Design, synthesis, and biological activity of methoctramine-related polyamines as putative Gi protein activators

Article abstract of DOI:10.1021/jm0155594

The universal template approach provided a prospect of modifying methoctramine (2) structure. Thus, polyamines 3-7 were designed in which the flexibility of the diaminohexane spacer of 2 was replaced by a bipiperidinyl moiety. In electrically stimulated g

Full text of DOI:10.1021/jm0155594

J . Med. Chem. 2001, 44, 4035-4038
4035
Ch a r t 1
Design , Syn th esis, a n d Biologica l Activity
of Meth octr a m in e-Rela ted P olya m in es a s
P u ta tive Gi P r otein Activa tor s
Carlo Melchiorre,*,† Maria L. Bolognesi,†
Roberta Budriesi,† Carla Ghelardini,‡
Alberto Chiarini,† Anna Minarini,† Michela Rosini,†
Vincenzo Tumiatti,† and Erik J . Wade§
Sch em e 1a
Department of Pharmaceutical Sciences, University of
Bologna, Via Belmeloro 6, 40126 Bologna, Italy, Department
of Preclinical and Clinical Pharmacology, University of
Florence, Viale G. B. Morgagni 65, 50134 Florence, Italy,
and Department of Molecular Pharmacology, Gru¨nenthal
GmbH, P.O. Box 500444, 52088 Aachen, Germany
Received August 2, 2001
Abstr a ct: The universal template approach provided a pros-
pect of modifying methoctramine (2) structure. Thus, polyamines
3-7 were designed in which the flexibility of the diamino-
hexane spacer of 2 was replaced by a bipiperidinyl moiety. In
electrically stimulated guinea pig left atria, these novel
polyamines, unlike prototype 2, displayed a potent intrinsic
activity, which was in contrast with the muscarinic antago-
nism shown in binding studies by some of them (3 and 4) and
was inhibited by benzalkonium chloride, an inhibitor of Gi
proteins.
In t r od u ct ion . It has been suggested that a poly-
methylene tetraamine backbone may represent a uni-
versal template on which suitable pharmacophores can
be inserted to achieve selectivity for any given recep-
tor.1,2 Accordingly, we have demonstrated that benex-
tramine (1) (Chart 1), developed as an irreversible
(nonequilibrium in the kinetic sense) antagonist at both
R1 and R2-adrenoreceptors,3 could be used as a lead
compound for the design of polyamines to achieve
specific recognition of muscarinic receptors. This re-
search led to the discovery of methoctramine (2),4 which
is widely used as a pharmacological tool for muscarinic
receptor subtype characterization (Chart 1).5 In turn,
appropriate structural modifications performed on the
structure of 2 have allowed us to obtain new polyamines
endowed with high affinity and selectivity for muscar-
inic receptor subtypes6 and nicotinic receptors as well.7
Furthermore, using 1 as the focus, polyamines have
been designed to achieve specific recognition for differ-
ent biological targets such as neuropeptide Y receptors8
and acetylcholinesterase.9
Tetraamine 2 can assume many low-energy confor-
mations in an aqueous environment because of its
flexible polymethylene chain. Therefore, more rigid
analogues are needed to determine whether flexibility
is an important determinant of potency with respect to
muscarinic receptors. In this regard, we have demon-
strated that changes in flexibility may enable one to
design polyamine-containing compounds with specificity
for nicotinic receptors over muscarinic receptors.7
a
Boc ) Me3COCO-.
To further investigate the effect of reduction in
flexibility, we have synthesized tetraamine 3 in which
the diaminohexane spacer of 2 has been replaced by a
bipiperidinyl moiety. Since we have already verified that
the inner amine functions of tetraamines can be trans-
formed into amide groups without affecting the affinity
toward muscarinic receptors,2 we have also investigated
the corresponding diamine diamide 4.
Polyamines 2, 3, and 4 are potent competitive an-
tagonists at muscarinic receptors. However, polyamines
3 and 4, but not 2, display a potent inotropic effect in
atrial preparations. It is well known that a negative
inotropic response in the atrium is typical for muscarinic
agonists but not for antagonists, which do not possess
intrinsic activity. Such an effect is clearly in contrast
with the competitive antagonism observed in binding
assays on cloned muscarinic receptors. In an attempt
to explain this unexpected biological behavior of poly-
amines 3 and 4, we investigated also compounds 5-7,
obtained by splitting 3 and 4 into two halves. The
rationale for this choice stands on the observation that
the diamines obtained by performing a similar modifi-
cation on the structure of prototype 2 produced very
weak antagonists toward muscarinic M2 receptors.6
Consequently, we hoped that 5-7 would retain the
intrinsic activity of parent compounds 3 and 4 in atrial
tissue while loosing affinity for muscarinic receptors.
Ch em istr y. The compounds used in this investiga-
tion were synthesized by standard procedures as shown
in Scheme 1.10
* Corresponding author: Carlo Melchiorre, Department of Phar-
maceutical Sciences, University of Bologna, Via Belmeloro 6, 40126
Bologna, Italy. Tel: +39-051-2099706. Fax: +39-051-2099734. E-
mail: camelch@kaiser.alma.unibo.it.
Diamine 5 was obtained by reaction of 9 with 2-meth-
oxybenzyl chloride followed by removal of the protecting
group. Diamine 6 and tetraamine 3 were obtained by
† University of Bologna.
‡ University of Florence.
§ Gru¨nenthal GmbH.
10.1021/jm0155594 CCC: $20.00 © 2001 American Chemical Society
Published on Web 10/27/2001

4036 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24
Letters
Ta ble 1. Negative Inotropic Responses, Expressed as pEC50
Values, Induced by Polyamines 3-7, APE, and 8 in Electrically
Paced (1 Hz) Guinea Pig Left Atria
relative efficacy
(APE ) 100)
a
no.
pEC50
slope
APE
3
4
5
8.15 ( 0.03
9.65 ( 0.04
9.40 ( 0.03
9.28 ( 0.06
9.22 ( 0.03
8.89 ( 0.05
9.47 ( 0.03
1.15 ( 0.09
0.72 ( 0.05b
0.70 ( 0.05b
0.75 ( 0.08
1.15 ( 0.09
0.80 ( 0.07
0.98 ( 0.05
100
84
85
75
70
79
82
6
7
8c
a
pEC50 ) -log EC50. EC50 values are the means ( SE of at
least four independent experiments and were calculated by a
nonlinear regression curve-fitting computer program.22 Signifi-
b
cantly different from unity (p < 0.01). c N-Dodecyl lysine amide.21
Ta ble 2. Antagonist Affinities, Expressed as pKB Values, of
Atropine and Tripitramine in Electrically Paced (1 Hz) Guinea
Pig Left Atria Using APE, 3, and 8 as Agonists
a
pKB
compound
APE
3
8b
atropine
tripitramine
9.15 ( 0.11
9.61 ( 0.06
7.18 ( 0.13
7.32 ( 0.04
7.45 ( 0.09
a
The dissociation constants, expressed as pKB values ( SE of
four experiments, were calculated at one antagonist concentration
(0.1 µM; 1 h incubation) by the equation pKB ) - log {[antagonist]/
F igu r e 1. Cumulative concentration-response curves (a) for
APE, 3, and 4 and (b) for APE in the absence and presence of
3 in electrically paced (1 Hz) guinea pig left atria. Each point
is the mean ( SE of four experiments.
b
(DR - 1)}.22 N-Dodecyl lysine amide.21
by APE. Interestingly, concentration-response curves
of 3 and 4 were shallower than that of APE as revealed
by their Hill slope values (0.72 ( 0.05 and 0.70 ( 0.05
for 3 and 4, respectively) that were lower than unity
(Table 1). Moreover, the effects produced by these
compounds, unlike APE, could not be reversed following
extensive washing (up to 3 h) of tissues (data not
shown). This prevented the study of the effect of 3 or 4
and muscarinic antagonists on the same preparation.
To circumvent this problem, one tissue was incubated
with the test antagonist whereas another tissue was
used as control. After the incubation period, concentra-
tion-response curves to 3 were obtained on both prep-
arations, allowing calculation of the EC50 value in the
presence and the absence of the antagonist. The affinity
values obtained for atropine, a nonselective muscarinic
antagonist, and tripitramine, a selective muscarinic M2
receptor antagonist, were totally different from the
affinity observed using APE as agonist (Table 2). This
finding clearly suggests that 3 hardly interacts with the
site where APE and the two antagonists bind.
To clarify the site of action of these novel polyamines
we performed binding assays in CHO-K1 cells express-
ing human cloned muscarinic M1-M5 receptors. It turned
out that both 3 and 4 are effective ligands for muscarinic
receptor subtypes whereas diamines 5 and 6 were not
able to displace [3H]NMS, even at relavtively high
concentrations (Table 3). To verify whether 3 and 4 are
muscarinic agonists the increase of GTPγS-binding in
cell membranes from CHO-K1 cells transfected with
human cloned muscarinic M2 and M4 receptors was
investigated. Both compounds behaved as antagonists
in the dose range that was examined (0.1 nM-100 µM),
whereas carbachol, a muscarinic agonist, showed the
expected increase in GTPγS-binding (Figure 2a). To
detect an allosteric agonism of these substances, a
concentration-response curve to carbachol was deter-
mined in the absence and in the presence of a fixed
alkylation of 5 with formaldehyde/formic acid and 1,8-
dibromooctane, respectively. Amine amide 7 and di-
amine diamide 4 were synthesized by amidation of 5
with acetyl chloride and suberic acid, respectively.
Biology. Functional activity of the compounds under
investigation was determined in driven guinea pig and
rat left atria (1 Hz). Tetraamine 3 was investigated also
in left atria from rats pretreated with pertussis toxin.
These methods have been described in detail earlier.6,10
To allow comparison of the results, arecaidine propargyl
ester (APE), a muscarinic agonist, was used as a positive
control. The biological results are expressed as pEC50
values.
The muscarinic receptor subtype selectivity was as-
sessed by employing receptor binding assays in CHO-
K1 cells expressing human cloned muscarinic M1-M5
receptors as reported previously.11 Methoctramine (2)
was used as standard.
Resu lts a n d Discu ssion . Polyamines 3 and 4 and
their truncated analogues 5-7, unlike 2, did not behave
as muscarinic antagonists in atrial preparations but
showed an unexpected intrinsic activity. These novel
polyamines displayed potent negative inotropic effects
that were even more pronounced than the inotropic
responses elicited by APE as revealed by their pEC50
values (Figure 1a, Table 1). Furthermore, the onset was
definitely different for 3-7 in comparison to APE
because the maximum effect elicited by the addition of
any concentration of drug was reached after at least 30
min incubation of polyamine and after only a few
seconds following APE addition. Similar results were
obtained in tissues from rat (not shown). A slow onset
of action was reported also by others for unexpected
muscarinic agonists with an unusual chemical struc-
ture.12 Apparently, these novel polyamines behaved like
partial muscarinic agonists because their responses
were within 70-85% of the maximal response elicited

Letters
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24 4037
Ta ble 3. Affinity Estimates, Expressed as Ki (nM) Values, of
Polyamines 3-7 and 8 for the Five Human Cloned Muscarinic
Receptor Subtypes Expressed in CHO-K1 Cells
antagonists rather than agonists. Consequently, the
negative inotropic effect observed in atrial preparations
remains to be explained. Thus, our attention was
focused on other targets, among which we considered
the possibility that 3 and 4 could interact directly with
Gi proteins, triggering the negative inotropic response
without the activation of the muscarinic M2 receptor to
which they are coupled. In other words, 3 and 4 might
behave as Gi protein activators. This view is in agree-
ment with the finding that polyamines are able to
interact with G proteins.13-15 The prototype methoctra-
mine (2) was found to activate Gi proteins in mast cells,
while inhibiting Gi protein in pig atrial membrane
preparation enriched in muscarinic M2 receptors.16,17
Since it is known that pertussis toxin uncouples mus-
carinic receptors from inhibiting adenylate cyclase by
alkylating a cysteine residue near the carboxy terminus
of Gi and Go R subunits,18 we have investigated the
inotropic effect elicited by 3 in rat atrial tissue pre-
treated with pertussis toxin. It turned out that 3 was
devoid of activity in atrial preparations from pretreated
animals with pertussis toxin (not shown), as one would
expect if 3 interacts directly with Gi proteins to produce
its effects. This finding also rules out the possibility that
3 might interact with a different target, such as a K+
channel, which is connected to muscarinic M2 receptors
and, consequently, to Gi proteins in atria.19 Clearly, if
3 was able to activate directly K+ channels, pretreat-
ment with pertussis toxin should not affect its negative
inotropic effect in atrial tissue.
Ki,a (nM)
no.
M1
M2
M3
M4
M5
2b
3
49.8 ( 6.2 14.3 ( 2.2 277 ( 27
38.0 ( 3.5 313 ( 22
19.8 ( 9.2 8.6 ( 1.1
108 ( 7.8 9.2 ( 1.0
422 ( 112 15.3 ( 7.5 105 ( 26
97.2 ( 5.6 16.6 ( 3.3 183 ( 80
4
5c
6c
22%
17%
49%
35%
13%
12%
12%
17%
28%
25%
0%
29%
25%
24%
8c,d 17%
a
Ki values are the means ( SE of two to three experiments,
each performed in triplicate, and were calculated from IC50 values
with the equation of Cheng and Prusoff.23
Data taken from
b
ref 18. c Results are the percent reduction of binding of the radio-
labeled ligand with the test compound at a concentration of 10
µM. N-Dodecyl lysine amide.21
d
To gain further insight into the potential target of our
compounds, we used benzalkonium chloride, an inhibi-
tor of Gi proteins,20 to antagonize the effects produced
by 3 and APE (see Supporting Information). The ino-
tropic effect induced by APE was not antagonized by
benzalkonium chloride whereas the responses elicited
by 3 were noncompetitively blocked with an IC50 value
of 22.9 ( 0.6 µM in agreement with the hypothesis that
3 interacts with Gi proteins. However, if 3 is an activator
of Gi proteins, an intriguing question arises: Why does
a compound that directly activates Gi proteins not
increase GTPγS binding (see Figure 2)? A possible
explanation could be that 3 binds to muscarinic M2
receptors with high affinity as well (Table 3). The
interaction of 3 with the receptor would stabilize the
complex between GDP and R, â, and γ subunits of Gi
proteins, preventing the dissociation of the R subunit
from the receptor and from âγ dimer and, as a conse-
quence, the regulatory cycle of Gi proteins. This reason-
ing may find support in the observation that 3 produced
a concentration-response curve with a Hill slope value
significantly lower than unity, which might be the result
of a negative cooperativity by way of a dual mode of
action of 3, that is, antagonism at the receptor and
“agonism” at the Gi protein. The interaction of 3 with
the receptor would induce a conformational change in
Gi protein structure, decreasing its affinity for the
binding site on the Gi protein. Another piece of evidence
supporting the view that 3 may be a Gi protein activator
came from the observation that pretreatment of guinea
pig left atria with 10 pM 3 (a concentration 1000-fold
lower than the observed Ki value at muscarinic M2
receptors) caused a significant leftward shift of the
concentration-response curve to APE (Figure 1b).
F igu r e 2. Effect of carbachol, 3, and 4 on GTPγS binding of
CHO-K1 cells expressing human M2 mAChR. (a) GTPγS
binding in the presence of increasing concentrations of carba-
chol (9), 3 (1), or 4 ([) and of carbachol in the presence of 100
nM 3 (4) or 100 nM 4 (]). (b) GTPγS binding in the presence
of increasing concentrations of carbachol (9) and of 3 (2), 4
(1), or scopolamine ([) in the presence of a fixed concentration
of carbachol (5 µM). Data represent mean values from dupli-
cate determinations.
concentration (that was more than 10 times higher than
the observed Ki values) of 3 and 4. A rightward shift of
the concentration-response curve to carbachol was
observed in both cases, confirming that 3 and 4 behave
like competitive antagonists (Figure 2a). To exclude a
potential allosteric agonism at other concentrations of
3 and 4, we combined a fixed concentration of carbachol
(5 µM) with increasing concentrations of 3 and 4 (0.1
nM-100 µM). It was observed that both compounds
were antagonists, because they were only able to inhibit
the carbachol stimulated GTPγS-binding. Scopolamine
was used as a reference compound and showed the same
behavior of 3 and 4 (Figure 2b).
Taken together, the functional studies in cloned
receptors suggest clearly that 3 and 4 are muscarinic

4038 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24
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Clearly, tetraamine 3, by interacting with Gi proteins,
would stabilize, by way of a conformational change, the
activated state of the receptor thus increasing the
affinity of APE for its site.
To test the hypothesis that 3 is a Gi protein activator
we have investigated (a) diamines 5 and 6 because they
displayed a very weak, if any, affinity for muscarinic
M2 receptors while having a potent inotropic effect in
atrium (Tables 1 and 3) and (b) N-dodecyl lysine amide
(8) because it was reported to be a Gi protein activator.21
Interestingly, 8 showed a potent intrinsic activity
comparable to that of both 5 and 6 and 3 as well (Table
1); this effect was antagonized by benzalkonium chloride
(IC50 ) 35.7 ( 1.1 µM) and the muscarinic antagonists
atropine and tripitramine in a manner similar to that
observed for 3 (Figure 3 and Table 2). Furthermore,
compounds 5, 6, and 8, unlike 3 and 4, produced
concentration-response curves with Hill slope values
not significantly different from unity (Table 1), suggest-
ing that the lack of interaction with muscarinic M2
receptors did not affect the interaction with Gi proteins.
This reasoning rationalizes rather well the results
obtained in functional assays. However, it does not give
an answer to the question of why 5 and 8, which have
almost no affinity for muscarinic receptors in CHO-K1
cells (Table 3), behaved like 3 rather than to give a
synergistic activation of the carbachol signal in GTPγS
binding assays (not shown), as one would expect if they
are supposed to interact directly with Gi proteins. Work
is in progress to gain a better understanding of the
intriguing trends noted above.
In conclusion, although methoctramine (2) is a potent
muscarinic M2 receptor antagonist in both functional
and binding assays, its constrained analogues 3 and 4
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8, a Gi protein activator, but this effect was different
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that four basic nitrogen atoms are not required for
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amine amide 7, obtained by truncating in two halves 3
and 4, respectively, were almost as active as or slightly
less potent than their parent compounds. Interestingly,
diamines 5 and 6 were almost inactive as muscarinic
antagonists. Consequently, appropriate structural modi-
fication of methoctramine structure can afford com-
pounds endowed with affinity for muscarinic receptors
or for different biological targets such as, in the present
case, Gi proteins.
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Ack n ow led gm en t. This research was supported by
grants from the University of Bologna, the European
Community (BMH4-CT97-2395), and MURST.
Su p p or tin g In for m a tion Ava ila ble: Synthesis of com-
pounds 3-7 and effects of benzalkonium chloride antagonism
are available free of charge via the Internet at http://
pubs.acs.org.
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J M0155594