Systematic development of high affinity bis(ammonio)alkane-type allosteric enhancers of muscarinic ligand binding

Article abstract of DOI:10.1021/jm021017q

Bis(ammonio)alkane compounds carrying lateral phthalimidopropyl substituents on the nitrogen atoms belong to the archetypal muscarinic allosteric agents. Herein, a series of symmetrical and nonsymmetrical compounds was synthesized in which the phthalimide residues were replaced by differently substituted imide moieties. The allosteric action was measured in porcine heart muscarinic M₂ receptors using [³H]N-methylscopolamine (NMS) as a ligand for the orthosteric receptor site in equilibrium binding and dissociation experiments. 1,8-Naphthalimido residues conferred an up to 100-fold gain in affinity leading into the low nanomolar range, while the inhibition of NMS binding was maintained. Additional propyl chain methylation was accompanied by an allosteric elevation of orthosteric ligand binding. In general, the gain in allosteric activity achieved by ring variation plus propyl chain methylation on one side of the molecule could not be augmented by symmetrical variations. The elevation of the ligand binding can be explained by different quantitative structure - activity relationships for the affinities to the free and the orthoster-liganded receptor.

Full text of DOI:10.1021/jm021017q

J . Med. Chem. 2003, 46, 1031-1040
1031
System a tic Develop m en t of High Affin ity Bis(a m m on io)a lk a n e-typ e Alloster ic
En h a n cer s of Mu sca r in ic Liga n d Bin d in g
Mathias Muth,† Wiebke Bender,† Olaf Scharfenstein,† Ulrike Holzgrabe,†,* Edith Balatkova,‡
Christian Tra¨nkle,‡ and Klaus Mohr‡
Institute of Pharmacy and Food Chemistry, University of Wu¨rzburg, Am Hubland, D-97074 Wu¨rzburg,
and Pharmacology and Toxicology, Institute of Pharmacy, University of Bonn,
An der Immenburg 4, 53121 Bonn, Federal Republic of Germany
Received August 2, 2002
Bis(ammonio)alkane compounds carrying lateral phthalimidopropyl substituents on the nitrogen
atoms belong to the archetypal muscarinic allosteric agents. Herein, a series of symmetrical
and nonsymmetrical compounds was synthesized in which the phthalimide residues were
replaced by differently substituted imide moieties. The allosteric action was measured in porcine
heart muscarinic M2 receptors using [3H]N-methylscopolamine (NMS) as a ligand for the
orthosteric receptor site in equilibrium binding and dissociation experiments. 1,8-Naphthalimido
residues conferred an up to 100-fold gain in affinity leading into the low nanomolar range,
while the inhibition of NMS binding was maintained. Additional propyl chain methylation
was accompanied by an allosteric elevation of orthosteric ligand binding. In general, the gain
in allosteric activity achieved by ring variation plus propyl chain methylation on one side of
the molecule could not be augmented by symmetrical variations. The elevation of the ligand
binding can be explained by different quantitative structure-activity relationships for the
affinities to the free and the orthoster-liganded receptor.
In tr od u ction
radioligand used as a probe, the alloster binding con-
stant for the orthoster-occupied receptor depends on the
type of orthosteric ligand. Often, [3H]N-methylscopola-
mine ([3H]NMS) has been used by others and by us as
the orthosteric radioligand, but structure-activity-
relationships for cooperative interactions with this
orthosteric probe have not yet been unravelled.
Many allosteric modulators of the muscarinic receptor
protein influencing the binding of the antagonist [3H]-
NMS are structurally symmetrical, for example alcu-
ronium,14 caracurine V derivatives,15 tubocurarine,16,17
methoctramine,18 obidoxime,11 and a wide range of the
bis(ammonio)alkane derivatives, such as W84,19 and its
congeners dimethyl-W84,20 CHIN 3/6,21 and IWDUO22
(see Scheme 1).
Within the series of symmetrical compounds having
an bis(ammonio)alkane middle chain, the lateral moi-
eties, mostly aromatic imides, were found to govern the
allosteric potency, i.e., the retardation of [3H]NMS-
dissociation from M2 muscarinic receptors. In the fol-
lowing, the term allosteric potency refers to the experi-
mentally observed effects of an allosteric agent on
orthosteric ligand binding; the term allosteric affinity
is used when effects are interpreted in terms of binding
affinities.
Variation of the lateral moieties resulted in the
following structure-activity relationships: saturation
of the aromatic imide and shortening of the imide at
both ends of the molecule resulted in a substantial
decrease in potency.23 Replacement of one carbonyl of
the imide with alkoxy, benzyl, or benzylidene groups,
again at both ends revealed the allosteric potency to be
dependent on a high polarizibility and the presence of
a sp2 hybridized carbon atom, and along with this,
rigidity in this position.24 The variation of the aromatic
Allosteric modulators of receptors are capable of
enhancing or decreasing ligand binding to receptors
which offers novel therapeutic perspectives.1,2 Intensive
efforts were and still are directed at the allosteric
modulation of muscarinic receptors opening new con-
cepts for the therapy of organophosphate poisoning,
pain, or Alzheimer’s disease.3-8
The effect on ligand binding depends on the type of
allosteric modulator, the type of ligand, and the subtype
of muscarinic receptor. To guide a rational design of
appropriate allosteric agents, structure-activity rela-
tionships have been elucidated. Often, the allosteric
inhibition of the dissociation of the orthosteric ligand
serves to indicate occupancy of the allosteric site.9-11
In addition it is of high interest to know whether the
allosteric agents elevate or reduce orthosteric ligand
equilibrium binding or leave it unchanged. According
to the ternary complex model of allosteric interactions12
as formulated by Ehlert,13 the cooperativity factor R is
the alloster binding constant for the interaction with
the orthoster (agonist or antagonist) occupied receptor
divided by the alloster binding constant for the free
receptor. Thus, R < 1, R ) 1, and R > 1 indicate a
positive, neutral, or negative cooperativity, respectively,
i.e., an enhanced, unchanged, or diminished equilibrium
binding of the orthosteric ligand in the presence of the
allosteric agent.
Whereas the alloster binding constant for the free
muscarinic receptor is independent of the orthosteric
* Corresponding author. Tel. +49 931-888-5460. Fax +49 931-888-
5494. E-mail: holzgrab@pharmazie.uni-wuerzburg.de.
† University of Wu¨rzburg.
‡ University of Bonn.
10.1021/jm021017q CCC: $25.00 © 2003 American Chemical Society
Published on Web 02/07/2003

1032 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6
Muth et al.
ivity with [3H]N-methylscopolamine. However, in
phthalimidopropyl-substituted bis(ammonio)alkane se-
ries we recently discovered induction of positive coop-
erativity by methylation of the lateral propyl chains28
or by replacement of one quaternary nitrogen by a
silicon atom.29
Sch em e 1. Structural Formulas of Various Allosteric
Modulators
To study structure-activity relationships (SAR) within
the bis(ammonio)alkane series in detail and in an
attempt to tailor a highly potent and positive coopera-
tive muscarinic allosteric modulator, we set out to
synthesize and to test symmetrical and nonsymmetrical
novel compounds 2d , 3b-d , 5a -d , 6a , 6b, 6d , 7, 9, 14-
20 carrying on charged nitrogens various substituted
and unsubstituted aromatic imidopropyl moieties. In
addition, also some already known compounds 1a , 1b,
1d , 2a -c, 3a , 4a , 4b, 4d , 8, 10-13 were synthesized to
be included in the study.
The methylation of the propyl chain was made on the
most active compounds and the lead compound W84
with the aim to induce positive cooperativity with [3H]-
NMS. SARs will be derived from the observed effects
concerning the affinities of the allosteric agents to
interact with free and NMS liganded muscarinic M2
receptors, respectively, because these affinities underly
the extent and direction of cooperativity with the
orthosteric ligand.
Ch em istr y
To synthesize the series of symmetrical bis(ammonio)-
alkane compounds 14-20 and the nonsymmetrical
analogues 7-13 the commercially nonavailable starting
anhydrides were synthesized by conversion of the 1,2-
dicarboxylic acids by acetic anhydride according to
procedures described in the literature.30-33 The phthal-
imido-like propylamine derivatives 21-27, 29, and 31-
32, and 1,2- and 1,8-naphthalimidopropylamines 28, 30,
and 33 were obtained by refluxing, in toluene and
catalytic amount of p-toluenesulfonic acid, equimolar
amounts of the phthalic-like anhydrides and the naph-
thalic anhydrides, respectively, with the corresponding
N1,N1-dimethylpropane-1,3-diamines, using a water
separator (Scheme 2). To obtain the nonsymmetric W84-
derivatives, the phthalimidopropylamines 22, 25, and
31-33, respectively, were stirred with 1,6-dibromohex-
ane for several days at room temperature and 50 °C,
respectively, to give the alkylated compounds 34-38
(Scheme 2). By using a huge excess of the alkylating
agent a double amination of dibromohexane could be
avoided.25 Next, equimolar amounts of 21-33 and 34-
38 were reacted by refluxing in acetonitrile for several
days to give the nonsymmetrical compounds 1b, 2a -d ,
3a -d , 4b, 5a -d , 6b, 7-13 in varying yields (Scheme
2). The symmetrical bis(ammonio)alkane-type com-
pounds 1a , 1d , 4a , 4d , 6a , 6d , and 14-20 were obtained
by refluxing 2 mol of 21-33 and 1 mol of 1,6-dibromo-
hexane in acetonitrile for several days (Scheme 2). The
identities were established by one- and two-dimensional
1H and 13C NMR spectroscopic experiments (see Tables
6-8 in Supporting Information).
imide moiety within a unilaterally varied series showed
the dependence of the potency on the volume of this
skeleton.25 Till now only one comparison of a corre-
sponding contralateral variation of the imides, i.e., a
comparison of symmetrical versus nonsymmetrical com-
pounds, has been performed, actually within the WDUO
series26,27 which is derived from the acetylcholinesterase
reactivators obidoxime and TMB-4. The agents carry
phthalimido substituents at both ends of the bispyri-
dinium middle chain, which were stepwise replaced with
dichlorobenzyl residues. The bisphthalimido compound
WDUO (see Scheme 1) reduces the dissociation of [3H]-
NMS from the receptor protein half-maximally at an
EC50 value of 0.7 µM. Replacement of one phthalimido
residue with a dichlorobenzyl substituent increases the
EC50 value to 1.3 µM and the replacement of both
phthalimido substituents further elevates the EC50
value to 4.7 µM. This finding indicates a superiority of
the symmetrical imide compounds in this series. Yet,
none of the aforementioned bis(ammonio)alkane-type
compounds have shown considerable positive cooperat-
P h a r m a cology
Binding experiments were carried out in homogenates
of porcine heart ventricles containing muscarinic ace-
tylcholine receptors of the M2 subtype. The applied

Allosteric Enhancers of Muscarinic Ligand Binding
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6 1033
Sch em e 2. Synthesis Pathwaya
a
The detailed substitution pattern of the compounds 1-20 is given in Table 1 and Table 2, respectively.
buffer (2.7 mM MgHPO4, 45 mM TrisHCl, 37 °C, pH
7.3) yields allosteric activities that are similar to those
found under organ bath conditions.15,34 We used the
orthosteric antagonist [3H]N-methylscopolamine ([3H]-
NMS) as a probe to measure the interaction of the test
compounds with the allosteric site of the M2 receptors.
[3H]NMS-dissociation experiments revealed the allos-
teric stabilization of the [3H]NMS-receptor complexes
and allowed computation of the incubation time that is
required for the equilibrium binding experiments with
[3H]NMS in the presence of the allosteric agents.
connection with the substitution pattern are sum-
marized in Tables 1 and 2.
Figure 1 illustrates essential aspects of the present
study. The effect of three test compounds on [3H]NMS
equilibrium binding is shown. The parent compound 1a
reduced [3H]NMS binding as was expected from previ-
ous studies (e.g., ref 28). Analysis of the curve according
to the ternary complex model of allosteric interactions
yields the affinity value pKA for the binding of 1a to
free M2 receptors, in which the orthosteric site is not
occupied by [3H]NMS (Table 2). Furthermore, the factor
R of cooperativity between 1a and [3H]NMS is derived
from the curve. In Table 2 we indicate the minus log
value of R, because R is log-normally distributed35 and
because the minus sign indicates the direction of coop-
erativity. The value p(RKA) reflects the binding affinity
of 1a for [3H]NMS-occupied M2 receptors. This affinity
Resu lts a n d Discu ssion
All compounds synthesized were subjected to the
pharmacological evaluation. The pEC50,diss values, p(RKA)
values, pKA values, and the cooperativity factors pR in

1034 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6
Muth et al.
Ta ble 1. Substitution Pattern and Parameters Characterizing the Allosteric Interactions of the Compounds 1a , 2a , 3a , 4a , 6a , and
7-20 with the Binding of [3H]N-Methylscopolamine at Porcine Heart M2 Receptors (mean values ( SEM from 3-12 independent
experiments; for details, see text)
nonsym
pR
6.25 ( 0.18 -0.59 ( 0.03 5.66 ( 0.21 5.79 ( 0.05 14 5.44 ( 0.16c
sym
substituent Ar
pyr
no.
pKA
p(RKA)
pEC50,diss
no.
pKA
pR
p(RKA)
pEC50,diss
7
-
8
9
-0.44 ( 0.02 5.00 ( 0.19 5.20 ( 0.12c
-0,47 ( 0.06 5.73 ( 0.16 5.87 ( 0.06
-0.39 ( 0.09 6.08 ( 0.21 6.13 ( 0.10
-0.22 ( 0.05 6.15 ( 0.12 6.35 ( 0.12
phth
-
-
-
-
1a 6.19 ( 0.13
5,6-difluoro-phth
5-CF3-phth
5-mephth
tert.-butyl-phth
Diels-ald
6.68 ( 0.18 -0.37 ( 0.02 6.31 ( 0.18 6.34 ( 0.06b 15 6.47 ( 0.19
6.32 ( 0.08 -0.21 ( 0.06 6.12 ( 0.09 6.30 ( 0.10 16 6.37 ( 0.09
2a 6.73 ( 0.08 -0.31 ( 0.02 6.42 ( 0.09 6.49 ( 0.04b 4a 7.08 ( 0.10a,c -0.33a
6.75a
6.77 ( 0.03a,c
10 7.25 ( 0.05 -0.81 ( 0.04 6.43 ( 0.02 6.51 ( 0.03b 17 7.38 ( 0.04
11 7.78 ( 0.07 -1.21 ( 0.04 6.56 ( 0.08 6.69 ( 0.12 18 8.12 ( 0.08c
12 7.27 ( 0.12 -0.47 ( 0.04 6.80 ( 0.16 6.84 ( 0.08b 19 7.62 ( 0.09
-0.89 ( 0.02 6.49 ( 0.51 6.52 ( 0.08
-1.38 ( 0.03 6.74 ( 0.09 6.40 ( 0.05
-0.85 ( 0.01 6.77 ( 0.10 6.69 ( 0.10
-0,29 ( 0.06 6.57 ( 0.08 6.64 ( 0.07c
-0.28 ( 0.03 7.83 ( 0.11c 7.86 ( 0.07c
1,2-naphth
5,6-dichloro-phth 13 7.08 ( 0.18 -0.17 ( 0.04 6.90 ( 0.13 6.94 ( 0.05b 20 6.83 ( 0.11
1,8-naphth
3a 7.24 ( 0.13 -0.20 ( 0.05 7.04 ( 0.12 6.94 ( 0.03 6a 8.11 ( 0.13c
a
b
Data taken from ref 28. Data taken from ref 25. c Significantly different from the parameter value of the nonsymmetrical compound,
p < 0.05.
Ta ble 2. Substitution Pattern and Parameters Characterizing the Allosteric Interactions of the Compounds 1-6 with the Binding of
[3H]N-Methylscopolamine at Porcine Heart M2 Receptors (mean values ( SEM from 3-12 independent experiments; for details,
see text)
no.
Ar1
X1
H
X2
H
Ar2
pKA
pR
p(RKA)
pEC50,diss
1a
phth
phth
6.19 ( 0.13
n ) 4
-0.47 ( 0.06c
n ) 4
5.73 ( 0.16
n ) 4
5.87 ( 0.06
n ) 4
1b
1d
2a
2b
2c
2d
4a
4b
4d
3a
3b
3c
3d
5a
5b
5c
5d
6a
6b
6d
phth
CH3
CH3
H
H
CH3
H
phth
phth
6.90 ( 0.03a
n ) 3
-0.04 ( 0.03a
n ) 3
6.86 ( 0.01a
n ) 3
6.87 ( 0.04a
n ) 3
phth
7.08 ( 0.09a
n ) 3
-0.21 ( 0.02a,c
n ) 3
6.87 ( 0.08a
n ) 3
6.75 ( 0.04a
n ) 3
5-mephth
5-mephth
5-mephth
5-mephth
5-mephth
5-mephth
5-mephth
1,8-naphth
1,8-naphth
1,8-naphth
1,8-naphth
1,8-naphth
1,8-naphth
1,8-naphth
1,8-naphth
1,8-naphth
1,8-naphth
1,8-naphth
phth
6.73 ( 0.08
n ) 3
-0.31 ( 0.02c
n ) 3
6.42 ( 0.09
n ) 3
6.49 ( 0.04
n ) 3
CH3
H
H
phth
7.26 ( 0.03a
n ) 3
0.19 ( 0.02a,b
n ) 3
7.45 ( 0.03a
n ) 3
7.26 ( 0.02a
n ) 3
CH3
CH3
H
phth
7.20 ( 0.22a
n ) 3
-0.13 ( 0.02a,b
n ) 3
7.07 ( 0.24a
n ) 3
7.15 ( 0.11a
n ) 3
CH3
H
phth
6.73 ( 0.24
n ) 4
0.17 ( 0.02c
n ) 4
6.90 ( 0.23
n ) 4
6.92 ( 0.08
n ) 3
5-mephth
5-mephth
5-mephth
phth
7.08 ( 0.10a
n ) 3
-0.33a
6.75a
6.77 ( 0.03a
n ) 2-5
7.44 ( 0.06a
n ) 3
n ) 3
n ) 3
CH3
CH3
H
H
7.41 ( 0.02a
n ) 3
0.03 ( 0.02a
n ) 3
7.44 ( 0.0001a
n ) 3
CH3
H
7.21 ( 0.11a
n ) 3
0.17 ( 0.02a,b
n ) 3
7.38 ( 0.09a
n ) 3
7.33 ( 0.10a
n ) 3
7.24 ( 0.13
n ) 4
-0.20 ( 0.05b
n ) 4
7.04 ( 0.12
n ) 4
6.94 ( 0.30
n ) 6
CH3
H
H
phth
8.29 ( 0.18
n ) 8
0.37 ( 0.08c
n ) 8
8.66 ( 0.14
n ) 8
8.36 ( 0.09
n ) 11
CH3
CH3
H
phth
7.28 ( 0.002
n ) 3
-0.005 ( 0.004
n ) 3
7.28 ( 0.00
n ) 3
7.28 ( 0.08
n ) 4
CH3
H
phth
7.41 ( 0.09
n ) 5
0.56 ( 0.08c
n ) 5
7.97 ( 0.10
n ) 5
7.73 ( 0.09
n ) 5
5-mephth
5-mephth
5-mephth
5-mephth
1,8-naphth
1,8-naphth
1,8-naphth
8.01 ( 0.12
n ) 3
-0.22 ( 0.03b
n ) 3
7.79 ( 0.10
n ) 3
7.60 ( 0.05
n ) 3
CH3
H
H
7.90 ( 0.15
n ) 3
0.32 ( 0.07b
n ) 3
8.23 ( 0.13
n ) 3
8.12 ( 0.10
n ) 7
CH3
CH3
H
7.48 ( 0.02
n ) 3
-0.04 ( 0.02
n ) 3
7.44 ( 0.002
n ) 3
7.44 ( 0.07
n ) 3
CH3
H
7.90 ( 0.16
n ) 3
0.29 ( 0.06b
n ) 3
8.19 ( 0.11
n ) 3
7.85 ( 0.09
n ) 4
8.11 ( 0.13
n ) 3
-0.28 ( 0.03b
n ) 3
7.83 ( 0.11
n ) 3
7.86 ( 0.07
n ) 12
CH3
CH3
H
8.07 ( 0.06
n ) 3
0.17 ( 0.02b
n ) 3
8.24 ( 0.08
n ) 3
8.11 ( 0.10
n ) 5
CH3
7.51 ( 0.08
n ) 3
0.53 ( 0.06b
n ) 3
8.04 ( 0.07
n ) 3
8.07 ( 0.10
n ) 4
a
c
Data taken from ref 28. b,c Cooperativity is significantly different from neutral (pR different from zero, bp < 0.05, p < 0.01).
value can also be derived from the separate [3H]NMS-
dissociation experiments as pEC50,diss; EC50,diss denotes
the concentration of the allosteric agent at which [3H]-
NMS-dissociation is half-maximally retarded. For all
compounds, the respective values for p(RKA) and pEC50,diss
were equal, thus supporting the validity of the ternary

Allosteric Enhancers of Muscarinic Ligand Binding
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6 1035
and the volume of the lateral moiety. The data set has
now slightly changed: the pyridinimide compound was
replaced with pyrazinimide derivatives (7, 14) and
compounds substituted with a trifluoromethyl group in
position 5 (9, 16) were added. In analogy to the previous
study25 the lipophilicity (log P1, log P2), the volume (Vol1,
Vol2), the surface, the polarizibility (Pol), and the
refractivity (Ref) of the corresponding N-methylimides
were calculated for both lateral imide moieties sepa-
rately using the Hyperchem/Chemplus software pack-
age, and the quantatitive structure-activity relation-
ships (QSAR) of the series of symmetrical and non-
symmetrical compounds were analyzed. The best cor-
relations between the physicochemical parameters and
the affinities to the free and the NMS-occupied receptors
are displayed in Table 3 using the general equation
F igu r e 1. Effects of the indicated test compounds on the
equilibrium binding of [3H]N-methylscopolamine ([3H]NMS)
to porcine heart M2 receptors. Ordinate: specific [3H]NMS
binding in percent of the control value in the absence of test
compound as indicated by the starting value of the binding
curve. Abscissa: concentration of the test compound. Indicated
are mean values ( SEM, n ) 4-8 independent experiments.
Error bars are not visible when smaller than the symbols.
Curve fitting according to the ternary complex model of
allosteric interactions. For details, see text.
pBE ) â0 + âVol·xVol1 + âVol·xVol2 + âlogP1·xlogP1
+
âPol·xPol + âRef·xRef
pBE is the estimated negative logarithm of the corre-
sponding biological effect. R2 is the square of the
correlation coefficient, s the standard deviation, F the
ratio of explained to unexplained variance, Q2 is the
cross validated R2 by using the leave-one-out procedure
(Q2 can adopt values between 1 and 0), and sPRESS the
standard deviation from the predictive residual sum of
squares. Only equations with significant correlation
coefficients (â) are considered in the following discus-
sion. Interestingly, the correlations found with the
affinities for the free and the NMS-occupied receptors
are different from each other. The affinity for the free
receptor can be described using one parameter, the
polarizibility. Adding the lipophilicity reveals a slightly
weaker but nonsignificant correlation. In contrast, the
correlations found with the affinities to the NMS-
occupied receptor are much weaker. Using three pa-
rameters, i.e., the volume of one lateral moiety, the
lipophilicity, and the polarizibility, a significant cor-
relation for p(RKA) was found, but it was still weak.
Using a parabolic model for the regression analysis37
did not improve the situation. However, the divergent
correlations clearly indicate that the binding sites of the
free and NMS-liganded receptor are structurally differ-
ent and the affinity of this series of compounds for the
free M2 receptor is always higher than for the NMS-
liganded receptor, thus leading to a negative cooperat-
ivity.
The 21 compounds carrying either phthalimides,
methylphthalimides, or naphthalimides at the ends and
having a systematic introduction of methyl groups in
the propyl chains will be considered in the following.
Table 2 clearly shows that the introduction of one
methyl group always increases the p(RKA) value in
comparison to the nonalkyl substituted compounds 1a ,
2a , 3a , 4a , 5a , and 6a , with the exception of compound
5c. However, in the series of nonsymmetrical com-
pounds the extent by which the affinity to the NMS-
occupied receptor (Table 2) surpasses the affinity to the
free receptor depends sensitively on the locus of methyl-
ation: if the 2,2-dimethylpropyl chain is neighbored
upon the imide which induces a higher affinity to the
occupied receptor in comparison to the affinity to the
free receptor the cooperativity is positive, and vice versa,
if the 2,2-dimethylpropyl chain is neighbored upon the
complex model as a quantitative mechanism underlying
the experimental findings. The molecular basis for the
negative cooperativity between 1a and [3H]NMS is that
1a has a higher affinity for free receptors, pKA ) 6.19,
than for [3H]NMS-occupied receptors, p(RKA) ) 5.73.
Compound 3a , in which one of the phthalimide
residues is replaced by naphthalimide, has a 10-fold
higher affinity for free M2 receptors than 1a (Table 2).
The cooperativity with [3H]NMS is negative, i.e., pKA
> p(RKA), cf. Table 2. Propyl-chain methylation in 3a
next to the naphthalimide residue leads to 3b; compared
with 3a , the affinity for free receptors is increased by a
factor of about 10, and most remarkably, the negative
cooperativity with [3H]NMS is switched into a positive
cooperative interaction. On a molecular level this means
that the affinity of 3b is higher for [3H]NMS-occupied
receptors, p(RKA) ) 8.66, compared with free receptors,
pKA ) 8.29 (Table 2). Comparison of 3b with 1a and 3a
reveals that the above-mentioned structural modifica-
tions increased the affinity for [3H]NMS-occupied recep-
tors, p(RKA), to a greater extent than the affinity for
free receptors, pKA. In other words, cooperativity is a
composite parameter36 and, for the compounds described
here, the underlying molecular events are characterized
by divergent structure-activity relationships.
With regard to structure-activity relationships, the
symmetrical and nonsymmetrical compounds (1a , 2a ,
3a , 4a , 6a , and 7-20) having no methyl groups in the
lateral propyl chains will be discussed first. The affinity
for NMS-occupied receptors of the nonsymmetrical
compounds was found to be in a range of p(RKA) ) 5.7
to 7.0 whereas the affinity range of the symmetrical
compounds is slightly larger (5.0 to 7.8). However,
comparing pairs of nonsymmetrical and symmetrical
compounds reveals, with the exception of the 3a /6a pair,
that there are no significant differences in the affinity
to bind to NMS-liganded receptors. Logically, with the
exception of the 5-phthalimidomethylated compounds
2a and 4a , ranking of both series with respect to the
affinity for NMS-occupied receptors gave the same
order. In a previous study,25 that was focused on
nonsymmetrical compounds, we found a parabolic cor-
relation between the affinity for NMS-occupied receptors

1036 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6
Muth et al.
Ta ble 3. Results of the Multidimensional Linear Regression Analysis Representing the Best Correlations between the
Physicochemical Parameters and the Affinities for the Free (y ) pKA) and the NMS-Occupied Receptor (y ) p[RKA]) of the Compounds
1a , 2a , 3a , 4a , 6a , and 7-20
y ) p(RKA)
Vol1 Vol2 logP1 Pol
âVol1
âVol2
âlogP1
âPol
â0
R2
s
F
Q2
sPRESS
-
-
+
+
+
-
-
-
-
+
+
+
+
+
+
-
+
-
+
+
-
-
-
-
-
-
0.443 ( 0.22 -
5.519 ( 0.51 0.510 0.432 17.696 0.378 0.487
0.327 ( 0.33 0.0349 ( 0.073 5.029 ( 1.15 0.540 0.432 9.372 0.335 0.519
0.000742 ( 0.0046
-0.0145 ( 0.0072
-0.0150 ( 0.0074 0.00102 ( 0.0024 0.611 ( 0.27 0.251 ( 0.12
0.494 ( 0.39
-
5.850 ( 2.10 0.514 0.444 8.449 0.345 0.515
8.469 ( 1.89 0.792 0.299 19.131 0.621 0.405
8.250 ( 2.01 0.805 0.301 14.375 0.631 0.414
0.611 ( 0.27 0.251 ( 0.12
y ) pKA
Vol1 logP1 Pol Ref
âVol1
âlogP1
âPol
âRef
â0
R2
s
F
Q2 sPRESS
+
-
-
-
+
+
+
+
-
-
+
+
-
+
+
+
-
+
-
+
+
-
+
+
-
-
-
-
-
-
-
+
0.00693 ( 0.0025
-
-
-
-
-
-
-
-
-
-
2.875 ( 1.48 0.669 0.414 34.381 0.600 0.455
3.970 ( 0.80 0.792 0.327 65.013 0.734 0.371
5.846 ( 0.57 0.548 0.483 20.634 0.404 0.555
4.150 ( 0.86 0.808 0.324 33.808 0.678 0.421
4.630 ( 1.62 0.804 0.328 32.962 0.712 0.398
3.456 ( 1.96 0.687 0.414 17.594 0.541 0.502
5.792 ( 1.86 0.852 0.295 28.594 0.696 0.422
-
0.143 ( 0.037
-
-
0.535 ( 0.25
-
0.135 ( 0.25 0.121 ( 0.055
-
-0.00313 ( 0.0067
0.200 ( 0.13
-
0.00536 ( 0.0043 0.166 ( 0.36
-0.00693 ( 0.0071 0.270 ( 0.27 0.224 ( 0.12
-0.00964 ( 0.0070 0.250 ( 0.24 0.240 ( 0.11 0.0208 ( 0.021 5.970 ( 1.70 0.887 0.267 27.409 0.761 0.388
Ta ble 4. Results of the Multidimensional Linear Regression Analysis Representing the Best Correlations between the
Physicochemical Parameters and the Affinities to the Free (y ) pKA) and the NMS-Occupied Receptor (y ) p[RKA]) of the
Compounds 1-6
y ) pR
Vol1 Vol2 Met1 Met2
âVol1
âVol2
âMet1
âMet2
â0
R2
s
F
Q2 sPRESS
-
-
+
+
+
+
-
-
-
+
-
+
+
+
+
+
+
+
-
+
-
-
+
+
-
-
-
-
-
0.432 ( 0.180 -
-0.220 ( 0.14 0.573 0.194 25.574 0.485 0.213
0.408 ( 0.170 0.143 ( 0.170 -0.268 ( 0.14 0.635 0.184 15.645 0.511 0.213
0.00380 ( 0.0014
0.455 ( 0.110
-
-
-2.402 ( 0.81 0.848 0.119 50.213 0.793 0.139
-2.063 ( 0.86 0.889 0.112 38.765 0.789 0.144
0.00440 ( 0.0015 -0.00130 ( 0.0015 0.466 ( 0.110
0.00367 ( 0.0012
0.00418 ( 0.0014 -0.00107 ( 0.0014 0.446 ( 0.096 0.105 ( 0.096 -2.089 ( 0.77 0.904 0.100 37.940 0.815 0.139
-
0.435 ( 0.099 0.117 ( 0.099 -2.365 ( 0.72 0.889 0.105 45.101 0.815 0.135
y ) p(RKA)
Vol1 Vol2 Met1 Met2
âVol1
âVol2
âMet1
âMet2
â0
R2
s
F
Q2 sPRESS
+
+
+
+
+
+
-
+
-
-
+
+
-
-
+
+
+
+
-
-
-
+
-
+
0.0135 ( 0.0054 -
-
-
-
-0.294 ( 3.10 0.588 0.463 27.130 0.476 0.523
-1.145 ( 3.52 0.613 0.462 14.240 0.449 0.551
-1.111 ( 1.86 0.863 0.274 57.102 0.816 0.319
0.0121 ( 0.0061 0.00315 ( 0.0062 -
0.0142 ( 0.0032 -
0.0143 ( 0.0033 -
0.0133 ( 0.0037 0.00185 ( 0.0037 0.715 ( 0.26 -
0.731 ( 0.25 -
0.753 ( 0.26 -0.120 ( 0.26 -1.149 ( 1.87 0.870 0.274 38.277 0.800 0.341
-1.593 ( 2.10 0.872 0.273 38.680 0.798 0.343
0.0136 ( 0.0038 0.00162 ( 0.0038 0.734 ( 0.27 -0.103 ( 0.27 -1.568 ( 2.13 0.878 0.276 28.621 0.775 0.374
y ) pKA
Vol1 Vol2 Met1 Met2
âVol1
âVol2
âMet1
âMet2
â0
R2
s
F
Q2 sPRESS
+
+
+
+
+
+
-
+
-
-
+
+
-
-
+
+
+
+
-
-
-
+
-
+
0.01010 ( 0.0041 -
-
-
-
1.594 ( 2.33 0.590 0.347 27.247 0.479 0.391
0.625 ( 2.53 0.646 0.331 16.411 0.483 0.400
1.285 ( 2.21 0.659 0.325 17.421 0.533 0.380
0.00851 ( 0.0044 0.00359 ( 0.0044 -
0.01040 ( 0.0038 -
0.01070 ( 0.0037 -
0.00895 ( 0.0042 0.00314 ( 0.0042 0.249 ( 0.29 -
0.277 ( 0.30 -
0.318 ( 0.29 -0.238 ( 0.29 1.210 ( 2.11 0.709 0.309 13.856 0.537 0.390
0.469 ( 2.41 0.702 0.313 13.344 0.510 0.401
0.00940 ( 0.0041 0.00268 ( 0.0042 0.290 ( 0.29 -0.210 ( 0.29 0.521 ( 2.33 0.740 0.301 11.393 0.508 0.414
imide which produces the lower affinity to the NMS-
liganded receptor the cooperativity remains negative or
only changes to neutral. Compare, for example, the
nonsubstituted compound 3a (pR ) -0.2) with com-
pound 3b where the affinity-increasing naphthalimide
moiety is attached to the methylated propyl chain
resulting in a positive cooperativity (pR ) 0.37) and with
compound 3c in which the alkylated propyl chain is
connected to the “less potent” phthalimide resulting in
a neutral cooperativity (pR ) -0.005). The same is true
for the series 2a , 2b, and 2c and the series 5a , 5b, and
5c. In addition, the highest affinity for the allosteric site
in conjunction with positive cooperativity is found with
compounds having at least one naphthyl group attached
to a 2,2-dimethylpropyl chain.
the aforementioned physicochemical parameters. As an
additional parameter we introduced two indicator vari-
ables (Met1, Met2) which represent the presence or
absence of the methyl residues in the propyl chains
adopting values of either one or zero. The best correla-
tions are summarized in Table 4 using the general
equation
pBE ) â0 + âVol1·xVol1 + âVol2·xVol2 + âMet1·xMet1
+
âMet2·xMet2
Again different correlations were found for pKA and
p(RKA). The affinity to the NMS-liganded receptor,
p(RKA), cannot be characterized with either volume or
methylation but it can be described with the volume in
connection with the adjacent methyl group with high
significance (R2 ) 0.86 and Q2 ) 0.82). The combination
To quantify the SARs it was tried to correlate the
affinities to the free and NMS-occupied receptors with

Allosteric Enhancers of Muscarinic Ligand Binding
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6 1037
is in accordance with the qualitative SAR discussed
above. Using further parameters leads to weaker cor-
relations. In contrast to this observation and to the
findings revealed for the series of symmetrical and
nonsymmetrical compounds with a missing methylation
(see Table 1) much weaker correlations were found for
the affinities to the free receptor. There is no significant
difference when using both volumes, the volume and the
adjacent methylation, or combinations of three or four
of these parameters. These results clearly indicate the
importance of the combination of the affinity generating
lateral imide moiety with an adjacent methylation with
regard to positive cooperativity. A contralateral methyl-
ation is not able to induce a positive cooperativity, at
the very best a neutral cooperativity is attained.
The affinity of an allosteric agent for an orthosteric
ligand-occupied receptor is influenced by the structure
of the orthosteric ligand.42 Therefore, cooperative inter-
actions may depend in a specific fashion on the type of
orthosteric ligand. In other words, the findings described
here for the antagonist NMS cannot be generalized for
other orthosteric ligands such as the endogenous agonist
acetylcholine. The dependence of cooperativity on the
type of orthosteric ligand is an interesting field for
systematic investigations in order to understand the
molecular basis of cooperativity.
Con clu sion
Taken all results together it can be stated that the
lateral imide moieties are able to modulate the affinities
to free and NMS-liganded receptors within a rather
wide range of 3 orders of magnitude. No relevant
differences were found between symmetrical and non-
symmetrical compounds. Negative cooperativity with
NMS could consistently be abolished or even switched
into positive cooperativity by alkylation of the lateral
propyl chains for all imide moieties studied in this
regard. This effect results from a more pronounced gain
in affinity in the NMS-occupied receptor than in the free
receptor. As a consequence, the methylated compounds
show higher allosteric potency in the nanomolar range
of concentration. From these results the question arises
whether a monoalkylation of the propyl chain would
produce the same effects and whether monoalkylation
yields enantioselectivity and whether ethylation, pro-
pylation, etc., are characterized by a “cutoff” phenom-
enon. This work is in progress.
Previous molecular modeling and QSAR studies re-
vealed the pharmacophore for a high affinity to the
NMS-occupied receptor to be composed of two positively
charged nitrogens in a distance of about 10 Å and two
aromatic imides all together arranged in an S-shaped
conformation.38-40 This pharmacophore hypothesis pre-
dicts symmetrical compounds to surpass nonsymmetri-
cal compounds in allosteric potency. However, inspection
of the data obtained for the symmetrical/nonsymmetri-
cal collection of compounds (Table 1) generally revealed
similar affinities in both series (except 3a /6a ). Further-
more, in the series of compounds displayed in Table 2
(1-6) the highest allosteric affinities are not found with
the symmetrical compounds 1a , 1d , 4a , 4d , 6a , and 6d .
These are found with compounds having at least one
naphthyl group attached to a 2,2-dimethylpropyl chain,
indicating that this moiety may fit perfectly into a
binding pocket of the allosteric site. Phthalimide and
methylphthalimide skeletons connected to a 2,2-di-
methylpropyl chain seem to have less ability to interact
with the binding pocket resulting in lower affinities;
compare e.g., 1b, 2b, 2c, 4b, with 3b. Furthermore,
when the phthalimide or the methylphthalimide with
a 2,2-dimethylpropyl chain are combined with a naph-
thyl group attached to a 2,2-dimethylpropyl chain (i.e.,
3d and 5d ), the allosteric potency is found to be less
than for 3b and 5b, respectively, having only a phthal-
imidopropyl or a methylphthalimidopropyl chain, but
no dimethylation on that side. Thus, the phthalimide
and methylphthalimide skeletons by connection with a
2,2-dimethylpropyl chain seem to gain the competence
to compete for the binding pocket that is used by the
corresponding naphthalimide resulting in a lower af-
finity to the allosteric site.
Exp er im en ta l Section
Melting points were determined with a Gallenkamp and a
Dr. Tottoli melting point apparatus (Bu¨chi, Switzerland) and
were not corrected. 1H and 13C NMR spectra (Supporting
Information) were recorded on a Varian XL 300 (1H 299.956
MHz; 13C 75.433 MHz) and on a Bruker AV 400 instrument
(1H 400.132 MHz; 13C 100.613 MHz). Abbreviations for data
quoted are: s, singlet; d, doublet; t, triplet; qu, quartet; quin,
quintet; dd, doublet of doublets; m, multiplet; br, broad. The
centers of the peaks of CDCl3 and DMSO-d6 were used as
internal references. IR spectra were obtained using a Biorad
PharmalyzIR FT-IR spectrometer and a Perkin-Elmer 298
spectrometer. Dry solvents were used throughout. Chemicals
were of analytical grade and purchased from Lancaster
(Mu¨hlheim, Germany) and Aldrich (Steinheim, Germany).
1a (W84) was synthesized according to ref 18, 4a (dimethyl-
W84) according to ref 20, 1b, 1d , 2a -c, and 4d according to
ref 28, and 3a , 8, and 10-13 according to ref 25. The
intermediate compounds 22, 25, 31, 32, as well as 34, 36, and
37, were obtained according to ref 28. Moreover the intermedi-
ate compounds 27 and 30 are known.25
Gen er a l P r oced u r e for th e Syn th esis of 6-(3-Dim eth y-
la m in op r op yl)p yr r olo[3,4-b]p yr a zin e-5,7-d ion e (21), 2-(3-
Dim et h yla m in op r op yl)-5,6-d iflu or oisoin d ole-1,3-d ion e
(23), 2-(3-Dim eth yla m in op r op yl)-5-tr iflu or om eth ylisoin -
d ole-1,3-d ion e (24), 5-ter t-Bu t yl-2-(3-(d im et h yla m in o-
)p r op yl)isoin d ole-1,3-d ion e (26), 2-(3-Dim et h yla m in o-
p r op yl)-4,9[1‘2‘]-b en zen o-1H -b en z[f]isoin d ole-1,3-d ion e
(27), 2-(3-Dim eth yla m in op r op yl)ben zo[f]isoin d ole-1,3-d i-
on e (28), 5,6-Dich lor o-2-(3-(d im eth yla m in o)p r op yl)isoin -
d ole-1,3-d ion e (29), 2-(3-Dim eth yla m in op r op yl)ben zo-
[d e]isoqu in olin e-1,3-d ion e (30), a n d 2-(3-Dim eth yla m in o-
2,2-d im eth ylp r opyl)ben zo[d e]isoqu in olin e-1,3-d ion e (33).
A mixture of the appropriate anhydride (40 mmol), N1,N1-
dimethylpropane-1,3-diamine or 2,2,N1,N1-tetramethylpropane-
1,3-diamine (40 mmol) and catalytic amount of p-toluene-
Since all affinities to the NMS-occupied receptor were
determined in Mg/Tris-buffered medium and magne-
sium cations were found to be weak allosteric modula-
tors competing with the hexamethonium compounds for
the allosteric binding site,41 the high-affinity compound
6a was additionally evaluated in 5 mM Na+/K+-
phosphate-buffered medium, resulting in a pKA ) 9.06
( 0.02, pR ) -0.03 ( 0.02, p(RKA) ) 9.03 ( 0.01 (n )
3) and a pEC50,diss ) 9.03 ( 0.05 (n ) 4). Compared with
4a (pKA ) 8.03 ( 0.12, pR ) -0.04 ( 0.05, p(RKA) )
8.36 ( 0.10 (n ) 3) and pEC50,diss ) 8.46 ( 0.09 (n ) 5))
as well as alcuronium (pEC50,diss ) 8.40 in Na+/K+/Pi),10
the bisnaphthalimide compound 6a is found to have the
highest affinity reaching the subnanomolar range of
concentrations.

1038 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6
Muth et al.
Ta ble 5. Reaction Conditions and Analytical Data of Compounds 2d , 3a -d , 5a -d , 6a , 6b, 6d , 7-21, 23, 24, 26-30, 33, 35, and 38
reaction time:
hours (h), days (d)
yield,
%
mp,
°C
compd
formula (mol wt)
reagents
IR (ν, cm-1
)
2d a
3a
C37H54Br2N4O4 (778.7)
31 + 37
12 d
2 d
2 d
1 d
15 d
1 d
3 d
4 d
12 d
2 d
2 d
12 d
2 d
2 d
2 d
2 d
2 d
2 d
2 d
2 d
2 d
2 d
2 d
2 d
2 d
2 d
12 h
12 h
2 h
12 h
12 h
2 d
2 d
3 h
9 h
2 d
7
206-207
236
733, 1080, 1366, 1711, 1769, 2972
C
36H46Br2N4O4 (758.6)
C38H50Br2N4O4 (786.7)
38H50Br2N4O4 (786.7)
30 + 34
56
42
51
14
78
19
59
8
725, 780, 1660, 1710, 1770, 2670, 2950, 3010
723, 780, 1236, 1339, 1587, 1657, 1707, 1768, 2940
730, 780, 1610, 1660, 1710, 1780, 2960
729, 781, 1237, 1341, 1589, 1658, 1712, 1775, 2935
744, 782, 1230, 1344, 1660, 1709, 1769, 2869, 2947
739, 781, 1236, 1338, 1587, 1657, 1699, 1765, 2945
740, 770, 1380, 1620, 1660, 1710, 1770, 2960
750, 779, 1236, 1338, 1587, 1655, 1705, 1770, 2937
780, 1230, 1345, 1660, 1700, 2950, 3000
775, 1238, 1336, 1382, 1587, 1649, 1693, 2960
781, 1238, 1341, 1587, 1655, 1703, 2931
730, 1405, 1710, 1770, 2950
725, 750, 1295, 1400, 1710, 1765, 2950, 3000
692, 725, 1137, 1172, 1321, 1380, 1710, 3006
725, 750, 1375, 1400, 1705, 1770, 2960, 3010
730, 765, 1400, 1700, 1770, 2950, 3000
725, 770, 1370, 1700, 1760, 2950, 3010
725, 1375, 1400, 1705, 1770, 2950
3ba
3c
22 + 38
222-224
198-200
201-202
256-257
202-204
204-207
198-199
255
219
248
165
226
228-231
241
258
253
228
114
241
246-247
176
278
261
227
oil
93
54
oil
163
97
146
C
30 + 36
3d a
5a a
5b
5c
C40H54Br2N4O4 (814.7)
C37H48Br2N4O4 (772.6)
31 + 38
30 + 35
C
C
39H52Br2N4O4 (800.7)
39H52Br2N4O4 (800.7)
25 + 38
30 + 37
5d a
6a
C41H56Br2N4O4 (828.7)
40H48Br2N4O4 (808.7)
32 + 38
C
30
29
73
34
26
44
35
17
43
51
10
17
46
49
34
59
26
21
38
76
70
62
82
34
60
88
61
84
55
6ba
6d a
7
C42H52Br2N4O4 (836.7)
C44H56Br2N4O4 (864.8)
30 + 38
33
C
C
30H42Br2N6O4 (710.5)
32H42Br2F2N4O4 (744.5)
21 + 34
8
23 + 34
9a
C33H43Br2F3N4O4 (776.5)
24 + 34
10
11
12
13
14
15
16a
17
18
19
20
21
23
24a
26
27
28
29
30a
33a
35a
38a
C
C
C
C
C
C
36H52Br2N4O4 (764.6)
42H54Br2N4O4 (838.7)
36H46Br2N4O4 (758.6)
32H42Br2Cl2N4O4 (777.3)
28H40Br2N8O4 (712.5)
32H40Br2F4N4O4 (780.5)
26 + 34
27 + 34
28 + 34
29 + 34
21
23
24
26
27
28
29
-
-
-
-
-
-
-
-
-
25
33
700, 1195, 1445, 1535, 1665, 2950
745, 770, 1400, 1490, 1710, 1775, 2950, 3060
691, 744, 1120, 1164, 1317, 1717, 1776, 3004
750, 1375, 1400, 1705, 1770, 2960
770, 1400, 1460, 1700, 1775, 2960, 3000
770, 1375, 1700, 1760, 2950, 3010
745, 1380, 1400, 1705, 1770, 3100
730, 1470, 1530, 1660, 2950, 3300
745, 1200, 1405, 1690, 2770, 3050
694, 750, 1126, 1320, 1699, 2767, 2949
750, 1370, 1710, 1770, 2930, 3470
770, 1690, 1765, 2770, 2980, 3450
770, 1385, 1690, 1760, 2770, 2940
750, 1640, 1710, 1775, 2770, 2950, 3300
775, 1232, 1340, 1587, 1651, 1695, 2762, 2942
780, 1236, 1330, 1588, 1662, 1699, 2763, 2960
738, 1391, 1698, 1762, 2858, 2941
C34H42Br2F6N4O4 (844.5)
C
C
C
C
C
C
40H60Br2N4O4 (820.7)
52H62Br2N4O4 (966.9)
40H48Br2N4O4 (808.6)
32H40Br2Cl4N4O4 (846.3)
11H14N4O2 (234.3)
13H14F2N2O2 (268.3)
C14H15F3N2O2 (300.3)
C
C
C
C
17H24N2O2 (288.4)
23H24N2O2 (360.5)
17H18N2O2 (282.4)
13H14Cl2N2O2 (301.2)
C17H18N2O2 (282.4)
C19H22N2O2 (310.4)
C20H30Br2N2O2 (490.3)
C25H34Br2N2O2 (554.4)
119
112-114
145-146
178-180
11 d
781, 1237, 1338, 1586, 1659, 1705, 2950
a
Recorded on a Biorad PharmalyzIR FT-IR spectrometer.
sulfonic acid were refluxed in toluene (100 mL) using a water
separator. After a reaction time of 2 h to 2 days the solvent
was evaporated. Compound 33 was obtained from a solid
residue that was recrystallized by a mixture of ethanol and a
few drops of petroleum ether. Concerning compounds 21, 23,
24, 26, and 28-30, their oily residues were purified by means
of column chromatography (silica gel, eluent CH2Cl2:MeOH )
1:1). With exception of compounds 21 and 26 the obtained oils
crystallized after a few hours at room temperature.
zin -6-y l)p r o p y l]a m m o n iu m }h e x y l)-1,1-d im e t h y la m -
m on io]p r op yl}p yr r olo[3,4-b]-p yr a zin e-5,7-d ion e Dibr o-
m id e (14), 2-{3-[1-(6-{1,1-Dim eth yl-1-[3-(5,6-d iflu or o-1,3-
dioxo-1,3-dih ydr oisoin dol-2-yl)pr opyl]am m o-n iu m}h exyl)-
1,1-d im et h yla m m on io]p r op yl}-5,6-d iflu or oisoin d olin e-
1,3-d ion e Dibr om id e (15), 2-{3-[1-(6-{1,1-Dim et h yl-1-[3-
(1,3-d ioxo-1,3-d ih yd r o-5-t r iflu or o-m e t h yl-isoin d ol-2-
y l)p r o p y l]a m m o n i u m }h e x y l)-1,1-d i m e t h y la m m o n -
io]p r op yl}-5-t r iflu or om et h yl-isoin d olin e-1,3-d ion e Di-
br om id e (16), 2-{3-[1-(6-{1,1-Dim eth yl-1-[3-(5-ter t-bu tyl-
1,3-d io x o -1,3-d ih y d r o is o in d o l-2-y l)p r o p y l]a m m o n -
iu m }h exyl)-1,1-d im et h yla m m on io]p r op yl}-5-ter t-b u t yl-
isoin d olin e-1,3-d ion e Dib r om id e (17), 2-{3-[1-(6-{1,1-
Dim et h yl-1-[3-(1,3-d ioxo-1,3-d ih yd r o-4,9[1‘2‘]b en zen o-
1H -b en z[f]isoin d ol-2-yl)p r op yl]a m m on iu m }h exyl)-1,1-
d i m e t h y la m m o n i o ]p r o p y l}-(4,9[1‘2‘]-b e n z e n o -1H -
ben z[f])isoin d olin e-1,3-d ion e Dibr om id e (18), 2-{3-[1-(6-
{1,1-Dim eth yl-1-[3-(1,3-dioxo-1,3-dih ydr oben zo[f]isoin dol-
2-yl)p r op yl]a m m on iu m }h e xyl)-1,1-d im e t h yla m m on -
io]p r op yl}ben zo[f]isoin d olin e-1,3-d ion e Dibr om id e (19),
a n d 2-{3-[1-(6-{1,1-Dim eth yl-1-[3-(5,6-d ich lor o-1,3-d ioxo-
1,3-d ih yd r oisoin d ol-2-yl)p r op yl]a m m on iu m }h exyl)-1,1-
d im et h yla m m on io]p r op yl}-5,6-d ich lor o-isoin d olin -1,3-
d ion e Dibr om id e (20). Two equivalents of 21, 23, 24, 26-
30, and 33 (2 mmol) and 1 equiv of 1,6-dibromohexane (1
mmol) were dissolved in acetonitrile (50 mL), and a catalytic
amount of KI and K2CO3 (1:1) was added. The reaction solution
was refluxed for 2 to 12 days. After the reaction was completed
(TLC-monitoring: silica gel, mobile phase ) CH3OH:0.2 M
NH4NO3 solution (aqueous) ) 3:2), the solution was allowed
to cool to room temperature and a white to light yellow
precipitate was collected by filtration. It was washed several
times with acetonitrile and pentane and dried in vacuo.
For analytical data see Table 5, for spectroscopic data see
Table 8 (Supporting Information).
Gen er a l P r oced u r e for th e Syn th esis of (6-Br om o-
h exyl)d im eth yl-[3-(5-m eth yl-1,3-d ioxo-1,3-d ih yd r oisoin -
d ol-2-yl)p r op yl]a m m on iu m Br om id e (35) a n d (6-Br om o-
h exyl)-[3-(1,3-d ioxo-1H ,3H -b en zo[d e]isoq u in olin -2-yl)-
2,2-d im eth ylp r op yl]d im eth yla m m on iu m Br om id e (38).
Precursor propylamines 25 and 33 (10 mmol) were stirred in
a 10-fold excess of 1,6-dibromohexane (100 mmol) without any
solvent. Compound 25 reacted at room temperature, whereas
compound 33 was heated to 50 °C. After a reaction time of 2
and 11 days, respectively, a voluminous white precipitate was
collected by filtration. To remove the 1,6-dibromohexane
excess, the solid was suspended in petroleum ether, refluxed,
cooled, filtered, and dried in vacuo.
For analytical data see Table 5, for spectroscopic data see
Table 8 (Supporting Information).
Gen er a l P r oced u r e for th e Syn th esis of 2-{3-[1-(6-{1,1-
Dim eth yl-1-[3-(1,3-d ioxo-1,3-d ih yd r oben zo[d e]isoqu in o-
lin -2-yl)pr opyl]am m on iu m }h exyl)-1,1-dim eth ylam m on io]-
p r op yl}ben zo[d e]isoqu in olin e-1,3-d ion e Dibr om id e (6a ),
2-{3-[1-(6-{1,1-Dim eth yl-1-[3-(1,3-d ioxo-1,3-d ih yd r oben zo-
[d e]isoq u in olin -2-yl)-2,2-d im et h ylp r op yl]a m m on iu m }-
h exyl)-1,1-dim eth ylam m on io]-2,2-dim eth ylpr opyl}ben zo-
[d e]isoqu in olin e-1,3-d ion e Dibr om id e (6d ), 6-{3-[1-(6-{1,1-
Dim eth yl-1-[3-(5,7-d ioxo-5,7-d ih yd r op yr r olo[3,4-b]-p yr a -
For analytical data see Table 5, for spectroscopic data see
Tables 6 and 7 (Supporting Information).

Allosteric Enhancers of Muscarinic Ligand Binding
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6 1039
Gen er a l P r oced u r e for th e Syn th esis of 2-{3-[1-(6-{1,1-
Dim eth yl-1-[3-(1,3-d ioxo-1,3-d ih yd r o-5-m eth ylisoin d ol-2-
yl)-2,2-d im eth ylp r op yl]a m m on iu m }h exyl)-1,1-d im eth yl-
a m m on io]-2,2-d im et h ylp r op yl}-5-m et h ylisoin d ole-1,3-
d ion e Dibr om id e (2d ), 2-{3-[1-(6-{1,1-Dim eth yl-1-[3-(1,3-
dioxo-1,3-dih ydr oisoin dol-2-yl)pr opyl]am m on iu m}h exyl)-
1,1-d im e t h y la m m o n io ]-2,2-d im e t h y lp r o p y l }b e n z o -
[d e]isoqu in olin e-1,3-d ion e Dibr om id e (3b), 2-{3-[1-(6-{1,1-
Dim et h yl-1-[3-(1,3-d ioxo-1,3-d ih yd r oisoin d ol-2-yl)-2,2-
d i m e t h y lp r o p y l]a m m o n i u m }h e x y l)-1,1-d i m e t h y l-
a m m on io]p r op yl}b en zo[d e]isoq u in olin e-1,3-d ion e Di-
br om id e (3c), 2-{3-[1-(6-{1,1-Dim eth yl-1-[3-(1,3-d ioxo-1,3-
d ih y d r o is o in d o l-2-y l)-2,2-d im e t h y lp r o p y l]a m m o n -
i u m }h e x y l )-1 ,1 -d i m e t h y l a m m o n i o ]-2 ,2 -d i m e t h y l -
pr opyl}- ben zo[de]isoqu in olin e-1,3-dion e Dibr om ide (3d),
2-{3-[1-(6-{1,1-Dim eth yl-1-[3-(1,3-dioxo-1,3-dih ydr o-5-m eth -
y l-i s o i n d o l-2-y l)p r o p y l]a m m o n i u m }h e x y l)-1,1-d i -
m et h yla m m on io]p r op yl}b en zo[d e]isoq u in olin e-1,3-d i-
on e Dibr om id e (5a ), 2-{3-[1-(6-{1,1-Dim eth yl-1-[3-(1,3-
d i o x o -1,3-d i h y d r o -5-m e t h y l-i s o i n d o l-2-y l)p r o p y l]-
a m m o n i u m }h e x y l )-1 ,1 -d i m e t h y l a m m o n i o ]-2 ,2 -d i -
m et h ylp r op yl}b en zo[d e]isoq u in olin e-1,3-d ion e Dib r o-
m id e (5b ), 2-{3-[1-(6-{1,1-Dim et h yl-1-[3-(1,3-d ioxo-1,3-
d ih yd r o-5-m e t h yl-isoin d ol-2-yl)-2,2-d im e t h ylp r op yl]-
a m m o n i u m }h e x y l ) -1 , 1 -d i m e t h y l a m m o n i o ] p r o -
pyl}ben zo[de]isoqu in olin e-1,3-dion e Dibr om ide (5c), 2-{3-
[1-(6-{1,1-Dim eth yl-1-[3-(1,3-d ioxo-1,3-d ih yd r o-5-m eth yl-
isoin d ol-2-yl)-2,2-d im eth ylp r op yl]a m m on iu m }h exyl)-1,1-
d i m e t h y l a m m o n i o ] -2 ,2 -d i m e t h y l p r o p y l }b e n z o -
[d e]isoqu in olin e-1,3-d ion e Dibr om id e (5d ), 2-{3-[1-(6-{1,1-
D i m e t h y l -1 -[ 3 -( 1 ,3 -d i o x o -1 ,3 -d i h y d r o b e n z o [ d e ] -
isoqu in olin -2-yl)-2,2-dim eth ylpr opyl]am -m on iu m }h exyl)-
1,1-d im et h yla m m on io]p r op yl}-b en zo[d e]isoq u in olin e-
1,3-d ion e Dibr om id e (6b), 2-{3-[1-(6-{1,1-Dim eth yl-1-[3-
(1,3-d io x o -1,3-d ih y d r o is o in d o l-2-y l)p r o p y l]a m m o n -
iu m }h exyl)-1,1-d im et h yla m -m on io]p r op yl}p yr r olo[3,4-
b]-p yr a zin e-5,7-d ion e Dib r o-m id e (7), 2-{3-[1-(6-{1,1-
Dim eth yl-1-[3-(1,3-dioxo-1,3-di-h ydr oisoin dol-2-yl)pr opyl]-
a m m on iu m }h e xyl)-1,1-d im e t h yla m m on io]p r op yl}-5-
trifluoromethylisoindole-1,3-dione Dibromide (9).Equimolar
amounts (2 mmol) of 21-33 and 34-38 were dissolved in
acetonitrile (50 mL), and a catalytic amount of KI and K2CO3
(1:1) was added. The reaction solution was refluxed for 1 to
15 days. After the reaction was completed (TLC-monitoring:
silica gel, mobile phase ) CH3OH:0.2 M NH4NO3 solution
(aqueous) ) 3:2), the solution was allowed to cool to room
temperature. If there was no precipitate the solution was
cooled to 4 °C for several days. The obtained precipitates were
collected by filtration and washed several times with little
amounts of acetonitrile and with pentane and dried in vacuo.
scintillation fluid (Ready protein, Beckman, Palo Alto, CA) the
radioactivity was measured by liquid scintillation counting in
a Beckman LS 6000 counter.
Two types of experiments were applied to evaluate the effect
of the test compounds on the dissociation of [3H]NMS. In the
case of “complete dissociation experiments” membranes were
preincubated with 0.2 nM [3H]NMS over a period of 30 min in
a volume of about 23 mL before 1 µM atropine was added alone
or together with a test compound to initiate the reaction.
Aliquots of 1 mL were drawn from the incubation medium at
appropriate intervals over a period of 120 min and processed
as described above. Data were analyzed by means of nonlinear
regression analysis; [3H]NMS dissociation was monophasic in
the absence and in the presence of test compound and was
characterized by the apparent rate constant of dissociation k-1
.
In “two-point kinetic experiments” (cf. ref 36) membranes were
incubated with the radioligand for 30 min to obtain the binding
equilibrium. Specific [3H]NMS binding was measured before
(t ) 0 min) and after (t ) 10 min) addition of 1 µM atropine
alone or in combination with various concentrations of a test
compound. Specific [3H]NMS binding at t ) 0 min and t ) 10
min served to characterize the monoexponential time course
of [3H]NMS dissociation and to obtain the apparent rate
constant of dissociation k-1. The half time of dissociation
amounted to t1/2 ) 3.9 ( 0.9 min (mean value ( SEM, 118
experiments). To obtain concentration-effect-curves for the
allosteric delay of [3H]NMS dissociation, the k-1-values were
plotted over the log drug concentration. Curve fitting was
based on a four parameter logistic function (GraphPad Prism
Version 3.00; GraphPad, San Diego, CA). Applying a partial
F-test (p < 0.05 taken to indicate a significant difference), we
checked whether it was possible to set the parameters “top”
to a value k-1 ) 100%, “bottom” to a value k-1 ) 0% and “slope
factor” to nH ) -1; this was always the case.
Allosteric effects of the test compound on [3H]NMS equilib-
rium binding was measured with 0.2 nM [3H]NMS and
increasing concentrations of a test compound. The incubation
time required to obtain binding equilibrium was calculated
based on the eq 31 in the study of Lazareno and Birdsall3 and
amounted to 180-530 min. Data for test compound effects on
specific [3H]NMS equilibrium binding were analyzed by non-
linear regression according to Ehlert13 using eq 3 in ref 34.
Statistical comparisons between affinity parameters were
performed applying a two-tailed unpaired t-test with Welch
correction when necessary (p < 0.05 taken to indicate a
significant difference).
QSAR An a lysis. The lateral varied N-methylimides were
built up using the model builder in HyperChem 5.1 (Hypercube
Inc. Waterloo, Ont. Canada 1997). Geometry optimization was
achieved using the MM+ program implemented in Hyper-
Chem. By means of ChemPlus (Hypercube Inc.) the following
physicochemical properties were calculated: the octanol/water
partition coefficient (log P), the steric parameters volume and
surface area (grid) as well as polarizibility and refractivity, a
chameleon parameter comprising both steric bulk and polar-
izibility. The allosteric potencies (EC50) were transformed in
the QSAR form pEC50,diss. The QSARs were constructed by
performing a multidimensional linear regression analysis
using the BILIN software developed by Kubinyi.45
For analytical data see Table 5, for spectroscopic data see
Tables 6 and 7 (Supporting Information).
P h a r m a cology. Radioligand binding experiments were
carried out with domestic pig heart ventricle homogenates in
a buffer composed of 2.7 mM MgHPO4, 45 mM Tris-HCl, pH
7.3 at 37 °C. As indicated in the text, selected compounds were
additionally applied in a Na+/K+/Pi buffer which allows a high
allosteric potency (4 mM Na2HPO4, 1mM KH2PO4; 23 °C; pH
7.4). The procedure to prepare the homogenates has been
described previously.10,43 Protein content was determined
according to Lowry et al.44 (1951) and amounted to 4.89 ( 0.49
mg/mL (mean value ( SEM; n ) 8). The radioligand used was
[3H]N-methylscopolamine ([3H]NMS, PerkinElmer Life Sci-
ences, Boston, MA) with a specific activity of 70-83.5 Ci/mmol
and at a concentration of 0.20 nM. Nonspecific [3H]NMS-
binding was determined in the presence of 1 µM atropine and
was smaller than 10% of the total binding. [3H]NMS binding
affinity amounted to pKD ) 9.50 ( 0.04 (mean value ( SEM;
n ) 8), and the concentration of receptors in the cardiac
homogenates was Bmax ) 76.47 ( 10.6 fmol/mg membrane
protein (means ( SEM; n ) 8). Membranes were separated
by a rapid vacuum filtration procedure (glass filters No. 6;
Schleicher and Schu¨ll, Dassel, Germany). Filters were washed
twice with 5 mL of distilled water. After adding 5 mL of
Ack n ow led gm en t. Thanks are due to the DFG (HO
1368/7-1; MO 821/1-2), to the Fonds der Chemischen
Industrie for financial support and to Dr. Knut Bau-
mann, University of Wu¨rzburg, for valuable discussions.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR and 13C
NMR data. This material is available free of charge via the
Internet at http://pubs.acs.org.
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