Evaluation of 1,2,5-thiadiazoles as modulators of M1/M 5 muscarinic receptor subtypes

Article abstract of DOI:10.1016/j.bmc.2014.01.049

Studies have demonstrated the presence of allosteric binding sites on each of the muscarinic acetylcholine receptor (mAChR) subtypes. Since most drugs targeting muscarinic receptors bind to the highly conserved orthosteric binding site, they fail to achieve appreciable subtype selectivity. Targeting non-conserved allosteric sites may provide a new way of enhancing selectivity for individual subtypes of muscarinic receptor. Tetra(ethyleneglycol)(3-methoxy- 1,2,5-thiadiazol-4-yl)[3-(1-methyl-1,2,5,6-tetrahydropyrid-3-yl)-1,2, 5-thiadiazol-4-yl] ether, CDD-0304 (10), was found to be a M_1/2/4 selective muscarinic agonist and might prove useful in treating the symptoms associated with schizophrenia (J. Med. Chem. 2003, 46, 4273). It was hypothesized that the observed subtype selectivity demonstrated by 10 may be due to its ability to function as a bitopic ligand (J. Med. Chem. 2006, 49, 7518). To further investigate this possibility, a novel series of compounds was synthesized using a 1,2,5-thiadiazole moiety along with varying lengths of a polyethylene glycol linker and terminal groups, for evaluation as potential allosteric modulators of muscarinic receptors. Preliminary biological studies were performed using carbachol to stimulate M₁ and M₅ receptors. No significant agonist activity was observed at either M₁ or M₅ receptors for any of the compounds. Compound 18, 2-(4-methoxy-1,2,5-thiadiazol-3-yloxy)-N,N-dimethylethanamine fumarate (CDD-0361F) was found to block the effects of carbachol at M₅ muscarinic receptors.

Full text of DOI:10.1016/j.bmc.2014.01.049

Bioorganic & Medicinal Chemistry 22 (2014) 1838–1844
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Evaluation of 1,2,5-thiadiazoles as modulators of M1/M5 muscarinic
receptor subtypes
Aditya Maheshwari a, P. S. S. Rao a, William S. Messer Jr. a,b,
⇑
a Department of Medicinal and Biological Chemistry, MS 1015, The University of Toledo, College of Pharmacy and Pharmaceutical Sciences, 3000 Arlington Ave., Toledo, OH
43614, United States
b Department of Pharmacology, MS 1015, The University of Toledo, College of Pharmacy and Pharmaceutical Sciences, 3000 Arlington Ave., Toledo, OH 43614, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Studies have demonstrated the presence of allosteric binding sites on each of the muscarinic acetylcho-
line receptor (mAChR) subtypes. Since most drugs targeting muscarinic receptors bind to the highly
conserved orthosteric binding site, they fail to achieve appreciable subtype selectivity. Targeting
non-conserved allosteric sites may provide a new way of enhancing selectivity for individual subtypes
of muscarinic receptor. Tetra(ethyleneglycol)(3-methoxy-1,2,5-thiadiazol-4-yl)[3-(1-methyl-1,2,5,6-tetr-
ahydropyrid-3-yl)-1,2,5-thiadiazol-4-yl] ether, CDD-0304 (10), was found to be a M1/2/4 selective musca-
rinic agonist and might prove useful in treating the symptoms associated with schizophrenia (J. Med.
Chem. 2003, 46, 4273). It was hypothesized that the observed subtype selectivity demonstrated by 10
may be due to its ability to function as a bitopic ligand (J. Med. Chem. 2006, 49, 7518). To further inves-
tigate this possibility, a novel series of compounds was synthesized using a 1,2,5-thiadiazole moiety
along with varying lengths of a polyethylene glycol linker and terminal groups, for evaluation as potential
allosteric modulators of muscarinic receptors. Preliminary biological studies were performed using car-
bachol to stimulate M1 and M5 receptors. No significant agonist activity was observed at either M1 or M5
receptors for any of the compounds. Compound 18, 2-(4-methoxy-1,2,5-thiadiazol-3-yloxy)-N,N-dimeth-
ylethanamine fumarate (CDD-0361F) was found to block the effects of carbachol at M5 muscarinic
receptors.
Received 21 October 2013
Revised 20 January 2014
Accepted 30 January 2014
Available online 7 February 2014
Keywords:
Muscarinic receptor
M5 antagonist
Drug abuse
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
allosterism) or a decrease (negative allosterism) in the affinity of
the receptor for muscarinic agonists, such as ACh, and/or classical
Muscarinic receptors contain a classical acetylcholine binding
site (orthosteric binding site), with high sequence homology
among receptor subtypes, and allosteric binding sites which are
topographically distinct from the orthosteric binding site, and less
highly conserved between muscarinic receptor subtypes.3 The
orthosteric site is responsible for acetylcholine (ACh) binding and
most drugs that bind to the highly conserved orthosteric site of
muscarinic receptors fail to achieve subtype selectivity. Most
reportedly selective muscarinic agonists exhibit some binding
affinity for all five muscarinic receptor subtypes, but show func-
tional selectivity in activating one or more subtypes preferentially.
In contrast, the binding of an allosteric ligand to an allosteric
binding site may alter the conformation of the orthosteric site on
muscarinic receptors and may result in either an increase (positive
antagonists. With neutral cooperativity, allosteric compounds bind
to the receptor but have no effect on the binding of a primary li-
gand at any concentration. Two likely sites for allosteric modula-
tion have been identified with at least one site located on the
extracellular surface of muscarinic receptors near the orthosteric
binding site.4,5 The influence of allosteric binding on the function
of the classical binding site can be measured by determining
changes in the association, dissociation, and equilibrium binding
of muscarinic agonists and competitive antagonists,6 or by measur-
ing the biochemical responses produced by muscarinic agonists in
the absence and presence of the putative allosteric ligand. Alloste-
ric modulation can be helpful in increasing subtype-specificity of
an agonist as well.7,8 Allosteric muscarinic drugs might be more
efficacious and have fewer side effects than classic muscarinic ago-
nists and antagonists.
Several novel monovalent muscarinic agents that bind to their
target receptors through allosteric mechanisms are reported in
the literature (Fig. 1). N-heterocyclic derivatives of quinolone
Abbreviations: ACh, acetylcholine; CCh, carbachol.
Corresponding author. Tel.: +1 4195302291; fax: +1 4193831909.
E-mail address: william.messer@utoledo.edu (W.S. Messer).
⇑
http://dx.doi.org/10.1016/j.bmc.2014.01.049
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

A. Maheshwari et al. / Bioorg. Med. Chem. 22 (2014) 1838–1844
1839
F
F
O
N
O
O
F3CO
O
N
OH
O
N
AC-42
OCH3
2
1
3
N
N
NH
N
N
O
N
N
N
HN
Cl
TBPB
5
N
H
N-desmethylclozapine
4
Cl
Cl
O
O
O
O
N
N
N
N
N
Cl
N
N
Cl
O
O
W84 (A=B=BENZO)
Duo3
7
W84 analog (A=B=1,8 naphth
6
Figure 1. Selective muscarinic agonists: allosteric compounds.
carboxylic acid (1) recently were reported to be M1 selective posi-
tive allosteric modulators.9 VU0238429 (2) provides >30 fold selec-
tivity for M5 versus the other four receptor subtypes and
represents the first highly selective M5 positive allosteric modula-
tor.10 AC-42 (4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-
piperidine hydrogen chloride) (3) and N-desmethylclozapine (4)
activate M1 mAChRs with a considerable degree of selectivity by
interacting with non-orthosteric receptor binding sites.11,12 TBPB
(1,3-dihydro-1-[10-[(2-methylphenyl)methyl][1,40-bipiperidin]-4-
yl]-2H-benzimidazol-2-one) (5) was found to be a selective alloste-
ric activator for M1 receptors. It displayed antipsychotic like activ-
ity in rodent models and also decreased the production of Ab
in vitro by increasing non-amyloidogenic processing of APP.13
The bisbenzyl bispyridinium (DUO series) and W-84 have been
shown to be potent allosteric modulators of muscarinic M2 musca-
rinic receptors14–19 (compounds 6 and 7). Hence novel selective
allosteric compounds provide a new way of enhancing selectivity
for individual muscarinic receptor subtypes and reducing the po-
tential for adverse effects.
Xanomeline (8) is reported to display two different modes of
binding at M1 muscarinic receptors, with the first a reversible bind-
ing through interaction with the orthosteric site and the second a
wash-resistant and very stable binding that takes place at a differ-
ent position which is accompanied by marked allosteric modula-
tion of competitive ligands with the receptor7 (Fig. 2). Aceclidine
analogs carrying a 1,2,5-thiadiazole moiety have also been identi-
fied as potent M1 receptor agonists.20 Another selective M1 agonist
is the bivalent compound tetra(ethylene glycol) di[3-(1-methyl-
1,2,5,6-tetrahydropyrid-3-yl)-1,2,5-thiadiazol-4-yl] ether, CDD-
0273 (9), which contains two xanomeline pharmacophores
connected through a tetraethylene glycol linker21 (Fig. 2). Binding
of one agonist pharmacophore increases the affinity for the other
at M1 receptors and hence results in high selectivity and activity
at M1 receptors. It may be possible for both 9 (CDD-0273) and 10
(CDD-0304) to behave as bitopic ligands, which contain separate
orthosteric and allosteric moieties separated by a tetraethylene
glycol linker, and can, theoretically, bind to both the orthosteric
and allosteric sites (Fig. 2). For these molecules, interactions with
the allosteric site at M1 receptors may help in achieving subtype
selectivity and interactions at orthosteric sites may promote recep-
tor activation.
Receptor binding studies at wild-type and mutant (Thr192Ala)
M1 receptors studies were helpful for identifying the interaction
of muscarinic agonists with amino acid residues present in the
transmembrane domain of M1 receptors. Thr192 was found to
be an important amino acid residue involved in the binding of
the muscarinic agonists xanomeline22 and 10. Since xanomeline
shares many structural features with compounds 9 and 10, the
data suggest that the pharmacophore common between these
compounds might be interacting with transmembrane domains
of M1 receptors. Chimeric receptor data and site directed muta-
genesis studies suggest that amino acids found in the second
and third extracellular loops of the M1 receptor are critical for
binding and activity of 10.2 In particular Glu170 and Gln185
present in second extracellular loop were identified as important
residues contributing to the potency and activity of 10 at M1
receptors as compared with M5 receptors (Fig. 3). The tetraethyl-
ene glycol linker present in 10 may position the 3-methoxy-
1,2,5-thiadiazole to allow interactions with amino acids present
in loop regions.
N
S
S
N
S
N
N
N
S
S
N
N
N
O
O
O
O
O
N
N
O
O
O
O
O
O
OCH3
Xanomeline
8
CDD-0273
9
CDD-0304
10
N
N
N
N
Figure 2. Selective muscarinic agonists: bitopic ligands.

1840
A. Maheshwari et al. / Bioorg. Med. Chem. 22 (2014) 1838–1844
3-Methylsulfonyl-4-methoxy-1,2,5-thiadiazole offered two advan-
tages over 3-chloro-4-methoxy-1,2,5-thiadiazole (CMT). Firstly, it
has a melting point of 110.5 °C and hence is more compatible than
CMT (which sublimates at 52.5 °C) for use at high temperatures.
Additionally, sulfone is a much better leaving group than the
chloro group and hence can be more easily replaced by other
nucleophiles.
Compound 14 was converted to compound 15 using 2-(tetrahy-
dro-2H-pyran-2-yloxy)-ethanol and then the tetrahydropyranyl
group was removed under mild acidic conditions to provide 16
(CDD-0360). Compound 17 was synthesized from compound 14
using commercially available N,N-dimethylethanolamine and then
converted to the fumarate salt 18 (CDD-0361F). Compound 17 also
was converted to the quaternary amine 19 (CDD-0362), using
methyl iodide in quantitative yield. Compound 20 (CDD-0363)
was synthesized from compound 14 using di(ethylene glycol) ben-
zyl ether and potassium tert butoxide. The benzyl group could not
be removed under hydrogenation even using high pressures. It
could be removed however, under 1,2-dichloro-4,5-dicyanobenzo-
quinone (DDQ)/acetonitrile/80°C24 but the reaction was not clean
and the resulting compound could not be purified. Compound 14
was treated with triethylene glycol and potassium tert butoxide
to yield 21 (CDD-0364) as the major product in 45% yield and 22
(CDD-0365) as a minor product in 15% yield. Similarly, compound
14 was converted to 23 (CDD-0366) as the major product in 41%
yield and 24 (CDD-0367) as a minor product in 8% yield using tet-
raethylene glycol. The 1H and 13C spectra (D2O) of 19 were compa-
rable with commercially available acetylcholine chloride due to
high structural resemblance.25
Figure 3. Schematic depiction of the M1 muscarinic acetylcholine receptor.
To explore the possibility that this interaction is allosteric, sev-
eral novel analogs of 3-methoxy-1,2,5-thiadiazole containing vary-
ing lengths of a polyethylene glycol linker were synthesized. If this
interaction is allosteric then these compounds could modulate the
binding of ligands that interact only with the orthosteric site of
muscarinic receptors, like carbachol.
2. Materials and methods
2.1. Chemistry
As shown in Scheme 1, compound 121 was synthesized in
10% yield and converted to 14 using established methods.23
S
S
S
S
b
43%
N
N
a
10%
N
N
c
52%
N
N
N
N
(E)
(E)
(E)
(E)
(E)
O (E)
(E)
(E)
S
O
14
Cl
O
O
S
Cl
Cl
O
11
12
13
S
S
N
N
d
e
(E)
(E)
N
N
O
OH
(E)
(E)
O
62%
O
O
O
O
16
15
(CDD-0360)
S
S
N
N
f
(E)
g
(E)
N
N
HOOC
N
N
O
O
(E)
(E)
90%
O
O
17
COOH
18 (CDD-0361F)
h
S
N
(E)
S
N
N
N
N
(E)
O
(E)
(E) O
I
O
O
S
19
(CDD-0362)
O
14
S
N
H2
C
i
(E)
N
O
O
(E)
O
71%
O
20
(CDD-0363)
S
N
S
O
N
N
(E)
j
N
(E)
N
O
(E)
O
O
OH
O
(E)
O
O
(E)
O
+
O
(E) N
N
O
O
S
22 (CDD-0365)
21 (CDD-0364)
S
S
S N
N
(E)
(E)
N
N
(E)
N
k
O
O
O
O
O
+
(E)
(E)
O
O
(E)
OH
O
O
O
O
23
(CDD-0366)
24 (CDD-0367)
Scheme 1. Synthesis of compounds 16 through 24. Reagents and conditions: (a) Methanol, sodium hydride, THF, overnight; (b) sodium hydrosulfide hydrate, iodomethane,
DMF, rt; (c) potassium peroxymonosulfate, water, 5 h; (d) 2-(tetrahydro-2H-pyran-2-yloxy)-ethanol, potassium tert-butoxide, THF, rt, overnight; (e) p-toluene sulfonic acid,
methanol, overnight; (f) N,N-dimethylethanolamine, potassium tert-butoxide, THF, rt, overnight; (g) fumaric acid, ethanol; (h) iodomethane, acetone, 5 h; (i) di(ethylene
glycol) benzyl ether, potassium tert-butoxide, THF, rt, overnight; (j) potassium tert-butoxide, triethylene glycol, THF, rt, overnight; (k) potassium tert-butoxide, tetraethylene
glycol, THF, rt, overnight.

A. Maheshwari et al. / Bioorg. Med. Chem. 22 (2014) 1838–1844
1841
2.2. Pharmacology
During this incubation, 80 ll of YSi-SPA beads (1 mg/80 ll
water) was added to a 96 well white plate. After incubation, the
For in vitro biological evaluations, a phosphatidylinositol (PI)
turnover assay (for Gq coupled receptors) was employed to explore
the potential allosteric activity of novel compounds. The procedure
for determining levels of inositol phosphates following the stimu-
lation of muscarinic receptors has been described previously.1,2 In
brief, A9 L cell lines stably expressing human muscarinic receptors
(M1 or M5 receptors through plasmids obtained from Missouri S&T
cDNA Resource Center) were seeded in 96-well tissue culture
plate containing cells was tapped on all sides to obtain a homoge-
neous cell lysate. 20
to corresponding wells containing SPA beads (white plate). In order
to get a blank reading corresponding to formic acid, 20 l of formic
ll of the cell extract in formic acid was added
l
acid also was assessed for activity. The 96 well (white) plates were
sealed using TopSeal A and contents were mixed via shaking for 1 h
at 4 °C at 200 rpm using an orbital shaker. After standing at 4 °C for
2 h or overnight, radioactivity in counts per minute (CPM) was
determined using the Topcount NXT system. Activity was pre-
sented either as the percentage activation above basal levels
(CPM-CNTRL/CNTRL-BLANK ⁄ 100) or counts per minute. Experi-
ments were repeated three times to confirm the activity of all
the tested compounds. Carbachol served as a positive control for
muscarinic receptor activation in each assay. Two factor analysis
of variance (ANOVA) was used to assess the impact of varying con-
centrations of carbachol and synthesized compounds on phospho-
plates (100 ll with approximately 30,000 cells).
Cells were incubated for 24 h at 37 °C in an incubator condi-
tioned with 5% CO2. The following day, the plates were rinsed twice
with 200
DMEM supplemented with
(4 mM), BSA (0.6%) and [3H]-inositol (10
l
l PBS. To radiolabel the cells, 100
-glucose (25 mM),
Ci/ml) was dispensed
l
l of inositol free (IF)
D
L-glutamine
l
into each well. The labeled cells were incubated overnight at
37 °C with 5% CO2.
The next day, test ligand dilutions were prepared in HBSS buffer
supplemented with LiCl (10 mM) and HEPES (20 mM). Receptor
inositide metabolism, with a set at 0.05.
activation was initiated by addition of 100 ll of the appropriate
3. Results and discussion
concentration of test ligands (with buffer serving to measure basal
[control] levels) to each well in triplicate sets. The cells were incu-
bated subsequently at 37 °C (5% CO2) for 1 h. Receptor stimulation
was terminated by rapid removal of all media. Then, 100 ll of ice-
cold formic acid (50 mM) was added to each well prior to incuba-
Compound 18 (Fig. 4) did not elicit a pharmacological response
at either M1 or M5 receptors. At M1 muscarinic receptors, the car-
bachol response was not significantly different from the response
produced by carbachol in the presence of 1
lM and 100 lM 18 (P
tion at room temperature for 20 min.
>0.05). However, at M5 muscarinic receptors, 18 inhibited the
Figure 4. Stimulation of phosphoinositide metabolism by carbachol, 18 and 19 (separately and combined) at M1 and M5 muscarinic receptors expressed in A9 L cells. Data
represent the mean ( SEM) of three experiments each performed in triplicate. ⁄Significantly different from carbachol + HEPES values (P <0.05); ^significantly different from
compound (1 l lM) + CCh values (P <0.05).
M) + carbachol values (P <0.05); #significantly different from compound (100

1842
A. Maheshwari et al. / Bioorg. Med. Chem. 22 (2014) 1838–1844
Figure 5. Stimulation of phosphoinositide metabolism by carbachol, 16, 21 and 23 (separately and combined) at M1 and M5 muscarinic receptors expressed in A9 L cells. Data
represent the mean ( SEM) of three experiments each performed in triplicate. ⁄Significantly different from CCh + DMSO values (P <0.05); ^significantly different from
compound (1 l lM) + CCh values (P <0.05).
M) + CCh values (P <0.05); #significantly different from compound (100
response of carbachol at both concentrations (P <0.05) consistent
with potential antagonist activity.
the response of carbachol at either the 1 lM or 100 lM concentra-
tion (P >0.05).
Compound 19 (Fig. 4) was inactive at M1 and M5 receptors by
itself. When tested along with carbachol at both M1 and M5 recep-
tors, it inhibited the response of carbachol at the 100 lM concen-
tration, suggesting it may act as an antagonist. The statistical
analysis indicated that the (drug + carbachol) response was signif-
Compound 21 (Fig. 5) did not possess any intrinsic activity at
M1 and M5 receptors, and did not significantly alter carbachol re-
sponses at either concentration of 21 (P >0.05). Compound 20
along with the bivalent ligands 22 and 24 exhibited poor solubility,
even in a 50% DMSO solution, and hence could not be tested for
activity at muscarinic receptors.
To further understand the mode of antagonism (competitive or
allosteric) for 18, a Schild regression analysis was performed at M1
and M5 receptors. This analysis is based on the principle that with
icantly different from that of carbachol itself at 100
<0.05).
lM of 19 (P
Compounds 16 and 23 (Fig. 5), on the other hand, were unable
to stimulate M1 or M5 receptors. Moreover, 16 and 23 did not alter

A. Maheshwari et al. / Bioorg. Med. Chem. 22 (2014) 1838–1844
1843
Overall, the synthesized analogs of 3-methoxy-1,2,5-thiadia-
zole, carrying varying lengths of a polyethylene glycol linker, did
not activate either M1 or M5 muscarinic acetylcholine receptors.
On the other hand, compounds such as 18 and 19 possessed antag-
onist activities which, given their resemblance in structure to car-
bachol, could be attributed to possible interactions with the
orthosteric site. Increasing concentrations of compound 18 did
not shift the dose response curves for carbachol to the right in a
parallel manner, however, suggesting that compound 18 is not a
competitive antagonist. Further studies, including radioligand
binding assays, are needed to evaluate the nature of the interaction
of compound 18 with muscarinic receptors.
4. Conclusions
Figure 6. The dose–response curve for carbachol in the absence and presence of 18
(1 M, 10 M, 100 M and 300 M) at M1 muscarinic receptors.
Out of five compounds (16, 18, 19, 21 and 23) tested so far, 18
and 19 were found to possess antagonistic properties at the M5
receptor subtype. By themselves, both these compounds were
inactive at M1 and M5 muscarinic receptors. The data suggest that
18 and 19 behave as antagonists at M5 receptors. These com-
pounds might interact with the orthosteric binding site rather
than at allosteric sites. The structural similarity of these com-
pounds with carbachol might allow them to compete with carba-
chol for the orthosteric binding site and hence behave as
antagonists at muscarinic receptors. Since the logarithmic plot
was not linear in the Schild regression analysis on either M1 or
M5 receptors, it was not possible to define the mode of antago-
nism. Further evaluation of these compounds at other receptor
subtypes (M2, M3, and M4 receptors) will help determine their
selectivity. In this regard, studies focusing on assessing the bind-
ing affinity of these compounds for muscarinic receptors subtypes
would be insightful. In addition, future studies will assess the
ability of the compounds to modulate acetylcholine activity at
M5 receptors.
l
l
l
l
Figure 7. The dose–response curve for carbachol in the absence and presence of 18
(1 M, 10 M, 100 M and 300 M) at M5 muscarinic receptors.
Acknowledgment
l
l
l
l
The authors would like to thank Karen Papadakis for providing
secretarial assistance.
increasing concentrations of an antagonist, the dose–response
curve of the agonist will shift to the right in a parallel manner
for competitive antagonism. That is, a higher concentration of ago-
nist would be required to achieve the same level of activation in
the presence of an antagonist. Dose–response curves for carbachol
were obtained in the absence and presence of fixed concentrations
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.01.049.
of 18 (1 lM, 10 lM, 100 lM or 300 lM). The response was mea-
sured using the well-established PI turnover assay.
References and notes
At M1 receptors (Fig. 6), 18 did not produce a shift in the carba-
chol response at the 1
100 M, 18 did produce a shift along with an increase in the base-
line value. Similarly, at the 300 M concentration of 18, the dose–
response curve for carbachol further shifted towards right but the
baseline increased dramatically to give almost a straight line.
These observations rendered calculations for two data points unfit
for the logarithmic plot, thereby limiting the usefulness of the data
for a Schild analysis.
lM concentration. However, at 10 lM and
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l
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produced by low concentrations of carbachol suggesting a more
complex interaction with M5 receptors. Therefore, similar to the re-
sults from the interaction of compound 18 at M1 receptors, the log-
arithmic plot did not permit an evaluation of the type of
interaction that compound 18 exerts at M5 receptors in the pres-
ence of carbachol.

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