Synthesis and analgesic evaluation of a series of proline-typed spiro cyclic quaternary ammoniums

Article abstract of DOI:10.1007/s00044-013-0681-6

A series of proline derivated spirocyclic quaternary ammoniums were synthesized and evaluated via in vivo analgesic activities. Compounds 9a showed the best analgesic effect with 84 % inhibition in mice. These compounds showed only micromolar Ki value by in vitro α7 nAChR binding test, which may indicate the form of active metabolite play a role in their in vitro analgesic activities.

Full text of DOI:10.1007/s00044-013-0681-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:862–869
DOI 10.1007/s00044-013-0681-6
ORIGINAL RESEARCH
Synthesis and analgesic evaluation of a series of proline-typed
spiro cyclic quaternary ammoniums
•
Songwen Lin Na Wang Shuo Zhao Qi Sun
•
•
•
•
Weiwei Zhang Jia Ye Tieming Cheng Runtao Li
•
•
Received: 23 March 2013 / Accepted: 25 July 2013 / Published online: 8 August 2013
Ó Springer Science+Business Media New York 2013
Abstract A series of proline derivated spirocyclic qua-
ternary ammoniums were synthesized and evaluated via
in vivo analgesic activities. Compounds 9a showed the best
analgesic effect with 84 % inhibition in mice. These
compounds showed only micromolar Ki value by in vitro
a7 nAChR binding test, which may indicate the form of
active metabolite play a role in their in vitro analgesic
activities.
receptors (nAChR) and several compounds with high
affinity have been reported (Jones and Dunlop 2007).
Nevertheless, most of them still have significant side
effects due to their unselective action on the other sub-
populations of nAChRs. For example, tebanicline (1, ABT-
594), a famous a4b2 agonist with significant antinocicep-
tive effects, failed in phase II trials because of gastroin-
testinal adverse effects (Holladay et al., 1998; Ji et al.,
2007).
Keywords Spirocyclic quaternary ammonium ꢀ
SAR ꢀ Synthesis ꢀ Analgesic activity ꢀ a7 nAChR
The quaternary ammonium salts, which are easily sol-
uble in water, were regarded that they cannot easily pass
the blood–brain barrier (BBB). However, some of them
exhibit significant affinity for nAChRs. For example,
Gualtieri and co-workers found that most of N1,N1-dime-
thyl-N4-phenylpiperazinium iodide 2 (DMPP, a well-
known nicotinic agonist (Kizawa and Takayanagi, 1984;
Lippiello and Fernandes, 1986); Fig. 1) derivatives showed
excellent affinity for a4b2 nAChR after systematically
studying the structure–activity relationships of DMPP
Introduction
Pain is a pervasive public health problem. Opioid agents
and non-steroidal anti-inflammatory drugs (NSAIDs) are
the two major clinic analgesics, which have undesirable
side effects associated with their use (Decker and Meyer
1999). This prompted medicinal chemists a search for
mechanistically different analgesic agents (Kowaluk et al.,
2000). Recently, considerable attention has been focused
on the identification of ligands for nicotinic acetylcholine
(Romanelli et al., 2001). Gu
¨ndisch et al. showed that the
phenylcarbamate derivatives 3 (R = F, a7 Ki = 62 nM;
R = CF3, a7 Ki = 29 nM, Fig. 2) have significantly
higher affinity for a7 nAChR than for a4b2 subtype
(Gu¨ndisch et al., 2004). Gotti et al. also reported that
choline ether 4 (a7 Ki = 57 nM, Fig. 2) is a a7-selective
antagonist with little affinity (Ki = 39 lM) for b2-con-
taining nAChR (Gotti et al., 1998, 2000).
S. Lin ꢀ N. Wang ꢀ S. Zhao ꢀ Q. Sun (&) ꢀ T. Cheng ꢀ R. Li
State Key Laboratory of Natural and Biomimetic Drugs, School
of Pharmaceutical Sciences, Peking University, Beijing 100191,
People’s Republic of China
Our group has been involved in the development of
quaternary ammonium salts as analgesics for many years
(Li et al., 1996, 1998, 1999a, b; Wang et al., 2003; Li et al.,
2007). A series of novel monospirocyclopiperazinium salts
5 (MSPZ; Fig. 2; Gao et al., 2003) and compounds 6
(LXM–10, acetic acid writhing test with 65 % inhibition,
Fig. 2, Sun et al., 2007), 7a (LXM–15, 61 % inhibition,
Fig. 2, Sun et al., 2007), 7b (LXM–19, 56 % inhibition,
e-mail: sunqi@bjmu.edu.cn
R. Li
e-mail: lirt@bjmu.edu.cn
W. Zhang ꢀ J. Ye
Department of Molecular and Cellular Pharmacology, School of
Pharmaceutical Sciences, Peking University, Beijing 100191,
People’s Republic of China
123

Med Chem Res (2014) 23:862–869
863
NH
2010; Zhao et al., 2011). As a result, they produced neither
central side effects nor typical side effects associated with
AChR agonists. In continuing our long-term research on
spiro cyclic quaternary ammoniums as analgesics, we
herein reported the synthesis and analgesic evaluation of a
new series of compounds 9a–9m (Fig. 3) according to
structure modification of compound 7.
O
N
N
N
I
Cl
1, ABT-594
2, DMPP
Fig. 1 Structure of a4b2 agonists
O
H
N
N
O
Results and discussion
N
I
I
O
The preparation of analogs 9 from racemic Boc-proline and
anilines is shown in Scheme 1. Intermediate 12 was
obtained by the condensation reaction of proline 10 with
various substituted aniline, followed by removal of the
Boc-group in presence of CF3COOH in CH2Cl2. Column
chromatography afforded the desired product with a yield
of 45–85 % in two steps. Compounds 9a–9m were
prepared through the reaction of intermediate 12 with
1,4–dibromobutane, 1,5-dibromo-pentane or N-substituted-
bis(2-chloro ethyl)amine in presence of excess NaHCO3
(Sun et al., 2007) with a yield of 14–50 %. The low yields
may be due to weak basicity of 1-NH and the hindrance of
the 2-amide group in compound 12.
R
4
3
N
N
O
N
NR
X
Cl
6 (LXM-10)
5 (MSPZ)
R
N
N
MeO
N
N
Br
7a (LXM-15 R = 3-NO )
7b (LXM-19 R = 4-MeO)
8
Cl
2
On the basis of our previous experimental results, the
preliminary analgesic activities of compounds 9a–9m were
assessed at doses of 20 lM/kg, sc, by acetic acid writhing
test in mice. Furthermore, receptor binding assays were
used to evaluate for their abilities to compete for [3H]MLA
binding sites (a7 nAChR). All the results were listed in
Table 1.
Fig. 2 Quaternary ammonium-typed a7nAChRagonists/antagonists
HN
modification
R
R
N
N
N
O
Br
X
Y
Firstly, we explored the influence of the substituted
groups of aniline in 9 on analgesic activity (Table 1).
Compared to compound 9a (84 %, entry 2, Table 1)
without any substituted group, both electron-donating
group and electron-withdrawing group were detrimental to
the analgesic activity. Large groups at ortho-positon (9i,
18 %, entry 10, Table 1) and meta-position (9g, 19 %,
entry 8 and 9j, 0 %, entry 11, Table 1), which might make
the molecular conformation unsuitable for receptor binding
7
9a-9m
Fig. 3 The design of compounds 9a–9m
Fig. 2, Sun et al., 2007) and 8 (63 % inhibition, Fig. 2, Lin
et al., 2011) were identified to show significant in vivo
analgesic activities. Pharmacological studies revealed that
this kind of compounds could selectively bind to peripheral
a7 nAChR and M4 mAChR (Yue et al., 2007; Xiong et al.,
Scheme 1 The synthetic route
of compounds 9a–9m. Reagents
and reaction conditions:
HN
O
HN
O
a
b
R
COOH
R
N
Boc
N
Boc
N
a subsituted aniline,
H
ClCOOC2H5, Et3N, THF,
0 °C?r.t., 16 h, then reflux,
3 h; b CF3COOH:CH2Cl2=1:1,
r.t., 1 h; c Br(CH2)4Br or
Br(CH2)5Br or N-substituted-
bis(2-chloroethyl) amine,
NaHCO3, EtOH, reflux, 24 h
10
11a-11j
12a-12j
HN
O
c
R
R = H,Cl, NO2, OMe, OBn
Y = bond, CH2, N(CH2)2Ph, N-(4-OMe)Ph
X = Cl, Br
N
X
Y
9a-9m
123

864
Med Chem Res (2014) 23:862–869
Table 1 The in vivo activities of compounds 9a–9m
HN
O
R
N
X
Y
9a-9m
Entry
Compd.
R
Y
–
X
Analgesic activities (%)a,b
1
NS
9a
9b
9c
9d
9e
9f
–
–
2
H
Bond
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Cl
Cl
84**c
3
p-NO2
m-NO2
m-Cl
o-Cl
p-Cl
m-OMe
p-OMe
o-OMe
o-OBn
H
Bond
Bond
Bond
Bond
Bond
Bond
Bond
Bond
Bond
CH2
25
4
27
5
31**c
30**c
34**c
19
6
7
8
9g
9h
9i
9
33*d
10
11
12
13
14
a
18
9j
0
9k
9l
46**c
31**c
32**c
H
PhCH2CH2N
4-MeOPhN
9m
H
Drugs were dissolved in isotonic saline solution (NaCl 0.9 %) immediately before use. Drug concentrations were prepared in such a way that
the necessary dose could be administered in a volume of 10 mL/kg. Mice were administered with drugs (20 lmol/kg, sc) 30 min prior to
injection of 0.6 % acetic acid (10 mL/kg, i.p.). The number of stretching movements was recorded for 10 min, starting 5 min after acetic acid
injection
b
Percent analgesia was expressed by following formula: % Inhibition = 100 - (A/B 9 100), where A incidence of writhing in the treated group
and B incidence of writhing in the control group
c
P \ 0.01
d
P \ 0.05
site. It was also found that chloro-atom at any position
Table 2 The in vitro receptor binding test of compounds 9a and 9k
decreased analgesic activity slightly, which maybe con-
Entry
Compd.
Ki(lM)a
tribute to its smaller steric hindrance and electron-donating
conjugated effect.
1
2
3
a
9a
3.5
The influence of the spiro ring was obvious, which was
demonstrated by the activity of compound 9k (46 %, entry
12, six member ring, Table 1) compared to 9a (84 %, entry
2, five member ring, Table 1). Compounds 9l and 9m
(entries 13, 14, Table 1), which was enlightened by pre-
viously obtained compounds 6 and 7a, were designed and
synthesized. Unfortunately, only moderate analgesic
activities were got. It indicated the ring number of mono-
spirocyclic quaternary ammonium salts is critical for the
activities.
9k
5.8
MLA
0.003
Competition binding assays with [3H] MLA were done according to
reference (Wang et al., 2005)
metabolites. Further research about the metabolites and the
binding target of compound 9a is in due course.
We also explored the receptor binding test of com-
pounds 9a and 9k against a7 nAChR (Table 2). However,
compounds 9a and 9k showed only micromolar Ki value
(9a: 3.5 lM and 9k: 5.8 lM) compared to MLA (3nM).
Considering it potent in vivo analgesic activities, these
compounds may play a role in the form of active
Conclusions
In summary, a series of monospirocyclic quaternary
ammonium salts derived from proline were designed and
synthesized to search for a peripherally-acting analgesic
drug with low side effects. Extensive SAR studies revealed
123

Med Chem Res (2014) 23:862–869
865
that substitution of aniline ring of 9 by both electron-
donating and electron-withdrawing groups were detrimen-
tal to the analgesic activity, especially large groups at
ortho- and meta-position. The influence of the spiro ring
size on the analgesic activity was also demonstrated. As a
novel kind of monospirocyclic quaternary ammonium salts,
compound 9a showed good analgesic activities, it would be
very valuable for further investigation of this program.
8.22 (d, J = 9.0 Hz, 2H), 10.32 (s, 1H). 13C NMR (CDCl3,
100 MHz): d 173.46, 143.58, 143.35, 125.01, 118.82,
60.98, 47.32, 30.67, 29.68, 26.19.
N-(3-Nitrophenyl)pyrrolidine-2-carboxamide 12c
1
Yellow oil in a 70 % yield, H NMR (CDCl3, 300 MHz):
d 1.87 (m, 2H), 2.02–2.12 (m, 2H), 2.26–2.36 (m, 1H),
3.12–3.20 (m, 3H), 4.13 (m, 1H), 6.01 (brs, 1H), 7.43 (t,
J = 8.1 Hz, 1H), 7.87–7.93 (m, J = 8.1 Hz, J = 2.1 Hz,
2H), 8.46(t, J = 2.1 Hz, 1H), 10.39 (s, 1H). 13C NMR
(CDCl3, 100 MHz): d 174.31, 148.37, 139.01, 129.60,
124.98, 118.23, 113.86, 60.94, 47.27, 30.68, 26.26.
Experimental
General experimental procedures
All reagents and solvents were from commercial sources.
Melting points were determined on X4 microscope. 1H
NMR and 13C NMR spectra were recorded on JOEL AL
300 (300 MHz) and Bruker AVANCEIII 400 (400 MHz)
instruments. Elemental analyses were performed on a
Vario EL III (Germany) instrument.
N-(3-Chlorophenyl)pyrrolidine-2-carboxamide 12d
Yellow oil in a 85 % yield, 1H NMR (CDCl3, 300 MHz): d
1.70–1.79 (m, 2H), 1.95–2.05 (m, 1H), 2.14–2.26 (m, 1H),
2.86 (s, 1H), 2.94–3.12 (m, 2H), 3.87 (dd, J = 5.1 Hz,
J = 9.3 Hz, 1H), 7.04 (dd, J = 0.9 Hz, J = 8.1 Hz, 1H),
7.21 (t, J = 8.1 Hz, 1H), 7.40-7.43 (m, 1H), 7.73 (t,
J = 2.1 Hz, 1H), 9.87 (s,1H). 13C NMR (CDCl3,
100 MHz): d 171.49, 138.84, 134.39, 129.89, 124.24,
119.72, 117.74, 60.68, 46.91, 30.49, 29.67, 25.51.
General procedure for the synthesis of compounds
12a–12j
To a solution of N-Boc-proline (9.3 mmol, 2.0 g) and tri-
ethylamine (9.3 mmol, 0.94 g) in THF at 0 °C was added
ethyl chloroformate (9.3 mmol, 0.89 mL) and the mixture
was stirred for 30 min. Substituted aniline (9.3 mmol) was
added at 0 °C and the mixture was stirred at rt for 16 h, and
then refluxed for 3 h. After filtration and condensation, the
obtained crude product 11a–11j was dissolved in 50 %
TFA-DCM solution and stirred for 30 min. The solvent
was then removed under reduced pressure and neutralized
with saturated NaHCO3 solution, and the aqueous layer
was extracted with ethyl acetate. The combined organic
layer was washed with saturated NaCl, dried over anhy-
drous Na2SO4, filtered and concentrated. Column chro-
matography afforded the desired product 12.
N-(2-Chlorophenyl)pyrrolidine-2-carboxamide 12e
Yellow oil in a 63 % yield, 1H NMR (CDCl3, 400 MHz): d
1.72–1.83 (m, 2H), 2.02–2.09 (m, 1H), 2.17–2.31 (m, 2H),
3.01–3.14 (m, 2H), 3.89–3.92 (m, 1H), 6.99–7.03 (m, 1H),
7.24–7.28 (m, 1H), 7.35 (d, J = 8.0 Hz, 1H), 8.46 (d,
J = 8.0 Hz, 1H), 10.45 (s, 1H). 13C NMR (CDCl3,
100 MHz): d 173.76, 134.66, 127.43, 126.40, 124.23,
123.16, 120.78, 61.24, 47.21, 30.77, 26.18.
N-(4-Chlorophenyl)pyrrolidine-2-carboxamide 12f
Yellow oil in a 58 % yield,1H NMR (CDCl3, 400 MHz): d
1.84–1.91 (m, 2H), 2.03–2.08 (m, 1H), 2.28–2.38 (m, 1H),
3.18–3.21 (m, 2H), 4.27–4.31 (m, 1H), 7.23 (d,
J = 8.8 Hz, 2H), 7.48 (d, J = 8.8 Hz, 2H), 10.16 (s, 1H).
13C NMR (DMSO, 100 MHz): d 168.23, 137.30, 128.59,
127.43, 120.91, 59.76, 45.81, 29.92, 24.02.
N-Phenylpyrrolidine-2-carboxamide 12a
Yellow oil in a 75 % yield,1H NMR (CDCl3,300 MHz):
d 1.80 (m, 2H), 2.00–2.10 (m, 1H), 2.13–2.23 (m, 2H),
2.92–3.08 (m, 2H), 3.85 (m, 1H), 7.09 (m, 1H), 7.29 (m,
2H), 7.60 (m, 2H), 9.75 (s, 1H). 13C NMR (CDCl3,
100 MHz): d 170.35, 136.71, 127.90, 123.23, 118.62,
59.72, 45.99, 29.50, 28.68, 24.68.
N-(3-Methoxyphenyl)pyrrolidine-2-carboxamide 12g
Yellow oil in a 53 % yield, 1H NMR (CDCl3, 400 MHz): d
1.66–1.70 (m, 2H), 1.85–1.91 (m, 1H), 2.14–2.17 (m, 1H),
2.92–2.97 (m, 1H), 3.03 (m, 2H), 3.61 (s, 3H), 4.09 (d,
J = 8.0 Hz, 1H), 6.37 (brs, 1H), 6.49 (d, J = 8.0 Hz, 1H),
6.95 (d, J = 8.0 Hz, 1H), 7.03 (t, J = 8.0 Hz, 1H), 7.14 (s,
1H), 10.00 (s, 1H). 13C NMR (CDCl3, 100 MHz): d
N-(4-Nitrophenyl)pyrrolidine-2-carboxamide 12b
1
Yellow oil in a 45 % yield, H NMR (CDCl3, 300 MHz):
d 1.80 (m, 2H), 2.00–2.10 (m, 1H), 2.23–2.32 (m, 1H),
3.01–3.20 (m, 3H), 3.91 (m, 1H), 7.79 (d, J = 9.0 Hz, 2H),
123

866
Med Chem Res (2014) 23:862–869
1-((4-Nitrophenyl)carbamoyl)-5-azaspiro[4.4]nonan-5-
170.68, 160.04, 138.80, 129.58, 112.15, 110.17, 105.63,
60.65, 55.15, 46.79, 30.42, 25.27.
ium bromide 9b
Yellow solid in a 34 % yield, mp: 235 °C. 1H NMR (D2O,
300 MHz): d 2.06–2.48 (m, 8H), 3.48–3.80 (m, 6H), 4.35
(m, 1H), 7.56 (d, J = 9.0 Hz, 2H), 8.11 (d, J = 9.0 Hz,
2H).Anal calcd. for C15H20BrN3O3(?0.8H2O): C, 46.84;
H, 5.66; N, 10.92. Found: C, 46.85; H, 5.20; N, 10.76.
N-(4-Methoxyphenyl)pyrrolidine-2-carboxamide 12h
1
Yellow oil in a 50 % yield, H NMR (CDCl3, 400 MHz):
d 1.67–1.78 (m, 2H), 1.96–2.04 (m, 1H), 2.12–2.20 (m,
1H), 2.32 (brs, 1H), 2.92–2.97 (m, 1H), 3.01–3.07 (m, 1H),
3.76–3.82 (m, 4H), 6.84 (d, J = 8.0 Hz, 2H), 7.50 (d,
J = 8.0 Hz, 2H), 9.61 (s, 1H). 13C NMR (CDCl3,
100 MHz): d 170.56, 155.23, 130.00, 120.12, 113.02,
59.67, 54.39, 46.00, 29.57, 28.66, 24.76.
1-((3-Nitrophenyl)carbamoyl)-5-azaspiro[4.4]nonan-5-
ium bromide 9c
1
Yellow solid in a 32 % yield, mp: 244–246 °C. H NMR
(D2O, 300 MHz): d 2.06–2.43 (m, 8H), 3.49 –3.74(m, 6H),
N-(2-Methoxyphenyl)pyrrolidine-2-carboxamide 12i
7.42 (m, 1H), 7.55 (m, 1H), 7.89 (m, 1H), 8.16 (m, 1H). 13
C
Yellow oil in a 56 % yield, 1H NMR (CDCl3, 400 MHz): d
1.68–1.79 (m, 2H), 1.99–2.06 (m, 1H), 2.13–2.22 (m, 2H),
2.96–3.05 (m, 2H), 3.79–3.86 (m, 4H), 6.86 (d, J = 8.0 Hz,
1H), 6.94 (t, J = 8.0 Hz, 1H), 7.02 (t, J = 8.0 Hz, 1H), 8.42
(d, J = 8.0 Hz, 1H), 10.11 (s, 1H). 13C NMR (CDCl3,
100 MHz): d 173.52, 148.58, 127.53, 123.60, 120.96,
119.52, 110.09, 61.47, 55.75, 47.34, 30.89, 29.69, 26.25.
NMR (D2O, 75 MHz): d21.18, 21.82, 22.66, 27.44, 60.45,
63.67, 65.35, 74.05, 116.54, 121.16, 128.01, 130.76, 137.66,
148.49, 167.06. Anal calcd. for C15H20BrN3O3(?0.7H2O):
C, 47.06; H, 5.63; N, 10.98. Found: C, 47.31; H, 5.34; N,
10.80.
1-((3-Chlorophenyl)carbamoyl)-5-azaspiro[4.4]nonan-5-
ium bromide 9d
N-(2-Benzyloxyphenyl)pyrrolidine-2-carboxamide 12j
White solid in a 14 % yield, mp: 172–174 °C.1H NMR
(D2O, 300 MHz): d 1.97–2.15 (m, 7H), 2.45 (m, 1H),
3.35–3.62 (m, 6H), 4.42 (d, J = 6.6 Hz, 1H), 6.84 (d,
J = 7.5 Hz, 1H), 7.05 (t, J = 8.1 Hz, 1H), 7.19 (d,
J = 7.8 Hz, 1H), 7.50 (s, 1H). 13C NMR (D2O, 75 MHz): d
21.38, 21.93, 22.77, 27.58, 60.34, 63.66, 65.24, 73.79,
119.56, 121.11, 125.91, 130.93, 134.42, 138.40, 166.27.Anal
calcd. for C15H20BrClN2O: C, 50.09; H, 5.60; N,7.732.
Found: C, 50.08; H, 5.647; N,7.732.
Yellow oil in a 60 % yield, 1H NMR (CDCl3, 300 MHz): d
1.70–1.79 (m, 2H), 1.99–2.09 (m, 1H), 2.14–2.27 (m, 1H),
2.39 (s, 1H), 2.90–3.09 (m, 2H), 3.89 (dd, J = 5.1 Hz,
J = 9.3 Hz, 1H), 5.13 (s, 2H), 6.95–7.06 (m, 3H),
7.32–7.43 (m, 3H), 7.49–7.51 (m, 2H), 8.44 (dd,
J = 1.8 Hz, J = 7.5 Hz, 1H), 10.35 (s,1H). 13C NMR
(CDCl3, 100 MHz): d 172.59, 146.65, 135.79, 127.46,
126.92, 126.63, 125.95, 122.53, 120.45, 118.51, 110.65,
69.34, 60.34, 46.21, 29.88, 25.23.
1-((2-Chlorophenyl)carbamoyl)-5-azaspiro[4.4]nonan-5-
ium bromide 9e
General procedure for the synthesis of compounds 9
1
White solid in a 35 % yield, mp: 176–178 °C. H NMR
A mixture of compound 12, bis-halogenated alkane, and
NaHCO3 (20 mmol) was refluxed for 10 h. After filtration,
the filtrate was concentrated, and crystallized to afford the
desired product 9.
(D2O, 400 MHz): d 2.29–2.54 (m, 7H), 2.66–2.76 (m, 1H),
3.71–3.93 (m, 6H), 4.68–4.71 (m, 1H), 7.39–7.47 (m, 2H),
7.52 (d, J = 7.2 Hz, 1H), 7.61 (d, J = 7.2 Hz, 1H).13C
NMR (D2O, 100 MHz): d 21.31, 21.92, 22.76, 27.52,
60.44, 63.61, 65.24, 73.11, 128.18, 128.41, 129.60, 129.67,
130.44, 132.35, 167.23.Anal calcd. for C15H20BrClN2O: C,
50.09; H, 5.60; N, 7.79. Found: C, 50.08; H, 5.589; N,
7.789.
1-(Phenylcarbamoyl)-5-azaspiro[4.4]nonan-5-ium
bromide 9a
1
White solid in a 46 % yield, mp: 192–194 °C. H NMR
(D2O, 400 MHz): d 2.04–2.28 (m, 7H), 2.42–2.47 (m, 1H),
3.43–3.70 (m, 6H), 4.33–4.36 (m, 1H), 7.14–7.18 (t,
J = 7.2 Hz, 1H), 7.29–7.37 (m, 1H).13C NMR (D2O,
100 MHz): d 20.72, 21.31, 22.12, 26.82, 59.84, 63.12,
64.75, 73.50, 121.86, 126.41, 129.37, 135.84, 166.10. Anal
calcd. for C15H21BrN2O: C, 55.39; H, 6.51; N, 8.62.
Found: C, 55.48; H, 6.609; N, 8.516.
1-((4-Chlorophenyl)carbamoyl)-5-azaspiro[4.4]nonan-5-
ium bromide 9f
1
White solid in a 43 % yield, mp: 234–236 °C. H NMR
(D2O, 400 MHz): d 2.23–2.48 (m, 7H), 2.62–2.64 (m, 1H),
3.68–3.88 (m, 6H), 4.53–4.54 (m, 1H), 7.44–7.47 (m,
123

Med Chem Res (2014) 23:862–869
867
4H).13C NMR (D2O, 100 MHz): d 20.64, 21.26, 22.07,
26.78, 59.85, 63.14, 64.74, 73.53, 123.28, 129.15, 130.84,
134.47, 166.25. Anal calcd. for C15H20BrClN2O: C, 49.92;
H, 5.542; N, 7.692. Found: C, 50.09; H, 5.60; N, 7.79.
13C NMR (D2O, 100 MHz): d 21.98, 22.47, 23.26, 27.87,
60.91, 63.97, 65.46, 71.80, 73.96, 114.03, 121.75, 126.07,
126.57, 128.83, 129.32, 129.47, 129.62, 137.99, 153.21,
166.92. Anal calcd. for C22H27BrN2O2 (?0.5H2O):
C,60.32; H, 6.388; N,6.155. Found: C,60.00; H, 6.41; N,
6.36.
1-((3-Methoxyphenyl)carbamoyl)-5-azaspiro[4.4]nonan-5-
ium bromide 9g
1-(Phenylcarbamoyl)-5-azaspiro[4.5]decan-5-ium bromide
1
White solid in a 39 % yield, mp: 144–145 °C. H NMR
9k
(D2O, 400 MHz): d 2.12–2.36 (m, 7H), 2.47–2.52 (m, 1H),
3.50–3.78 (m, 9H), 4.33–4.36 (m, 1H), 6.80 (d,
J = 8.0 Hz, 1H), 6.95 (dd, J = 0.6 Hz, J = 8.0 Hz, 1H),
7.05 (s, 1H), 7.25–7.27 (m, 1H).13C NMR (D2O,
100 MHz): d 20.74, 21.30, 22.13, 26.87, 55.47, 59.82,
63.06, 64.72, 73.45, 107.13, 111.48, 113.86, 130.22,
137.32, 159.33, 165.87. Anal calcd. for C16H23BrN2O2: C,
54.01; H, 6.517; N, 7.897. Found: C, 54.09; H, 6.53; N,
7.89.
White solid in a 29 % yield, mp: 182–184 °C. 1H
NMR(D2O, 400 MHz): d 1.47–1.52 (m, 1H), 1.57–1.62 (m,
1H), 1.72–1.83 (m, 4H), 2.13–2.44 (m, 4H), 3.28–3.43 (m,
4H), 3.58–3.63 (m, 1H), 3.69–3.74 (m, 1H), 4.32–4.35 (m,
1H), 7.17 (t, J = 7.2 Hz, 1H), 7.30–7.37 (m, 4H).13C NMR
(D2O, 100 MHz): d 19.90, 20.66, 20.78, 21.36, 25.48,
57.34, 60.89, 61.30, 74.10, 121.99, 126.56, 129.35, 135.67,
165.46. Anal calcd. for C16H23BrN2O: C, 56.68; H, 6.714;
N, 8.258. Found: C, 56.64; H, 6.83; N, 8.26.
1-((4-Methoxyphenyl)carbamoyl)-5-azaspiro[4.4]nonan-5-
ium bromide 9h
8-Phenethyl-1-(phenylcarbamoyl)-5,8-
diazaspiro[4.5]decan-5-ium chloride 9l
1
White solid in a 50 % yield, mp: 172–173 °C. H NMR
White solid in a 27 % yield, mp: 180–182 °C.1H
NMR(D2O, 400 MHz): d 2.30–2.46 (m, 3H), 2.58–2.94 (m,
8H), 3.08–3.13 (m, 1H), 3.35 (s, 1H), 3.59–3.69 (m, 3H),
3.78–3.87 (m, 2H), 3.97–4.04 (m, 1H), 7.14–7.27 (m,
6H),7.35 (t, J = 8.0 Hz, 2H), 7.68 (d, J = 8.0 Hz, 2H).
13C NMR (D2O, 100 MHz): d 19.88, 25.52, 32.83, 47.68,
47.89, 56.47, 58.46, 59.93, 120.38, 124.73, 125.76, 128.03,
128.34, 128.52, 138.22, 139.71, 164.32.Anal calcd. for
C23H30ClN3O (?1H2O): C, 66.42; H, 7.442; N, 10.06.
Found: C, 66.09; H, 7.72; N, 10.05.
(D2O, 400 MHz): d 2.06–2.32 (m, 7H), 2.46–2.52 (m, 1H),
3.45–3.68(m, 9H), 4.39 (t, J = 4.4 Hz, 1H), 6.86(d,
J = 8.8 Hz, 2H), 7.31(d, J = 8.8 Hz, 2H).13C NMR (D2O,
100 MHz): d 20.81, 21.38, 22.18, 26.83, 55.70, 59.86,
63.17, 64.75, 73.43, 114.51, 123.63, 129.16, 156.95,
165.88.Anal calcd. for C16H23BrN2O2: C, 53.98; H, 6.402;
N, 7.929. Found: C, 54.09; H, 6.53; N, 7.89.
1-((2-Methoxyphenyl)carbamoyl)-5-azaspiro[4.4]nonan-5-
ium bromide 9i
White solid in a 42 % yield, mp: 229–230 °C. 1H
NMR(D2O, 400 MHz): d 2.12–2.38 (m, 7H), 2.50–2.52 (m,
1H), 3.53–3.78 (m, 9H), 4.42–4.46 (m, 1H), 6.93–6.97 (m,
1H), 7.05 (d, J = 8.0 Hz, 1H), 7.23–7.27 (m, 1H), 7.42 (d,
J = 8.0 Hz, 1H).13C NMR (D2O, 100 MHz): d 20.76,
21.32, 22.10, 26.78, 55.96, 59.84, 63.13, 64.71, 73.15,
112.18, 120.87, 123.90, 124.91, 128.33, 152.07, 166.57.
Anal calcd. for C16H23BrN2O2: C, 53.95; H, 6.453; N,
7.785. Found: C, 54.09; H, 6.53; N, 7.89.
8-(4-Methoxyphenyl)-1-(phenylcarbamoyl)-5,8-
diazaspiro[4.5]decan-5-ium chloride 9m
White solid in a 29 % yield, mp: 138–140 °C. 1H
NMR(D2O, 400 MHz): d 2.24–2.51 (m, 4H), 3.20–3.46 (m,
3H), 3.50–3.67 (m, 3H), 3.70–3.88 (m, 7H), 4.47 (dd,
J = 5.2 Hz, J = 7.6 Hz, 1H), 6.86–6.97 (m, 4H), 7.19 (t,
J = 7.2 Hz, 1H), 7.32–7.40 (m, 4H). 13C NMR (D2O,
100 MHz): d 20.01, 25.51, 45.53, 46.05, 55.68, 55.85,
59.73, 60.66, 74.76, 114.96, 119.18, 121.89, 126.53,
129.38, 135.73, 142.92, 154.38, 164.86. Anal calcd. for
C22H28ClN3O2(?1.4CH3OH): C, 62.77; H, 7.296; N,
9.368. Found: C, 62.90; H, 7.58; N, 9.40.
1-((2-Benzyloxyphenyl)carbamoyl)-5-azaspiro[4.4]nonan-
5-ium bromide 9j
White solid in a 46 % yield, mp: 149–150 °C. 1H
NMR(D2O, 400 MHz): d 1.96–2.03 (m, 2H), 2.14–2.29 (m,
5H), 2.53–2.58(m, 1H), 3.39–3.45(m, 1H), 3.64–3.72 (m,
4H), 3.78–3.85 (m, 1H), 4.80–4.82 (m, 1H), 5.14 (s, 2H),
6.97 (t, J = 7.2 Hz, 1H), 7.16(d, J = 8.4 Hz, 1H),
7.25(t, J = 7.6 Hz, 1H), 7.33–7.42(m, 3H), 7.53(m, 3H).
Acetic acid writhing test
Drugs were dissolved in isotonic saline solution (NaCl
0.9 %) immediately before use. Drug concentrations were
123

868
Med Chem Res (2014) 23:862–869
Holladay MW, Wasicak JT, Lin NH, He Y, Ryther KB, Bannon AW,
Buckley MJ, Kim DJ, Decker MW, Anderson DJ, Campbell JE,
Kuntzweiler TA, Donnelly-Roberts DL, Piattoni-Kaplan M,
Briggs CA, Williams M, Arneric SP (1998) Identification and
initial structure–activity relationships of (R)-5-(2-azetidinyl-
methoxy)-2-chloropyridine (ABT-594), a potent, orally active,
non-opiate analgesic agent acting via neuronal nicotinic acetyl-
choline receptors. J Med Chem 41(4):407–412
prepared in such a way that the necessary dose could be
administered in a volume of 10 mL/kg. Mice were
administered with drugs (20 lmol/kg, s.c.) 30 min prior to
injection of 0.6 % acetic acid (10 mL/kg, i.p.). The number
of stretching movements was recorded for 10 min, starting
5 min after acetic acid injection. Percent analgesia was
expressed by following formula: % Inhibition = 100 -
(A/B 9 100), where A incidence of writhing in the treated
group and B incidence of writhing in the control group.
Ji JG, Bunnelle WH, Anderson DJ, Faltynek C, Dyhring T, Ahring
PK, Rueter LE, Curzon P, Buckley MJ, Marsh KC, Kempf-Grote
A, Meyer MD (2007) A-366833: a novel nicotinonitrile-
substituted 3,6-diazabicyclo[3.2.0]-heptane a4b2 nicotinic ace-
tylcholine receptor selective agonist: Synthesis, analgesic effi-
cacy and tolerability profile in animal models. Biochem
Pharmacol 74:1253–1262
[3H] MLA binding assay
Jones PG, Dunlop J (2007) Targeting the cholinergic system as a
therapeutic strategy for the treatment of pain. Neuropharmacol-
ogy 53:197–206
Kizawa Y, Takayanagi I (1984) Nicotinic stimulant, DMPP (1,1-
dimethyl-4-phenylpiperazinium iodide) on the isolated bronchial
smooth muscle preparation of guinea-pig. Gen Pharmacol
15(2):149–152
Kowaluk EA, Lynch KJ, Jarvis MF (2000) Recent advances in
development of novel analgesics. Annu Rep Med Chem
35:21–30
Li RT, Cao SL, Chen HC, Yang JZ, Cai MS (1996) Synthesis and
pharmacological activities of 1,1-dialkyl-4-(3-bromopropio-
nyl)piperazinium bromides. Acta Pharm Sinica 31(10):757–760
Li RT, Cui JL, Cai MS (1998) Synthesis and biological activities of
1-alkyl-1-benzyl-4-(3-chlororo-2-hydroxy)propyl piperazinium
halides. Acta Pharm Sinica 33(1):28–33
Li RT, Cai JC, Tang XC, Cai MS (1999a) Synthesis and biological
activities of dibenzyl dipiperazine diquaternary ammonium salts.
Arch Pharm 332(5):179–181
Li RT, Cui JL, Cheng TM, Cai MS (1999b) Synthesis and biological
activities of 1,10-diacyl-4,4,40,40-tetraalkyl dipiperazinium deriv-
atives. Acta Pharm Sinica 34(9):666–668
Li A, Wang X, Yue CQ, Ye J, Li CL, Li RT (2007) Unique
spirocyclopiperazinium salt. Part 4: modification of dispirocy-
clopiperazinium (DSPZ) salts as analgesics. Bioorg Med Chem
Lett 17:5078–5081
Lin SW, Sun Q, Ge ZM, Wang X, Ye J, Li RT (2011) Synthesis and
structure–analgesic activity relationships of a novel series of
monospirocyclopiperazinium salts (MSPZ). Bioorg Med Chem
Lett 21:940–943
Competition binding assays with [3H] MLA were done
according to previous reports (Li et al., 1998). Briefly,
hippocampus membranes (containing 0.4 mg of protein)
were incubated in borosilicate glass tubes with 2 nM [3H]
MLA (100 Ci/mmol) in a final volume of 0.5 mL for 2 h at
4 °C, in the absence or presence of increasing concentra-
tions of compound 9 from 1.00 9 10-9 to 1.00 9 10-5 M.
Nonspecific binding was determined from tubes containing
1 mM nicotine. Incubation buffer consisted of phosphate
buffer (20 mM Na2HPO4, 5 mM KH2PO4, 150 mM NaCl,
pH 7.4) supplemented with 0.1 % (w/v) BSA. Incubation
was completed by rapid filtration under vacuum through
Whatman GF/B glass fibrefilters (Whatman Intl. Ltd.,
Maidstone, UK) pre-soaked in 0.3 % (v/v) polyethylene-
imine overnight. Tubes and filters were washed rapidly
three times with 3 mL ice-cold PBS. Filters were then dried
and counted on liquid scintillation spectrometry (Tri-Carb
2100TR, PerkinElmer, USA).
Acknowledgments This research was supported by the fund of
National Science Foundation of China (NSFC 20802004). Biological
activities were completed by Department of Molecular and Cellular
Pharmacology, School of Pharmaceutical Sciences, Peking University.
Lippiello PM, Fernandes KG (1986) The binding of L-[3H]nicotine to
a single class of high affinity sites in rat brain membranes. Mol
Pharmacol 29(5):448–454
References:
Romanelli MN, Manetti D, Scapecchi S, Borea PA, Dei S, Bartolini
A, Ghelardini C, Gualtieri F, Guandalini L, Varani K (2001)
Structure–affinity relationships of a unique nicotinic ligand: N1-
dimethyl-N4-phenylpiperazinium iodide (DMPP). J Med Chem
44(23):3946–3955
Sun Q, Yue CQ, Ye J, Li CL, Cheng TM, Li RT (2007) Unique
spirocyclopiperazinium salt III: further investigation of mono-
spirocyclopiperazinium (MSPZ) salts as potential analgesics.
Bioorg Med Chem Lett 17:6245–6249
Wang X, Gao FL, Piao HB, Cheng TM, Li RT (2003) Unique
spirocyclopiperazinium salt. Part 2: synthesis and structure-
activity relationship of dispirocyclopiperazinium salts as anal-
gesics. Bioorg Med Chem Lett 13:1729–1732
Wang Y, Su DM, Wang RH, Liu Y, Wang H (2005) Antinociceptive
effects of choline against acute and inflammatory pain. Neuro-
science 132:49–56
Decker MW, Meyer MD (1999) Therapeutic potential of neuronal
nicotinic acetylcholine receptor agonists as novel analgesics.
Biochem Pharmacol 58:917–923
Gao FL, Wang X, Zhang HM, Cheng TM, Li RT (2003) Unique
spirocyclopiperazinium salt I: synthesis and structure-activity
relationship of spirocyclopiperazinium salts as analgesics. Bio-
org Med Chem Lett 13:1535–1537
Gotti C, Balestra B, Moretti M, Rovati GE, Maggi L, Rossoni G, Berti
F, Villa L, Pallavicini M, Clementi F (1998) 4-Oxystilbene
compounds are selective ligands for neuronal nicotinic aBunga-
rotoxin receptors. Br J Pharmacol 124:1197–1206
Gotti C, Carbonnelle E, Moretti M, Zwart R, Clementi F (2000)
Drugs selective for nicotinic receptor subtypes: a real possibility
or a dream? Behav Brain Res 113:183–192
¨
Gu¨ndisch D, Andra M, Munoz L, Tilotta MC (2004) Synthesis and
Xiong YL, Zhao X, Sun Q, Li RT, Li CL, Ye J (2010) Antinoci-
ceptive mechanism of the spirocyclopiperazinium compound
evaluation of phenylcarbamate derivatives as ligands for nico-
tinic acetylcholine receptors. Bioorg Med Chem 12:4953–4962
123

Med Chem Res (2014) 23:862–869
869
LXM-10 in mice and rats. Pharmacol Biochem Behav 95(2):
192–197
Yue CQ, Ye J, Li CL, Li RT, Sun Q (2007) Antinociceptive effects of
the novel spirocyclopiperazinium salt compound LXM-10 in
mice. Pharmacol Biochem Behav 86:643–650
Zhao X, Ye J, Sun Q, Xiong YL, Li RT, Jiang YM (2011)
Antinociceptive effect of spirocyclopiperazinium salt compound
LXM-15 via activating peripheral a7 nAChR and M4 mAChR in
mice. Neuropharmacology 60(2–3):446–452
123