Synthesis and SAR studies of 2-oxoquinoline derivatives as CB2 receptor inverse agonists

Article abstract of DOI:10.1021/jm050879z

The highly CB2 selective cannabinoid receptor inverse agonist, 7-methoxy-2-oxo-8-pentyloxy-1,2-dihydroquinoline-3-carboxylic acid N-benzo[1,3]dioxol-5-ylmethyl)amide (JTE-907; 9b), served as the lead compound for investigating the structure-activity relat

Full text of DOI:10.1021/jm050879z

2022
J. Med. Chem. 2006, 49, 2022-2027
Synthesis and SAR Studies of 2-Oxoquinoline Derivatives as CB2 Receptor Inverse Agonists
Katri H. Raitio,†,* Juha R. Savinainen,‡ Jouko Vepsa¨la¨inen,§ Jarmo T. Laitinen,‡ Antti Poso,† Tomi Ja¨rvinen,† and
Tapio Nevalainen†
Department of Pharmaceutical Chemistry, Department of Physiology, and Department of Chemistry, UniVersity of Kuopio,
P.O. Box 1627, FI-70211 Kuopio, Finland
ReceiVed September 6, 2005
The highly CB2 selective cannabinoid receptor inverse agonist, 7-methoxy-2-oxo-8-pentyloxy-1,2-
dihydroquinoline-3-carboxylic acid N-benzo[1,3]dioxol-5-ylmethyl)amide (JTE-907; 9b), served as the lead
compound for investigating the structure-activity relationships of its analogues and in the search for more
potent and effective CB2 receptor inverse agonists. A series of aromatic amides of 7-methoxy-2-oxo-8-
pentyloxy-1,2-dihydroquinoline-3-carboxylic acid 6 was synthesized, and the CB2 receptor activities of the
compounds were determined by a [35S]GTPγS-binding assay using membranes of CHO cells stably transfected
with the human CB2 receptor. As a result, all the compounds were defined as full CB2 receptor inverse
agonists, and additionally, except for two 3,4-dihydroxyphenylalkylamides, they were found to be equally
potent as SR144528.
Introduction
presented in the patent,10 the selected backbone structure was
considered as one of the most potential starting points for
nontraditional cannabinoid receptor ligand development with a
special focus on CB2 selectivity.
The peripheral cannabinoid receptor CB2 was molecularly
identified in 1993 by Munro et al.,1 and their work in
determining the distribution pattern of CB2 was subsequently
complemented by Galie`gue et al.2 The two signaling pathways
utilized by the CB2 receptor are inhibition of adenylate cyclase
through activation of the Gi/o protein3 and activation of mitogen-
activated protein (MAP) kinase resulting in induction of the
growth-related gene Krox-24 expression.4
The immunomodulatory properties of the cannabinoids
present in the plant Cannabis satiVa have been proposed to be
mediated via the CB2 receptor. This hypothesis is supported
by the abundant expression of CB2 in cells of the immune
system, such as those in spleen, tonsils, and thymus, natural
killer cells, T cells, and B cells.2 Therefore, modification of
immune functions by CB2 receptor ligands has become a focus
of particular interest. In drug development, the mainly peripheral
distribution of CB2 suggests that CB2-selective ligands will not
have cannabinoid-related psychoactive side effects, i.e., drugs
can be selectively targeted at the peripheral cannabinoid
receptor.5 The CB2 selective ligands developed so far have
In this study we have improved the previously published
synthesis pathway10 for compound 6 (see Scheme 1) and coupled
it with a series of phenylalkylamines carrying variable substitu-
tion patterns to produce a series of amides. Also the effect of
inversion of the amide bond has been studied, since we also
prepared the 9b inverse amide 9a (Scheme 2). CB2 receptor
activities of these compounds were determined by using a
functional assay monitoring G protein activation, assessed by
[35S]GTPγS binding to Chinese hamster ovary (CHO) cell
membranes stably expressing the human CB2 receptor (hCB2).
Our previous study has established that the CB2 receptor is
constitutively active in this model and therefore allows detection
of agonist, neutral antagonist, and inverse agonist ligand
activities.11 As far as we are aware, these results have permitted
us to derive the first structure-activity relationships of 2-oxo-
quinoline derivatives as CB2 receptor inverse agonists.
Results and Discussion
6
recently been reviewed by Raitio et al.
This study stems from the Japan Tobacco invention of the
CB2 selective inverse agonist 9b (JTE-907), which binds to the
CB2 receptor with nanomolar affinity (Ki 35.9 nM) and
possesses antiinflammatory properties in vivo.7 The concept of
inverse agonism can be explained with the prevailing two-state
receptor model theory, according to which the G-protein coupled
receptors may fluctuate between active (R*) and inactive (R)
states. Inverse agonists possess higher affinity to the R form,
agonists to R* and neutral agonists bind with similar affinity to
both receptor states. Therefore, inverse agonists decrease the
proportion of R* in the receptor population, and this results in
reduced constitutive activation of G-proteins by receptors in the
absence of agonist.8,9 9b contains several functionalities which
might be important for its action. Based on the affinity data
Chemistry. The synthesis pathway for 6 is presented in
Scheme 1. Nitration of isovanillin 1 was carried out with
nitronium tetrafluoroborate12 since the typical nitration reagent
(HNO3/H2SO4) used in the patent10 only gave 2 as a side product
with approximately 10% yield. The phenolic hydroxyl group
of 2 was alkylated with pentylbromide by using the Williamson
ether synthesis10 with satisfactory yields. The reduction of the
nitro group with iron powder in EtOAc/AcOH/H2O-mixture with
a catalytic amount of HCl13 was found to be a rapid and efficient
method to reduce the nitro group prior to the aldehyde group.
Reaction of 4 with dimethylmalonate in the presence of
piperidine and AcOH using EtOH as solvent produced the
transesterified 2-oxoquinoline as an ethyl ester 5 at high yield.
Hydrolyzation of 5 in aqueous EtOH/2M HCl yielded the
directly crystallized carboxylic acid 6 with high purity and at
relatively high yield.14
* To whom correspondence should be addressed. Tel: +358-17-163662.
Fax: +358-17-162456. E-mail: Katri.Raitio@uku.fi.
† Department of Pharmaceutical Chemistry.
‡ Department of Physiology.
As presented in Scheme 2, compound 9a, the inverse amide
of 9b, was prepared from the acid chloride of 6 with concen-
trated NH4OH in dioxane.15 The amide was subjected to
§ University of Kuopio.
10.1021/jm050879z CCC: $33.50 © 2006 American Chemical Society
Published on Web 02/22/2006

CB2 Receptor InVerse Agonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 2023
Scheme 1a
a Reagents:(i) NO2BF4, DCM; (ii) 1-bromopentane, K2CO3, DMF; (iii) Fe-powder, concentrated HCl, EtOH/AcOH/water; (iv) dimethyl malonate, piperidine,
AcOH, EtOH; (v) 2 M HCl, EtOH.
Scheme 2a
a Reagents: (i) Concentrated NH4OH, dioxane; (ii) Br2, NaOH, water; (iii) (1) 10, SOCl2, toluene; (2) Et3N, DCM; (iv) (1) SOCl2, toluene; (2) R-NH2,
Et3N, DCM.
None of the compounds 9a-m stimulated G protein activity
via the CB2 receptor but instead inhibited basal [35S]GTPγS
binding in CB2 expressing but not in control CHO cell
membranes. The responses were compared to that of 1 µM
SR144528, which corresponds to the maximal inhibitory effect
(Imax) of SR144528. Accordingly, relative Imax values as
percentages of SR144528 Imax were produced (Table 1).
Furthermore, statistical analysis of the Imax-values did not reveal
any differences between compounds 9a-m and SR144528.
Figure 1. The chemical structure of SR144528, a full inverse agonist
at the CB2 receptor.
Consequently, as SR144528 is commonly accepted to be a full
inverse agonist of the CB2 receptor, compounds 9a-m were
likewise concluded to evoke full CB2 receptor inverse agonist
responses in the assay employed in our study.
bromine-catalyzed Hofmann rearrangement under alkaline con-
ditions16 yielding decarbonylated 8, which was reacted with 3,4-
(methylenedioxy)phenylacetyl chloride, prepared from the cor-
responding carboxylic acid 10 with SOCl2, to produce compound
9a. To yield amides 9b-m (except for 9i, for the synthesis of
which see below), the carboxylic acid 6 was converted to the
corresponding acid chloride, which was then reacted with the
appropriate amines in the presence of triethylamine.17 Compound
9i was synthesized from 9f by using the same reduction
procedure as in the preparation of 4. The final products as well
as most of the intermediates were purified with flash chroma-
tography using suitable solvent systems.
First, we synthesized compound 9c, in which the benzo[1,3]-
dioxole ring of 9b was replaced with a phenyl ring. This
modification affected neither efficacy nor potency. Conse-
quently, compounds 9d and 9e with ethylene and propylene
linkers between the amide bond and the phenyl ring, respec-
tively, were synthesized. As a result, 100-fold increases in
potency without any losses of efficacy were achieved with both
9d and 9e when compared to 9c (Table 1).
Compound 9d being less lipophilic than 9e was chosen as
the template for studying the effects of phenyl ring para-
substitution on CB2 activity. Accordingly, the p-nitro-substituted
9f was found to be significantly less potent than 9d (Table 1)
whereas none of the chloro, hydroxyl, or amino substituents
(in 9g, 9h, and 9i, respectively) caused any significantly notable
chances in Imax or in IC50 values when compared to 9d. The
preliminary results from docking 9f to our rhodopsin based CB2
model18 (data not shown) indicated that the negatively charged
nitro group might repulsively interact with the amino acids at
the entrance of the receptor binding cavity, which has been
suggested to carry a negative charge.19 In addition, loss of phenyl
ring electron density to the electron-withdrawing nitro group
might partly explain the potency decrease through diminishing
Structure-Activity Relationship. The CB2 receptor activi-
ties of the compounds were determined by using the [35S]GTPγS
binding assay with membranes of stably hCB2-transfected CHO
cells. We have previously shown that the human CB2 receptor
is constitutively active in this particular assay, allowing potency
and efficacy determinations for the entire spectrum of CB2
receptor ligands, including full (CP-55,940) and partial
(WIN55212-2) agonists, neutral antagonists (WIN55212-3) and
inverse agonists (SR144528, Figure 1).11 In this system, agonists
typically stimulate [35S]GTPγS binding responses by 2-3-fold,
the neutral antagonist WIN55212-3 per se has no effect and
the inverse agonist SR144528 inhibits [35S]GTPγS basal binding
by 20-40%.11 The biological data are summarized in Table 1.

2024 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Table 1. CB2 Receptor Activities of the Synthesized Compoundsa
Raitio et al.
a The Imax values are presented as percentages of the Imax of SR144528. CB1 antagonist activities were determined by antagonizing HU-210 (10 nM)
response as described in Experimental Section. Values are from at least three independent experiments performed in duplicate. b Significantly higher inhibition
(P < 0.05) when compared to 9a-9m. c Significantly higher inhibition (P < 0.05) when compared to 9a-9m except for 9d, 9e and 9j. d Significantly lower
potency (P < 0.05) when compared to 9d. e Significantly lower potency (P < 0.05) when compared to SR144528. f Significantly lower potency (P < 0.05)
when compared to 9b.
aromatic stacking interactions between the ligand and aromatic
amino acids of the receptor.
compounds 9a-m seem to be more CB2 selective than
SR144528. At 10 µM concentration compounds 9a-m blocked
15-41% of the HU-210 response (Table 1). Considering the
IC50 values of compounds 9a-m as CB2 inverse agonists and
their low ability to cause CB1 response at 1 µM concentration,
we conclude that these compounds, excluding the less potent
9j and 9k, are at least 10-1000-fold CB2 selective.
Another phenyl ring substitution pattern affecting potency
was the catechol group, whose potency-decreasing effect was
shown by comparing compound 9j with 9d. Both catechol group
bearing derivatives 9j and 9k were also shown to be significantly
less potent than SR144528 (and 9k also less potent than 9b),
while all of the other synthesized compounds possessed poten-
cies similar to that of SR144528 (Table 1). The low potency of
9j and 9k could be accounted for by the ionization of one of
the catechol hydroxy groups and moreover with intramolecular
hydrogen bond formation providing stabilization of the negative
charge. The possible potency-lowering mechanisms based on
the negative charge have been discussed above in the case of
the nitro group and are also likely to be valid for the catechol
derivatives.
To confirm the engagement of the CB2 receptor in mediating
the response, compounds 9a-m were further tested at a 10 µM
concentration in native CHO cell membranes (data not shown).
Thereby, the inverse agonist responses were shown to be CB2-
mediated as none of the compounds showed any activity in the
control cells. All the synthesized compounds were also tested
at 10 µM concentration for their CB1 agonist activity and at 1
µM and 10 µM concentrations for their ability to antagonize
agonist (10 nM HU-210) response in rat cerebellar membranes.
No CB1 agonist activities were detected in these studies (data
not shown). At 1 µM concentration, maximum 13% decreases
in 10 nM HU-210-induced CB1 agonist activity were detected,
whereas for SR144528 a significantly higher (23%) decrease
was measured (Table 1). The data therefore suggests that
Conclusions
We have synthesized a series of CB2 receptor ligands by
replacing the piperonylamide end of 9b with a variety of
aromatic amide structures. In the [35S]GTPγS-binding assay, all
the compounds elicited full CB2 inverse agonist responses
similar to SR144528. Therefore, we conclude that the changes
we made to the structure of 9b did not affect efficacy. However,
differences in potency were observed. The unsubstituted phen-
ylethylamide 9d showed significantly higher potency than the
benzylamide 9c and was therefore chosen as the probe for
examination of para-substitution on CB2 activity. As a result,
only nitro substitution (9f), but not chloro, hydroxy, and amino
substitutions (9g, 9h, and 9i, respectively), decreased potency
when compared to 9d. Additionally, the catechol group bearing
amides 9j and 9k both exhibited lower potencies than SR144528.
Experimental Section
Chemistry. 1H and 13C NMR spectra were recorded on a Bruker
Avance instrument operating at 500.1 and 125.8 MHz, respectively.
Chemical shifts are reported as δ values (ppm) relative to an internal
standard of tetramethylsilane (TMS). Electrospray ionization mass
spectra were determined on Finnigan MAT LCQ quadrupole ion

CB2 Receptor InVerse Agonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 2025
trap mass spectrometer. Elemental analyses were performed on
ThermoQuest CE instrument (EA 1110 CHNS-O).
All chemicals and solvents were of commercial quality and were
used without further purification. Most intermediate and end
products were purified by flash chromatography using 30-60 µm
silica gel and an appropriate eluent.
Procedure A: Preparation of a Carboxylic Acid Chloride
from the Parent Carboxylic Acid. To a solution of carboxylic
acid (1.0 mmol) in anhydrous toluene (10-20 mL) was added
thionyl chloride (3.5 mmol), and the reaction mixture was refluxed
under argon for 3 h. The mixture was allowed to cool to room
temperature, and the solvent was evaporated. Anhydrous toluene
(10 mL) was added, evaporation repeated, and the residue dried
under high vacuum.
mL) was added 2 M HCl (25 mL), and the mixture was stirred at
60 °C overnight, cooled, and filtered. The solids were dried under
vacuum to give 6 as a white powder (950 mg, 82% yield, typically
1
varying from 62% to 87%). H NMR (DMSO).
7-Methoxy-2-oxo-8-pentyloxy-1,2-dihydroquinoline-3-carbox-
amide (7). The acid chloride prepared from 6 (200 mg, 0.66 mmol)
according to procedure A was added dropwise in dioxane (10 mL)
to concentrated NH4OH (11 mL). The reaction mixture was stirred
at room temperature for 3.5 h and evaporated to dryness, and the
residue was purified by flash chromatography eluting with a gradient
from petroleum ether/EtOAc/concentrated NH3 (aq) 100:100:1 to
petroleum ether/EtOAc/concentrated NH3 (aq) 100:200:1, yielding
1
7 as white crystals (170 mg, 85%). H NMR (CDCl3).
3-Amino-7-methoxy-8-pentyloxy-1H-quinolin-2-one (8). Com-
pound 7 (140 mg, 0.46 mmol) was suspended with cold (0 °C)
hypobromide solution (15 mL), prepared from bromine (0.25 mL,
4.88 mmol), NaOH (1.9 g, 47.51 mmol), and water (100 mL). The
reaction mixture was heated to 65 °C within 75 min and extracted
with EtOAc (3 × 20 mL). The combined organic layers were
washed with water (2 × 20 mL) and saturated NaCl (15 mL) and
dried over Na2SO4. The brown solid crude product was purified
by flash chromatography using petroleum ether/EtOAc 3:1 as eluent
to yield 8 a whitish solid product (50 mg, 39%). 1H NMR (DMSO).
3,4-(Methylenedioxy)phenylacetyl Chloride. Prepared accord-
ing to procedure A starting from 3,4-(methylenedioxy)phenyl acetic
acid 10 (49 mg, 0.27 mmol). The reaction was assumed to proceed
quantitatively, and the product was used without purification.
2-Benzo[1,3]dioxol-5-yl-N-(7-methoxy-2-oxo-8-pentyloxy-1,2-
dihydroquinolin-3-yl)acetamide (9a). Acid chloride 10 was added
dropwise in anhydrous DCM (5 mL) to a solution of 8 (50 mg,
0.18 mmol) and triethylamine (0.038 mL, 0.27 mmol) in anhydrous
DCM (3 mL) at 0 °C. The ice bath was removed, and the reaction
mixture was stirred at room temperature for 36 h. Saturated NaCl
(20 mL) was added to the mixture, the layers were separated, and
the aqueous layer was extracted with DCM (3 × 30 mL). The
combined organic layers were washed with saturated NaCl (20 mL),
dried over Na2SO4, and purified by flash chromatography using
petroleum ether/EtOAc 3:1 as eluent yielding 9a as a yellow solid
Procedure B: Coupling of a Carboxylic Acid Chloride and
an Amine under Basic Conditions. The acid chloride was added
dropwise in anhydrous dichloromethane (DCM) at 0 °C to a solution
of the amine (1.5 mmol) and triethylamine (1.5-3.5 mmol) in
anhydrous DCM (10-20 mL). The ice bath was removed, and the
reaction mixture stirred at room temperature under argon for 1-2
h. Saturated NaCl (20-30 mL) was added to the mixture, and the
aqueous layer was extracted with DCM. The combined organic
layers were washed with saturated NaCl, dried over Na2SO4, and
purified by flash chromatography.
3-Hydroxy-4-methoxy-2-nitrobenzaldehyde (2). Isovanillin 1
(5.0 g, 32.86 mmol) and nitronium tetrafluoroborate (6.55 g, 49.29
mmol) were reacted in anhydrous DCM (100 mL) at -20 °C for
20 h. Water (30 mL) was added dropwise, and the mixture was
allowed to reach room temperature. The combined diethyl ether
extracts (200 mL) were washed with water (2 × 25 mL) and
saturated NaCl (50 mL), dried over Na2SO4, and purified by flash
chromatography using petroleum ether/EtOAc 1:1 as eluent to yield
a yellow solid product (4.1 g, 63%, purity ca. 90%), which was
used without further purification. Typical yields varied from 40%
1
to 81%. H NMR (DMSO).
4-Methoxy-2-nitro-3-pentyloxybenzaldehyde (3). 1-Bromopen-
tane (4.4 mL, 35.51 mmol) was added dropwise to a solution of 2
(2.0 g, 10.14 mmol) and potassium carbonate (4.91 g, 35.51 mmol)
in anhydrous DMF (40 mL), and the reaction mixture was stirred
vigorously under argon at 100 °C for 1 h followed with filtration.
The combined organic layers from extractions with EtOAc:/hexane
1:1 (3 × 30 mL) were washed with water (2 × 20 mL) and saturated
NaCl (20 mL), dried over Na2SO4, and purified by flash chroma-
tography, eluting with a gradient from petroleum ether/EtOAc 6:1
to petroleum ether/EtOAc 1:1, yielding 3 as a yellow oil (1.8 g,
66%). Typical yields varied from 62% to 79%. 1H NMR (DMSO).
2-Amino-4-methoxy-3-pentyloxybenzaldehyde (4). Iron pow-
der (0.65 g, 11.6 mmol) and concentrated HCl (0.2 mL) were added
to the solution of 3 (1.05 g, 3.7 mmol) in EtOH/AcOH/water 2:2:1
(25 mL). The reaction mixture was refluxed for 15 min and then
stirred at room temperature for 40 min, filtered, and extracted with
EtOAc (3 × 50 mL). The organic layer was washed with saturated
NaHCO3 (3 × 30 mL) and saturated NaCl (40 mL), dried over
Na2SO4, and then purified by flash chromatography using petroleum
ether/EtOAc 10:1 as eluent to produce 4 as an oily product (800
mg, 91%). Typical yields varied from 73% to 91%. 1H NMR
(CDCl3).
1
(30 mg, 38%). H and 13C NMR (DMSO); ESI-MS: m/z ) 439
(M + H) +. Anal. (C24H26N2O6‚0.18hexane) C, H, N.
7-Methoxy-2-oxo-8-pentyloxy-1,2-dihydroquinoline-3-carbox-
ylic Acid (Benzo[1,3]dioxol-5-ylmethyl)amide (9b). Prepared
according to procedures A and B starting from 6 (300 mg, 0.98
mmol) and 3,4-methylenedioxybenzylamine (0.18 mL, 1.45 mmol).
Flash chromatography with a gradient from hexane/EtOAc 3:1 to
hexane/EtOAc 1:1 as eluent yielded 9b as white crystals (165 mg,
1
38%). H and 13C NMR (CDCl3); ESI-MS: m/z ) 439 (M + H)
+. Anal. (C24H26N2O6) C, H, N.
7-Methoxy-2-oxo-8-pentyloxy-1,2-dihydroquinoline-3-carbox-
ylic Acid Benzylamide (9c). Prepared as 9b from 6 (150 mg, 0.49
mmol) and benzylamine (0.081 mL, 0.74 mmol). Flash chroma-
tography with petroleum ether/EtOAc 2:1 as eluent yielded 9c as
1
a light solid product (129 mg, 67%). H and 13C NMR (DMSO);
ESI-MS: m/z ) 395 (M + H) +. Anal. (C23H26N2O4) C, H, N.
7-Methoxy-2-oxo-8-pentyloxy-1,2-dihydroquinoline-3-carbox-
ylic Acid Phenethylamide (9d). Prepared as 9b from 6 (200 mg,
0.66 mmol) and phenethylamine (0.13 mL, 1.04 mmol). Recrystal-
lizing from methanol yielded 9d as white crystals (150 mg, 56%).
1H and 13C NMR (CDCl3); ESI-MS: m/z ) 409 (M + H) +. Anal.
(C24H28N2O4) C, H, N.
7-Methoxy-2-oxo-8-pentyloxy-1,2-dihydroquinoline-3-carbox-
ylic Acid Ethyl Ester (5). Dimethyl malonate (5.16 mL, 45.15
mmol), piperidine (4.47 mL, 45.15 mmol), and AcOH (0.1 mL)
were added to a solution of 4 (4.3 g, 18.06 mmol) in EtOH (80
mL), and the reaction mixture was refluxed under argon overnight.
After being cooled to room temperature, the mixture was treated
with water (50 mL) and extracted with EtOAc (3 × 50 mL). The
combined organic layers were washed with water (2 × 25 mL)
and saturated NaCl (50 mL), dried over Na2SO4, and purified by
flash chromatography eluting with a gradient from petroleum ether/
EtOAc 2:1 to EtOAc, yielding 5 as white crystals (4.9 g, 85%).
7-Methoxy-2-oxo-8-pentyloxy-1,2-dihydroquinoline-3-carbox-
ylic Acid (3-phenylpropyl)amide (9e). Prepared as 9b from 6 (150
mg, 0.49 mmol) and 3-phenylpropylamine (0.105 mL, 0.74 mmol).
Flash chromatography with petroleum ether/EtOAc 2:1 as eluent
1
yielded 9e as a white solid product (134 mg, 65%). H and 13C
NMR (DMSO); ESI-MS: m/z ) 423 (M + H) +. Anal. (C25H30N2O4)
C, H, N.
7-Methoxy-2-oxo-8-pentyloxy-1,2-dihydroquinoline-3-carbox-
ylic Acid [2-(4-nitrophenyl)ethyl]amide (9f). Prepared as 9b from
6 (300 mg, 0.98 mmol) and 2-(4-nitrophenyl)ethylamine hydro-
chloride (298 mg, 1.47 mmol). Flash chromatography using
1
Typical yields varied from 80% to 85%. H NMR (DMSO).
7-Methoxy-2-oxo-8-pentyloxy-1,2-dihydroquinoline-3-carbox-
ylic Acid (6). To a solution of 5 (1.26 g, 3.78 mmol) in EtOH (40

2026 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Raitio et al.
petroleum ether/EtOAc 1:2 as eluent yielded 9f as a yellow solid
product (140 mg, 32%). 1H and 13C NMR (CDCl3); ESI-MS: m/z
) 454 (M + H) +. Anal. (C24H27N3O6‚0.18 hexane‚0.16EtOAc)
C, H, N.
clone) in Ham’s F-12 nutrient mixture (Euroclone), containing 10%
fetal calf serum (Euroclone), 100 U/mL penicillin, and 100 µg/mL
streptomycin (Euroclone) at 37 °C in a humidified atmosphere of
5% CO2/95% air. CHO cell membranes were prepared as previously
described.17 Rat cerebellar membranes were prepared as previously
described.20All animal experiments were conducted according to
the Declaration of Helsinki and were approved by the local ethics
committee.
7-Methoxy-2-oxo-8-pentyloxy-1,2-dihydroquinoline-3-carbox-
ylic Acid [2-(4-chlorophenyl)-ethyl]-amide (9g). Prepared as 9b
from 6 (150 mg, 0.49 mmol) and 2-(4-chlorophenyl)ethylamine
(0.103 mL, 0.74 mmol). Purification by flash chromatography using
petroleum ether/EtOAc 1:1 as eluent yielded 9g as a whitish solid
[35S]GTPγS-Binding Assays. Incubations with CHO cell mem-
branes were carried out as previously described.11 Briefly, the final
incubation contained 5 µg of membrane protein, 55 mM Tris-HCl
pH 7.4, 1.1 mM EDTA, 100 mM NaCl, 5 mM MgCl2, 0.5% (w/v)
BSA, 11 µM GDP, ∼150 pM [35S]GTPγS with the studied ligands
in ethanol (final concentration 1%, v/v) or in DMSO (final
concentration 0.5%, v/v). Incubations for measuring CB1 receptor
activities with rat cerebellar membranes were conducted under
optimized conditions, essentially as previously described.21
Data Analysis. The experimental data from at least three
independent experiments performed in duplicate have been ex-
pressed as means and variability as SEM. Values for IC50, maximal
effects (Imax) and for the SEM have been calculated by nonlinear
regression analysis using the equation for a sigmoidal concentra-
tion-response curve (GraphPad Prism 4). Statistical differences
between groups have been compared using one-way analysis of
variance (ANOVA) followed by Tukey’s multiple comparison test
with P value < 0.05 considered to be significant.
1
product (95 mg, 44%). H and 13C NMR (DMSO); ESI-MS: m/z
) 443 (M + H) +. Anal. (C24H27N2O4Cl) C, H, N.
7-Methoxy-2-oxo-8-pentyloxy-1,2-dihydroquinoline-3-carbox-
ylic Acid [2-(4-hydroxyphenyl)ethyl]amide (9h). Prepared as 9b
from 6 (150 mg, 0.49 mmol) and tyramine hydrochloride (127 mg,
0.73 mmol). Flash chromatography with petroleum ether/EtOAc
2:3 as eluent yielded 9h as a whitish solid product (112 mg, 54%).
1H and 13C NMR (CDCl3); ESI-MS: m/z ) 425 (M + H)+. Anal.
(C24H28N2O5‚0.12H2O) C, H, N.
7-Methoxy-2-oxo-8-pentyloxy-1,2-dihydroquinoline-3-carbox-
ylic Acid [2-(4-aminophenyl)ethyl]amide (9i). Concentrated HCl
(0.1 mL) and Fe-powder (0.65 g, 11.6 mmol) were added to the
solution 9f (110 mg, 0.24 mmol) in EtOH/AcOH/water 2:2:1 (20
mL). The reaction mixture was refluxed for 15 min and stirred at
room temperature for 1 h. The mixture was filtered, and the filtrate
was extracted with EtOAc (3 × 20 mL). The organic layer was
washed with saturated NaHCO3 (3 × 15 mL) and saturated NaCl
(20 mL), dried over Na2SO4, and then purified by flash chroma-
tography using petroleum ether/EtOAc 1:2 as eluent yielding 9i
(59 mg, 58%).1H and 13C NMR (CDCl3); ESI-MS: m/z ) 424
(M + H) +. Anal. (C24H27N3O6‚0.75H2O) C, H, N.
7-Methoxy-2-oxo-8-pentyloxy-1,2-dihydroquinoline-3-carbox-
ylic Acid [2-(3,4-dihydroxyphenyl)ethyl]amide (9j). Prepared as
9b from 6 (200 mg, 0.66 mmol) and dopamine (150 mg, 0.98
mmol). Flash chromatography eluting with 2% 2-propanol in DCM
yielded 9j as a white solid product (62 mg, 21%). 1H and 13C NMR
(DMSO); ESI-MS: m/z ) 441 (M + H) +. Anal. (C24H28N2O6) C,
H, N.
3,4-Dihydroxybenzylamine. BBr3 (0.40 mL, 4.23 mmol) was
added to a solution of 3,4-dimethoxybenzylamine in anhydrous
DCM (15 mL) at -78 °C. The reaction mixture was stirred at room
temperature under argon for 24 h, water (10 mL) was added
dropwise at 0 °C, and the aqueous layer was washed with DCM (3
× 20 mL). The aqueous layer was evaporated, and the brown solid
crude product was dried under vacuum. The reaction was assumed
to have proceeded quantitatively, and the product (670 mg) was
not separated from the mainly inorganic impurities.
Acknowledgment. The authors are grateful to Ms. Miia
Reponen, Ms. Minna Glad, Ms. Katja Tiainen, Ms. Helly
Rissanen, and Ms. Tiina Koivunen for their outstanding technical
assistance. We also thank Outi Salo, M.Sc (Pharm.) for her
consulting regarding SARs. This study was financially supported
by the National Technology Agency of Finland, the Emil
Aaltonen Foundation and the Academy of Finland (grant #
107300).
Supporting Information Available: Elemental analysis data
and spectroscopic data. This material is available free of charge
via the Internet at http://pubs.acs.org.
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