Fluorinated cannabinoid CB2 receptor ligands: Synthesis and in vitro binding characteristics of 2-oxoquinoline derivatives

Article abstract of DOI:10.1016/j.bmc.2011.07.062

Cannabinoid receptor 2 (CB2) plays an important role in human physiology and the pathophysiology of different diseases, including neuroinflammation, neurodegeneration, and cancer. Several classes of CB2 receptor ligands, including 2-oxoquinoline derivatives, have been previously reported. We report the synthesis and results of in vitro receptor binding of a focused library of new fluorinated 2-oxoquinoline CB2 ligands. Twelve compounds, 13-16 18, 19, 21-24, 27, and 28 were synthesized in good yields in multiple steps. Human U87 glioma cells expressing either hCB1 (control) or hCB2 were generated via lentiviral transduction. In vitro competitive binding assay was performed using [³H]CP-55,940 in U87hCB1 and U87hCB2 cells. Inhibition constant (K_i) values of compounds 13-16, 18, 19, 21-24, 27, and 28 for CB2 were >10,000, 2.8, 5.0, 2.4, 22, 0.8, 1.4, >10,000, 486, 58, 620, and 2400 nM, respectively, and those for CB1 were >10,000 nM. Preliminary in vitro results suggest that six of these compounds may be useful for therapy of neuropathic pain, neuroinflammatory diseases and immune disorders. In addition, compound 19, with its subnanomolar K_i value, could be radiolabeled with ¹⁸F and explored for PET imaging of CB2 expression.

Full text of DOI:10.1016/j.bmc.2011.07.062

Bioorganic & Medicinal Chemistry 19 (2011) 5698–5707
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Fluorinated cannabinoid CB2 receptor ligands: Synthesis and in vitro
binding characteristics of 2-oxoquinoline derivatives
Nashaat Turkman, Aleksander Shavrin, Roman A. Ivanov , Brian Rabinovich, Andrei Volgin,
⇑
Juri G. Gelovani, Mian M. Alauddin
Department of Experimental Diagnostic Imaging, The University of Texas, M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Cannabinoid receptor 2 (CB2) plays an important role in human physiology and the pathophysiology of
different diseases, including neuroinflammation, neurodegeneration, and cancer. Several classes of CB2
receptor ligands, including 2-oxoquinoline derivatives, have been previously reported. We report the syn-
thesis and results of in vitro receptor binding of a focused library of new fluorinated 2-oxoquinoline CB2
ligands. Twelve compounds, 13–16 18, 19, 21–24, 27, and 28 were synthesized in good yields in multiple
steps. Human U87 glioma cells expressing either hCB1 (control) or hCB2 were generated via lentiviral
transduction. In vitro competitive binding assay was performed using [3H]CP-55,940 in U87hCB1 and
U87hCB2 cells. Inhibition constant (Ki) values of compounds 13–16, 18, 19, 21–24, 27, and 28 for CB2
were >10,000, 2.8, 5.0, 2.4, 22, 0.8, 1.4, >10,000, 486, 58, 620, and 2400 nM, respectively, and those for
CB1 were >10,000 nM. Preliminary in vitro results suggest that six of these compounds may be useful
for therapy of neuropathic pain, neuroinflammatory diseases and immune disorders. In addition, com-
pound 19, with its subnanomolar Ki value, could be radiolabeled with 18F and explored for PET imaging
of CB2 expression.
Received 10 May 2011
Revised 28 June 2011
Accepted 4 July 2011
Available online 5 August 2011
Keywords:
Central nervous system
Cannabinoid receptor 2
Multiple sclerosis
Neuroinflammation
Neurodegeneration
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
tissue taken from a patient with Alzheimer’s disease. Microglia clus-
tering at b-amyloid plaques expressed both CB2 receptor proteins
The hemp plant, Cannabis sativa L., known as marijuana, has
been used for centuries as a therapeutic agent and recreational
drug.1,2 The active components of marijuana and their derivatives
are classified as cannabinoids.2 Two subtypes of the mammalian
cannabinoid receptors have been identified: the CB1 and CB2
receptors. The CB1 receptor is primarily located in the central ner-
vous system,3,4 and the CB2 receptor is expressed in cells of the
immune system, spleen, tonsils, and lymph nodes.3,5,6 The CB1
and CB2 receptors are both G-protein coupled receptors and are
involved in the inhibition of adenylate cyclase.
and fatty acid amine hydrolase.7 Ramirez et al.12 replicated this find-
ing and investigated the role of CB2 in the pathogenesis of a mouse
model of Alzheimer’s disease. CB2 receptor expression was also
found to be up-regulated in sub-populations of microglia in a rat
model of Huntington’s disease.13 CB2 receptors have been reported
to be over-expressed in several cancer cells, including breast can-
cer,14 skin cancer,15 prostate,16 and hepatocellular carcinoma.17
Experimental evidence has shown that cannabinoids inhibit the
growth of tumor xenografts in mice, tumor angiogenesis, and
directly induce apoptosis in neoplastic cells.14,15
CB2 is believed to be devoid of psychoactivity, and has significant
anti-inflammatory functions,7 and inflammation is known to be a
critical part of many types of neurodegenerative diseases.8 CB2
receptors have been implicated in a range of leukocyte functions,
and studies of CB2 receptors in leukocytes have shown results con-
sistent with an anti-inflammatory and immunosuppressive role.
This has also been supported by demonstrations that CB2 regulates
inflammation in a diverse range of animal models, including gastro-
intestinal,9 acute hindpaw,10 and pulmonary inflammation.11 CB2
expression in microglia in the brain was studied using human brain
The CB2 receptor is an important target for therapeutic immune
intervention, and, as such, research is currently focused on the
development of CB2 selective ligands. WIN-55,212-2 was the first
reported CB2 selective ligand, and it has been thoroughly investi-
gated.1 As a result, there are several different documented values
for the binding affinity of WIN-55,212-2 at both CB1 and CB2
receptors.18 While WIN-55,212-2 is extensively used for evaluating
the receptor binding of potential cannabinoid ligands, it is not very
useful pharmacologically as a selective ligand for either cannabi-
noid receptor because it has high affinity for both receptors.1,9
Subsequent to WIN-55,212-2, several other CB2 selective
ligands have been synthesized, including cannabimimetic indoles,
such as 1-(2,3-dichlorobenzoyl)-2-methyl-3-(2-[1-morpholino]et
hyl)-5-methoxyindole (L768242)19 and 1-propyl-2-methyl-3-
⇑
Corresponding author. Tel.: +1 713 563 4872; fax: +1 713 563 4894.
E-mail address: alauddin@mdanderson.org (M.M. Alauddin).
Present address: SiPix Imaging, Inc., Fremont, CA.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.07.062

N. Turkman et al. / Bioorg. Med. Chem. 19 (2011) 5698–5707
5699
(1-naphthoyl)indole (JWH-015).20 Indole L768242 has exception-
ally high selectivity for the CB2 receptor (146-fold) with signifi-
cantly high affinity at CB2 (inhibition constant [Ki] = 14 nM). The
Huffman group has synthesized a variety of CB2 selective 1-deoxy
receptor, and these compounds may be suitable for treatment of
neuropathic pain, inflammation, and immune disorders.
2. Results and discussion
2.1. Chemistry
and 1-methoxy-
D
8-THC analogues, which include 1-deoxy-
3-(10,10-dimethylbutyl)-
D
8-THC (JWH-133), one of the most highly
selective ligands for the CB2 receptor (Ki = 677 132 nM at CB1
and Ki = 3.4 1.0 nM at CB2).21 1-Methoxy-3-(10,10-dim-
ethylhexyl)-D8-THC (JWH-229) is another highly selective ligand,
The scheme for synthesis of compounds 13, 14, 15, and 16 is
shown in Figure 1 (Synthetic Scheme 1).
Compounds 2–12 were prepared following previously pub-
lished methods34,39,40 as shown in the synthetic Scheme 1 (Fig. 1)
with minor modifications. Compound 2 was obtained in 80% yield
and compounds 3 and 4 were obtained in 70% and 60% yields,
respectively. Reduction of 3 and 4 using iron (Fe) powder and
concd HCl produced compounds 5 and 6 in 80% and 83% yields,
respectively. Compounds 7 and 8 were obtained from 5 and 6,
respectively, in 80% yields. The ethyl esters 7 and 8 were hydro-
lyzed by 2 M HCl acid to produce 9 and 10 in 73% and 75% yields,
respectively. Intermediate compounds 2–10 were fully character-
ized by spectroscopic methods, and the 1H NMR spectra were con-
sistent with those reported previously.39 Detailed experimental
procedures for compounds 2–12 are presented in the Supplemen-
tary data.
Compounds 11 and 12 were prepared in situ and used without
isolation and purification. Compounds 13, 14, 15, and 16 were pre-
pared from the intermediate acid chlorides 11 and 12 by reaction
with commercially available 4-fluorobenzyl amine and 4-fluoro-
phenyl ethylamine (Scheme 1). The yields for 13, 14, 15 and 16
were 76%, 70%, 86% and 96%, respectively.
Compound 17 was synthesized in house according to a previ-
ously reported method41 and reacted with the acid chlorides 11
and 12 as shown in Figure 2. The desired products 18 and 19 were
obtained in 60% and 85% yields, respectively.
Synthesis of the compounds 20 and 21 is shown in Figure 3
(Synthetic Scheme 3).
Compound 20 was prepared by reaction of 12 with 4-amino-
phenyl ethylamine and obtained in 80% yield. The experimental
details are identical as those described for preparation of com-
pounds 13–16 (Scheme 1). Compound 20 was used for an alterna-
tive synthesis of 16 and synthesis of 21.
Compound 16 was synthesized by two different methods:
Method 1: The acid chloride 12 was reacted with 4-fluorophenyl
ethylamine to produce 16 (as shown in Fig. 1). In this method,
the yield of 16 was 97%; however, the method is not suitable for
radiosynthesis using 18F, because 18F should be inserted into the
benzene ring by a special method. Therefore, we explored an alter-
native method, as shown in Figure 3, which should be suitable for
radiosynthesis of 16. Method 2: In this method, diazotization of 20
was performed to produce an azo-salt compound in situ, which
was converted to 16 by reaction with HF in pyridine. This reaction
produced a mixture of two products, 16 and 21, in 40% and 36%
yields, respectively. The formation of 21 is quite interesting; in
addition to fluorination of the aromatic ring, C6-hydrogen was
substituted by a nitro group. Thus, compound 21 was obtained as
a by-product during diazotization of 20, followed by reaction with
HF in pyridine.
with more than 170-fold selectivity for the CB2 receptor
(Ki = 3134 110 nM at CB1 and Ki = 18 2 nM at CB2).22 This group
has also synthesized a large number of indole derivatives to iden-
tify CB2-specific ligands.1,2,6,23 A variety of cannabimimetic indole
derivatives have also been developed by the pharmaceutical com-
panies, including Bristol-Myers Squibb and Abbott Laborat
ories.24,25
In addition to traditional dibenzopyran-based cannabi-
noids1,2,21,22 and cannabimimetic indoles,6,24–26 a variety of potent
cannabinoid ligands that are resorcinol derivatives27,28 have been
synthesized and reported. Two series of resorcinol dimethyl ethers
were also reported by Wiley and others.28 One of these com-
pounds, O-1966A has very high (220-fold) selectivity for the CB2
receptor combined with very low affinity for the CB1 receptor
(Ki = 5055 984 nM at CB1 and Ki = 23 2.1 nM at CB2). Mussinu
et al.29 described several highly CB2 selective tricyclic pyrazoles
based upon CB1 antagonists SR141716A (RimonabantÒ)30 and the
CB2 inverse agonist SR144528.31,32 These pyrazoles are among
the most highly selective ligands for the CB2 receptor to be re-
ported to date, with selectivity ranging from 32- to 9810-fold
depending on the substituent at the aromatic ring.
Iwamura et al. have synthesized several 2-oxoquinoline ana-
logues with exceptionally high selectivity for the CB2 receptor.33
Compound JTE-907 (29, Table 2, Supplementary data) in particular,
is described as an inverse agonist in vitro for the CB2 receptor and
possesses anti-inflammatory properties in vivo. JTE-907 (29) has
modest affinity (Ki = 35.9 nM)1 for the CB2 receptor, with an excep-
tionally high selectivity of 2760-fold at this receptor. Structural
variations in these 2-oxoquinolines include an N-methyl substitu-
ent at the quinoline ring; variable alkoxy substituents in the 6, 7,
and 8 positions of the quinoline moiety and substituents at the
3-position that vary from carboxylic acid groups to various amide
and ester groups.34 Compound 30 (Table 2, Supplementary data) in
particular has very high CB2 receptor affinity (Ki = 0.014 nM) and
extremely high selectivity (262,202-fold) for this receptor. Raitio
et al. synthesized a new series of CB2 inverse agonists with re-
sponses comparable to SR144528,34,35 based on the structure of
2-oxoquinoline JTE-907 (29, Table 2, Supplementary data) in which
the piperonylamide end has been replaced with a variety of aro-
matic amide structures (32–37, Table 2, Supplementary data).34
Several other classes of compounds, including tricyclic pyra-
zoles,30–32 sulfamoyl benzamides,36 triarylbis-sulfones,37 and aryl-
sulfonamide,38 have been synthesized and reported to be CB2
receptor ligands. However, among all the classes of CB2 ligands,
2-oxoquinoline derivatives appear to be the most efficient inverse
agonists, with high binding affinity and selectivity for CB2
receptor.34,35
Synthesis of the compounds 22–24 is shown in Figure 4
(Synthetic Scheme 4).
In order to develop novel CB2-specific ligands for therapy of
neurological diseases and non-invasive imaging of CB2 receptor
expression, we have rationally designed a focused library of poten-
tial CB2-specific compounds. We report synthesis of a new library
of fluorinated analogues of 2-oxoquinoline as CB2 receptor ligands
and results of their in vitro receptor binding in cell lines genetically
engineered to express the CB1 or CB2 receptors. These studies
demonstrate that some of the fluorinated 2-oxoquinoline ana-
logues have high binding efficiency and selectivity for CB2
The acid chlorides 11 and 12 were prepared in situ from 9 and
10 as described in Scheme 1, then coupled with the corresponding
fluoroalkyl amines with two different carbon-chain lengths to
produce compounds 22–24. The yields of these compounds were
90–95%, almost quantitative.
Synthesis of compounds 27 and 28 is presented in Figure 5
(Synthetic Scheme 5).

5700
N. Turkman et al. / Bioorg. Med. Chem. 19 (2011) 5698–5707
O
O
O
O
O
O
d
OC2H5
b
c
H
H
H
H
a
H3CO
H3CO
H3CO
NO2
NH2
H3CO
H3CO
N
H
NO2
OH
OR
OR
OH
OR
7 R=CH3
8 R=C4H9
3 R=CH3
4 R=C4H9
5 R=CH3
6 R=C4H9
2
1
e
O
O
O
N
H
n
F
OH
g
Cl
f
H3CO
N
H3CO
N
H
O
O
H3CO
N
H
O
H
OR
OR
OR
13 R=CH3, n=1
14 R=C4H9, n=1
15 R=CH3, n=2
16 R=C4H9, n=2
11 R=CH3
12 R=C4H9
9
R=CH3
10 R=C4H9
Figure 1. Scheme for synthesis of 7-methoxy-8-alkoxy-2-oxo-1,2-dihydroquinoline-3-carboxylic acid 4-fluoro-benzylamides 13, 14 and 4-fluoro-phenethylamides 15 and
16. Reagents and conditions: (a) DCM, ꢀ20 °C, NO2BF4, 24 h; (b) DMF, K2CO3, MeI or 1-bromobutane, 80 °C 24 h; (c) EtOH, Fe, HCl, H2O, reflux 15 min; (d) EtOH, CH2(CO2CH3)2,
piperidine, AcOH, reflux 16 h; (e) EtOH, HCl/H2O, 60 °C, 16 h; (f) toluene, SOCl2, reflux 3 h; (g) DCM, Et3N, amine, rt, 1 h.
F
NH2
O
O
O
O
O
O
OH
Cl
17
H3CO
NH
F
H3CO
N
H
H3CO
N
H
N
H
b
a
OR
OR
OR
11 R=CH3
12 R=C4H9
9
R=CH3
18 R=CH3
19 R=C4H9
10 R=C4H9
Figure 2. Scheme for synthesis of 7-methoxy-8-alkoxy-2-oxo-1,2-dihydroquinoline-3-carboxylic acid 2-fluoro-2-phenethylamides 18 and 19. Reagents and conditions: (a)
toluene, SOCl2, reflux 3 h; (b) DCM, Et3N, 17, rt, 1 h.
O
O
O
O
NH2
Cl
N
H
a
H3CO
N
H
H3CO
N
H
OBu
12
OBu
20
b
O
O
F
F
O2N
N
N
H
H
+
H3CO
N
H3CO
N
O
O
H
H
OBu
OBu
21
16
Figure 3. Scheme for synthesis of 7-methoxy-8-butyloxy-2-oxo-1,2-dihydroquinoline-3-carboxylic acid 4-amino-phenethylamide 20, 7-methoxy-8-butoxyoxy-2-oxo-1,2-
dihydroquinoline-3-carboxylic acid 4-fluoro-phenethylamide 16 and 6-nitro-7-methoxy-8-butyloxy-2-oxo-1,2-dihydroquinoline-3-carboxylic acid 4-fluoro-phenethylamide
21. Reagents and conditions: (a) DCM, Et3N, 4-aminophenylethylamine, rt, 30 min; (b) NaNO2, pyridineꢁHF, rt, 1 h, 85 °C, 1 h.
The acid chloride 12 was prepared in situ as shown in Scheme 1,
then coupled with the alkyne–amines following a previously pub-
lished methodology42 to produce 25 and 26. Both compounds 25
and 26 were obtained in quantitative yields. Fluroethylazide was
prepared following a previously reported method43 and used with-
out purification. Compounds 25 and 26 were then reacted with

N. Turkman et al. / Bioorg. Med. Chem. 19 (2011) 5698–5707
5701
O
O
O
F
N
H
n
Cl
H2N
CH2Cl2, Et3N
rt, 10 min
F
n
+
H3CO
N
H
H3CO
N
H
O
OR
OR
22 R=CH3, n=1
23 R= C4H9, n=1
24 R=C4H9, n=2
11 R=CH3
12 R=C4H9
Figure 4. Scheme for synthesis of 7-methoxy-8-alkoxy-2-oxo-1,2-dihydroquinoline-3-carboxylic acid fluoro-alkylamides 22–24.
O
O
O
O
O
O
N
F
N
Cl
N
H
n
n
H2N
a
N
H
n
N3
b
N
F
H3CO
N
H
H3CO
N
H
H3CO
N
H
OBu
OBu
27 n=1
OBu
25 n=1
26 n=2
28 n=2
Figure 5. Scheme for synthesis of 7-methoxy-8-butoxy-2-oxo-1,2-dihydroquinoline-3-carboxylic acid-(fluoroethyl-triazolalkyl)amides 27 and 28. Reagents and conditions:
(a) CH2Cl2, Et3N, rt; (b) CuSO4, Na-ascorbate, DMF, rt.
fluoroethylazide in the presence of CuSO4 and Na-ascorbate at room
temperature using click chemistry to produce 27 and 28. The yields
in this step were 55% and 58%, respectively, for 27 and 28.
affinity than 28. Compounds 14–16, 18, 19, and 21 did not have
any significant competitive binding to CB1 receptor-expressing cell
membranes (Ki > 10 lM), suggesting that these compounds are not
All new compounds were fully characterized by 1H, 13C, and 19
F
CB1-specific receptor ligands. By contrast, the Ki values of some of
these compounds for the CB2 receptor are in the low nanomolar
and sub-nanomolar range, which suggests that they have high
affinity to the CB2 receptor. In terms of selectivity, compounds
14, 15, 16, 18, 19, and 21 have better selectivity for CB2, and 19
is the most selective one. Compound 21, with a nitro substitution
at the C6-position, did not affect any binding efficiency to the
CB2 receptor, but the substitution increased its specificity for
CB2. The Ki values for CB2 and CB1 for compounds 13–16, 18, 19,
21–24, 27, and 28 and their selectivity for CB2 are summarized
in Table 1.
Multiple 2-oxo-quinoline derivatives containing various sub-
stituents at the R1-, R2-, and R3-position have been previously re-
ported34,35 (Table 2, Supplementary data), which showed that
most of the compounds had Ki values of sub-nanomolar concentra-
tions,35,44 much lower than those of our compounds. For example,
compounds 29–31, 41–43 and 46–49 have Ki values 0.01–0.08 nM,
which is >10-fold lower than we found for our compounds. How-
ever, the Ki value of compound 29 was also reported to be
35.9 nM in another report1 as opposed to 0.087 nM in separate re-
ports.35,44 Furthermore, the Ki value of the potent CB2 specific in-
dole derivative L-768242 has been reported to be 14 nM23; and
those of the traditional tetrahydrocannabibnoids (THC) JWH229
and JWH133 were reported to be 18.0 and 3.4 nM, respectively.21
All these Ki values for the indole derivative and traditional THC
derivatives are comparable to those of our fluorinated 2-oxoquin-
iline derivatives. For further confirmation, we performed an
in vitro binding assay of compound 20, which has an amino group
at the para-position of the aromatic ring and a butyl group at the
C8-position, similar to the reported compound 39, which has a pen-
tyl group at C8.34 The Ki value of 20 was 22.3 nM, comparable to
that of our fluorinated compound 18 (22 nM). In one paper, the
binding efficiency of 39 was reported in terms of ꢀlog IC50; we
have also calculated the ꢀlog IC50 of compound 20 for comparison.
The reported ꢀlog IC50 of 39 is 8.5 0.3, and that of our compound
20 is 8.8 0.4, which are comparable to those reported previ-
ously.34 Therefore, Ki values of our compounds are reliable and
consistent with previously reported results.
NMR spectroscopy and high-resolution mass spectrometry. All
compounds were analyzed by high-performance liquid chromatog-
raphy (HPLC) for chemical purity and found to be greater than 98%
pure. These compounds were used for an in vitro binding assay
with the CB2 and CB1 receptors. HPLC chromatograms of the
new compounds are presented in the Supplementary data.
2.2. In vitro studies
2.2.1. CB1 and CB2 expressing U87 glioblastoma cells lines
To evaluate the specific binding of the aforementioned fluori-
nated compounds to hCB2, U87 cells expressing either hCB1 (con-
trol) or hCB2 were generated via lentiviral transduction. The red
fluorescent co-reporter, mKateS158A, was used for cells sorting.
The resulting populations were 84% and 98% positive for CB1 and
CB2, respectively, (Fig. 6).
2.2.2. In vitro receptor binding assay
This study was performed on the compounds 13–16, 18, 19, 21–
24, 27, and 28 using [3H]CP-55,940, [side chain-2,3,4-3H(N)], a
radiolabeled CB2 receptor ligand, following a literature method.6
The percent inhibition against [3H]CP-55,940 and the Ki values
were calculated using GraphPad Prism v4 software. The dissocia-
tion constant (Kd) and maximum binding (Bmax) for [3H]CP-
55,940 were determined to be 4.9 nM and 791 fmol/mg protein,
respectively (Fig. 7A), in CB2-expressing cells, and these values
were in agreement with previously reported data.6 The Kd and Bmax
for [3H]CP-55,940 were determined to be 16.8 nM and 1773 fmol/
mg protein in CB1-expressing cells (Fig. 7B).
Compounds 14–16, 18, 19, and 21 demonstrated high binding
efficiency to CB2-expressing cell membrane preparations, as evi-
denced by inhibition of binding of [3H]CP-55,940 (Fig. 7C). Other
compounds, 13, 22–24, 27, and 28, did not show good binding
for either CB2 or CB1; in particular, compounds 13 and 22 did
not have any affinity for the CB2 receptor. Compounds 23, 24, 27,
and 28 demonstrated low levels of binding affinity for the CB2
receptor. Among these compounds, 23, 24, and 27, had better

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N. Turkman et al. / Bioorg. Med. Chem. 19 (2011) 5698–5707
Figure 6. Expression of CB1 and CB2 in transduced U87 cells. U87 cells transduced with hCB1 (upper row) or hCB2 (lower row) and the red fluorescent co-reporter
mKateS158A (y-axis), were assessed for expression of hCB1 and hCB2 (x-axis) via flow cytometry, respectively. Density plots are shown for each. Two-step staining was
performed with an allophycocyanine (APC)-conjugated goat anti-mouse secondary antibody plus either an isotype control antibody (left column), anti-hCB1 (middle column)
or an anti-hCB2 (right column).
Figure 7. (A) Specific binding of [3H]CP-55,940 to CB2-expressing cell membranes, (B) specific binding of [3H]CP-55,940 to CB1-expressing cell membranes, (C) competitive
inhibition of [3H]CP-55,940 binding and Ki values for compounds 14–16, 18, 19, and 21.
2.2.3. Structure–activity relationship
the substituent at C8-position makes a significant difference in
binding to CB2 for a compound with a methylene linker between
the amide bond and phenyl ring. It has been reported that an eth-
ylene linker between the amide bond and the phenyl ring (R3) had
100-fold increase in potency compared with a methylene linker.34
In the current series of newly synthesized fluorinated compounds,
no such significant difference in binding efficiency was observed
due to the different carbon-chain (methylene vs ethylene) between
the amide and aromatic ring. For example, the Ki values of com-
pounds 14 with a methylene and 16 with an ethylene linker but
a butyl group at C8 are comparable (2.8 vs 2.4 nM). A large differ-
ence in binding was observed between the methylene and ethylene
linker only when the size of the substituent at the C8-position was
changed from the methyl to the butyl group.
As described above (Table 1 and Fig. 7C), compounds 14–16, 18,
19, and 21 demonstrated high binding affinity in the low-to-subn-
anomolar concentration range and high selectivity to the CB2
receptor. Compounds 13 and 22 did not show any binding affinity
for CB2; however, compounds 23, 24, 25, and 28 had some low lev-
els of binding affinity for CB2. Compounds 13 and 14, with a 4-flu-
oro-benzyl amide group (R3) have in common a one-carbon chain
between the amide bond and aromatic ring; only alkyl substituents
(methyl vs butyl) at the C8-position makes a significant difference
in binding efficiency (Ki > 10,000 vs 2.8 nM) between these two
compounds. This suggests that the larger group at the C8-position
enhances the binding affinity. By contrast, compounds 15 and 16,
with a two-carbon chain between the amide and the aromatic ring
and a different alky substituent at the C8-position, did not show a
significant difference in binding efficiency. Therefore, the size of
The position of fluorine (in the aromatic ring vs side chain) also
made a difference in binding efficiency. For example, compound 19

N. Turkman et al. / Bioorg. Med. Chem. 19 (2011) 5698–5707
5703
Table 1
Inhibition constant (Ki) values of compounds 13–16, 18, 19, 21–24, 27, and 28 using [3H]CP-55,940 a radioligand for hCB1 and hCB2 cannabinoid receptors
O
R2
R4O
R3
N
O
OR1 R5
Compd
R1
R2
H
R3
R4
R5
H
hCB2 Ki (nM)
hCB1 Ki (
lM)
Selectivity CB2/CB1
13
Methyl
F
F
CH3
>10,000
>10
>1
HN
14
15
16
Butyl
H
H
H
CH3
CH3
CH3
H
H
H
2.8 0.4
5.0 1.1
2.4 0.7
>10
>10
>10
>3570
>2000
>4166
HN
HN
F
F
Methyl
Butyl
HN
HN
F
F
18
Methyl
H
CH3
H
22 4.1
>10
>454
HN
HN
19
21
Butyl
Butyl
H
CH3
CH3
H
H
0.8 0.3
1.4 0.4
>10
>10
>12500
>7143
F
F
NO2
F
F
22
23
24
Methyl
Butyl
H
H
H
CH3
CH3
CH3
H
H
H
>10,000
486 96
58 14
>10
>10
>10
>1
HN
>20.5
>172
HN
HN
Butyl
F
N
N
27
28
Butyl
Butyl
H
H
CH3
CH3
H
H
620 114
>10
>10
>16
>4
HN
HN
N
N
N
2400 680
F
N
had a much higher binding efficiency (Ki = 0.8 nM) than compound
16 (Ki = 2.4 nM). The major difference between these two com-
pounds is the position of fluorine: in the side chain rather than
in the aromatic ring. Compounds with fluorine in the side chain
and different substituents at the C8-position had significantly dif-
ferent binding efficiency; for example, when the R1 was changed
from the butyl to methyl group, the Ki increased from 0.8 nM for
19 to 22 nM for 18. This observation suggests that a larger group
at the C8-position (R1) has better binding capability than a smaller
group, irrespective of the other substituents, possibly due to the
lipophylic nature of these compounds. Fluorine on the side chain
rather than in the aromatic ring may also play a significant role
Substitution of methyl for butyl at the C8-position makes a signif-
icant difference in binding efficiency for the methylene-containing
compounds (13 and 14), but no such difference was observed in
the ethylene-containing compounds 15 and 16. By contrast, there
was a small difference in binding efficiency between the compounds
containing a methylene and ethylene linker with a butyl group at the
C8-position, that is, an ethylene-containing compound had slightly
better binding efficiency than those with a methylene-containing
compound. There was a general trend that compounds containing
a C8-butyl substituent have better binding efficiency than those con-
taining C8-methyl. A nitro group at the C6-position in compound 21
had a small influence that improved the binding efficiency com-
pared with that of the C6-unsubstituted compound 16.
in the
p-stacking interactions between the ligand and aromatic
amino acids of the receptor. Compounds with a 4-substituted-
phenylethyl amide (R3) with pentyl substituent at C8-position
(R1) have been developed and reported earlier.34,35 The presence
of an electron withdrawing nitro group at the para-position of
the aromatic ring has been shown to decrease potency through
Substitution at the amide bond (R3) makes a significant differ-
ence in binding efficiency of these 2-oxo-quinoline derivatives.
Compounds with a straight chain substituent, such as fluoroethyl,
showed no binding affinity; however, binding affinity increased
with increasing carbon-chain length, which is consistent with the
hydrocarbon-substituted compounds reported previously.34 To
further explore the structure–activity relationship with regard to
diminishing aromatic p-stacking interactions compared with other
substituents at the para-position.34 Thus, in our compounds, the
presence of electronegative fluorine at the para-position of the aro-
p-stacking and charge or polarity on the R3, we synthesized tria-
matic ring may withdraw electron density of the
p
-electrons, re-
zol-containing derivatives of 2-oxoquiniline. If these compounds
had demonstrated improved binding characteristics to CB2, the ra-
pid radiolabeling of these compounds could be achieved with click
chemistry approaches using an alkyne derivative of 2-oxoquinoline
and 18F-labeled fluoroethylazide. However, the binding efficiency
of these compounds was quite low. Thus, compounds 27 and 28
have Ki values of 620 and 2400 nM, respectively. These results sug-
gest that substitution at the amide bond is critical for the binding
efficiency of these compounds. In terms of specificity, compound
19 has the highest specificity for the CB2 receptor, and compounds
21, 16, and 14 are the next selective compounds for the CB2 recep-
tor. Thus, given their binding efficiency and specificity to the CB2
duce the stacking interaction, and diminish the potency of the
compound compared with a fluorine in the benzylic position,
which is consistent with the results in the literature.34 As a result,
compound 19 is 3-fold more potent than 16. Our results suggest
that substitution at the C6-position improves selectivity for CB2
over CB1. Compound 21 has a nitro group at the C6-postion, and
this substitution makes the compound more selective for CB2,
maintaining a low Ki value. This result is also consistent with the
literature, because other C6-substituted compounds 30, 41, and
42 (Table 2, Supplementary data) have been reported to be highly
selective and sensitive CB2 ligands.35,44

5704
N. Turkman et al. / Bioorg. Med. Chem. 19 (2011) 5698–5707
receptor, the selected compounds 14, 15, 16, 19, and 21 require
further in vivo studies in animal models.
cooling bath was removed, and the mixture was stirred for an addi-
tional 20 min at rt. The solvent was removed under reduced pres-
sure and the residue was purified by flash chromatography on a
silica gel column using 5% MeOH/CH2Cl2. The solvent was evapo-
rated on a rotary evaporator, and the product 13 was obtained as
a white solid (powder) in 76% yield. 1H NMR (CDCl3) d:10.04 (t,
1H, NH), 9.36 (s, 1H, NH), 8.92 (s, 1H, C4–H), 7.50 (d, J = 8.4 Hz,
1H, C5–H), 7.36 (m, 2H, aromatic), 7.03 (m, 2H, aromatic), 6.97
(d, J = 8.4 Hz, 1H, C6-H), 4.66 (d, J = 6.6 Hz, 2H, N–CH2), 4.02 (s,
3H, OMe), 3.99 (s, 3H, OMe). 13C NMR (CDCl3) d: 163.66, 162.28,
162.05 (d, J1-F = 243.8 Hz), 154.51, 145.13, 134.47, 134.45, 133.28
(d, J4-F = 5.0 Hz), 129.33 (d, J3-F = 8.8 Hz), 125.63, 119.49, 115.38
(d, J2-F = 21.3 Hz), 114.29, 109.11, 61.08, 56.32, 42.81.19F NMR
(CDCl3) d: ꢀ114.66 (septet, AA’MM’X, J = 15.0 Hz, J = 9.0 Hz,
J = 6.0 Hz, J = 3.0 Hz). High-resolution MS: calculated for
Previously, a few ligand docking, mutation, and modeling stud-
ies have been reported for the CB2 receptor, that have used the
structure of rhodopsin as a template to construct a model of the
CB2 receptor for use in docking studies.45 Based on docking studies
with CB2 selective ligands, it was reported that hydrophobic and
aromatic stacking interactions were more important than
hydrogen bonding for CP-55,940 and WIN-55,212-2, and numer-
ous aromatic stacking interactions were observed for the binding
of WIN-55,212-2 at the CB2 receptor site.46 It appears that hydro-
phobic and aromatic
p-stacking interactions play similar roles in
the newly synthesized library of compounds in binding with the
CB2 receptor, and our results are in agreement with those
previously reported for CP-55,940 and WIN-55,212-2.46
C19H17FN2O4 (M+H) 356.1172, found 356.1169.
Compound 14 was purified on a silica gel column eluted with
3. Conclusion
70% EtOAc/hexane. The product 14 was obtained as a white solid
in 50% yield. 1H NMR (CDCl3) d: 8.55 (s, 1H, C4–H), 7.60 (d,
J = 8.4 Hz, 1H, C5–H), 7.42–7.37 (m, 3H, aromatic), 7.10-7.04 (m,
3H, aromatic), 4.70 (d, J = 6.6 Hz, 2H, NCH2), 4.27 (t, J = 6.6 Hz,
2H, OCH2), 4.11 (s, 3H, OMe), 4.10 (s, 3H, OMe), 1.85–1.80 (m,
2H), 1.58–1.55 (m, 2H), 0.98 (t, J = 7.2 Hz, 3H, CH3). 13C NMR
(CDCl3) d: 163.67, 162.21, 162.05 (d, J1-F = 243.8 Hz), 154.51,
145.13, 134.47, 134.45, 133.28 (d, J4-F = 5.0 Hz), 129.33 (d, J3-
F = 8.8 Hz), 125.63, 119.49, 115.38 (d, J2-F = 21.3 Hz), 114.29,
109.13, 73.54, 56.27, 42.79, 32.21, 32.21, 19.13, 13.82. 19F NMR
We have synthesized and tested a library of new fluorinated
2-oxoquinoline derivatives as CB2 receptor ligands. The results of
our in vitro competitive radioligand binding studies suggest that
some of these compounds are highly active and specific CB2 recep-
tor ligands in the low nanomolar to sub-nanomolar affinity range
and no significant binding to CB1 receptors. Therefore, these com-
pounds may be useful for therapy of neuropathic pain, inflamma-
tion, and immune disorders and should be evaluated further in
corresponding disease models in animals. In addition, compound
19 could be radiolabeled with 18F and explored for PET imaging
of CB2 expression. The studies are currently in progress.
(CDCl3) d: ꢀ114.4
J = 6.0 Hz, J = 3.0 Hz). High-resolution MS: calculated for
22H23FN2O4 (M+H) 398.1642, found 398.1649.
6 (septet, AA’MM’X, J = 15.0 Hz, J = 9.0 Hz,
C
4. Experimental
4.1. Chemistry
4.1.2.2. Preparation of 7-methoxy-8-alkoxy-2-oxo-1,2-dihydro-
quinoline-3-carboxylic acid-(4-fluorophenylethyl)amide: 15,
16. Both compounds 15 and 16 were synthesized following the
same methodology; a representative method is described here.
To a solution of the 4-fluorophenyl ethylamine (0.187 mmol) in
4.1.1. Reagents and instrumentation
All reagents and solvents were purchased from Aldrich Chemical
Co. (Milwaukee, WI), and used without further purification unless
otherwise specified. Tritiated CP-55,950 ([3H]CP-55,940), with a
DCM (3.0 mL) was added triethylamine (37 lL), and the mixture
was cooled to 0 °C. The acid chloride 11 or 12 (0.280 mmol) in
DCM (0.5 mL) was added slowly and the mixture was stirred for
10 min; then the cooling bath was removed, and the mixture was
stirred for an additional 20 min at room temperature. The solvent
was removed under reduced pressure, and the residue was purified
by flash chromatography on a silica gel column using 5% MeOH/
CH2Cl2. The solvent was evaporated on a rotary evaporator and
the product 15 was obtained as a white solid in 86% yield. 1H
NMR (CDCl3) d: 9.70 (s, 1H, NH), 9.32 (s, 1H, NH), 8.88 (s, 1H, C4–
H), 7.50 (d, J = 8.4 Hz, 1H, C5–H), 7.22–7.28 (m, 2H, aromatic),
6.95–7.05 (m, 3H, aromatic), 4.01 (s, OCH3), 3.99 (s, 3H, OCH3),
3.72 (q, 2H, J = 7.2 Hz, N–CH2), 2.94 (t, J = 7.2 Hz, 2H, benzylic).
13C NMR (CDCl3) d: 163.66, 162.28, 162.05 (d, J1-F = 243.8 Hz),
154.51, 145.13, 134.47, 134.45, 133.28 (d, J4-F = 5.0 Hz), 129.33 (d,
J3-F = 8.8 Hz), 125.63, 119.49, 115.38 (d, J2-F = 21.3 Hz), 114.29,
109.11, 61.08, 56.32, 56.2, 42.81. High-resolution MS: calculated
for C20H19N2FNaO4 (M+Na) 393.1227, found 393.1244.
specific activity of 174.6 mCi/lmol was purchased from PerkinEl-
mer (USA). Thin-layer chromatography (TLC) was performed on
pre-coated Kieselgel 60 F254 (Merck, Darmstadt, Germany) glass
plates. Proton, 13C, and 19F NMR spectra were recorded on a Brucker
300 MHz spectrometer or a 600 MHz spectrometer using tetrameth-
ylsilane as an internal reference and hexafluorobenzene as an exter-
nal reference, respectively, at The University of Texas M. D. Anderson
Cancer Center. High-resolution mass spectra were obtained on a
Brucker BioTOF II mass spectrometer at the University of Minnesota
using the electrospray ionization (ESI) technique.
HPLC was performed with an 1100 series pump (Agilent,
Germany), with a built-in UV detector operated at 214 or 254 nm,
and a radioactivity detector with single-channel analyzer (Bioscan,
Washington DC) with a C18 reverse-phase analytical column
(4.6 ꢂ 250 mm; Alltech, Econosil). An acetonitrile/water (MeCN/
H2O) solvent system with various composition (depending on com-
pound), was used for quality control analyses at a flow of 1 mL/min.
Compound 16 was obtained in 96% yield. 1H NMR (CDCl3) d:
9.65 (s, 1H, NH), 9.36 (s, 1H, NH), 8.85 (s, 1H, C4–H), 7.43 (d,
J = 8.4 Hz, 1H, C5–H), 7.22–7.20 (m, 2H, aromatic), 6.96–7.00 (m,
2H, aromatic), 6.92 (d, J = 9.0 Hz, 1H, C6–H), 4.12 (t, J = 6.6 Hz, 2H,
OCH2), 3.95 (s, 3H, OMe), 3.70–3.67 (m, 2H, N–CH2), 2.90 (t,
J = 7.2 Hz, 2H, benzylic), 1.80–1.75 (m, 2H), 1.52–1.45 (m, 2H),
0.97 (t, J = 7.2 Hz, 3H, CH3). 13C NMR (CDCl3) d: 163.5, 162.4,
162.2, 160.7, 154.4, 144.9, 134.8 (2C), 133.4, 132.4, 130.2 130.1,
125.2, 119.3, 115.3, 115.2, 114.3, 109.1, 73.5, 56.2, 41.0, 34.9,
32.1, 19.1, 13.8. 19F NMR (CDCl3) d: -116.9. High-resolution MS:
calculated for C23H25N2FNaO4 (M+Na) 435.1718, found 435.1788.
4.1.2. Chemical synthesis
4.1.2.1. Preparation of 7-methoxy-8-alkoxy-2-oxo-1,2-dihydro-
quinoline-3-carboxylic acid-(4-fluorobenzyl)amide: 13, 14. Both
compounds 13 and 14 were synthesized following the same meth-
odology; a representative method is described here. To a solution
of the 4-fluorobenzylamine (0.187 mmol) in DCM (3.0 mL) was
added triethylamine (37 lL), and the mixture was cooled to 0 °C.
The acid chloride 11 or 12 (0.280 mmol) in DCM (0.5 mL) was
added slowly and the mixture was stirred for 10 min; then the

N. Turkman et al. / Bioorg. Med. Chem. 19 (2011) 5698–5707
5705
4.1.2.3. Preparation of 2-fluoro-2-phenylethylamine: 17. Com-
pound 17 was prepared following a literature method41 with mod-
ifications. Briefly, to a stirred solution of mandelonitrile (1.33 g,
1.0 mmol, 1.0 equiv) in dry DCM (5 mL) at 0 °C was added diethyla-
minosulfure trifluoride (DAST) (1.77 g, 1.1 mmol, 1.1 equiv) in
DCM (2 mL). The resulting mixture was stirred at 0 °C and stirred
for 30 min, diluted with DCM (15 mL), and treated with saturated
NaHCO3. The organic layer was collected and dried over MgSO4.
After removal of the solvent under vacuum, the crude product
was chromatographed on a silica gel column and eluted with 10%
for compounds 13–16 (Scheme 1). Briefly, the acid chloride 12
(1.0 equiv) in anhydrous DCM was added drop-wise to a solution
of 4-aminophenylethylamine (1.5 equiv) and triethylamine
(2.0 equiv) in dry DCM at 0 °C. The ice bath was removed in
15 min and the reaction mixture stirred at room temperature (rt)
for 16 h. The reaction was quenched in a brine solution and the
product was extracted with DCM, dried over MgSO4, and then con-
centrated under vacuum at ambient temperature. The crude resi-
due was chromatographed on a silica gel column eluting with
10% MeOH/CH2Cl2 to give the desired product 20 in 96% yield. 1H
NMR (CDCl3) d: 9.67 (s, 1 H), 9.13 (s, 1H), 8.88 (s, 1H), 7.48 (d,
J = 8.4 Hz, 1H), 7.10 (d, J = 7.8 Hz, 2H), 6.95 (d, J = 8.4 Hz, 1H),
6.69 (d, J = 7.2, 2H), 4.16 (t, J = 6.6 Hz, 2H), 4.00 (s, 3H), 3.70–3.69
(m, 2H), 3.61 (br s, 2H), 2.86 (t, J = 7.2 Hz, 2 H), 1.83–1.80 (m,
2H), 1.55–1.52 (m, 2H), 1.02 (t, J = 7.2 Hz, 3H). 13C NMR (CDCl3)
d: 163.4, 162.1, 154.1, 144.7, 144.6, 133.4, 132.3, 129.5 (2C),
129.3, 125.2, 120.0, 115.7 (2C), 114.2, 109.0, 73.7, 56.3, 41.4,
35.0, 29.8, 28.1, 22.4, 13.8. MS: calculated for C22H25N3O4 (M+Na)
395.1643, found 395.1648.
EtOAc/hexane to afford
a-fluorophenylacetonitrile as yellow oil
(0.88 g) in 65% yield. The 1H NMR spectrum was consistent with
the literature,41 and 19F NMR showed a doublet at ꢀ167.4 ppm
with J = 48 Hz. Finally, the cyano-group was reduced to the corre-
sponding amine by treatment with 1 M borane–THF solution in
THF at 0 °C for 40 min and quenched the reaction mixture with
ethanolic hydrochloric acid, then concentrated under reduced
pressure. The desired 2-fluorophenylethyl amine was triturated
with CH3CN and then recrystallized from ethanol/acetonitrile to af-
ford 17 as colorless crystals in 49% yield. 1H NMR (CDCl3) d: 7.50
(m, 5H, aromatic), 5.8 (dddd, J = 48.0, 8.4, 7.2, 4.5, 3.0 Hz, 1H, ben-
zylic), 3.50 (m, 2H, methylene). 19F NMR (CDCl3) d: ꢀ183 (dm). MS:
154 (M+H).
4.1.2.6. Preparation of 7-methoxy-8-butyloxy-2-oxo-1,2-dihy-
droquinoline-3-carboxylic acid-(4-fluorphenethyl)amide and 6-
nitro-7-methoxy-8-butyloxy-2-oxo-1,2-dihydroquinoline-3-
carboxylic acid-(4-fluorphenethyl)amide: 16, 21. Compound 16
was prepared by two different methods.
Method 1: This method is a direct coupling of the 4-fluorophenyl
ethylamine with the acid chloride 12, similar to the preparation of
other compounds, 13–16. Method 2: To a solution of 7-methoxy-8-
butyloxy-2-oxo-1,2-dihydroquinoline-3-carboxylic acid p-amino-
4.1.2.4. Preparation of 7-methoxy-8-alkoxy-2-oxo-1,2-dihydro-
quinoline-3-carboxylic acid-(2-fluoro-2-phenyl ethyl)amide:
18, 19. Both compounds 18 and 19 were prepared using the same
methodology, a representative method is described here. To a solu-
tion of the 2-fluoro-2-phenylethylamine (0.187 mmol) in DCM
(3.0 mL) was added triethylamine (37
lL), and the mixture was
phenethylamide 20 (20 mg, 1 equiv) in 125
l
L of pyridineꢁHF was
cooled to 0 °C. The acid chloride 11 or 12 (0.280 mmol) in DCM
(0.5 mL) was added slowly and the mixture was stirred for
10 min; then the cooling bath was removed and the mixture was
stirred for an additional 20 min at rt. The solvent was removed un-
der reduced pressure, and the residue was purified by flash chro-
matography on a silica gel column using 5% MeOH/CH2Cl2. The
solvent was evaporated on a rotary evaporator, and the product
18 was obtained as a white solid (powder) in 75% yield. 1H NMR
(CDCl3) d: 10.00 (t, J = 6 Hz, 1H, NH), 9.32 (s, 1H, NH), 8.89 (s, 1H,
C4–H), 7.35–7.55 (m, 6H, aromatic H & C5–H), 6.97 (d, J = 9.0 Hz,
1H, C6–H), (dddd, J = 48.0 Hz, 8.4 Hz, 7.2 Hz,4.5 Hz, 3.0 Hz, 1 H,
benzylic), 4.05–4.20 (m,1H, N–CH2), 4.03 (s, 3H, OMe), 4.00 (s,
3H, OMe), 3.58–3.84 (m, 1H, N–CH2). 13C NMR (CDCl3) d: 163.4,
162.1, 154.1, 144.7, 144.6, 133.4, 132.3, 129.5 (2C), 129.3, 125.2,
120.0, 115.7 (2C), 114.2, 109.0, 73.7, 56.3, 41.4, 35.0, 29.8, 28.1,
22.4, 13.8. 19F NMR (CDCl3) d: ꢀ182.71 (dt, J = 47.6 Hz,
J = 30.8 Hz, J = 14.0 Hz). High-resolution MS: calculated for
added 5.5 mg of sodium nitrite (0.08 mmol, 1.5 equiv). The reac-
tion mixture was stirred at rt for 1 h and then at 85 °C for an addi-
tional hour. The reaction mixture was quenched with ice-water
(10 mL), and the product was extracted with ethyl acetate, dried
over MgSO4, and then the solution was concentrated under vac-
uum. The crude residue was chromatographed on silica gel column
eluting with 2:1 EtOAc/hexane to give 8.0 mg of the desired prod-
uct 16 (40% yield) and 8 mg of its 6-nitro derivative 21 (36% yield).
The NMR and mass spectra of 16 produced by this method were
identical with those of 16 obtained from method 1.
Compound 21 was isolated as a by-product in 36% yield during
the preparation of 16 using method 2. 1H NMR (CDCl3) d: 9.63 (s,
1H, NH), 9.48 (s, 1H, NH), 9.29 (s, 1H, C4–H), 7.76 (s, 1H, C5–H),
7.22–7.19 (m, 2H, aromatic), 6.98 (t, J = 8.4 Hz, 2H, aromatic),
4.32 (t, J = 7.2 Hz, 2H, O–CH2), 4.03 (s, 3H, OMe), 3.71–3.68 (m,
2H, N–CH2), 2.91 (t, J = 7.2 Hz, 2H, benzylic), 1.81–1.76 (m, 2H),
1.51–1.45 (m, 2H), 0.98 (t, J = 7.2 Hz, 3H, CH3). 13C NMR (CDCl3)
d: 162.4 (2C), 160.8 (2C), 151.3, 141.2, 140.1, 136.4, 134.7 (2C),
133.4 130.2, 130.1, 123.2, 115.3, 115.2, 108.0, 107.3, 74.2, 56.8,
41.2, 34.8, 29.7, 19.0, 13.7. 19F NMR (CDCl3) d: ꢀ116.8. High-reso-
lution MS: calculated for C23H24N3FO6Na (M+Na) 480.1718, found
480.1593.
C20H19N2FNaO4 (M+Na) 393.1227, found 393.1258.
Compound 19 was purified on silica gel column using 70%
EtOAc/Hexane. The product 19 was obtained as a white solid in
50% yield. 1H NMR (CDCl3) d: 10.00 (t, J = 6 Hz, 1H, NH), 9.17 (s,
1H, NH), 8.88 (s, 1H, C4-H), 7.28–7.49 (m, 6H, aromatic & C5–H),
6.95 (d, J = 9.0 Hz, 1H, C6–H), 5.70 (dm, JHF = 48 Hz 1H, benzylic),
4.14–4.19 (m, 3H, O-CH2 & N–CH2), 3.99 (s, 3H, O–CH3), 3.65–
3.85, (m, 1H, N–CH2), 1.76-1.84 (m, 2H), 1.50–1.57 (m, 2H), 1.02
(t, J = 7.2 Hz, 3H, CH3). 13C NMR (CDCl3) d: 163.96, 162.02,
154.45, 145.06, 137.78, 137.63, 133.56, 132.42, 128.67, 128.37,
125.64, 125.58, 125.36, 119.35, 114.2, 109.09, 92.71 (d, J1-
F = 172.5 Hz), 73.56, 56.30, 45.40 (d, J2-F = 24.0 Hz), 32.26, 19.15,
13.8. 19F NMR (CDCl3) d: ꢀ183.11 (dt, J=47.6 Hz, J = 30.8 Hz,
J = 14.0 Hz). High-resolution MS: calculated for C23H25N2FNaO4
(M+Na) 435.1696, found 435.1702.
4.1.2.7. Preparation of 7-methoxy-8-alkoxy-2-oxo-1,2-dihydro-
quinoline-3-carboxylic acid-(fluoroalkyl)amide: 22–24. Com-
pounds 22–24 were synthesized following the same
methodology as described in Scheme 4; a representative method
is described here. To
(0.187 mmol) in DCM (3.0 mL) was added triethylamine (37
a
solution of the 2-fluoroethylamine
L),
l
and the mixture was cooled to 0 °C. The acid chloride 11 or 12
(0.280 mmol) in DCM (0.5 mL) was added slowly, the mixture
was stirred for 10 min; then the cooling bath was removed, and
the mixture was stirred for an additional 30 min at rt. The solvent
was removed under reduced pressure, and the residue was purified
by flash chromatography on a silica gel column using 5% MeOH/
CH2Cl2. The solvent was evaporated on a rotary evaporator and
4.1.2.5. Preparation of 7-methoxy-8-butyloxy-2-oxo-1,2-dihy-
droquinoline-3-carboxylic
acid
4-amino-phenethylamide:
20. Compound 20 was prepared by similar method as described

5706
N. Turkman et al. / Bioorg. Med. Chem. 19 (2011) 5698–5707
the product 22 was obtained as a white solid (powder) in 90% yield.
1H NMR (CDCl3) d: 9.94 (br t, 1H, NH), 9.55 (s, 1H, NH), 8.88 (s, 1H,
C4–H), 7.48 (d, J = 9.0 Hz, 1H, C5–H), 6.97 (d, J = 9.0 Hz, 1H, C6–H),
4.65 (dt, J1-F = 47.4 Hz, J2 = 4.8 Hz, 2H, F–CH2), 3.99 (s, 6H, OMe),
3.85 (q, J1 = 10.5 Hz, J2 = 5.4 Hz, 1H, N–CH2), 3.76 (q, J1 = 10.5 Hz,
J2 = 5.4). 13C NMR (CDCl3) d: 164.01, 162.26, 154.55, 145.03,
133.40, 133.30, 125.63, 119.34, 114.23, 109.03, 82.45 (d, J1-
F = 161.3 Hz), 61.10, 56.32, 40.00 (d, J3-F = 21.1 Hz). 19F NMR
(CDCl3) d: ꢀ223.06 (m). High-resolution MS: calculated for
4.1.2.10. Preparation of 7-methoxy-8-butoxy-2-oxo-1,2-dihy-
droquinoline-3-carboxylic acid-((1-(2-fluoroethyl)-1H-1,2,3-
triazol)alkyl)amide: 27, 28. Compounds 27 and 28 were prepared
from 25 and 26, respectively, following a method similar to that
described in Scheme 5. A representative procedure is described
here. To a solution of fluoroethylazide (1.5 mL, 2.8 mM, in DMF)
was added propargyl amide 25. In a separate flask was dissolved
copper(II) sulfate pentahydrate (25.0 mg, 0.10 mmol) in water
(1.0 mL) followed by addition of sodium ascorbate (56 mg, mmol).
This solution was transferred to the above reaction mixture and
stirred at rt for 12 h. The solvent was removed under a stream of
air; the residue was redissolved in DCM and subjected to column
chromatography using 10% MeOH/CH2Cl2. The solvent was evapo-
rated on a rotary evaporator and the product 27 was obtained as a
white solid in 55% yield. 1H NMR (CDCl3) d: 10.00 (br t, 1H, NH),
9.14 (s, 1H, NH), 8.89 (s, 1H, C4-H), 7.58 (s, 1H, triazole-H), 7.49
(d, J = 9.0 Hz, 1H, C5–H), 6.60 (d, J = 9.0 Hz, 1H, C6–H), 4.88 (t,
J = 4.7 Hz 1H, N–CH), 4.81 (m, 2H), 4.70 (m, 2H), 4.60 (m, 1H, F–
CH), 4.16 (t, J = 6.6 Hz, 2H, OCH2), 3.99 (s, 3H, OMe), 1.81 (m,
2H), 1.52 (m, 2H), 1.00 (t, J = 7.2 Hz, 3H, CH3). 13C NMR (CDCl3) d:
163.84, 161.93, 154.45, 145.84, 145.03, 133.54, 132.39, 125.35,
123.20, 119.34, 114.19, 109.09, 81.51 (d, J1-F = 171.0 Hz), 73.55,
56.29, 50.50 (d, J2-F = 20.0 Hz), 35.26, 32.22, 19.12, 13.33. 19F
NMR (CDCl3) d: ꢀ221.6 (m). High-resolution MS: calculated for
C14H15FN2O4Na (M+Na) 317.0914, found 317.0909.
Compound 23 was also purified on a silica gel column eluted
with 70% EtOAc/hexane and obtained as a white solid in 92% yield.
1H NMR (CDCl3) d: 9.91 (br t, 1H, NH), 9.17 (s, 1H, NH), 8.88 (s, 1H,
C4–H), 7.47 (d, J = 9.0 Hz, 1H, C5–H), 6.96 (d, J = 9.0 Hz, 1H, C6–H),
4.60 (dt, J1-F = 47.6 Hz, J2 = 5.7 Hz, 2H, F-CH2), 4.17 (t, J = 6.6 Hz,
2H, OCH2), 3.99 (s, 3H, OMe), 3.62 (q, J1 = 12.9 Hz, J2 = 6.9 Hz, 2H,
N-CH2), 1.80 (m, 2H), 1.58 (m, 2H), 0.98 (t, J = 7.2 Hz, 3H, CH3).
13C NMR (CDCl3) d: 164.00, 162.08, 154.45, 145.03, 133.55,
132.38, 125.36, 119.28, 114.20, 108.07, 82.43 (d, J1-F = 168.3 Hz),
73.54, 56.28, 39.97(d, J2-F = 21.1 Hz), 32.21, 19.13, 13.83. 19F NMR
(CDCl3) d: ꢀ223.18 (m). High-resolution MS: calculated for
C17H21FN2O4 Na (M+Na) 336.1485, found 336.1485.
Compound 24 was obtained as a white solid in quantitative
yield after purification. 1H NMR (CDCl3) d: 9.75 (br t, 1H, NH),
9.16 (s, 1H, NH), 8.87 (s, 1H, C4–H), 7.47 (d, J = 9.0 Hz, 1H, C5–H),
6.95 (d, J = 9.0 Hz, 1H, C6–H), 4.60 (dt, J1-F = 47.6 Hz, J2 = 5.7 Hz,
2H, F–CH2), 4.17 (t, J = 6.6 Hz, 2H, OCH2), 3.99 (s, 3H, OMe), 3.62
(q, J1 = 12.9 Hz, J2 = 6.9 Hz, 2H, N-CH2), 2.13–2.00 (m, 2H), 1.81
(m, 2H), 1.52 (m, 2H), 1.00 (t, J = 7.2 Hz, 3H, CH3). 13C NMR (CDCl3)
C
20H24FN5O4Na (M+Na) 440.1710, found 440.1694.
Compound 28 was obtained as a white solid in 58% yield. 1H
NMR (CDCl3) d: 9.79 (br t, 1H, NH), 9.17 (s, 1H, NH), 8.86 (s, 1H,
C4–H), 7.58 (s, 1H, triazole-H), 7.46 (d, J = 9.0 Hz, 1H, C5–H), 6.95
(d, J = 9.0 Hz, 1H, C6–H), 4.87 (t, J = 4.7 Hz 1H, N–CH), 4.70 (m, over-
lapped, 2H, N–CH–CH–F), 4.60 (m, 1H, F-CH), 4.15 (t, J = 6.6 Hz, 2H,
OCH2), 3.98 (s, 3H, OMe), 3.82 (q, J1 = 12.9 Hz, J2 = 6.9 Hz, 2H, N-
CH2), 3.10 (t, J = 6.9 Hz, 2H), 1.81 (m, 2H), 1.52 (m, 2H), 1.00 (t,
J = 7.2 Hz, 3H, CH3). 13C NMR (CDCl3) d: 163.71, 162.07, 154.36,
145.74, 144.86, 133.45, 132.37, 125.31, 122.33, 119.49, 114.24,
109.06, 81.61 (d, J1-F = 172.0 Hz), 73.53, 56.28 50.41 (d, J2-
F = 20.0 Hz), 33.04, 32.22, 26.02, 19.12, 13.33. 19F NMR (CDCl3) d:
ꢀ221.4 (m). High-resolution MS: calculated for C21H26FN5O4Na
(M+Na) 454.1867, found 454.1869.
d: 169.79, 162.15, 154.36, 144.36, 133.45, 132.38, 125.32, 119.43,
114.26, 109.07, 82.01 (d, J1-F = 164.3 Hz), 73.54, 56.27, 35.92 (d,
J3-F = 5.2 Hz), 32.22, 30.55 (d, J2-F = 19.6 Hz), 19.13, 13.83. 19F
NMR (CDCl3) d: ꢀ226.82 (m). High-resolution MS: calculated for
C18H23FN2O4Na (M+Na) 373.1540, found 373.1573.
4.1.2.8. Preparation of 7-methoxy-8-butoxy-2-oxo-1,2-dihydro-
quinoline-3-carboxylic acid-(alkyl-alkyne)amide: 25, 26. Com-
pounds 25 and 26 were prepared by the same method as
described in Scheme 5 following a previously published method.42
The acid chloride 12 (0.280 mmol) in DCM (0.5 mL) was added
slowly to a solution of either 2-propyne amine or 3-butyne amine
(0.187 mmol) in DCM (3.0 mL), which was cooled to 0 °C, and the
mixture was stirred for 10 min. The cooling bath was removed,
and the reaction mixture was stirred for an additional 3 h at rt.
The solvent was removed under reduced pressure, and the residue
was purified by flash chromatography on a silica gel column using
70% EtOAc/hexane. Compound 25 was obtained in quantitative
yield; its 1H NMR spectrum was consistent with that reported in
the literature.42
Compound 26 was obtained as a white solid in quantitative
yield. 1H NMR (CDCl3) d: 9.90 (br t, 1H, NH), 9.18 (s, 1H, NH),
8.87 (s, 1H, C4–H), 7.46 (d, J = 9.0 Hz, 1H, C5–H), 6.95 (d,
J = 9.0 Hz, 1H, C6–H), 4.15 (t, J = 6.9 Hz, 2H, OCH2), 3.99 (s, 3H,
OMe), 3.65 (q, J1 = 12.9 Hz, J2 = 6.6 Hz, 2H, N–CH2), 2.54 (ddd,
J = 9.3 Hz, J = 6.9 Hz, J = 2.7 Hz, 2H), 2.06 (t, J = 2.7 Hz, 2H), 1.81
(m, 2H), 1.52 (m, 2H), 1.00 (t, J = 7.2 Hz, 3H, CH3). 13C NMR (CDCl3)
4.2. In vitro studies
4.2.1. Engineering and flow cytometric evaluation of human
CB1 and CB2-expressing U87 glioblastoma cells
pCR4-TOPO-hCB1 and pPCR-Script Amp SK(+)-hCB2, encoding
the human CB1 (accession number NM016083) and hCB2 (acces-
sion number BC069722) were purchased from Open Biosystems
(Huntsville, AL), respectively. CB1 and CB2 were subcloned into
pDONR222 via BP reaction following amplification with attB1
and attB2 flanked primers and then recombined into a Gateway-
adapted lentivirus encoding an internal mscv LTR (Ref = http://
www.ncbi.nlm.nih.gov/pubmed/20730500) and the red fluores-
cent protein mKateS158A downstream of an emcv IRES
(pLV4312-CB2). Virus was packaged in 293-FT cells using pMD2.G
(VSV.G env) and pCMV-deltaR8.91 and concentrated 50ꢂ using
Amicon Ultra-15 100,000 NMWL centrifugal concentration units
(Millipore, Billerica, MA). Concentrated viral supernatants were
used to transduce the human glioblastoma cell line U87 (ATTC,
Manassas, VA) via spinfection for 2 h at 2200 RPM/30 °C. CB1+/
mKateS158A+ and CB2+/mKateS158A+ U87 cells were sorted on
a FACSAria cell sorter (BD Biosciences, San Jose, CA) based on
expression of the co-reporter mKateS158A. Cell surface expression
of hCB1 and hCB2 was assessed via flow cytometry on a FACSCal-
ibur (BD Biosciences, San Jose, CA). Primary antibodies specific
for CB1 (Clone 368302) and CB2 (Clone 352114) were obtained
from R&D Systems (Minneapolis, MN). Secondary staining was
d: 163.67, 162.11, 154.37, 144.93, 133.47, 132.36, 125.33, 119.44,
114.25, 109.06, 81.70, 73.54, 69.76, 56.28, 38.36, 32.24, 19.51,
19.13, 13.83. High-resolution MS: calculated for C19H22N2O4Na
(M+Na) 365.1477, found 365.1475.
4.1.2.9. Preparation of 2-fluorethylazide. This compound was
prepared according to a previously reported method.43 Briefly,
2-fluoroethyl bromide (0.028 mmol) was reacted with sodium
azide (0.029 mmol) in DMF (10 mL) for 48 h at rt. The solution
was filtered and used without further purification.

N. Turkman et al. / Bioorg. Med. Chem. 19 (2011) 5698–5707
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This study was performed on the compounds 13–16, 18, 19,
21–24, 27, and 28 using [3H]CP-55,940, [side chain-2,3,4-3H(N)],
a radiolabeled CB2 receptor ligand, as follows. U87 cells (trans-
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Supplementary data
Supplementary data (experimental details on compounds 2–12,
HPLC chromatograms of the new compounds, and a list of com-
pounds with Ki values previously published in the literature (with
references)) associated with this article can be found, in the online
version, at doi:10.1016/j.bmc.2011.07.062.
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