Synthesis of novel and functionally selective non-competitive muscarinic antagonists as chemical probes

Article abstract of DOI:10.1111/cbdd.13059

Muscarinic receptors are known to play important biological roles and are drug targets for several human diseases. In a pilot study, novel muscarinic antagonists were synthesized and used as chemical probes to obtain additional information of the muscarinic pharmacophore. The design of these ligands made use of current orthosteric and allosteric models of drug–receptor interactions together with chemical motifs known to achieve muscarinic receptor selectivity. This approach has led to the discovery of several non-competitive muscarinic ligands that strongly bind at a secondary receptor site. These compounds were found to be non-competitive antagonists that completely abolished carbachol activation in functional assays. Several of these compounds antagonized functional response to carbachol with great potency at M₁ and M₄ than at the rest of receptor subtypes.

Full text of DOI:10.1111/cbdd.13059

PROF. JOHN BOULOS (Orcid ID : 0000-0001-8561-6418)
Article type : Research Article
TITLE PAGE
TITLE
SYNTHESIS OF NOVEL AND FUNCTIONALLY SELECTIVE NON-COMPETITIVE
MUSCARINIC ANTAGONISTS AS CHEMICAL PROBES
SHORT TITLE
Muscarinic Antagonists as probes
KEY WORDS
PD- Parkinson Disease
GPCR- G-Protein-coupled receptor
mAChR- Muscarinic acetylcholine receptor
PAM- Positive allosteric modulator
NMS- N-methylscopolamine
AUTHORS
John F. Boulos, Department of Physical Sciences, Barry University, Miami Shores, Florida 33161,
Tel. 305-899-3432, Fax 305-899-3479, email: jboulos@barry.edu
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13059
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CO-AUTHORS
Jan Jakubik, Department of Neurochemistry, Institute of Physiology of the Academy of Sciences of
the Czech Republic, v.v.i., 142 20 Prague, Czech Republic, Tel. +420 2 4106 2620, Fax +420 2 4106
2488, email: jan.jakubik@fgu.cas.cz
John M. Boulos, University of North Carolina, Eshelman School of Pharmacy, Chapel Hill, North
Carolina, 27559, Tel. 305-332-7232, email: jmboulos12@gmail.com
Alena Randakova, Department of Neurochemistry, Institute of Physiology of the Academy of
Sciences of the Czech Republic, v.v.i., 142 20 Prague, Czech Republic, Tel. +420 2 4106 2620, Fax
+420 2 4106 2488,
Alena.randakova@fgu.cas.cz
Jelena Momirov, Department of Physical Sciences, Barry University, Miami Shores, Florida 33161,
Tel. 305-899-3432, Fax 305-899-3479, email: Jelena.momirov@mymail.barry.edu
SYNTHESIS OF NOVEL AND FUNCTIONALLY SELECTIVE NON-COMPETITIVE
MUSCARINIC ANTAGONISTS AS CHEMICAL PROBES
John F. Boulos, Jan Jakubik, John M. Boulos, Jelena Momirov, Alena Randakova
ABSTRACT: Muscarinic receptors are known to play important biological roles and are drug targets
for several human diseases. In a pilot study, novel muscarinic antagonists were synthesized and used
as chemical probes to obtain additional information of the muscarinic pharmacophore. The design of
these ligands made use of current orthosteric and allosteric models of drug-receptor interactions
together with chemical motifs known to achieve muscarinic receptor selectivity. This approach has
led to the discovery of several non-competitive muscarinic ligands that strongly bind at a secondary
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receptor site. These compounds were found to be non-competitive antagonists that completely
abolished carbachol activation in functional assays. Several of these compounds antagonized
functional response to carbachol with great potency at M1 and M4 than at the rest of receptor subtypes.
INTRODUCTION
Muscarinic receptor subtypes provide effective therapeutic targets for a number of neurological and
[1-3]
psychiatric diseases such as Alzheimer’s (AD), Schizophrenia (Sc) and Parkinson (PD).
Considerable biochemical and pharmacological evidence have validated the cholinergic hypothesis of
memory dysfunction and led to the development of numerous selective allosteric and orthosteric
muscarinic ligands. One such ligand, Xanomeline (1), M1/M4- functionally selective agonist,
demonstrated beneficiary effects on cognitive decline in several AD patients and a similar therapeutic
profile to antipsychotics drugs such as clozapine and olanzapine. [4-6]
Classical approaches to G-protein coupled receptor (GPCR) drug design have targeted the orthosteric
receptor binding site and most drugs are known to interact with this endogenous ligand-binding
pocket. Orthosteric ligands must overcome many limitations which make the development of subtype-
[7]
specific agonists and antagonists very difficult.
Some G-protein coupled receptors (including
muscarinic receptors) also contain less conserved allosteric binding sites that are targeted to attain
receptor selectivity and to elicit distinct signaling profiles. Allosteric ligands exert their effects by
[8]
modulating the binding affinity and downstream efficacy of the orthosterically bound ligand. The
use of allosteric muscarinic ligands, gallamine and alcuronium, in site-directed mutagenesis studies
[9]
provided evidence for the “common” allosteric site. The existence of a second allosteric site was
proposed when indolocarbazole (KT5720) was found to allosterically enhance the binding of
[10]
acetylcholine and N-methylscopolamine (NMS) at the M1-M4 receptors.
Numerous selective
muscarinic ligands have been identified, including positive allosteric modulators (PAMs) and putative
bitopic agonists. Benzyl quinolone carboxylic (BQCA, 2) was the first M1 muscarinic allosteric ligand
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[11]
exhibiting absolute subtype selectivity and high degree of cooperativity.
Several allosteric
modulators including heptane-1,7-bis(dimethyl-3'-phthalimidopropyl) ammonium bromide (C7/3-
phth), gallamine and alcuronium were found to form cation-π interactions with the “common site” of
[12]
the M2 receptor.
These findings provide a structural basis for the rational design of allosteric
modulators. The recent publications of crystallographic structures of muscarinic receptors will mostly
help with the determination of actual binding sites and specific drug-receptor interactions. [13-15]
There exists substantial evidence for bitopic ligands, molecules that simultaneously bind to both the
allosteric and orthosteric sites. [16-17] Xanomeline was shown to bind to the receptor in a wash-resistant
manner, suggesting that it acts both competitively as well as allosterically. [18] Pharmacological studies
revealed that compound AC-42 (3), a M1-selective agonist, exhibits characteristics suggestive of both
[19]
allosteric and orthosteric modes of action.
The potential of linking orthosteric and allosteric
pharmacophores to yield bitopic ligands may lead to novel compounds with high potency and receptor
selectivity that can elicit distinct signaling profiles.
We have synthesized and tested muscarinic ligands with the general formula 4 and structural features
similar to both known agonists 1 and 3. Several of these compounds were found to be functionally
selective non-competitive antagonists. Compounds contain a para-disubstituted alkyl or alkoxy (R1)
phenyl group linked to a tetrahydropyridinyl ring. From measured radioligand binding and functional
data, we concluded that the positively charged, tetrahydropyridinyl group, most likely interacts with
the orthosteric receptor site whereas the hydrophobic phenyl moiety interacts more strongly with the
secondary receptor site.
Compounds 9b (R1= n-C4H9, R2= H), 10a (R1= O-n-C4H9, R2= Me) and 10b (R1= n-C4H9, R2= Me)
(scheme 1) were found to be non-competitive M1/M4 functionally selective antagonists. These
compounds antagonized response to carbachol at M1 and M4 receptors better than at other subtypes.
Other compounds 5 of similar structural features, with a substituted piperidinyl ring, were also
synthesized and found to exhibit similar biological profiles. The bitopic antagonist 13a (scheme 2),
our lead compound which led to the synthesis of all other analogs was found to slow down the
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dissociation of both N-methyl scopolamine (NMS) and acetylcholine to the extent comparable to
classical allosteric modulators gallamine and alcuronium at M1-M5 muscarinic receptors. [20] We plan
on synthesizing other structural analogs with alkyl and alkoxy groups at both ortho and meta positions
and others with an ether moiety, in place of the ester, to fully explore the large body of available
information.
M1 receptors were found to participate in the overall regulation of basal ganglia function and
[21]
antiparkinsonian effects of muscarinic antagonists.
The competitive orthosteric M1 selective
antagonist VU0255035 was found to inhibit induction of generalized seizures by the agonist
pilocarpine without the cognitive impairing effects of the antagonist scopolamine. [22] M4 muscarinic
receptors, expressed in different regions of the forebrain and co-expressed with D1 dopamine
[23]
receptors, were found to play a critical role in modulating dopamine-dependent activities.
Based
on those observations, we believe that our more functionally selective M1/M4 analogs may provide a
viable approach for the treatment of certain central nervous system disorders including Parkinson
[3]
(PD).
However, muscarinic antagonists may have somewhat limited clinical applications due to
central and peripheral adverse effects.
METHODS AND MATERIALS
General Information
Reagents were purchased from Aldrich Chemical Company (St. Louis, MO) unless otherwise noted,
and all starting liquid materials were distilled before use. NMR spectra were recorded on a Varian 300
MHz spectrometer. GC-MS spectra were recorded on a Perkin Elmer Clarus 500 and 560S system.
Elemental analyses were carried out by Galbraith Laboratories (Knoxville, TN) and biological assays
were conducted at the Institute of Physiology of the Czech Academy of Sciences in Prague. Melting
points were recorded on a Digimelt MPA160 purchased from Stanford Research Systems and are
uncorrected. Refractive indexes were recorded on r2 i300 digital refractometer from Reichert
Technologies corrected for 20 oC. The radioligands [3H]-N-methylscopolamine chloride ([3H]-NMS),
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and [3H]-myo-inositol were purchased from American Radiolabeled Chemicals (St. Louis, MO,
USA). Carbachol, dithiothreitol, ethylendiaminotetraacetic acid (EDTA), and N-methylscopolamine
chloride (NMS) were purchased from Sigma (St. Louis, MO, USA).
Synthesis
1-(2-hydroxyethyl) pyridinium bromide (6) A solution of 10.07 g of 2-bromoethanol (0.0805 mol)
in 20 mL of acetonitrile was added slowly to another solution containing 6.36 g of pyridine (0.0804
mol) and 20 mL of acetonitrile. After addition, the mixture was allowed to stir for 6 days and then
refluxed for one hour. The mixture was then concentrated and residue recrystallized from n-butanol to
o
1
yield 11.79 g (72.3%) of white crystals, m.p. 102.5-103.0 C. H-NMR (D2O): δ 8.75 (2H, d), 8.45
(1H, t), 7.95 (2H, t), 4.6 (2H, t), 3.9 (2H, t). MS (m/z): 79.01 base [C5H5N]+.
1-(2-hydroxyethyl)-1,2,5,6-tetrahydropyridine (7) To a mixture containing 7.10 g of 6 (0.0348 mol)
and 100 mL of methanol, a solution of sodium borohydride (5.25 g, 0.139 mol) in 60 mL of 0.1 M
sodium hydroxide was added slowly with external cooling (ice-bath). The mixture was then allowed
to stir at room temperature for an additional 30 minutes. 6 M hydrochloric acid was added to pH 5 and
the solution was then brought to pH 8 with 3 M of sodium hydroxide. The mixture was extracted with
dichloromethane, dried over anhydrous magnesium sulfate and concentrated to yield 1.24 g (28.0%).
1H-NMR (CD3COCD3): δ5.75 (1H, m), 5.65 (1H, m), 4.0-4.2 (1H,bs), 3.7 (2H, t), 3.05 (2H, t), 2.65
(4H, m), 2.15 (2H, m). MS (m/z): M+ 127.08, 96.03 base [C6H10N]+, 82.00 [C5H8N]+.
2-(1,2,5,6-tetrahydropyridine-N-ethyl)-4-butoxybenzoate 8(a) 2.07 g of 4-butoxy benzoylchloride
(0.00974 mol) in 5 mL of diethyl ether was added slowly to a solution of 7 (1.24 g, 0.00974 mol) in
10 mL of anhydrous ether and 1.0 gram (0.0094 mol) of sodium carbonate. The mixture was stirred at
room temperature for one hour. Diethyl ether was added and the excess carbonate was filtered off.
The filtrate was adjusted to pH 8 with 6M sodium hydroxide and the mixture extracted with ether.
Combined ether extracts was dried over magnesium sulfate and concentrated to afford 1.84 g (62.4%).
1H-NMR (CDCl3): δ 7.9 (2H, d), 6.9 (2H, d), 6.9 (1H, m), 6.7 (1H, m), 4.7 (2H, t), 4.0 (2H, t), 3.5
(2H, t), 3.2 (2H, d), 3.1 (2H, t), 2.4 (2H, m), 1.8 (2H, m), 1.5 (2H, m), 1.0 (3H, t). MS (m/z): M+
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303.18, 96.05 base [C6H10N]+,109.05 [C7H11N]+, 82.00 [C5H8N]+.
2-(1,2,5,6-tetrahydropyridine-N-ethyl)-4-butoxybenzoate hydrochloride 9(a) Hydrogen chloride
gas was bubbled through a solution containing 0.50 g (0.00165 mol) of 8a and 5 mL of acetonitrile.
The solution was concentrated to afford 0.55 g of a solid residue. The solid was recrystallized from n-
butanol to yield 0.42 g (75.0%), m.p. 145-147 oC. 1H-NMR (D2O): δ 7.8 (2H, d), 6.9 (2H, d), 5.8 (1H,
m), 5.5 (1H, m), 4.5 (2H, t), 3.9 (2H, d), 3.7 (2H, t), 3.5 (2H, t), 3.3 (2H, t), 2.3 (2H, m), 1.6 (2H, m),
1.3 (2H, m), 0.7 (3H, t). Elemental analysis calculated (%) for C18H26NO3Cl (H2O): C 60.44, H 7.83,
N 3.91, Cl 9.91. Found: C 60.58, H 7.34, N 3.76, Cl 10.02.
2-(1,2,5,6-tetrahydropyridine-N-ethyl)-4-butoxybenzoate N-methyl iodide 10(a) To a solution of
1.00 g of 8a (0.0033 mol) in 10 mL of acetonitrile, 0.57 g of methyl iodide (0.00395 mol) was added
and stirred overnight. The mixture was concentrated and the residue washed with anhydrous ether to
promote crystallization. The resulting salt was recrystallized from n-hexanol and vacuum dried to
1
yield 1.037 g (70.45%) of an off-white powder, m.p. 99-100 °C. H-NMR (D2O): δ 7.9 (2H, d), 6.9
(2H, d), 5.9 (1H, m), 5.6 (1H, m), 4.6 (2H, t), 4.0 (2H, t), 3.7 (2H, d), 3.6 (2H, t), 3.5 (2H, t), 3.1 (3H,
s), 2.4 (2H, m), 1.6 (2H, m), 1.4 (2H, m), 0.9 (3H, t). Elemental analysis calculated (%) for
C19H28NO3I: C 51.24, H 6.34, N 3.15, I 28.50. Found: C 50.80, H 6.18, N 2.92, I 28.72.
2-(1,2,5,6-tetrahydropyridine-N-ethyl)-4-butylbenzoate 8(b). Reagents used: 1.16 g of 4-butyl
benzoylchloride (0.0059 mol) dissolved 3 mL of anhydrous ether, 0.75 g of 7 (0.0059 mol) dissolved
in 3 mL of anhydrous ether, one gram of sodium carbonate (0.0094 mol). 1.5 g was recovered (89%).
1
Purity was determined by GC-MS. H-NMR (CDCl3) δ7.95 (2H, d), 7.3(2H, d), 5.7 (1H, m), 5.65
(1H, m), 4.5 (2H, t), 3.1 (2H, d), 2.9 (2H, t), 2.7 (2H, t), 2.6 (2H, m), 2.2 (2H, t), 1.6 (2H, m), 1.35
(2H, m), 0.9 (3H, t). MS (m/z): M+ 287.13, 161.12 [C11H13O]+, 109.11 [C5H8N]+, 96.06 base
[C6H10N]+, 82.03 [C5H8N]+.
2-(1,2,5,6-tetrahydropyridine-N-ethyl)-4-butylbenzoate hydrochloride 9(b) Reagents used: 0.5 g
(0.00174mol) of 8b, hydrogen chloride, 5 mL of acetonitrile. Solid residue was recrystallized from a
mixture of n-butanol, carbon tetrachloride and ether to afford 0.402 g (71.4%), mp 115.3-116.5 °C.
The sample was first dissolved in just enough warm 1-butanol, carbon tetrachloride was added to
double the volume. This mixture was then cooled and anhydrous diethyl ether was added until
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solution turned cloudy with white crystals precipitating out with further cooling. 1H-NMR (D2O): δ7.8
(2H, d), 7.25 (2H, d), 5.85 (1H, m), 5.6 (1H, m), 4.56 (2H, t), 3.8 (2H, t), 3.7 (2H, d), 3.54 (2H, t),
2.55 (2H,t), 2.3 (2H, m), 1.45 (2H, m), 1.15 (2H, m), 0.75 (3H, t). Elemental analysis calculated (%)
for C18H26NO2Cl (H2O): C 66.76, H 8.09, N 4.33, Cl 10.95. Found: C 66.59, H 7.99, N 4.32, Cl 10.83.
2-(1,2,5,6-tetrahydropyridine-N-ethyl)-4-butylbenzoate N-methyl iodide 10(b) A solution
containing 0.86 g (0.0030 mol) of 8(b), 0.509 g (0.00448 mol) of CH3I and 5 ml of acetonitrile was
stirred at room temperature overnight. The solution was concentrated and solid recrystallized from n-
butanol to yield 0.96 g (75 %), mp 110.3-111.6 oC. 1H-NMR (D2O) δ7.8 (2H, d), 7.2 (2H, d), 5.9 (1H,
m), 5.5 (1H, m), 4.0 (2H, d), 3.8 (2H, m), 3.7 (2H, t), 3.5 (2H, t), 3.1 (3H, s), 2.6 (2H, t), 2.4 (2H, m),
1.5 (2H, m), 1.1 (2H, m), 0.7 (3H, t). Elemental analysis calculated (%) for C19H28NO2I: C 53.15%, H
6.57%, N 3.26%, I 29.56. Found: C 52.85%, H 6.44%, N 3.22%, I 29.63%.
2-(1,2,5,6-tetrahydropyridine-N-ethyl)-4-hexoxybenzoate 8(c) Reagents used: 1.022 g of 4-hexoxy
benzoylchloride (0.00425 mol) dissolved 5 mL of anhydrous ether, 0.54 g of 7 (0.00425 mol)
dissolved in 5 mL of anhydrous ether, one gram of sodium carbonate (0.0094 mol). 0.94 g of was
1
recovered (67.1%). Purity was determined by GC-MS. H-NMR (CD3COCD3) δ7.95 (2H, d),
7.05(2H, d), 5.7-5.6 (2H, m), 4.0 (2H, t), 4.1 (2H, t), 3.1 (2H, d), 2.8 (2H, t), 2.7 (2H, t), 2.1 (2H, m),
1.8 (2H, m), 1.5 (2H, m), 1.4 (4H, m), 0.9 (3H, t).MS (m/z): M+ 331.18, 205.16 [C13H17O2]+, 109.14
[C7H11N]+, 96.07 base [C6H10N]+, 82.02 [C5H8N]+.
2-(1,2,5,6-tetrahydropyridine-N-ethyl)-4-hexoxybenzoate hydrochloride 9(c) Hydrogen chloride
gas was passed to a solution containing 0.47 g (0.00142 mol) of 8c in 5 mL of CH2Cl2 for one minute.
The solution was concentrated and residue recrystallized from n-butanol to afford 0.25 g of 9c
1
(48.0%), m.p. 126-126.5 OC. H-NMR (D2O) δ7.8 (2H, d), 6.8 (2H, d), 5.9 (1H, m), 5.6 (1H, m), 4.5
(2H, t), 3.9 (2H, t), 3.7 (2H, t), 3.5 (2H, t), 3.4 (2H, t), 2.38 (2H, m), 1.6 (2H, m), 1.35-1.05 (6H, m),
0.75 (3H, t). Elemental analysis calculated (%) for C20H30NO3Cl: C 65.33%, H 8.16%, N 3.80%.
Found: C 65.03%, H 8.13%, N 3.77%.
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2-(1,2,5,6-tetrahydropyridine-N-ethyl)-4-hexoxybenzoate N-methyl iodide 10(c) A solution
containing 0.47 g (0.00142 mol) of 8c, 1.14 g (0.0074 mol) of CH3I and 5 ml of acetonitrile was
stirred at room temperature overnight. The solution was concentrated and solid residue recrystallized
o
1
from n-butanol to yield 0.46 g (69 %), mp 107.5-108.5 C. H-NMR (CD3COCD3) δ8.1 (2H, d), 7.1
(2H, d), 6.1 (1H, m), 5.9 (1H, m), 4.9 (2H, t), 4.4 (2H, dd), 4.3 (2H, t), 4.0 (2H, t), 3.6 (2H, t), 2.8
(3H, s), 2.7 (2H, m), 2.1 (2H, m), 1.8 (2H, m), 1.5 (2H, m), 1.4 (2H, m), 0.9 (3H, t). Elemental
analysis calculated (%) for C21H32NO3I: C 53.31%, H 6.76%, N 2.96%, I 26.82. Found: C 52.88%, H
6.66%, N 2.98%, I 29.87%.
1-(2-hydroxyethyl) piperidine (11) To a mixture containing 10.38 g of piperidine (0.122 mol),
10.818 g of sodium carbonate (0.122 mol) and 50 mL of acetonitrile was added slowly another
mixture containing 15.25 g of 2-bromoethanol (0.121 mol) and 50 mL of acetonitrile. The reaction
mixture was allowed to stir for 7 additional days at room temperature. The mixture was then
concentrated and the residue washed several times with ether. Ether solution was concentrated and
residue distilled to afford 5.15 g (33.0%), colorless liquid, bp 59 oC/5 mm Hg, nD 1.4787. MS m/z: M+
129.03, 98.06 base [C6H12N]+, 31 [CH2=OH]+. Literature value: bp 70-75/10 mm Hg, nD 1.4794. 1H-
NMR (CD3COCD3): δ 1.40-1.45 (2H, m), 1.5-1.6 (4H, m), 2.45-2.40 (6H, m), 3.55 (2H, t).
2-(N-Piperidine ethyl) p-butylbenzoate 12(b) A solution of 0.50 g (0.0039 mol) of 11 in 5 mL of
acetonitrile was added to another mixture containing 0.76 g of 4-butyl benzoyl chloride (0.0039 mol),
0.50 g of sodium carbonate (0.00472 mol) and 5 mL of acetonitrile. After 2 hours of stirring, at room
temperature, the sodium carbonate was filtered off and filtrate then made alkaline with 6M sodium
hydroxide to pH 8. The mixture was then extracted with ether, dried over magnesium sulfate and
concentrated to afford 0.605 g (54%). 1H-NMR (CD3COCD3): δ 7.95 (2H, d), 7.35 (2H, d), 4.4 (2H,
t), 2.7 (4H, m), 2.5 (4H, m), 1.65 (2H, m), 1.55 (4H, m), 1.4 (4H, m), 0.9 (3H, t). MS (m/z): M+ 289,
205.25 [C13H17O2]+, 98.17 base [C6H12N]+, 111.17 [C7H13N]+.
2-(N-Piperidine ethyl) p-butylbenzoyl ester N-methyl iodide 13(b) 0.80 mL of iodomethane (0.013
mol) was added to a mixture of 0.30 g of 12b (0.00104 mol) dissolved in 6 mL of dichloromethane.
The solution was allowed to stand overnight at room temperature, concentrated to yield a solid
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residue. The solid was then recrystallized from a mixture of n-butanol and ether to yield 0.245 g
(54.4%), mp 113.3-114.7 oC. 1H-NMR (D2O): δ 8.0 (2H, d), 7.4 (2H, d), 4.9 (2H, t), 4.25 (2H, t), 3.9
(4H, t), 2.8 (3H, s), 2.75 (2H, t), 2.1 (6H, m), 1.6 (2H, m), 1.4 (2H, m), 1.0 (3H, t). Elemental analysis
calculated (%) C19H30NO2I: C 52.93, H 6.96, N 3.25, I 29.44. Found: C 52.67, H 7.14, N 3.18, I
30.17.
2-(N-Piperidine ethyl) p-tertbutylbenzoate 12(c) A solution of 0.50 g (0.0039 mol) of 11 in 5 mL of
acetonitrile was added to another mixture containing 0.76 g of 4-tertbutyl benzoyl chloride (0.0039
mol), 0.50 g of sodium carbonate (0.00472 mol) and 5 mL of acetonitrile. After 2 hours of stirring, at
room temperature, the sodium carbonate was filtered off and filtrate then made alkaline with 6M
sodium hydroxide to pH 8. The mixture was then extracted with ether, dried over magnesium sulfate
and concentrated to afford 0.67 g (60%). 1H-NMR (CD3COCD3): δ 7.95 (2H, d), 7.6 (2H, d), 4.4 (2H,
t), 2.75 (2H, t), 2.5 (4H, t), 1.6 (6H, m), 1.4 (9H, s).
2-(N-Piperidine ethyl) p-tertbutylbenzoyl ester N-methyl iodide 13(c) same procedure as in 13b
with 0.305 gram (0.00106 mol) of 12c in 5 ml of CH2Cl2 to afford 0.21 g of 13c (46%) after
O
1
recrystallization from n-butanol, m.p. 152.8-154.2 C. H-NMR (D2O): δ 7.82 (2H, d), 7.5 (2H, d),
4.7 (2H, t), 3.75 (2H, t), 3.5-3.3 (4H, t), 3.1 (3H, s), 1.9-1.7 (4H, m), 1.4-1.35 (2H, m), 1.1 (9H, s).
Elemental analysis calculated (%) for C19H30NO2I: C 52.90, H 6.96, N 3.24. Found: C 52.48, H 7.07,
N 3.12.
2-(N-Piperidine ethyl) p-butylbenzoyl ester N-methyl iodide 14(b) HCl gas was passed through a
mixture of 0.30 g of 12b (0.001014 mol) for several minutes. The solution was concentrated to afford
0.39 g of a solid residue. The solid was then recrystallized from a mixture of n-butanol and ether to
yield 0.136 g (40.0%), mp 159.6-160.8 oC. 1H-NMR (D2O): δ 7.8 (2H, d), 7.3 (2H, d), 4.5 (2H, t), 3.5
(4H, m), 3.0 (2H, bm), 2.6 (2H, t), 2.75 (2H, t), 1.8-1.6 (6H, m), 1.45 (2H, m), 1.2 (2H, m), 0.75 (3H,
t). Elemental analysis calculated (%) for C18H28NO2Cl: C 66.39, H 8.60, N 4.30, Cl 10.89. Found: C
66.24, H 8.36, N 4.33, Cl 10.74.
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Cell Culture and Membrane Preparation
Chinese hamster ovary cells stably transfected with the genes of human variants of muscarinic
receptors and cDNA coding G15 G-protein were purchased from Missouri S&T cDNA Resource
Center (Rolla, MO, USA). M2 or M4 receptors were connected to phospholipase C pathway by G15 G-
[26]
protein that was cotransfected into CHO cells.
Stable cell lines were established by selection of
hygromycine B resistant clones. Cell cultures and crude membranes were prepared as described
[27]
previously.
Cells were grown to confluence in 75 cm2 flasks in Dulbecco’s modified Eagle’s
medium supplemented with 10% fetal bovine serum and 2 million of cells were sub-cultured to
100 mm Petri dishes. Medium was supplemented with 5 mM butyrate for the last 24 hours of
cultivation to increase receptor expression. Cells were detached by mild trypsinization on day 5 after
subculture. Detached cells were washed twice in 50 ml of phosphate-buffered saline and 3 min
centrifugation at 250 × g. Washed cells were suspended in 20 ml of ice-cold incubation medium
(100 mM NaCl, 20 mM Na-HEPES, 10 mM MgCl2, pH = 7.4) supplemented with 10 mM EDTA and
homogenized on ice by two 30 s strokes using Polytron homogenizer (Ultra-Turrax; Janke & Kunkel
GmbH & Co. KG, IKA-Labortechnik, Staufen, Germany) with a 30 s pause between strokes. Cell
homogenates were centrifuged for 30 min at 30,000 × g. Supernatants were discarded, pellets
suspended in fresh incubation medium, incubated on ice for 30 minutes and centrifuged again.
Resulting membrane pellets were kept at - 80 °C until assayed within 10 weeks at a maximum.
Radioligand Binding Experiments
All radioligand binding experiments were optimized and carried out as described earlier. Briefly,
membranes were incubated in 96-well plates at 30 oC in the incubation medium described above.
Incubation volume was 400 μl or 800 μl for competition and saturation experiments with [3H]NMS,
respectively. Approximately 30 μg of membrane proteins per sample were used.
N-methylscopolamine binding was measured directly in saturation experiments using six
concentrations (30 pM to 1000 pM) of [3H]NMS for 1 hour. For calculations of equilibrium
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dissociation constant (KD), concentrations of free [3H]NMS were calculated by subtraction of bound
radioactivity from total radioactivity in the sample and fitting equation 1 (data analysis section).
Binding of tested ligands was determined in competition experiments with 1 nM [3H]NMS and
inhibition constant KI was calculated according Eq. 3. Nonspecific binding was determined in the
presence of 1 μM unlabeled atropine. Incubations were terminated by filtration through Whatman
GF/C glass fiber filters (Whatman) using a Brandel cell harvester (Brandel, Geithesburg, MD, USA).
In kinetic experiments membranes were pre-incubated with 1nM [3H]NMS for 1 hour (M2), 3 hours
(M1, M3, M4) or 5 hours (M5). Then dissociation was started by addition of atropine to final
concentration of 10 µM. Atropine was added either alone or in mixture with antagonist at final
concentration of 100 µM. Dissociation was terminated by filtration after 5 min (M2), 30 min (M1, M3),
40 min (M4) or 2 hours (M5) Filters were dried in microwave oven and then solid scintillator Meltilex
A was melted on filters (105 °C, 90 s) using a hot plate. The filters were cooled and counted in Wallac
Microbeta scintillation counter.
Accumulation of Inositol phosphates
[28]
Accumulation of inositol phosphates was measured according to Michal et al.
Inositolphosphates
formation was determined in cells pre-labeled overnight by 0.5 µM [3H]myo-inositol in 0.3 ml of
DMEM (3 μCi/ml) at 37 °C. Cells were then washed with fresh medium and pre-incubated in 0.4 ml
of DMEM containing 10 mM LiCl and
tested compound for 15 min at 37°C. Then agonist
carbachol was added and samples were incubated for additional 20 min in final volume 0.5 ml.
Incubation was stopped by removal of incubation buffer and addition 0.2 ml 20% trichloracetic acid
(TCA). After 1 h incubation at 4 °C, 100 µl of TCA extracts were taken for measurement. Rest of the
TCA was removed, and TCA precipitates were washed with 200 μl TCA and dissolved in 300 μl of 1
M NaOH. After 1 h incubation at 4 °C, 100 µl of NaOH lysates were taken for measurement.
Radioactivity in TCA extracts and NaOH lysates were determined by liquid scintillation counting. The
rate of inositolphosphates accumulation was calculated as a percentage of the soluble (released)
inositolphosphates from the total incorporated radioactivity (sum of radioactivity in NaOH lysates and
TCA extracts).
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Binding and activation parameters of CHO cells
Equilibrium dissociation constants (KD) and maximum binding capacities (BMAX) of [3H]NMS were
obtained by fitting Equation 1 (data analysis section) to the data from saturation binding experiments
(table 1). KD values are expressed as negative logarithms and BMAX values as pmol of binding sites
per mg of membrane protein. Inhibition constants Ki of carbachol were calculated according
equation 3 (data analysis section) and are expressed as negative logarithms. % low denotes
percentage of low affinity binding sites for carbachol. Half-efficient concentrations (EC50) and
maximal effects (EMAX) of carbachol were obtained by fitting equation 4 (data analysis section) to
the data from measurements of accumulation of inositol phosphates. EC50 values are expressed as
negative logarithms and EMAX values as folds over basal. Values are means SD.
Determination of Acetylcholinesterase Activity
Acetylcholinestarase activity in samples (figure 1) mimicking binding and functional or assay
conditions was determined by decrease in recovery of 14C-acetylcholine in organic phase of
tetraphenyl borate precipitation assay. [29] Acetylcholine radiolabeled by 14C in the acetate was added
to either wells containing only buffer or also washed adherent cells or cell membranes to final
concentration of 50 nM. After 20 min incubation at 37 ºC (adherent cells) or 3 hours at 30 ºC
(membranes), membranes were spin down, supernatant was taken out and combined 1:1 with sodium
tetraborate in 3-heptanone (15 mg/ml). Samples were vigorously vortexed, centrifuged 15 min at
3,000 × g to separate the organic and aqueous phases. Aliquots of the organic phase were taken for
scintillation counting.
Data analysis
Data from biological evaluation experiments were processed in Libre Office, analyzed and plotted
[30]
[31]
using program Grace.
Statistical analysis was performed using statistical package R.
For non-
linear regression analysis following equations were used:
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[3H]NMS saturation binding
BMAX ∗ x
y=
Eq. 1
x+ KD
where y is specific binding at free concentration x, BMAX is maximum binding capacity, and KD is
equilibrium dissociation constant.
Competition binding
100 ∗ x
x + IC50
Eq. 2
y =100 −
where y is specific radioligand biding at concentration x of competitor expressed as percent of binding
in the absence of competitor, IC50 is concentration causing 50 % inhibition of radioligand binding.
Inhibition constant KI was calculated as:
IC50
Eq. 3
KI =
[ ]
D
1+
KD
where IC50 is concentration causing 50 % inhibition of [3H]NMS binding calculated according Eq. 2
from competition binding data, [D] is concentration of [3H]NMS used, and KD is its equilibrium
dissociation constant calculated according Eq. 1 from saturation binding data. [32]
Concentration response
(
EMAX −1
)
∗ xnH
xnH + EC50
y =1+
Eq. 4
where y is response normalized to basal (in the absence of carbachol) at ligand concentration x, EMAX
is maximal effect, EC50 is concentration causing half-maximal effect, and nH is Hill coefficient.
Equilibrium dissociation constant of antagonism of functional response KB was calculated from the
shift in EC50 of functional response to carbachol by antagonist according Eq. 5 (determination from a
single antagonist concentration) or Eq. 6 (determination from six concentration of antagonist)
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lo g (DR− 1) = log (KB) − log ([ B])
[B](1− α)
Eq. 5
Eq. 6
log (DR− 1) = log [
]
α[B ]+ KB
where DR is ratio of EC50 of the functional response in the presence to the absence of antagonist B
and α is a factor of cooperativity between carbachol and antagonist.
RESULTS
Synthesis
Compound 6 (scheme 1) was synthesized by reacting pyridine with 2-bromoethanol and then
[24]
selectively reduced with sodium borohydride to produce compound 7.
Reaction of 7 with p-
substituted benzoyl chlorides afforded compounds 8a-c which were then treated with hydrogen
chloride gas and iodomethane to afford salts 9a-c and 10a-c, respectively.
Compound 11 (scheme 2) was formed by reacting piperidine with 2-bromoethanol and then treated
with several p-substituted benzoyl chlorides to yield compounds 12a-c. Compounds 12a-c reacted
with iodomethane and hydrogen chloride to produce the corresponding salts 13a-c and 14a-b,
respectively.
Biological Evaluation
Affinity of compounds (schemes 1 & 2) was assessed in competition experiments with 1 nM
[3H]NMS. All tested compounds completely inhibited [3H]NMS binding. Inhibition constants KI
(expressed as pKI), calculated according to equation 3 (data analysis section), micromolar range, are
summarized in table 2.
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Our novel ligands (9a-14b) bind to all receptor subtypes and display the same affinity for all receptors
with some exceptions. Compound 13a displays slightly (three to eight fold) higher affinity for M2
receptor, 9b displays slightly (two to three-fold) higher affinity for M1 receptor, 14b displays slightly
(two to five fold) higher affinity for the M2 receptor, 9a, 13(b-c) display slightly (two to four fold)
higher affinity for the M2 receptor, 9c displays slightly (1 to 3 fold) for M2 receptor, and compound
10c displays slightly higher affinity (one to three fold) for the M2 and M5 receptors. In general,
analogs with a quaternary ammonium group bind with higher affinity than those with a tertiary
cationic group at all receptor subtypes with the exception of 13c with R= tert-C4H9. Thus potency of
10b > 9b, 10a > 9a, and 13b > 14b> 13c. Furthermore, compound 10c with a para-substituted O-n-
C6H13 group displays appreciably higher affinity than other compounds at all receptors.
Ability of tested compounds to antagonize functional response at muscarinic receptors was
determined in measurements of carbachol induced accumulation of inositol phosphates. Antagonism
of tested compounds was determined at single high concentration of 0.1 mM. All tested compounds
antagonized functional response to carbachol and increased carbachol half-efficient concentration
(EC50). Based on shift in the EC50 by antagonist equilibrium dissociation constants (KB) of antagonists
were calculated according equation 5 (data analysis section) and are summarized in table 3. The
most potent antagonist among tested compounds was 10c displaying KB in nanomolar range, with
exception of M2 receptor.
A full Schild regression analysis (figure 2) for representative antagonist 10a was performed with pKB
and alpha (α) values shown in table 4.
All Compounds slowed down in [3H]NMS dissociation as summarized in table 5 and figure 3. The
existence of the secondary binding site is supported by slow-down in [3H]NMS dissociation as any
change of the ligand kinetic is conditioned by concurrent binding of two ligands to the receptor and
concurrent binding of two ligands may occur only to two distinct sites.
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Acetylcholinestarase activity in samples (figure 1) mimicking binding and functional or
assay conditions was determined by decrease in recovery of 14C-acetylcholine in organic
phase of tetraphenyl borate precipitation assay.
DISCUSSION
The major finding of this pilot study is the identification of M1/M4 functionally selective muscarinic
antagonists structurally related to agonist AC-42. All five muscarinic receptor subtypes share high
sequence homology, especially in the orthosteric binding site. Current crystallographic structures of
M2 and M3 receptors show that homology in the secondary and tertiary structure extends even beyond
the orthosteric binding site.25 This high homology hinders the discovery of ligands that bind
selectively to specific receptor subtypes. This study describes various functionally selective ligands
(ligands that preferentially activate only some receptor subtypes or signaling pathways) that bind to
all receptor subtypes with the same affinity. Functionally selective ligands thus appear to be possible
way for selective modulation of function of muscarinic receptors.
Complete inhibition of the orthosteric antagonist [3H]NMS suggests steric interaction (mutual
exclusivity of binding) between tested compounds and [3H]NMS. On the other hand, in some cases,
antagonism of carbachol induced functional response by compounds suggests non-competitive
interaction between compounds and carbachol as potency of functional antagonism (pKB) is greater
than affinity of the compounds (pKI) (table 3 versus table 2). A full Schild regression analysis of
representative compound 10a shows inhibition of carbachol response is indeed allosteric with factor
cooperativity alpha (α) of about 0.001 (potency limits to about 1000-fold decrease) and KB values
about 2-fold underestimated, so the difference between KB and Ki is even greater (table 4, figure 2).
This discrepancy is explained by existence of the secondary binding site to which new compounds
bind with higher affinity than to the orthosteric binding site. From this secondary site, these
compounds block receptor activation but do not inhibit [3H]NMS binding. The existence of the
secondary binding site is supported by slow-down in [3H]NMS dissociation (table 5, figure 3) as any
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change of the ligand kinetic is conditioned by concurrent binding of two ligands to the receptor and
concurrent binding of two ligands may occur only to two distinct sites.
Interestingly, compounds 9b, 10a and 10b are much more potent antagonists at M1 and M4 receptors
than at the rest of subtypes. These compounds are considered M1/M4 functionally selective antagonists.
The most potent antagonist among tested compounds was 10c displaying KB in nanomolar range with
exception of M2 receptor (table 3). These novel prototypical compounds of functionally selective
M1/M4 antagonists may provide a viable approach for the treatment of certain central nervous system
disorders including Parkinson disease (PD).
Finally, acetylcholinestarase activity in samples (figure 1) mimicking binding and functional or assay
conditions was determined by decrease in recovery of 14C-acetylcholine in organic phase of
tetraphenyl borate precipitation assay. Recovery of [14C] acetylcholine in the organic phase is the
same under all experimental conditions suggesting that no detectable acetylcholinesterase activity in
the samples.
ACKNOWLEGMENTS
This work was supported by the Academy of Sciences of the Czech Republic support
[RVO:67985823], the Grant Agency of the Czech Republic grants [14-05696S] and [P304/12/G069]
and by the Department of Physical Sciences at Barry University.
FIGURE LEGENDS
Scheme 1. Synthesis of N-Substituted 4-Benzoyl Tetrahydropyridinyl Salts
Scheme 2. Synthesis of N-Substituted p-Benzoyl Piperidinyl Salts
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Table 1. Equilibrium dissociation constants (KD) and maximum binding capacities (BMAX) of
[3H]NMS
Table 2. Binding equilibrium constants (expressed as pKI) calculated from equation 3 and expressed
as negative logarithms. (*) Higher than at other subtypes (p<0.05, ANOVA, Dunnet's post-test), values
are means SD.
Table 3. Equilibrium dissociation constants of antagonist competition of functional response to
carbachol. Equilibrium dissociation constants KB obtained by fitting Eq. 5 to the data from functional
response assays in the presence of antagonist and expressed as negative logarithms. (*) Greater than
inhibition constant pKi in table 2 (p<0.05, t-test).
Table 4. Equilibrium dissociation constants of compound 10a competition of functional response to
carbachol. Equilibrium dissociation constants KB and factors of cooperativity obtained by fitting
Eq. 6 to the data from functional response assays in the presence of antagonist. KB values expressed
as negative logarithms. Data are means SD from 3 independent experiments performed in triplicates.
(*) Different from other subtypes (p<0.05, ANOVA, Tukey HSD post-test).
Table 5. Rate dissociation constants koff of [3H]NMS dissociation in the absence (control) or presence
of competitor in the final concentration of 0.1 mM expressed in min-1. (*) Different from control
(p<0.05, t-test), values are means SD.
Figure 1. Acetylcholineterase Activity. [14C] acetylcholine recovery in the organic phase expressed as
percent of added radioactivity. Values are means SD from 3 independent experiments performed in
duplicates.
Figure 2. Schild regression analysis of compound 10a antagonism. Logarithms of dose ratios (DR) of
inositol phosphates accumulation induced by carbachol in the presence to absence of compound 10a
at individual subtypes of muscarinic receptors plotted against used concentration of 10a. Data are
means SD from 3 independent experiments preformed in triplicates.
Figure 3. Effect of antagonists on [3H]NMS dissociation. Membranes incubated with 1nM [3H]NMS
for 1 hour (M2), 3 hours (M1, M3, M4) or 5 hours (M5). Dissociation started by addition of atropine to
final concentration of 10 µM. Atropine added either alone or in mixture with antagonist at final
concentration of 100 µM. Dissociation terminated by filtration after 5 min (M2), 30 min (M1, M3), 40
min (M4) or 2 hours (M5). Data are means SD from 3 independent experiments preformed in
quadruplicates.
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CONFLICT OF INTEREST
I have no conflict of interest
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