Synthesis of tropeines and allosteric modulation of ionotropic glycine receptors

Article abstract of DOI:10.1021/jm040814g

Twenty esters of 3α- and 3β-hydroxy(nor)tropanes and two amides of 3α-aminotropane were prepared with substituted benzoic acids. These (nor)tropeines inhibited [³H]strychnine binding to glycine receptors in synaptosomal membranes of rat spinal cord. A ternary allosteric model was applied to determine the dissociation constants (K_A) of the tropeines having strong negative cooperativities with [³H]strychnine binding (α > 10). K_A values about 10 nM are well below those of known allosteric agents. Low concentrations (0.1K_A) of the (nor)tropeines potentiated the displacing effects of glycine. Positive cooperativity with glycine (β1) decreased with the increase in concentration and binding affinity of tropeines. Displacing potencies were also measured for [³H]granisetron binding to 5-HT₃ type serotonin receptors of rat cerebral cortex. Selectivities to glycine receptors versus 5-HT₃ receptors varied within 4 orders of magnitude. Nortropeines might serve as a lead to high-affinity selective allosteric modulators of glycine receptors.

Full text of DOI:10.1021/jm040814g

6384
J. Med. Chem. 2004, 47, 6384-6391
Synthesis of Tropeines and Allosteric Modulation of Ionotropic Glycine
Receptors
Ga´bor Maksay,*,† Pe´ter Nemes,‡ and T´ımea B´ıro´†
Molecular Pharmacology Group, Institute of Biomolecular Chemistry, Chemical Research Center,
Hungarian Academy of Sciences, H-1525, Budapest, Post Office Box 17, Hungary, and Department of Chemistry,
Faculty of Veterinary Sciences, Szent Istva´n University, H-1400, Budapest, Post Office Box 2, Hungary
Received March 18, 2004
Twenty esters of 3R- and 3â-hydroxy(nor)tropanes and two amides of 3R-aminotropane were
prepared with substituted benzoic acids. These (nor)tropeines inhibited [3H]strychnine binding
to glycine receptors in synaptosomal membranes of rat spinal cord. A ternary allosteric model
was applied to determine the dissociation constants (KA) of the tropeines having strong negative
cooperativities with [3H]strychnine binding (R > 10). KA values about 10 nM are well below
those of known allosteric agents. Low concentrations (0.1KA) of the (nor)tropeines potentiated
the displacing effects of glycine. Positive cooperativity with glycine (â < 1) decreased with the
increase in concentration and binding affinity of tropeines. Displacing potencies were also
measured for [3H]granisetron binding to 5-HT3 type serotonin receptors of rat cerebral cortex.
Selectivities to glycine receptors versus 5-HT3 receptors varied within 4 orders of magnitude.
Nortropeines might serve as a lead to high-affinity selective allosteric modulators of glycine
receptors.
Introduction
to be a promising lead to develop high-affinity and
subunit-selective allosteric agents of GlyRs. In vitro
receptor assays can facilitate the development of such
agents. GlyR binding10 of the competitive antagonist
[3H]strychnine and chloride ionophore activity11 can
differentiate the structure-activity requirements of
“Gly positive” and “Gly negative” agents with positive
and negative cooperativity with glycine, respectively.
“Gly positive” tropeines exert high-affinity (nanomolar)
displacement of [3H]strychnine binding potentiated by
glycine.10 We have recently applied a ternary allosteric
model for the quantitative characterization of binding
affinities and cooperativities for GlyRs.12 This model
permits the simultaneous binding of an allosteric agent
and an orthosteric ligand, [3H]strychnine or glycine.
Here, we describe some nortropeines with binding
affinities exceeding those of known potentiating agents
of GlyRs by 1-2 orders of magnitude while they have
lower activities on 5-HT3 receptors.
Glycine is the major inhibitory neurotransmitter in
mammalian spinal cord acting via ionotropic glycine
receptors (GlyRs). Pentamers of R1-4 and â GlyR sub-
units form chloride channels. Glycinergic neurotrans-
mission plays predominant roles in muscle tone regu-
lation and in the processing of sensory signals.1 Since
allosteric modulation is most suitable for the therapeu-
tical fine-tuning of neurotransmitter receptors, positive
modulators of GlyRs might exert beneficial muscle
relaxant, anticonvulsant, analgesic, or anesthetic ef-
fects.2 Still, GlyRs are referred to as “therapeutic
orphans”,3 having no high-affinity allosteric modulators.
Although GlyRs are modulated by Zn2+,4 alcohols,5 and
tropeines (esters of 3R-hydroxytropane and amides of
3R-aminotropane),6 these agents act on other ionotropic
receptors as well. Overlapping affinities to ionotropic
receptors can be partly attributed to the structural
similarities within the superfamily of GlyRs, nicotinic
acetylcholine, 5-HT3-type serotonin, and GABAA recep-
tors. Thus it is a challenge to medicinal chemists to get
high affinity and selective allosteric modulators of
GlyRs. As to high-affinity agents, some tropeines po-
tentiate GlyR activity in nanomolar concentrations,
while they inhibit it in micromolar concentrations.6 As
to selectivity, subunit-specific modulation of GlyRs by
some tropeines7,8 and neurosteroids9 has been promis-
ing. Inhibitory potencies of the tropeines (tropisetron
and atropine) versus glycine activation of recombinant
GlyRs vary for different R1-2(â) subunits and R1 mutants
by 4 orders of magnitude.7 In subunit selectivity the â
subunit plays a critical role in the potentiation of GlyR
ionophore function by tropisetron.8 So tropeines seem
Synthesis
Tropane alkaloids occur as esters of relatively simple
carboxylic acids and tropine, ψ-tropine, or nortropine.
Due to their biological activity and pharmacological
significance, they have been subject to intensive stere-
ochemical and synthetic investigations. In this paper
we describe the synthesis of substituted benzoates of
tropine, ψ-tropine, and nor derivatives of some tropeines.
The esters of tropine and ψ-tropine with substituted
benzoic acids can be prepared in simple acylation
processes,13,14 which can be carried out in different
solvents such as toluene, chloroform, dioxane, or pyri-
dine. Acylation of tropine is more difficult because of
the sterically hindered hydroxyl group and results in
lower yields than the ester formation of ψ-tropine. In
our synthesis we used the corresponding substituted
benzoyl chlorides, which were added to a toluene solu-
* Corresponding author: phone (361) 325-7900/282; fax (361) 325-
7554; e-mail maksay@chemres.hu.
† Chemical Research Center.
‡ Szent Istva´n University.
10.1021/jm040814g CCC: $27.50 © 2004 American Chemical Society
Published on Web 10/29/2004

Tropeine Synthesis and Modulation of Glycine Receptors
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25 6385
Scheme 1
Scheme 2
Scheme 3
tion of tropine in the presence of TEA. Several methods
are known for the demethylation of tropane deriva-
tives.15,16 By use of ethyl chloroformate, demethylation
takes place readily under the formation of methyl
chloride and N-ethoxycarbonyl derivatives. However,
the removal of the ethoxycarbonyl group requires vigor-
ous reaction conditions. Therefore the use of 2,2,2-
trichloroethyl chloroformate is much more appropriate
for the demethylation of tropeine.16 The removal of the
trichloroethoxycarbonyl group can be achieved with zinc
in acetic acid solution (Scheme 1). The procedure does
not affect the ester group, and nortropeines can be
obtained in good yield.
Since 3R-(3′-nitrobenzoyloxy)tropane did not undergo
demethylation with 2,2,2-trichloroethyl chloroformate
under the usual conditions, a different synthetic route
was elaborated to obtain 3R-(3′-aminobenzoyloxy)nor-
tropane (4e). N-Benzylnortropinone was prepared from
benzylamine, acetonedicarboxylic acid, and succinic
aldehyde following the classical Robinson-Scho¨pf bio-
mimetic route.17 The ketone was then hydrogenated
stereoselectively in the presence of Raney-Ni catalyst
under pressure, producing N-benzylnortropine (5).18
Acylation of 5 gave the 3-nitrobenzoyl derivative 6,
which was subjected to repeated hydrogenation at
ambient temperature, resulting in debenzylation and
reduction of the nitro group simultaneously to yield
directly 4e under mild conditions (Scheme 2). 3â-
Benzoyloxynortropane (8) was alkylated with ethyl
bromide at room temperature to yield N-ethyl-3â-
benzoyloxynortropane (9). 3R-Aminotropane was pre-
pared from tropinone in a palladium-catalyzed one-pot
stereoselective synthesis with ammonium formate as
nitrogen and hydrogen source.19 Acylation with benzoyl
chloride was carried out at room temperature to result
in the amide 10 in high yield20 (Scheme 3).
Receptor Binding
Modulation of GlyRs. The concentration-dependent
displacing effects of the tropeines were examined on [3H]-
strychnine binding to GlyRs of rat spinal cord. All
tropeines exerted great maximal displacements of spe-
cific [3H]strychnine binding but possessed different
potencies. This is illustrated in Figure 1 with three
representative tropeines. According to the ternary al-
losteric model and eq 2, they have strong negative
Figure 1. Displacement of [3H]strychnine binding by three
representative tropeines: 4d ([), 8 (O), and 2b (/). Points are
mean ( SEM of 3-4 experiments. Curve fitting according to
eq 2 resulted in KA and R values in Table 1.

6386 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25
Maksay et al.
Table 1. Binding Constants of (nor)Tropeines As Determined for [3H]Strychnine Binding to GlyRs of Rat Spinal Cord and Displacing
Potencies for [3H]Granisetron Binding to 5-HT3 Serotonin Receptors of Rat Cerebral Cortex
GlyR
5-HT3Ra
b
tropeines
-log KA
âc
-log IC50
nH
selectivity, IC50/KA
3R-benzoyloxytropane (2a)
6.387 ( 0.022 0.49 ( 0.09
5.770 ( 0.037 0.57 ( 0.05
5.337 ( 0.063 0.15 ( 0.02
5.292 ( 0.055 0.20 ( 0.03
4.532 ( 0.046 0.13 ( 0.02
7.081 ( 0.056 0.27 ( 0.04
6.310 ( 0.057 0.12 ( 0.01
5.845 ( 0.041 0.20 ( 0.07
5.824 ( 0.046 0.12 ( 0.01
5.975 ( 0.034 0.09 ( 0.01
6.525 ( 0.105 0.57 ( 0.09
6.302 ( 0.047 0.88 ( 0.10
6.739 ( 0.083 1.05 ( 0.09
5.978 ( 0.018 1.07 ( 0.12
0.78
0.30
0.04
0.20
3â-benzoyloxytropane (7)
3R-(4′-ethylbenzoyloxy)tropane (2b)
3R-(4′-nitrobenzoyloxy)tropane (2c)
3R-(4′-t-butylbenzoyloxy)tropane (2d)
3R-(3′-methoxybenzoyloxy)tropane(2e)
3R-(3′-chlorobenzoyloxy)tropane (2f)
3R-(3′-nitrobenzoyloxy)tropane (2g)
3R-[3′-(trifluoromethyl)benzoyloxy]tropane (2h)
3R-(3′,4′,5′-trimethoxybenzoyloxy)tropane (2i)
7.149 ( 0.032 1.26 ( 0.03
7.168 ( 0.102 1.05 ( 0.21
0.87
0.15
6.637 ( 0.086 0.95 ( 0.13
0.23
3R-(3′,5′-dichlorobenzoyloxy)tropane (bemesetron) (2j) 5.991 ( 0.065d 0.06 ( 0.004d
3â-benzoyloxynortropane (8)
3R-benzoyloxynortropane (4a)
3R-(3′-methoxybenzoyloxy)nortropane (4b)
3R-(3′-chlorobenzoyloxy)nortropane (4c)
3R-(3′-aminobenzoyloxy)nortropane (4e)
3R-(2′,3′-benzofuran-5′,5′-dimethyl-8′-chloro-6′-
carbonyloxo)tropane (2k)
3R-(2′,3′-benzofuran-5′,5′-dimethyl-8′-chloro-6′-
carbonyloxo)nortropane (4d)
5.959 ( 0.068 4.3 ( 0.4
7.983 ( 0.039 0.30 ( 0.04
8.114 ( 0.035 0.39 ( 0.07
7.646 ( 0.080 0.23 ( 0.02
7.233 ( 0.044 1.4 ( 0.4
6.633 ( 0.142 0.06 ( 0.01
5.998 ( 0.119 1.00 ( 0.17
5.806 ( 0.086 1.03 ( 0.23
6.581 ( 0.063 0.90 ( 0.10
6.160 ( 0.090 1.17 ( 0.18
6.239 ( 0.099 0.91 ( 0.02
8.144 ( 0.148 0.87 ( 0.13
0.98
161.1
33.5
33.7
10.3
0.03
7.742 ( 0.106 0.13 ( 0.02
7.250 ( 0.127 0.90 ( 0.10
6.145 ( 0.025 1.01 ( 0.10
3.37
0.98
3â-N-ethylbenzoyloxynortropane (9)
3R-benzoylamidotropane (10)
3R-(2′,3′-benzofuran-5′,5′-dimethyl-8′-chloro-6′-
carbonylamido)tropane (zatosetron)
6.137 ( 0.064 0.55 ( 0.08
4.796 ( 0.052 0.22 ( 0.03
6.569 ( 0.089d
a Displacement curves were fitted with variable slopes (nH). Data are mean ( SEM of 3-6 experiments. b KA values were determined
with eq 2. c Tropeines were applied at a concentration of 0.1KA with different glycine concentrations for the determination of â with eq
1. The values of R are .10 for all compounds except for 2d, 2g, and 8 with R ) 14 ( 2, R ) 14 ( 7, and R ) 18 ( 9, respectively. d Taken
from Maksay and B´ıro´.12
cooperativity with strychnine binding (R > 10). This is
shown in Figure 1 for 4d with maximal displacement
approaching nonspecific [3H]strychnine binding. Table
1 summarizes the dissociation constants of the tropeines
with KA values between 8 nM and 30 µM. For most
compounds the allosteric (eq 2) and competitive (eq 3)
binding models result in identical KA values, with the
exception of three tropeines with R < 20 (Table 1).
We concentrated on 3R-esters since (1) C3 showed
marked stereoselectivity, that is, 100 times preference
for 3R in 4a over 3â in 8, and (2) the ester 2a was about
30 times more potent than its amide analogue 10. Ring
substituents in the para position decreased the affinities
parallel with size regardless of their electronic proper-
ties. Some substituents, for example, m-methoxy, were
advantageous in 2e and 4b; most others were disad-
Figure 2. Displacement of [3H]strychnine binding by glycine
vantageous. Nortropeines exerted highest potency, as
(control, O) and the effects of 0.1 µM (/) and 0.3 µM (b) 2j.
shown by the 40 times potency of N-demethylated 4a
Points are mean ( SEM of 3-5 experiments. The data set for
over N-methylated 2a.
0.1 µM 2j is taken from Maksay and B´ıro´12 and was deter-
mined under identical conditions. Curve fitting to control via
The concentration-dependent displacement of [3H]-
strychnine binding by glycine was also examined be-
cause we have found this as an optimal test to evaluate
the cooperativity with glycine (â).11 Figure 2 shows the
concentration-dependent positive cooperative effects of
bemesetron 2j with â < 1. This is an extension of our
previous analysis of the effect of 2j. With increasing
concentrations of “Gly positive” agents such as 2j,
potentiation turns into inhibition of GlyRs. This is
accompanied with decreasing positive cooperativity with
glycine and increasing â values as shown for 2j in Figure
3. Compound 2k, an ester analogue of zatosetron with
high (nanomolar) affinity, also shifted the displacement
curve of glycine to indicate positive cooperativity (Figure
4). Even lower (2 nM and 20 nM) concentrations of its
nor derivative 4d displayed positive cooperativity with
glycine (â < 1, Figure 3). Since cooperativity with
eq 1 resulted in KL ) 20.1 ( 1.0 µM glycine; for 0.3 µM 2j, R
. 10 and KA ) 0.4 µM. The â values are shown in Figure 3.
Control data (without glycine) are indicated on the abscissa
at -9.
glycine (â) depended on the concentration of the allo-
steric agents, we compared the â values at low identical
occupancies by the tropeines (0.1KA). Table 1 sum-
marizes the â values calculated with concentrations at
0.1KA. Most tropeines have positive cooperativities with
glycine (â < 1). The 3â nortropeine 8 not only has 40
times less affinity than 3R 4a but also its cooperativity
with glycine becomes negative (Table 1). The cooperat-
ivity of some nortropeines such as 4e is about neutral
(Table 1). Figure 5 shows a logarithmic plot of the
affinities of (nor)tropeines (KA) and cooperativities with
glycine (â). It can be observed for some structural groups

Tropeine Synthesis and Modulation of Glycine Receptors
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25 6387
substitution of 2a (with H) up to tert-butyl (2d) de-
creased the binding affinity but increased the cooper-
ativity with glycine. This correlation (r2 ) 0.88) can be
probably attributed mainly to steric effects because
ethyl and nitro derivatives (2b and 2c) have similar
sizes and different electronic properties yet overlapping
activities (Figure 5, inset).
5-HT3 Receptor Activity. The displacing effects of
selected (nor)tropeines were measured on [3H]granis-
etron binding to 5-HT3 type serotonin receptors of rat
cerebral cortex. Table 1 summarizes the displacing
potencies (IC50). The slope factors nH of most tropeines
were close to unity, supporting the view that the
compounds are competitive antagonists of 5-HT3 recep-
tors. The selectivity ratios IC50/KA vary by 4 orders of
magnitude, demonstrating different structure-activity
requirements for GlyRs versus 5-HT3 receptors. N-
Demethylation of 2a leads to the greatest preference for
GlyRs with a selectivity ratio of 161 for 4a. All
nortropeines have selectivity ratios beyond 10 and might
thus serve as a promising lead toward selective allo-
steric modulators of GlyRs.
Figure 3. Concentration-dependent effects of 2j (b) and 4d
([) on their positive cooperativities (â) with glycine.
Discussion
Allosteric interactions with glycine for [3H]strychnine
binding have been shown to correlate with the modula-
tion of GlyR function.10 This correlation between GlyR
binding and function has been confirmed for various
allosteric agents of known structures.10-12 The ternary
allosteric model enables us to describe structure-
activity relationships for GlyR binding in a quantitative
manner.12 We have applied now this model for the
development of new GlyR-selective allosteric modulators
with tropeine structures. Most “Gly positive” allosteric
agents such as tropeines exert biphasic effects on GlyRs.
Potentiation at low concentrations is followed by in-
hibition at high (micromolar) concentrations of the
tropeines.11 This is manifested in a concentration-
dependent shift in â from positive to negative cooper-
ativity with glycine binding. Therefore we measured this
functionally most relevant parameter (â) at low identical
occupancies (0.1KA) of GlyRs. Allosteric potentiation of
the chloride ionophore function of GlyRs also requires
low occupancy by glycine.8 Accordingly, some of these
nortropeines potentiated the effects of low glycine
Figure 4. Displacement of [3H]strychnine binding by glycine
(control, O) and the effects of 23 nM (]) and 230 nM (9) 2k.
Points are mean ( SEM of 3-4 experiments. Curve fitting to
control via eq 1 resulted in KL ) 24.5 ( 1.6 µM glycine; for 23
nM 2k, â ) 0.08 ( 0.01; for 230 nM 2k, R ) 5.8 ( 2.4 and â
) 0.26 ( 0.06. The effect of 23 nM 2k without glycine was
excluded from the fitting because of its high affinity displacing
effect, and R ≡ 1 was applied. Control data (without glycine)
are indicated on the abscissa at -9.
that a decrease in binding affinity is accompanied with
stronger positive cooperativity. The subset of para-
substituted benzoate esters is replotted on the inset of
Figure 5 and labeled with the para-substituents. Para-
Figure 5. Correlation between the dissociation constants (KA) of tropeines (b) and nortropeines (/) and the extent of cooperativity
(â) with glycine. (Inset) Subset of para-substituted 3R-benzoyloxytropane derivatives labeled with their moieties and the parent
compound 2a (H) show linear correlation (r2 ) 0.88).

6388 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25
Maksay et al.
The most striking example is the 3â-oriented nortropeine
8 with 40 times less affinity and opposite cooperativity
in comparison to 3R 4a. The hydrophobic interaction of
the phenyl ring seems to be of low specificity because
of its low sensitivity to the electronic properties of its
para substituents. The phenyl ring seems to fit in a
hydrophobic region sterically restricted toward the para
position. The ester group might be close to a hydrogen-
bond donor as suggested by the 30 times lower affinity
of the amide analogue 10. The tropane ring has struc-
turally most distinctive interactions. It has been shown
that its substitutions or replacement strongly decrease
the binding affinity and/or positive cooperativity with
glycine (efficacy).10 Moreover, demethylation of the basic
nitrogen leads to much more potent nortropeines. This
is a feature distinctive from the pharmacophore models
of 5-HT3 receptor antagonists, where demethylation
results in loss of activity.21 Consequently, distinct
structure-activity requirements of the pharmacophores
of the (nor)tropeine modulators of GlyRs versus 5-HT3
receptor antagonists lead to selectivity differences of 4
orders of magnitude between the two receptors. This is
promising for the development of GlyR-selective allo-
steric agents.
Figure 6. Pharmacophore model of tropeine binding to
GlyRs: stick and ball representation of 4d as a template.
Circles indicate essential structural elements. The basic
nitrogen may form an elecrostatic interaction, the carbonyloxy
moiety accepts an H-bond, and the aromatic ring interacts with
a hydrophobic region. Substituents are tolerated in the meta
position, while para substituents create steric hindrance.
concentrations on recombinant human GlyRs (G. Mak-
say, unpublished results). Most (nor)tropeines have
positive cooperativity with glycine (â < 1) and they
possess most different binding affinities (KA). However,
the greatest affinities of the tropeines are accompanied
by minor positive cooperativities with glycine.
Experimental Section
Chemistry. The structures of the final products as free
bases were confirmed by 1H and 13C NMR. Spectra were
recorded on a Bruker Avance 250 (250 MHz) spectrometer, in
CDCl3 solutions. Chemical shifts (δ) are expressed in parts per
million (ppm) relative to the internal standard tetramethyl-
silane (TMS). Infrared spectra were obtained on a Perkin-
Elmer 1605 FT-IR spectrometer. The purity of the compounds
was controlled with microanalyses, which were carried out on
a Heraeus Micro Rapid CHN. Melting points were determined
on a Bu¨chi SMP 20 apparatus.
Compounds 2a-d, 2i, 2j, 4a, 7, 8, and 9 are described in
the literature,14,22-27 while 2e-g, 2h, 2k, 4b-e, and 10 are
new. 5-Chloro-2,3-dihydro-2,2-dimethylbenzofuran-7-carboxy-
lic acid was synthesized according to a previously described
procedure.20
General Procedures: Acylation of Tropine. Tropine
(0.01 mol) and 0.011 mol of TEA were dissolved in 15 mL of
toluene, and 0.011 mol of acyl chloride was added dropwise,
at reflux temperature. The solution was then boiled for 3 h,
cooled, and extracted with saturated NaHCO3 solution. The
organic phase was separated, dried with Na2SO4, and evapo-
rated at reduced pressure. The oily residue was crystallized
from appropriate solvent, or it was converted into the hydro-
chloride salt in acetone with ethereal HCl solution. Yield: 40-
55%.
Demethylation of Tropeines. Tropeine (0.005 mol) was
dissolved in 10 mL of toluene, and a catalytic amount of
K2CO3 was added. The solution was boiled and 0.0075 mol of
trichloroethyl chloroformate in 5 mL of toluene was added
dropwise in 20 min while stirring. The mixture was refluxed
for additional 2 h, cooled, and extracted twice with 10% acetic
acid solution. The organic phase was separated, dried over
Na2SO4, and evaporated. The oily material was crystallized
from diisopropyl ether and used without further purification.
Yield: 70-80%.
Apparently full maximal displacement of specific [3H]-
strychnine binding by high concentrations of most
tropeines can also be reconciled with apparently com-
petitive interactions. Accordingly, eqs 2 and 3 resulted
in identical KA values for tropeines with R . 10. This
means that strong negative cooperativity (R . 10)
between the tropeines and [3H]strychnine binding can-
not be distinguished from apparently competitive dis-
placement. At high concentrations the tropeines com-
pete apparently also with glycine bound in the same
cavity as strychnine. Accordingly, atropine, a “Gly
negative” agent, resulted in a parallel rightward shift
of the chloride response curve of glycine for recombinant
GlyRs.7 With increasing concentrations even “Gly posi-
tive” tropeines become “Gly negative” in accordance with
increasing â values in the present binding assays. In
conclusion, while high concentrations of the tropeines
might apparently compete at the orthosteric binding site
of GlyRs, low concentrations of “Gly positive” agents
allosterically potentiate the binding and function of
glycine.
A consensus structure of “Gly positive” tropeines has
emerged from the structure-activity analysis of a
limited number of well-known tropeines.10 This high-
lights the importance of aromatic and tropane rings,
linked via an ester or amide moiety.10 The present data
lead to the development of a pharmacophore model of
tropeine binding to GlyRs, similar to those of 5-HT3
receptor antagonists.21 This is illustrated by Figure 6
showing 4d as a template. The pharmacophore is
composed of (a) an aromatic ring harbored by a lipophilic
region of the binding site, (b) an attached ester group
fixed via hydrogen bonds, and (c) a basic nitrogen of the
tropane ring interacting electrostatically with the recep-
tor. These three attachments form a triangle. Distortion
of this triangle leads to serious deterioration of binding
affinity (potency) and/or positive cooperativity (efficacy).
Removal of the Trichlorocarboethoxy Group. N-
(Trichlorocarboethoxy)nortropeine (0.0047 mol) dissolved in 47
mL of acetic acid was added dropwise to a suspension of 1.46
g (0.022 mol) of zinc dust at room temperature. After being
stirred for 2 h, the mixture was cooled to 10 °C and was made
alkaline by addition of 50 g of NaOH and 15 g of potassium-
sodium tartarate dissolved in 300 mL of water. The solution
was then saturated with K2CO3, and extracted three times
with CH2Cl2. The organic phases were combined, and the

Tropeine Synthesis and Modulation of Glycine Receptors
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25 6389
solution was dried and evaporated to give oily materials that
could be either crystallized or converted into their hydrochlo-
ride salts. Yield: 50-61%.
NMR Data of the New Compounds. 3r-Benzoyl-
oxytropane (2a): mp (hydrochloride) 248 °C; Anal.
(C15H20ClNO2) C, H, N; 1H NMR (CDCl3), δ 7.90 (m, 2H),
7.31-7.60 (m, 3H), 5.33 (t, 1H), 3.82 (bs, 2H), 2.27 (s, 3H),
1.71-2.15 (m, 8H); 13C NMR δ 166.44 (CdO), 133.28, 131.61,
128.59, 128.42 (aromatics), 68.37 (C3), 60.55 (C1,5), 40.58
(N-CH3) 37.33 (C2,4), 26.23 (C6,7).
6H); 13C NMR δ 165.44 (CdO), 158.23, 132.36, 130.47, 129.81,
125.24, 113.65 (aromatics), 89.69 (C2′-O), 66.18 (C3), 60.25
(C1,5), 42.33 (C3′), 40.31 (N-CH3), 33,95 (C2,4), 28.53 (2CH3),
26.09 (C6,7).
3r-Benzoyloxynortropane (4a): mp 232 °C (hydrochlo-
ride); Anal. (C14H18ClNO2) C, H, N; 1H NMR δ 9.28 (br s, 1H),
7.85 (m, 2H), 7.3-7.6 (m, 3H), 5.22 (t, 1H), 3.98 (br s, 2H),
2.48 (m, 2H), 1.97-2.14 (m, 6H); 13C NMR δ 165.27 (CdO),
133.20, 131.42, 128.98, 128.62 (aromatics), 67.28 (C3), 55.02
(C1,5), 34.22 (C2,4), 26,62 (C6,7).
3r-(4′-Ethylbenzoyloxy)tropane (2b): mp 56 °C; Anal.
(C17H23NO2) C, H, N; 1H NMR δ 7.92 (d, 2H), 7.24 (d, 2H),
5.22 (t, 1H), 3.11 (bs, 2H), 2.67 (q, 2H), 2.28 (s, 3H), 1.76-
2.15 (m, 8H), 1.22 (t, 3H); 13C NMR, δ 166.24 (CdO), 149.99,
129.91, 128.60, 128.31 (aromatics), 68.16 (C3), 60.18 (C1,5),
40.85 (N-CH3) 37.12 (C2,4), 29,29 (4′-CH2CH3) 26.17 (C6,7),
15.61 (4′-CH2CH3).
3r-(3′-Methoxybenzoyloxy)nortropane (4b): mp 255 °C
(hydrochloride); Anal. (C15H20ClNO3) C, H, N; 1H NMR δ 9.22
(br s, 1H), 7.68 (m, 3H), 7.38 (m, 1H), 5.24 (t, 1H), 3.94 (s,
3H), 3.85 (br s, 2H), 1.70-2.26 (m, 8H); 13C NMR δ 164.98
(CdO), 159.66, 131.43, 130.51, 121.59, 119.60, 114.28 (aromat-
ics), 66.37 (C3), 55.66 (C1,5), 53,23 (OCH3), 33.34 (C2,4), 26.12
(C6,7).
3r-(4′-Nitrobenzoyloxy)tropane (2c): mp 133 °C; Anal.
3r-(3′-Chlorobenzoyloxy)nortropane (4c): mp 215 °C
(hydrochloride); Anal. (C14H17Cl2NO2) C, H, N; 1H NMR δ 9.60
(br s, 1H), 7.85 (m, 2H), 7.71 (m, 1H), 7.58 (m, 1H), 5.16 (t,
1H), 3.97 (br s, 2H), 1.97-2.56 (m, 8H); 13C NMR δ 164.04
(CdO), 133.88, 133.59, 132.18, 128.91, 128.91, 128.04 (aromat-
ics), 67.20 (C3), 52.91 (C1,5), 33.15 (C2,4), 26.18 (C6,7).
1
(C15H18N2O4) C, H, N; H NMR δ 8.22 (d, 2H), 8.11 (d, 2H),
5.22 (t, 1H), 3.12 (br s, 2H), 2.24 (s, 3H), 1.76-2.18 (m, 8H);
13C NMR δ 164.25 (CdO), 150.73, 136.46, 130.81, 123.94
(aromatics), 69.78 (C3), 59.98 (C1,5), 40.76 (N-CH3) 36.95 (C2,4),
26.13 (C6,7).
3r-(4′-t-Butylbenzoyloxy)tropane (2d): mp 79-80 °C;
3r-(2′,3′-Benzofuran-5′,5′-dimethyl-8′-chloro-6′-carbo-
nyloxo)nortropane (4d): mp 247 °C (hydrochloride); Anal.
(C18H23Cl2NO3) C, H, N; 1H NMR δ 9.68 (br s, 1H), 7.63 (s,
1H), 7.21 (s, 1H), 5.33 (t, 1H), 4.05 (br s, 2H), 2.95 (s, 2H),
2.0-2.7 (m, 8H) 1.46 (s, 6H); 13C NMR δ (ppm) 164.37 (CdO),
158.15, 132.12, 130.37, 129.85, 125.03, 113.57 (aromatics),
89.64 (C2′-O), 66.00 (C3), 54.02 (C1,5), 42.27 (C3′), 34.05 (C2,4),
28.51 (2CH3), 26.38 (C6,7).
N-Benzyl-(3′-nitrobenzoyloxy)nortropane (6). The com-
pound was prepared from N-benzylnortropine (5) and 3-ni-
trobenzoyl chloride according to the standard acylation pro-
cedure described above: mp 105 °C; Anal. (C21H22N2O4) C, H,
N; 1H NMR δ 8.76 (s, 1H), 8.25-8.35 (m, 2H), 7.59 (t, 1H),
7.16-7.33 (m, 5H), 5.27 (t, 1H), 3.51 (s, 2H), 3.18 (br s, 2H),
1.75-2.26 (m, 8H); 13C NMR δ 164.18 (CdO), 148.74, 139.82,
135.42, 132.95, 130.14, 128.99, 128.68, 127.68, 127.37, 124.80
(aromatics), 70.58 (C3), 58.19 (C1,5), 57.13 (N-CH2), 37.38 (C2,4),
26.66 (C6,7).
3r-(3′-Aminobenzoyloxy)nortropane (4e). Compound 6
(0.005 mol, 1.83 g) was hydrogenated in 20 mL of ethanol in
the presence of palladium catalyst on charcoal (10%) at
atmospheric pressure and room temperature. After consump-
tion of the calculated amount of hydrogen the catalyst was
filtered, and the solvent was evaporated to yield 1.80 g of 4 as
an oil, which was converted into its hydrochloride: mp 256
°C; Anal. (C14H20Cl2N2O2) C, H, N; 1H NMR δ 9.55 (br s, 1H),
7.13-7.37 (m, 3H), 6.80 (m, 1H), 5.23 (t, 1H), 3.78 (br s, 2H),
3.50 (br s, 2H), 1.78-2.15 (m, 8H); 13C NMR δ 164.21 (CdO),
144.72, 13.01, 127.528, 117.82, 117.46, 113.78 (aromatics),
66.75 (C3), 52.53 (C1,5), 35.67 (C2,4), 27.56 (C6,7).
3â-Benzoyloxytropane (7): mp 52 °C; Anal. (C15H19NO2)
C, H, N; 1H NMR δ 7.93 (m, 2H), 7.30-7.52 (m, 3H), 5.17 (m,
1H), 3.18 (br s, 2H), 2.28 (s, 3H), 1.6-2.1 (m, 8H); 13C NMR δ
166.45 (CdO), 133.17, 130.90, 129.02, 128.63 (aromatics), 68.16
(C3), 60.65 (C1,5), 39.16 (N-CH3), 36.16 (C2,4), 26,89 (C6,7).
1
Anal. (C19H27NO2) C, H, N; H NMR δ 7.89 (d, 2H), 7.40 (d,
2H), 5.18 (t, 1H), 3.08 (br s, 2H), 2.24 (s, 3H), 1.72-2.21 (mm,
8H), 1.27 (s, 9H); 13C NMR δ 166.20 (CdO), 156.83, 129.68,
128.60, 125.78 (aromatics), 68.11 (C3), 60.23 (C1,5), 40.86 (N-
CH3), 37.14 (C2,4), 35.43 [4′-C(CH3)3], 31.49 [4′-C(CH3)3], 26.18
(C6,7).
3r-(3′-Methoxybenzoyloxy)tropane (2e): mp 113 °C;
Anal. (C16H21NO3) C, H, N; 1H NMR δ 7.61-7.69 (m, 3H), 7.40
(m, 1H), 5.33 (t, 1H), 3.94 (s, 3H), 3.25 (br s, 2H), 2.27 (s, 3H),
1.74-2.25 (m, 8H); 13C NMR δ 165.38 (CdO), 159.90, 131.21,
130.03, 121.64, 119.62, 114.59 (aromatics), 65.50 (C3), 62.41
(C1,5), 55.69 (OCH3), 39.47 (N-CH3), 34.85 (C2,4), 24.75 (C6,7).
3r-(3′-Chlorobenzoyloxy)tropane (2f): mp 71 °C; Anal.
1
(C15H18ClNO2) C, H, N; H NMR δ 7.90 (t, 1H), 7.82 (m, 1H),
7.45 (m, 1H), 7.32 (t, 1H), 5.17 (t, 1H), 3.11 (br s, 2H), 2.25 (s,
3H), 1.74-2.21 (m, 8H); 13C NMR δ 164.99 (CdO), 134.95,
133.23, 132.90, 130.14, 129.87, 127.84 (aromatics), 69.02 (C3),
60.13 (C1,5), 40.74 (N-CH3), 36.94 (C2,4), 26.15 (C6,7).
3r-(3′-Nitrobenzoyloxy)tropane (2g): mp 204 °C; Anal.
1
(C15H18N2O4) C, H, N; H NMR δ 8.74 (s, 1H), 8.34 (m, 2H),
7.55 (t, 1H) 5.24 (t, 1H), 3.16 (br s, 2H), 2.26 (s, 3H), 1.74-
2.24 (mm, 8H); 13C NMR δ 164.35 (CdO), 148.72, 135.23,
132.92, 130.25, 12.90, 127.68 (aromatics), 69.90 (C3), 58.90
(C1,5), 40.65 (N-CH3), 37.45 (C2,4), 26.40 (C6,7).
3r-[3′-(Trifluoromethyl)benzoyloxy]tropane (2h): mp
1
71 °C; Anal. (C16H18F3NO2) C, H, N; H NMR δ 8.21 (s, 1H),
8.13 (d, 1H), 7.74 (d, 1H), 7.53 (t, 1H), 5.22 (t, 1H), 3.12 (br s,
2H), 2.25 (s, 3H), 1.76-2.24 (m, 8H); 13C NMR δ (ppm) 164.86
(CdO), 132.86, 132.04 (aromatics), 131.6 (C-CF3, q, 2JC,F ) 81
Hz), 129.7, (q, 3JC,F ) 3.6 Hz), 129.6 (aromatic), 126.7 (q, 3JC,F
1
) 3.6 Hz), 124.05 (CF3,q, JC,F ) 275 Hz), 69.02 (C3), 60.13
(C1,5), 40.74 (N-CH3), 36.94 (C2,4), 26.15 (C6,7).
3r-(3′,4′,5′-Trimethoxybenzoyloxy)tropane (2i): mp 116
°C; Anal. (C18H25NO5) C, H, N; 1H NMR δ 7.24 (s, 2H), 5.17 (t,
1H), 3.84 (s, 9H), 3.10 (br s, 2H), 2.24 (s, 3H), 1.73-2.20 (m,
8H); 13C NMR δ 165.62 (CdO), 153.27, 142.35, 126.17, 107.12
(aromatics), 68.47 (C3), 61.21 (OCH3), 60.16 (C1,5), 56.45
(OCH3), 40.82 (N-CH3), 37.04 (C2,4), 26.06 (C6,7).
3r-(3′,5′-Dichlorobenzoyloxy)tropane (2j): mp 165 °C;
Anal. (C15H17Cl2NO2) C, H, N; 1H NMR δ 7.87 (s, 2H), 7.46 (s,
1H), 5.21 (t, 1H), 3.19 (br s, 2H), 2.27 (s, 3H), 1.75-2.22 (m,
8H); 13C NMR δ 164.88 (CdO), 135.14, 133.91, 133.32, 128.37
(aromatics), 69.25 (C3), 60.23 (C1,5), 40.94 (N-CH3) 36.90 (C2,4),
26.25 (C6,7).
3â-Benzoyloxynortropane (8): mp 252 °C (hydrochlo-
ride); Anal. (C14H18ClNO2) C, H, N; 1H NMR δ 9,32 (br s, 1H),
7.90 (m, 2H), 7.35-7.60 (m, 3H), 5.12 (m, 1H), 3.88 (br s, 2H),
1.97-2.34 (m, 8H); 13C NMR δ 165.88 (CdO), 133.41, 131.82,
129.07, 128.75 (aromatics), 68.19 (C3), 55.45 (C1,5), 35.21 (C2,4),
27,62 (C6,7).
N-Ethyl-3â-benzoyloxynortropane (9): mp 55 °C; Anal.
1
(C16H21NO2) C, H, N; H NMR δ 7.97 (m, 2H), 7.32-7.49 (m,
3H), 5.25 (m, 1H), 3.34 (br s, 2H), 2.48 (q, 2H), 1.65-2.01 (m,
8H), 1.08 (t, 3H); 13C NMR δ 166.37 (CdO), 133.10, 131.01,
129.84, 128.61 (aromatics), 68.58 (C3), 57.92 (C1,5), 44.41 (N-
CH2), 35.53 (C2,4), 27,24 (C6,7), 14.27 (N-CH2CH3).
3r-(2′,3′-Benzofuran-5′,5′-dimethyl-8′-chloro-6′-carbo-
nyloxo)tropane (2k): mp 141-142 °C; Anal. (C19H24ClNO3)
C, H, N; 1H NMR δ 7.65 (s, 1H), 7.28 (s, 1H), 5.22 (t, 1H), 3.95
(br s, 2H), 2.98 (s, 2H), 2.27 (s, 3H), 1.0-2.7 (m, 8H), 1.43 (s,
3r-Benzoylamidotropane (10): mp 138 °C; Anal.
1
(C15H20N2O) C, H, N; H NMR δ 7.62 (m, 2H), 7.30 (m, 3H),

6390 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25
Maksay et al.
6.51 (d, 1H), 4.13 (q, 1H), 3.10 (br s, 2H), 2.22 (s, 3H), 1.65-
2.22 (m, 8H); 13C NMR δ 166.83 (CdO), 135.31, 131.67, 128.95,
127.01 (aromatics), 60.35 (C1,5), 42.20 (C3), 40.63 (N-CH3),
36.71 (C2,4), 26.17 (C6,7).
(KA) were also calculated according to the Cheng-Prusoff
equation:
IC50
KA )
(3)
Receptor Binding: GlyRs. Synaptosomal membranes
were prepared from rat spinal cord as described.10 Briefly,
spinal cords of male Wistar rats were homogenized in 5 mM
Tris-HCl buffer (pH 7.4) by Ultra-Turrax for 2 × 10 s. The
homogenates were centrifuged at 30000g for 20 min, and the
pellets were suspended in 50 mM Tris-HCl buffer (pH 7.4),
washed by four similar centrifugations, and frozen. Before the
binding assay, the thawed suspensions were centrifuged in 50
mM Tris-HCl buffer containing 200 mM KSCN (pH 7.4) at
10000g for 10 min. Membrane suspensions (about 0.1 mg of
protein/mL) in 50 mM Tris-HCl buffer containing 200 mM
KSCN were incubated with 3 nM [3H]strychnine (10 µCi/mmol,
DuPont-NEN) for 40-50 min at 0 °C. Different concentrations
of the tropeines were coincubated to measure cooperativity
with [3H]strychnine binding (R). Different concentrations of
glycine were also incubated in the presence and absence of
tropeine concentrations at 0.1KA to measure cooperativity with
glycine (â). Nonspecific binding was determined in the pres-
ence of 2 mM glycine. Duplicate aliquots were filtered on
Whatman GF/B filters under vacuum.
1 + [S]/KS
where IC50 values represent 50% displacement of [3H]strych-
nine binding by the tropeines.
Displacement of [3H]granisetron binding was fitted via
GraphPad Prism 4 to get IC50 and slope values (nH) of
displacement.
Acknowledgment. This study was supported by
Grant NKFP (MediChem 2). T.B. has been supported
by the QLK2-CT-2002-90436 EU project for Center of
Excellence in Biomolecular Chemistry. Contribution to
the pharmacophore drawing is acknowledged to Dr.
Zsolt Bika´di (CRC-HAS, Budapest). Our thanks are due
to Mr. Zolta´n Vincze for registration of the NMR spectra.
Supporting Information Available: Results from el-
emental analysis. This material is available free of charge via
the Internet at http://pubs.acs.org.
5-HT3 Type Serotonin Receptors. Synaptosomal mem-
branes were prepared from cerebral cortex plus hippocampus
of male Wistar rats as described.28 Briefly, tissues were
homogenized in 10 mM HEPES buffer containing 140 mM
NaCl (pH 7.5) in an Ultra-Turrax for 2 × 10 s, centrifuged at
30000g for 20 min, washed by centrifugation at 30000g for 10
min, and frozen. Before the binding assay, the thawed suspen-
sions were centrifuged in 10 mM HEPES buffer containing
140 mM NaCl (pH 7.5) at 10000g for 10 min, incubated with
0.3 nM [3H]granisetron (85 µCi/mmol [3H]BRL 43694, Du-
Pont-NEN) for 2.5 h on ice. Different concentrations of the
tropeines were applied to measure their displacing potencies
and 10 µM granisetron was used for nonspecific binding.
Duplicate aliquots were filtered under vacuum on Whatman
GF/B filters presoaked in 0.1% poly(ethylenimine) solution.
Data Analysis. Nonlinear statistical regression program
NLREG (PH Sherrod, Nashville, TN) and GraphPad Prism
Version 4 (San Diego, CA) were used for fitting. The ternary
allosteric model applied was described previously.12 It contains
three dissociation constants (KS, KL, and KA) for the binding
of [3H]strychnine (S), glycine (L), and the allosteric agents (A)
as well as the cooperativity factors R and â. The cooperativity
factors greater or smaller than unity increase or decrease,
respectively, the dissociation of the ligands. Thus, R and â
values greater than unity represent negative cooperativity of
the allosteric agents with [3H]strychnine and glycine binding,
respectively. Equation 1 expresses the ratio of specific [3H]-
strychnine binding in the presence of three ligands (BSAL) over
control, in the presence of [3H]strychnine (BS):
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JM040814G