Synthesis and in vivo evaluation of 5-methoxy-2-(phenylethynyl)quinoline (MPEQ) and [11C]MPEQ targeting metabotropic glutamate receptor 5 (mGluR5)

Article abstract of DOI:10.5012/bkcs.2014.35.8.2304

The synthesis and in vivo evaluation of 5-methoxy-2-(phenylethynyl) quinoline (MPEQ) 3 as a potential mGluR5 selective radioligand is described. We have identified MPEQ 3 exhibiting the analgesic effect in the neuropathic pain animal model. The effect of mGluR5 on neuronal activity in rat brain was evaluated through FDG/PET imaging in the presence of MPEQ 3. In addition, the PET study of [¹¹C]MPEQ 3 proved that accumulation of [ ¹¹C]MPEQ 3 in rat brain was correlated to the localization of the mGluR5.

Full text of DOI:10.5012/bkcs.2014.35.8.2304

2304 Bull. Korean Chem. Soc. 2014, Vol. 35, No. 8
Ji Young Kim et al.
http://dx.doi.org/10.5012/bkcs.2014.35.8.2304
Synthesis and In vivo Evaluation of 5-Methoxy-2-(phenylethynyl)quinoline (MPEQ)
and [11C]MPEQ Targeting Metabotropic Glutamate Receptor 5 (mGluR5)
Ji Young Kim,†,‡ Myung-Hee Son,†,§ Kihang Choi,‡ Du-Jong Baek,§ Min Kyung Ko,† Eun Jeong Lim,† Ae Nim Pae,†,#
Gyochang Keum,†,# Jae Kyun Lee,† Yong Seo Cho,†,# Hyunah Choo,†,# Youn Woo Lee,¶ Byung Seok Moon,¶
Byung Cheol Lee,¶ Ho-Young Lee,¶,$,* and Sun-Joon Min†,#,*
†Center for Neuro-Medicine, Brain Science Institute, Korea Institute of Science and Technology (KIST),
Seoul 136-791, Korea. *E-mail: sjmin@kist.re.kr
‡Department of Chemistry, Korea University, Seoul 136-701, Korea
§Department of Chemistry, College of Natural Sciences, Sangmyung University, Seoul 110-743, Korea
#Department of Biological Chemistry, University of Science and Technology (UST), Daejeon 305-333, Korea
¶Department of Nuclear Medicine, Seoul National University Bundang Hospital, Seongnam 463-707, Korea
$Cancer Research Institute, Seoul National University, Seoul 110-779, Korea. *E-mail: debobkr@gmail.com
Received April 10, 2014, Accepted April 15, 2014
The synthesis and in vivo evaluation of 5-methoxy-2-(phenylethynyl)quinoline (MPEQ) 3 as a potential
mGluR5 selective radioligand is described. We have identified MPEQ 3 exhibiting the analgesic effect in the
neuropathic pain animal model. The effect of mGluR5 on neuronal activity in rat brain was evaluated through
FDG/PET imaging in the presence of MPEQ 3. In addition, the PET study of [11C]MPEQ 3 proved that
accumulation of [11C]MPEQ 3 in rat brain was correlated to the localization of the mGluR5.
Key Words : MPEQ, Metabotropic glutamate receptor 5 (mGluR5), in vivo, PET imaging, Neuropathic pain
Introduction
zation of glutamate receptors in vivo.7 However, only a
limited number have been successfully used in in vivo
studies. Among the mGluR5 PET tracers, [11C]ABP-688 (1)
turned out to be the most successful radioligand for PET
imaging, demonstrating relatively high ligand uptake in
mGluR5-rich brain regions such as the anterior cingulate,
medial temporal lobe, amygdala, caudate and putamen.8
In parallel to our discovery project aiming at development
of mGluR5 negative allosteric modulators (NAMs) for treat-
ment of neuropathic pain, we further extended our study to
indentify a PET tracer, which will allow us to better under-
stand mGluR5 and its role in neuropathic pain. Recently, we
reported that 2-alkynylquinoline 2 exhibited favorable
analgesic effects in spinal nerve ligation (SNL) model of
neuropathic pain.9 This result suggested that a radiotracer
based on this chemical structure would be suitable for
measuring its brain uptake and distribution, which is useful
for elucidating the effect of mGluR5 on neuronal activity in
the brain of rat neuropathic pain model.
Glutamate is the major excitatory neurotransmitter in the
central nervous system (CNS) and regulates signal trans-
mission between neurons through diverse sets of receptors.1
The metabotropic glutamate receptors (mGluR) belong to
the subfamily class C of G-protein coupled receptors (GPCRs).
They consist of eight subtypes, which have been divided into
three groups based on their structural similarity, localization
and signaling pathway. mGluR5, one of the Group I receptors,
is mainly expressed in brain regions. This receptor has been
implicated in a variety of neurological and psychiatric dis-
orders such as anxiety disorders,2 schizophrenia,3 Parkinson’s
disease,4 fragile X syndrome,5 and neuropathic pain.6 Given
the importance of mGluR5 in such disease states, in vivo
evaluation of mGluR5 in the CNS would validate the role of
this receptor in physiological and pathological conditions.
Thus, potent and selective mGluR5 radioligands could pro-
vide valuable tools for monitoring disease progression as
well as evaluating the pharmacokinetics of therapeutics.
During the past decade, a large number of radioligands
have been identified as PET tracer candidates for visuali-
Due to a lack of labeling substituents on compound 2, we
investigated in vitro inhibitory activity of other analogues
against mGluR5. Considering the several candidates, we
Figure 1. Structures of 1 (ABP688), 2, 3 (MPEQ) and 4.

Synthesis and In vivo Evaluation of 5-Methoxy-2-(phenylethynyl)quinoline Bull. Korean Chem. Soc. 2014, Vol. 35, No. 8 2305
Table 1. In vitro inhibitory activity and safety profile of 2-alkynylquinolines 3 and 4
% Inhibition (mGluR5)a
% Inhibition (mGluR1)a
Compds
mGluR5 IC50 (µM)
10 µM
1 µM
10 µM
1 µM
3 (MPEQ)
68.03
65.17
63.58
28.20
12.05
16.24
−4.73
0.41 0.07
4
2.13
−
aCa2+ flux assay using glutamate as agonist.
have selected compounds 3 and 4 containing either methoxy
group or fluorine at the 5-position of quinoline, which would
be easily converted to their corresponding PET ligands.
Based on the in vitro activities of both compounds (Table 1),
5-methoxy-2-(phenylethynyl)quinoline 3 (MPEQ) was
identified as a relevant radioligand candidate with regard to
potency and selectivity. Herein, we report on the in vitro
pharmacological characteristic of MPEQ as an mGluR5
antagonist and its preliminary evaluation for labeling mGluR5
in vivo in living rats.
Table 2. Optimization of methylation reaction
Entry
Condition
Time (h)
Yield
1
2
3
Me2SO4, K2CO3, acetone, rt
MeI, KOH, MeOH, 50 °C
MeI, TBAH, MeOH, 50 °C
1
31%
56%
47%
0.5
0.25
Result and Discussion
The synthesis of compound 3 and its labeling precursor 9
started from commercially available 5-hydroxyquinoline 5.
Methylation of 5 with TMSCHN2 gave 5-methoxyquinoline
6a, which was converted to 2-chloroquinoline derivative 7a
via N-oxidation and chlorination. The Sonogashira reaction
of 7a with phenylacetylene produced the target quinoline 3
in 50% yield. Next, we were able to synthesize the precursor
9 by using the similar reaction sequences. Thus, hydroxy-
quinoline 5 was protected as pivaloate 6b, which was sub-
jected to chlorination and alkynylation to afford 2-alkynyl-
quinoline 7b. Finally, removal of pivaloyl group was
performed by LiAlH4 reduction to give the corresponding 5-
hydroxy-2-alkynyl quinoline 9 in good yield (Scheme 1).
Methylation reactions of the precursor 9 are summarized
in Table 2. Initially, we treated compound 9 with dimethyl-
sulfate in the presence of potassium carbonate for 1 h to
obtain 3 in 31% yield. However, it is necessary to reduce the
reaction time within 30 min to generate reasonable amount
of PET probes. We performed further optimization using
methyl iodide as an electrophile. The reaction of 6 with
methyl iodide and tetrabutylammonium hydroxide in meth-
anol at 50 °C in 15 min yielded the target compound 3 in
47% yield. Therefore, we decided to attempt incorporation
of a [11C] methyl group to form the 11C-labeled MPEQ 3
under this optimized condition.
[11C]MPEQ 3 was synthesized from 9 by [11C]methylation
using [11C]CH3I in the TRACERlab FX C-pro module (GE
Healthcare). [11C]CO2 was produced at the KOTRON-13
cyclotron (Samyoung Unitech Co., Korea) by irradiation of
a gas target containing N2 (99.9999%) using the 14N(p,a)11C
nuclear reaction. [11C]CH3I, converted from [11C]CO2 using
the gas phase conversion in an automated module, was
bubbled by a flow of He gas into a solution of acetone (0.4
mL) containing 9 (1 mg) and tetrabutylammonium hydroxide
(40 wt % solution in water, 1.5 μL) at –20 °C. When radio-
activity had peaked, the solution was heated to 70 ºC and
kept at this temperature for 4 min. After cooling down to
room temperature by He flow, the reaction mixture was
quenched by addition of water (1.4 mL) and subsequently
transferred to the 2 mL of injection loop of HPLC system.
The reaction mixture was purified by reverse phase HPLC
(Waters, Xterra RP-18, 10 μm, 10 × 250 mm with guard
column (10 × 10 mm); eluent: 70% CH3CN/H2O; flow rate:
3 mL/min) equipped with a gamma-ray detector and a UV
detector at 254 nm (Figure 2). The desired fraction of
[11C]MPEQ 3 collected from HPLC at around 11.7 min was
diluted with 40 mL of water. The resulting solution was then
Scheme 1. Reagents and conditions: (a) TMSCHN2, MeOH, rt, 42% (R = Me) or PvCl, pyr, CH2Cl2, 84% (R = Pv); (b) i) mCPBA, DCM, 0
°C to rt; ii) POCl3, CH2Cl2, reflux, 32% (R = Me) or 37% (R = Pv); (c) phenylacetylene, PdCl2(PPh3)2, CuI, Et3N, 80 °C, 50% (R = Me) or
100% (R = Pv); (d) LiAlH4, THF, 0 °C to rt, 49%.

2306 Bull. Korean Chem. Soc. 2014, Vol. 35, No. 8
Ji Young Kim et al.
sure and oral bioavailability. In particular, the brain to
plasma ratios in intravenous and oral administration were
significantly high, which indicated that MPEQ 3 has ex-
cellent characteristics of brain penetration.
The in vivo efficacy of 3 was further evaluated in the rat
neuropathic pain model developed by Chung et al. (Figure
3).10 In order to induce a neuropathic pain state to rats, tight
ligation of the L5 spinal nerve at a site distal to the dorsal
root ganglion (DRG) was executed. After surgical operation,
the pain was fully stimulated for 14 days and behavioral tests
for mechanical allodynia and cold allodynia were perform-
ed. The rats were treated orally with 100 mg/kg of 3 or
gabapentin (a positive control). In fact, MPEQ 3 exhibited
highest analgesic effect in both behavior tests at 3 h.
Although the in vivo efficacy of 3 is not as high as that of
gabapentin in this behavior test, we believed that it was
effective enough to investigate in vivo imaging targeting
mGluR5 in the rat neuropathic pain model.
To evaluate the effect of MPEQ 3 in cerebral neuronal
activity, we acquired [18F] fluorodeoxyglucose (FDG) PET/
CT image of five Sprague Dawley (SD) rats with and with-
out intravenous administration of compound 3. Practically,
MPEQ 3 was intravenously administrated to rats 30 minutes
prior to FDG injection and the PET/CT image was acquired
60 minutes after FDG injection. Each image was spatially
normalized to rat template image and compared the images
obtained with and without administration of 3 were com-
pared using statistical parametric mapping 5 (SPM5), by
paired t-test (uncorrected p < 0.001 with permutation 10,000).
Based on this experiment, we verified that MPEQ 3 signi-
ficantly decreased regional glucose metabolism in both
primary somatosensory cortices (Figure 4).
Figure 2. Representative HPLC profile from the TRACERlab FX
C-pro module (upper: UV-254 nm; bottom: gamma-ray).
loaded into a C18 plus Sep-Pak cartridge to trap only
[11C]MPEQ 3 and to remove the HPLC solvent. After
rinsing the cartridge with 10 mL of water, the pure product
was eluted with 1.5 mL of ethanol and 16 mL of saline for
adjustment of 8.5% ethanol/saline. Consequently, the isolated
radiochemical yield was 19.1 4.9% (n = 6, decay corrected)
and produced approximately 1.7-2.2 GBq per batch. An
aliquot of the formulated solution was checked by analytical
HPLC (Waters, Xterra RP-18, 5 μm, 3.9 × 250 mm; eluent:
70% CH3CN/H2O; flow rate: 1 mL/min) for radiochemical
identity (Figure 2), radiochemical purity and specific activity.
The radiochemical purity was over 99% and specific radio-
activity was 137 52 GBq/μmol at the end of synthesis.
After establishing the synthesis of the labeled compound
3, the pharmacokinetic parameters for 3 following intraven-
ous and oral administration in rats are determined as shown
in Table 3. Although compound 3 has high value of the
mean volume distribution, it showed relatively good expo-
Next, we evaluated the effect of 3 in the neuropathic pain
rat model by the acquisition of FDG PET/CT image with
and without intravenous administration and compared the
Table 3. Mean ( SDa) pharmacokinetic parameters after intravenous (n = 5) administration and oral (n = 5) administration (10 mg/kg) of
MPEQ (3) to SD male rats
Plasma
Intravenous
Oral
AUC0–∞ (µg min/mL)
AUClast (µg min/mL)
Terminal half-life (min)
272.86 30.21
99.21 17.42
84.97 21.59
158.44 88.8
0.43 0.25
66 (30-120)b
−
250.60 22.54
170.68 28.52
Cmax (µg/mL)
−
−
Tmax (min)
CL (mL/mim/kg)
MRT (min)
−
Vss (mL/kg)
36.99 3.85
98.12 13.47
0.48 0.05
0.09 0.03
1.26 0.15
2.67
−
Ae (%)
0.12
Plasma conc (µg/mL) @ 2 h
Plasma conc (µg/mL) @ 8 h
Brain conc (µg/mL) @ 2 h
Brain-to-plasma ratio (B/P) @ 2 h
F (%)
0.31 0.13
0.05 0.02
0.84 0.24
3.03
36.4
Values are presented as mean (standard deviation in parentheses). AUC0–∞, total area under the plasma concentration–time curve from time zero to time
infinity; AUClast, total area under the plasma concentration–time curve from time zero to last measured time; Cmax, peak plasma concentration; Tmax
,
time to reach Cmax; CL, time-averaged total body clearance; MRT, mean residence time; Vss, apparent volume of distribution at steady state; Ae,
Excreted amount; F, bioavailability; aSD: Standard deviations; bMedian (range) for Tmax

Synthesis and In vivo Evaluation of 5-Methoxy-2-(phenylethynyl)quinoline Bull. Korean Chem. Soc. 2014, Vol. 35, No. 8 2307
Figure 3. Effect on mechanical allodynia (a and b) and cold allodynia (c and d) after oral administration of gabapentin (
○
, 100 mg/kg, n =
4) and 3 ( , 100 mg/kg, n = 3) to neuropathic pain-induced rats. Experimental time expressed as D for days after neuropathic injury (N)
●
*
and h for hours after gabapentin or 3 administration, *P < 0.05 (gabapentin), P < 0.05 (3) vs pre-administration value (paired t-test),
♣P < 0.05 gabapentin vs 3 (unpaired t-test).
Figure 4. Comparison FDG PET/CT of normal SD rat with and
without MPEQ 3 administration. (a) transaxial view of rat brain (b)
coronal view of rat brain. Colored lesions represent the area with
significantly lower neuronal activity after MPEQ 3 administration.
Those areas are the both somatosensory cortices (uncorrected
u < 0.001 with permutation 10,000).
Figure 5. Comparison FDG PET/CT of neuropathic model rat
with and without administration of 3. Neuronal activity was
reduced in the sensory motor region (a), PAG (periaqueductal
gray) region (b, c) and left thalamus (a, c) that are known as
related to the neuropathic pain pathway (uncorrected u < 0.001
with permutation 10,000).
different states (Figure 5). After intravenous administration
of 3, reduction of neuronal activity was observed in the
sensory motor region, PAG (periaqueductal gray) region and
left thalamus that are associated with the neuropathic pain
pathway.11 Taken together with the in vitro data (Figure 3),
this result indicated that MPEQ 3 exhibited the analgesic
effect in the SNL rat model by modulating the pain signaling

2308 Bull. Korean Chem. Soc. 2014, Vol. 35, No. 8
Ji Young Kim et al.
Figure 6. Summation image of [11C]MPEQ 3 dynamic PET/CT image. (a) coronal image (b) sagittal image (c) transaxial image of
[11C]MPEQ 3 dynamic PET/CT image. [11C]MPEQ 3 well penetrates blood brain barrier. [11C]MPEQ 3 distributed in cerebal cortex and
mid brain area. However, there is low uptake in the cerebellum that is known as area with lower mGluR5 expression.
pathway through mGluR5 antagonism.
graphy (TLC) was performed using glass plates precoated
with silica gel (0.25 mm). TLC plates were visualized by
exposure to UV light (UV), and then were visualized with a
p-anisaldehyde stain followed by brief heating on hot plate.
Flash column chromatography was performed using silica
gel 60 (230-400 mesh, Merck) with the indicated solvents.
1H and 13C spectra were recorded on Bruker 300, Bruker 400
Finally, the possibility of [11C]MPEQ 3 as a probe for
imaging the mGuR5 is examined by analysis of brain PET
imaging. Following the procedure described above, [11C]MPEQ
3 was prepared and intravenously injected to normal rats.
Subsequent dynamic PET image was acquired until 90
minutes (Figure 6). The PET study of [11C]MPEQ 3 in rat
brain revealed that it effectively penetrated blood brain
barrier (BBB). In particular, accumulation of [11C]MPEQ 3
in rat brain was correlated to the localization of the mGluR5.
We observed relatively high level of [11C]MPEQ 3 uptake in
the cerebral cortex, olfactory tubercle, hippocampus and
striatum. On the other hand, the cerebella uptake was
considerably low, which supports the evidence that there is
low mGluR5 expression in the cerebellum region as reported
in the previous literature.12
1
or Varian 300 NMR spectrometers. H NMR spectra are
represented as follows: chemical shift, multiplicity (s =
singlet, d = doublet, t = triplet, q = quartet, m = multiplet),
1
integration, and coupling constant (J) in Hertz (Hz). H
NMR chemical shifts are reported relative to CDCl3 (7.26
ppm). 13C NMR was recorded relative to the central line of
CDCl3 (77.0 ppm). GC/MS and LC/MS analyses were respec-
tively performed on Agilent 6890N Network Gas system
with 5793N MSD and Applied Biosystems/MDS SCIEX
API3200 LC/MS/MS system.
Conclusion
5-Methoxyquinoline (6a):13 To a solution of 5-quinolinol
5 (164 mg, 1.13 mmol) in MeOH (7 mL) cooled to 0 °C was
slowly added trimethylsilyldiazomethane (6 mL, 0.5 M in
Et2O). The reaction mixture was allowed to warm to room
temperature and stirred for 26 h. After addition of water, the
reaction mixture was extracted with ethyl acetate (3 × 15
mL). The combined organic layer was washed with brine,
dried over anhydrous MgSO4, and concentrated under reduced
pressure. The crude oil was purified by column chromato-
graphy on silica gel (EtOAc/CH2Cl2/hexane = 1:2:4) to give
In this study, we synthesized 5-methoxy-2-(phenylethyn-
yl)quinoline (MPEQ) 3 as a potential mGluR5 selective
radioligand. The in vivo evaluation of MEPQ 3 in the SNL
model turned out that it showed significant analgesic effect
in behavior tests of mechanical allodynia and cold allodynia.
The reduction of neuronal activity in sensory motor region
of the neuropathic pain animal model was observed by FDG/
PET imaging variation before and after treatment of MPEQ
3. In addition, the PET study of [11C]MPEQ 3 proved that
[11C]MPEQ 3 was highly localized in the mGluR5 rich
region of rat brain. Although MPEQ 3 showed possibility as
an mGluR5 radiotracer, it should be further optimized as an
accurate radiotracer, which is useful for quantitative evalua-
tion of mGluR5 biodistribution.
1
5-methoxyquinoline 6a (76 mg, 42%) as a white solid: H
NMR (CDCl3, 300 MHz) δ 4.04 (s, 3H), 6.89 (d, J = 7.5 Hz,
1H), 7.41 (q, J = 4.2 Hz, 1H), 7.62-7.74 (m, 2H), 8.61 (dd, J
= 8.4, 1.2 Hz, 1H), 8.93 (dd, J = 4.2, 1.8 Hz, 1H).
2-Chloro-5-methoxyquinoline (7a):13 To solution of 5-
methoxyquinoline 6a (104 mg, 0.66 mmol) in CH2Cl2 (3
mL) was added meta-chloroperoxybenzoic acid (195 mg,
1.13 mmol) at 0 °C for 30 min. The mixture was allowed to
warm to room temperature and stirred for additional 3 h. The
reaction is queched with 4 N NaOH and extracted with
CH2Cl2. The combined organic extracts were washed with
brine, dried over anhydrous MgSO4 and concentrated under
reduced pressure to give the crude N-oxide, which was
directly used for the next step without purification. To solu-
tion of the resulting N-oxide in CH2Cl2 (2.5 mL) was adeed
Experimental Section
General. All reactions were carried out under dry nitro-
gen unless otherwise indicated. Commercially available
reagents were used without further purification. Solvents
and gases were dried according to standard procedures.
Organic solvents were evaporated with reduced pressure
using a rotary evaporator. Analytical thin layer chromato-

Synthesis and In vivo Evaluation of 5-Methoxy-2-(phenylethynyl)quinoline Bull. Korean Chem. Soc. 2014, Vol. 35, No. 8 2309
phosphorus oxychloride (0.09 mL, 0.99 mmol). The reaction
mixture was refluxed at 60 °C for 3 h, allowed to cool to
room temperature and poured into ice-water. The resulting
mixture was treated with 4 N aqueous NaOH until pH
reached to around 10. The organic phase was extracted with
CH2Cl2 (3 × 5 mL), washed with brine, dried over anhydrous
MgSO4, and concentrated under reduced pressure. The crude
residue was purified by column chromatography on silica
gel (EtOA/CH2Cl2/Hexane = 1:2:4) to give 2-chloro-5-meth-
oxy-chloroquinoline 7a (40.1 mg, 32%) as a white solid: 1H
NMR (CDCl3, 300 MHz) δ 4.01 (s, 3H), 6.88 (dd, J = 7.2,
1.5 Hz, 1H), 7.35 (d, J = 8.7 Hz, 1H), 7.59-7.67 (m, 2H),
8.51 (d, J = 8.7 Hz, 1H).
5-Methoxy-2-(phenylethynyl)quinoline (3): To a solution
of 2-chloro-5-methoxyquinoline (12.7 mg, 0.0656 mmole)
in THF (1.5 mL) was added Pd(PPh3)4 (5.3 mg, 0.00459
mmol), CuI (3.3 mg, 0.017 mmol). The reaction mixture was
stirred for 5 min and triethylamine (0.5 mL) and phenyl-
acetylene (0.01 mL, 0.131 mmol) were added. After the
resulting mixture was stirred at 80 °C for 24 h, it was allow-
ed to cool to room temperature and filtered through a pad of
Celite by the aid of EtOAc. The filtrate was treated with
water and extracted with EtOAc (3 × 10 mL). The organic
layer was washed with water and brine, dried over anhydr-
ous MgSO4, and concentrated under reduced pressure. The
crude oil was purified by column chromatography on silica
gel (EtOAc/hexane = 1:10) to give 5-methoxy-2-(phenyl-
ethynyl)quinoline 3 (8.6 mg, 50%) as a white solid: mp 92-
97 °C; 1H NMR (CDCl3, 300 MHz) δ 4.05 (s, 3H), 6.91 (d, J
= 7.5 Hz, 1H), 7.42-7.44 (m, 3H), 7.62-7.77 (m, 5H), 8.60
(d, J = 8.7 Hz, 1H); 13C NMR (CDCl3, 100 MHz) δ 55.8,
89.4, 89.9, 104.8, 119.6, 121.5, 122.2, 123.5, 128.4, 129.1,
130.0, 131.0, 132.3, 143.9, 149.1, 155.0; GC/MS (EI): m/z:
calcd for C18H13NO: 259.10, M+; found: 259.
Quinolin-5-yl pivalate (6b):14 To a solution of 5-quino-
linol 5 (197.2 mg, 1.36 mmol) in CH2Cl2 (2.8 mL) was
added pyridine (0.32 mL, 3.97 mmol) and pivaloyl chloride
(0.18 mL, 1.5 mmol). The reaction mixture was stirred at
room temperature for 17 h and quenched with saturated
ammonium chloride. The resulting solution was extracted
with EtOAc (3 × 10 mL) and the combined organic layer
was washed with brine, dried over MgSO4, and concentrated
under reduced pressure. The crude oil was purified by
column chromatography on silica gel (EtOAc/hexane = 1:1)
to give quinolin-5-yl pivalate 6b (261.2 mg, 84%) as a white
solid: 1H NMR (CDCl3, 300 MHz) δ 1.48 (s, 9H), 7.29 (dd, J
= 7.6, 0.9 Hz, 1H), 7.42 (dd, J = 8.5, 4.2 Hz, 1H), 7.70 (dd, J
= 8.5, 7.7 Hz, 1H), 8.00-8.03 (m, 1H), 8.17 (qd, J = 8.5, 0.8
Hz, 1H), 8.94 (dd, J = 4.2, 1.7 Hz, 1H); 13C NMR (CDCl3,
100 MHz) δ 27.2, 39.4, 118.4, 121.2, 122.3, 127.1, 128.7,
129.6, 146.3, 148.8, 150.6, 176.6.
solid: 1H NMR (CDCl3, 300 MHz) δ 1.48 (s, 9H), 7.31 (dd, J
= 7.7, 0.9 Hz, 1H), 7.41 (d, J = 8.8 Hz, 1H), 7.73 (t, J = 8.1
Hz, 1H), 7.92 (d, J = 8.6 Hz, 1H), 8.11 (d, J = 8.8 Hz, 1H);
13C NMR (CDCl3, 100 MHz) δ 27.3, 39.6, 119.1, 121.1,
122.6, 126.3, 130.2, 132.8, 146.5, 148.5, 151.3, 176.7.
2-(Phenylethynyl)quinolin-5-yl Pivalate (8): Following
the same procedure as that used for the synthesis of 3, the
reaction of -chloroquinolin-5-yl pivalate 7b (23.9 mg,
0.0906 mmol), CuI (3.7 mg, 0.019 mmol), Pd (PPh3)4 (7.5
mg, 0.000649 mmol), phenylacetylene (0.02 mL, 0.181
mmol) in triethylamine (0.6 mL) gave 2-(phenylethynyl)-
quinolin-5-yl pivalate 8 (29.8 mg, 100%) as a white solid:
1H NMR (CDCl3, 400 MHz) δ 1.49 (s, 9H), 7.29 (dd, J =
7.6, 0.9 Hz, 1H), 7.36-7.40 (m, 3H), 7.62 (d, J = 8.6 Hz, 1H),
7.66-7.69 (m, 2H), 7.72-7.75 (m, 1H), 8.02 (d, J = 8.6 Hz,
1H), 8.15 (dd, J = 8.7, 0.7 Hz, 1H); 13C NMR (CDCl3, 75
MHz) δ 27.36, 39.59, 89.12, 90.59, 119.10, 121.23, 122.00,
124.60, 127.09, 128.47, 129.35, 129.49, 129.97, 132.33,
144.11, 146.27, 148.92, 176.78; GC/MS (EI): m/z: calcd for
C21H18N2O2: 330.14, [M−H]+; found: 329.
2-(Phenylethynyl)quinolin-5-ol (9): To the solution of 2-
(phenylethynyl)quinolin-5-yl pivalate 8 (15.4 mg, 0.047
mmol) in THF (0.78 mL) was added lithium aluminum
hydride (0.06 mL, 1.0 M in THF) at 0 °C. The reaction was
allowed to warm to room temperature and stirred for 30 min.
The mixture was quenched with NH4Cl solution. The result-
ing mixture was extracted with diethyl ether (3 × 3 mL) and
the combined organic layer was washed with water and
brine, dried over MgSO4, and concentrated under reduced
pressure. The crude oil was purified by column chromato-
graphy on silica gel (EtOAc/hexane = 1:1) to give 2-(phen-
ylethynyl)quinolin-5-ol 9 (5.6 mg, 49%) as a yellow solid:
1H NMR (MeOD, 300 MHz) δ 6.92 (d, J = 0.84 Hz, 1H),
7.42-7.49 (m, 4H), 7.55-7.68 (m, 4H), 8.66 (d, J = 4.3 Hz,
1H); 13C NMR (MeOD, 75 MHz) δ 88.2, 89.9, 108.9, 118.0,
119.1, 121.8, 122.4, 128.4, 129.3, 130.8, 131.7, 132.2, 143.5,
153.5; LC/MS (ESI+): m/z: calcd for C17H12NO: 246.09,
[MI+H]+; found: 246.2.
In vivo Behavioral Test. Two behavioral tests (mech-
anical allodynia and cold allodynia) were conducted for rats
at 1 day prior to surgery and 14 days after surgery. After the
postoperative behavioral test, the animals were treated orally
with 100 mg/kg compound 3 or gabapentin. The tests were
re-evaluated at 1 h, 3 h, and 5 h after administration.
Mechanical Allodynia. Testing was performed according
to methods described previously.16 Rats were acclimated in a
transparent plastic boxes permitting freedom of movement
with a wire mesh floor to allow access to the planter surface
of the hind paws. A von Frey filament (Stoelting, Wood
Dale, IL) was applied 5 times (once every 3-4 s) to hind paw.
Von Frey filaments were used to assess the 50% mechanical
threshold for paw withdrawal. The 50% withdrawal threshold
was determined by using the up-down method and formula
given by Dixon17: 50% threshold = 10 (X + kd)/104, where
X is the value of the final von Frey hair used (in log units), k
is the tabular value for the pattern of positive/negative
responses modified from Dixon,17 and d is the mean differ-
2-Chloroquinolin-5-yl pivalate (7b):15 Following the
same procedure as that used for the synthesis of 7a, the
reaction of 6b (245 mg, 1.07 mmol), meta-chloroperoxy-
benzoic acid (368 mg, 2.13 mmol), phosphorus oxychloride
(0.15 mL, 1.60 mmol) in CH2Cl2 (10.0 mL) afforded 2-
chloroquinolin-5-yl pivalate 7b (104 mg, 37%) as a white

2310 Bull. Korean Chem. Soc. 2014, Vol. 35, No. 8
Ji Young Kim et al.
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Acknowledgments. This research was supported by a
grant of the Korea Health technology R&D Project, the
Ministry of Health and Welfare, Republic of Korea
(HI11C0998) and by the Korea Institute of Science and
Technology (KIST, 2E24670, 2E25023, and 2E24510).
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