Homologation as a lead modification approach en route to a series of lactone-based muscarinic ligands

Article abstract of DOI:10.1007/s00044-013-0692-3

Previous work identified the lactone ring as a useful scaffold for the design of muscarinic ligands and reported a lactone-based ligand with an IC ₅₀ of 340 nM. Using homologation as a lead modification approach, a new series of lactone-based c

Full text of DOI:10.1007/s00044-013-0692-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:1023–1030
DOI 10.1007/s00044-013-0692-3
ORIGINAL RESEARCH
Homologation as a lead modification approach en route to a series
of lactone-based muscarinic ligands
•
Rong Gao Richie R. Bhandare Daniel J. Canney
•
Received: 14 March 2013 / Accepted: 6 August 2013 / Published online: 22 August 2013
Ó Springer Science+Business Media New York 2013
Abstract Previous work identified the lactone ring as a
useful scaffold for the design of muscarinic ligands and
reported a lactone-based ligand with an IC50 of 340 nM.
Using homologation as a lead modification approach, a
new series of lactone-based compounds have been
designed, synthesized, and screened in muscarinic binding
assays. The approach provided a series of compounds with
improved % inhibition values and identified the highest
affinity lactone-based ligand reported to date. The results of
these efforts and the structure–activity relationship for this
series of lactones-based ligands are discussed.
treatment of various disorders including overactive blad-
der, Alzheimer’s disease, pain, cognitive impairment, drug
addiction, schizophrenia, and Parkinson’s disease (Abrams
et al., 2006; Wess et al., 2007). To date, efforts to develop
subtype selective ligands for muscarinic acetylcholine
receptors (mAChRs) have been hampered by a lack of
X-ray crystal structures of the proteins and the high degree
of homology among the receptor subtypes (Felder et al.,
2000). However, the recent availability of the X-ray crys-
tal structures for the M2 and M3 subtypes should be useful
in the design of future ligands (Haga et al., 2012; Kruse
et al., 2012). Our interest in the development of subtype-
selective muscarinic ligands led to previous reports
detailing the identification of substituted lactones as lead
muscarinic compounds (Ahungena and Canney, 1996;
Ahungena et al., 2003). The general pharmacophoric
elements of the lactone-based ligands are illustrated in the
Fig. 1 where the lactone oxygens serve as a H-bond
acceptor moieties while different nitrogen-containing het-
erocycles provide the requisite cationic group. These
groups may be separated by linkers of varying sizes. Later
work involved molecular modifications of those leads that
included the addition of aromatic groups with a variety of
substitution patterns. These efforts led to an increase in
receptor affinity (Bhandare and Canney, 2011) and pro-
duced a lactone-based muscarinic ligand with an IC50 of
340 nM.
Keywords Homologation Á Lactone Á
Muscarinic receptors
Introduction
The muscarinic acetylcholine receptor system is classified
into five different subtypes (M1–M5). In general, M1, M3,
and M5 receptor subtypes are coupled via Gq-like proteins;
while M2 and M4 subtypes are coupled to Gi-proteins
(Burstein et al., 1998; Koch et al., 2005). The receptors
have been targeted in drug discovery efforts for the
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-013-0692-3) contains supplementary
material, which is available to authorized users.
We herein report a continuation of that work with the
goal of further improving affinity for muscarinic receptors
and preliminary evaluation of specific ligands for subtype
selectivity. The design, synthesis, and evaluation of a
homologous series of lactone-based compounds as mus-
carinic ligands are included in the approach. The results of
these efforts and the structure–activity relationship for this
series of lactones-based ligands are discussed.
R. Gao Á R. R. Bhandare Á D. J. Canney (&)
Department of Pharmaceutical Sciences, Moulder Center
for Drug Discovery Research, Temple University School
of Pharmacy, 3307 N. Broad Street, Philadelphia,
PA 19140, USA
e-mail: canney@temple.edu
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Fig. 1 General structural
features of the newly designed
ligands
Hydrogen bonding Moeity
O
Cationic Center
R
O
Hydrophobic Site
X
N
n
n=1,2
X=C, N
R=CN, OH, NO2, etc.
Linker
Chemistry
profound effect on affinity but the electronic nature of the
substituents were not as relevant. For example, the unsub-
stituted compounds 9a and 9b inhibited specific binding of
radioligand by 16 and 74 %, respectively. The para-
substituted compounds 3a, 3b, 5a, 5b, 7a, 7b, 8a, and 8b
contain substituents with electron donating (3a, 3b; 5a, 5b)
and electron withdrawing (7a, 7b; 8a, 8b). In each case, the
homologous compound in the pair [3b (56 %), 5b (61 %),
7b (57 %), and 8b (70 %)] exhibits higher % inhibition
regardless of the electronic nature of the substituent
(Fig. 2). The para-substituted compounds were similar to or
less than the unsubstituted 9b in their ability to inhibit
specific binding. The ortho-substituted compounds, 1a, 1b,
4a, 4b, 6a, and 6b, show a similar trend with the homologs
1b (82 %), 4b (81 %), and 6b (83 %) exhibiting higher %
inhibition than the parent lactones regardless of the nature
of the substituent. However, the ortho-substituted com-
pounds were found to have slightly higher % inhibition
values than the corresponding unsubstituted 9b, suggesting
that ortho substitution may be preferred to para. The sub-
stitution on the ortho position may influence the orientation
of the aromatic ring with respect to the piperazine and this
may have effected in improvement in the % inhibition
values over the other positions on the ring.
The synthesis and testing of lactone-based compounds 1a–
9a, 11a–15a (Table 1) has been published previously
(Bhandare and Canney, 2011). Scheme 1 shows the syn-
thesis of a homologous series of lactone-based muscarinic
ligands beginning with the olefinic ester as starting mate-
rial. A previously published modified Prins reaction was
used to prepare precursor 2 from the olefinic ester in good
yield (Gao and Canney, 2009). Compound 2 was treated
with triethylamine and tosyl chloride in dichloromethane
(DCM) to afford intermediate tosylate 3 in 69 % yield.
Displacement reactions involving 3 and the appropriate
secondary amines under refluxing conditions (72 h) affor-
ded the target ligands 1b–15b in 50–75 % yield. Precursor
4 was synthesized using a previously described method
(Ahungena and Canney, 1996). Displacement of the iodine
of lactone 4 by 1-(4-fluorobenzyl)piperazine under reflux-
ing condition (48 h) afforded 10a in 12 % yield (Ahungena
et al., 2003; Ahungena and Canney, 1996).
Results and discussion
Previously reported lactone-based muscarinic ligands were
used in the design of a homologous series of compounds
(1b–15b Table 1). Novel homologs were prepared in
modest to good yield using a newly developed route
involving a modified Prins reaction followed by the dis-
placement of the corresponding tosylated intermediate by
secondary amines (Scheme 1). Binding studies were per-
formed at CEREP using rat cerebral cortex membranes as
per a previously published method (Richards, 1990).
Preliminary data presented in Table 1 and Fig. 2 repre-
sent the percent inhibition of specific binding of radioligand
at a single concentration (10 lM) of test compound. The
inclusion of the methylene spacer between the H-bonding
lactone ring and the amine-containing heterocyclic rings
(see Fig. 1) resulted in an increase in affinity for each of the
homologs tested. Based on the limited number of com-
pounds evaluated here, the substitution pattern on the aro-
matic rings of the test compounds was found to have a
Several additional piperazine (10a, 10b, 11a, 11b, and
15b) derivatives were also prepared and tested. In our
previous work, 11a was found to have a high % inhibi-
tion value and was chosen for further evaluation
(IC50 = 340 nM). Among this new series of lactones, 11b
showed the highest percent inhibition and was chosen for
further evaluation. The IC50 value (non-selective) for 11b
was determined to be 17 nM, the highest affinity of any of
the lactone-based muscarinic ligands reported to date. Due
to its high affinity in the general muscarinic assay, 11b
underwent further screening to evaluate possible subtype
selectivity (see Fig. 3). Compound 11b was tested for the
ability to inhibit the specific binding of radioligand to
muscarinic receptor subtypes hM1–hM5 at a concentration
of 10 nM. The % inhibition values for the five subtypes
were found to be 22, 56, 34, 62, and 14 % for hM1–hM5,
respectively, demonstrating that no receptor selectivity was
exhibited for the compound.
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Med Chem Res (2014) 23:1023–1030
1025
Table 1 Preliminary binding data of compounds 1a–15a and 1b–15b
O
O
R
n
( )
R
#
n
% Inhiba, b
R
#
n
% Inhiba, b
1a
1b
1
2
32
82
9a
9b
1
2
16
74
MeO
N
N
N
N
2a
2b
1
2
9
10a
10b
1
2
28
63
OMe
N
N
F
75
N
N
3a
3b
1
2
26
56
11a
11b
1
2
97
99
Ph
Ph
N
N
OMe
N
N
HO
N
4a
4b
1
2
46
81
12a
12b
1
2
68
86
N
N
5a
5b
1
2
7
13a
13b
1
2
46
66
N
N
N
OH
N
61
N
H
NC
N
6a
6b
1
2
31
83
14a
14b
1
2
44
57
N
N
7a
7b
1
2
18
57
15a
15b
1
2
5
O
N
N
CN
N
N
33
Ph
8a
8b
1
2
18
70
N
N
NO2
a
For details regarding the evaluation of results, see ‘‘Experimental’’ section
b
% Inhibition at 10 lM
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O
O
O
O
1. H2SO4, (CH2O)n,
AcOH, 70oC
Amines
Et3N, TosCl
O
O
O
O
O
THF
CH Cl ,
rt
2
2
2. 40% NaOH
3. H2SO4
R
OTos
OH
2
1b~15b
3
1. NaOH, EtOH, reflux
2. NaHCO3, I2
O
Amine
THF
O
O
R
I
10a
4
Scheme 1 Synthesis of a homologous series of lactone-based muscarinic ligands 10a, 1b–15b
Fig. 2 Comparison of %
inhibition values of homologs to
the parent lactones
Fig. 3 Subtype selectivity data
for hM1–hM5 receptor subtypes
for compound 11b
Conclusion
number of compounds screened here, it appears that the
position of aromatic substitution may affect receptor
affinity but the electronic nature of the substitution does not
have a strong effect. Compound 11b was identified as the
highest affinity lactone-based ligand reported to date
(IC50 = 17 nM). Compound 11b was evaluated for
In summary, a homologous series of lactone-based mus-
carinic ligands was synthesized using a facile-modified
Prins reaction followed by displacement of the protected
alcohol with the appropriate amine. Based on the limited
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Med Chem Res (2014) 23:1023–1030
1027
selectivity for muscarinic subtypes and found to show no
selectivity. While these data confirm that the lactone
nucleus is a useful scaffold for the design of muscarinic
ligands and that changes in aromatic substitution patterns
may be useful in improving affinity, additional lead mod-
ification techniques will be required to improve selectivity
for the target subtypes. Compounds 6b, 11b, and 12b will
be utilized in future lead modification efforts to design
subtype-selective muscarinic ligands.
specific binding. The bound radioactivity was measured
with scintillation counter. For interpretation of this type of
preliminary data, CEREP suggests the following guide-
lines: 50 % inhibition or higher represent significant effects
(i.e., 50 % is a common cut-off value for further investi-
gation; determination of IC50 or EC50 values from con-
centration response curves). Results showing an inhibition
between 20 and 50 % indicate weak to moderate effects;
inhibition less than 20 % are considered inactive.
Chemistry
Experimental
Procedure for intermediate 3
All the commercial chemicals were purchased from Sigma-
Aldrich or Fisher scientific. Dry solvents were purchased
from Fisher Scientific. Column Chromatography was run
using Silicycle SiliaFlash P60 Silica Gel. Analytical TLC
was performed using Silicycle Precoated 60-f-254 TLC.
Melting points were recorded on a Thomas-Hoover Uni-
melt Capillary melting point apparatus. The 1H NMR (300,
400 MHz) spectra were recorded on UnityINOVA 300 or
AVANCE 400 spectrometer. The 13C NMR (101 MHz)
spectra were recorded on AVANCE 400 spectrometer. The
data are presented as follows: chemical shift in parts per
million (ppm) on the d scale relative to internal tetra-
methylsilane (TMS) (coupling constant(s) in hertz). The
following abbreviations are used to denote signal patterns:
s = singlet, d = doublet, t = triplet, and m = multiplet.
The purity was analyzed using two mobile phases in
10–90 % acetonitrile in water solvent gradient containing
0.1 % formic acid (method A) and 10–90 % methanol in
water solvent gradient containing 0.1 % trifluroacetic acid
(method B). Elemental analysis was carried out by Atlantic
Microlab Inc, Atlanta, GA. LC–MS (ESI) analyses were
done on an Agilent technologies 1200 series instrument on
target compounds using 5–95 % acetonitrile in water
(containing 0.1 % formic acid) gradient solvent system and
was found to be [95 %.
Over a mixture of the corresponding 5-membered lactone
(2, 1.0 Eq), Et3N (1.5 Eq) in dry DCM, a solution of
p-TosCl (1.25 Eq) in DCM was added dropwise at 0 °C.
The resulting mixture was stirred at 0 °C for 1 h and
allowed to stir overnight at room temperature. Then, the
reaction mixture was diluted with DCM (100 mL), washed
with 10 % HCl, brine, dried over MgSO4, and concentrated
in vacuo to afford yellowish oil. This crude product was
then purified by flash chromatography (silica gel; 10–20 %
EtOAc/Hexane) to afford desired tosylate (69 % yield).
General procedure for the synthesis of 1b–15b
Tosylate 3 (1.0 Eq) was treated with appropriate amines
(3.0 Eq) in dry THF and refluxed for 72 h. The THF was
evaporated under reduced pressure, the residue was dis-
solved in DCM, washed with H2O and brine, then dried
over MgSO4, and concentrated in vacuo to give a crude
product which was purified by flash chromatography (silica
gel; 2–8 % MeOH in DCM) to afford pure product. For 1b,
2b, 4b, 5b, 7b–12b, and 14b, the purified product was then
dissolved in ether and treated with HCl solution (2.0 M in
diethyl ether) to afford the hydrochloride salt which was
recrystallized with isopropanol or a MeOH/Ether mixture.
Binding studies
Procedure for the synthesis of 10a
The target compounds 1a–b to 15a–b were evaluated in
radioligand binding assays performed by CEREP (86600
CELLE L’EVESCAULT, France) using rat cerebral cortex
membranes expressing muscarinic receptor subtypes
M1–M5. The competitive binding assays were performed
according to the previously reported method (Richards,
1990). The assays were done in duplicate and are presented
as supplied by CEREP. Briefly, [3H]-QNB (0.05 nM) and
the test compound (10 lM) were incubated with rat cere-
bral cortex membranes for 90 min at room temperature.
Following incubation, the reaction was terminated by fil-
tration. Atropine (1 lM) was used to determine non-
Iodomethyl lactone 4 (1.0 Eq) and the appropriate sec-
ondary amine (5.0 Eq) in anhydrous tetrahydrofuran
(35 mL) was stirred at reflux under a nitrogen atmosphere
for 48 h. After 48 h, the mixture was concentrated under
reduced pressure and the residue was dissolved in dichlo-
romethane and purified by flash silica gel chromatography
using methanol (0–3 %) in dichloromethane.
2-(4,4-Diethyl-5-oxotetrahydrofuran-2-yl)ethyl-4-methyl-
benzenesulfonate (3) Colorless crystal. Yield: 69 %. mp:
57–58 °C. 1H NMR (300 MHz, CDCl3) d 7.79 (d,
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Med Chem Res (2014) 23:1023–1030
J = 8.3 Hz, 2H), 7.36 (d, J = 8.0 Hz, 2H), 4.55–4.33 (m,
1H), 4.14 (dd, J = 6.5, 13.3 Hz, 3H), 2.46 (s, 3H),
2.21–1.84 (m, 3H), 1.83–1.68 (m, 1H), 1.58 (t, J = 7.4 Hz,
4H), 0.89 (dt, J = 7.5, 18.0 Hz, 6H); 13C NMR (101 MHz,
CDCl3) d 180.33, 145.30, 132.72, 130.15, 128.03, 77.68,
77.36, 77.04, 73.18, 66.95, 48.67, 37.53, 35.82, 29.14,
28.23, 21.76, 8.81, 8.74. Anal. Calcd for C17H24O5S: C,
59.98; H, 7.11; Found: C, 60.27; H, 7.25.
D2O) d 7.33 (d, J = 8.3 Hz, 1H), 7.25 (t, J = 7.8 Hz, 1H),
7.03 (t, J = 8.0 Hz, 2H), 4.72 (m, 1H), 3.95–3.36 (m, 10H),
2.37 (dd, J = 7.0, 13.5 Hz, 1H), 2.24 (dd, J = 10.0,
19.4 Hz, 2H), 2.02 (dd, J = 9.4, 13.5 Hz, 1H), 1.82–1.47
(m, 4H), 1.01–0.75 (m, 6H); 13C NMR (101 MHz, D2O) d
187.89, 152.26, 136.50, 131.01, 123.97, 123.39, 119.52,
79.51, 56.59, 54.12, 52.40, 51.58, 39.37, 32.82, 31.89, 30.67,
11.00, 10.87. Anal. Calcd for C20H32Cl2N2O3: C, 57.28; H,
7.69; N, 6.68; Found: C, 57.37; H, 7.64; N, 6.59.
Dihydrochloride salt of 3,3-diethyl-5-(2-(4-(2-methoxy-
phenyl)piperazin-1-l)ethyl)dihydrofuran-2(3H)-one (1b)
White solid. Yield: 55 %. mp: 228–229 °C. 1H NMR
(400 MHz, D2O) d 7.21–7.10 (m, 2H), 7.04 (d, J = 8.2 Hz,
1H), 6.97 (t, J = 7.6 Hz, 1H), 4.60 (m, 1H), 3.89 (s, 3H),
3.71 (m, 4H), 3.54–3.19 (m, 6H), 2.37–2.21 (m, 2H), 2.14 (s,
1H), 1.95 (dd, J = 9.4, 13.3 Hz, 1H), 1.78–1.52 (m, 4H),
1.09–0.80 (m, 6H); 13C NMR (101 MHz, D2O) d 183.21,
154.76, 139.55, 127.76, 123.21, 121.35, 114.18, 77.01,
57.07, 55.83, 54.06, 50.79, 50.50, 50.29, 50.21, 50.07, 49.86,
49.65, 49.43, 49.22, 39.21, 32.60, 30.91, 30.05, 9.86, 9.77.
Anal. Calcd for C21H34Cl2N2O3: C, 58.20; H, 7.91; N, 6.46;
Found: C, 58.05; H, 7.95; N, 6.39.
Dihydrochloride salt of 3,3-diethyl-5-(2-(4-(4-hydroxy-
phenyl)piperazin-1-yl)ethyl)dihydrofuran-2(3H)-one (5b)
White solid. Yield: 58 %. mp: 235 °C. 1H NMR (400 MHz,
D2O) d 7.16 (d, J = 9.0 Hz, 2H), 6.94 (d, J = 9.0 Hz, 2H),
4.71 (m, 1H), 3.46 (m, 10H), 2.36 (dd, J = 6.9, 13.5 Hz,
1H), 2.23 (dd, J = 9.2, 19.4 Hz, 2H), 2.01 (dd, J = 9.4,
13.5 Hz, 1H), 1.77–1.50 (m, 4H), 0.90 (dt, J = 7.5,
12.5 Hz, 6H); 13C NMR (101 MHz, D2O) d 187.92, 155.62,
143.21, 123.52, 119.36, 79.53, 56.53, 54.17, 52.42, 52.07,
39.38, 32.83, 31.92, 30.68, 11.00, 10.87. Anal. Calcd for
C20H32Cl2N2O3: C, 57.28; H, 7.69; N, 6.68; Found: C,
57.53; H, 7.74; N, 6.62.
Dihydrochloride salt of 3,3-diethyl-5-(2-(4-(3-methoxy-
phenyl)piperazin-1-yl)ethyl) dihydrofuran-2(3H)-one (2b)
White solid. Yield: 52 %. mp: 203–205 °C. 1H NMR
(400 MHz, DMSO) d 7.20 (t, J = 8.2 Hz, 1H), 6.66–6.45
(m, 3H), 4.58 (m, 1H), 3.77 (s, 3H), 3.61 (s, 2H), 3.26 (s,
2H), 3.16 (d, J = 8.9 Hz, 4H), 2.25 (dd, J = 6.8, 13.3 Hz,
3H), 1.86 (dd, J = 9.4, 13.1 Hz, 1H), 1.69–1.45 (m, 4H),
0.89 (dt, J = 7.5, 10.2 Hz, 6H); 13C NMR (101 MHz,
MeOH) d 183.23, 163.09, 150.73, 132.18, 77.00, 56.75,
55.73, 53.34, 50.78, 50.50, 50.29, 50.07, 49.86, 49.65,
49.43, 49.22, 39.17, 32.55, 30.89, 30.03, 9.86, 9.78. Anal.
Calcd for C21H34Cl2N2O3: C, 58.20; H, 7.91; N, 6.46;
Found: C, 58.24; H, 7.93; N, 6.46.
2-(4-(2-(4,4-Diethyl-5-oxotetrahydrofuran-2-yl)ethyl)pip-
erazin-1-yl)benzonitrile (6b) Yellowish oil. Yield: 50 %.
1H NMR (400 MHz, CDCl3)
d 7.50–7.40(m, 2H),
6.97–6.93 (m, 2H), 4.44–4.40 (m, 1H), 3.21–3.19 (m, 4H),
2.67–2.55 (m, 6H), 2.11–2.06, (m, 1H), 1.85–1.75 (m, 3H),
1.58–1.55, (m, 4H), 0.90–0.82, (m, 6H); 13C NMR
(101 MHz, CDCl3) d 181.02, 155.77, 134.63, 134.15,
122.23, 119.01, 118.70, 106.22, 75.70, 54.63, 53.41, 51.54,
48.90, 37.95, 33.80, 29.55, 28.59, 9.08, 9.01. MS (ESI) (m/
z) 356.1 (M ? H)?. HPLC analysis: [95 % purity using
two different mobile phases.
Hydrochloride salt of 4-(4-(2-(4,4-diethyl-5-oxotetrahy-
drofuran-2-yl)ethyl)piperazin-1-yl)benzonitrile (7b) White
1
3,3-Diethyl-5-(2-(4-(4-methoxyphenyl)piperazin-1-yl)ethyl)
dihydrofuran-2(3H)-one (3b) Yellowish oil. Yield: 53 %.
1H NMR (400 MHz, CDCl3) d 6.84-6.95 (m, 4H), 4.45–4.48
(m, 1H), 3.70 (s, 3H), 3.04–3.01 (m, 4H), 2.57–2.47 (m,
6H), 2.10–2.05 (m, 1H), 1.80–1.75 (m, 3H), 1.58–1.53 (m,
4H), 0.90–0.82 (m, 6H); 13C NMR (101 MHz, CDCl3) d
181.10, 154.13, 145.94, 118.49, 114.73, 75.91, 55.87, 54.79,
53.70, 50.89, 48.92, 38.00, 34.11, 29.55, 28.61, 9.10, 9.02.
MS (ESI) (m/z) 361.1 (M ? H)?. HPLC analysis: [95 %
purity using two different mobile phases.
solid. Yield: 56 %. mp: 213–214 °C; H NMR (400 MHz,
MeOH-d4) d 7.68–7.52 (m, 2H), 7.12 (d, J = 9.1 Hz, 2H),
4.58 (m, 1H), 4.33–2.97 (m, 10H), 2.35–2.20 (m, 2H),
2.20–2.07 (m, 1H), 1.95 (dd, J = 9.4, 13.3 Hz, 1H),
1.74–1.53 (m, 4H), 0.94 (dt, J = 7.5, 13.3 Hz, 6H). 13C
NMR (101 MHz, MeOH-d4) d 183.18, 154.60, 135.59,
121.26, 117.41, 103.85, 77.01, 55.79, 53.62, 50.77, 50.50,
50.29, 50.07, 49.86, 49.65, 49.44, 49.22, 46.77, 39.19,
32.59, 30.90, 30.03, 9.85, 9.77. Anal. Calcd for
C21H30ClN3O2: C, 64.35; H, 7.72; N, 10.72; Found: C,
64.46; H, 7.65; N, 10.65.
Dihydrochloride salt of 3,3-diethyl-5-(2-(4-(2-hydroxy-
phenyl)piperazin-1-l)ethyl)dihydrofuran-2(3H)-one (4b)
White solid. Yield: 49 %. mp: 252 °C. 1H NMR (400 MHz,
Hydrochloride salt of 3,3-diethyl-5-(2-(4-(4-nitrophenyl)pip-
erazin-1-yl)ethyl)dihydrofuran-2(3H)-one
(8b) Yellowish
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Med Chem Res (2014) 23:1023–1030
1029
solid. Yield: 47 %. mp: 184 * 185 °C. 1H NMR
(400 MHz, MeOH-d4) d 8.15 (d, J = 9.3 Hz, 2H), 7.11 (d,
J = 9.4 Hz, 2H), 4.59 (m, 1H), 4.37–3.08 (m, 10H),
2.36–2.21 (m, 2H), 2.21–2.08 (m, 1H), 1.95 (dd, J = 9.4,
13.2 Hz, 1H), 1.75–1.52 (m, 4H), 0.94 (dt, J = 7.5, 13.2 Hz,
6H); 13C NMR (101 MHz, MeOH-d4) d 183.19, 156.05,
141.97, 127.53, 116.10, 77.01, 55.81, 53.54, 50.77, 50.50,
50.29, 50.07, 49.86, 49.65, 49.43, 49.22, 46.59, 39.19, 32.57,
30.89, 30.03, 9.85, 9.77. Anal. Calcd for C20H30Cl1
N3O4Á0.5H2O: C, 57.07; H, 7.42; N, 9.98; Found: C, 57.02;
H, 7.26; N, 9.91.
D2O) d 7.67 (d, J = 7.2 Hz, 4H), 7.50 (dq, J = 7.1,
14.4 Hz, 6H), 4.67 (m, 1H), 3.82–3.35 (m, 10H), 2.33 (dd,
J = 6.9, 13.5 Hz, 1H), 2.18 (d, J = 33.6 Hz, 2H), 1.98 (dd,
J = 9.5, 13.5 Hz, 1H), 1.62 (ddd, J = 6.8, 14.3, 28.4 Hz,
4H), 0.98–0.79 (m, 6H); 13C NMR (101 MHz, D2O) d
187.81, 137.25, 132.85, 132.78, 130.96, 79.35, 78.44, 56.55,
52.34, 52.09, 51.50, 39.35, 32.76, 31.83, 30.61, 26.67, 10.97,
10.84. Anal. Calcd for C21H34Cl2N2O3Á0.5 H2O: C, 64.53;
H, 7.82; N, 5.57; Found: C, 64.49; H, 7.90; N, 5.57.
Hydrochloride salt of 3,3-diethyl-5-(2-(4-phenylpiperidin-1-
yl)ethyl)dihydrofuran-2(3H)-one (12b) White solid. Yield:
51 %. mp: 239.5 °C. 1H NMR (400 MHz, D2O) d 7.39 (tt,
J = 7.3, 14.3 Hz, 5H), 4.75–4.67 (m, 1H), 3.75–3.68 (m,
2H), 3.39–3.31 (m, 2H), 3.22–3.12 (m, 2H), 3.03–2.95 (m,
1H), 2.37 (dd, J = 6.9, 13.4 Hz, 1H), 2.31–2.10 (m, 4H),
2.02 (dd, J = 9.4, 13.5 Hz, 3H), 1.78–1.53 (m, 4H), 0.92
(dt, J = 7.5, 12.7 Hz, 6H); 13C NMR (101 MHz, D2O) d
187.89, 146.67, 131.85, 130.03, 129.64, 79.59, 52.33,
41.74, 39.25, 32.90, 31.85, 30.60, 10.89, 10.76. Anal.
Calcd for C21H32ClNO2 : C, 68.93; H, 8.81; N, 3.83;
Found: C, 68.87; H, 8.93; N,3.79.
Dihydrochloride salt of 3,3-diethyl-5-(2-(4-phenylpipera-
zin-1-yl)ethyl)dihydrofuran-2(3H)-one (9b) White solid.
Yield: 49 %. mp: decomposed over 239 °C. 1H NMR
(400 MHz, D2O) d 7.46 (t, J = 7.8 Hz, 2H), 7.31–7.11 (m,
3H), 4.70 (m, 1H), 3.85–3.44 (m, 10H), 2.34 (d,
J = 7.0 Hz, 1H), 2.23 (d, J = 29.4 Hz, 2H), 2.06–1.92 (m,
1H), 1.63 (dd, J = 10.1, 17.0 Hz, 4H), 0.90 (dt, J = 7.5,
12.1 Hz, 6H); 13C NMR (101 MHz, D2O) d 187.92,
150.20, 132.89, 127.03, 121.14, 79.53, 56.53, 54.13, 52.41,
50.87, 39.37, 32.81, 31.91, 30.68, 11.00, 10.87. Anal.
Calcd for C20H32Cl2N2O2: C, 59.55; H, 8.00; N, 6.94;
Found: C, 59.62; H, 8.11; N, 6.90.
5-(2-(4-(1H-benzo[d]imidazol-2-yl)piperidin-1-yl)ethyl)-3,3-
diethyldihydrofuran-2(3H)-one (13b) Yellowish semi-
solid. Yield: 43 %. 1H NMR (400 MHz, CDCl3) d
7.44–7.46 (m, 2H), 7.13–7.11 (m, 2H), 4.40–4.37 (m, 1H),
3.43 (broad s, 1H), 2.92–2.90 (m, 3H), 2.44–2.42 (m, 2H),
1.98–1.90 (m, 7H), 1.77–1.74 (m, 3H), 1.56–1.52 (m, 4H),
0.89–0.80 (m, 6H); 13C NMR (101 MHz, CDCl3) d 181.52,
157.99, 122.47, 76.18, 54.93. 53.98, 53.61, 48.99, 37.90,
36.93, 34.14, 31.11, 31.06, 29.52, 28.57, 9.09, 9.01. MS
(ESI) (m/z) 370.1 (M ? H)?. HPLC analysis: [95 %
purity using two different mobile phases.
3,3-Diethyl-5-[4-(4-fluorobenzyl)-piperazin-1-ylmethyl]-
dihydrofuran-2-one (10a) Pale yellow liquid. Yield:
12.32 %. 1H NMR (400 MHz, CDCl3) d 0.85–0.96 (m,
6H), 1.58–1.64 (m, 4H), 1.81–1.90 (m, 1H), 2.02–2.07 (m,
1H), 2.45–2.63 (m, 8H), 3.45 (d, 2H), 4.51–4.58 (m, 1H),
5.30 (s, 2H), 6.96–7.01 (m, 2H), 7.21–7.31 (m, 2H). LC–
MS (ESI) (m/z) 349.2 (M ? H)?. HPLC analysis: [95 %
purity using two different mobile phases.
Dihydrochloride salt of 3,3-diethyl-5-(2-(4-(4-fluorobenzyl)
piperazin-1-yl)ethyl)dihydrofuran-2(3H)-one (10b) White
solid. Yield: 52 %. mp: decompose over 213.5 °C. 1H NMR
(400 MHz, D2O) d 7.55 (dd, J = 5.3, 8.6 Hz, 2H), 7.26 (t,
J = 8.7 Hz, 2H), 4.70 (m, 1H), 4.45 (d, J = 7.1 Hz, 2H),
3.70–3.35 (m, 10H), 2.34 (dd, J = 6.9, 13.4 Hz, 1H),
2.30–2.08 (m, 2H), 1.98 (dd, J = 9.5, 13.4 Hz, 1H), 1.63
(dddt, J = 7.1, 14.0, 21.4, 28.4 Hz, 4H), 0.88 (dt, J = 7.4,
14.7 Hz, 6H); 13C NMR (101 MHz, D2O) d 187.84, 167.92,
165.46, 136.47, 136.38, 126.74, 119.43, 119.21, 79.41,
62.68, 56.69, 52.36, 51.85, 51.11, 39.37, 32.84, 31.85, 30.63,
26.67, 10.97, 10.85. Anal. Calcd for C21H33Cl2FN2O2: C,
57.93; H, 7.64; N, 6.43; Found: C, 57.71; H, 7.69; N, 6.32.
Hydrochloride salt of 5-(2-(3,4-dihydroisoquinolin-2(1H)-yl)
ethyl)-3,3-diethyldihydrofuran-2(3H)-one (14b) White solid.
Yield: 51 %. mp: 227.5 * 228.5 °C. 1H NMR (400 MHz,
MeOH-d4) d 7.39–7.17 (m, 4H), 4.63–4.54 (m, 1H), 4.49
(s, 2H), 3.75–3.54 (m, 2H), 3.51–3.40 (m, 2H), 3.22 (t,
J = 6.3 Hz, 2H), 2.36–2.24 (m, 2H), 2.23–2.08 (m, 1H),
1.95 (dd, J = 9.4, 13.3 Hz, 1H), 1.75–1.53 (m, 4H), 0.94
(dt, J = 7.5, 12.2 Hz, 6H); 13C NMR (101 MHz, MeOH-d4)
d 183.24, 132.92, 130.75, 130.38, 129.74, 129.17, 128.70,
77.07, 55.67, 55.33, 55.28, 52.24, 50.78, 50.50, 50.29, 50.07,
49.86, 49.65, 49.43, 49.22, 39.25, 32.87, 30.89, 30.02, 27.35,
9.85, 9.77. Anal. Calcd for C19H28ClNO2: C, 67.54; H, 8.35;
N, 4.15; Found: C, 67.60; H, 8.36; N, 4.14.
Dihydrochloride salt of 5-(2-(4-benzhydrylpiperazin-1-
5-(2-(4-Benzoylpiperazin-1-yl)ethyl)-3,3-diethyldihydrofu-
ran-2(3H)-one (15b) Yellowish oil. Yield: 52 %. 1H
NMR (400 MHz, CDCl3) d 7.34–7.30 (m, 4H), 4.44–4.38
yl)ethyl)-3,3-diethyldihydrofuran-2(3H)-one (11b) White
1
solid. Yield: 50 %. mp: 213–215 °C. H NMR (400 MHz,
123

1030
Med Chem Res (2014) 23:1023–1030
furanone as subtype- selective muscarinic leads. Med Chem Res
12(9):481–511
(m, 1H), 3.70 (broad s, 2H); 3.37 (broad s, 2H); 2.32–2.48
(m, 6H); 2.08–2.04 (m 1H), 1.78–1.72 (m, 3H), 1.58–1.51
(m, 4H), 0.89–0.80 (m, 6H); 13C NMR (101 MHz, CDCl3)
180.92, 170.47, 135.94, 129.90, 128.68, 127.22, 75.53,
54.53, 48.78, 37.83, 33.88, 29.40, 28.45, 8.98, 8.90. MS
(ESI) (m/z) 359.1 (M ? H)?. HPLC analysis: [95 %
purity using two different mobile phases.
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Acknowledgments The authors are grateful to Temple University
(R. G., University Fellowship; R.R.B., Teaching Assistantship; D.
J. C., Faculty Senate grant-in-aid) and the Dean’s Office, School of
Pharmacy (R. G., R. B., D. J. C) for generous financial support; and to
Dr. Abou-Gharbia and Rogelio Martinez (Moulder Center for Drug
Discovery Research) for access to instrumentation and helpful
discussions.
Koch HJ, Haas S, Jurgens T (2005) On the physiological relevance of
muscarinic acetylcholine receptors in alzheimer’s disease. Curr
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