Diphenidol-related diamines as novel muscarinic M4 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2008.03.061

A series of hydrochloride derivatives 2a-9a and quaternary ammonium derivatives 3b-9b of diphenidol have been synthesized and characterized in receptor binding and cellular functional assays versus human muscarinic M₁-M₅ receptors expressed in CHO cells. Compound 8b, a methiodide derivative with a bipiperidinyl moiety and a second diphenidol framework, showed a potent and selective M₄ activity as competitive antagonist. Moreover 8b, acting as an allosteric modulator, was able to retard the dissociation rate of [³H]-N-methylscopolamine from CHO-M₄ cell membranes exposed to atropine. Taken together, these data suggest that 8b might open new avenues to the discovery of novel multivalent antagonists for the muscarinic receptors.

Full text of DOI:10.1016/j.bmcl.2008.03.061

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2972–2976
Diphenidol-related diamines as novel muscarinic M4
receptor antagonists
Lucilla Varoli,a,* Aldo Andreani,a Silvia Burnelli,a Massimiliano Granaiola,a
Alberto Leoni,a Alessandra Locatelli,a Rita Morigi,a Mirella Rambaldi,a
Andrea Bedini,b Nicola Faziob and Santi Spampinatob
aDepartment of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, I-40126 Bologna, Italy
bDepartment of Pharmacology, University of Bologna, Via Irnerio 48, I-40126 Bologna, Italy
Received 8 February 2008; revised 19 March 2008; accepted 20 March 2008
Available online 23 March 2008
Abstract—A series of hydrochloride derivatives 2a–9a and quaternary ammonium derivatives 3b–9b of diphenidol have been syn-
thesized and characterized in receptor binding and cellular functional assays versus human muscarinic M1–M5 receptors expressed
in CHO cells. Compound 8b, a methiodide derivative with a bipiperidinyl moiety and a second diphenidol framework, showed a
potent and selective M4 activity as competitive antagonist. Moreover 8b, acting as an allosteric modulator, was able to retard
the dissociation rate of [3H]-N-methylscopolamine from CHO-M4 cell membranes exposed to atropine. Taken together, these data
suggest that 8b might open new avenues to the discovery of novel multivalent antagonists for the muscarinic receptors.
Ó 2008 Elsevier Ltd. All rights reserved.
Five distinct gene sequences coding for muscarinic M1–
M5 receptors, members of the superfamily of G-protein
coupled receptors, have been cloned. Muscarinic recep-
tors are expressed in a complex, overlapping fashion in
most tissues and modulate the activity of a large number
of functions. These receptors display a high degree of se-
quence identity, and subtype-selective binding of ago-
nists and antagonists is likely based on conformational
dissimilarities.1 Studies using knockout mice have at-
tempted to clarify the main role of each muscarinic
receptor subtype in different physiological functions.
However, the loss of one of these subtypes can be com-
pensated as a result of functional redundancy.2 These
complications have contributed to slow the discovery
of selective and reversible M4 antagonists, since so far
only PD 1028073 and few other benzoxazine analogues4
have been described. The need of M4-selective ligands is
crucial to better explore the role played by this receptor
subtype in the central nervous system5 and in the periph-
eral tissues6 and to investigate them as potential, novel
drugs that may overcome the limitations of available,
non-selective compounds.
In our recent work, we have synthesized novel musca-
rinic antagonists derived from diphenidol (1,1-diphe-
nyl-4-piperidin-1-yl-butan-1-ol, 1), an antiemetic agent
used in the treatment of vomiting and vertigo and bind-
ing to M1–M4 receptors with superimposable affinity.
Appropriate structural modifications of 1 led us to ob-
tain M2, M3 as well as M4 antagonists.7 On the basis
of the findings described in our previous works, we syn-
thesized a novel series of diphenidol derivatives and
characterized them in receptor binding and cellular
functional assays versus human muscarinic M1–M5
receptors expressed in CHO cells. Thereon, we turned
our attention to the basic centre and replaced the piper-
idine ring with some diamines, in order to evaluate the
influence on affinity and/or selectivity of a second basic
centre. At first, we considered the piperazine moiety,
which may be used as a suitable basic portion for mus-
carinic antagonists,8 and introduced some substituents
on the second nitrogen atom, including a second diphen-
idol framework. Thereafter, we modified the flexibility
and the length of the spacer linking the nitrogen atoms
of the two diphenidol frameworks (Fig. 1).
The title compounds 2–9 were synthesized by direct
mono- or di-alkylation with 4-chloro-1,1-diphenyl-bu-
tan-1-ol9 (10) of the proper diamine (piperazine for 2
and 5, 1-methylpiperazine for 3, 1-phenylpiperazine
for 4, N,N0-dimethyl-ethane-1,2-diamine for 6, N,N0-
Keywords: M4-selective muscarinic antagonists; Diphenidol diamine
derivatives; Muscarinic receptor binding assays; Functional studies;
Allosteric modulation.
*
Corresponding author. Tel.: +39 051 2099717; fax: +39 051
2099715; e-mail: lucilla.varoli@unibo.it
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.03.061

L. Varoli et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2972–2976
2973
Ph OH
Ph
OH Ph
Ph
Ph OH
Ph
N
N
N
R1
R1
R2
(CH2)n
1a,b
6-7a,b
Ph OH
Ph
Ph OH
Ph
N
N R
R2
N
N R
R2
R1
2a, 3-5a,b
R1
8-9a,b
R
R1
H
R2
-
R
n
2
2
3
3
-
R1
H
R2
H
1a
1b
2a
3a
3b
4a
4b
5a
5b
-
6a
6b
7a
7b
8a
8b
9a
9b
-
-
-
-
-
CH3
H
-
CH3 CH3
H
H
H
CH3
-
H
H
CH3
CH3
Ph
Ph
H
CH3 CH3
-
(CH2)3C(OH)Ph2
(CH2)3C(OH)Ph2
CH3
H
H
H
-
CH3 CH3
CH3
H
-
-
H
H
(CH2)3C(OH)Ph2
(CH2)3C(OH)Ph2
H
-
CH3
-
CH3 CH3
CH3
Figure 1. Structure of diphenidol and target compounds.
dimethyl-propane-1,3-diamine for 7, 4,40-bipiperidine
for 8 and 1-methyl-4,40-bipiperidine10 for 9). The ob-
tained tertiary amines11 were converted to hydrochlo-
rides 2a–9a with hydrochloric acid12 and to the
corresponding methiodides 3b–9b, bearing permanent
positive charges, with methyl iodide13 (Scheme 1). It
was not possible to obtain compound 2b in a pure form
suitable for pharmacological assays.
ences in affinity at human M1–M5 receptors, compared
with 5a. Interestingly, one-carbon homologation of the
chain linking the two nitrogen atoms (7a) increased
affinity of about or more than 2 orders of magnitude
at human M1–M5 receptor subtypes. With respect to
methiodide derivatives 3b–7b, when compared to the
corresponding tertiary amines, compounds 3b–6b
showed higher binding affinity at human M1–M5 recep-
tors, while 7b was less active. The better profile of the
activity of quaternary ammonium derivatives is a com-
mon feature for most of the muscarinic antagonists.14
All the synthesized compounds were tested for affinity
versus human M1–M5 stable transfectants of CHO-K1
cells, using [3H]-N-methylscopolamine chloride ([3H]-
NMS) as labeled ligand. Binding profile of the reference
diphenidol hydrochloride (1a) and methiodide (1b)7b
was also investigated in the present study. Binding data
are reported as pKi values (affinity) in Table 1. All the
competition curves obtained with assayed compounds
were compatible with the competitive inhibition of tra-
cer binding.
Compound 8a, bearing a bipiperidinyl moiety and a sec-
ond diphenidol framework, displayed a relevant affinity
at the human M4 receptor subtype (pKi = 7.13), with a
12-fold enhanced affinity with respect to 1a at this recep-
tor subtype. Compound 8a maintained a moderate affin-
ity at the other receptor subtypes, comparable to those
of 1a. The corresponding quaternary derivative 8b was
the most interesting compound of this series, since it
exhibited a noteworthy affinity at human M4 receptor
(pKi = 7.90), similar to that found for the reference M4
antagonist PD 102807 (ethyl 9-methoxy-2-methyl-
3,11,12,14-tetrahydro-6aH-indolo[40,50:5,6][1,3]oxazino[2,
3-a]isoquinoline-1-carboxylate),3,4 maintaining a moder-
ate affinity at the other muscarinic receptor subtypes.
The following selectivity ratios were found: M4/
M1 = 48, M4/M2 = 29, M4/M3 = 30 and M4/M5 = 47. A
decrease in binding affinity was observed in derivative
9a, where 4,40-bipiperidine was preserved as diamine
and a methyl group replaced the second diphenidol
framework. The corresponding quaternary derivative
9b showed affinity values similar to 9a.
Reference compound 1a showed a moderate affinity to
human M1–M5 muscarinic receptors; however, it was
not able to discriminate among the different muscarinic
receptor subtypes. An appreciable increase of binding
affinity to all muscarinic receptor subtypes was evi-
denced for the corresponding quaternary ammonium
salt 1b.
Replacement of the piperidine moiety with the pipera-
zine ring (2a) did not cause any significant change in
binding affinity at human M1–M5 subtypes, and the
introduction of a substituent on 4-nitrogen atom of
the piperazine (compounds 3a–5a) was detrimental and
led to drastic decrease of the binding affinity at all the
five muscarinic subtypes.
The affinity profile of derivatives 8a and 8b towards the
human M4 muscarinic receptor subtype was confirmed
in functional assays. First, we investigated the effects
of these compounds on GTP binding in membranes ob-
Opening the piperazine ring, while maintaining the sec-
ond diphenidol framework (6a), resulted in slight differ-

2974
L. Varoli et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2972–2976
OH
N
N R
2-5
OH
OH
OH
a
N
(CH2)n
N
N
Cl
10
6, 7
OH
N R
8, 9
b
2a-9a
3b-9b
2-9
c
Scheme 1. Reagents and conditions: (a) diamine, 100–200 °C; (b) HCl, CHCl3/Et2O; (c) CH3I, CHCl3/acetone or CHCl3/Et2O.
Table 1. Binding affinities (pKi) of the antagonists 1a, 1b, 2–9a, 3–9b and PD 102807 to human muscarinic receptors M1–M5
Compound
pKi SEMa
hM1
hM2
hM3
hM4
hM5
1a
1b
6.37 0.09
6.70 0.05
6.22 0.07
5.11 0.24
5.53 0.27
3.44 0.34
4.76 0.10
3.07 0.26
5.15 0.26
3.61 0.16
4.58 0.17
5.87 0.14
5.14 0.14
6.10 0.16
6.22 0.06
5.24 0.17
5.08 0.15
5.64 0.16
5.55 0.26
6.56 0.21
5.14 0.24
4.45 0.11
4.63 0.19
3.34 0.53
4.59 0.19
3.75 0.43
5.64 0.39
3.67 0.23
4.32 0.09
5.26 0.14
4.92 0.26
6.06 0.11
6.44 0.22
5.40 0.20
5.36 0.23
5.96 0.27
5.95 0.13
6.56 0.21
6.18 0.12
5.13 0.34
5.39 0.26
4.15 0.23
5.12 0.47
4.06 0.19
5.26 0.35
3.42 0.53
4.36 0.23
5.76 0.19
4.91 0.29
6.14 0.32
6.43 0.09
5.28 0.15
5.44 0.11
6.14 0.09
6.04 0.08
6.54 0.09
5.93 0.28
5.01 0.15
5.32 0.19
4.00 0.25
4.64 0.14
4.65 0.10
5.91 0.30
3.79 0.20
4.95 0.14
6.21 0.15
4.95 0.14
7.13 0.09
7.90 0.07
5.93 0.11
5.57 0.11
8.19 0.15
5.89 0.25
6.15 0.20
5.85 0.30
4.87 0.21
5.02 0.22
3.91 0.31
5.16 0.26
4.63 0.21
5.81 0.18
3.64 0.26
4.74 0.45
6.08 0.13
5.12 0.16
6.11 0.33
6.23 0.25
5.54 0.25
4.59 0.32
5.74 0.22
2a
3a
3b
4a
4b
5a
5b
6a
6b
7a
7b
8a
8b
9a
9b
PD 102807
a Obtained in CHO-K1 cells stably expressing the human muscarinic M1–M5 receptors (mean of three experiments performed in triplicate). Further
details are reported in Supplementary data.
tained from CHO cells stably expressing human M1–M5
receptors. We used a concentration-ratio analysis to esti-
mate functionally the affinity constants (pKb) of 8a and
8b and of the M4 antagonist PD 102807,3,4 at human
M1–M5 receptors. The functional response was the stim-
the assay measures a functional consequence of receptor
occupancy at one of the earliest receptor-mediated
events. The assay allows for traditional pharmacological
parameters of potency, efficacy and antagonist affinity.15
As reported in Table 2, 8b displayed a noteworthy po-
tency to prevent carbachol-stimulated binding of Eu-
GTP in CHO/human M4 cells. Compound 8b showed
a potency superimposable to that observed for PD
102807 whereas 8a was less effective. As expected, in
agreement with receptor binding data, 8a, 8b and PD
102807 were less effective in antagonizing carbachol-in-
duced Eu-GTP binding to human M1, M2, M3 and M5
ulation by fixed concentrations of carbachol (10ꢀ12
–
10ꢀ4 M) in the presence of europium-labeled GTP ana-
logue (Eu-GTP) binding to cell membranes. The Eu-
GTP assay measures the level of G-protein activation
following agonist occupation of a G-protein coupled
receptor, by determining the binding of the non-hydro-
lyzable analogue Eu-GTP to Galpha subunits. Thus,

L. Varoli et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2972–2976
2975
Table 2. Affinity constants (pKb values) of 8a, 8b and PD 102807 in antagonizing carbachol-induced stimulation of Eu-GTP binding to CHO-M1–M5
cell membranes
Compound
pKb SEMa
hM1
hM2
hM3
hM4
hM5
8a
8b
5.92 0.21
6.04 0.11
5.74 0.18
5.82 0.27
6.10 0.22
5.55 0.35
6.14 0.32
6.25 0.13
5.63 0.16
7.21 0.14
7.83 0.18
7.90 0.12
6.40 0.13
6.02 0.16
5.84 0.21
PD 102807
a Obtained in CHO-K1 cells expressing the human muscarinic M1–M5 receptors (mean of three experiments performed in triplicate). Further details
are reported in Supplementary data.
receptors. Compounds 8a, 8b and PD 102807 failed to
affect basal Eu-GTP binding to CHO cell membranes
expressing human M1–M5 (data not shown). Thereafter,
to confirm the antagonist profile of 8a and 8b towards
the human M4 receptor, we evaluated the regulation of
cAMP formation in CHO cells expressing the human
M4 receptor. Forskolin itself (10 lM) increased the rate
of cAMP more than 12-fold in these cells. The musca-
rinic agonist carbachol (10ꢀ12–10ꢀ4 M) inhibited, in a
concentration-related manner, the forskolin-stimulated
synthesis of cAMP (EC50 = 1870 nM). As shown in Ta-
ble 3, 8b and PD 102807 were very potent to reverse the
inhibitory effect elicited by 5 lM carbachol whereas 8a
was less potent. All the other compounds were less effec-
tive (IC50 > 1 lM; data not shown). Compounds 8a and
8b or PD 102807 (from 0.1 nM to 1 lM), added to CHO
cells, expressing human M4 receptor, in the absence of
carbachol did not modify forskolin-stimulated cAMP
accumulation; thus, they do not exhibit any activity as
muscarinic agonists (data not shown). Taken together,
functional assay data confirmed the M4-selective profile
of compounds 8a and 8b and showed that they behave
like competitive antagonists at M4 receptor subtype.
Indeed, muscarinic receptors can be modulated by li-
gands capable to interact with the acetylcholine binding
pocket (the orthosteric site) and/or with allosteric sites.19
The above-mentioned alkane-bis-ammonium com-
pounds share a common pharmacophore consisting of
two positively charged nitrogens, bridged by six or seven
methylene groups, and two lateral aromatic moieties as
crucial elements to obtain a high allosteric potency.
Based upon these considerations, we evaluated the bind-
ing of 8b to any allosteric site of the M4 receptor. We
measured the dissociation rate of [3H]-NMS at the hu-
man M4 muscarinic receptor, expressed in CHO cells,
in experiments in which cell membranes were incubated
with the radioligand and 8b before radioligand dissocia-
tion was revealed by atropine (5 lM). In fact, an altered
dissociation rate of the radioligand is unequivocally
indicative of an allosteric action.20 As expected, 8b
(10 nM), added to membranes incubated for 60 min in
the presence of [3H]-NMS, acted as a competitive antag-
onist and at this concentration almost halved the bind-
ing of [3H]-NMS at the M4 muscarinic receptor;
interestingly, it was also able to retard the observed dis-
sociation rate of the radioligand produced by atropine
(Fig. 2). Under control conditions, the apparent dissoci-
ation rate constant of [3H]-NMS (kꢀ1 = ln2/t1/2) was
0.03 0.004 minꢀ1 (mean standard error; n = 4) and
it was decelerated in the presence of 8b (kꢀ1 = 0.01
0.003 minꢀ1; mean standard error; n = 4). These re-
sults are consistent with an allosteric interaction be-
tween 8b and the M4 muscarinic receptor.
The M4 receptor binding affinity of compounds 8a and
8b, with a bipiperidinyl moiety and a second diphenidol
framework, suggests that supplementary pharmaco-
phores, such as bulky hydrophobic groups and a hydro-
xyl group, enhance the affinity by interaction with
additional binding sites at M4 muscarinic receptor sub-
type. In fact, compounds 9a and 9b, lacking the second
4-hydroxy-4,4-diphenyl-butyl moiety, showed a 16- and
214-fold decrease in affinity, respectively. Moreover, a
proper spacer linking the two nitrogen atoms is crucial.
Compounds 6a and 6b, which present a two-carbon
chain as link between the nitrogen atoms of the two
diphenidol units, exhibited very low affinity values. A
one-carbon chain elongation (7a and 7b) resulted in
higher affinity values, even if modest. Considering M1,
M2, M3 and M5 subtypes, the presence of the second
diphenidol skeleton does not influence receptor interac-
tion with respect to diphenidol, both as tertiary base and
quaternary salt.
In summary, we have synthesized a series of diphen-
idol derivatives, which were evaluated both as hydro-
chlorides and as methiodides for their in vitro
activity as muscarinic M1–M5 antagonists. Com-
pounds 8a and 8b, bearing a bipiperidinyl moiety
and a second diphenidol framework, showed an inter-
esting selective M4 antagonist activity. The quater-
nary ammonium derivative 8b was the most potent
M4 antagonist and displayed, in receptor binding
and in functional assays, a potency superimposable
to that elicited by the reference M4 antagonist PD
102807. Considering this also the corresponding
hydrochloric salt 8a exhibited a similar receptor pro-
file, and the results obtained in our study provide the
basis for further drug development. In addition, this
idea is strengthened by the observation that 8b, bind-
ing to the orthosteric and to a distinct allosteric site
of the M4 muscarinic receptor, might open new ave-
nues to the discovery of novel multivalent antago-
nists21 for the muscarinic receptors.
Interestingly, compound 8b shows structural sim-
ilarity to several alkane-bis-ammonium compounds, as
W84 (hexamethylenebis[dimethyl(3-phthalimidopropyl)
ammonium] dibromide), dimethyl-W84, the related
DuoW and the eptane analogue C7/30-phth, which have
been characterized as allosteric modulators of musca-
rinic receptors,17 including M4 muscarinic receptor.18

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L. Varoli et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2972–2976
Table 3. EC50 values and their 95% confidence limits (CL95) of 8a, 8b
and PD 102807 in antagonizing carbachol-induced inhibition of
3. Augelli-Szafran, C. E.; Jaen, J. C.; Moreland, D. W.;
Nelson, C. B.; Penvose-Yi, J. R.; Schwarz, R. D. Bioorg.
Med. Chem. Lett. 1998, 8, 1991.
4. Bo¨hme, T. M.; Augelli-Szafran, C. E.; Hallak, H.; Pugsley,
T.; Serpa, K.; Schwarz, R. D. J. Med. Chem. 2002, 45, 3094.
5. (a) Tzavara, E. T.; Bymaster, F. P.; Felder, C. C.; Wade,
M.; Gomeza, J.; Wess, J.; McKinzie, D. L.; Nomikos, G.
C. Mol. Psychiatry 2003, 8, 673; (b) Ukai, M.; Okuda, A.;
Mamiya, T. Eur. J. Pharmacol. 2004, 492, 183.
forskolin-stimulated cAMP accumulation in CHO cells stably express-
ing hM4 receptors
a
Compound
EC50 (nM) and CL95
8a
8b
138 (82–163)
67 (42–82)
54 (22–78)
PD 102807
6. (a) Takeuchi, T.; Fujinami, K.; Goto, H.; Fujita, A.;
Taketo, M. M.; Manabe, T.; Matsui, M.; Hata, F.
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Borda, L.; Reina, S.; Borda, E. S. Auton. Autacoid
Pharmacol. 2005, 25, 93.
The EC50 values and their 95% confidence limits (CL95) were generated
from dose–response curves using a computer program based on the
method of Litchfield and Wilcoxon16 (see also Supplementary data).
a Data are means ( SEM) of three experiments with incubations per-
formed in triplicates.
7. (a) Varoli, L.; Burnelli, S.; Budriesi, R.; Guarnieri, A.;
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11. Compound 3 was previously synthesized and evaluated for
antiemetic activity: Kaiser, C.; Pavloff, A. M.; Garvey, E.;
Fowler, P. J.; Tedeschi, D. H.; Zirkle, C. L.; Nodiff, E. A.;
Saggiomo, A. J. J. Med. Chem. 1972, 15, 665.
12. Dihydrochlorides were obtained for all derivatives, except
for compound 4a, due to the N-phenil substituent.
Compound 3a was previously synthesized and a weak
antispastic activity was reported: Zaugg, H. E.; Michaels,
R. J.; Glenn, H. J.; Swett, L. R.; Freifelder, M.; Stone, G.
R.; Weston, A. W. J.A.C.S. 1958, 80, 2763.
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methyl iodide, gave monoquaternary salts, as previously
reported for 3b: (a) Zaugg, H. E.; Michaels, R. J.; Glenn,
H. J.; Swett, L. R.; Freifelder, M.; Stone, G. R.; Weston,
A. W. J.A.C.S. 1958, 80, 2763; (b) Zaugg, H. E.; Michaels,
R. J. J.A.C.S. 1958, 80, 2768. Monoquaternary com-
pounds 3b–5b separated in a pure form from reaction
mixture.
Figure 2. Effect of 8b (10 nM) on the rate of [3H]-NMS dissociation
from CHO cell membranes expressing the human M4 muscarinic
receptor. Membranes were preincubated with 0.5 nM [3H]-NMS for
120 min at 25 °C; then, radioligand dissociation was measured after the
addition of 5 lM atropine (s). 8b was added to the tubes 60 min before
atropine (d). Data points represent means SEM of four experiments
conducted in duplicate. [3H]-NMS specific binding was calculated by
subtracting the non-specific binding from the total binding as reported
in Supplementary data. For the sake of clarity, error bars are not shown
where they do not exceed 10% of the respective mean value.
14. Wess, J.; Buhl, T.; Lambrecht, G.; Mutschler, E.. In
Comprehensive Medicinal Chemistry; Hansch, C., Sammes,
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Hurskainen, P.; Scheinin, M.; Hemmila¨, I. Assay Drug
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Acknowledgment
This work was supported by a grant from MIUR (Ital-
ian Ministry of University and Research).
16. Tallarida, R. J.; Murray, R. B. Manual of Pharmacological
Calculation with Computer Programs, 2nd ed; Springer-
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