, the limited amounts of (6S)-BGT-A available from the nat-
ural resources and the poor yield of total synthesis of ( )-BGT-A make
it necessary to find an alternative of (6S)-BGT-A through bioactive
screening of BGT-A analogs.6–10 Certain ( )-3-substituted-6b-acet-
oxyl tropane analogs of BGT-A were found to be mAChR agonist or
antagonist.
and 3 (( )-3
antagonistic activity, while 4 (( )-3
2
(( )-3
-paranitrobenzoyloxy-6b-acetoxytropane) have higher
-benzenesulfonyloxy-6b-acet-
a-parachlorobenzoyloxy-6b-acetoxytropane)
a
a
oxytropane) (Fig. 1) agonistic activity. The structure-activity relation-
ship (SAR) study indicated that 6b-acetoxyl and the absolute
2. Results and discussion
2.1. Chemistry
* Corresponding authors. Tel./fax: +86 21 64674721.
E-mail addresses: yaoli@shsmu.edu.cn (H.-Z. Chen), huaxue@shsmu.edu.cn,
luyangssmu@yahoo.com (Y. Lu).
These authors contributed equally to this work.
As the absolute configuration of C2 or C3 is in agreement with that of C6 for
à
(ꢀ)-5 ((ꢀ)-3a-hydroxy-6b-acetoxytropane) and (+)-5, two
discussed compounds in this article, the absolute configurations of their enantiomers
are simply expressed as 6S or 6R for convenience in the following discussion.
enantiomers, were acquired by resoluting ( )-5 with (+) or (ꢀ)-
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.10.047

10252
Y.-Y. Niu et al. / Bioorg. Med. Chem. 16 (2008) 10251–10256
CH3
CH3
CH3
H
OH
N
N
N
N
2
O
O
AcO
AcO
NO2
AcO
O
S
AcO
4
3
6
Cl
O
O
O
1
2
4
3
O
Figure 1. Structures of 1–4.
Figure 2. Dynamic chromatograms of A. enantiomers of 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: 4.7 ml/min; column temperature: 25 °C. The peak-time of (6S)-5, (6R)-5 are 9.69 min, 11.28 min, respectively.
2,3-dibenzoyl tartaric acid in isopropanol. The absolute configura-
tion 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 the chiral tropane com-
pounds.16 The resoluted (6S)-5 and (6R)-5 were examined to be
optically pure by HPLC (Fig. 2). Carbonylating or sulfonylating
(6S)-5 and (6R)-5 respectively gave three pairs of optically pure
enantiomers of 2, 3, 4, (6S)-2, (6S)-3, (6S)-4, (6R)-2, (6R)-3, and
(6R)-4 (Chart 1).
2.2.2. Effect of carbachol on the contraction of isolated iris
muscle under pre-incubation of (6S)-2, (6R)-2, (6S)-3, (6R)-3
(6S)-2 and (6S)-3 could inhibit the contraction of isolated iris
muscle induced by carbachol, but their enantiomers (6R)-2, (6R)-
3 could not do, indicating that 6R configuration was not benefit
for 2, 3 to elicit antagonistic activity to mAChRs. (6S)-2, (6S)-3,
shifted the carbachol-induced response curves to the right with a
parallel manner (Fig. 4), generating pA2 values of 5.86 0.24 and
6.11 0.17 against carbachol, respectively (Table 1).
2.2. Pharmacology
2.2.3. [3H]NMS binding to submandibulary glands
[3H]NMS (3H-labelled N-methylscopolamine) binding studies
were performed with a crude membrane fraction prepared from
the submandibulary glands. Specific binding of [3H]NMS to mAChRs
of submandibulary glands was saturable. The dissociation equilib-
rium constant (Kd) and receptor density (Bmax) were 0.71 0.20 nM
and 70.50 12.20 fmol/mg protein (n = 3), respectively (Fig. 5).
The binding affinities of (6S)-2, (6R)-2, (6S)-3, (6R)-3, (6S)-4, and
(6R)-4 were examined. As shown in Figure 6, [3H]NMS binding to
mAChRs was strongly inhibited by (6S)-2, (6S)-3, (6S)-4 and weakly
inhibited by (6R)-2, (6R)-3 in a log concentration-dependent man-
ner, but could not be inhibited by (6R)-4. The binding affinity of
2.2.1. Effect of carbachol, (6S)-4 and (6R)-4 on the contraction
of isolated iris muscle
(6S)-4 like carbachol, a typical muscarinic agonist, could stimu-
late the contraction of isolated iris muscle, whereas, (6R)-4 could
not, indicating that 6S configuration was crucial for 4 to elicit mus-
carinic agonistic activity. Cumulative addition of mAChR agonists
carbachol or (6S)-4 to isolated iris muscle produced a log concen-
tration-dependent contractile response (Fig. 3). The pEC50 and Emax
values for carbachol and (6S)-4 are shown in Table 1. (6S)-4 pro-
duces 87% maximum contraction compared to that of carbachol,
and the potency of (6S)-4 (pEC50 = 6.06) is lower than that of carba-
chol (pEC50 = 6.57).
(6S)-4 is the largest (Ki, 0.04 lM), and the binding affinity of
(6S)-2 (Ki, 9
lM), (6S)-3 (Ki, 3.7
lM) are 8-fold, 120-fold as that
CH3
N
CH3
N
carbonylate or
sulfonylate
AcO
AcO
6
6
CH3
HO
(6R)-(+)-5
RO
(6R)-2, 3, 4
N
R
resolution
AcO
2 p-ClPhCO
3 p-NO2PhCO
4 PhSO2
HO
CH3
( )-5
CH3
N
N
carbonylate or
sulfonylate
AcO
AcO
6
6
HO
(6S)-(-)-5
RO
(6S)-2, 3, 4
Chart 1. Synthesis of the enantiomers of 2, 3, and 4.

Y.-Y. Niu et al. / Bioorg. Med. Chem. 16 (2008) 10251–10256
10253
caused by the tests of control to the right pupil as reference. By com-
parison, (6R)-2, 3, 4 elicited little influence on the rabbit’s pupil
(changing percentage in the scale of < 5%). These results indicated
(6S)-2, 3 had muscarinic antagonistic activity, while (6S)-4 agonistic
activity, and (6R)-2, 3, 4 showed little mAChR activity.
3. Conclusions
The synthesis from two chiral starting materials (6S)-5, (6R)-5
has afforded six optically pure BGT-A analogs, (6S)-2, (6S)-3, (6S)-
4, (6R)-2, (6R)-3, and (6R)-4 with definite absolute configurations.
The six enantiomeric-pure compounds were applied to study the
relationship between the absolute configuration and mAChR activ-
ities through functional studies in vitro, in vivo and radioreceptor
binding assays. The results of functional studies showed the 6S
enantiomers of 2, 3, and 4 elicited obvious muscarinic agonistic
or antagonistic effect, while their 6R enantiomers showed little
biological effect, indicating 6S configuration was benefit for BGT-
A analogs to elicit muscarinic activity. It was in good agreement
with that of our recent studies concerning the absolute configura-
tion of active BGT-A analogs.17 The radioreceptor binding assays
registered that affinity of 6S enantiomers of 2, 3, and 4 was greatly
larger than that of their 6R enantiomers, and the larger the affinity
of 6S enantiomer of active BGT-A analog is, the smaller the affinity
for its corresponding 6R enantiomer be. All these pharmalogical re-
sults imply that the absolute configuration of BGT-A analogs plays
an important role in evaluating their mAChR activities, and provide
the basis for the screening of effective muscarinic agonists or
antagonists in clinical use.
Figure 3. Effects of mAChR agonists carbachol and (6S)-4 on the contractile
response of isolated guinea-pig iris muscle. The contractile response of iris muscle
to each dose of the agonists is expressed as percentage of that induced by 10ꢀ4 M
carbachol. The data points represent mean SEM (n = 3–5).
Table 1
Results of (6S)-2, (6R)-2, (6S)-3, (6R)-3, (6S)-4, and (6R)-4 to dynamic test on mAChRs
of the isolated guinea-pig iris muscle (n = 3–5).
Compound
pEC50
Emax
%
pA2
Carbachol
(6S)-2
(6R)-2
(6S)-3
(6R)-3
(6S)-4
(6R)-4
6.57 0.08*
100 5.67*
—
—
—
—
—
5.86 0.24*
—
—
—
—
6.11 0.17*
—
—
—
6.06 0.07*
—
87.10 3.8*
—
pEC50: Negative logarithm of the concentration of agonists causing a half-maximal
response.
4. Experimental
Emax: maximal response, expressed relative to that of carbachol.
pA2: a logarithmic measure of the potency of the antagonist. *P < 0.05.
‘‘—”: not detected.
4.1. Instruments and materials
Meltingpointswere recordedon a WRS-1Ameltingpointappara-
tus. NMR spectra were recorded in CDCl3 on a Bruck AM-400 NMR
spectrophotometer operating. Mass spectra (MS) were determined
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 polarim-
eter at 20 2 °C. Reactions were monitored by TLC using silica gel
type HSG (F254) of Qingdao Ocean Chemical Plant and visualized
with iodine vapors or Dragendorff reagent. Column chromatography
of (6R)-2 (Ki, 73 lM), (6R)-3 (Ki, 450 lM) respectively (Table 2),
indicating that 6S configuration was benefit for 2, 3, and 4 to com-
bine with mAChRs. The affinity of (6S)-3 is great than that of (6S)-2,
while (6R)-3 less than (6R)-2, suggesting that the stronger the
binding of 6S enantiomer of active BGT-A analog to mAChRs is,
the weaker the binding for its corresponding 6R enantiomer be.
2.2.3.1. Mydriatic effects of(6S), (6R)-2, (6S), or(6R)-3 and myotic
effects of (6S) or (6R)-4 on conscious guinea-pig in vivo.
The
mean mydriatic or myotic percentages caused by both enantiomers
of 2, 3, and 4 in different groups were recorded in Table 3. Obvious
mydriatic effect of (6S)-2, 3 (mydriatic percentage > 30%, P < 0.05)
and myotic effect of (6S)-4 (myotic percentage < ꢀ40%, P < 0.05) in
test groups were observed, compared to 3.7% mydriatic percentage
wasperformedon silicagel(QingdaoOcean, 10–40 lm). Solvents for
synthesis were redistilled. ( )-5 was obtained from Hangzhou
Minsheng Pharmaceutical Factory (China), and (+), (ꢀ)-2,3-diben-
zoyl tartaric acids were purchased from Aldrich Chemical Inc.
(USA). Tris-[hydroxymethyl] amino methane (Tris) was purchased
Figure 4. Effects of muscarinic agonist carbachol on iris contraction (n = 3–5).

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Y.-Y. Niu et al. / Bioorg. Med. Chem. 16 (2008) 10251–10256
from Toronto Research Chemicals (Canada). Carbachol (79H0110)
and atropine (69H0545) were purchased from Sigma corporation
(USA.), and [3H]NMS (spec. act. 43 Ci/mM) from Amersham Inc.
(England). (6S)-2, (6R)-2, (6S)-3, (6R)-3, (6S)-4, and (6R)-4 were dis-
solved in distilled water as the test drugs and preserved under 4 °C
for functional in vitro studies and radioreceptor binding assays.
4.2. Resolution of ( )-3a-hydroxy-6b-acetoxytropane (5)
6.270 g (31.5 mmol) ( )-5 and 10.991 g (30.7 mmol) (+)-2,3-
dibenzoyl- -tartaric acid were dissolved in 48 ml isopropanol,
D
placed at room temperature for 24 h. Precipitate was collected
and recrystallized in absolute alcohol. 7.725 g of white needle crys-
tals were obtained, mp.183-184 °C, ½a D20
ꢂ
+63.8° (c, 0.95, H2O). The
salt was treated in usual manner to give the base 2.633 g (42%)
Figure 5. Saturation isotherms of [3H]NMS binding to submandibulary glands.
[3H]NMS with increasing concentrations were incubated in rat submandibulary
glands and specific binding (SB) was defined as the difference between total binding
(TB) and nonspecific binding (NSB) observed in the presence of 10 lM atropine.
Points represent mean SEM of three experiments each performed in duplicate. In
(6S)-5 as colorless oil, ½a D20
ꢀ20.3° (c, 1.02, CHCl3). The mother li-
ꢂ
quor was evaporated in vacuo. The residue was dissolved in water
and adjusted with concentrated ammonium hydroxide to pH 9–10.
The solution was extracted with 30 ml CH2Cl2, and the organic
layer was dried over sodium carbonate and evaporated to dryness.
some points, the error deviation is hidden inside the symbol. (n = 3–5).
Figure 6. Specific binding of BGT-A analogs as muscarinic ligands competing against [3H]NMS. Glandular protein was incubated with 0.33 nM [3H]NMS and increasing
concentrations of (6S)-2, (6S)-3, (6S)-4, (6R)-2, or (6R)-3.

Y.-Y. Niu et al. / Bioorg. Med. Chem. 16 (2008) 10251–10256
10255
Table 2
(s, 3H, CH3N), 1.54–3.3 (m, 6H, 2, 4, 7-H), 3.40–3.80 (m, 2H, 1, 5-H),
5.40–5.90 (m, 2H, 3, 6-H), 8.20–8.60 (m, 4H, Ph-H).
Binding affinities for rat submandibulary gland of (6S)-2, (6R)-2, (6S)-3, (6R)-3, (6S)-4,
or (6R)-4.
Compound
Submandibulary glandsa
Ki ( M)
4.3.3. (6S), (6R)-3a-Benzenesulfonyloxy-6b-acetoxytropane (4)
Pale yellow oil, (6S)-4, (168 mg, 35%), ½a D20
ꢂ
ꢀ6.1°(c, 1.60, CHCl3);
l
(6R)-4, (155 mg, 27%), ½a D20
ꢂ
+5.9° (c, 1.35, CHCl3). IR (KBr) cmꢀ1
:
(6S)-2
(6R)-2
(6S)-3
(6R)-3
(6S)-4
(6R)-4
9.0 1.1
73 22
3.70 0.51
450 34
0.040 0.003
—
2948, 2858, 1733, 1586. EI-MS m/z: 339 (M+, 8.56), 182, 122, 94,
82, 43. 1H NMR (CDCl3, 400 MHz) d: 1.76–2.60 (m, 6H, 2,4,7-H),
2.04 (s, 3H, CH3CO), 2.54 (s, 3H, CH3N), 3.17 (s, 1H, 5-H), 3.42 (m,
1H, 1-H), 4.80 (m, 1H, 3-H), 5.45 (dd, J = 3.0, 6.4 Hz, 1H, 6-H),
7.34–7.70 (m, 3H, Ph-H), 7.86–7.95 (m, 2H, Ph-H).
a
The Kd (dissociation constant), Bmax (maximal number of binding sites) and Hill
coefficient of H-labelled N-methylscopolamine are: 0.71 nM, 70.5 (fmol/mg wet
weight), 0.92. ‘‘—”: not detected.
4.4. Functional in vitro studies
(6S)-4 and (6R)-4 were tested for stimulating the contraction
of isolated iris muscle as agonistic activity, while (6S)-2, (6R)-2,
(6S)-3, and (6R)-3 for inhibiting the carbachol-induced contrac-
tion of isolated iris muscle as antagonistic activity. Guinea-pigs
(250–350 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 longitudinal muscle was rap-
idly removed and gently cleaned of adhering connective tissue
in a prewarmed (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 longitudinal muscle (1.5 cm) prepared were transferred to
10 ml organ baths and loaded with a tension of 500 mg. The prep-
aration was allowed to equilibrate for 30 min, changing the bath
fluid every 10 min. Contractions were recorded isotonically with
an electromechanical transducer connected to Bridge amplifier
and Powerlab system recorder. Cumulative concentration–re-
sponse curves were obtained for carbachol, (6S)-4, (6R)-4. The
concentration of agonist in the organ bath was increased approx-
imately 3-fold at each step, with each addition being made only
after the response to the previous addition had attained a maxi-
mal level and remained steady. The contractile responses of iris
muscle to each dose of the muscarinic agonists were expressed
as percentages of the maximum effect induced by carbachol.
For the antagonistic test, after stable concentration-response
curves for carbachol was obtained, (6S)-2, (6R)-2, (6S)-3, or
(6R)-3 was added and the tissue was stimulated cumulatively
with carbachol as before. Three different concentrations of (6S)-
2, (6R)-2, (6S)-3, or (6R)-3 were investigated.
Table 3
Mydriatic or myotic effect caused by (6S)-2, (6R)-2, (6S)-3, (6R)-3, (6S)-4, or (6R)-4.
Compound
Percentage of the pupil
diameter changing (%)
Control
(6S)-2
(6R)-2
(6S)-3
(6R)-3
(6S)-4
(6R)-4
2.7 0.5*
32.2 1.9*
3.0 1.5
35.1 2.1*
3.7 1.3
ꢀ44.0 3.1*
ꢀ4.7 2.2
The positive values represent mydriatic effect, while the negative myotic effect. The
left pupil diameters (mm) of the rabbits were measured under constant light, and
readings were taken before and at 10 min after dropping 0.05 ml agent, while the
right for the control as reference. *P < 0.05.
The residue 2.980 g with 5.442 g (15.2 mmol) (ꢀ)-2,3-dibenzoyl-
L-
tartaric acid in 20 ml isopropanol, treated as mentioned above,
7.022 g (40%) salt as white needle crystals were obtained, mp
180–181 °C, ½a 2D0
ꢀ63.2° (c, 0.95, H2O), followed by getting the col-
ꢂ
orless oil 2.320 g (37%), (6R)-5, ½a D20
ꢂ
+19.9° (c, 1.02, CHCl3).
4.3. General procedure for the preparation of enantiomers of 2,
3, and 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
(5ꢁ 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. Dichloromethane/
methanol (25:1) eluted the pure ester.
4.5. Radioreceptor binding assays
The binding of (6S)-2, (6R)-2, (6S)-3, (6R)-3, (6S)-4, or (6R)-4 to
mAChR was determined using the submandibulary glands of the
rat. A male SD rat (220–240 g) was killed by cervical dislocation.
The submandibulary glands were removed, cleaned adhering tis-
sue in ice-cold 50 mM Tris buffer (pH 7.4). Homogenisation of
the submandibulary glands was carried out in 1 g:20 ml (w:v) vol-
ume ice-cold 0.32 M sucrose in Tris buffer using a Waring blender
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 20,000g to yield a mem-
brane 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 assay, membranes (0.1 mg protein) were
incubated vibrantly at 37 °C for 30 min in 0.078–1.55 nM
4.3.1. (6S), (6R)-3a-Parachlorobenzoyloxy-6b-acetoxytropane
(2)
Pale yellow oil, (6S)-2, (191 mg, 65%), ½a D20
ꢂ
+20.2° (c, 0.40,
CHCl3); (6R)-2 (231 mg, 71%), ½a D20
ꢀ19.8° (c, 0.53, CHCl3). IR
ꢂ
(KBr) cmꢀ1: 2939, 2857, 1732, 1592. EI-MS m/z: 337 (M+), 182,
122, 94, 82, 43. 1H NMR (CDCl3, 400 MHz) d: 1.98 (s, 3H, CH3CO),
2.56 (s, 3H, CH3N), 1.18–2.77 (m, 6H, 2, 4, 7-H), 3.2–3.6 (m, 2H,
1, 5-H), 5.1–5.4 (m, 1H, 3-H), 5.53 (dd, 1H, J = 4.0, 8.0 Hz, 6-H),
7.38 (d, 2H, J = 14 Hz, Ph-H), 7.92 (d, 2H, J = 14 Hz, Ph-H).
[3H]NMS with or without 10
lM atropine sulfate in a total volume
4.3.2. (6S), (6R)-3a-Paranitrobenzoyloxy-6b-acetoxytropane (3)
Pale yellow oil, (6S)-3, (268 mg, 51%), ½a D20
ꢂ
+30.9° (c, 1.05, CHCl3);
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 stan-
(6R)-3 (202 mg, 66%), ½a D20
ꢂ
ꢀ30.7° (c, 1.15, CHCl3). IR (KBr) cmꢀ1
:
2938, 2857, 1732, 1609. EI-MS m/z: 348 (M+, 8.07), 182, 149, 122,
94, 82, 43. 1H NMR (CDCl3, 400 MHz) d: 2.34 (s, 3H, CH3CO), 2.82

10256
Y.-Y. Niu et al. / Bioorg. Med. Chem. 16 (2008) 10251–10256
dard. For competition binding assays, iris muscle membranes
(0.1 mg protein) were incubated with 0.4 nM [3H]NMS at 37 °C
for 60 min with increasing concentrations of (6S)-2, (6R)-2, (6S)-
3, (6R)-3, (6S)-4, or (6R)-4 in total volume of 0.4 ml. All the dilu-
tions for the test compounds were made in Tris buffer. Assays were
performed in duplicate.18
Acknowledgments
This study was supported by National Natural Science Founda-
tion of China (Nos. 30672441, 30701018), Key Project of Shanghai
Municipal Science and Technology Commission (Nos. 03JC14064,
34319230). The program of Shanghai Subject Chief Scientist (No.
06XD14011), Shanghai Municipal Natural Science Foundation
(No. 05ZR14054), Grant from Shanghai Municipal Committee
(No. J50201) and Doctor Innovation Fund of School of Medicine
in Shanghai Jiao Tong University (No. BXJ0707).
4.6. Functional in vivo studies
0.10–0.15 ml 1 M chloric acid was added respectively to the
solutions of 0.02 M of (6S)-2, (6R)-2, (6S)-3, (6R)-3, (6S)-4, or
(6R)-4 to pH 6, which were used as test drugs. Twenty health
rabbits (2.5–3.0 kg) of either sex provided by animal experimen-
tal center of Shanghai Jiao Tong University were randomly di-
vided into four groups, five in each group. The left pupil
diameters of the rabbits were measured under constant light,
and readings were taken before and at 10 min after 0.05 ml of
agent dropping, while the right for the control as reference.
Mydriatic or myotic effects were expressed as the percentage
of diameter changing value after application of agent to the
diameter before application of agent.
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