Synthesis and in vitro biological evaluation of carbon-11-labeled quinoline derivatives as new candidate PET radioligands for cannabinoid CB2 receptor imaging

Article abstract of DOI:10.1016/j.bmc.2010.02.011

Cannabinoids have been recently proposed as a new family of potential antitumor agents, and cannabinoid receptor 2 (CB2) is believed to be over-expressed in tumor cells. This study was designed to develop new radioligands for imaging of CB2 receptor in ca

Full text of DOI:10.1016/j.bmc.2010.02.011

Bioorganic & Medicinal Chemistry 18 (2010) 2099–2106
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and in vitro biological evaluation of carbon-11-labeled quinoline
derivatives as new candidate PET radioligands for cannabinoid CB2
receptor imaging
Mingzhang Gao a, Min Wang a, Kathy D. Miller b, Gary D. Hutchins a, Qi-Huang Zheng a,
*
a Department of Radiology and Imaging Sciences, Indiana University School of Medicine, 1345 West 16th Street, L3-208, Indianapolis, IN 46202, USA
b Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Cannabinoids have been recently proposed as a new family of potential antitumor agents, and cannabi-
noid receptor 2 (CB2) is believed to be over-expressed in tumor cells. This study was designed to develop
new radioligands for imaging of CB2 receptor in cancer using biomedical imaging technique positron
emission tomography (PET). Carbon-11-labeled 2-oxoquinoline and 2-chloroquinoline derivatives,
Received 5 January 2010
Revised 4 February 2010
Accepted 5 February 2010
Available online 10 February 2010
[
[
11C]6a–d and [11C]9a–d, were prepared by O-[11C]methylation of their corresponding precursors using
11C]CH3OTf under basic conditions and isolated by a simplified solid-phase extraction (SPE) method in
Keywords:
40–50% radiochemical yields based on [11C]CO2 and decay corrected to end of bombardment (EOB).
The overall synthesis time from EOB was 15–20 min, the radiochemical purity was >99%, and the specific
Carbon-11-labeled quinoline derivatives
Positron emission tomography (PET)
Cannabinoid receptor 2 (CB2)
Radioligands
activity at end of synthesis (EOS) was 111–185 GBq/lmol. Radioligand binding assays indicated com-
pounds 6f, 6b, and 9f display potent in vitro binding affinities with nanomolar Ki values and at least
100–2000-fold selectivity for CB2.
Cancer imaging
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
developed as potent CB2 receptor inverse agonists.22 To radiolabel
therapeutic agents as diagnostic agents for noninvasive imaging of
The endogenous cannabinoid system consists of cannabinoid
receptors, cannabinoid receptor 1 (CB1) and cannabinoid receptor 2
(CB2), and they are G protein-coupled receptors. CB1 is found predo-
minantlyinthebrain,CB2 isexpressedmainlyonimmune tissues and
cancer cells, and thus cannabinoid receptors are associated with
various brain, cardiovascular, and cancer diseases.1,2 Cannabinoid re-
ceptors provide an attractive target for the development of therapeu-
tic agents, and many CB1 antagonists and CB2 agonists have been
developed and described in the literature.3 Recent attention has
turned to CB2 receptors, since CB2-selective inverse agonists have
beenproposedas a new family of potential antitumor agents.4 Canna-
binoid receptors also provide a particularly attractive target for the
development of imaging agents, and numerous papers have reported
the synthesis andevaluation of radioligands for imaging of CB1 recep-
tor in the brain using the biomedical imaging technique positron
cancer and monitoring of therapeutic efficacy, we report the design,
synthesis, labeling, and in vitro biological evaluation of quinoline de-
rivatives, 7-[11C]methoxy-2-oxo-8-pentyloxy-1,2-dihydroquinoline-
3-carboxylicacid(2-phenylethyl)amide([11C]6a),7-[11C]methoxy-2-
oxo-8-pentyloxy-1,2-dihydroquinoline-3-carboxylic acid [2-(4-
chlorophenyl)ethyl]amide([11C]6b), 7-[11C]methoxy-2-oxo-8-penty-
loxy-1,2-dihydroquinoline-3-carboxylic acid (3-phenylpropyl)a-
mide ([11C]6c), 7-methoxy-2-oxo-8-pentyloxy-1,2-dihydroquino-
line-3-carboxylic acid [2-(4-[11C]methoxyphenyl)ethyl]amide
([11C]6d),
2-chloro-7-[11C]methoxy-8-pentyloxy-1-quinoline-3-
carboxylic acid (2-phenylethyl)amide ([11C]9a), 2-chloro-7-
[
11C]methoxy-8-pentyloxy-1-quinoline-3-carboxylic acid [2-(4-
chlorophenyl)ethyl]amide ([11C]9b), 2-chloro-7-[11C]methoxy-8-
pentyloxy-1-quinoline-3-carboxylic acid (3-phenylpropyl)amide
([11C]9c), and 2-chloro-7-methoxy-8-pentyloxy-1-quinoline-3-car-
boxylic acid [2-(4-[11C]methoxyphenyl)ethyl]amide ([11C]9d), as
new +candidate PET radioligands for CB2 receptor imaging.
emission tomography (PET).5–14 However, only
a few papers
described ligands for imaging of CB2 receptor in cancer.15–17 We are
interested in the development of enzyme-based and/or receptor-
based PET cancer imaging agents. Several CB2 receptor agonists were
found to induce apoptosis in differenttumor cells.18–21 Therefore, CB2
receptor has become a valuable clinical target for treating cancer dis-
eases. Recently a novel series of 2-oxoquinoline derivatives have been
2. Results and discussion
2.1. Chemistry
Synthesis of 2-oxoquinoline derivative precursors and stan-
dards was accomplished using a modification of the previously
* Corresponding author. Tel.: +1 317 278 4671; fax: +1 317 278 9711.
E-mail address: qzheng@iupui.edu (Q.-H. Zheng).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.02.011

2100
M. Gao et al. / Bioorg. Med. Chem. 18 (2010) 2099–2106
reported procedures.22 As depicted in Scheme 1, nitration of isova-
nillin was directly carried out with HNO3/EtOAc to give compound
1 in 62% yield, instead of expensive nitration reagent nitronium
tetrafluoroborate used in the literature,22 and other typical nitra-
tion reagent (HNO3/H2SO4) used in the patent,23 which only gave
1 as a side product with approximately 10% yield. Nitration of iso-
vanillin with HNO3 mainly gave 1 (2-position nitration) in ester
solvent (EtOAc), whereas it mainly gave 4-position nitration prod-
uct in acid solvent (H2SO4 or HOAc). Alkylation of 1 with bromo-
pentane using the Williamson ether synthesis23 provided ether 2
in 80% yield. Reduction of 2 with iron powder afforded compound
3 in 85% yield. Transesterification reaction of 3 with dimethyl mal-
onate in the presence of piperidine and HOAc using EtOH as sol-
vent in 2 days produced ethyl ester 4 in 86% yield. The literature
procedure (overnight reaction)22 gave a mixture of ethyl ester 4
and a methyl ester analog. Thus we extended the reaction time
to 2 days, since the longer reaction time would favor complete
transesterification to form ethyl ester product 4. Hydrolysis of 4
in HCl formed acid 5 in 84% yield. Then, the two-step literature
procedure22 was modified to a one-step reaction, and acid 5 was
directly reacted with amines in the presence of the coupling re-
derivative 6 in very poor yield. Desmethylation of 6a–c with LiCl
in DMF produced phenolic hydroxyl precursors 7a–c in 80–83%
yield. In comparison with the results reported in the literature,22
several improvements in the synthetic methodology for prepara-
tion of 2-oxoquinoline derivatives have been made. They included
inexpensive nitration reagent system for regioselective nitration,
longer reaction time for complete transesterification, modified
synthetic approaches with moderate to excellent chemical yields,
and synthesis of new 2-oxoquinoline derivatives 6d, 6f, and 7a–c.
The unsuccessful attempt to prepare 2-oxoquinoline derivatives
using the literature method22 has resulted in the synthesis of new
2-chloroquinoline derivatives. As indicated in Scheme 2, acid 5 was
reacted with SOCl2 in toluene22 to give a minor product of 2-oxo-
quinoline acid chloride, and the major product was 2-chloroquino-
line acid chloride 8. The direct reaction of acid 5 with SOCl2
without solvent toluene easily formed 8 in 99% yield. Compound
8 was reacted with amines to give 2-chloroquinoline derivatives
9a–g in 87–93% yield. Likewise, desmethylation of 9a–c with LiCl
in DMF yielded phenolic hydroxyl precursors 10a–c in 78–83%
yield.
Compounds 7a–c and 6e are the precursors for positron emit-
ting radionuclide carbon-11 labeling at 7-methoxy position and
methoxyphenyl position to prepare 2-oxoquinoline carbon-11
radioligands, and the corresponding reference standards are 6a–c
and 6d. Compounds 10a–c and 9e are the precursors for carbon-
11 labeling at 7-methoxy position and methoxyphenyl position
to prepare 2-chloroquinoline carbon-11 radioligands, and the cor-
responding reference standards are 9a–c and 9d. Compounds 6g
and 9g, and 6f and 9f can be used as nitro-precursors and fluoro-
standards for another positron emitting radionuclide fluorine-18
labeling to prepare corresponding 2-oxoquinoline and 2-chloro-
quinoline fluorine-18 radioligands. Although the nitrobenzene
agent,
(benzotriazol-1-yloxy)tris(dimethylamino)phosphonium
hexafluorophosphate (BOP), and N,N-diisopropylethylamine (DIEA)
to produce amides 6a–g in 88–93% yield. Different coupling re-
agents including BOP, 1-[3-(dimethylamino)propyl]3-ethylcarbo-
diimide (EDC) and N,N0-dicyclohexylcarbodiimide (DCC) were
tested in different coupling reactions, and BOP was identified as
the best coupling reagent. This modification significantly improved
the yields and simplified the work-up procedures. Following the
literature procedure,22 we have converted 5 into corresponding
acid chloride with SOCl2, which was then reacted with amines in
the presence of triethylamine, but we obtained 2-oxoquinoline
CHO
CHO
CHO
HNO3
Bromopentane
EtOAc
K2CO3, DMF
H3CO
NO2
H3CO
H3CO
NO2
OC5H11
2
OH
1
OH
Fe-powder, HCl
EtOH, HOAc, H2O
O
O
CHO
O
dimethyl malonate
piperidine, HOAc, EtOH
H3CO
2 M HCl, EtOH
H3CO
N
H
NH2
OC5H11
OC5H11
4
3
O
X
O
O
RNH2, BOP
DIEA, DMF
OH
N
n
H
H3CO
N
H
O
6a, n=1, X=H
6b, n=1, X=Cl
6c, n=2, X=H
H3CO
N
OC5H11
H
OC5H11
5
6d, n=1, X=OCH3
6e, n=1, X=OH
6f, n=1, X=F
LiCl
DMF
6g, n=1, X=NO2
X
O
N
H
n
HO
N
O
7a, n=1, X=H
7b, n=1, X=Cl
7c, n=2, X=H
H
OC5H11
Scheme 1. Synthesis of 2-oxoquinoline derivative precursors and standards.

M. Gao et al. / Bioorg. Med. Chem. 18 (2010) 2099–2106
2101
O
O
O
RNH2, Et3N
CH2Cl2
OH
Cl
SOCl2
H3CO
N
H3CO
N
Cl
H
OC5H11
OC5H11
5
8
X
X
O
O
N
H
n
N
H
n
LiCl, DMF
HO
N
Cl
10a, n=1, X=H
10b, n=1, X=Cl
10c, n=2, X=H
H3CO
N
OC5H11
Cl
9a, n=1, X=H
9b, n=1, X=Cl
9c, n=2, X=H
OC5H11
9d, n=1, X=OCH3
9e, n=1, X=OH
9f, n=1, X=F
9g, n=1, X=NO2
Scheme 2. Synthesis of 2-chloroquinoline derivative precursors and standards.
group is not activated, it is possible to substitute the nitro group by
fluorine-18. Recently an effective nucleophilic aromatic [18F]fluori-
nation of nitrobenzene without activating (electron-withdrawing)
groups has been developed by using the fluorine-18 labeling re-
agent Cs[18F]F or TBA[18F]F in TBAOMs solvent instead of K[18F]F/
Kryptofix2.2.2 in acetonitrile.24
which would not stick to the C-18 Sep-Pak. The cartridge was
washed with water to remove non-reacted [11C]CH3OTf, phenolic
hydroxyl precursor and reaction solvent, and then the final labeled
product was eluted with ethanol. Overall synthesis time was
15–20 min from EOB. SPE technique is fast, efficient and convenient
and works very well for the O-methylated tracer purification in this
case using a free phenolic hydroxyl group in the precursor for radio-
labeling.27 The radiosynthesis was performed in an automated mul-
ti-purpose 11C-radiosynthesis module, allowing measurement of
specific activity during synthesis.28,29 The specific activity was esti-
2.2. Radiochemistry
Synthesis of carbon-11 labeled 2-oxoquinoline derivatives [11C]
6a–c and [11C]6d is indicated in Scheme 3. Phenolic hydroxylprecur-
sors 7a–c and 6e were labeled by a reactive [11C]methylating agent,
mated in a range of 111–185 GBq/lmol at the end of synthesis (EOS)
based on other radiotracers produced using the same targetry condi-
tions in our PET chemistry facility which have been measured by the
on-the-fly technique.29,30 The specific activity is dependent on the
production of [11C]CO2. The cyclotron we used was Siemens radio-
nuclide delivery system (Eclipse RDS-111). The typical proton-beam
current and irradiation time for the production of [11C]CO2 were
[
11C]methyl triflate ([11C]CH3OTf)25,26 prepared from [11C]CO2, un-
der basic conditions (2 N NaOH) in acetonitrile through the O-
[
11C]methylation and isolated by a simplified solid-phase extraction
(SPE) method27 to provide target tracers [11C]6a–d in 40–50% radio-
chemical yields, decay corrected to end of bombardment (EOB),
based on [11C]CO2. The large polarity difference between the sodium
salt of the phenolic hydroxyl precursor and the labeled O-methyl-
ated ether product permitted the use of SPE technique for purifica-
tion of the labeled product from the radiolabeling reaction
mixture. Either a C-18 Plus Sep-Pak cartridge or a semi-prep C-18
guard cartridge column was used in SPE purification technique.
The crude reaction mixture was treated with sodium bicarbonate
and loaded onto the cartridge by gas pressure. Any non-reacted pre-
cursor was actually converted into the corresponding sodium salt,
55
l
A ꢀ 30 min. The actual measurement of specific activity at
EOS by analytical HPLC31 is in agreement with this estimation.
Chemical purity and radiochemical purity were determined by
analytical HPLC.31 The chemical purity of the precursors and
reference standards was >96%. The radiochemical purity of the
target tracers was >99% determined by radio-HPLC through
c-ray (PIN diode) flow detector, and the chemical purity of the
target tracers was >95% determined by reversed-phase HPLC
through UV flow detector.
X
X
O
O
[
11C]CH3OTf, 2 N NaOH
N
H
n
CH3CN
N
H
n
HO
N
O
H311CO
7a, n=1, X=H
7b, n=1, X=Cl
7c, n=2, X=H
11C]6a, n=1, X=H
11C]6b, n=1, X=Cl
11C]6c, n=2, X=H
N
O
[
[
[
H
H
OC5H11
OC5H11
OH
O
11CH3
O
O
O
[
11C]CH3OTf, 2 N NaOH
CH3CN
N
H
n
N
n
H
H3CO
N
O
H3CO
N
H
H
6e, n=1
OC5H11
11C]6d, n=1
OC5H11
[
Scheme 3. Synthesis of carbon-11-labeled 2-oxoquinoline derivatives.

2102
M. Gao et al. / Bioorg. Med. Chem. 18 (2010) 2099–2106
Synthesis of carbon-11 labeled 2-chloroquinoline derivatives
say.17 The biological data are summarized in Table 1. These
in vitro data indicate some interesting information about the struc-
ture–activity relationship (SAR). Overall, 2-oxoquinoline deriva-
tives (nM Ki values) are superior to 2-chloroquinoline derivatives
[
11C]9a–c and [11C]9d is indicated in Scheme 4, according to the
same procedure for preparation of [11C]6a–c and [11C]6d. The de-
cay corrected radiochemical yields were 40–50%. The specific
activity was in a range of 111–185 GBq/lmol at EOS. The chemical
(lM Ki values for compounds 9b, 9c, and 9d). In comparison with
purity of the precursors and reference standards was >97%. The
radiochemical purity of the target tracers was >99%, and the chem-
ical purity of the target tracers was >95%.
the data of compounds 6a, 6c, 9a, and 9c, the shorter chain length
is better for high affinity. Compared with the data of compounds
6b, 6f, 9b, and 9f, the fluoro-containing compounds are better for
high affinity and selectivity than the chloro-containing com-
pounds. The biological data suggest that compounds 6f (Ki value
of 5.4 nM for CB2 and CB1/CB2 ratio of Ki > 1851.8), 6b (Ki value
of 9.37 nM for CB2 and CB1/CB2 ratio of Ki > 1067.2) and 9f (Ki va-
lue of 23.80 nM for CB2 and CB1/CB2 ratio of Ki > 120.79) are the
best candidates for in vivo imaging in terms of their affinity and
selectivity, since they are potent (nM Ki values) and selective (at
least 100–2000-fold)22 CB2 ligands.
2.3. Lipophilicity
The measured HPLC lipophilicity coefficient (Log P) is an impor-
tant parameter in selecting PET ligand candidates for further eval-
uations. We calculated Log P values of compounds 6a–d, 6f and
9a–d, 9f based on their retention times that were measured by
C-18 HPLC method.32,33 Retention times in the analytical HPLC sys-
tem for compounds 6a–d, 6f and 9a–d, 9f were 5.90, 7.77, 7.16,
5.32, 6.48, 5.45, 5.60, 6.25, 3.62, and 5.49 min, respectively. The
calculation results showed the Log P values for compounds 6a–d,
6f and 9a–d, 9f were 2.68, 3.04, 2.94, 2.53, 2.81, 2.57, 2.60, 2.76,
1.88, and 2.58, respectively.
3. Materials and methods
3.1. General
All commercial reagents and solvents from Sigma–Aldrich and
Fisher were used without further purification. [11C]CH3OTf was
prepared according to a literature procedure.25 Melting points
were determined on a MEL-TEMP II capillary tube apparatus and
were uncorrected. 1H NMR spectra were recorded on Varian
2.4. In vitro binding studies
The CB1 and CB2 receptors activities of the compounds 6a–d, 6f
and 9a–d, 9f were determined by using [3H]CP55940 binding as-
X
X
O
O
[
11C]CH3OTf, 2 N NaOH
CH3CN
N
n
N
H
n
H
H311CO
11C]9a, n=1, X=H
11C]9b, n=1, X=Cl
11C]9c, n=2, X=H
N
Cl
HO
N
Cl
[
[
[
10a, n=1, X=H
10b, n=1, X=Cl
10c, n=2, X=H
OC5H11
OC5H11
O11CH3
OH
O
O
[
11C]CH3OTf, 2 N NaOH
CH3CN
N
n
N
H
n
H
H3CO
N
Cl
H3CO
N
Cl
[
11C]9d, n=1
OC5H11
9e, n=1
OC5H11
Scheme 4. Synthesis of carbon-11-labeled 2-chlorooquinoline derivatives.
Table 1
CB1 and CB2 binding affinity and selectivity of quinoline derivatives
Compounds
CB1
Secondary binding Kib (nM)
CB2
Secondary binding Kib (nM)
CB1/CB2 ratio of Kic
Primary bindinga (%)
Primary bindinga (%)
6a
6b
6c
6d
6f
9a
9b
9c
9d
9f
63.4
ꢁ9.0
51.4
68.7
26.3
73.7
34.4
66.3
71.0
64.8
196.0 15
—
323.0 17
733.0 47
—
>10,000
—
>10,000
>10,000
2875 220
102.6
102.0
104.1
102.0
100.6
86.3
74.1
59.2
65.9
103.6
6.92 0.14
9.37 0.64
28.32
>1067.2
31.29
10.32 0.46
27.95 1.18
5.4 0.22
496.7 27.66
1008.0 107.21
1068.0 114.18
1330.0 135.33
23.80 1.24
26.22
>1851.8
>20.13
>9.92
>9.36
>7.51
120.79
a
For primary assays, the PDSP has a low threshold for ‘hits’ at primary assays. We perform a large number of secondary assays using a relatively low threshold in order not
to miss potential high-affinity compounds. The likelihood of an actual, high-affinity, ‘hit’ is still very low. Thus, one should not over-interpret results from primary assays.
Data represent mean% inhibition (N = 4 determinations) for compound tested at receptor subtypes. Significant inhibition is considered >50%. In cases where negative
inhibition (ꢁ) is seen, this represents a stimulation of binding. Occasionally, compounds at high concentrations will non-specifically increase binding. The default concen-
tration for primary binding experiments is 10 lM.
b
For secondary assays, data represent Ki (nM) values obtained from non-linear regression of radioligand competition binding isotherms. Ki values are calculated from best
fit IC50 values using the Cheng–Prusoff equation.
c
CB1 Ki values of compounds 6b, 6f, and 9b be regarded as >10,000.

M. Gao et al. / Bioorg. Med. Chem. 18 (2010) 2099–2106
2103
Gemini 2000 200 MHz FT-NMR and Bruker Avance II 500 MHz
NMR spectrometers using tetramethylsilane (TMS) as an internal
standard. Chemical shift data for the proton resonances were re-
ported in parts per million (ppm, d scale) relative to internal stan-
dard TMS (d 0.0), and coupling constants (J) were reported in hertz
(Hz). Low resolution mass spectra (LRMS) were obtained using a
Bruker Biflex III MALDI-Tof mass spectrometer, and high resolution
mass spectra (HRMS) were obtained using a Thermo MAT 95XP-
Trap spectrometer. Chromatographic solvent proportions are indi-
cated as volume: volume ratio. Thin-layer chromatography (TLC)
was run using Analtech Silica Gel GF uniplates (5 ꢀ 10 cm2). Plates
were visualized under UV light. Preparative TLC was run using
Analtech Silica Gel UV 254 plates (20 ꢀ 20 cm2). Normal phase
flash column chromatography was carried out on EM Science Silica
Gel 60 (230–400 mesh) with a forced flow of the indicated solvent
system in the proportions described below. All moisture- and/or
air-sensitive reactions were performed under a positive pressure
of nitrogen maintained by a direct line from a nitrogen source.
Analytical HPLC was performed using a Prodigy (Phenomenex)
Celite 521, and extracted with EtOAc. The organic layer was
washed with saturated NaHCO3 and brine, dried over Na2SO4,
and then purified by column chromatography (20% EtOAc/hex-
anes) to produce 3 (7.54 g, 85%) as a yellow oil, Rf = 0.50 (1:3
EtOAc/hexanes). 1H NMR (CDCl3): d 0.93 (t, J = 7.0 Hz, 3H, CH3),
1.33–1.48 (m, 4H, CH2CH2), 1.70–1.84 (m, 2H, CH2), 3.89 (s, 3H,
OCH3), 3.93 (t, J = 6.6 Hz, 2H, OCH2), 6.27 (s, 2H, NH2), 6.34 (d,
J = 8.8 Hz, 1H, Ph-H), 7.18 (d, J = 8.8 Hz, 1H, Ph-H), 9.73 (s, 1H,
CHO). MS (ESI): 238 ([M+H]+, 100%).
3.5. 7-Methoxy-2-oxo-8-pentyloxy-1,2-dihydroquinoline-3-
carboxylic acid ethyl ester (4)
Dimethyl malonate (9.74 g, 73.7 mmol), piperidine (6.28 g,
73.7 mmol), and acetic acid (0.1 mL) were added to a solution of
3 (7.0 g, 29.5 mmol) in EtOH (80 mL). The reaction mixture was re-
fluxed under N2 for 48 h. Subsequently the reaction mixture was
cooled to rt, treated with water (50 mL) and extracted with EtOAc
(3 ꢀ 80 mL). The combined organic layers were washed with water
and brine, dried over Na2SO4, and purified by column chromatog-
raphy (20% EtOAc/hexanes) to obtain 4 (8.45 g, 86%) as a colorless
solid, mp 116–118 °C, Rf = 0.31 (1:1 EtOAc/hexanes). 1H NMR
(CDCl3): d 0.93 (t, J = 7.2 Hz, 3H, CH3), 1.37–1.44 (m, 7H, CH2CH2
and CH3), 1.73–1.83 (m, 2H, CH2), 3.96 (s, 3H, OCH3), 4.10 (t,
J = 6.8 Hz, 2H, OCH2), 4.38 (q, J = 7.2 Hz, 2H, OCH2CH3), 6.85 (d,
J = 8.8 Hz, 1H, Ph-H), 7.32 (d, J = 8.8 Hz, 1H, Ph-H), 8.44 (s, 1H,
CH@), 9.01 (s, 1H, CONH). MS (ESI): 234 ([M+H]+, 100%).
5
l
m C-18 column, 4.6 ꢀ 250 mm; 3:1:1 CH3CN:MeOH: 20 mM,
pH 6.7 phosphate (buffer solution) mobile phase; flow rate
1.5 mL/min; and UV (254 nm) and -ray (PIN diode) flow detectors.
c
C-18 Plus Sep-Pak cartridges (WAT020515) were obtained from
Waters Corporate Headquarters, Milford, MA. Semi-prep C-18
guard cartridge column 1 ꢀ 1 cm was obtained from E. S. Indus-
tries, Berlin, NJ, and part number 300121-C18-BD 10 lm. Sterile
Millex-GS 0.22 lm vented filter unit was obtained from Millipore
Corporation, Bedford, MA.
3.6. 7-Methoxy-2-oxo-8-pentyloxy-1,2-dihydroquinoline-3-
carboxylic acid (5)
3.2. 3-Hydroxy-4-methoxy-2-nitrobenzaldehyde (1)
To a solution of isovanillin (50.0 g, 0.33 mol) in EtOAc (800 mL)
was slowly added nitric acid (28.8 g, 90%, 0.41 mol) at 0 °C, and the
mixture was stirred at room temperature (rt) for 2.5 h. Then the
reaction mixture was added additional EtOAc (600 mL), washed
with water, saturated NaHCO3 and brine, and dried over Na2SO4.
The organic layer was concentrated under reduced pressure, and
the crude product was purified by column chromatography (1:1
EtOAc/hexanes) to give 1 (40.1 g, 62%) as a yellow solid, mp 147–
148 °C, Rf = 0.40 (1:1 EtOAc/hexanes). 1H NMR (DMSO-d6): d 3.99
(s, 3H, OCH3), 7.32 (d, J = 8.5 Hz, 1H, Ph-H), 7.56 (d, J = 8.5 Hz, 1H,
Ph-H), 9.76 (s, 1H, CHO), 10.77 (s, 1H, OH). MS (ESI): 198
([M+H]+, 100%).
HCl (2 N, 125 mL) was added to a solution of 4 (6.3 g,
18.9 mmol) in EtOH (180 mL). The mixture was stirred at 70 °C
for 12 h, cooled to rt, and filtered. The solid was dried in air to af-
ford pure 5 (4.85 g, 84%) as a white solid, mp 181–182 °C, Rf = 0.14
(1:19 MeOH/CH2Cl2). 1H NMR (CDCl3): d 0.90 (t, J = 7.2 Hz, 3H,
CH3), 1.31–1.42 (m, 4H, CH2CH2), 1.74–1.83 (m, 2H, CH2), 3.96 (s,
3H, OCH3), 3.99 (t, J = 6.8 Hz, 2H, OCH2), 7.24 (d, J = 8.8 Hz, 1H,
Ph-H), 7.78 (d, J = 8.8 Hz, 1H, Ph-H), 8.89 (s, 1H, CH@), 12.3 (s,
1H). MS (ESI): 306 ([M+H]+, 100%).
3.7. General procedure for preparation of compound 6
BOP (265 mg, 0.6 mmol) and DIEA (155 mg, 1.2 mmol) were
added to the solution of 5 (153 mg, 0.5 mmol) and an amine
(0.5 mmol) in anhydrous DMF (20 mL). The reaction mixture was
stirred at rt under N2 for 15 h. Water (30 mL) was added to the
mixture, and the mixture was extracted with dichloromethane
(3 ꢀ 60 mL), washed with brine, dried over Na2SO4, and then puri-
fied by column chromatography (40% EtOAc/hexanes) to give 6 as a
white solid in 88–93% yield. Rf = 0.36–0.47 (1:1 EtOAc/hexanes).
3.3. 4-Methoxy-2-nitro-3-pentyloxybenzaldehyde (2)
1-Bromopentane (34.5 g, 228.4 mol) was added dropwise to a
solution of 1 (15.0 g, 76.1 mmol) and K2CO3 (31.55 g, 228.3 mmol)
in anhydrous DMF (200 mL). The reaction mixture was stirred un-
der N2 at 100 °C for 3 h, and then followed by filtration. The solid
residue was washed with additional DMF. The combined organic
layers were washed with water, extracted with EtOAc, washed
with brine, dried over MgSO4, and then purified by column chro-
matography (30% EtOAc/hexanes) to afford 2 (16.3 g, 80%) as a
brown solid, mp 26–27 °C, Rf = 0.57 (1:1 EtOAc/hexanes). 1H NMR
(CDCl3): d 0.92 (t, J = 7.0 Hz, 3H, CH3), 1.34–1.41 (m, 4H, CH2CH2),
1.66–1.76 (m, 2H, CH2), 3.99 (s, 3H, OCH3), 4.10 (t, J = 6.6 Hz, 2H,
OCH2), 7.08 (d, J = 8.4 Hz, 1H, Ph-H), 7.61 (d, J = 8.6 Hz, 1H, Ph-H),
9.78 (s, 1H, CHO). MS (ESI): 268 ([M+H]+, 70%), 198 (100%).
3.7.1. 7-Methoxy-2-oxo-8-pentyloxy-1,2-dihydroquinoline-3-
carboxylic acid (2-phenylethyl)amide (6a)
Mp 116–118 °C. 1H NMR (CDCl3): d 0.94 (t, J = 7.2 Hz, 3H, CH3),
1.37–1.46 (m, 4H, CH2CH2), 1.74–1.84 (m, 2H, CH2), 2.95 (t,
J = 7.2 Hz, CH2), 3.73 (dd, J = 7.0, 13.5 Hz, 2H, NCH2), 3.97 (s, 3H,
OCH3), 4.12 (t, J = 7.2 Hz, 2H, OCH2), 6.92 (d, J = 8.8 Hz, 1H, Ph-H),
7.21–7.24 (m, 1H, Ph-H), 7.27–7.39 (m, 4H, Ph-H), 7.44 (d,
J = 8.8 Hz, 1H, Ph-H), 8.86 (s, 1H), 9.16 (s, 1H), 9.70 (s, 1H, CONH).
MS (ESI): 409 ([M+H]+, 100%).
3.4. 2-Amino-4-methoxy-3-pentyloxybenzaldehyde (3)
Iron powder (6.68 g, 119.7 mmol) and concentrated HCl (37%,
1.5 mL) were added to a solution of 2 (10.0 g, 37.4 mmol) in
EtOH/AcOH/H2O (96 mL/96 mL/48 mL). The reaction mixture was
refluxed for 30 min and then stirred at rt for 1 h, filtered through
3.7.2. 7-Methoxy-2-oxo-8-pentyloxy-1,2-dihydroquinoline-3-
carboxylic acid [2-(4-chlorophenyl)ethyl]amide (6b)
Mp 137–139 °C. 1H NMR (CDCl3): d 0.94 (t, J = 7.0 Hz, 3H, CH3),
1.34–1.48 (m, 4H, CH2CH2), 1.78–1.84 (m, 2H, CH2), 2.92

2104
M. Gao et al. / Bioorg. Med. Chem. 18 (2010) 2099–2106
(t, J = 7.0 Hz, CH2), 3.69 (dd, J = 7.0, 13.6 Hz, 2H, NCH2), 3.97 (s, 3H,
OCH3), 4.13 (t, J = 6.8 Hz, 2H, OCH2), 6.91 (d, J = 8.8 Hz, 1H, Ph-H),
7.17–7.30 (m, 4H, Ph-H), 7.43 (d, J = 8.8 Hz, 1H, Ph-H), 8.86 (s,
1H), 9.15 (s, 1H), 9.68 (s, 1H, CONH). MS (ESI): 443 ([M+H]+, 100%).
chromatography (40% EtOAc/hexanes) to give 7a–c as a white solid
in 80–83% yield. Rf = 0.30–0.34 (1:1 EtOAc/hexanes).
3.8.1. 7-Hydroxy-2-oxo-8-pentyloxy-1,2-dihydroquinoline-3-
carboxylic acid (2-phenylethyl)amide (7a)
Mp 99–101 °C. 1H NMR (CDCl3): d 0.92 (t, J = 6.8 Hz, 3H, CH3),
1.32–1.46 (m, 4H, CH2CH2), 1.75–1.87 (m, 2H, CH2), 2.95 (t,
J = 7.2 Hz, CH2), 3.73 (dd, J = 7.0, 13.5 Hz, 2H, NCH2), 4.07 (t,
J = 7.0 Hz, 2H, OCH2), 6.94 (d, J = 8.8 Hz, 1H, Ph-H), 7.21–7.38 (m,
6H, Ph-H), 7.62 (s, 1H, OH), 8.84 (s, 1H, CH@), 9.11 (s, 1H, CONH),
9.78 (s, 1H, CONH). MS (ESI): 395 ([M+H]+, 100%). HRMS (ESI) calcd
for C23H26N2O4H ([M+H]+), 395.1971; found 395.1955.
3.7.3. 7-Methoxy-2-oxo-8-pentyloxy-1,2-dihydroquinoline-3-
carboxylic acid (3-phenylpropyl)amide (6c)
Mp 84–86 °C. 1H NMR (CDCl3): d 0.94 (t, J = 6.8 Hz, 3H, CH3),
1.34–1.48 (m, 4H, CH2CH2), 1.75–1.85 (m, 2H, CH2), 1.90–2.05
(m, 2H, CH2), 2.73 (t, J = 7.8 Hz, CH2), 3.48 (dd, J = 7.0, 13.0 Hz,
2H, NCH2), 3.97 (s, 3H, OCH3), 4.13 (t, J = 6.8 Hz, 2H, OCH2), 6.90
(d, J = 8.8 Hz, 1H, Ph-H), 7.13–7.32 (m, 5H, Ph-H), 7.42 (d,
J = 8.8 Hz, 1H, Ph-H), 8.86 (s, 1H), 9.15 (s, 1H), 9.68 (s, 1H, CONH).
MS (ESI): 423 ([M+H]+, 100%).
3.8.2. 7-Hydroxy-2-oxo-8-pentyloxy-1,2-dihydroquinoline-3-
carboxylic acid [2-(4-chlorophenyl)ethyl]amide (7b)
Mp 121–123 °C. 1H NMR (CDCl3): d 0.92 (t, J = 6.8 Hz, 3H, CH3),
1.32–1.46 (m, 4H, CH2CH2), 1.76–1.87 (m, 2H, CH2), 2.88–2.97 (m,
2H, CH2), 3.70 (dd, J = 6.8, 13.2 Hz, 2H, NCH2), 4.09 (t, J = 7.0 Hz, 2H,
OCH2), 6.94 (d, J = 8.8 Hz, 1H, Ph-H), 7.16 (d, J = 8.6 Hz, 2H, Ph-H),
7.24 (d, J = 8.6 Hz, 2H, Ph-H), 7.33 (d, J = 8.8 Hz, 1H, Ph-H), 7.93
(s, 1H, OH), 8.83 (s, 1H, CH@), 9.15 (s, 1H, CONH), 9.79 (t,
J = 5.6 Hz, 1H, CONH). MS (ESI): 429 ([M+H]+, 100%). HRMS (ESI)
calcd for C23H25N2O4ClH ([M+H]+), 429.1581; found 429.1583.
3.7.4. 7-Methoxy-2-oxo-8-pentyloxy-1,2-dihydroquinoline-3-
carboxylic acid [2-(4-methoxyphenyl)ethyl]amide (6d)
Mp 131–133 °C. 1H NMR (CDCl3): d 0.94 (t, J = 6.8 Hz, 3H, CH3),
1.35–1.48 (m, 4H, CH2CH2), 1.75–1.85 (m, 2H, CH2), 2.89 (t,
J = 7.0 Hz, CH2), 3.68 (dd, J = 7.0, 13.2 Hz, 2H, NCH2), 3.78 (s, 3H,
OCH3), 3.97 (s, 3H, OCH3), 4.12 (t, J = 6.8 Hz, 2H, OCH2), 6.83 (d,
J = 8.4 Hz, 2H, Ph-H), 6.90 (d, J = 8.8 Hz, 1H, Ph-H), 7.17 (d,
J = 8.4 Hz, 2H, Ph-H), 7.42 (d, J = 8.8 Hz, 1H, Ph-H), 8.86 (s, 1H),
9.14 (s, 1H), 9.67 (s, 1H, CONH). MS (ESI): 439 ([M+H]+, 100%).
HRMS (ESI) calcd for C25H30N2O5H ([M+H]+), 439.2233; found
439.2229.
3.8.3. 7-Hydroxy-2-oxo-8-pentyloxy-1,2-dihydroquinoline-3-
carboxylic acid (3-phenylpropyl)amide (7c)
Mp 128–130 °C. 1H NMR (CDCl3): d 0.92 (t, J = 6.8 Hz, 3H, CH3),
1.32–1.47 (m, 4H, CH2CH2), 1.77–1.87 (m, 2H, CH2), 1.91–2.05 (m,
2H, CH2), 2.73 (t, J = 7.4 Hz, 2H, CH2), 3.49 (dd, J = 6.8, 13.2 Hz, 2H,
NCH2), 4.10 (t, J = 7.0 Hz, 2H, OCH2), 6.95 (d, J = 8.6 Hz, 1H, Ph-H),
7.16–7.31 (m, 5H, Ph-H), 7.33 (d, J = 8.8 Hz, 1H, Ph-H), 7.76 (s,
1H, OH), 8.85 (s, 1H, CH@), 9.18 (s, 1H, CONH), 9.82 (t, J = 5.6 Hz,
1H, CONH). MS (ESI): 409 ([M+H]+, 100%); HRMS (ESI) calcd for
C24H28N2O4H ([M+H]+), 409.2127; found 409.2107.
3.7.5. 7-Methoxy-2-oxo-8-pentyloxy-1,2-dihydroquinoline-3-
carboxylic acid [2-(4-hydroxyphenyl)ethyl]amide (6e)
Mp 143–145 °C. 1H NMR (CDCl3): d 0.93 (t, J = 7.0 Hz, 3H, CH3),
1.35–1.48 (m, 4H, CH2CH2), 1.76–1.84 (m, 2H, CH2), 2.87 (t,
J = 6.6 Hz, CH2), 3.69 (m, 2H, NCH2), 3.97 (s, 3H, OCH3), 4.12 (t,
J = 6.8 Hz, 2H, OCH2), 6.78 (d, J = 8.2 Hz, 2H, Ph-H), 6.92 (d,
J = 8.8 Hz, 1H, Ph-H), 7.08 (d, J = 8.2 Hz, 2H, Ph-H), 7.43 (d,
J = 8.8 Hz, 1H, Ph-H), 8.89 (s, 1H), 9.27 (s, 1H), 9.79 (s, 1H, CONH).
MS (ESI): 425 ([M+H]+, 100%).
3.9. 2-Chloro-7-methoxy-8-(pentyloxy)quinoline-3-carbonyl
chloride (8)
3.7.6. 7-Methoxy-2-oxo-8-pentyloxy-1,2-dihydroquinoline-3-
carboxylic acid [2-(4-fluorophenyl)ethyl]amide (6f)
To 5 (3.05 g, 10 mmol) was added thionyl chloride (20 mL), and
the reaction mixture was refluxed for 3 h. The mixture was cooled
down to rt, and the excess thionyl chloride was evaporated. The
residue was added anhydrous toluene (20 mL), and evaporation
was repeated. The residue was dried under vacuum to afford 8
(3.38 g, 99%) as a yellow solid, mp: 44–46 °C. 1H NMR (CDCl3): d
0.94 (t, J = 7.0 Hz, 3H, CH3), 1.31–1.60 (m, 4H, CH2CH2), 1.79–1.93
(m, 2H, CH2), 4.06 (s, 3H, OCH3), 4.28 (t, J = 6.8 Hz, 2H, OCH2),
7.42 (d, J = 9.0 Hz, 1H, Ph-H), 7.69 (d, J = 9.0 Hz, 1H, Ph-H), 8.95
(s, 1H, CH@).
Mp 106–108 °C. 1H NMR (CDCl3): d 0.94 (t, J = 6.8 Hz, 3H, CH3),
1.34–1.48 (m, 4H, CH2CH2), 1.75–1.85 (m, 2H, CH2), 2.92 (t,
J = 7.0 Hz, CH2), 3.69 (dd, J = 6.8, 13.0 Hz, 2H, NCH2), 3.97 (s, 3H,
OCH3), 4.13 (t, J = 6.8 Hz, 2H, OCH2), 6.91–7.03 (m, 3H, Ph-H),
7.19–7.26 (m, 2H, Ph-H), 7.43 (d, J = 8.8 Hz, 1H, Ph-H), 8.86 (s,
1H), 9.14 (s, 1H), 9.66 (s, 1H, CONH). MS (ESI): 427 ([M+H]+,
100%). HRMS (ESI) calcd for C24H27N2O4FH ([M+H]+), 427.2033;
found 427.2017.
3.7.7. 7-Methoxy-2-oxo-8-pentyloxy-1,2-dihydroquinoline-3-
carboxylic acid [2-(4-nitrophenyl)ethyl]amide (6g)
3.10. General procedure for preparation of compound 9
Mp 170–172 °C. 1H NMR (CDCl3): d 0.94 (t, J = 6.8 Hz, 3H, CH3),
1.34–1.48 (m, 4H, CH2CH2), 1.74–1.84 (m, 2H, CH2), 3.06 (t,
J = 7.0 Hz, CH2), 3.75 (dd, J = 7.0, 13.2 Hz, 2H, NCH2), 3.97 (s, 3H,
OCH3), 4.13 (t, J = 6.8 Hz, 2H, OCH2), 6.92 (d, J = 8.8 Hz, 1H, Ph-H),
7.41–7.47 (m, 3H, Ph-H), 8.15 (d, J = 8.8 Hz, 2H, Ph-H), 8.84 (s,
1H), 9.12 (s, 1H), 9.73 (s, 1H, CONH). MS (ESI): 454 ([M+H]+, 100%).
The solution of 8 (0.34 g, 1.0 mmol) in dichloromethane (10 mL)
was added at 0 °C to a solution of an amine (1.0 mmol) and trieth-
ylamine (0.20 g, 2.0 mmol) in dichloromethane (20 mL). The reac-
tion mixture was allowed to warm up to rt and stirred for 2 h.
Saturated NaCl (30 mL) was added to the mixture, and the aqueous
layer was extracted with dichloromethane (3 ꢀ 30 mL). The com-
bined organic layers were washed with saturated NaCl, dried over
Na2SO4, and purified by column chromatography (30% EtOAc/hex-
anes) to obtain 9 as a white solid in 87–93% yield. Rf = 0.50–0.58
(1:1 EtOAc/hexanes).
3.8. General procedure for preparation of compound 7
Lithium chloride (0.34 g, 8.0 mmol) was added to a solution of
6a–c (1.0 mmol) in DMF (20 mL) under nitrogen, and then the
reaction mixture was refluxed for 18 h. The reaction mixture was
cooled down to rt, water (30 mL) was added. The mixture was ex-
tracted with EtOAc (3 ꢀ 50 mL), washed with brine, and dried over
MgSO4. The organic layer was evaporated, and purified by column
3.10.1. 2-Chloro-7-methoxy-8-pentyloxy-1-quinoline-3-
carboxylic acid (2-phenylethyl)amide (9a)
Mp 73–74 °C. 1H NMR (CDCl3): d 0.93 (t, J = 7.0 Hz, 3H, CH3),
1.33–1.56 (m, 4H, CH2CH2), 1.77–1.88 (m, 2H, CH2), 2.99

M. Gao et al. / Bioorg. Med. Chem. 18 (2010) 2099–2106
2105
(t, J = 7.2 Hz, 2H, CH2), 3.78 (dd, J = 6.6, 12.8 Hz, 2H, NCH2), 4.01 (s,
3H, OCH3), 4.25 (t, J = 6.6 Hz, 2H, OCH2), 6.66 (s, 1H, CONH), 7.21–
7.30 (m, 4H, Ph-H), 7.33 (d, J = 9.0 Hz, 2H, Ph-H), 7.54 (d, J = 9.0 Hz,
1H, Ph-H), 8.47 (s, 1H, CH@). MS (ESI): 427 ([M+H]+, 100%). HRMS
(ESI) calcd for C24H27N2O3ClH ([M+H]+), 427.1788; found 427.1772.
CH2), 3.12 (t, J = 7.0 Hz, 2H, CH2), 3.83 (dd, J = 7.0, 13.0 Hz, 2H,
NCH2), 4.03 (s, 3H, OCH3), 4.25 (t, J = 7.0 Hz, 2H, OCH2), 6.86 (s,
1H, CONH), 7.37 (d, J = 9.0 Hz, 1H, Ph-H), 7.45 (d, J = 8.5 Hz, 2H,
Ph-H), 7.56 (d, J = 9.0 Hz, 1H, Ph-H), 8.18 (d, J = 8.5 Hz, 2H, Ph-H),
8.48 (s, 1H, CH@). MS (ESI): 472 ([M+H]+, 100%). HRMS (ESI) calcd
for C24H26N3O5ClNa ([M+Na]+), 494.1459; found 494.1461.
3.10.2. 2-Chloro-7-methoxy-8-pentyloxy-1-quinoline-3-
carboxylic acid [2-(4-chlorophenyl)ethyl]amide (9b)
Mp 105–107 °C. 1H NMR (CDCl3): d 0.93 (t, J = 7.0 Hz, 3H, CH3),
1.30–1.56 (m, 4H, CH2CH2), 1.78–1.92 (m, 2H, CH2), 2.96 (t,
J = 7.0 Hz, 2H, CH2), 3.75 (dd, J = 6.8, 13.0 Hz, 2H, NCH2), 4.01 (s,
3H, OCH3), 4.25 (t, J = 6.8 Hz, 2H, OCH2), 6.69 (s, 1H, CONH),
7.18–7.32 (m, 4H, Ph-H), 7.34 (d, J = 9.0 Hz, 1H, Ph-H), 7.55 (d,
J = 9.0 Hz, 1H, Ph-H), 8.47 (s, 1H, CH@). MS (ESI): 461 ([M+H]+,
100%). HRMS (ESI) calcd for C24H26N2O3Cl2H ([M+H]+), 461.1399;
found 461.1377.
3.11. General procedure for preparation of compound 10
10a–c were prepared from 9a–c using the same procedure de-
scribed for 7a–c as a white solid in 78–83% yield. Rf = 0.72–0.78
(1:1 EtOAc/hexanes).
3.11.1. 2-Chloro-7-hydroxy-8-pentyloxy-1-quinoline-3-
carboxylic acid (2-phenylethyl)amide (10a)
Mp 55–57 °C. 1H NMR (CDCl3): d 0.93 (t, J = 6.8 Hz, 3H, CH3),
1.35–1.46 (m, 4H, CH2CH2), 1.77–1.87 (m, 2H, CH2), 2.99 (t,
J = 7.2 Hz, CH2), 3.78 (dd, J = 6.8, 12.6 Hz, 2H, NCH2), 4.46 (t,
J = 7.0 Hz, 2H, OCH2), 6.44 (s, 1H, OH), 6.63 (s, 1H, CONH), 7.24–
7.32 (m, 6H, Ph-H), 7.74 (d, J = 9.0 Hz, 1H, Ph-H), 8.45 (s, 1H,
CH@). MS (ESI): 413 ([M+H]+, 100%). HRMS (ESI) calcd for
C23H25N2O3ClH ([M+H]+), 413.1632; found 413.1623.
3.10.3. 2-Chloro-7-methoxy-8-pentyloxy-1-quinoline-3-
carboxylic acid (3-phenylpropyl)amide (9c)
Mp 85–87 °C. 1H NMR (CDCl3): d 0.93 (t, J = 7.0 Hz, 3H, CH3),
1.33–1.56 (m, 4H, CH2CH2), 1.78–1.92 (m, 2H, CH2), 1.98–2.05
(m, 2H, CH2), 2.77 (t, J = 7.6 Hz, 2H, CH2), 3.57 (dd, J = 7.0,
13.2 Hz, 2H, NCH2), 4.01 (s, 3H, OCH3), 4.26 (t, J = 6.8 Hz, 2H,
OCH2), 6.68 (s, 1H, CONH), 7.18–7.30 (m, 5H, Ph-H), 7.33 (d,
J = 9.0 Hz, 1H, Ph-H), 7.53 (d, J = 9.0 Hz, 1H, Ph-H), 8.44 (s, 1H,
CH@). MS (ESI): 463 ([M+Na]+, 100%). HRMS (ESI) calcd for
C25H29N2O3ClNa ([M+Na]+), 463.1765; found 463.1766.
3.11.2. 2-Chloro-7-hydroxy-8-pentyloxy-1-quinoline-3-
carboxylic acid [2-(4-chlorophenyl)ethyl]amide (10b)
Mp 106–108 °C. 1H NMR (CDCl3): d 0.95 (t, J = 7.2 Hz, 3H, CH3),
1.37–1.50 (m, 4H, CH2CH2), 1.81–1.87 (m, 2H, CH2), 2.97 (t,
J = 7.2 Hz, CH2), 3.76 (dd, J = 7.0, 13.0 Hz, 2H, NCH2), 4.46 (t,
J = 7.0 Hz, 2H, OCH2), 6.66 (s, 1H, CONH), 7.20 (d, J = 8.5 Hz, 2H,
Ph-H), 9.29 (dd, J = 1.5, 6.5 Hz, 2H, Ph-H), 7.32 (d, J = 8.5 Hz, 1H,
Ph-H), 7.50 (d, J = 8.5 Hz, 1H, Ph-H), 8.47 (s, 1H, CH@). MS (ESI):
447 ([M+H]+, 100%). HRMS (ESI) calcd for C23H24N2O3ClH
([M+H]+), 447.1242; found 447.1227.
3.10.4. 2-Chloro-7-methoxy-8-pentyloxy-1-quinoline-3-
carboxylic acid [2-(4-methoxyphenyl)ethyl]amide (9d)
Mp 83–84 °C. 1H NMR (CDCl3): d 0.95 (t, J = 7.0 Hz, 3H, CH3),
1.37–1.42 (m, 2H, CH2), 1.50–1.3 (m, 2H, CH2), 1.82–1.87 (m, 2H,
CH2), 2.94 (t, J = 7.0 Hz, 2H, CH2), 3.75 (dd, J = 7.0, 12.5 Hz, 2H,
NCH2), 3.80 (s, 3H, OCH3), 4.02 (s, 3H, OCH3), 4.26 (t, J = 7.0 Hz,
2H, OCH2), 6.73 (s, 1H, CONH), 6.87 (dd, J = 2.0, 6.5 Hz, 2H, Ph-H),
7.19 (dd, J = 2.0, 6.5 Hz, 2H, Ph-H), 7.35 (d, J = 9.0 Hz, 1H, Ph-H),
7.56 (d, J = 9.0 Hz, 1H, Ph-H), 8.46 (s, 1H, CH@). MS (ESI): 457
([M+H]+, 100%). HRMS (ESI) calcd for C25H29N2O4ClH ([M+H]+),
457.1894; found 457.1880.
3.11.3. 2-Chloro-7-hydroxy-8-pentyloxy-1-quinoline-3-
carboxylic acid (3-phenylpropyl)amide (10c)
Mp 127–129 °C. 1H NMR (CDCl3): d 0.93 (t, J = 7.0 Hz, 3H, CH3),
1.38–1.50 (m, 4H, CH2CH2), 1.77–1.91 (m, 2H, CH2), 1.97–2.03 (m,
2H, CH2), 2.76 (t, J = 7.6 Hz, CH2), 3.53 (dd, J = 7.0, 13.0 Hz, 2H,
NCH2), 4.47 (t, J = 7.0 Hz, 2H, OCH2), 6.49 (s, 1H, OH), 6.66 (s, 1H,
CONH), 7.15–7.32 (m, 6H, Ph-H), 7.74 (d, J = 8.8 Hz, 1H, Ph-H),
8.40 (s, 1H, CH@). MS (ESI): 449 ([M+Na]+, 100%). HRMS (ESI) calcd
for C24H27N2O3ClNa ([M+Na]+), 449.1608; found 449.1609.
3.10.5. 2-Chloro-7-methoxy-8-pentyloxy-1-quinoline-3-
carboxylic acid [2-(4-hydroxyphenyl)ethyl]amide (9e)
Mp 150–152 °C. 1H NMR (CDCl3): d 0.91 (t, J = 7.0 Hz, 3H, CH3),
1.36–1.40 (m, 2H, CH2), 1.47–1.51 (m, 2H, CH2), 1.81–1.87 (m, 2H,
CH2), 2.90 (t, J = 7.0 Hz, 2H, CH2), 3.74 (dd, J = 6.5, 12.5 Hz, 2H,
NCH2), 4.01 (s, 3H, OCH3), 4.25 (t, J = 7.0 Hz, 2H, OCH2), 6.74 (s,
1H, CONH), 6.78 (d, J = 8.5 Hz, 2H, Ph-H), 7.09 (d, J = 8.5 Hz, 2H,
Ph-H), 7.35 (d, J = 9.0 Hz, 1H, Ph-H), 7.55 (d, J = 9.0 Hz, 1H, Ph-H),
8.46 (s, 1H, CH@). MS (ESI): 443 ([M+H]+, 100%). HRMS (ESI) calcd
for C24H27N2O4ClNa ([M + Na]+), 465.1557; found 465.1550.
3.12. General procedure for preparation of target tracers
[
11C]CO2 was produced by the 14N(p, 11C nuclear reaction in
a)
small volume (9.5 cm3) aluminum gas target (CTI) from 11 MeV pro-
ton cyclotron on research purity nitrogen (+1%O2) ina Siemens radio-
nuclidedeliverysystem. The proton-beam current was55 lA, andthe
irradiation time was 30 min. The phenolic hydroxyl precursor 7a, 7b,
3.10.6. 2-Chloro-7-methoxy-8-pentyloxy-1-quinoline-3-
carboxylic acid [2-(4-fluorophenyl)ethyl]amide (9f)
7c, 6e, 10a, 10b, 10c, or 9e (0.1–0.3 mg) was dissolved in CH3CN
(300 lL). To this solution was added 2 N NaOH (2 lL). The mixture
Mp 69–71 °C. 1H NMR (CDCl3): d 0.93 (t, J = 7.0 Hz, 3H, CH3),
1.36–1.41 (m, 2H, CH2), 1.48–1.54 (m, 2H, CH2), 1.81–1.87 (m,
2H, CH2), 2.97 (t, J = 7.0 Hz, 2H, CH2), 3.76 (dd, J = 7.0, 13.0 Hz,
2H, NCH2), 4.01 (s, 3H, OCH3), 4.25 (t, J = 7.0 Hz, 2H, OCH2), 6.86
(s, 1H, CONH), 6.99–7.03 (m, 2H, Ph-H), 7.22–7.25 (m, 2H, Ph-H),
7.35 (d, J = 9.0 Hz, 1H, Ph-H), 7.56 (d, J = 9.0 Hz, 1H, Ph-H), 8.48
(s, 1H, CH@). MS (ESI): 445 ([M+H]+, 100%). HRMS (ESI) calcd for
C24H26N2O3FClH ([M+H]+), 445.1694; found 445.1673.
was transferred to a small reaction vial. No-carrier-added (high spe-
cific activity) [11C]CH3OTf that was produced by the gas-phase pro-
duction method25 from [11C]CO2 through [11C]CH4 and [11C]CH3Br
with silver triflate (AgOTf) column was passed into the reaction vial
at rt until radioactivity reached a maximum (ꢂ2 min), and then the
reaction vial was isolated and heated at 80 °C for 3 min. The contents
of the reaction vial were diluted with NaHCO3 (1 mL, 0.1 M). The reac-
tion tube was connected to either a C-18 Plus Sep-Pak cartridge or a
semi-prep C-18 guard cartridge column. The labeled product mixture
solution was passed onto the cartridge for SPE purification by gas
pressure. The cartridge was washed with H2O (2 ꢀ 3 mL), and the
aqueous washing was discarded. The product was eluted from the
column with EtOH (2 ꢀ 3 mL), and then passed onto a rotatory
3.10.7. 2-Chloro-7-methoxy-8-pentyloxy-1-quinoline-3-
carboxylic acid [2-(4-nitrophenyl)ethyl]amide (9g)
Mp 110–112 °C. 1H NMR (CDCl3): d 0.95 (t, J = 7.0 Hz, 3H, CH3),
1.37–1.43 (m, 2H, CH2), 1.49–1.55 (m, 2H, CH2), 1.83–1.88 (m, 2H,

2106
M. Gao et al. / Bioorg. Med. Chem. 18 (2010) 2099–2106
evaporator. The solvent was removed by evaporation under vacuum.
(NIMH PDSP). The NIMH PDSP is directed by Bryan L. Roth MD,
PhD at the University of North Carolina at Chapel Hill and Project
Officer Jamie Driscol at NIMH, Bethesda MD, USA. The referees’
criticisms and editor’s comments for the revision of the manuscript
are greatly appreciated.
The labeled product [11C]6a, [11C]6b, [11C]6c, [11C]6d, [11C]9a, [11C]9b,
[
11C]9c, or [11C]9d was formulated with saline, sterile-filtered
through a sterile vented Millex-GS 0.22 lm cellulose acetate mem-
brane and collected into a sterile vial. Total radioactivity was assayed
and the totalvolumewasnoted for tracerdosedispensing. The overall
synthesis time including SPE purification and formulation was 15–
20 min. The radiochemical yields decay corrected to EOB, from
References and notes
[
11C]CO2, were40–50%. Retentiontimes intheanalyticalHPLC system
1. Diaz, P.; Phatak, S. S.; Xu, J.; Astruc-Diaz, F.; Cavasotto, C. N.; Naguib, M. J. Med.
Chem. 2009, 52, 433.
2. Diaz, P.; Xu, J.; Astruc-Diaz, F.; Pan, H.-M.; Brown, D. L.; Naguib, M. J. Med. Chem.
2008, 51, 4932.
3. Muccioli, G.; Lambert, D. M. Expert Opin. Ther. Patents 2006, 16, 1405.
4. Cianchi, F.; Papucci, L.; Schiavone, N.; Lulli, M.; Magnelli, L.; Vinci, M. C.;
Messerini, L.; Manera, C.; Ronconi, E.; Romagnani, P.; Donnini, M.; Perigli, G.;
Trallori, G.; Tanganelli, E.; Capaccioli, S.; Masini, E. Clin. Cancer Res. 2008, 14,
7691.
5. Donohue, S. R.; Krushinski, J. H.; Pike, V. W.; Chernet, E.; Phebus, L.;
Chesterfield, A. K.; Felder, C. C.; Halldin, C.; Schaus, J. M. J. Med. Chem. 2008,
51, 5833.
were: tR 7a = 3.90 min, tR 6a = 5.90 min, tR
7b = 4.80 min, tR 6b = 7.77 min, tR
11C]6b = 7.77 min; tR 7c =
4.55 min, tR 6c = 7.16 min, tR
11C]6c = 7.16 min; tR 6e = 2.71 min, tR
6d = 5.32 min, tR
11C]6d = 5.32 min; tR 10a = 3.96 min, tR 9a =
5.45 min, tR
11C]9a = 5.45 min; tR 10b = 2.18 min, tR 9b = 5.60 min,
tR
[
11C]6a = 5.90 min; tR
[
[
[
[
[
11C]9b = 5.60 min; tR 10c = 4.50 min, tR 9c = 6.25 min, tR
[
[
11C]9c = 6.25 min; and tR 9e = 2.38 min, tR 9d = 3.62 min, tR
11C]9d = 3.62 min.
6. Donohue, S. R.; Pike, V. W.; Finnema, S. J.; Truong, P.; Andersson, J.; Gulyas, B.;
Halldin, C. J. Med. Chem. 2008, 51, 5608.
3.13. Radioligand binding assays
7. Nojiri, Y.; Ishiwata, K.; Qinggeletu; Tobiishi, S.; Sasada, T.; Yamamoto, F.;
Mukai, T.; Maeda, M. Biol. Pharm. Bull. 2008, 31, 1274.
Radioligand binding assays were performed by the National
Institute of Mental Health’s Psychoactive Drug Screening Program
(NIMH/PDSP). The receptors were CB1 and CB2. The radioligand
was [3H]CP55940. The reference was CP55940. The assay buffer
was cannabinoid binding buffer, 50 mM Tris–HCl, 1 mM EDTA,
3 mM MgCl2, 5 mg/mL fatty acid-free bovine serum albumin
(BSA), pH 7.4.
8. Terry, G.; Liow, J. S.; Chernet, E.; Zoghbi, S. S.; Phebus, L.; Felder, C. C.; Tauscher,
J.; Schaus, J. M.; Pike, V. W.; Halldin, C.; Innis, R. B. Neuroimage 2008, 41, 690.
9. Goffin, K.; Bormans, G.; Casteels, C.; Bosier, B.; Lambert, D. M.; Grachev, I. D.;
Van Paesschen, W.; Van Laere, K. Neuropharmacology 2008, 54, 1103.
10. Liu, P.; Lin, L. S.; Hamill, T. G.; Jewell, J. P.; Lanza, T. J., Jr.; Gibson, R. E.; Krause, S.
M.; Ryan, C.; Eng, W.; Sanabria, S.; Tong, X.; Wang, J.; Levorse, D. A.; Owens, K.
A.; Fong, T. M.; Shen, C. P.; Lao, J.; Kumar, S.; Yin, W.; Payack, J. F.; Springfield, S.
A.; Hargreaves, R.; Burns, H. D.; Goulet, M. T.; Hagmann, W. K. J. Med. Chem.
2007, 50, 3427.
11. Burns, H. D.; Van Laere, K.; Sanabria-Bohorquez, S.; Hamill, T. G.; Bormans, G.;
Eng, W. S.; Gibson, R.; Ryan, C.; Connolly, B.; Patel, S.; Krause, S.; Vanko, A.; Van
Hecken, A.; Dupont, P.; De Lepeleire, I.; Rothenberg, P.; Stoch, S. A.; Cote, J.;
Hagmann, W. K.; Jewell, J. P.; Lin, L. S.; Liu, P.; Goulet, M. T.; Gottesdiener, K.;
Wagner, J. A.; de Hoon, J.; Mortelmans, L.; Fong, T. M.; Hargreaves, R. J. Proc.
Natl. Acad. Sci. U.S.A. 2007, 104, 9800.
12. Yasuno, F.; Brown, A. K.; Zoghbi, S. S.; Krushinski, J. H.; Chernet, E.; Tauscher, J.;
Schaus, J. M.; Phebus, L. A.; Chesterfield, A. K.; Felder, C. C.; Gladding, R. L.;
Hong, J.; Halldin, C.; Pike, V. W.; Innis, R. B. Neuropsychopharmacology 2008, 33,
259.
13. Horti, A. G.; Fan, H.; Kuwabara, H.; Hilton, J.; Ravert, H. T.; Holt, D. P.;
Alexander, M.; Kumar, A.; Rahmim, A.; Scheffel, U.; Wong, D. F.; Dannals, R. F. J.
Nucl. Med. 2006, 47, 1689.
14. Willis, P. G.; Pavlova, O. A.; Chefer, S. I.; Vaupel, D. B.; Mukhin, A. G.; Horti, A. G.
J. Med. Chem. 2005, 48, 5813.
4. Conclusions
An efficient and convenient synthesis of new quinoline deriva-
tive radioligands, carbon-11-labeled 2-oxoquinoline and 2-chloro-
quinoline derivatives, has been well developed. The synthetic
methodology employed classical organic chemistry such as nitra-
tion, alkylation (Williamson ether synthesis), reduction, transeste-
rification, hydrolysis, coupling reaction, and desmethylation to
prepare a series of 2-oxoquinoline and 2-chloroquinoline deriva-
tive precursors and standard compounds. The target radioligands
were prepared by O-[11C]methylation of their corresponding phe-
nolic hydroxyl precursors using a reactive [11C]methylating agent,
15. Evens, N.; Bosier, B.; Lavey, B. J.; Kozlowski, J. A.; Vermaelen, P.; Baudemprez,
L.; Busson, R.; Lambert, D. M.; Van Laere, K.; Verbruggen, A. M.; Bormans, G. M.
Nucl. Med. Biol. 2008, 35, 793.
[
11C]CH3OTf, and isolated by a simplified SPE purification proce-
16. Bai, M.; Sexton, M.; Stella, N.; Bornhop, D. J. Bioconjug. Chem. 2008, 19, 988.
17. Evens, N.; Muccioli, G. G.; Houbrechts, N.; Lambert, D. M.; Verbruggen, A. M.;
Van Laere, K.; Bormans, G. M. Nucl. Med. Biol. 2009, 36, 455.
18. Sugiura, T.; Oka, S.; Gokoh, M.; Kishimoto, S.; Waku, K. J. Pharmacol. Sci. 2004,
96, 367.
19. Gutierrez, T.; Farthing, J. N.; Zyonok, A. M.; Makriyannis, A.; Hohmann, A. G. Br.
J. Pharmacol. 2007, 150, 153.
20. Herrera, B.; Carracedo, A.; Diez-Zaera, M.; Guzman, M.; Velasco, G. FEBS Lett.
2005, 579, 5084.
21. Blazquez, C.; Carracedo, A.; Barrado, L.; Real, P. J.; Fernandez-Luna, J. L.;
Velasco, G.; Malumbres, M.; Guzman, M. FASEB J. 2006, 20, 2633.
22. Raitio, K. H.; Savinainen, J. R.; Vepsalainen, J.; Laitinen, J. T.; Antti, P.; Jarvinen,
T.; Nevalainen, T. J. Med. Chem. 2006, 49, 2022.
dure in high radiochemical yields, short overall synthesis time,
and great specific radioactivities. In vitro radioligand binding as-
says indicated the compounds 6f, 6b, and 9f display potent
in vitro binding affinities with nanomolar Ki values and at least
100–2000-fold selectivity for CB2. These chemistry and biology re-
sults combined with the reported in vitro and in vivo biological
data17,22 encourage further in vivo biological evaluation of new
carbon-11-labeled 2-oxoquinoline and 2-chloroquinoline deriva-
tives as candidate PET radioligands for imaging of CB2 in cancer.
23. Inaba, T.; Kaya, T.; Iwamura, H. WO Patent 00/40562, 2000.
24. Seo, J. W.; Hong, E. P.; Lee, B. S.; Lee, S. J.; Oh, S. J.; Chi, D. Y. J. Labelled Compd.
Radiopharm. 2007, 50, S164.
25. Mock, B. H.; Mulholland, G. K.; Vavrek, M. T. Nucl. Med. Biol. 1999, 26, 467.
26. Jewett, D. M. Int. J. Rad. Appl. Instrum. A 1992, 43, 1383.
27. Gao, M.; Wang, M.; Hutchins, G. D.; Zheng, Q.-H. Appl. Radiat. Isot. 2008, 66,
1891.
28. Mock, B. H.; Zheng, Q.-H.; DeGrado, T. R. J. Labelled Compd. Radiopharm. 2005,
48, S225.
29. Mock, B. H.; Glick-Wilson, B. E.; Zheng, Q.-H.; DeGrado, T. R. J. Labelled Compd.
Radiopharm. 2005, 48, S224.
30. Zheng, Q.-H.; Gao, M.; Mock, B. H.; Wang, S.; Hara, T.; Nazih, R.; Miller, M. A.;
Receveur, T. J.; Lopshire, J. C.; Groh, W. J.; Zipes, D. P.; Hutchins, G. D.; DeGrado,
T. R. Bioorg. Med. Chem. Lett. 2007, 17, 2220.
31. Zheng, Q.-H.; Mock, B. H. Biomed. Chromatogr. 2005, 19, 671.
32. Fei, X.; Zheng, Q.-H. J. Liq. Chromatogr. Related Technol. 2005, 28, 939.
33. Gao, M.; Mock, B. H.; Hutchins, G. D.; Zheng, Q.-H. Nucl. Med. Biol. 2005, 32, 543.
Acknowledgments
This work was partially supported by the Breast Cancer Re-
search Foundation and Indiana Genomics Initiative (INGEN) of
Indiana University, which is supported in part by Lilly Endowment
Inc. The authors would like to thank Dr. Bruce H. Mock and Barbara
E. Glick-Wilson for their assistance in production of [11C]CH3OTf.
1H NMR spectra were recorded on a Bruker Avance II 500 MHz
NMR spectrometer in the Department of Chemistry and Chemical
Biology at Indiana University Purdue University Indianapolis (IU-
PUI), which is supported by a NSF-MRI Grant CHE-0619254. The
primary binding inhibition and secondary binding Ki data were
generously provided by the National Institute of Mental Health’s
Psychoactive Drug Screening Program, Contract # NO1MH32004