Carbachol dimers as homobivalent modulators of muscarinic receptors

Article abstract of DOI:10.1016/j.bcp.2016.03.012

A series of homodimers of the well-known cholinergic agonist carbachol have been synthesized, showing the two agonist units symmetrically connected through a methylene chain of variable length. The new compounds have been tested on the five cloned muscarinic receptors (hM_1-5) expressed in CHO cells by means of equilibrium binding studies, showing an increase in affinity by rising the number of methylene units up to 7 and 9. Functional experiments on guinea-pig ileum and assessment of ERK1/2 phosphorylation on hM₁, hM₂ and hM₃ on CHO cells have shown that the new compounds are endowed with muscarinic antagonistic properties. Kinetic binding studies have revealed that some of the tested compounds are able to slow the rate of dissociation of NMS, suggesting a bitopic behavior. Docking simulations, performed on the hM₁ and hM₂ receptors, give a sound rationalization of the experimental data revealing how these compounds are able to interact with both orthosteric and allosteric binding sites depending on the length of their connecting chain.

Full text of DOI:10.1016/j.bcp.2016.03.012

Biochemical Pharmacology xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Biochemical Pharmacology
journal homepage: www.elsevier.com/locate/biochempharm
Carbachol dimers as homobivalent modulators of muscarinic receptors
a
b
b
b
a
Rosanna Matucci a, , Marta Nesi , Maria Vittoria Martino , Cristina Bellucci , Dina Manetti , Elisa Ciuti ,
Angelica Mazzolari c, Silvia Dei b, Luca Guandalini b, Elisabetta Teodori b, Giulio Vistoli c,
Maria Novella Romanelli b
⇑
a Department of Neuroscience, Psychology, Drug Research and Child’s Health, Section of Pharmacology and Toxicology, University of Florence, Viale G. Pieraccini 6, 50139 Firenze, Italy
b Department of Neuroscience, Psychology, Drug Research and Child’s Health, Section of Pharmaceutical and Nutraceutical Sciences, University of Florence, Via Ugo Schiff 6,
50019 Sesto Fiorentino, Italy
c Department of Pharmaceutical Sciences, University of Milan, Via Mangiagalli 25, 20133 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of homodimers of the well-known cholinergic agonist carbachol have been synthesized, showing
the two agonist units symmetrically connected through a methylene chain of variable length. The new
compounds have been tested on the five cloned muscarinic receptors (hM1–5) expressed in CHO cells
by means of equilibrium binding studies, showing an increase in affinity by rising the number of methy-
lene units up to 7 and 9. Functional experiments on guinea-pig ileum and assessment of ERK1/2 phospho-
rylation on hM1, hM2 and hM3 on CHO cells have shown that the new compounds are endowed with
muscarinic antagonistic properties. Kinetic binding studies have revealed that some of the tested com-
pounds are able to slow the rate of dissociation of NMS, suggesting a bitopic behavior. Docking simula-
tions, performed on the hM1 and hM2 receptors, give a sound rationalization of the experimental data
revealing how these compounds are able to interact with both orthosteric and allosteric binding sites
depending on the length of their connecting chain.
Received 21 December 2015
Accepted 15 March 2016
Available online xxxx
Chemical compounds studied in this article:
Carbachol chloride (PubChem CID: 5831)
Acetylcholine chloride (PubChem CID:
6060)
Hexamethonium chloride (PubChem CID:
93550)
Gallamine triethiodide (PubChem CID:
6172)
Atropine sulfate (PubChem CID: 64663)
Ó 2016 Elsevier Inc. All rights reserved.
Keywords:
Muscarinic acetylcholine receptor
Carbachol
Bivalent ligand
Allosteric modulation
1. Introduction
disease or schizophrenia, whose activity was associated to many
adverse effects arising from lack of selectivity [3,4]. The discovery
Muscarinic acetylcholine receptors are a group of five mem-
brane proteins (M1–M5) belonging to class A G-Protein Coupled
Receptors (GPCR), involved in a large number of physiological pro-
cesses. Several drugs targeting these proteins are in clinical use for
diseases, such as chronic obstructive pulmonary disease, overac-
tive bladder and Sjögren’s syndrome, but muscarinic modulators
may be potentially useful in several other disorders, inside and
outside the central nervous system [1,2].
So far the main problem in the development of muscarinic mod-
ulators is the high homology found in the M1–M5 orthosteric bind-
ing site. This has hampered the development of drugs such as
xanomeline, a muscarinic agonist potentially useful for Alzheimer’s
of allosteric modulators has opened a new avenue in the develop-
ment of selective drugs, since these compounds interact with sites
which are less conserved within the five muscarinic receptors, thus
allowing a selective modulation of only one subtype [5,6].
New intriguing perspectives have been associated also to the
discovery of dualsteric (bitopic) ligands, i.e. divalent compounds
in which two pharmacophoric units, connected by a suitable
spacer, are able to interact at the same time both with the orthos-
teric site and allosteric binding areas, thus exploiting the favorable
characteristics of both sites [7–10]. Originally divalent ligands have
been designed to study receptor dimerization [11], but in many
instances the length of the linker allowed only the bridging of
two neighboring interaction sites on the same protein [9]. Several
examples of dualsteric molecules have been reported in the litera-
ture, showing interesting properties in terms of subtype selectivity,
⇑
Corresponding author.
E-mail address: rosanna.matucci@unifi.it (R. Matucci).
http://dx.doi.org/10.1016/j.bcp.2016.03.012
0006-2952/Ó 2016 Elsevier Inc. All rights reserved.
Please cite this article in press as: R. Matucci et al., Carbachol dimers as homobivalent modulators of muscarinic receptors, Biochem. Pharmacol. (2016),
http://dx.doi.org/10.1016/j.bcp.2016.03.012

2
R. Matucci et al. / Biochemical Pharmacology xxx (2016) xxx–xxx
such as THRX-160209 [12], or biased signaling, such as iper-8-naph
[13]. In principle, bitopic ligands may interact with the othosteric
or the allosteric binding site, or with both, within a monomeric
receptor [8]; very recently it has been shown a bitopic interaction
also within a dimeric receptor [14].
Atropine sulfate salt monohydrate and (ꢀ)-Scopolamine methyl-
bromide were purchased from Sigma–Aldrich SRL, Milano, Italy;
[3H]N-methylscopolamine chloride specific activity range 2590–
3200 GBq/mmol from Perkin-Elmer Life and Analytical Science,
Monza, Milano, Italy. All other test compounds were purchased
from Sigma–Aldrich SRL (Milano, Italy) unless stated otherwise.
Several muscarinic homodivalent ligands, i.e. divalent com-
pounds carrying two identical pharmacophoric units, have also
been disclosed, such as the M2 selective antagonist methoctramine
[15,16] or the dimers of agonists such as xanomeline [17,18] or
arecaidine propargyl ester (APE) [19]. These compounds displayed
a different range of potency, affinity and intrinsic activity, depend-
ing on the pharmacophoric structure and the linker, as the phar-
macophoric doubling did not always result in an increased
affinity or potency. In addition, homo or heterobivalent ligands
carrying at least one agonist unit were not always endowed with
receptor activation properties (see, for instance, Refs. [20,21]).
On this basis we thought it interesting to see the effect of
homodimerization on carbachol, the well-known cholinergic ago-
nist: the idea underlying this approach was the possibility to bind
two different sites in the same receptor, either orthosteric and
allosteric, or two orthosteric sites in a dimeric receptor. Although
carbachol itself does not display allosteric properties, the allosteric
site of muscarinic receptors can bind compounds carrying choline
residues: gallamine is an important example. Therefore, we
designed a series of compounds where the two agonist units are
symmetrically connected through a methylene chain of variable
length, linking the carbamic nitrogen atoms (general formula A,
compounds 1a–f, 2a–f, Fig. 1A), with the aim to find the optimal
length of the spacer; both tertiary amines and ammonium deriva-
tives were prepared. At first, compounds 1a–e and 2a–e were syn-
thesized, showing an odd number of carbon units (i.e. n = 3, 5, 7, 9,
11; Fig. 1A); later also compounds 1f and 2f (n = 6) were prepared.
To check the importance of the second pharmacophoric unit, 2-
(dimethylamino)ethyl undecylcarbamate 3 and its quaternary
ammonium derivative 4 have been also synthesized and tested.
The compounds were tested for their muscarinic activity in binding
and functional studies; the results have been rationalized by
means of computational methods.
2.2. Reagents and synthetic methods
All chemicals were of the highest quality commercially avail-
able. All melting points were taken on a Büchi apparatus and are
uncorrected (Büchi Italia, Milano, Italy). NMR spectra were
recorded on a Brucker Avance 400 spectrometer (400 MHz for 1H
NMR, 100 MHz for 13C) (Brucker Italia SRL, Milano, Italy). Chro-
matographic separations were performed on a silica gel column
by gravity chromatography (Kieselgel 40, 0.063–0.200 mm; Merck
KGaA, Darmstadt, Germany) or flash chromatography (Kieselgel
40, 0.040–0.063 mm; Merck). Yields are given after purification,
unless differently stated. When reactions were performed under
anhydrous conditions, the mixtures were maintained under nitro-
gen atmosphere. Compounds were named following IUPAC rules as
applied by Reaxys software (www.reaxys.com). In the 1H NMR
spectra the following abbreviations are used: s, singlet; bs, broad
singlet; d, doublet; t, triplet; q, quartet; m, multiplet.
2.2.1. General procedures for the synthesis of bis(2-(dimethylamino)
ethyl)-alkane-a,x-diyldicarbamate (1a–f) and 2-(dimethylamino)
ethyl undecylcarbamate 3
The suitable dicarboxylic acid (n = 5, 7, 9, 11, Fig. 1; 1.5 mmol)
was dissolved in CH2Cl2 (15 ml) and SOCl2 (15 mmol) was added;
the mixture was left stirring at room temperature until it became
clear. The solvent was removed under vacuum, then the residue
was treated with hexane and the solvent removed again: this step
was repeated three times. The residue was dissolved in acetone
(4 mL) and treated with a saturated solution of NaN3 (2 ml); after
0.5 h stirring at room temperature, the mixture is diluted with
H2O and extracted three times with CH2Cl2 (10 ml). Drying
(Na2SO4) and removal of the solvent gave a residue which was
dissolved in toluene (12 ml) and heated to reflux for 4 h; after this
time the transformation of the acyl azide (IR: 2135 cmꢀ1, N3) into
isocyanate (IR: 2275 cmꢀ1, NCO) was complete. The isocyanate
[22] was not isolated; after cooling, dimethylaminoethanol
(3 mmol) was added and the solution was first heated at 60 °C
for 4 h then left stirring at room temperature overnight. The
solvent was removed under vacuum leaving a residue which was
2. Material and methods
2.1. Drugs
The following drugs were used: Carbachol chloride, Acetyl-
choline chloride, Hexamethonium chloride, Gallamine triethiodide,
Fig. 1. (A) Chemical structure of the new compounds synthesized in this work; n = 3 (a), 5 (b), 7 (c), 9 (d), 11 (e), 6 (f) (see also Table 1). (B) Synthetic pathway to obtain
compounds 1a–f, 2a–f, 3 and 4. Reagents and conditions: (a) SOCl2; (b) NaN3, acetone; (c) heating, toluene; (d) Me2NCH2CH2OH; (e) MeI.
Please cite this article in press as: R. Matucci et al., Carbachol dimers as homobivalent modulators of muscarinic receptors, Biochem. Pharmacol. (2016),
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R. Matucci et al. / Biochemical Pharmacology xxx (2016) xxx–xxx
3
dissolved in diethyl ether and filtered. Removal of solvent gave the
desired product, which was purified by flash chromatography,
using abs. EtOH/CH2Cl2/pet. Ether/NH4OH 65:340:60:8 as eluent.
The reaction with dimethylaminoethanol was performed also on
1,3-diisocyanatopropane (n = 3), prepared according to King [23],
and on the commercially-available 1,6-diisocyanatohexane (n = 6)
and 1-undecane isocyanate. The following compounds were
prepared.
bis-(2-(Dimethylamino)ethyl)-propane-1,3-diyldicarbamate
1a: oil; yields: 30%. [1H] NMR (CDCl3) d: 1.58 (quintet, J = 6.2 Hz,
2H, CH2CH2NH); 2.22 (s, 12H, 4CH3); 2.49 (t, J = 5.6 Hz, 4H,
CH2N); 3.15 (q, 4H, J = 6.2 Hz, CH2NH); 4.08 (t, J = 5.6 Hz, 4H,
CH2O); 5.36 (s, 2H, NH) ppm. [13C] NMR (CDCl3, APT) d: 30.28,
37.65 (CH2); 45.08 (CH3); 58.07 (CH2); 61.9 (CH2); 156.81 (CO)
ppm. Anal. (C13H28N4O4); calcd: C 51.30, H 9.27, N 18.41; found:
C 50.96, H 8.94, N 18.08.
bis-(2-(Dimethylamino)ethyl)-pentane-1,3-diyldicarbamate
1b: oil; yields: 23%. [1H] NMR (CDCl3) d: 1.34–1.37 (m, 2H,
CH2CH2CH2NH); 1.47–1.52 (m, 4H, CH2CH2NH); 2.46 (s, 12H,
4CH3); 2.76 (t, J = 5.2 Hz, 4H, CH2N); 3.14–3.18 (m, 4H, CH2NH);
4.23 (t, J = 5.2 Hz, 4H, CH2O); 5.25 (s, 2H, NH) ppm. [13C] NMR
(CDCl3, APT) d: 23.68, 29.43 40.67 (CH2); 45.03 (CH3); 59.08
(CH2); 61.73 (CH2); 156.48 (CO) ppm. Anal. (C15H32N4O4); calcd:
C 54.19, H 9.70, N 16.85; found: C 53.85, H 9.34, N 16.51.
bis-(2-(Dimethylamino)ethyl)-heptane-1,3-diyldicarbamate 1c:
Oil; yields: 22%. [1H] NMR (CDCl3) d: 1.27–1.31 (m, 6H, 3CH2);
1.47–1.49 (m, 4H, CH2CH2NH); 2.54 (s, 12H, 4CH3); 2.86 (t,
J = 5.2 Hz, 4H, CH2N); 3.12–3.17 (m, 4H, CH2NH); 4.27 (t,
J = 5.2 Hz, 4H, CH2O); 5.31 (s, 2H, NH) ppm. [13C] NMR (CDCl3,
APT) d: 26.47, 28.72, 29.70 40.81 (CH2); 45.36 (CH3); 58.10 (CH2);
61.7 (CH2); 156.40 (CO) ppm. Anal. (C17H36N4O4); calcd: C 56.64,
H 10.07, N 15.54; found: C 56.27, H 9.76, N 15.20.
bis (2-(Dimethylamino)ethyl)-nonane-1,3-diyldicarbamate 1d:
low melting solid; yields: 22%. [1H] NMR (CDCl3) d: 1.18–1.24
(m, 10H, 5CH2); 1.40–1.43 (m, 4H, CH2CH2NH); 2.25 (s, 12H,
4CH3); 2.52 (t, J = 5.2 Hz, 4H, CH2N); 3.09–3.13 (m, 4H, CH2NH);
4.08–4.13 (m, 4H, CH2O); 4.85 (s, 2H, NH) ppm. [13C] NMR (CDCl3,
APT) d: 26.66, 29.13, 29.35, 40.99 (CH2); 45.52 (CH3); 58.25 (CH2);
61.94 (CH2); 156.60 (CO) ppm. Anal. (C19H40N4O4); calcd: C 58.73,
H 10.38, N 14.42; found: C 58.39, H 10.02, N 14.47.
2.2.2. General procedure for the synthesis of methiodides
An excess of CH3I was added to a solution of the suitable amine
(1a–f, 3) dissolved in anhydrous diethyl ether (30 ml each mmol of
reagent). The mixture was left stirring at room temperature over-
night in the dark. The solid was collected and dried. By this way
the following compounds were prepared.
bis-(2-(Dimethylamino)ethyl)-propane-1,3-diyldicarbamate
methiodide 2a: solid, m.p. 168–170 °C. Yields: 50%. [1H] NMR
(CD3OD) d: 1.69 (quintet, J = 6.8 Hz, 2H, CH2CH2NH,); 3.18 (t,
J = 6.8 Hz, 4H, CH2NH); 3.22 (s, 18H, 6CH3); 3.70–3.73 (m, 4H,
CH2N); 4.50–4.52 (m, 4H, CH2O) ppm. [13C] NMR (CD3OD, APT) d:
29.27, 37.32 (CH2); 53.21 (CH3); 57.86 (CH2); 65.22 (CH2); 155.90
(CO) ppm. Anal. (C15H34I2N4O4); calcd: C 30.63, H 5.83, N 9.52;
found: C 30.32, H 5.46, N 9.43.
bis-(2-(Dimethylamino)ethyl)-pentane-1,3-diyldicarbamate
methiodide 2b: hygroscopic solid. Yields: 20%. [1H] NMR (CD3OD)
d: 1.36–1.39 (m, 2H, CH2CH2CH2NH); 1.59–1.49 (m, 4H,
CH2CH2NH,); 3.12 (t, 4H, J = 6.8 Hz, CH2NH); 3.24 (s, 18H, 6CH3);
3.70–3.72 (m, 4H, CH2N); 4.48–4.52 (m, 4H, CH2O) ppm. [13C]
NMR (CD3OD, APT) d: 23.61, 29.06, 40.40 (CH2); 53.27 (CH3);
53.31 (CH3); 57.76 (CH2); 65.23 (CH2); 156.50 (CO) ppm. Anal.
(C17H38I2N4O4); calcd: C 33.13, H 6.21, N 9.09; found: C 32.98, H
6.54, N 9.37.
bis-(2-(Dimethylamino)ethyl)-heptane-1,3-diyldicarbamate
methiodide 2c [25]: hygroscopic solid. Yields: 87%. [1H] NMR
(CD3OD) d: 1.32–1.35 (m, 6H, 3CH2); 1.49–1.53 (m, 4H, CH2CH2NH);
3.11 (t, J = 6.8 Hz, 4H, CH2NH); 3.26 (s, 18H, 6CH3); 3.73–3.75 (m,
4H, CH2N); 4.50–4.52 (m, 4H, CH2O) ppm. [13C] NMR (CD3OD,
APT) d: 26.33, 28.60, 29.30, 40.49 (CH2); 53.41 (CH3); 57.83
(CH2); 65.25 (CH2); 156.03 (CO) ppm. Anal. (C19H42I2N4O4); calcd:
C 35.41, H 6.57, N 8.69; found: C 35.09, H 6.44, N 8.82.
bis-(2-(Dimethylamino)ethyl)-nonane-1,3-diyldicarbamate
methiodide 2d: solid, m.p. 80–82 °C. Yields: 26%. [1H] NMR
(CD3OD) d: 1.24–1.31 (m, 10H, 5CH2); 1.46–1.51 (m, 4H,
CH2CH2NH); 3.09 (t, 4H, J = 6.8 Hz, CH2NH); 3.22 (s, 18H,
6CH3); 3.65–3.70 (m, 4H, CH2N); 4.37–4.50 (m, 4H, CH2O)
ppm. [13C] NMR (CD3OD, APT) d: 26.41, 28.91, 29.16, 29.40,
40.53 (CH2); 53.41 (CH3); 57.81 (CH2O); 65.25 (CH2N); 156.50
(CO) ppm. Anal. (C21H46I2N4O4); calcd: C 37.51, H 6.90, N 8.33;
found: C 37.88, H 6.72, N 8.56.
bis (2-(Dimethylamino)ethyl)-undecane-1,3-diyldicarbamate
1e: solid, m.p. 87–89 °C. Yields: 46%. [1H] NMR (CDCl3) d: 1.20–
1.24 (m, 14H, 7CH2); 1.43–1.46 (m, 4H, CH2CH2NH); 2.26 (s, 12H,
4CH3); 2.52 (t, 4H, J = 5.4 Hz, CH2N); 3.05–3.13 (m, 4H, CH2NH);
4.12 (t, 4H, J = 5.4 Hz, CH2O; 4–93 (bs, 1H, NH); 5.63 (bs, 1H, NH)
ppm. [13C] NMR (CDCl3, APT) d: 25.44, 26.62, 29.11, 29.16, 29.29,
29.40, 29.81, 35.86, 40.96 (CH2); 45.44 (CH3); 58.18 (CH2); 61.86
(CH2); 156.6 (CO) ppm. Anal. (C21H44N4O4); calcd: C 60.54, H
10.65, N 13.45; found: C 60.22, H 10.31, N 13.10.
bis-(2-(Dimethylamino)ethyl)-undecane-1,3-diyldicarbamate
methiodide 2e: solid, m.p. 106–108 °C. Yields: 34%. [1H] NMR
(CD3OD) d: 1.26–1.35 (m, 14H, 7CH2); 1.48–1.53 (m, 4H,
CH2CH2NH,); 3.09 (t, 4H, J = 6.8 Hz, CH2NH); 3.22 (s, 18H, 6CH3);
3.67–3.70 (m, 4H, CH2N); 4.45–4.50 (m, 4H, CH2O) ppm. [13C]
NMR (CD3OD) d: 26.41, 28.91, 29.16 (CH2); 29.40 (CH2CH2NH);
40.53 (CH2NH); 53.41 (CH3); 57.81 (CH2O); 65.25 (CH2N); 156.43
(CO) ppm. Anal. (C23H50I2N4O4); calcd: C 39.44, H 7.19, N 8.00;
found: C 39.51, H 6.87, N 8.24.
bis-(2-(Dimethylamino)ethyl)-hexane-1,3-diyldicarbamate 1f
[24]: solid, m.p. 73–75 °C. Yields 87%. [1H] NMR (CDCl3) d: 1.30–
1.36 (m, 4H, CH2CH2CH2CH2NH); 1.46–1.49 (m, 4H, CH2CH2NH);
2.29 (s, 12H, 4CH3); 2.56 (t, 4H, J = 5.6 Hz, CH2N); 3.12–3.17 (m,
4H, CH2NH); 4.15 (t, J = 5.6 Hz, 4H, CH2O); 4.92 (s, 2H, NH) ppm.
bis-(2-(Dimethylamino)ethyl)-hexane-1,3-diyldicarbamate
methiodide 2f [24,25]: solid, m.p. 178–180 °C. Yields: 28%. [1H]
NMR (CD3OD) d: 1.33–1.36 (m, 4H, 2CH2); 1.49–1.53 (m, 4H,
CH2CH2NH,); 3.11 (t, J = 6.8 Hz, 4H, CH2NH); 3.26 (s, 18H, 6CH3);
3.73–3.75 (m, 4H, CH2N); 4.50–4.52 (m, 4H, CH2O) ppm. [13C]
NMR (CD3OD, APT) d: 25.98, 29.28, 40.36 (CH2); 53.4 (CH3); 57.8
(CH2); 65.3 (CH2); 156.0 (CO) ppm. Anal. (C18H40I2N4O4); calcd: C
34.30, H 6.40, N 8.89; found: C 33.98, H 6.56, N 8.50.
2-(Dimethylamino)ethyl undecylcarbamate methiodide 4:
white solid, m.p. 86–88 °C. Yields: 70%. [1H] NMR (CD3OD) d:
0.90 (t, J = 6.8 Hz, 3H, CH3); 1.16–1.29 (m, 16H, 8CH2); 1.48–1.50
(m, 2H, CH2CH2NH); 3.10 (t, J = 7.0, 2H, CH2NH); 3.21 (s, 9H,
CH3N); 3.66–3.68 (m, 2H, CH2N); 4.50 (m, 2H, CH2O) ppm. [13C]
NMR (CD3OD, APT) d: 13.05 (CH3); 22.33, 26.48, 29.33, 31.66,
40.56 (CH2); 53.35 (CH3N); 57.8 (CH2); 65.3 (CH2); 155.89 (CO)
ppm. Anal. (C17H37IN2O2); calcd: C 47.66, H 8.71, N 6.54; found:
C 47.42, H 8.77, N 6.33.
[
13C] NMR (CDCl3) d: 26.43, 29.91, 41.82 (CH2); 45.65 (CH3);
58.43 (CH2); 58.91 (CH2); 155.94 (CO) ppm. Anal. (C16H34N4O4);
calcd: C 55.47, H 9.89, N 16.17; found: C 55.52, H 9.63, N 16.01.
2-(Dimethylamino)ethyl undecylcarbamate 3: hygroscopic
solid; yields: 90%. [1H] NMR (CD3Cl3) d: 0.87 (t, J = 6.8 Hz, 3H,
CH3C); 1.18–1.33 (m, 16H, 8CH2); 1.45–1.49 (m, 2H, CH2CH2NH);
2.30 (s, 6H, CH3N); 2.55 (t, J = 5.4 Hz, 2H, CH2N); 3.13–3.18 (m,
2H, CH2NH); 4.16 (t, J = 5.4 Hz, 2H, CH2O); 4.81 (bs, 1H, NH)
ppm. [13C] NMR (CD3Cl3, APT) d (ppm): 14.09 (CH3); 22.67,
26.75, 29.31. 31.89, 41.06 (CH2); 45.48 (CH3N); 58.25 (CH2);
61.88 (CH2); 155.9 (CO) ppm. Anal. (C16H34N2O2); calcd: C 67.09,
H 11.96, N 9.78; found: C 67.29, H 11.80, N 9.95.
Please cite this article in press as: R. Matucci et al., Carbachol dimers as homobivalent modulators of muscarinic receptors, Biochem. Pharmacol. (2016),
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R. Matucci et al. / Biochemical Pharmacology xxx (2016) xxx–xxx
2.3. Binding assays
scintillation counting (TRI-CARB 1100, Perkin-Elmer Life and Ana-
lytical Science, Monza, Italy). The determination of nonspecific
binding, the filtration method and the counting procedure were
the same in all the [3H]NMS dissociation kinetic assays described
subsequently in this section.
2.3.1. Cell culture and membrane preparation
Chinese Hamster Ovary (CHO) cells stably expressing the
human muscarinic receptors (hM1–hM5) were routinely cultured
in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with
10% fetal bovine serum (FBS), 100 units/ml each of penicillin G and
streptomycin, 4 mM glutamine (Sigma–Aldrich, Milano, Italy),
non-essential aminoacid solution 100x (Sigma–Aldrich, Milano,
2.3.3.2. One point kinetic assays. Off-rate assays were performed to
estimate the affinity of compounds 1b–f, 2b–f, 3, 4 and gallamine
for the [3H]NMS occupied receptor. In subsequent experiments,
designed to investigate the effects of a range of modulator concen-
trations on the [3H]NMS dissociation rate, a ‘‘one point kinetic
assay” approach was used, where the effect on radioligand dissoci-
ation of each test ligand was determined at 0 min and at one time
point, which was chosen to be ca 2.5 dissociation half-lives of [3H]
NMS alone [28]. A high concentration of hM1–hM5 CHO membranes
Italy) and 50 lg/ml of geneticin (Gibco, Grand Iland, NY).
Membrane preparations were conducted as described previ-
ously [26]. Briefly, confluent CHO cell lines were scraped, washed
with PBS buffer (25 mM sodium phosphate, containing 5 mM MgCl2
at pH 7.4) and homogenized for 30 s using an Ultra-Turrax (setting
5) (IKA-Werke GmbH & Co. KG, Staufen, Germany). The pellet was
sedimented 17,000g for 15 min at 4 °C and the membranes were
resuspended in the same buffer, re-homogenized with Ultra-
Turrax and stored at 80 °C. The protein content was measured
according to the method of Bradford [27] using the Bio-Rad protein
assay reagent (Bio-Rad Laboratories, Munchen, Germany).
(50–75
(2 nM) for about 60 min at room temperature. Then, 100
were distributed into tubes which contained 10 l of 100
l
g/ml) was incubated with a high concentration of [3H]NMS
l aliquots
M atro-
l
l
l
pine alone, or in the presence of different concentrations of test
compounds, and diluted in 1 ml total volume of buffer. The time
zero data point was obtained using only 100 ll of the mixture con-
2.3.2. Equilibrium radioligand binding assays
taining membranes plus [3H]NMS. At a fixed time point later
(80 min for hM1, 20 min for hM2, 100 min for hM3, 90 min for
hM4, 70 min for hM5) the samples were filtered as described above.
Radioligand equilibrium binding experiments with membrane
homogenates from CHO cells expressing one of the five muscarinic
subtypes were conducted in a 25 mM PBS containing 5 mM MgCl2
at pH 7.4, at room temperature as described previously [26].
2.4. Functional assays
Briefly, membranes (25–70
[3H]N-methylscopolamine chloride ([3H]NMS) and different con-
centrations (usually 0.1 nM–1000 M) of unlabeled test com-
lg/ml) were incubated with 0.2 nM
2.4.1. Guinea-pig ileum
l
This preparation was set up according to Dei [26] on guinea pig
ileum. The terminal portion of ileum was taken at about 1.5 cm
from the ileum–cecum junction from male guinea-pigs (200–
300 g); segments 2 cm in length were carefully removed, washed
and then mounted isotonically under a tension of 0.5 g in 10 ml
organ baths filled with Krebs–Henseleit solution (composition
mM: NaCl 118.0, KCl 4.7, CaCl2 2.52, KH2PO4 1.18, MgSO4.7H2O
1.18, NaHCO3 25.0 and glucose 11.1), gassed with 95% O2: 5%CO2
and maintained at 37 °C.
pounds in PBS buffer plus MgCl2 in polypropylene 96-well plates
(Sarstedt, Verona, Italy) in a final volume of 0.25 ml for 2 h.
At the end of the binding reaction, free radioligand was sepa-
rated from bounded ligand by rapid filtration through UniFilter
GF/C plates (Perkin-Elmer Life and Analytical Science, Monza, Italy)
using a FilterMate Cell Harvester (Perkin-Elmer Life and Analytical
Science, Monza, Italy). After filtration, the filters were washed sev-
eral times with ice-cold milliQ water and dried at room tempera-
ture under air flow. 25
l
l of scintillation liquid Microscint 20
After one hour to allow for equilibration, non-cumulative con-
centration–response curves to carbachol (CCh), acetylcholine
(ACh) and the new compounds were constructed. The tissues were
exposed to drugs for 30 s until maximum contraction was reached
and then washed. The contractions were recorded by means of a
force displacement transducer (Ugo Basile SRL, Varese, Italy) con-
nected to the recorder Unirecord 7050 (Ugo Basile SRL, Varese,
Italy).
(Perkin-Elmer Life and Analytical Science, Monza, Italy) was added
to each well and the quantity of membrane-bound radioligand was
measured by TopCount NXT Microplate Scintillation Counter
(Perkin-Elmer Life and Analytical Science, Monza, Italy) after 4 h.
Stock solutions of tested compounds were made in DMSO, and
dilutions were usually made in the incubation buffer. DMSO at the
highest concentration used (10%) had no effect on binding. In all
radioligand binding assays nonspecific binding was defined using
The responsiveness of each piece of tissue was assessed by con-
sidering the responses to the same concentration of ACh or CCh at
the beginning and end of the experiment. Agonist efficacy
10 lM atropine. In all assays, the total binding was always less
than 10% of the total counts added.
(expressed as pD2 = ꢀlogEC50
) and maximum response were
2.3.3. Dissociation kinetic assays
determined.
2.3.3.1. Full time course. For these experiments, a high concentra-
When the compounds were tested as antagonists, a dose–
response curve to the agonists ACh was repeated after 45 min incu-
bation with the test compounds (1 lM). Antagonist potency was
expressed as pKB (for the definition of pKB see Section 2.6.2).
All animal experiments were conducted in accordance with the
principles for the care and use of laboratory animals for scientific
purposes contained in the European Union regulations (Directive
2010/63/EU).
tion of CHO cell membranes (50–100
2 nM [3H]NMS in PBS buffer for 60 min at room temperature. Then,
100 l aliquots of this mixture were distributed into tubes, which
contained 10 l of 100 M atropine to prevent radioligand reasso-
lg/ml) was equilibrated with
l
l
l
ciation to receptors (final total volume 1 ml), at various time points
(0, 1, 10, 20, 40, 80 and 160 min). At the appropriate time, samples
were filtered. Incubation was terminated by rapid filtration
through Whatman GF/C filters (Brandel, Gaithersburg, MD, USA),
that had been presoaked in a 0.05% polyethylenimine (PEI) solution
for at least 1 h, using a Brandell cell harvester (Biomedical Research
and Development Laboratory, Inc Atlas Drive, Gaithersburg, MD,
USA). Filters were washed three times with 3 ml aliquots of ice-
cold milliQ water and dried before the addition of 4.5 ml of scintil-
lation cocktail (Filter Count, Perkin-Elmer Life and Analytical
Science, Monza, Italy). The radioactivity was determined using
2.4.2. Extracellular Signal-Regulated Kinase (ERK1/2) Phosphorylation
Assays
These assays were performed using the Cellul’ERK kit (Cisbio
Bioassays, Codolet, France). CHO cells stably transfected with the
human muscarinic receptors (M1–M3) were plated into 96-well
plates (Sarstedt, Verona, Italy) at a density of 40,000 cells/well
and grown for at least 4 h in DMEM medium containing 10% FBS,
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R. Matucci et al. / Biochemical Pharmacology xxx (2016) xxx–xxx
5
100 U/ml penicillin, and 100
l
g/ml streptomycin, with
within the first hM2 structure and the hM1 homology model was
focused on a region obtained combining a 10.0 Å radius sphere
around Asp105 (hM1) or Asp103 (hM2) plus a 10.0 Å radius sphere
around Tyr179 (hM1) or Tyr177 (hM2) thus completely encompass-
ing both binding cavities. In contrast, the search within the second
hM2 structure was focused on a region obtained combining a
10.0 Å radius sphere around Tyr177 keeping the bound QNB antag-
onist. For each ligand, speed 1 was used and 10 poses were gener-
ated and scored using the ChemPlp function. The so obtained best
complexes were finally optimized by a minimization keeping all
atoms fixed apart from those included within a 10.0 Å radius
sphere around the bound ligand.
L-glutamine as required for the cell line used. Cells were then
washed twice with PBS and were starved by replacing the cell
growth media with similar media that did not contain FBS and
incubating at 37 °C overnight to allow FBS-stimulated phosphory-
lated ERK1/2 levels to subside.
For stimulation experiments, agonists were diluted into FBS-
free media and applied to the cells for 5–8 min. For inhibition
experiments, a similar protocol was followed, but the cells were
preincubated with the inhibitor for 30 min prior to stimulation.
The Cellul’ERK kit assay was performed in accordance with the
manufacturer’s instructions, using only reagents supplied with the
kit. Briefly, media containing the compounds were removed from
96-well plates, 50
ll of lysis buffer was added to the wells and
2.6. Data analysis
the plates were incubated for 30 min at room temperature with
shaking to lyse the cells.
2.6.1. Binding assays
Lysate mixture (16
(Perkin-Elmer Life and Analytical Science, Monza, Italy), then con-
jugate working solution (4 l) was added and the plate was sealed,
wrapped in metal foil to shield from light, and incubated for 2 h at
room temperature. The fluorescence signal in the wells was deter-
mined with a Flex Station 3 Microplate Reader (Molecular Devices,
Sunnyvale, CA, USA).
All data were expressed as a percentage of ERK1/2 phosphory-
lation produced after a 5 min exposure to Dulbecco’s modified
Eagle’s medium containing 10% FBS.
l
l) was transferred to a 384-well Proxiplate
Data are presented as mean S.E.M., unless otherwise noted.
Data from equilibrium binding studies were corrected for non
specific binding and were analyzed by computer-aided nonlinear
regression analysis using a four parameter logistic equation in
GraphPad Prism 5.02 (GraphPad Software, San Diego, CA). IC50 val-
ues were, irrespective of the slope factor of the curve, converted to
binding constants Ki using the Cheng–Prusoff correction.
Radioligand dissociation rates were analyzed by nonlinear
regression according to the following equation for mono-
exponential decay using GraphPad Prism 5.02:
l
Y ¼ Y0 ꢁ expðꢀk ꢁ XÞ þ Plateau
ð1Þ
2.5. Computational studies
where Y0 is the specific binding of the radioligand at time = 0, before
dissociation was initiated. Y is bound radioligand (specific binding)
after dissociation for time X. The equation starts at (Y0 + Plateau) at
time = 0 and decays to Plateau with a rate constant k. The half-life is
0.69/k. k is the koff and denotes the observed radioligand dissocia-
tion rate constant in the absence or presence (koffobs) of the allos-
teric modulator. For monoexponential and fully reversible binding
it is predicted that the curve would decay to Plateau = 0.
Docking simulations involved the recently resolved structures
of the hM2 subtype in complex with both the agonist iperoxo
and the allosteric modulator LY2119620 (PDB Id: 4MQT) as well
as in complex with the antagonists QNB (PDB Id: 3UON). The
choice of the first hM2 structure is justified by the bound ligands
which should assure that both binding cavities (allosteric and
orthosteric sites) are finely optimized for ligand recognition. The
second hM2 structure was selected to simulate receptor conditions
similar to those experienced during the kinetic experiments. Again,
docking studies involved the hM1 (Entry Id: P11229, Entry name:
ACM1_HUMAN) homology model as generated using the first
hM2 structure as the template. Briefly, the homology modeling
was performed by Modeller 9.10 using the default parameters
[29]; among the 20 generated models, the best structure was
selected according to the computed scores (i.e. DOPE and GA341)
as well as to the percentage of residues falling in the allowed
regions of the Ramachandran (91.2%) and chi plots (95.8%). The
completed model was carefully checked to avoid unphysical occur-
rences such as cis peptide bonds, wrong configurations, improper
bond lengths, non-planar aromatic rings or colliding side-chains.
The so obtained hM1 and hM2 structures were then completed
by adding hydrogen atoms and to remain compatible with physio-
logical pH, Asp, Glu, Lys and Arg residues were considered in their
ionized form while His and Cys were maintained neutral by
default. Finally, the structures were optimized by a minimization
made up by two phases: a first minimization without constraints
until RMS = 0.1 kcal molꢀ1 Åꢀ1 and then a second minimization
with backbone fixed until RMS = 0.01 kcal molꢀ1 Åꢀ1 to preserve
their folding.
According to the detailed protocol developed by [28], one point
kinetic data were analyzed in order to obtain estimates of the affin-
ity of an allosteric agent for the [3H]NMS-occupied receptor (Kocc
)
in a single step. Eq. (1) was modified by substituting k (=koffobs) by:
K
off =ð1 þ Kocc ꢁ XÞ ð2Þ
where koff is the dissociation rate constant of the radioligand in the
absence of X. X refers to the log concentration of the allosteric ligand
and logKocc is its log affinity constant for the occupied receptor. The
resulting equation directly gives the values of [3H]NMS rate con-
stants in the absence (koff) and in the presence of allosteric modula-
tors (koffobs) and an estimate of logKocc by analyzing the amount of
[3H]NMS remaining at certain times at different concentrations of
the allosteric modulators (see [28]).
2.6.2. Functional assays
Results are given as mean SEM of n experiments and a two-
tailed Student’s t-test was used for comparison of mean values.
The agonist concentration–response curves were analyzed using
computer GraphPad Prism 5.02 (GraphPad Software, San Diego,
CA) that fits the data directly with a logistic function, providing
the EC50 value (the concentration required for an agonist to pro-
duce a half-maximal response: ꢀlogEC50 = pD2), the maximum
response (Emax).
The antagonist potency (pKB) was estimated using the following
Eq. (3) [32] where a single concentration of antagonist was used.
Agonist EC50 values in the absence and presence of antagonists
were determined graphically for the calculation of dose ratios (DR).
All ligands were simulated in their protonated state since this is
involved in receptor recognition. The conformational profile was
investigated by MonteCarlo simulations (as implemented in the
VEGA program [30], which produced 1000 minimized conforma-
tions by randomly rotating the rotatable bonds and the so com-
puted lowest energy structure underwent docking simulations.
Docking simulations were carried out using PLANTS [31] with
default settings and without geometric constraints. The search
pKB ¼ logðDR ꢀ 1Þ ꢀ logðconcentration of antagonistÞ
ð3Þ
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6
R. Matucci et al. / Biochemical Pharmacology xxx (2016) xxx–xxx
range as the orthosteric ligand CCh; on the contrary compounds
3. Results
3.1. Chemistry
with n P 7 have pKi values higher than 7 with few exceptions
(1c, n = 7 and 1d, n = 9, on hM2, and 1e, n = 11 on hM2, hM3, hM4
and hM5). Actually, two-methylene elongation of the spacer brings
about a difference in affinity which is usually below 1 log unit,
except when going from n = 5–7.
This finding prompted us to synthesize and test the analogs
with n = 6, 1f and 2f, which were found able to fully inhibit the
binding of [3H]NMS to hM1–hM5 receptors, with pKi values inter-
mediate between those of the compounds with 5 and 7 methylene
units (Table 1).
In order to determine if the second carbachol unit could affect
the affinity to muscarinic receptors, compound 3 and its methio-
dide 4, carrying on the carbamic nitrogen atom a long but not func-
tionalized alkyl chain, has been prepared and tested: their pKi
values (Table 1) were found in the same range as those of the biva-
lent compounds with long chain (n P 7). Similarly to their analogs,
they were not able to discriminate among receptor subtypes.
Compounds 1a–f and 3 were obtained by reacting the suitable
isocyanate or diisocyanate with dimethylaminoethanol as shown
in Fig. 1B. 1,6-Diisocyanatohexane and 1-isocyanatoundecane
were commercially available; 1,3-diisocyanatopropane was
obtained according to King [23], while the other diisocyanates
were prepared from commercially available alkanedicarboxylic
acids, which were transformed into the acyl azides by reaction first
with thionyl chloride and then with sodium azide in acetone; Cur-
tius rearrangement gave the desired compounds. Reaction of car-
bamates 1a–f and 3 with methyl iodide gave the corresponding
quaternary ammonium analogs 2a–f and 4, respectively.
3.2. [3H]N-methyl scopolamine equilibrium binding studies
The compounds were tested in equilibrium binding studies on
membrane preparations from CHO-K1 cells stably expressing the
five human muscarinic receptors (hM1–hM5), using [3H]NMS as
radioligand and carbachol (CCh) as reference [26].
3.3. Functional studies
All the compounds displaced, in a concentration-dependent
manner, 0.2 nM [3H]NMS from the five mAChRs, with different
potency but without subtype selectivity (pKi values are shown in
Table 1).
3.3.1. Isolated guinea-pig ileum
Preliminary evaluation of the functional muscarinic properties
of compounds 1a–f and 2a–f was assessed on isolated guinea pig
ileum, a functional model readily available to us, according to an
already published protocol [33].
Compounds 1a,b, 2a,b and 2f (n = 3–6) evoked weak smooth
muscle contractions, even at the highest concentration tested
Compounds 1c–e and 2c–e strongly and completely inhibited
the binding of 0.2 nM [3H]NMS, while 1a,b and 2a,b fully displaced
the radioligand only from some subtypes, even at the highest con-
centrations tested (1 mM), showing pKi values <5. As an example,
Fig. 2 shows the inhibition curves of the methiodides 2a–e, mea-
sured on hM1 (Fig. 2A) and hM2 (Fig. 2B); CCh is used as reference.
The hM3–hM5 subtypes gave similar graphs (data not shown).
Table 1 shows that affinity is usually higher for methiodides
with respect to tertiary amines, although the difference is below
one log unit, and increases with the length of the methylene chain
up to n = 7 or n = 9, depending on the receptor subtype or on the
(100 lM) (Table 1); only 2b (n = 5) and 2f (n = 6) displayed a
dose-dependent effect (pD2 = 4.93 0.09 and pD2 = 5.10 0.10,
respectively, Fig 4A). Compound 1f (n = 6) as well as the higher
homologs (n P 7, both amines and methiodides) were totally
unable to contract guinea pig smooth muscle preparation.
In order to verify the involvement of the muscarinic system in
the agonistic effect of compounds 2b and 2f, dose–response curves
were performed after 30 min preincubation with atropine 1 nM.
While, as expected [34,35], atropine completely inhibited ACh
and CCh induced contractions, it failed to significantly modify the
contractile response induced by 2b and 2f, which, on the contrary,
amine–ammonium functionality, with
a slight decline when
n = 11; at hM2 receptor subtype the lowest affinities were revealed.
Interestingly, compounds 1a–e and 2a–e can be divided into
two groups according to their pKi values (Fig. 3A and B). The affin-
ity of compounds 1a,b and 2a,b (n 6 5) is low, although in the same
was prevented by pretreatment with 30
not shown).
lM hexamethonium (data
Table 1
Equilibrium binding affinity and functional activity of the tested compounds. Inhibition binding constants (pKi) of the compounds for human cloned muscarinic receptors
expressed in CHO-K1 cells membranes. Agonist or antagonist potencies (pD2 or pKB, respectively) from functional tests on guinea-pig ileum preparations. Values are reported as
mean of 3–4 experiments. n.a. = not active.
n
X
hM1
hM2
hM3
hM4
hM5
Guinea pig ileum
1a
2a
1b
2b
1c
2c
1d
2d
1e
2e
1f
2f
3
4
3
3
5
5
7
7
9
9
11
11
6
6
–
NMe2
NMe3I
NMe2
NMe3I
NMe2
NMe3I
NMe2
NMe3I
NMe2
NMe3I
NMe2
NMe3I
NMe2
NMe3I
4.68 0.08
4.91 0.04
4.98 0.11
4.95 0.07
7.45 0.05
7.75 0.05
7.56 0.05
7.93 0.03
7.16 0.06
7.59 0.05
6.18 0.05
6.16 0.04
7.50 0.04
8.19 0.02
4.42 0.01
4.14 0.08
4.65 0.04
4.84 0.06
4.95 0.05
6.42 0.07
7.20 0.05
6.53 0.06
7.35 0.04
6.29 0.05
7.38 0.04
5.37 0.03
5.73 0.03
6.80 0.05
7.73 0.04
5.92 0.07
<4
<4
<4
<4
<4
<4
pD2 < 4
pD2 < 4
pD2 < 4
4.46 0.10
4.79 0.07
7.76 0.05
8.14 0.04
7.89 0.05
8.02 0.02
6.89 0.04
7.50 0.04
6.16 0.07
6.28 0.04
7.34 0.04
7.85 0.05
4.36 0.01
4.24 0.08
<4
4.41 0.09
4.84 0.07
8.01 0.05
8.19 0.03
7.68 0.05
7.91 0.06
6.76 0.05
7.35 0.03
6.12 0.06
6.36 0.05
7.30 0.02
8.00 0.05
4.16 0.09
pD2 = 4.93 0.09
pKB = 5.61 0.12
pKB = 6.82 0.13
pKB = 5.31 0.08
pKB = 6.66 0.05
pKB = 5.33 0.03
pKB = 6.67 0.09
n.a.
pD2 = 5.10 0.10
pKB = 6.40 0.10
pKB = 7.41 0.12
pD2 = 6.68 0.01
7.27 0.04
7.70 0.04
7.48 0.04
7.78 0.05
6.80 0.09
7.49 0.06
5.78 0.06
5.87 0.05
7.05 0.03
7.69 0.04
5.20 0.07
–
Carbachol
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R. Matucci et al. / Biochemical Pharmacology xxx (2016) xxx–xxx
7
Fig. 2. Inhibition of [3H]NMS specific binding by CCh and 2a–f in membrane homogenates of CHO cells stably expressing the hM1 (A) and hM2 (B) mAChRs. Curves from single
experiments were fitted using the standard four parameter logistic equation and are the mean S.E.M. from three to six independent experiments.
Fig. 3. The pKi values of compounds 1a–f (A) and 2a–f (B) from the equilibrium binding studies are shown as a function of the length of the spacer. Values represent the
mean S.E.M. from three to six independent experiments and the error bars are smaller than symbols used.
Fig. 4. (A) Concentration–response curve for contractions evoked by 2b (n = 5) and 2f (n = 6) in guinea-pig ileum. (B) Inhibition effect of 1 lM 2d on ACh induced contractions
in guinea-pig ileum. The effect is expressed as percentage of the maximum, normalized to the maximum response given by carbachol in the same experiment (100% of
contraction). The data points represent the mean SEM of 2–4 experiments.
In order to assess the functional behavior of compounds with
n P 7, as well as that of 1f, ACh dose–response curves were con-
ducted in the absence (pD2 = 7.84 0.26) and in the presence of
Surprisingly, compound 1f, despite its micromolar affinity
assessed in radioligand binding studies, did not show functional
activity in this test.
fixed concentrations of these molecules (1–30 lM). Compounds
1c–e and 2c–e behaved as antagonists, shifting rightward the
dose–response curves of ACh (a representative dose–response
curve is reported in Fig. 4B for 2d): their potency is reported in
Table 1 as pKB calculated according to the Van Rossum equation
[32]. Quaternary ammonium compounds 2c–e were about one
order of magnitude more potent than the corresponding tertiary
amines 1c–e. The pKB values reported in Table 1 show that the
antagonist potency of amines 1c–e and methiodides 2c–e-does
not depend on the chain length.
In the same test, also N-undecanyl derivatives 3 and 4 behaved
as antagonist, as they were able to shift rightward in a parallel way
ACh dose–response curves, with and pKB values of 6.40 0.10 and
7.41 0.12, respectively (Table 1).
3.3.2. ERK1/2 phosphorylation assays
To further investigate functional activity, avoiding the
interference of nicotinic receptors, selected compounds (2b, 2c,
2f, 4 and 1f) were evaluated in the ERK1/2 phosphorylation assay
in intact CHO cells stably expressing M1, M2, and M3 human
mAChRs.
At first, each compound (10–100 lM) was tested alone to assess
intrinsic agonism. ACh (10 nM), taken as reference, mediated a
robust stimulation of ERK1/2 phosphorylation, that peaked at
5 min in CHO cells and was prevented by preincubation with
10 lM atropine. In contrast, none of the tested compounds stimu-
lated ERK1/2 phosphorylation, failing to elicit a functional response
at the hM1–hM3 mAChRs (data not shown).
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R. Matucci et al. / Biochemical Pharmacology xxx (2016) xxx–xxx
Given the lack of agonist activity of these compounds, we inves-
tigated their antagonist properties toward ACh. The compounds
were preincubated for 30 min at 100 M before adding ACh
l
10 nM [36]. Fig. 5 shows the results on hM1 receptors: 2c and 4
confirmed the antagonistic behavior shown on guinea-pig ileum
(Table 1), completely preventing ACh-induced ERK1/2 phosphory-
lation (comparable effect to that of atropine); 2b, 2f and also 1f
showed a significant reduction. Analogous results were found also
in hM2 and hM3 receptor subtypes (data not shown), pointing to a
muscarinic antagonistic profile of this class of compounds.
Fig. 6. Representative one point kinetic assay curves of the dissociation of [3H]NMS
3.4. Kinetic binding studies
(0.2 nM) from hM1–5 receptor subtypes. Re-association is blocked with an excess of
atropine (10 lM). Data shown are the mean SEM from 3 to 11 experiments
performed in duplicate.
To study the potential interactions of this series of molecules
with a secondary site on muscarinic receptors, kinetic binding
experiments were performed. Since it is known that ligands that
bind to a secondary (allosteric) site on GPCRs modify the dissocia-
tion rate of the ligand bound to the primary (orthosteric) site [7],
the ability of the tested compounds to affect [3H]NMS dissociation
rate was investigated.
data; the parameters obtained for all tested compounds are sum-
marized in Table 3.
Like most other allosteric agents at muscarinic receptors, gal-
lamine inhibited the dissociation of [3H]NMS from all receptor sub-
types almost completely and so gave rise to an increase in residual
[3H]NMS binding at the given time point (Fig. 7). Log Kocc values are
in good agreement with the literature, confirming the preference
for hM2 subtype (logKocc 5.16 0.06) [37]. The orthosteric agonist
CCh was tested as negative control and, as expected, it didn’t pro-
duce any effect on the dissociation of [3H]NMS.
In general, all tested compounds, although with different and in
some cases very low potency, are able to slow down the rate of dis-
sociation of [3H]NMS at all subtypes. Compounds 1b–e and 2b–e
reveal higher logKocc values for hM1, hM2 and hM4 with respect
to hM3 and hM5 subtypes. The affinity of the tested compounds
for the occupied receptor increases with linker’s elongation, this
effect being more evident for the tertiary amines compared to
the corresponding quaternary ammonium analogs (Fig. 8). On the
contrary, 1f and 2f, both carrying a hexamethylene chain, display
low affinity for the allosteric site at all five subtypes (Table 3), devi-
ating from the trend shown in Fig. 8. In addition, also the monova-
lent molecules 3 and 4, carrying only one carbachol unit, display a
different behavior, presenting lower affinity for hM1 compared to
hM2–hM5 (Table 3).
To determine the time course of [3H]NMS dissociation, prelim-
inary assays in full time mode were performed as described in the
Methods section (Fig. 6). The mean rate of dissociation of [3H]NMS
(koff) and the mean half-life of dissociation (t1/2) were calculated
for each receptor subtype and their values are reported in Table 2.
Since [3H]NMS dissociation was found to follow a monoexpo-
nential time course, a one point kinetic protocol was applied
[28]. The residual binding of [3H]NMS at only one time point was
measured in the absence and presence of different concentrations
of putative allosteric ligands and a single-point estimate of [3H]
NMS (koff) was determined.
The incubation times necessary to equilibrate [3H]NMS binding
to all five muscarinic subtypes in the presence of an allosteric mod-
ulator were calculated around 2–3 times the estimated half-lives
reported in Table 2: 80 min (hM1), 20 min (hM2), 100 min (hM3),
90 min (hM4) and 70 min (hM5). Gallamine, CCh and the synthe-
sized compounds were tested at several concentrations (0.01–
100 lM), to assess their potency in inhibiting the radioligand dis-
sociation from the five receptor subtypes. Untransformed data
were analyzed in order to obtain estimates of the affinity of an
allosteric agent for the [3H]NMS-occupied receptor (logKocc) in a
single step. Representative graphs of the one point kinetic assay
experiment for Gallamine and CCh (Fig. 7A and B, for hM1 and
hM2 subtypes respectively) show the good fit to the experimental
3.5. Docking studies
To rationalize the results of binding and functional experiments,
docking studies were performed on the recently resolved hM2
structure in complex with the agonist iperoxo and the allosteric
modulator LY2119620 [38], for both ammonium derivatives 2a–f
and ammines 1a–f in the protonated form; since both classes inter-
act in a similar way, only the methiodides are discussed here. In
detail, the short-chain derivatives (n = 3 or 5) can assume two
distinct binding modes.
In the first pose (as shown in Fig. 9A for 2a, n = 3), the ligands
are completely accommodated in the orthosteric cavity, where
they stabilize the following set of interactions: (i) one ammonium
head is engaged in the key ion-pairing with Asp103, reinforced by a
set of charge transfer interactions with surrounding aromatic resi-
dues (e.g., Tyr80, Trp99, Tyr426, Tyr430); (ii) the two carbamate
moieties elicit clear H-bonds with Tyr104, Ser107 and Tyr403
and (iii) the second ammonium head approaches Asn108,
Phe195, Trp400, and Asn404. In the second binding mode, the
ligands assume a central pose by which they contact both the
orthosteric and the allosteric sites. Specifically, both ammonium
heads are engaged in ion-pairs, one with Asp103 in the orthosteric
cavity and the other with Glu172 and Glu175 in the allosteric site,
Fig. 5. Determination of ERK1/2 phosphorylation in intact cells stably transfected
with hM1 subtype receptors. Concentrations used are: ACh (10 nM), tested
compounds (100 lM) and atropine (10 lM). Data are normalized to the response
mediated by 10% serum and are presented as mean SEM of 3–6 experiments, each
one performed in quadruplicate. Significantly different (*p 6 0.0001, ^p 6 0.01) from
the control value ACh 10 nM.
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R. Matucci et al. / Biochemical Pharmacology xxx (2016) xxx–xxx
9
Table 2
Half-time dissociation (t½) of [3H]NMS and rate constants of dissociation (koff) observed at hM1–5 receptor subtypes. Values are the mean SEM derived from 3 to 11 experiments
performed in duplicate.
hM1
34.95 1.98
0.020 0.001
hM2
7.09 0.28
0.098 0.004
hM3
36.64 0.64
0.019 0.000
hM4
36.43 1.99
0.019 0.001
hM5
23.71 3.13
0.030 0.004
t½ (min)
koff (minꢀ1
)
the latter being further reinforced by charge transfer interactions
with Tyr177. The two carbamate moieties elicit again H-bond
interactions, but with Tyr426 and Asn419 in the orthosteric and
the allosteric sites, respectively. For the short-chain derivatives,
the first binding mode is markedly favored in terms of both calcu-
lated docking scores and relative abundance among the computed
poses. This finding is in agreement with the affinity of these com-
pounds which are comparable with that of carbachol thus under-
lining their incapacity to conveniently interact with both sites.
When extending the linker, the first binding mode becomes
progressively less favored to disappear for n = 6 due to obvious
steric hindrance. As exemplified in Fig. 9B for 2d (n = 9), the
long-chain analogs show only the second binding mode and
assume a deeper pose compared to that shown by short-chain
derivatives by which the dimethylaminoethylcarbamate moiety
within the orthosteric cavity contacts Asn404 while retaining the
already mentioned interactions with Asp103 plus the surrounding
aromatic residues. The interactions established by the second car-
bachol unit, accommodated in the allosteric site, are similar to
those already seen for the short-chain analogs, consisting in ion
pairing between the ammonium moiety and Glu172 and Glu175,
reinforced by a set of H-bonds with Tyr83, Tyr177, Asn419. How-
ever, it is worth noting that all derivatives, when assuming this
second binding mode, are unable to insert the carbachol unit into
the orthosteric cavity in a pose comparable to that shown by
CCh: as a matter of fact, the linker constrains the dimethy-
laminoethylcarbamate moiety in an inverted arrangement which
prevents the carbamate group to contact Asn404. In such a second
binding mode the linker is inserted in a constrained channel lined
by aromatic residues. Compounds 3 and 4, endowed with only one
carbachol moiety, insert it into the orthosteric site where it stabi-
lizes the same interactions previously described for long-chain
derivatives while the alkyl chain reaches the allosteric site where
it can at most establish hydrophobic contacts. Taken together,
these results can provide an explanation for the antagonistic
Fig. 7. Representative graphs of the one point kinetic assay for the determination of
the affinity of the ligands gallamine and CCh at [3H]NMS-occupied hM1 (A) and hM2
(B) receptor. These graphs show the increase in the [3H]NMS bound to the receptor
as the concentrations of the modulators increase. This is due to the reduction of the
rate of the dissociation of [3H]NMS. The range of concentrations used for the agents
was 0.01–100 lM in addition of 10 lM of atropine. The data point at log[agent]
= ꢀ1 represents the [3H]NMS bound in the absence of added atropine and
muscarinic ligand. The curves represent the non-linear square fits to the experi-
mental data using an equation (Section 2.6) that estimates the affinity when the
receptor is occupied by an orthosteric ligand.
Table 3
Log affinity values (logKocc) for the tested compounds for the [3H]NMS-occupied muscarinic receptors obtained from elaboration with Eq. (2) of experimental data. Values are
reported as mean SEM of at least three experiments performed in duplicate. (ND, not detectable).
n
X
hM1
hM2
hM3
hM4
hM5
1b
2b
1c
2c
1d
2d
1e
2e
1f
2f
3
4
5
5
7
7
9
9
11
11
6
NMe2
NMe3I
NMe2
NMe3I
NMe2
NMe3I
NMe2
NMe3I
NMe2
NMe3I
NMe2
NMe3I
3.43 0.05
4.04 0.03
4.24 0.07
4.11 0.05
5.00 0.09
5.25 0.12
5.05 0.22
5.43 0.08
3.13 0.14
3.61 0.06
3.92 0.15
3.84 0.06
4.11 0.06
ND
3.71 0.11
4.64 0.05
4.57 0.09
4.62 0.07
4.76 0.05
4.98 0.10
4.72 0.11
5.12 0.15
3.00 0.08
3.95 0.07
4.02 0.17
4.46 0.09
5.16 0.06
ND
3.36 0.10
3.58 0.15
3.73 0.09
3.71 0.10
4.14 0.04
4.34 0.02
3.43 0.16
4.10 0.07
3.50 0.07
3.66 0.04
4.19 0.19
4.40 0.06
4.19 0.06
ND
3.36 0.14
4.34 0.05
4.14 0.03
4.15 0.04
5.54 0.16
5.36 0.14
5.16 0.25
5.50 0.12
3.00 0.12
3.58 0.06
4.29 0.08
4.57 0.15
4.56 0.08
ND
3.39 0.09
3.41 0.11
3.69 0.05
3.53 0.10
3.94 0.08
4.31 0.03
3.76 0.04
4.35 0.03
3.62 0.04
3.59 0.08
4.27 0.07
4.40 0.04
4.30 0.07
ND
6
–
–
Gallamine
Carbachol
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R. Matucci et al. / Biochemical Pharmacology xxx (2016) xxx–xxx
Fig. 8. Relationship between logKocc values and linker’s length. Connecting lines were drawn for hM1–5 mAChR to visualize trends (A, tertiary amines; B, quaternary
ammonium compounds).
Fig. 9. Docking poses of selected ligands into the hM1 and hM2 receptors. A: 2a (n = 3) within the orthosteric cavity of hM2. B: 2d (n = 9) docked between the orthosteric and
allosteric sites of hM2. C: 2d docked between the orthosteric and allosteric sites of hM1. D: 2b (n = 5) into the allosteric site of the NMS-occupied hM2 receptor.
activity of the long-chain derivatives, since they do achieve a rich
interaction pattern while being unable to elicit the key contacts
usually established by the agonists within the orthosteric site.
Since the compounds show higher affinity for the hM1 receptor,
compared to hM2 subtype, docking simulations were also per-
formed on a hM1 homology model as generated using the above
mentioned recently resolved hM2 structure as the template. Also
on this subtype, docking studies suggest significant differences
on the binding mode of the compounds, depending on the length
of the linker. In detail, the short-chain derivatives tend to remain
in the allosteric site where one carbachol moiety elicits ionic inter-
actions with Glu397 plus a set of H-bonds with Tyr85, Tyr179,
Gln177, while the second carbachol group approaches the orthos-
teric site without reaching it. On the contrary, as exemplified by 2d
(n = 9) in Fig. 9C, the long-chain derivatives assume a central pose
by which they occupy both the orthosteric and the allosteric sites.
Such a pose brings to mind that previously described for the hM2
subtype, although these ligands appear to be more fittingly
accommodated in the hM1 receptor. In fact, while the key contacts
within the allosteric cavity are similar in both subtypes, the carba-
chol moiety within the orthosteric site assumes an optimal orien-
tation being able to elicit the same key interactions established by
CCh, namely the ammonium head with Asp105 plus the surround-
ing aromatic residues and the carbamate moiety with Asn382.
Moreover the alkyl linker appears to be suitably inserted in a tight
channel completely lined by aliphatic residues, which can form a
rich set of apolar contacts with the ligand. Also on this subtype,
compounds 3 and 4 show a binding mode similar to that of long-
chain carbachol dimers, clearly lacking the polar contacts stabi-
lized within the allosteric cavity.
Docking simulations were repeated using the M2 resolved
structure in complex with the QNB antagonist in order to mimic
an experimental condition comparable with that experienced dur-
ing kinetic binding studies. The obtained results reveal that all
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R. Matucci et al. / Biochemical Pharmacology xxx (2016) xxx–xxx
11
compounds with two carbachol units are conveniently
accommodated in the allosteric binding cavity regardless of the
length of the linker. Thus Fig. 9D depicts the putative complex
for 2b (n = 5) which shows on hM2 subtype the highest logKocc
value. The ligand is completely harbored in the allosteric site
where it can elicit a rich set of polar contacts. In detail, (i) one
ammonium head is engaged in the key ion-pairing with Glu175
reinforced by charge transfer interactions with surrounding aro-
matic residues (e.g., Tyr83 and Tyr88); (ii) the second ammonium
head elicits only charge transfer interactions with Tyr177 and
Phe181; (iii) both carbamate moieties are involved in several H-
bonds with Tyr80, Thr187, Tyr403, Asn419, Thr423, and Tyr426;
(iv) the linker elicits hydrophobic contacts with Trp422. All exam-
ined ligands show a very similar binding mode, permitted by their
folding degree which increases with the length of the linker.
Notably, some tyrosine residues which normally belong to the
orthosteric site (i.e. Tyr403 and Tyr426) are here slightly shifted
due to the presence of the antagonist and can participate to the
interaction in the allosteric site, thus suggesting that these aro-
matic residues act as a watershed to divide the two considered
binding sites.
hM1, as demonstrated by previous studies [39]. Third, while
assuming comparable pose, the long-chain derivatives are
predicted to elicit a more favorable pattern of interactions with
hM1 receptors compared to hM2. This difference is due to the
contacts established by the carbachol moiety within the orthos-
teric site as well as to the hydrophobic interactions stabilized by
the linker.
Functional studies have shown that carbachol dimers have lost
the muscarinic agonist properties of the parent compound. In fact,
although some compounds were able to induce a contraction on
guinea-pig ileum, this effect was not prevented by atropine, but
it was blocked by hexamethonium, pointing to an involvement of
the ganglionic nicotinic receptor. This agonistic activity on nico-
tinic receptor could be in a way expected since CCh is able to acti-
vate both muscarinic and nicotinic receptor, although, surprisingly,
we found in the literature that compound 2f (n = 6) and its con-
geners with general formula A (Fig. 1) and n = 4, 8, 10 have been
described as neuromuscular blocking agents with no ganglionic
stimulating activity [24,40]. To avoid interference with nicotinic
activity, the functional properties of selected compounds (1f, 2b,
2c, 2f and 4) were tested for their ability to increase ERK1/2 phos-
phorylation in CHO cells, transfected with muscarinic receptors. All
the tested compounds were found unable to activate the hM1, hM2
and hM3 muscarinic receptors, while 2b, 2c, 2f and 4 were able to
prevent the increase in ERK1/2 phosphorylation induced by ACh.
The different docking behavior can suggest a possible explanation,
since the compounds fit into the muscarinic receptor binding site
without eliciting the same pattern of interaction usually estab-
lished by agonists within the orthosteric site.
The possible interaction with the allosteric site, suggested by
the docking approach, is supported by the kinetic binding experi-
ments: compounds 1b–e and 2b–e slow the dissociation rate of
[3H]NMS from the five muscarinic receptors; logKocc values, calcu-
lated according to [28] were mostly in the same range as gal-
lamine. With respect to equilibrium binding studies, a different
preference is evidenced: higher affinity is usually found for the
[3H]NMS-occupied hM2/hM4 receptors, and also on hM1 for com-
pounds 1d,e and 2d,e, with respect to hM3/hM5. The hexam-
ethylene derivatives 1f and 2f display very low affinity for the
allosteric site, as well as the ‘‘short” amine 1b. As far as the undecyl
derivatives 3 and 4 are concerned, the different trend of their
logKocc values suggest a different binding mode; in addition, their
lower affinity for the [3H]NMS-occupied hM1 and hM2 receptors,
compared to that of the long dimers 1d,e and 2d,e (n = 9, 11, i.e.
with the length of the linker approaching that of the undecyl resi-
due), suggests an active role of the second carbachol unit in the
interaction with the allosteric site.
4. Discussion
In this paper we report the characterization of a series of diva-
lent ligands carrying two carbachol units. It is well known that the
agonist carbachol is not endowed with subtype selectivity, but
only with a small preference for M2/M4 as shown by our equilib-
rium binding studies (Table 1) [26]. On the contrary, the binding
profile of the carbachol homodimers reported in this study is com-
pletely changed: from equilibrium binding studies their affinity is
higher for hM1, hM3 and hM5 than for hM2 and hM4 with the
exception of the ‘‘short” derivatives 1a,b and 2a,b. Affinity
increases with the linker’s length, suggesting a contribution by this
moiety, probably by hydrophobic interactions. However, the
increase in pKi is not smooth, since a ‘‘jump” in the affinity values
can be seen going from the compounds with n 6 5 to those with
n P 7. The difference in affinity depends on the receptor subtype
and the presence of the amine/ammonium functionality: for exam-
ple, the increase in affinity going from methiodides 2b to 2c varies
from about 180 times in hM2 subtype to more than 5000 times in
hM4 subtype. Moreover, differences are larger for methiodides 2b,c
compared to tertiary amines 1b,c. This behavior may be explained
by a change in the binding mode of the compounds, which allows a
better fit within the receptor, as if, for instance, a gap could be
bridged between the binding sites accommodating the two phar-
macophoric units. Indeed, docking simulations, performed on the
recently resolved hM2 structure [38], revealed significant differ-
ences between the simulated compounds depending on the length
of their linker, suggesting a change in binding mode for n = 6.
While short compounds (n 6 5) may interact within the orthosteric
binding site or between the orthosteric and allosteric sites, by
increasing the length of the linker only the bitopic interaction is
possible due to steric hindrance.
In conclusion, we have prepared a series of homodimers of the
well-known cholinergic agonist carbachol. The compounds have
been analyzed by means of equilibrium and kinetic binding stud-
ies; docking simulations on the hM1 and hM2 receptors give a
sound rationalization of the experimental data. Functional experi-
ments show that homodimerization, connecting two carbachol
units through the carbamic nitrogen atoms, gave derivatives which
lost agonistic activity. Studies are ongoing on other carbachol
homodimers, where the two units are linked in a different way,
to further explore the structural requirements to bridge the orthos-
teric and allosteric binding sites.
The comparison of the docking results obtained for hM1 and
hM2 allows some considerations which correlate with the out-
comes of equilibrium binding studies. First, in both receptors the
binding mode depends on the length of the linker and only the
long-chain analogs are able to occupy both binding sites. Second,
the short-chain derivatives reveal significant differences between
the two receptor subtypes: in fact, when docked on hM2 receptor
they tend to be completely harbored within the orthosteric site
while on the hM1 subtype they remain in the allosteric cavity. This
different behavior is easily explained by considering that the hM2
orthosteric pocket is larger and more flexible than the one in
Acknowledgments
This work was supported by grants from MIUR – Italy (PRIN
2009, 2009ESXPT2_002). We thank professor Fulvio Gualtieri for
helpful discussion. This work is dedicated to our dear colleague
Serena Scapecchi, who began this research but departed this life
too early.
Please cite this article in press as: R. Matucci et al., Carbachol dimers as homobivalent modulators of muscarinic receptors, Biochem. Pharmacol. (2016),
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12
R. Matucci et al. / Biochemical Pharmacology xxx (2016) xxx–xxx
oxotremorine-related hybrid-type allosteric modulators of muscarinic
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Please cite this article in press as: R. Matucci et al., Carbachol dimers as homobivalent modulators of muscarinic receptors, Biochem. Pharmacol. (2016),
http://dx.doi.org/10.1016/j.bcp.2016.03.012