Synthesis and pharmacological properties of 5-alkyl substituted nicotine analogs

Article abstract of DOI:10.1002/cjoc.201200952

This paper describes a concise and practical route to enantiomerically enriched 5-alkyl substituted nicotine analogs. The Vilsmeier reaction was used to construct the nicotinaldehydes ring followed by the introduction of the chiral homoallylic alcohol by organic boron reagent and the cyclization of the pyrrolidine ring through the reduction of a chiral azide. 17 analogs have been synthesized and their corresponding biological activities were tested, in which compounds 10d and 10g exhibit excellent IC₅₀ values against RD and SY-SY5Y. Copyright

Full text of DOI:10.1002/cjoc.201200952

FULL PAPER
DOI: 10.1002/ cjoc.201200952
Synthesis and Pharmacological Properties of
5-Alkyl Substituted Nicotine Analogs†
Wang, Jing(王静)
Li, Xi(李蹊)
Yuan, Qianjia(袁乾家)
Ren, Jiangmeng*(任江萌)
Huang, Jin(黄瑾)
Zeng, Bubing*(曾步兵)
Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China University of Science and
Technology, Shanghai 200237, China
This paper describes a concise and practical route to enantiomerically enriched 5-alkyl substituted nicotine ana-
logs. The Vilsmeier reaction was used to construct the nicotinaldehydes ring followed by the introduction of the
chiral homoallylic alcohol by organic boron reagent and the cyclization of the pyrrolidine ring through the reduction
of a chiral azide. 17 analogs have been synthesized and their corresponding biological activities were tested, in
which compounds 10d and 10g exhibit excellent IC50 values against RD and SY-SY5Y.
Keywords nicotine analog, asymmetric synthesis, Vilsmeier reaction, biological activities
Introduction
Scheme 1 Retrosynthesis of (S)-nicotine analogs
Nicotine attracted considerable attention from me-
dicinal chemists because of its benefit on patients suf-
fering from Pakinson’s disease, anxiety, Alzheimer's
disease, schizophrenia, ulcerative colitis and other CNS
disorders.[1] However, the detrimental effects, such as
actions on the cardiovascular and gastrointestinal sys-
tems, sleep disturbance and addiction, limited the use of
nicotine as a therapeutic reagent.[2] As a consequence,
researchers focused on synthesizing nicotine derivatives
which exhibit the same effects with lower toxicity.
Synthesis of enantiomerically pure substituted nico-
tine derivatives especially with the variations at C-5
position is current research interests. SIB-1508Y, which
progressed as far as Phase II clinical trials for the treat-
ment of Parkinson’s disease, confirmed this issue.[3]
Nevertheless, regioselective substitution of the pyridine
ring of nicotine especially at C-5 is difficult to achieve
using known pyridine substitution chemistry.[4] Thus,
we select an alternative route to obtain a series of nico-
tine derivatives from non-pyridine ring.
R
R
N
N3
R'
N
X
N
X
R
CHO
X
R
OH
N
N
X
be constructed. Considering the lack of reactivity of
pyridines toward electrophilic substitution reactions, the
introduction of a formyl group onto the pyridine ring is
difficult to achieve. This problem was resolved by
Vilsmeier reaction that could construct the pyridine ring
as well as introduce the C-5 substituent and the formyl
group.[5] Therefore, various enmides 3 were prepared by
condensing corresponding aldehydes with benzylamine
to form initial Schiff bases and then reacted with acetic
anhydride. The following Vilsmeier reaction was con-
ducted to construct the desired nicotinaldehydes 4 using
DMF and triphosgene in toluene at 95 ℃ (Scheme 2).
With the desired nicotinaldehydes in hand, Roush
reagent was firstly used to introduce the chiral homoal-
lylic alcohol.[6] However, this reaction was found diffi-
cult to handle together with low yield. As a consequence,
(＋)-B-allyldiisopinocampheyborane was applied to get
From the retrosynthesis (Scheme 1), it is easy to see
that the difficulties rely on three aspects: (1) the con-
struct of the C-5 substituted nicotinaldehydes; (2) the
introduction of the chiral homoallylic alcohol; (3) build-
ing the pyrrolidine ring from the chiral azide.
Results and Discussion
First, the C-5 substituted nicotinaldehydes need to
*
E-mail: renjm@ecust.edu.cn; zengbb@ecust.edu.cn; Tel.: 0086-021-64253689; Fax: 0086-021-64253689
Received September 27, 2012; accepted November 25, 2012; published online December 13, 2012.
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cjoc.201200952 or from the author.
Dedicated to the 60th Anniversary of East China University of Science and Technology.
†
Chin. J. Chem. 2012, 30, 2813—2818
© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2813

Wang et al.
FULL PAPER
Scheme 2 Construction of the pyridine ring
Overall, 17 nicotine analogs were successfully syn-
thesized through the route we developed and ready for
the biological test. Up to date, 17 nicotinic acetylcholine
receptors subunits have been identified and nicotine
receptors are pentamers of these subunits. In our ex-
periment, we were interested in two types of nicotinic
acetylcholine receptors, human neuromuscular α1β1γδ
receptors and ganglionic (α7)5 receptors, to test the ef-
fects of the compounds. As references reported, RD
rhabdomyosarcoma cells expressed human neuromus-
cular α1β1γδ receptors,[10] and SH-SY5Y neuroblastoma
cells expressed human ganglionic (α7)5 receptors.[11]
Therefore, the corresponding biological activities of
those nicotine analogs against RD and SY-SY5Y were
tested and the results were summarized in Table 1.
Acetic anhydride
TEA, 0 oC
Benzylamine
R
R
CHO
Ph
N
KOH, 0 oC
2
1
R
O
R
CHO
Cl
R = isopropyl, ethyl, methyl
Triphosgene
DMF, 90 oC
N
N
Ph
3
4
compound 5 with moderate yield (49%).[7]
Compound 5 was mesylated with MsCl in the pres-
ence of triethylamine to afford compound 6. The unsta-
ble compound 6 was directly treated with sodium azide
in DMF at 60 ℃ to access compound 7 in 85% yield
over two steps. Finally, borane dimethyl sulfide com-
plex was used to reduce the azide group followed by
cyclization of pyrrolidine ring to provide chiral com-
pound 8. At the same time, (—)-B-allyldiisopinocam-
pheyborane was used to obtain the corresponding
(R)-nicotine analogs (Scheme 3).
Table 1 Structures and biological activities of the nicotine ana-
logs
a
b
SY-SY5Y IC50
/
RD IC50
/
No.
Structure
－
(μmol•L )
－
(μmol•L )
1
1
17.25
62.49
9a
N
Scheme 3 Construction of the pyrrolidine
N
N
Cl
OH
R
CHO
Cl
(+)-Ipc2BCH2CH=CH2
Et2O
R
15.75
62.08
29.04
85.70
10a
10b
N
N
N
5
Cl
-78 oC
OMs
Bn
4
Cl
Cl
R
MsCl, Et3N
CH2Cl2
0 oC
NaN3, DMF
0 oC
N
Cl
N
6
N
N3
Cyclohexene
R
R
.
BH3 Me2S
N
H
THF
N
Cl
N
Cl
8
N
7
19.94
35.65
4.88
77.96
43.58
22.26
10c
9b
R = isopropyl, ethyl, methyl
N
Cl
Selective N-alkylation was a challenge as nicotine’s
two nitrogen atoms are nucleophilic and can compete
for electrophiles. [8] Finally, the methylation of the nico-
tine ring was carried out using Mannich reaction[9] and
other alkyl groups were introduced by sodium hydride
(Scheme 4).
N
N
N
Cl
Cl
10d
N
Bn
Scheme 4 N-Alkylation of nicotine pyrrolidine ring
R
HCOOH, HCHO
80 oC
N
N
14.71
21.12
27.07
62.98
10e
10f
N
9
Cl
Cl
N
Cl
R
N
H
N
Cl
alkyl halides
NaH
R
8
N
N
R'
N
R = isopropyl, ethyl, methyl
R' = allyl, propargyl, benzyl
N
Cl
10
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© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Synthesis and Pharmacological Properties of 5-Alkyl Substituted Nicotine Analogs
figuration. Based on the SAR analysis, our next genera-
Continued
tion targets were concentrated on the synthesis of vari-
ous N-substituted aromatic or hetero-aromatic drugs.
a
b
SY-SY5Y IC50
/
RD IC50
/
No.
Structure
－
(μmol•L )
－
(μmol•L )
1
1
Conclusions
N
63.23
91.42
9c
In summary, an eight-step synthetic route was de-
veloped to a series of enantiomerically enriched 5-alkyl
substituted nicotine analogs. The key steps involved the
construction of the nicotinaldehydes ring through Vils-
meier reaction, the introduction of the chiral homoally-
lic alcohol by an organic boron reagent and the cycliza-
tion of the pyrrolidine ring through the reduction of a
chiral azide. According to this method, 17 nicotine ana-
logs were synthesized, in which compounds 10d and
10g exhibited good activities against RD and SY-SY5Y
cell.
N
N
Cl
Cl
N
5.97
22.81
69.17
10g
10h
Bn
N
26.16
N
Cl
Experimental
N
34.95
94.28
62.41
45.72
112.35
37.74
10i
9d
9e
Chemistry
N
Cl
Unless stated otherwise, all reagents and solvents
were obtained from commercial sources without further
purification. Column chromatography was carried out
N
on silica gel (300—400 µm). H NMR and 13C NMR
1
N
Cl
spectra were recorded on a spectrometer (400 and 100
MHz, respectively) using TMS as internal standard.
HRMS data were determined by EI ionization.
N
N
Cl
(E)-N-Benzyl-N-(3-methylbut-1-enyl) acetamide
(3)[5] Isovaleraldehyde (14.3 mL, 0.13 mol) was added
to benzyl amine (10.9 mL, 0.10 mol) dropwise at 0 ℃
under nitrogen. Then, solid potassium hydroxide (2.8 g,
0.05 mol) was added. The resulting mixture was stirred
for 2 h and the additional potassium hydroxide (1.0 g)
was added. The mixture was cooled to 0 ℃ for 12 h
and the supernatant liquid (compound 2) was taken out
for the next step.
Compound 2 was cooled to 0 ℃ before the addition
of triethylamine (18.1 mL, 0.13 mol) and acetic anhy-
dride. Then, the mixture was warmed to r.t. and kept
stirring for 3 h. Compound 3 was gained by distillation
(142 ℃/10 mmHg) as colorless oil (16.2 g, 75% for 2
steps).
N
69.05
88.22
10j
N
Cl
N
14.71
12.74
32.40
60.63
10k
10l
N
Cl
N
N
Cl
2-Chloro-5-isopropylnicotinaldehyde (4)[5] To a
solution of triphosgene (154.9 g, 521.8 mmol) in tolu-
ene (300 mL) was added DMF (40.6 mL, 521.8 mmol)
at 0 ℃. The mixture was stirred for 30 min before the
solution of compound 3 (16.2 g, 74.5 mmol) in toluene
(30 mL) was added. The mixture was stirred for 2 h be-
fore heated to 95 ℃ and kept stirring for another 3 h.
Upon cooling, the organic layer was discarded. The
aqueous layer was extracted with DCM (60 mL×3) and
washed with water. The organic layer was dried with
anhydrous sodium sulfate and concentrate under vac-
uum. The crude product was further purified by column
chromatography to get compound 4 as yellow oil (7.9 g,
Mecamylamine
DMSO
1.25
0
26.50
0
a All compounds showed negative agonist activities against RD.
b All compounds showed negative agonist activities against SY-
SY5Y.
The data showed that all of the compounds had
negative agonist activities against RD and SY-SY5Y.
Among these compounds, 10d and 10g showed exciting
IC50 values toward these two cell lines which were close
to that of Mecamylamine. We also found that the mole-
cules bearing methyl or ethyl group at the Py-5 position
generally had lower IC50 value than those of isopropyl
at the same position. All the compounds with S con-
figuration had better IC50 values than those with R con-
1
58%). H NMR (400 MHz, CDCl3) δ: 10.40 (s, 1H),
8.44 (d, J＝2.4 Hz, 1H), 8.05 (d, J＝2.4 Hz, 1H),
3.03—2.96 (m, 1H), 1.28 (d, J＝7.2 Hz, 6H).
Chin. J. Chem. 2012, 30, 2813—2818
© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Wang et al.
FULL PAPER
(R)-1-(2-Chloro-5-isopropylpyridin-3-yl)but-3-en-
1-ol (5) The allyl magnesium bromide (6.4 g, 12.0
mmol) was added to the solution of (＋)-Ipc2BCl in di-
ethyl ether at －78 ℃. After stirring for 15 h at －78
℃, the mixture was warmed to 25 ℃ and stirred for
another 1 h. Then, it was cooled to －78 ℃ again and
the solution of 4 (1.0 g, 5.4 mmol) in diethyl ether (20
mL) was added. After stirring for 1 h, the mixture was
warmed to r.t. and kept stirring for another 1 h. The
mixture was then added aqueous NaOH (8.8 mL, 3
mol/L) with H2O2 (30% in water, 3.6 mL) and heated to
reflux for 1 h. After the solvent was removed under
vacuum, the residue was dissolved in ethyl acetate (50
mL). It was then washed with brine and dried over an-
hydrous sodium sulfate. After concentration under vac-
uum, the crude product was further purified by column
chromatography to get compound 5 as yellow oil (540
BH3•Me2S in THF (2 mol/L, 5.47 mL, 10.95 mmol) at 0
℃ under nitrogen. The mixture was stirred at 0 ℃ for
1 h to get a white suspension. The mixture was cooled
to －15 ℃ and the solution of compound 7 (860 mg,
3.65 mmol) in THF (10 mL) was added into the reaction
mixture. Then, it was slowly warmed to r.t. and contin-
ued to stir for another 12 h before quenched with
methanol. After the solvent was removed under reduced
pressure, the residue was dissolved in ethyl acetate (30
mL) and washed with hydrochloric acid (1 mol/L, 20
mL). The organic layer was discarded, the aqueous
phase was adjusted to pH＝13—14 with the solution of
sodium hydroxide (30%). Extracted with DCM (20
mL×3), the organic layer was washed with brine and
dried over anhydrous sodium sulfate. After concentra-
tion under vacuum, the crude product was further puri-
fied by column chromatography to get compound 8 as
yellow oil (720 mg, 88%). [α]2D5 ＋1.9 (c 1.0, MeOH);
1H NMR (400 MHz, CDCl3) δ: 8.11 (d, J＝2.4 Hz, 1H),
7.86 (d, J＝2.4 Hz, 1H), 4.48 (t, J＝7.6 Hz, 1H), 3.22—
3.09 (m, 2H), 2.97—2.90 (m, 1H), 2.42—2.37 (m, 1H),
1.91—1.86 (m, 2H), 1.59—1.54 (m, 2H), 1.28 (d, J＝
6.8 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ: 147.4,
145.9, 143.1, 138.5, 134.3, 58.2, 46.9, 32.8, 31.3, 25.2,
23.6; ESI-MS: 224.8 [M＋1]. HRMS (EI) calcd for
C12H17ClN2: 224.1080, found 224.1078.
1
mg, 45%). [α]2D5 ＋18.0 (c 1.0, EtOH); H NMR (400
MHz, CDCl3) δ: 8.16 (d, J＝2.4 Hz, 1H), 7.79 (d, J＝
2.4 Hz, 1H), 5.91—5.85 (m, 1H), 5.23 (s, 1H), 5.20—
5.08 (m, 2H), 2.99—2.93 (m, 1H), 2.71—2.68 (m, 1H),
2.38—2.31 (m, 1H), 1.28 (d, J＝6.8 Hz, 6H); 13C NMR
(100 MHz, CDCl3) δ: 146.7, 145.9, 143.3, 137.2, 134.2,
133.7, 119.2, 69.0, 41.8, 31.3, 23.6, 23.5; ESI-MS:
225.8 [M＋1]. HRMS (EI) calcd for C12H16ClNO:
225.0920, found 225.0924.
(S)-3-(1-Azidobut-3-enyl)-2-chloro-5-isopropylpyri-
dine (7) To a solution of 5 (0.50 g, 2.21 mmol) in
DCM (30 mL) was added triethylamine (0.61 mL, 4.42
mmol) and methylsulfonyl chloride (0.26 mL, 3.32
mmol) successively at 0 ℃. Then, the mixture was
warmed to r.t. and kept stirring for 2 h. It was quenched
with water and extracted with DCM (10 mL×3). The
organic layer was washed with brine and dried over an-
hydrous sodium sulfate. After concentration under vac-
uum, the crude product was used for the next step di-
rectly.
To the solution of pervious crude product in DMF
(10 mL) was added sodium azide (220 mg, 3.32 mmol)
at r.t. Then, the mixture was heated to 60 ℃ and stirred
for 2 h. The mixture was cooled to r.t. and quenched
with water. Extracted with ethyl acetate (10 mL×3), the
organic layer was washed with brine and dried with an-
hydrous sodium sulfate. After concentration under vac-
uum, the crude product was further purified by column
chromatography to get compound 7 as yellow oil (470
mg, 85% over two steps). [α]2D5 ＋1.74 (c 1.0, CHCl3);
1H NMR (400 MHz, CDCl3) δ: 8.22 (d, J＝2.0 Hz, 1H),
7.61 (d, J＝2.4 Hz, 1H), 5.86—5.76 (m, 1H), 5.18 (s,
1H), 5.15—5.02 (m, 2H), 3.03—2.92 (m, 1H), 2.66—
2.47 (m, 2H), 1.30 (d, J＝6.8 Hz, 6H); 13C NMR (100
MHz, CDCl3) δ: 147.5, 146.8, 143.5, 134.7, 133.3,
132.5, 119.2, 61.5, 39.4, 31.2, 23.5; ESI-MS: 250.8
[M＋1]. HRMS (EI) calcd for C12H15ClN4: 250.0985,
found 250.0993.
(S)-2-Chloro-5-isopropyl-3-(1-methylpyrrolidin-
2-yl) pyridine (9a) The mixture of compound 8 (61.6
mg, 0.27 mmol), formic acid (6 mL) and formaldehyde
(3 mL) was heated to 80 ℃ and kept stirring for 4 h.
Then, the formic acid was removed under reduced
pressure. Diluted hydrochloric acid was used to adjust
the solution to pH＝2 and then washed with DCM (20
mL). The aqueous layer was adjusted to pH＝12 with
diluted sodium hydroxide solution and extracted with
DCM (10 mL×3). The organic layer was washed with
brine and dried over anhydrous sodium sulfate. After
concentration under vacuum, the crude product was
further purified by column chromatography to get com-
pound 9a as yellow oil (44.6 mg, 69%). [α]2D5 －83.8 (c
1
1.80, CHCl3); H NMR (400 MHz, CDCl3) δ: 8.12 (d,
J＝2.8 Hz, 1H), 7.80 (d, J＝2.4 Hz, 1H), 3.52 (t, J＝8.4
Hz, 1H), 3.28—3.23 (m, 1H), 2.99—2.89 (m, 1H),
2.46—2.35 (m, 2H), 2.22 (s, 1H), 1.90—1.81 (m, 2H),
1.54—1.50 (m, 1H), 1.27 (d, J＝7.2 Hz, 6H); 13C NMR
(100 MHz, CDCl3) δ: 148.1, 145.9, 143.4, 137.1, 134.8,
66.5, 56.8, 40.5, 33.3, 31.3, 23.7, 23.6, 22.6; ESI-MS:
238.9 [M ＋ 1]. HRMS (EI) calcd for C13H18ClN2:
238.1237, found 238.1235.
(S)-3-(1-Benzylpyrrolidin-2-yl)-2-chloro-5-iso-pro-
pylpyridine (10a) To the solution of compound 8 (50
mg, 0.22 mmol) in DMF (2 mL) was added sodium hy-
dride (60%, 35.6 mg, 0.89 mmol) at r.t. The mixture was
stirred for 1 h before benzyl bromide (0.08 mL, 0.67
mmol) was added. Quenched with water until the start-
ing material was consumed, the mixture was extracted
with ethyl acetate (10 mL×3). The organic layer was
washed with brine and dried over anhydrous sodium
(S)-2-Chloro-5-isopropyl-3-(pyrrolidin-2-yl)pyri-
dine (8) To the solution of cyclohexene (2.14 mL,
21.89 mmol) in THF (3 mL) was added the solution of
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Chin. J. Chem. 2012, 30, 2813—2818

Synthesis and Pharmacological Properties of 5-Alkyl Substituted Nicotine Analogs
sulfate. After concentration under vacuum, the crude
product was further purified by column chromatography
to get compound 10a as yellow oil (65 mg, 94%). [α]D25
－14.4 (c 1.30, CHCl3); 1H NMR (400 MHz, CDCl3) δ:
8.14 (d, J＝2.4 Hz, 1H), 8.04 (d, J＝2.4 Hz, 1H),
7.32—7.31 (m, 4H), 7.27—7.25 (m, 1H), 3.87 (t, J＝8.4
Hz, 1H), 3.80 (d, J＝12.8 Hz, 1H), 3.23 (d, J＝13.2 Hz,
1H), 3.19—3.14 (m, 1H), 3.02—2.92 (m, 1H), 2.47—
2.42 (m, 1H), 2.38—2.32 (m, 1H), 1.87—1.84 (m, 2H),
1.59—1.54 (m, 1H), 1.30 (d, J＝6.8 Hz, 6H); 13C NMR
(100 MHz, CDCl3) δ: 148.1, 146.1, 143.3, 139.2, 137.8,
134.9, 128.5, 128.2, 126.9, 64.7, 58.6, 53.6, 33.1, 31.3,
23.8, 23.6, 22.7; ESI-MS: 314.9 [M＋1]. HRMS (EI)
calcd for C19H23ClN2: 314.1550, found 314.1551.
rum, 100 U/mL penicillin and 100 μg/mL streptomycin.
The cells were cultured at 37 ℃ in a humidified at-
mosphere containing 5% CO2.
Membrane potential measurements The assay
was carried out according to the FLIPR Membrane Po-
tential Assay Kit Protocol (Molecular Devices). RD
cells were seed at a density of 30 000 cells/well in a
black 384 well plate 24 h prior to the day of the assay.
25 μL membrane potential dye solution was added to
the cells and the plate was incubated for 60 min at 37
℃. The plate was removed from incubator, allowed to
reach room temperate for 20 min. Antagonists were in-
cubated with cells for 10 min, and the plate was trans-
ferred to FLIPR (Molecular Devices). The excitation
and emission wavelengths were set to 535 nm and 560
nm, respectively, and with a cutoff of 550 nm. Fluores-
cence baseline was measured for 10 s followed by the
addition of 100 μmol/L nicotine. Then the measurement
was carried out for 120 s at 1 s intervals. The inhibitory
rates of the antagonists were expressed in the percent
relative to the response induced by nicotine.
(S)-3-(1-Allylpyrrolidin-2-yl)-2-chloro-5-isopropyl-
pyridine (10b) The procedure was same as compound
1
10a. [α]2D5 －48.4 (c 1.0, CHCl3); H NMR (400 MHz,
CDCl3) δ: 8.12 (d, J＝2.4 Hz, 1H), 7.87 (d, J＝2.0 Hz,
1H), 5.88—5.80 (m, 1H), 5.19—5.05 (m, 2H), 3.74 (t,
J＝8.0 Hz, 1H), 3.35—3.30 (m, 1H), 3.20 (dd, J＝5.2,
13.6 Hz, 1H), 2.98—2.91 (m, 1H), 2.80—2.75 (m, 1H),
2.43—2.31 (m, 2H), 1.88—1.83 (m, 2H), 1.54—1.50
(m, 1H), 1.27 (d, J＝7.2 Hz, 6H); 13C NMR (100 MHz,
CDCl3) δ: 148.0, 145.9, 143.3, 137.8, 135.7, 134.9,
116.6, 64.0, 57.1, 53.6, 33.0 31.2, 23.8, 23.5, 22.7;
ESI-MS: 264.8 [M ＋ 1]. HRMS (EI) calcd for
C15H21ClN2: 264.1393, found 264.1397.
Calcium fluorescence measurements
The
changes of calcium flux were evaluated in SH-SY5Y
cells using FLIPR Calcium 5 Assay Kit (Molecular De-
vices). According to the manufacturer's instructions, this
assay was conducted in a similar method to the mem-
brane potential assay with modifications. Briefly, after
overnight cell culture, 25 μL calcium 5 d solution, sup-
plemented with probenecid at a final concentration of
2.5 mmol/L, was added to the cells. The plate was in-
cubated for 60 min at 37 ℃ and 20 min at room tem-
perate, and then the antagonists were added to the cells
for 10 min incubation. After the addition of 20 μmol/L
nicotine, the fluorescence measurements were taken as
before, but with excitation and emission wavelengths set
to 485 and 525 nm, respectively, and with a cutoff of
515 nm.
(S)-2-Chloro-5-isopropyl-3-(1-(prop-2-ynyl)pyrrolidin-
2-yl)pyridine (10c) The procedure was same as com-
1
pound 10a. [α]2D5 －37.4 (c 1.0, CHCl3); H NMR (400
MHz, CDCl3) δ: 8.14 (d, J＝2.0 Hz, 1H), 7.79 (s, 1H),
4.01 (s, 1H), 3.47 (d, J＝17.2 Hz, 1H), 3.25—3.16 (m,
2H), 2.98—2.91 (m, 1H), 2.78 (d, J＝8.4 Hz, 1H),
2.48—2.39 (m, 2H), 2.22 (s, 1H), 1.90 (d, J＝6.4 Hz,
2H), 1.57 (d, J＝2.0 Hz, 1H), 1.28 (d, J＝6.8 Hz, 6H);
13C NMR (100 MHz, CDCl3) δ: 148.2, 146.1, 143.4,
136.9, 134.7, 78.8, 72.8, 61.8, 52.4, 40.7, 33.3, 31.3,
23.7, 23.6, 22.7; ESI-MS: 262.9 [M＋1]. HRMS (EI)
calcd for C15H19ClN2: 262.1237, found 262.1238.
Acknowledgement
Financial support of this work from Shanghai Foun-
dation of Science and Technology (No. 09JC1404200),
the National "863" Project of China (No.
2007AA02Z301), "111" Project and the Fundamental
Research Funds for the Central University (No.
WY1113007) are gratefully acknowledged.
Biology
(－)-Nicotine hydrogen tartrate, mecamylamine and
probenecid were purchased from Sigma. FLIPR Mem-
brane Potential Assay Kit and FLIPR Calcium 5 Assay
Kit were purchased from Molecular Devices. Tissue
culture media and fetal bovine serum (FBS) were ob-
tained from GIBCO.
Cell lines RD rhabdomyosarcoma cells expressing
endogenous human neuromuscular α1β1γδ receptors[10]
and SH-SY5Y neuroblastoma cells expressing human
ganglionic (α7)5 receptors[11] were bought from Institute
of Biochemistry and Cell Biology, Shanghai Institute for
Biological Sciences, CAS, China. RD cells were grown
in Dulbecco's modified Eagle's media supplemented
with 10% fetal bovine serum, 100 U/mL penicillin and
100 μg/mL streptomycin. SH-SY5Y cells were grown in
a 1∶1 mixture of DMEM and Ham F12 medium
(DMEM/F12) supplemented with 20% fetal bovine se-
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