Bisquaternary dimers of strychnine and brucine. A new class of potent enhancers of antagonist binding to muscarinic M2 receptors

Article abstract of DOI:10.1016/S0968-0896(03)00146-9

Bisquaternary dimers of strychnine and brucine were synthesized and their allosteric effect on muscarinic acetylcholine M₂ receptors was examined. The compounds retarded the dissociation of the antagonist [³H]N-methylscopolamine ([³H]NMS) from porcine cardiac cholinoceptors. This action indicated ternary complex formation. All compounds exhibited higher affinity to the allosteric site of [³H]NMS-occupied M₂ receptors than the monomeric strychnine and brucine, while the positive cooperativity with NMS was fully maintained. SAR studies revealed the unchanged strychnine ring as an important structural feature for high allosteric potency.

Full text of DOI:10.1016/S0968-0896(03)00146-9

Bioorganic & Medicinal Chemistry 11 (2003) 2627–2634
Bisquaternary Dimers of Strychnine and Brucine. A New Class of
Potent Enhancers of Antagonist Binding to Muscarinic M2
Receptors
a,
b
a
b
D. P. Zlotos, * S. Buller, U. Holzgrabe and K. Mohr
a
Pharmaceutical Institute, University of Wu¨rzburg, Am Hubland, 97074Wu¨rzburg, Germany
Department of Pharmacology and Toxicology, Institute of Pharmacy, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany
b
Received 20 December 2002; accepted 28 February 2003
Abstract—Bisquaternary dimers of strychnine and brucine were synthesized and their allosteric effect on muscarinic acetylcholine
3
3
2
M receptors was examined. The compounds retarded the dissociation of the antagonist [ H]N-methylscopolamine ([ H]NMS) from
porcine cardiac cholinoceptors. This action indicated ternary complex formation. All compounds exhibited higher affinity to the
3
allosteric site of [ H]NMS-occupied M receptors than the monomeric strychnine and brucine, while the positive cooperativity with
2
NMS was fully maintained. SAR studies revealed the unchanged strychnine ring as an important structural feature for high allo-
steric potency.
#
2003 Elsevier Science Ltd. All rights reserved.
Introduction
Since the neuromuscular blocker alcuronium has been
shown to increase the binding of NMS to the M subtype
2
1
3
All five muscarinic receptor subtypes have allosteric
binding domains in addition to the orthosteric conven-
of muscarinic receptors, a number of other positively
cooperative compounds have been identified. The alka-
1
ꢀ5
14,15
16
17
tional ligand binding site.
appears to be especially sensitive to allosteric modula-
The cardiac M receptor
loids strychnine,
series of quaternary strychnine and brucine analogues
brucine, eburnamonine and a
1
2
6
2
,6,7
8ꢀ10
its allosteric site is well-defined and affinity
data for a number of structurally different compounds
tion;
were reported to be positively cooperative with NMS at
M receptors. However, their affinities for liganded M
2
2
3
have been reported for M receptors occupied by [ H]N-
2
receptors were considerably lower than that of alcur-
onium, which limits their usefulness as research tools.
Recently, some very potent pentacyclic carbazolones
3
11
methylscopolamine ([ H]NMS).
Orthosteric and
allosteric ligands and the receptor interact with each
other according to the ternary complex model of allo-
also showed positive cooperativity with NMS at M
18
2
1
,12
steric interactions.
The allosteric effect on ligand
receptors. The poor allosteric potency of strychnine
equilibrium binding reflects the cooperativity of inter-
action. Negative cooperativity is indicated by a reduc-
tion of equilibrium binding; neutral cooperativity means
no change of equilibrium binding upon formation of the
ternary complex; positive cooperativity is indicated by
an increase of equilibrium binding. In the case of mus-
carinic allosteric compounds, the formation of a ternary
complex generally results in an inhibition of dissociation
3
of the orthosteric ligand, such as [ H]NMS. Thus, an
3
inhibition of [ H]NMS dissociation is indicative of an
allosteric interaction.
*
5
Corresponding author. Tel.: +49-931-888-5489; fax: +49-931-888-
494; e-mail: zlotos@pharmazie.uni-wuerzburg.de
0
968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00146-9

2628
D. P. Zlotos et al. / Bioorg. Med. Chem. 11 (2003) 2627–2634
and its quaternary analogues compared to alcuronium is in
accordance with the pharmacophore model for highly
1
9,20
potent ligands at NMS-occupied M receptors.
The
pharmacophoric elements are two positively charged
2
˚
nitrogens at a distance of about 10A surrounded by two
aromatic ring systems. A monoquaternary strychnine
analogue, which in simplest terms can be regarded as a half
of alcuronium, has only one positively charged nitrogen
and one aromatic indole ring and should, thus, interact
with fewer receptor binding domains than alcuronium.
Bis(ammonio)alkane-type allosteric modulators derived
2
1ꢀ23
from the lead compound W84
were for a long time
regarded as negatively cooperative with NMS at M2
receptors. Even the highly potent analogue dimethyl-
3
Figure 1. NOE interactions (CDCl , 600 MHz) indicating the chair
conformation of the piperidine ring in dihydrostrychnine (1).
9
W84 revealed a weak inhibitive effect on NMS binding.
Recently, we reported that an introduction of methyl
groups into the propyl side chain of W84 and its deri-
selectively from the less hindered side of the strychnine
ring system (Scheme 1). The absolute configuration of
the new chiral atom C-20was determined by a 6 00 MHz
NOESY experiment. A strong NOE effect between
H-14b (d 2.14), which has an axial orientation to the
piperidine ring, and H-20( d 2.35) revealed the axial
position of H-20in the piperidine ring. The resulting
2
4,25
vatives
charged nitrogen by a silicon atom switched the allo-
steric action from negative to positive cooperativity.
as well as the exchange of one positively
2
6
(
R)-configuration for C-20is in agreement with the ste-
reochemistry of the C19-C20bond determined in 19,20-
2
8
dihydrostrychnine crystals by X-ray analysis. More-
over, like in the crystal structure, the piperidine ring
conformation has changed upon hydrogenation from a
boat form in strychnine to a chair form in 1 as indicated
by NOE interactions displayed in Figure 1.
22-Oximinobrucine (2) was synthesized by isonitrosa-
tion of brucine with tert-butyl nitrite/t-BuOK in toluene
following the procedure reported for oxohomoeburnane
2
9
(Scheme 2). The major E-oxime (2a) (89%) could be
separated from the Z-oxime (2b) (2%) by column chro-
matography on silica gel. The stereochemistry was
In this paper, we examined the effect of quaternization
of two strychnine, dihydrostrychnine, brucine and 22-
oximinobrucine molecules with various dibromohalo-
genides on the affinity and cooperativity with the
antagonist NMS at muscarinic M2 receptors. The
resulting bisquaternary strychnine and brucine dimers
include all pharmacophoric elements for highly potent
allosteric modulators and are therefore expected to
exhibit a higher affinity to the allosteric binding site
than strychnine, brucine and their monoquaternary
analogues. With respect to cooperativity, the new com-
pounds may take advantage of two structural features
found in other positively cooperative agents. They
comprise strychnine or brucine units and can be regar-
ded as W84 analogues with completely rigid side chains.
1
13
determined by H and C NMR spectroscopy. The
H-17 resonance signal of the major isomer appeared at
d 5.00 ppm, downfield by 0.31 ppm from the corre-
sponding signal of the minor isomer due to the aniso-
3
0
tropic deshielding effect of the oxime hydroxy group.
The E-configuration of the major isomer was confirmed
by the chemical shift of C-17 at d 75.2 ppm, upfield by
4.7 ppm from the corresponding signal of the Z-isomer
owing to g-interaction between the hydroxy group and
3
1
C-17 in the E-oxim (2a).
The bisquaternary title compounds 3–10 were synthe-
sized by refluxing an excess of strychnine, brucine and
dihydrostrychnine (1) with the respective dibromo-
alkane in dry acetonitrile for 14 h in 58–90% yield.
Due to a low solubility of oxime (2a) in acetonitrile,
Results and Discussion
Chemistry
1
9,20-Dihydrostrychnine (1) was obtained by catalytic
hydrogenation of strychnine in 10% aqueous acetic acid
using Pd/C 10% in 95% yield according to the proce-
2
7
dure of Oxford et al. Due to single sets of signals in
1
13
both H and C NMR spectra of 1, addition of hydro-
gen to the C19–C20double bond proceeded diastereo-
Scheme 1. (i) H /Pd/C 10%, 10% AcOH, 48 h.
2

D. P. Zlotos et al. / Bioorg. Med. Chem. 11 (2003) 2627–2634
2629
its quaternization was carried out in a 1:2 mixture of
chloroform and acetonitrile giving compound 10 in
27% yield. The identities of the NMR-spectroscopically
pure compounds 1–10 were established by elemental
1
13
analysis (C, H, N) and H and C NMR studies. The
NMR assignments were based on HH-COSY, HMQC
and HMBC experiments.
Pharmacological studies
The allosteric action of the test compounds on the dis-
3
sociation of the orthosteric radioligand [ H]N-methyl-
3
3
scolamine ([ H]NMS) and their effect on [ H]NMS
equilibrium binding were studied in homogenates of
porcine heart ventricles. Dissociation assays were used
to determine the affinity of the allosteric agents at
receptors in which access to the orthosteric binding site
3
was blocked by [ H]NMS. It is thus ensured that only
3
allosteric effects are being observed. [ H]NMS dissociation
was monophasic both in the absence and in the presence of
the test compounds. All investigated compounds were able
3
to retard the dissociation of [ H]NMS concentration
dependently. To generate concentration-effect curves, the
apparent rate constant of dissociation k was expressed
ꢀ1
Scheme 2. (i) tert-Butylnitrite/t-BuOK/toluene, 2 h.
as a percentage of the value under control conditions.
3
Table 1. Parameters characterizing the allosteric interaction of the indicated test compounds with [ H]-N-methylscopolamine at porcine heart M
receptors (for details see text)
2
n
R
pEC0.5,diss
[3H]NMS equilibrium binding (%) at EC0.25,diss
a
Alcuronium
W84
Strychnine
—
—
—
6
7
8
7
—
6
7
8
—
—
—
H
H
H
H
—
OMe
OMe
OMe
OMe
8.61ꢁ0.05
—
—
b
7.67ꢁ0.04
c
6.28ꢁ0.06
124.1ꢁ3.7
121.5ꢁ5.4
122.8ꢁ6.5
125.2ꢁ4.3
111.0ꢁ3.4
114.8ꢁ2.9
107.8ꢁ4.0
114.3ꢁ4.6
112.0ꢁ3.1
114.0ꢁ1.8
d,e
3
4
5
7.99ꢁ0.07
8.30ꢁ0.05
8.38ꢁ0.06
7.87ꢁ0.03
5.49ꢁ0.04
7.54ꢁ0.03
7.53ꢁ0.04
7.57ꢁ0.04
7.24ꢁ0.05
d,f
e
k
f
9
Brucine
g
h
6
7
8
h,i
h
1
0l
7
i,j
a
Ref 34.
Ref 35.
b
c
n
H
=ꢀ0.79ꢁ0.08 [significantly different from unity (F-test, p <0.05)].
d
pEC0.5,diss of Compounds 3 and 4 significantly different (t-test, p <0.05).
e
pEC0.5,diss of Compounds 3 and 5 significantly different (t-test, p <0.05).
pEC0.5,diss of Compounds 4 and 9 significantly different (t-test, p <0.05).
n
f
g
H
=ꢀ0.73ꢁ0.05 [significantly different from unity (F-test, p <0.05)].
h
i
pEC0.5,diss of Compounds 6, 7 and 8 not significantly different from each other (t-test, p >0.05).
pEC0.5,diss of Compounds 7 and 10 significantly different (t-test, p <0.05).
Min=8.69ꢁ0.05% [significantly different from min=0% (F-test, p <0.05)].
j
k
l
Double bonds of both strychnine units hydrogenated.
E-oxime groups at C-22 of both brucine units.

2630
D. P. Zlotos et al. / Bioorg. Med. Chem. 11 (2003) 2627–2634
The concentration of an allosteric agent for a half-
maximum effect on orthosteric ligand dissociation
The influence of the length of the interquaternary alkyl
chain on the allosteric potency was different in both
series. While affinities of the brucine analogues 6–8
remained almost unchanged with increasing lengths
of the middle chain, the allosteric potency of the
strychnine derivatives with C and C middle chains
(
liganded receptors by the respective allosteric test com-
EC0.5,diss) corresponds to a 50% occupancy of the
8
pound. Thus, EC0
sociation constant (K ) of alloster binding to the
indicates the equilibrium dis-
.5,diss
D
7
8
ligand-occupied receptor and reflects the affinity of the
test compound to the allosteric binding site. pEC0.5,diss
4 and 5, respectively, were about 2-fold higher than
for the C analogue 3. The data in the strychnine series are
-
6
values (meansꢁSE, n=3 experiments in triplicate
in agreement with our previous studies in the W84,
WDUO and IWDUO series in which the minimal essen-
tial distance between the positive charges was found to be
determinations) are compiled in Table 1.
3
˚
10A, equivalent to seven methylene groups.
21
The effect of the allosteric test compounds on [ H]NMS
equilibrium binding was investigated at equieffective
concentrations EC0.25,diss. EC0.25,diss is the concentration
In order to examine which structural elements of the
strychnine ring system are responsible for high allosteric
potency, two chemical modifications of the strychnine
ring skeleton have been carried out: reduction of the
C19–C20double bond (compound 9) and introduction
of the oxime function at C-22 (compound 10). Both
structural changes resulted in a significant reduction of
binding affinity. Hydrogenation of both double bonds
in 4 led to about 3-fold decrease in affinity. A similar
decrease of binding affinity (2-fold) resulted from the
introduction of E-oxime groups at C-22 in both brucine
moieties of 7. The findings indicate that the unchanged
strychnine ring is an optimal structural feature respon-
sible for high allosteric potency. Since expansion of the
strychnine ring system by two methoxy groups and an
oxime function led to significant decrease of affinity, the
volume of the strychnine ring skeleton seems to be opti-
mal to fit into the binding pocket of the receptor protein.
3
of test compound at which the rate of [ H]NMS dis-
sociation is reduced to 25% of the control value. Equi-
librium binding data in the presence of allosteric
modulator were expressed as a percentage of the value
under control conditions, which was set as 100%.
3
[
H]NMS equilibrium binding greater than 100% indi-
cates positive cooperativity between the allosteric and
orthosteric ligand. The latter was found with all test
compounds in this series (Table 1; meansꢁSE, n=3–5
experiments in quadruplicate determinations). In the
3
case of W84, [ H]NMS binding is lowered indicating
3
35
negative cooperativity with [ H]NMS.
SAR discussion
3
The pEC0.5,diss values and the effect on [ H]NMS equi-
librium binding at equieffective concentrations EC0.25,diss
for all investigated compounds are given in Table 1.
Strychnine, brucine and their quaternary analogues are
known to be positively cooperative with NMS at M
The allosteric modulators in this series with highest
3
2
16
3
potency to affect [ H]NMS dissociation are the bisqua-
receptors. Screening of the [ H]NMS equilibrium
binding at equieffective concentrations showed that the
positive cooperativity with NMS remained unchanged
after bridging of two strychnine or brucine units with
C –C alkyl chains. This finding suggests that all com-
ternary double strychnines 3–5. Compounds 4 and 5
exhibited nearly the same affinity to NMS-occupied M2
receptors as alcuronium (EC0.5,diss=4 nM). While qua-
ternization of strychnine with simple alkyl groups (i.e.,
methyl, ethyl, propyl) was reported to result in virtually
6
8
pounds in this series have higher affinity for the ligand-
occupied receptor compared with the free receptor and
that the ratio of these affinities is hardly affected by the
structural modifications.
1
6
unchanged affinity to NMS-liganded M receptors, the
2
bridging of two strychnine moieties via C –C8 alkyl
6
chains carried out here caused a dramatic (50-fold for 3,
05-fold for 4 and 131-fold for 5) increase of affinity.
1
The considerably higher affinity of the bisquaternary
double strychnines relative to the monomeric analogues
can be explained based on the pharmacophore for
potent allosteric modulators consisting of two posi-
Conclusion
In this study we have been able to demonstrate that
bridging of two strychnine and brucine moieties with
C –C alkyl chains leads to a pronounced increase in
˚
tively charged groups at a distance of about 10A sur-
1
9,20
rounded by two aromatic ring systems.
The
6
8
presence of only two of these pharmacophoric ele-
ments, that is, one positive charge and one indole ring,
gives rise to limited interactions of the monoquaternary
strychnines with the receptor protein and to reduced
binding affinity.
affinity for the allosteric site of the muscarinic M2
receptors, while the positive cooperativity with the
antagonist NMS is fully maintained. SAR studies have
revealed the unchanged strychnine ring as an important
structural feature for high allosteric potency. Com-
pounds 4 and 5 (EC0.5,diss 5 or 4 nM, respectively) are
among the most potent enhancers of NMS binding to
The findings observed in the strychnine series also apply
for the brucine analogues. Thus, N-substitution of two
brucine units with C –C alkyl chains led to a nearly
M receptors and may serve as useful pharmacological
2
tools in exploring allosteric interactions at muscarinic
receptors. Apparently, the allosteric binding site of M2
receptors nicely accommodates bulky substituents such
as strychnine rings as lateral heterocyclic moieties of
bis(ammonio)alkane-type allosteric agents.
6
8
1
15-fold increase in affinity. However, brucine and its
bisquaternary dimer analogues 6–8 are about 5 times
less potent than strychnine and its corresponding bis-
quaternary derivatives.

D. P. Zlotos et al. / Bioorg. Med. Chem. 11 (2003) 2627–2634
2631
Experimental
suspended in CHCl /MeOH 4/1 (300 mL) and the mix-
3
ture was stirred for 30min. The residue obtained upon
filtration and evaporation of CHCl and MeOH was
General procedures
3
Melting points were determined with a Gallenkamp
melting point apparatus (Sanyo) and were not cor-
rected. Optical rotations were measured using a Perkin-
Elmer model 241 polarimeter. H and C NMR spectra
were recorded on Bruker AV 400 and AV 600 instruments.
TLC was carried out on silica gel 60F254 aluminium sheets;
Merck. Proton chemical shifts are referred to CHCl3
purified on a silica gel column, eluting with CHCl3/
MeOH/25%NH 130:10:1 to afford two oximes.
3
1
13
ꢃ
2b. (0.30 g, 2.4%), yellow crystals; mp >320 C (dec.);
20
R =0.24 (CHCl /MeOH/25% NH 130:10:1); ½ꢀŠ
f
3
3
D
ꢃ
2863, 1630, 1560, 1479, 1446, 1404, 1285, 1214, 1112,
ꢀ239.6 (c 0.25, CHCl /MeOH 1:3); IR (ATR) 2937,
3
ꢀ
1 1
(7.24 ppm) and DMSO-d (2.55ppm); carbon chemical
shifts are referred to CDCl (77.0ppm) and DMSO- d
1001, 848, 750 cm ; H NMR (400 MHz, DMSO-d ) d
6
6
13
1.41 (d, J=14.2 Hz, 1H, H-14a), 1.71 (ddd, J=10.6, 2.8,
2.8 Hz, 1H, H-16), 1.83 (m, 2H, CH -6), 2.34 (ddd,
3
6
(39.50ppm). IR spectra were obtained using a Biorad
2
PharmalyzIR FT-IR spectrometer. Elemental analyses
were performed by the microanalytical section of the Insti-
J=14.2, 4.3, 4.3 Hz, 1H, H-14b), 2.61 (d, J=14.4 Hz,
1H, H-21a), 2.73 (m, 1H, H-5a), 3.02 (m, 1H, H-5b),
3.15 (s, br, 1H, H-15), 3.63 (d, J=14.4 Hz, H-21b), 3.79
(s, 3H, –CH ), 3.81 (s, 3H, –CH ), 3.90(d, J=10.6 Hz,
tute of Inorganic Chemistry, University of Wu
¨
rzburg. All
reactions were carried out under an argon atmosphere.
3
3
1H, H-2), 3.92 (s, br., 1H, H-3), 4.21 (d, J=6.3 Hz, 2H,
CH2-18), 4.69 (d, J=2.8 Hz, 1H, H-17), 5.85 (m, 1H, H-
(
20R)-19,20-Dihydrostrychnine (1). One gramme Pd/C
1
0
0% was added to a solution of strychnine (10 g,
.03 mol) in 10% acetic acid (200 mL). The reaction
19), 7.08 (s, 1H, H-12), 7.74 (s, 1H, H-9), 13.56 (br, 1H,
3
1
OH); C NMR (100 MHz, DMSO-d ) d 26.1 (C-14),
6
mixture was shaken in a Paar-like hydrogenation appa-
ratus under the hydrogen pressure of 5 bar for 48 h. The
catalyst was filtered off and washed with 10% acetic
acid (2 ꢂ 20mL). The free base was precipitated from
the combined aqueous solutions by the dropwise addi-
tion of 20% NaOH under ice-cooling and the reaction
mixture was extracted with chloroform (3 ꢂ 100 mL).
The combined chloroform layers were washed with
30.8 (C-15), 42.1 (C-6), 46.0 (C-16), 49.1 (C-5), 51.3 (C-
7), 52.3 (C-21), 55.4, 55.7 (2 ꢂ CH ), 58.8 (C-2), 59.1
3
(C-3), 64.2 (C-18), 79.9 (C-17), 106.6 (C-9), 100.5
(C-12), 125.3 (C-8), 126.2 (C-19), 138.3 (C-20), 141.2 (C-
13), 145.3 (C-22), 146.2 (C-10), 148.3 (C-11), 157.4
(C-23). Anal. calcd for C H N O : C, 65.24; H, 5.95;
2
3
25
3
5
N, 9.92; found : C, 64.91; H, 6.05; N, 9.75.
ꢃ
water and dried over MgSO . Evaporation of the sol-
4
2a. (11.3 g, 89%); mp >320 C (MeOH/CH Cl ) (dec.);
2
2
20
vent afforded TLC and NMR pure, colorless crystals
9.5 g, 94%), which were used for the next step without
R =0.13 (CHCl /MeOH/25% NH 130:10:1); ½ꢀŠ
f
3
3
D
ꢃ
2900, 2870, 2847, 1655, 1492, 1298, 1274, 1117, 1035,
(
ꢀ186.8 (c 0.25, CHCl /MeOH 1:3); IR (ATR) 2951,
3
ꢃ
27
ꢃ
further purification; mp 223–225 C (lit. 220–222 C);
IR (ATR) 2924, 2872, 1663, 1597, 1475, 1390, 1099,
7
ꢀ
1 1
869, 744 cm ; H NMR (400 MHz, DMSO-d ) d 1.42
6
ꢀ
1 1
78 cm ; H NMR (600 MHz, CDCl ) d 1.45 (dddd,
(d, br., J=14.2 Hz, 1H, H-14a), 1.58 (ddd, J=10.6, 2.5,
2.5 Hz, 1H, H-16), 1.81 (m, 2H, CH -6), 2.33 (ddd,
3
J=15.1, 12.5, 5.3, 2.6 Hz, 1H, H-19a), 1.55 (ddd,
J=11.0, 3.7, 3.7 Hz, 1H, H-16), 1.65 (ddd, J=13.6, 3.1,
2
J=14.2, 4.3, 4.3 Hz, 1H, H-14b), 2.62 (d, J=14.2 Hz,
1H, H-21a), 2.74 (m, 1H, H-5a), 3.02 (dd, J=9.6,
7.3 Hz, 1H, H-5b), 3.09 (s, br., 1H, H-15), 3.63 (d,
3
1
2
.1 Hz, 1H, H-14a), 1.91–1.98 (m, 2H, H-6a and H-
9b), 2.14 (ddd, J=13.6, 3.3, 3.3 Hz, 1H, H-14b), 2.24–
.29 (m, 2H, H-15 and H-21a), 2.35 (m, 1H, H-20), 2.45
J=14.2 Hz, 1H, H-21b), 3.79 (s, 3H, –CH ), 3.81 (s, 3H,
3
(
ddd, J=13.9, 8.7, 8.7 Hz, 1H, H-6b), 2.61 (dd, J=15.9,
–CH ), 3.92 (s, br, 1H, H-3), 4.04 (d, J=10.6 Hz, 1H,
3
5
2
1
.2 Hz, 1H, H-22a), 2.83–2.91 (m, 2H, H-5a and H-
1b), 3.00 (dd, J=15.9, 8.6 Hz, 1H, H-22b), 3.12 (m,
H, H-5b), 3.45 (t, J=3.2 Hz, 1H, H-3), 3.57 (ddd,
H-2), 4.13 (dd, J=13.9, 6.1 Hz, 1H, H-18a), 4.18 (dd,
J=13.9, 7.1 Hz, 1H, H-18b), 5.00 (d, J=2.3 Hz, 1H, H-
17), 5.87 (m, 1H, H-19), 7.05 (s, 1H, H-12), 7.78 (s, 1H,
H-9), 12.79 (br, 1H, OH); C NMR (100 MHz, DMSO-
d6) d 26.4 (C-14), 31.4 (C-15), 42.4 (C-6), 44.7 (C-16),
1
3
J=12.8, 12.8, 3.3 Hz, 1H, H-18a), 3.95 (m, 1H, H-17),
.99 (d, J=11.0Hz, 1H, H-2), 3.99 (m, 1H, H-18b), 7.07
ddd, J=7.7, 7.4, 1.0Hz, 1H, H-10), 7.16 (d, J=7.7 Hz,
H, H-9), 7.21 (ddd, J=7.7, 7.4, 1.0Hz, 1H, H-11), 8.00
3
(
1
49.3 (C-5), 51.2 (C-7), 52.5 (C-21), 55.7, 55.9 (2 ꢂ CH ),
3
59.0(C-2), 59.1 (C-3), 65.3 (C-18), 75.2 (C-17), 1 06 .9 (C-
9), 100.3 (C-12), 124.5 (C-8), 126.6 (C-19), 135.6 (C-20),
140.0 (C-13), 149.2 (C-22), 146.3 (C-10), 148.5 (C-11),
158.9 (C-23). Anal. calcd for C H N O : C, 65.24; H,
1
3
(
d, J=7.7 Hz, 1H, H-12); C NMR (100 MHz, CDCl3)
d 30.2 (C-15), 31.4 (C-14), 32.7 (C-19), 35.0 (C-20), 41.2
C-22), 46.6 (C-6), 51.6 (C-7), 53.4 (C-16), 53.9 (C-5),
(
2
3
25
3
5
5
1
1
8.0(C-21), 62.7 (C-3), 67.8 (C-2), 68.6 (C-18), 76.6 (C-
7), 115.8 (C-12), 122.0(C-9), 124.4 (C-1 0) , 128.2 (C-
1), 135.2 (C-8), 140.9 (C-13), 169.6 (C-23).
5.95; N, 9.92; found: C, 64.86; H, 6.02; N, 9.80.
General procedure for the synthesis of compounds 3–10
E-22-Oximinobrucine (2a) and Z-22-oximinobrucine
2b). tert-Butyl nitrite (80mL) and tert-BuOK (8 g)
The solution of the alkaloid (1.25 mmol) and the
respective dibromoalkane (0.5 mmol) in dry acetonitrile
(50mL) was heated to reflux for 14 h. The precipitated
colorless crystalline product was collected by filtration,
washed with small amounts of acetonitrile and dried in
(
were added to the solution of brucine (11.8 g, 0.03 mol)
in dry toluene (500 mL). The reaction mixture was stir-
ꢃ
0% aqueous NH Cl solution (100 mL) was added and
red at 50 C for 2 h. After cooling to room temperature
1
ꢃ
vacuum at 80 C. No further purification was necessary
as indicated by TLC (silica gel, mobile phase MeOH/2 M
4
vigorous stirring was continued for 15 min. Water and
toluene were removed in vacuo, the yellow residue was
1
NH /2 M aqueous NH NO 84/24/12) and H NMR.
4
3
3

2632
D. P. Zlotos et al. / Bioorg. Med. Chem. 11 (2003) 2627–2634
0
82%) was obtained from strychnine and 1,6-dibromo-
hexane; mp >320 C (dec.); ½ꢀŠ +6.8 (c 0.5, DMSO);
ꢃ
H O 1:1); IR (ATR) 2927, 2836, 1649, 1649, 1501, 1466,
20
ane; mp >320 C (dec.); ½ꢀŠ +30.3 (c 0.33, MeOH/
ꢃ
(
1,6-Hexylene)-4,4 -distrychninium dibromide (3). 0.37 g
D
(
2
ꢃ
20
D
ꢃ
IR (ATR) 2955, 2847, 1681, 1595, 1480, 1458, 1388,
ꢀ1
1
1440, 1399, 1218, 1196, 1105, 845, 754 cm ; H NMR
(400 MHz, DMSO-d ) d 1.46 (ddd, J=10.9, 3.1, 3.1 Hz,
6
ꢀ
1
1
þ
1
327, 1112, 1051, 766 cm
;
H NMR (400 MHz,
2H, 2 ꢂ H-16), 1.51 (m, 4H, 2 ꢂ N CH2CH2CH2), 1.65
þ
DMSO-d ) d 1.52–1.58 (m, 6H, 2 ꢂ N CH2CH2CH2
(d, J=14.9 Hz, 2H, 2 ꢂ H-14a), 1.86–2.04 (m, 4H,
6
þ
and 2 ꢂ H-16), 1.70(d, J=15.4 Hz, 2H, 2 ꢂ H-14a),
2 ꢂ N CH CH ), 2.13–2.25 (m, 4H, CH -6), 2.62–2.73
2
2
2
þ
1
2
.90–2.10 (m, 4H, 2 ꢂ N CH2CH2), 2.26 (m, 4H,
ꢂ CH2-6), 2.71 (m, 2H, 2 ꢂ H-22a), 2.73 (m, 2H, 2 ꢂ
(m, 4H, 2 ꢂ H-14b, 2 ꢂ H-22a), 2.93 (dd, J=17.1,
8.1 Hz, 2H, 2 ꢂ H-22b), 3.38 (s, br, 2H, 2 ꢂ H-15), 3.44
þ
H-14b), 3.02 (dd, J=17.3, 7.8 Hz, 2H, 2 ꢂ H-22b), 3.55
(m, 2H, 2 ꢂ H-5a), 3.73 (m, 4H, 2 ꢂ N CH ), 3.75 (s,
2
(
ddd, J=12.9, 12.9, 6.1 Hz, 2H, 2 ꢂ H-5a), 3.38 (s, br,
6H, 2 ꢂ CH3), 3.82 (s, 6H, 2 ꢂ CH3), 3.90(m, 2H, 2 ꢂ
H-5b), 3.91 (d, J=13.4 Hz, 2H, 2 ꢂ H-21a), 4.12 (d,
J=10.9 Hz, 2H, 2 ꢂ H-2), 4.16 (dd, J=14.6, 5.6 Hz, 2H,
2 ꢂ H-18a), 4.24 (dd, J=14.6, 6.3 Hz, 2H, 2 ꢂ H-18b),
4.30(d, J=13.4 Hz, 2H, 2 ꢂ H-21b), 4.38 (m, 2H, 2 ꢂ
H-17), 4.46 (s, br, 2H, 2 ꢂ H-3), 6.37 (m, 2H, 2 ꢂ H-19),
þ
2
2
(
H, 2 ꢂ H-15), 3.75 (m, 4H, 2 ꢂ N CH2), 3.95 (m, 2H,
ꢂ H-5b), 3.97 (d, 2H, J=13.1 Hz, 2 ꢂ H-21a), 4.21
m, 2H, 2 ꢂ H-18a), 4.22 (d, J=10.9 Hz, 2H, 2 ꢂ H-2),
4
.29 (dd, J=14.3, 6.3 Hz, 2H, 2 ꢂ H-18b), 4.37 (d,
J=13.1 Hz, 2H, 2 ꢂ H-21b), 4.43 (m, 2H, 2 ꢂ H-17),
.53 (s, br, 2H, 2 ꢂ H-3), 6.43 (m, 2H, 2 ꢂ H-19), 7.22
t, J=7.6 Hz, 2H, 2 ꢂ H-10), 7.38 (dd, J=8.1, 7.6 Hz,
1
3
4
(
2
(
7.39 (s, 2H, 2 ꢂ H-9), 7.66 (s, 2H, 2 ꢂ H-12); C NMR
þ
(100 MHz, DMSO-d ) d 22.4 ðN CH2CH2Þ, 24.3 (C-
6
þ
H, 2 ꢂ H-11), 7.79 (d, J=7.6 Hz, 2H, 2 ꢂ H-9), 7.99
14), 25.3 ðN CH2CH2CH2Þ, 29.0(C-15), 38.4 (C-6),
1
3
d, J=8.1 Hz, 2H, 2 ꢂ H-12); C NMR (100 MHz,
40.3 (C-22), 46.1 (C-16), 51.9 (C-7), 55.7, 56.6
ð2 ꢂ CH3Þ, 57.5 (C-5), 58.4 (C-2), 60.4 (C-21), 63.2 (C-
þ
DMSO-d ) d 22.4 ðN CH2CH2Þ, 24.4 (C-14), 25.3
6
þ
þ
ðN CH2CH2CH2Þ, 29.0(C-15), 38.9 (C-6), 46.0(C-16),
18), 65.4 ðN CH2Þ, 73.2 (C-3), 75.8 (C-17), 100.2 (C-
4
2
1
1
1
0.9 (C-22), 51.9 (C-7), 58.1 (C-5), 58.2 (C-2), 60.7 (C-
12), 107.9 (C-9), 120.1 (C-8), 132.9 (C-20), 135.4 (C-19),
135.5 (C-13), 145.9 (C-10), 149.6 (C-11), 168.6 (C-23).
Anal. calcd for C H N O Br : C, 60.47; H, 6.25; N,
þ
1), 63.2 (C-18), 65.3 ðN CH2Þ, 73.0(C-3), 75.6 (C-17),
15.2 (C-12), 123.6 (C-9), 123.9 (C-10), 129.4 (C-11),
29.5 (C-8), 132.9 (C-20), 135.4 (C-19), 141.2 (C-13),
68.9 (C-23). Anal. calcd for C H N O Br : C, 63.16;
5
2
64
4
8
2
5.42; found : C, 60.09; H, 6.30; N, 6.34.
4
8
56
4
4
2
0
91%) was obtained from brucine and 1,6-dibromo-
H, 6.18; N, 6.14; found: C, 62.89; H, 6.42; N, 5.71.
(1,7-Heptylene)-4,4 -dibrucinium dibromide (7). 0.48 g
(
0
76%) was obtained from strychnine and 1,6-dibromo-
heptane; mp >320 C (dec.); ½ꢀŠ +11.4 (c 0.5, DMSO);
ꢃ
MeOH/H O 1:1); IR (ATR) 2937, 2827, 1659, 1591,
20
D
ꢃ
(
1,7-Heptylene)-4,4 -distrychninium dibromide (4). 0.35 g
heptane; mp >320 C (dec.); ½ꢀŠ +27.2 (c 0.5,
(
2
ꢃ
IR (ATR) 2943, 2838, 1653, 1499, 1445, 1398, 1284, 1215,
20
D
ꢃ
1507, 1471, 1452, 1367, 1284, 1201, 1042, 988, 867,
ꢀ
1 1
750cm ; H NMR (400 MHz, DMSO-d ) d 1.50(m,
6
ꢀ
1
1
þ
1
198, 1111, 847, 757 cm ; H NMR (400MHz, DMSO-
2H, 2 ꢂ N CH2CH2CH2CH2), chemical shifts and
þ
d ) d 1.52 (m, 2H, N CH2CH2CH2CH2), 2.20(m, 2H, 2
coupling constants for all other hydrogen atoms coin-
cide with the d values for the corresponding atoms of 6
6
ꢂ H-6a), 2.28 (m, 2H, 2 ꢂ H-6b), chemical shifts and
coupling constants for all other hydrogen atoms coin-
cide with the d values for the corresponding atoms of 3
1
3
within ꢁ0.05 ppm and ꢁ0.1 Hz, respectively; C NMR
þ
(100 MHz, DMSO-d ) d 28.0 ðN CH2CH2CH2CH2Þ,
6
1
3
within ꢁ0.02 ppm and ꢁ0.1 Hz, respectively; C NMR
chemical shifts for all other carbon atoms coincide with
the d-values for the corresponding atoms of 6 within
ꢁ0.1 ppm. Anal. calcd for C H N O Br : C, 60.80; H,
þ
(
chemical shifts for all other carbon atoms coincide with
100 MHz, DMSO-d ) d 28.0 ðN CH CH CH CH Þ,
6
2
2
2
2
5
3
66
4
8
2
the d values for the corresponding atoms of 3 within
6.35; N, 5.35; found: C, 60.45; H, 6.45; N, 5.20.
ꢁ
0.1 ppm. Anal. calcd for C H N O Br : C, 63.50; H,
.31; N, 6.04; found: C, 63.05; H, 6.09; N, 5.63.
49 58 4 4 2
0
65%) was obtained from brucine and 1,6-dibro-
6
(1,8-Octylene)-4,4 -dibrucinium dibromide (8). 0.35 g
(
0
ꢃ
20
D
MeOH/H O 1:1); IR (ATR) 2937, 2827, 1659, 1586,
ꢃ
(
(
1,8-Octylene)-4,4 -distrychninium dibromide (5). 0.37 g
78%) was obtained from strychnine and 1,6-dibro-
mooctane; mp >320 C (dec.); ½ꢀŠ +20.9 (c 0.5,
2
ꢃ
DMSO); IR (ATR) 2912, 2869, 1684, 1596, 1481, 1464,
20
D
ꢃ
ꢀ1
1
mooctane; mp >320 C (dec.); ½ꢀŠ +6.8 (c 0.5,
1469, 1445, 1402, 1283, 1198, 1107, 846, 754 cm ; H
NMR (400 MHz, DMSO-d ) chemical shifts and coup-
6
ꢀ
1 1
1
387, 1109, 1051, 762 cm ; H NMR (400MHz, DMSO-
ling constants for all hydrogen atoms coincide with the
d-values for the corresponding atoms of 7 within
þ
d ) d 1.50(m, 4H, 2 ꢂ N CH2CH2CH2CH2), 2.18 (m,
6
1
3
2
and coupling constants for all other hydrogen atoms
H, 2 ꢂ H-6a), 2.24 (m, 2H, 2 ꢂ H-6b), chemical shifts
ꢁ0.02 ppm and ꢁ0.1 Hz, respectively;
C NMR
(100 MHz, DMSO-d ) chemical shifts for all carbon
6
coincide with the d-values for the corresponding atoms of 3
atoms coincide with the d values for the corresponding
1
3
within ꢁ0.02 ppm and ꢁ0.1 Hz, respectively; C NMR
atoms of
C H N O Br : C, 60.80; H, 6.35; N, 5.35; found: C,
7
within ꢁ0.5 ppm. Anal. calcd for
þ
(
100MHz, DMSO-d ) d 28.5 ðN CH CH CH CH Þ,
6
2
2
2
2
53 66
4
8
2
chemical shifts for all other carbon atoms coincide with the
d values for the corresponding atoms of 3 within
60.45; H, 6.45; N, 5.20.
0
ꢁ
0.1 ppm. Anal. calcd for C H N O Br : C, 63.83; H,
4
.43; N, 5.95; found : C, 63.40; H, 5.91; N, 5.82.
(1,7-Heptylene)- 4,4 -bis[(20R)-19,20-dihydrostrychni-
nium] dibromide (9). 0.42 g (90%) was obtained from
50
60
4
2
6
ꢃ
+38.1 (c 0.33, DMSO); IR (ATR) 2947, 2908, 1672,
20
D
(
1) and 1,6-dibromoheptane; mp >320 C (dec.); ½ꢀŠ
0
85%) was obtained from brucine and 1,6-dibromohex-
ꢃ
1595, 1479, 1460, 1404, 1347, 1111, 745 cm ; H NMR
(
(
1,6-Hexylene)-4,4 -dibrucinium dibromide (6). 0.44 g
ꢀ
1 1

D. P. Zlotos et al. / Bioorg. Med. Chem. 11 (2003) 2627–2634
2633
ꢀ
6
(
400 MHz,
DMSO-d6)
d
1.46
(m,
2H,
binding was determined in the presence of 10 M atro-
pine and was less than 5% of the total binding. Mem-
branes were separated by vacuum filtration through
þ
N CH2CH2CH2CH2), 1.48 (m, 2H, 2 ꢂ H-19a), 1.50
þ
(
1
2
(
2
2
(
m, 4H, 2 ꢂ N CH2CH2CH2), 1.68 (m, 2H, 2 ꢂ H-14a),
þ
.72 (m, 2H, 2 ꢂ H-16), 1.80(m, 4H, 2 ꢂ N CH CH ),
¨
glass fibre filters (Schleicher and Schull, No. 6; Dassel,
2
2
.01 (m, 2H, 2 ꢂ H-19b), 2.14 (m, 2H, 2 ꢂ H-6a), 2.48
Germany) and membrane bound radioactivity was
determined by liquid scintillation counting. The pKD of
NMS binding amounted to 9.52ꢁ0.04 (meanꢁSEM,
m, 4H, 2 ꢂ H-14b and 2 ꢂ H-20), 2.76 (m, 2H, 2 ꢂ H-
2a), 2.78 (m, 2H, 2 ꢂ H-15), 2.86 (m, 2H, 2 ꢂ H-22b),
.89 (m, 2H, 2 ꢂ H-6b), 3.19 (m, 2H, 2 ꢂ H-5a), 3.56
3
n=3). [ H]NMS dissociation proceeded monophasically
m, 2H, 2 ꢂ H-5b), 3.61 (m, 2H, 2 ꢂ H-21a), 3.66 (m, 2H,
(t1=2;control ¼ 5:0 ꢁ 0:1 min; meanꢁSEM, n=60). To
þ
3
2
ꢂ N CHH), 3.72 (m, 2H, 2 ꢂ H-18a), 3.98 (m, 2H, 2 ꢂ
measure the effect of the test compounds on [ H]NMS
dissociation, cardiac membranes were prelabeled with
þ
H-21b), 4.00 (m, 4H, 2 ꢂ H-18b and 2 ꢂ N CHH), 4.13
3
(
m, 2H, 2 ꢂ H-17), 4.53 (s, br, 2H, 2 ꢂ H-3), 4.66 (d,
[ H]NMS for 30min, radioligand dissociation was then
revealed by the addition of 1 mM atropine, in the
absence or in the presence of allosteric modulator. Two-
J=11.3 Hz, 2H, 2 ꢂ H-2), 7.23 (t, J=7.6 Hz, 2H, 2 ꢂ H-
1
2
0), 7.37 (t, J=7.6 Hz, 2H, 2 ꢂ H-11), 7.82 (d, J=7.6 Hz,
1
3
32
H, 2 ꢂ H-9), 7.94 (d, J=7.6 Hz, 2H, 2 ꢂ H-12);
+
C
point kinetic experiments were applied and specific
3
þ
NMR (100 MHz, DMSO-d ) d 22.3 ðN CH2CH2Þ, 25.7
[ H]NMS binding was measured at t=0and t=10min.
6
(C-14), 26.01 (N CH CH CH CH ), 27.7 (C-20), 27.8
2
2 2 2
þ
3
ðN CH2CH2CH2Þ, 29.4 (C-15), 31.2 (C-19), 38.9 (C-6),
The effect of the allosteric test compounds on [ H]NMS
equilibrium binding was investigated at equieffective
concentrations EC0.25,diss. EC0.25,diss is the concentration
of test compound at which the rate of [ H]NMS dis-
sociation is reduced to 25% of the control. The appro-
priate time of incubation was determined according to
Lazareno and Birdsall (eq 31 therein). Five half-lives
were taken to ensure equilibrium conditions for binding
of the allosteric modulator to the allosteric site. Equili-
brium binding data (from 3 to 5 independent experi-
ments with quadruplicated values) in the presence of
allosteric modulator were expressed in percent relative
to the control binding in the absence of modulator,
which was set as 100%. Dissociation data were analysed
by nonlinear regression analysis using the Prism pro-
gram (version 3.02, GraphPAD Software, San Diego,
USA).
4
CH ), 57.0(C-5), 57.9 (C-21), 6 0. 7 (C-18), 64.6 (C-2),
0.0 (C-22), 50.0 (C-16), 51.1 (C-7), 55.7, 56.6 (2 ꢂ
3
þ
3
6
1
1
5.2 ðN CH2Þ, 70.6 (C-3), 74.0 (C-17), 114.9 (C-12),
23.6 (C-9), 124.4 (C-10), 129.2 (C-11), 132.1 (C-8),
40.5 (C-13), 169.3 (C-23). Anal. calcd for
3
3
C H N O Br : C, 63.23; H, 6.71; N, 6.02; found: C,
2
6
4
9
62
4
4
3.02; H, 6.70; N, 5.90.
0
mide (10). 0.15 g (27%) was obtained from 2a and 1,6-
(
1,7-Heptylene)- 4,4 -di-(E-22-oximinobrucinium) dibro-
dibromoheptane using chloroform/acetonitrile 2:1
(
ꢃ
54.0 (c 0.5, MeOH/H O 1:1); IR (ATR) 2940, 2820,
20
D
50mL) as a reaction solvent; mp >320 C (dec.); ½ꢀŠ
ꢃ
666, 1500, 1446, 1408, 1285, 1115, 1009 cm
ꢀ
2
ꢀ
1
1
1
;
H
NMR (400 MHz, DMSO-d ) d 1.44–1.60(m, 6H,
2
6
þ
ꢂ N CH2CH2CH2CH2), 1.74 (d, J=14.9 Hz, 2H, 2 ꢂ
H-14a), 1.79 (d, br, J=11.1 Hz, 2H, 2 ꢂ H-16), 1.94 (m,
þ
4
2
3
H, 2 ꢂ N CH CH ), 2.22 (m, 2H, 2 ꢂ H-6a), 2.27 (m,
2
2
H, 2 ꢂ H-6b), 2.77 (d, J=14.9 Hz, 2H, 2 ꢂ H-14b),
Acknowledgements
.38 (s, br, 2H, 2 ꢂ H-15), 3.49 (m, 2H, 2 ꢂ H-5a), 3.75
þ
(
2
4
m, 4H, 2 ꢂ N CH2), 3.81 (s, 6H, 2 ꢂ CH ), 3.87 (s, 6H,
Thanks are due to the Deutsche Forschungsgemein-
schaft (HO 1368/7; MO 821/1-2) and the Fonds der
Chemischen Industrie, Deutschland, for financial sup-
port, as well as to Mechthild Kepe, Institute of Pharmacy,
University of Bonn, for her skillful technical assistance
3
ꢂ CH ), 3.88 (m, 2H, H-21a), 3.89 (m, 2H, 2 ꢂ H-5b),
3
.29 (m, 4H, 2 ꢂ CH -18), 4.38 (d, J=10.9 Hz, 2H, 2 ꢂ
2
H-2), 4.34 (m, 2H, 2 ꢂ H-21b), 4.55 (s, br, 2H, 2 ꢂ H-3),
5
7
2
.12 (d, J=2.0Hz, 2H, H-17), 6.47 (m, 2H, 2 ꢂ H-19),
.43 (s, 2H, 2 ꢂ H-12), 7.79 (s, 2H, 2 ꢂ H-9), 12.86 (br,
¨
and to Dr. Matthias Grune and Elfriede Ruckdeschel,
Institute of Organic Chemistry, University of Wurzburg
1
3
H, 2 ꢂ OH); C NMR (100 MHz, DMSO-d ) d 22.7
¨
6
þ
þ
ðN CH CH Þ, 24.3 (C-14), 25.6 ð2 ꢂ N CH CH CH Þ,
for recording 600 MHz NMR spectra of 1.
2
þ
2
2
2
2
2
4
5
8.1 ðN CH2CH2CH2CH2Þ, 29.7 (C-15), 38.3 (C-6),
3.3 (C-16), 51.7 (C-7), 55.7, 56.6 (2 ꢂ CH ), 57.2 (C-5),
3
þ
7.5 (C-2), 60.6 (C-21), 64.5 (C-18), 65.4 ðN CH Þ, 73.2
2
References and Notes
(
C-3), 74.4 (C-17), 99.9 (C-12), 107.6 (C-9), 120.4 (C-8),
1
1
33.6 (C-20), 135.6 (C-13), 146.4 (C-10), 148.6 (C-22),
49.5 (C-22), 158.5 (C-23). Anal. calcd for
1. Stockton, J. M.; Birdsall, N. J. M.; Burgen, A. S. V.;
Hulme, E. C. Mol. Pharmacol. 1983, 23, 551.
2. Ellis, J.; Huyler, J.; Brann, M. R. Biochem. Pharmacol.
C H N O Br : C, 57.61; H, 5.84; N, 7.61; found: C,
5
5
3
64
6
10
2
7.23; H, 5.79; N, 7.27.
1
991, 42, 1927.
. Lee, N. H.; El-Fakahany, E. E. Biochem. Pharmacol. 1991,
3
42, 199.
Radioligand binding studies
4
5
3
6
. Holzgrabe, U.; Mohr, K. Drug Discov Today 1998, 3, 214.
. Christopoulos, A.; Kenakin, T. Pharmacol. Rev. 2002, 54,
23.
. Trankle, C.; Andresen, I.; Lambrecht, G.; Mohr, K. Mol.
¨
Porcine cardiac homogenates were prepared as descri-
8
3
bed previously. Binding of [ H]N-methylscopolamine
3
(
[ H]NMS) (0.2 nM; specific activity 70–83.5 Ci/mmol;
Perkin–Elmer Life Sciences, Boston, MA, USA) was
measured in a buffer composed of 4 mM Na HPO and
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