Pharmacomodulations around the 4-oxo-1,4-dihydroquinoline-3-carboxamides, a class of potent CB2-selective cannabinoid receptor ligands: Consequences in receptor affinity and functionality

Article abstract of DOI:10.1021/jm070387h

CB₂ receptor selective ligands are becoming increasingly attractive drugs due to the potential role of this receptor in several physiopathological processes. Thus, the development of our previously described series of 4-oxo-1,4-dihydroquinoline-3-carboxamides was pursued with the aim to further characterize the structure-affinity and structure-functionality relationships of these derivatives. The influence of the side chain was investigated by synthesizing compounds bearing various carboxamido and keto substituents. On the other hand, the role of the quinoline central scaffold was studied by synthesizing several 6-, 7-, or 8-chloro-4-oxo-1,4-dihydroquinolines, as well as 4-oxo-1,4-dihydronaphthyridine and 4-oxo-1,4-dihydrocinnoline derivatives. The effect of these modifications on the affinity and functionality at the CB₂ receptor was studied and allowed for the characterization of new selective CB₂ receptor ligands.

Full text of DOI:10.1021/jm070387h

J. Med. Chem. 2007, 50, 5471-5484
5471
Pharmacomodulations around the 4-Oxo-1,4-dihydroquinoline-3-carboxamides, a Class of
Potent CB2-Selective Cannabinoid Receptor Ligands: Consequences in Receptor Affinity and
Functionality
Eric Stern,†,‡ Giulio G. Muccioli,‡ Barbara Bosier,‡ Laurie Hamtiaux,‡ Re´gis Millet,† Jacques H. Poupaert,‡
Jean-Pierre He´nichart,† Patrick Depreux,† Jean-Franc¸ois Goossens,§ and Didier M. Lambert*,‡
Institut de Chimie Pharmaceutique Albert Lespagnol, UniVersite´ de Lille 2, EA 2692, 3 rue du Pr. Laguesse, B.P. 83, F-59006 Lille, France,
Unite´ de Chimie Pharmaceutique et de Radiopharmacie, Ecole de Pharmacie, Faculte´ de Me´decine, UniVersite´ catholique de LouVain, 73
aVenue E. Mounier UCL-CMFA (7340), B-1200 Bruxelles, Belgium, and Laboratoire de Chimie Analytique, EA 4034, Faculte´ des Sciences
Pharmaceutiques et Biologiques, UniVersite´ de Lille 2, 3 rue du Pr. Laguesse, B.P. 83, F-59006 Lille, France
ReceiVed April 3, 2007
CB2 receptor selective ligands are becoming increasingly attractive drugs due to the potential role of this
receptor in several physiopathological processes. Thus, the development of our previously described series
of 4-oxo-1,4-dihydroquinoline-3-carboxamides was pursued with the aim to further characterize the structure-
affinity and structure-functionality relationships of these derivatives. The influence of the side chain was
investigated by synthesizing compounds bearing various carboxamido and keto substituents. On the other
hand, the role of the quinoline central scaffold was studied by synthesizing several 6-, 7-, or 8-chloro-4-
oxo-1,4-dihydroquinolines, as well as 4-oxo-1,4-dihydronaphthyridine and 4-oxo-1,4-dihydrocinnoline
derivatives. The effect of these modifications on the affinity and functionality at the CB2 receptor was
studied and allowed for the characterization of new selective CB2 receptor ligands.
Introduction
modeling studies of a novel series of 4-oxo-1,4-dihydroquino-
line-3-carboxamide derivatives acting as selective and potent
CB2 receptor agonists (e.g., N3-(1-(3,5-dimethyl)adamantyl)-
4-oxo-1-pentyl-1,4-dihydroquinoline-3-carboxamide (ALICB179,
3), Chart 1).19
The cannabinoid CB2 receptor, a G protein-coupled receptor
(GPCR), is expressed throughout the immune cell system1,2 and
was recently described in the central nervous system (CNS)
under both pathological3 and physiological conditions.4 Along
with the CB1 cannabinoid receptor,5 they represent so far the
two cloned GPCRs of the endocannabinoid system (ECSa). The
ECS comprises, in addition to the two cannabinoid receptors,
several endocannabinoids (2-arachidonoylglycerol, N-arachi-
donoylethanolamine) as well as the enzymes responsible for their
production and inactivation (fatty acid amide hydrolases I and
II, monoglyceride lipase, and N-acylethanolamine acid ami-
dase).6 The therapeutic potential of modulating the activity of
the ECS is now well established, with selective receptor ligands
or enzyme inhibitors being tested clinically or preclinically.7,8
The interest for the CB2 cannabinoid receptors has been growing
recently, with proposed indications in alleviating pain9,10 and
inflammation,11,12 cough,13 and dermatitis14 and treating cancers
of different origins (glioblastomas,15 lymphomas16). In light of
these applications, the development of potent and selective CB2
ligands results in powerful therapeutic tools devoid of CB1
receptor-mediated psychotropic side effects. Quite surprisingly,
only a limited number of CB2 receptor selective ligands was
described so far. SR-144528 (1), JTE-907, and Sch225336 are
antagonist/inverse agonists based on a pyrazole, an oxoquinoline,
and a phenylsulfonyl scaffold, respectively. On the other hand,
CB2 receptor agonists were developed based on diterpenes
(HU-308, JWH-133 (2)) and indoles (AM1241, JWH-007,
GW405833).17,18 Along these lines, we previously described the
synthesis, pharmacological characterization, and molecular
Here, we report the synthesis and structure-activity relation-
ship studies of new 4-oxo-1,4-dihydroquinoline derivatives that
confirm the CB2 selectivity of this class of compounds as well
as the molecular modeling studies previously reported.19 As
shown in Chart 1, various structural modifications were realized
using the previously reported 3 as template. Replacement of
the 4-oxo-quinoline moiety by different naphthyridine isomers
was also investigated (Chart 2). The new chiral compounds
synthesized here confirm the described enantioselectivity of the
interaction.19 Interestingly enough, the [35S]-GTPγS data showed
that small changes in the position of the substituents around
the 4-oxo-1,4-dihydroquinoline core result in modifications of
the compounds functionality, suggesting that, similarly to the
pyrazole derivatives, both agonist and antagonists/inverse agonist
series can be developed from the 4-oxo-1,4-dihydroquinoline
derivatives.
Results
Chemistry. The synthetic route to obtain the target 4-oxo-
1,4-dihydroquinoline-3-carboxamide derivatives 10-25 was
previously described19 and is briefly outlined in Scheme 1.
Compounds 10-25 were obtained by a coupling reaction
between selected amines and 4-oxo-1,4-dihydroquinoline-3-
carboxylic acids 9a-i, using polystyrene-supported 1-hydroxy-
1H-benzotriazole (HOBt) as coupling reagent, obtained in three
steps following Gould-Jacobs’ procedure.20
The target 2-substituted-4-oxo-1,4-dihydroquinoline-3-car-
boxamide derivatives 29-35 were obtained by a similar
coupling reaction between amines and the desired 2-substituted
quinoline-3-carboxylic acids 28a,b, previously synthesized in
three steps (Scheme 2). N-Alkylation of isatoic anhydride with
1-bromopentane in anhydrous N,N-dimethylformamide (DMF),
in the presence of sodium hydride, gave 1-n-pentyl-1H-benzo-
* To whom correspondence should be addressed. Phone: +32-2-764-
7347. Fax: +32-2-764-7363. E-mail: didier.lambert@uclouvain.be.
† Institut de Chimie Pharmaceutique Albert Lespagnol, Universite´ de
Lille 2.
‡ Universite´ catholique de Louvain.
§ Laboratoire de Chimie Analytique, Universite´ de Lille 2.
a Abbreviations: ECS, endocannabinoid system; [35S]-GTPγS, [35S]-
guanosine-5′-(γ-thio)-triphosphate; hCB2, human cannabinoid receptor 2;
CHO, chinese hamster ovarian cells.
10.1021/jm070387h CCC: $37.00 © 2007 American Chemical Society
Published on Web 10/04/2007

5472 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
Stern et al.
Chart 1. Structural Modifications Considered on the 4-Oxo-1,4-dihydroquinoline-4-carboxamide Templatea
a Our lead compound, N3-(1-(3,5-dimethyl)adamantyl)-4-oxo-1-pentyl-1,4-dihydroquinoline-3-carboxamide (ALICB179, 3), is shown.
Chart 2. General Structures of 4-Oxo-1,4-dihydronaphthyridine,
4-Oxo-1,4-dihydrocinnoline, and
3-Aroyl-4-oxo-1,4-dihydroquinoline Derivatives
Chloro-4-oxo-1-pentyl-1,4-dihydroquinoline-3-carboxamide
derivatives 52-58 were synthesized by a general procedure
similar to the method described earlier for the synthesis of 10-
25 (Scheme 5) but starting from the corresponding chloroa-
niline.28 Enantiopure forms of 55 and 56 (noted 59-62) were
obtained by enantiomeric separation with HPLC on polysac-
charide-based chiral stationary phases (amylase carbamate
derivatives, Chiralpak AD) used in normal phase mode by
adapting an analytical procedure previously described.29
[1,5]-Naphthyridine-3-carboxamide (67) and [1,6]-naphthy-
ridine-3-carboxamide (68) derivatives (Scheme 6) were syn-
thesized by a general procedure similar to the synthetic approach
used to obtain 4-oxo-1,4-dihydroquinoline-3-carboxamide de-
rivatives 10-25 but starting from the corresponding aminopy-
ridine.30
[d][1,3]oxazine-2,4-dione (26).21 Selected â-ketoesters reacted
with 26 in anhydrous DMF in the presence of sodium hydride,
yielding the desired 4-oxo-1-pentyl-2-substituted-1,4-dihydro-
quinoline-3-carboxylic acid ethyl esters 27a,b.22 Hydrolysis in
10% aqueous NaOH resulted in the corresponding 4-oxo-1-
pentyl-2-substituted-1,4-dihydroquinoline-3-carboxylic acids
28a,b.23
As shown in Scheme 3, “retro-amide” isomers 38-40 were
synthesized starting from 4-oxo-1-pentyl-1,4-dihydroquinoline-
3-carboxylic acid 9a using a Curtius reaction in the presence
of diphenylphosphorylazide (DPPA).24 This reaction was con-
ducted in tert-butanol in the presence of potassium tert-
butanolate to obtain the corresponding amine derivative pro-
tected by a butoxycarbonyl moiety (36), which is easily cleaved
in acidic conditions. Treatment of 36 with hydrochloric acid in
isopropanol yielded the 3-amino-4-oxo-1,4-dihydroquinoline
hydrochloride 37. Addition of the desired acyl chloride, under
classical conditions, lead to the corresponding carboxamide
derivatives 38-40.
3-Aminomethyl-4-oxo-1-pentyl-1,4-dihydroquinoline deriva-
tives 42-44 were also synthesized starting from the carboxylic
acid 9a (Scheme 3). Compound 9a was first converted into its
carbaldehyde analogue 41, using tributyltin hydride in the
presence of tetrakis(triphenylphosphine)palladium(0) in dry
toluene,25 which was then engaged into a reductive amination
reaction with selected amines in the presence of sodium
cyanoborohydride in dry methanol to afford the target 3-ami-
nomethyl-quinoline derivatives (42-44).26
1-Pentyl-4-thioxo-1,4-dihydroquinoline-3-carboxamide de-
rivative 47 was obtained from 1-pentyl-4-thioxo-1,4-dihydro-
quinoline-3-carboxylic acid 46 using the same coupling proce-
dure (Scheme 4). To obtain the 4-thioxo-1,4-dihydroquinoline
nucleus, the 4-oxo-1-pentyl-1,4-dihydroquinoline-3-yl carboxylic
acid ethyl ester 8a was treated with phosphorus pentasulfide in
pyridine furnishing its 4-thioxo isomer 45 with very good
yield.27 Hydrolysis by lithium hydroxide in a tetrahydrofurane/
water mixture resulted in the corresponding 1-pentyl-4-thioxo-
1,4-dihydroquinoline-3-carboxylic acid 46.
The [1,8]-naphthyridine derivative 71 was synthesized using
the three-step procedure outlined in Scheme 7. 4-Oxo-1-pentyl-
1,4-dihydro-[1,8]-naphthyridine-3-carboxylic acid ethyl ester 69
was first formed using a one-pot method involving 2-chloroni-
cotinoyl chloride and dimethylaminoacrylate in dry acetonitrile.
This first step provided an acyclic intermediate that was
converted into the desired [1,8]-naphthyridine derivative 69 by
addition of n-pentylamine.31 Hydrolysis in aqueous NaOH
yielded the corresponding 4-oxo-1-pentyl-1,4-dihydro-[1,8]-
naphthyridine-3-carboxylic acid 70, which was coupled with
1-amino-3,5-dimethyladamantane to afford the 4-oxo-1-pentyl-
1,4-dihydro-[1,8]-naphthyridine-3-carboxamide 71.
The cinnoline derivative 76 was obtained using a five-step
synthetic route (Scheme 8). Diethylmalonate was added to a
freshly synthesized phenyl diazonium chloride, obtained by
action of sodium nitrite on aniline hydrochloride, in the presence
of sodium acetate in ethanol to give the malonate derivative
72, which was converted into its diacid analogue 73 by
hydrolysis in aqueous NaOH.32 In a one-pot procedure, the
diacid 73 was quantitatively converted into diacyl chloride using
thionyl chloride in 1,2-dichlorobenzene. After evaporation of
the excess of thionyl chloride, and without isolation of the diacyl
chloride intermediate, titanium tetrachloride was added to
conclude the cyclization and to furnish the 4-oxo-1,4-dihydro-
cinnoline-3-carboxylic acid 74. The introduction of the amide
substituent was performed using the desired amine and O-ben-
zotriazol-1-yl-tetramethyluronium hexafluorophosphate (HBTU)
as coupling reactant using triethylamine in dry DMF, affording
the 4-oxo-1,4-dihydrocinnoline-3-carboxamide 75.33 N-Alky-
lation of 75 with 1-bromopentane in anhydrous DMF, in the
presence of sodium hydride, gave the desired 4-oxo-1-pentyl-
1,4-dihydrocinnoline-3-carboxamide 76.

CB2-SelectiVe Cannabinoid Receptor Ligands
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22 5473
Scheme 1. Synthesis of the 4-Oxo-1,4-dihydroquinoline-3-carboxamide Derivatives 10-25a
a Reagents and conditions: (i) 100 °C, 91%; (ii) Ph-O-Ph, reflux, 77%; (iii) R-X, NaH, DMF, 90 °C, 50-96%; (iV) NaOH, EtOH, 100 °C, 65-90%;
(V) R′-NH2, PyBRoP, PS-HOBt (HL), DIEA, DMF, rt, 25-80%.
Scheme 2. Synthesis of the
define the structural elements required for the cannabinoid
receptor affinity of our derivatives, different pharmacomodu-
lations starting from our leading compound 3 (Ki CB2 ) 15.8
nM)19 have been carried out, as shown in Chart 1.
2-Substituted-4-Oxo-1,4-dihydroquinoline-3-carboxamide
Derivatives 29-35a
The adopted strategy was to prepare analogues by stepwise
introduction of structural modifications on positions 1-3 and
5-8 of the 4-oxo-quinoline template of compound 3. Replace-
ment of the N1-n-pentyl side chain by various aromatic, aryl-
alkyl, cycloalkyl-alkyl, or morpholino-alkyl moieties was
investigated. We previously showed that introducing a benzyl
group on the N1-nitrogen (cmpd 31 in ref 19, Ki ) 664 nM)
instead of the n-pentyl moiety resulted in a reduced affinity (44-
fold) for the CB2 cannabinoid receptor.19 Similarly, the intro-
duction of a 4-halogeno-benzyl substituent resulted in the same
reduction of affinity, as shown with 4-chloro- (11) and 4-bro-
mobenzyl (12)-substituted derivatives, which only displaced
48% and 30%, respectively, of the [3H]-CP-55,940 bound to
the hCB2 receptor. Only the 4-fluorobenzyl derivative (10)
preserves a nanomolar affinity (Ki ) 83 nM). Increasing the
distance between the phenyl and the 4-oxo-quinoline resulted
in an enhancement of the affinity as shown by 13 (phenylethyl)
and 14 (phenylpropyl), with Ki values of 333 nM and 160 nM,
respectively. Replacement of the 2-phenylethyl substituent (13)
by its nonaromatic analog 2-(cyclohexyl)ethyl resulted in a lower
affinity, as shown with compound 15, which only displaced 38%
of radioligand. Finally, the introduction of a 2-(morpholin-4-
yl)ethyl substituent (16-18), mimicking the morpholinoethyl
substituent characterizing WIN-55,212-2 (4), did not improve
the affinity. Indeed 16, with a Ki value of 221 nM, has a 14-
times lower affinity than the n-pentyl analogue 3. To confirm
the effect observed with the 2-(morpholin-4-yl)ethyl substituent,
we also synthesized compounds 17 and 18 bearing different
carboxamido substituents, 2-phenylethyl (17) and (-)-1-phe-
nylethyl (18), respectively. With these two amide substituents
too a marked decrease in affinity has been observed in
comparison with the n-pentyl analogs previously reported.19 For
example, with the (-)-1-(phenyl)ethyl substituent on the car-
boxamido link, we reported for the N1-n-pentyl derivative an
affinity of 37 nM (cmpd 32R in ref 19), whereas the Ki value
of the N1-2-(morpholin-4-yl)ethyl derivative (18) is up to 2000
nM. Taken together, these results clearly demonstrate that the
affinity is very sensitive to changes in the N1-substituent. Thus,
because the n-pentyl residue appears to be the preferred one,
the subsequent pharmacomodulations were performed while
keeping this N1-substituent constant.
a Reagents and conditions: (i) R-X, NaH, atm N2, rt, 65%; (ii) R′′-
CO-CH2-CO2Et, NaH, DMF, 120 °C, 75-76%; (iii) NaOH, EtOH,
100 °C, 70-76%; (iV) R′-NH2, PyBRoP, PS-HOBt (HL), DIEA, DMF, rt,
30-74%.
The synthetic route to obtain the target 1-alkyl-3-aroyl-1,4-
dihydroquinolin-4-one derivatives 80-98 was previously de-
scribed34 and is outlined in Scheme 9. The key step of the
1-alkyl-3-aroyl-1,4-dihydroquinolin-3-one synthesis was the
cyclization reaction of 2-((Z)-3-oxo-3-aryl-propenylamino)-
benzoic acid methyl esters 78a-g leading to the 3-aroyl-
quinolin-4-one derivatives 79a-g. This reaction was carried out
by refluxing 78a-g in a methanol/phenyl ether mixture (1:8
ratio) in the presence of sodium methanolate. The final
N-alkylation leading to the derivatives 80-98 was performed
using halogenoalkyl derivatives in dry DMF in the presence of
sodium hydride.
Pharmacology. Structure-Affinity Relationships. The
4-oxo-1,4-dihydroquinoline, 4-oxo-1,4-dihydronaphthyridine,
and 4-oxo-1,4-dihydrocinnoline derivatives were first screened
at 10 µM for their affinity toward the hCB2 and hCB1
cannabinoid receptors in a competitive binding experiment as
previously described.19 hCB2-CHO or hCB1-CHO cell mem-
branes were used in conjunction with [3H]-CP-55,940 and [3H]-
SR-141716A as radioligands for the hCB2 and hCB1 cannab-
inoid receptors, respectively. The results expressed as the
displacement percentages of the radioligand from its binding
site are summarized in Tables 1-3. The Ki values at the CB2
cannabinoid receptor were then determined for the compounds
exhibiting a displacement of the specific binding superior to
60%. Taken together, these results indicated that the tested
compounds possess fair to good selectivity for the hCB2
cannabinoid receptors.
The second step in our strategy was to study the effect of
substituting the position-2 of the 4-oxo-quinoline template. A
methyl or a phenyl group were introduced at this position leading
to compounds 29 and 30, which possess Ki values of 200 nM
and 119 nM, respectively. It seemed, therefore, that the
Our initial studies showed that 4-oxo-1,4-dihydroquinoline-
3-carboxamide derivatives constitute a suitable template in the
design of potent and selective CB2 cannabinoid ligands.19 To

5474 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
Stern et al.
Scheme 3. Synthesis of the Carboxylic Acid (4-Oxo-1,4-dihydroquinolin-3-yl)-amide Derivatives 38-40 and the
3-Aminomethyl-1H-quinolin-4-one Derivatives 42-44a
a Reagents and conditions: (i) DPPA, t-BuOK, t-BuOH, atm N2, reflux, 65%; (ii) HCl 6 N, isoOH, rt, 70%; (iii) R′-CO-Cl, Et3N, CH2Cl2, atm N2, 0
°C, 35-56%; (iV) (a) SOCl2, CH2Cl2; (b)HSnBu3, Pd(PPh3)4, toluene, atm N2, rt, 65%; (V) (a) R′-NH2, Et3N, MeOH, atm N2, 50 °C; (b) NaBH3CN, Et3N,
MeOH, atm N2, 50 °C, 32-35%.
Scheme 4. Synthesis of the
The two enantiopure forms of 23, referenced as 24 and 25,
were obtained as recently described29 and showed an affinity
of 14.0 nM and 202 nM, respectively. These results highlight a
stereoselective ligand-receptor interaction regardless of the
nature of the amide substituent. The affinity of 24 for the CB1
receptor was also determined to further assess the selectivity
of this potent CB2 cannabinoid receptor ligand. A Ki value of
416 nM, resulting in a selectivity ratio for the CB2 cannabinoid
receptor of 30, has been determined.
4-Thioxo-1,4-dihydroquinoline-3-carboxamide Derivative 47a
To investigate the hypothesis previously suggested by mo-
lecular modeling studies, that is, 3 interacts with the hCB2
receptor through a combination of hydrogen bonds with the
residue Ser-193 and aromatic/hydrophobic interactions,19 com-
pounds with different modifications of the carboxamide link
were synthesized. The amide link was first replaced by its “retro-
amide” isomer, giving compound 38. This modification did not
elicit a clear effect on the affinity, as 3 and 38 possess affinities
of the same magnitude (Ki value of 15.8 nM and 25.5 nM,
respectively). The carbonyl group of the amide link was also
replaced by a methylene, resulting in “reduced-analogues” of
the amide (42-44). This modification induced in a dramatic
reduction in affinity (up to 100-fold), as illustrated by compound
42 (Ki ) 1670 nM). These results support the importance of
the amide/Ser-193 hydrogen bond. The similar affinities found
for compounds 38 and 3 could be explained by the establishment
of a hydrogen bond between the amide hydrogen atom of 3
and the Ser-193 hydroxyl oxygen atom.
a Reagents and conditions: (i) P4S10, pyridine, reflux, 94%; (ii) LiOH,
THF/H2O, rt, 83%; (iii) 1-amino-3,5-dimethyladamantane, PyBRoP, PS-
HOBt (HL), DIEA, DMF, rt, 60%.
introduction of a substituent, leading to a marked reduction in
affinity (compared to 3), is unfavorable. To confirm this
hypothesis, compounds 31-35 have been synthesized and are
characterized by the presence of a substituent in position-2 and
by selected carboxamido substituents known to impart good
affinity in our series. In all cases, the affinity was strongly
decreased. Taken together, these data demonstrate that the
introduction of a substituent in position 2 results in a drastic
reduction of the affinity for the CB2 cannabinoid receptor.
Optically active 4-oxo-1,4-dihydroquinoline-3-carboxamide
derivatives previously reported showed a stereoselectivity in the
binding at the hCB2 receptor.19 In accordance with our previous
results, the eutomers of this novel series exhibit about 10-fold
higher affinity than the distomers. For instance, (+)-N3-(1-
(1,2,3,4-tetrahydronaphthyl))-4-oxo-1-pentyl-1,4-dihydroquino-
line-3-carboxamide (19) possesses a Ki value of 41.7 nM,
whereas the affinity of the (-)-enantiomer (20) is markedly
reduced (Ki ) 1010 nM). Because the adamantyl substituent
was shown to favor the affinity, we decided to combine this
structural requirement with a chiral center between the car-
boxamido function and the 1-adamantyl group (23). The steric
constraint and the distance imposed by the methyl group on
the methylene spacer does not affect the affinity as shown by
compound 23 (Ki ) 28.5 nM), which was first assayed as
racemate. The adamantylmethyl derivative (22) was also
synthesized as the comparison compound and showed a slight
decrease of affinity, with a Ki value of 50.6 nM.
Recently, Muccioli and co-workers showed, for some selec-
tive CB1 imidazolidine-2,4-diones, that the introduction of a
sulfur atom resulted in an enhanced affinity.35-37 Here, the
oxygen atom replacement by a sulfur atom, yielding the
4-thioxo-quinoline derivative (47), did not modify the affinity
for the hCB2 receptor because 47 possesses a Ki value of 18.2
nM compared to 15.8 nM for the oxo analogue (3).
It is well-established that the presence of halogen atoms on
the structure of cannabinoid ligands represents an important
requirement to increase the affinity (e.g. 1, tricyclic pyrazoles,
AM630). Besides the effect of introducing such halogen on the
N1-benzyl moiety (10-12), a set of chloro analogs of 3 were
synthesized to study the effect of introducing a halogen atom
on the 4-oxo-quinoline moiety (52-58, Table 2). Introduction
of a chlorine in position 6 (52) or 8 (54) resulted in a moderate
reduction of the affinities, Ki ) 54.6 nM or Ki ) 27.4 nM,
respectively. However, the introduction of a chlorine atom in

CB2-SelectiVe Cannabinoid Receptor Ligands
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22 5475
Scheme 5. Synthesis of the Chloro-4-oxo-1,4-dihydroquinoline-3-carboxamide Derivatives 52-58a
a Reagents and conditions: (i) 100 °C, 70-91%; (ii) Ph-O-Ph, reflux, 72-83%; (iii) R-X, NaH, DMF, 90 °C, 50-96%; (iV) NaOH, EtOH, 100 °C,
65-90%; (V) R′-NH2, PyBRoP, PS-HOBt (HL), DIEA, DMF, rt, 25-80%.
Scheme 6. Synthesis of the 4-Oxo-1,4-dihydro-[1,5]-naphthyridine-3-carboxamide Derivative 67 and the
4-Oxo-1,4-dihydro-[1,6]-naphthyridine-3-carboxamide Derivative 68a
a Reagents and conditions: (i) toluene, reflux, 90-95%; (ii) Ph-O-Ph, reflux, 85-90%; (iii) C5H11-Br, NaH, DMF, 90 °C, 85%; (iV) NaOH, EtOH,
100 °C, 70-75%; (V) 1-amino-3,5-dimethyladamantane, PyBRoP, PS-HOBt (HL), DIEA, DMF, rt, 25-30%.
Scheme 7. Synthesis of the
6-chloro derivative 59 (Ki values of 26.1 nM and 235 nM,
respectively). Because 61 displaced over 90% of the radioligand
bound to the CB1 receptor, the Ki value was also determined
and found to be of 615 nM, over 1 order of magnitude higher
than the CB2 receptor affinity.
4-Oxo-1,4-dihydro-[1,8]-naphthyridine-3-carboxamide Derivative
71a
Since a recent series of [1,8]-naphthyridine were published
as potent CB2-selective cannabinoid receptor ligands,38 the
influence of the second nitrogen atom present in the naphthy-
ridine nuclei was investigated with the syntheses of the [1,5]-,
[1,6]-, and [1,8]-naphthyridine derivatives of 3 (compounds 67,
68, and 71, respectively) as well as the cinnoline derivative 76
(Table 2). Binding data revealed that the introduction of a
nitrogen in position 5 (67, Ki ) 259 nM) resulted in a decrease
of the affinity (16-fold), whereas the [1,6]- (68, Ki ) 30.9 nM)
and [1,8]-naphthyridines (71, Ki ) 23.5 nM) gave affinities of
the same magnitude compared to that of 3. Finally, the cinnoline
76 exhibits a Ki value of 93.0 nM.
a Reagents and conditions: (i) (a) Et3N, acetonitrile, atm N2, reflux; (b)
C5H11-NH2, Et3N, acetonitrile, atm N2, reflux, 60%; (ii) NaOH, EtOH,
100 °C, 74%; (iii) 1-amino-3,5-dimethyladamantane, PyBRoP, PS-HOBt
(HL), DIEA, DMF, rt, 40%.
We next decided to keep the 4-oxo-1,4-dihydroquinoline
template constant and to replace the carboxamido link by a
ketone link, similar to the one present in 4. As for the amide
series, various structural modifications have been introduced,
starting with the substituent linked on the quinoline N1-position.
Different alkyl, aryl-alkyl, and cycloalkyl-alkyl were branched
on this new 3-(naphth-1-oyl)-4-oxo-1,4-dihydroquinoline tem-
plate. As shown in Table 3, the substitution of the N1-nitrogen
was mandatory to bind to the CB2 cannabinoid receptor because
79a (at 10 µM) only displaced 31% of the radioligand from its
position 7 of the quinoline template appears to be the more
favorable substitution, with 53 showing a Ki value of 5.3 nM.
The structure-affinity relationships on the introduction of the
chlorine were also confirmed with other carboxamido-substituted
quinoline derivatives (55-62). For instance, when considering
the (-)-1-(1-adamantyl)ethyl derivatives 59 and 61, the 7-chloro
derivative 61 showed an increased affinity compared to the

5476 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
Stern et al.
Scheme 8. Synthesis of the 4-Oxo-1,4-dihydro-cinnoline-3-carboxamide Derivative 76a
a Reagents and conditions: (i) (a) HCl 12 N, rt, quant; (b) NaNO2, H2O, 0 °C; (c) diethylmalonate, AcONa, H2O, EtOH, 0 °C to rt, 95%; (ii) NaOH,
EtOH, 100 °C, 81%; (iii) (a) SOCl2, 1,2-dichlorobenzene, 70 °C; (b) TiCl4, 1,2-dichlorobenzene, 90 °C, 65%; (iV) 1-amino-3,5-dimethyladamantane, HBTU,
Et3N, DMF, rt, 68%; (V) C5H11-Br, NaH, DMF, 90 °C, 40%.
Scheme 9. Synthesis of the 3-Aroyl-1,4-dihydroquinolin-4-one
Derivatives 80-98a
nM). Similarly, replacing the naphthyl moiety by a phenyl one
(92, Ki ) 446 nM) did not improve the affinity. And finally,
substitution of the phenyl with polar substituents (93-96)
induced in all cases a decrease in the affinity toward the CB2
cannabinoid receptor. Finally, as the ketone derivatives possess
lower affinities than their carboxamide analogues, it appears
that, in this quinoline chemical series, the presence of an H-bond
donor/acceptor group results in increased CB2 affinity.
Structure-Functionality Relationships. The derivatives
functionality was investigated by using a [35S]-GTPγS binding
assay as previously described.37 This assay constitutes a
functional measure of the direct interaction between the receptor
and the G protein, the first step in the activation of GPCRs.
Although the first series of 4-oxo-quinoline-3-carboxamide
derivatives behaved as agonists,19 quite unexpected results were
obtained with the new derivatives described here, which
comprised agonists, antagonists, and inverse agonists compounds
(Table 4).
a Reagents and conditions: (i) (a) EtONa, Et2O, rt; (b) C2H5-Br, DMF,
rt, 39-85%; (ii), methyl anthranylate, ZnCl2, THF, rt, 40-84%; (iii),
MeONa, MeOH/Ph-O-Ph (1/8), 120 °C or reflux, 37-66%; (iV) R-X,
NaH, DMF, 90 °C, 17-68%.
binding site. A first set of 11 compounds variously substituted
in position 1 was synthesized (80-90). In this series, as for the
carboxamide derivatives, the highest CB2 affinity was obtained
with an n-pentyl chain (81; Ki ) 154 nM). This compound
possesses a strong CB2 selectivity because it only displaced 56%
of the [3H]-SR-141716A CB1 specific binding at 10 µM.
Replacement of this alkyl chain by a benzyl (83, Ki ) 656 nM)
or a benzyl substituted by a chlorine (85, Ki ) 1120 nM) or
bromine (86, Ki ) 966 nM) resulted in reduction of the CB2
affinity. Note that the 4-fluorobenzyl derivative possesses a
higher affinity than the other benzyl derivatives (84, Ki ) 225
nM). In addition, the presence of a 4-halogenated-benzyl moiety
on the N1-position of the quinoline nucleus results in an
increased displacement of the [3H]-SR-141716A CB1 specific
binding (e.g., 84 at 10 µM displaced 70% of [3H]-SR-141716A
binding). Thus, the Ki value of 84 at the CB1 receptor was
determined and found to be higher than 2000 nM. However,
the significance of this selectivity is reduced by the lower CB2
affinities of these ketone derivatives.
Although increasing the spacer length between the quinoline
moiety and the phenyl from 1 (cmpd 31 in ref 19) to 3 (14)
methylenes resulted in an enhanced affinity, the functionality
of the resulting 3-phenylpropyl is also affected because 14
behaved as a neutral antagonist rather than as agonist. An
interesting switch in functionality was also observed when a
phenyl substituent was introduced in position 2 of the quinoline.
Indeed, 30 and 35 both behaved as inverse agonists at the CB2
receptor decreasing the [35S]-GTPγS binding by 75%, while
their unsubstituted analogues, 3039 and 28 in ref 19, activate
the receptor. Similarly, compound 47, characterized by a
4-thioxoquinoline template, significantly decreased the [35S]-
GTPγS binding (65% compared to control). Analogously, the
introduction of a chlorine in position 6 (52) or 7 (53) of the
N3-(1-(3,5-dimethyl)adamantyl)-quinoline-3-carboxamide moi-
ety completely modifies the derivatives functionality, as these
two compounds strongly reduced the [35S]-GTPγS binding, thus
acting as inverse agonists of the CB2 receptor (49% and 51%
of control, respectively). On the contrary, the 8-chloro-
substituted 54 retained an agonist profile (187% of control).
The introduction of an additional nitrogen in the quinoline
nucleus, leading to [1,5]-, [1,6]-, and [1,8]-naphthyridine (67,
68, and 71, respectively), did not influence the functionality
because all three compounds kept their agonist profile, with
some changes in the efficacy though because 68 and 71 act as
partial agonists. However, introduction of the nitrogen in
position 2 as for the cinnoline 76, resulted in a neutral antagonist
because no significant stimulation of the [35S]-GTPγS binding
was observed.
Increasing the benzyl methylene spacer into an ethyl (87) or
a propyl (88) did not result in an enhancement of the affinity.
Replacement of the aromatic moiety of 2-(phenyl)ethyl by a
2-cyclohexylethyl (89, Ki ) 1670 nM) or by a 2-(morpholin-
4-yl)ethyl (90) led to a loss of affinity. By keeping the N1-n-
pentyl side chain constant, a library of compounds with various
aroyl substituents in position 3 of the quinoline template has
been developed. The 1-naphthyl (81, Ki ) 154 nM) moiety
proved to be more favorable compared to its 2-naphthyl isomer
(91, Ki ) 1288 nM) or the bulkier 9-anthracenyl (98, Ki ) 1500

CB2-SelectiVe Cannabinoid Receptor Ligands
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22 5477
Table 1. Structure and Binding Data of Compounds 10-25, 29-35, 38-40, 42-44, and 47 and Reference Cannabinoid Ligands on hCB1 and hCB2
Cannabinoid Receptorsa
% of displacement
Ki (nM)
cmpd
R
R′′
R′
hCB1-R
hCB2-R
hCB2-R
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
29
30
31
32
33
34
35
38
39
40
42
43
44
47
4-fluorobenzyl
4-chlorobenzyl
4-bromobenzyl
2-phenylethyl
3-phenylpropyl
2-cyclohexylethyl
2-(morpholin-4-yl)ethyl
2-(morpholin-4-yl)ethyl
2-(morpholin-4-yl)ethyl
pentyl
pentyl
2-(morpholin-4-yl)ethyl
pentyl
pentyl
pentyl
pentyl
pentyl
pentyl
pentyl
pentyl
pentyl
pentyl
pentyl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
methyl
phenyl
methyl
methyl
methyl
methyl
phenyl
H
1-(3,5-dimethyl)adamantyl
1-(3,5-dimethyl)adamantyl
1-(3,5-dimethyl)adamantyl
1-(3,5-dimethyl)adamantyl
1-(3,5-dimethyl)adamantyl
1-(3,5-dimethyl)adamantyl
1-(3,5-dimethyl)adamantyl
2-phenylethyl
59.3 ( 2.9
38.7 ( 2.7
<10
93.2 ( 1.1
48.1 ( 1.8
30.5 ( 1.6
95.8 ( 1.4
101.2 ( 2.5
38.8 ( 2.4
95.6 ( 1.3
46.7 ( 3.7
66.5 ( 1.1
98.0 ( 1.6
90.9 ( 0.9
101.2 ( 5.1
87.2 ( 1.8
99.1 ( 1.5
101.0 ( 1.4
64.3 ( 3.5
96.7 ( 1.3
97.2 ( 0.5
57.3 ( 3.5
91.7 ( 3.2
62.6 ( 2.5
70.4 ( 3.1
94.9 ( 1.8
99.9 ( 0.1
83.4 ( 1.4
97.4 ( 1.4
81.3 ( 2.5
25.9 ( 4.6
63.9 ( 2.1
95.7 ( 9.9
83.4 ( 6.9
N.D.
N.D.
333 ( 51
160 ( 21
N.D.
221 ( 38
N.D.
>2000
41.7 ( 0.4
1010 ( 71
152 ( 30
50.6 ( 5.2
28.5 ( 1.4
14.0 ( 0.7
202 ( 65
200 ( 13
119 ( 8
N.D.
<20
<10
<10
<10
<10
(-)-1-phenylethyl
<10
(+)-1-(1,2,3,4-tetrahydronaphthyl)
(-)-1-(1,2,3,4-tetrahydronaphthyl)
1-adamantyl
82.1 ( 4.0
44.5 ( 5.3
<10
1-(adamantyl)methyl
(()-1-(adamantyl)ethyl
(-)-1-(adamantyl)ethyl
(+)-1-(adamantyl)ethyl
1-(3,5-dimethyl)adamantyl
1-(3,5-dimethyl)adamantyl
2-phenylethyl
36.7 ( 3.6
72.2 ( 0.7
83.6 ( 6.6
31.0 ( 1.8
39.8 ( 5.8
40.0 ( 4.1
<20
(-)-1-(phenylethyl)
27.2 ( 4.3
<20
663 ( 105
(-)-1-(2-naphthyl)ethyl
(-)-1-(1-naphthyl)ethyl
1-adamantyl
>2000
23.6 ( 4.3
29.1 ( 4.5
52.3 ( 3.3
28.1 ( 4.8
59.0 ( 3.5
27.0 ( 4.0
24.0 ( 6.3
41.0 ( 3.5
35.1 ( 3.0
>3000
338 ( 29
25.5 ( 1.3
1130 ( 102
265 ( 12
1670 ( 163
>4000
pentyl
pentyl
pentyl
pentyl
pentyl
pentyl
pentyl
1-adamantyl
2-phenylethyl
1-naphthyl
1-adamantyl
2-phenylethyl
(()-1-(1,2,3,4-tetrahydronaphthyl)
1-(3,5-dimethyl)adamantyl
H
H
H
H
H
H
>4000
18.2 ( 2.8
Reference Compounds
1
2
3
N.D.
N.D.
54.6 ( 2.4
N.D.
N.D.
N.D.
95.6 ( 5.4
N.D.
60.2 ( 5.5b
20.3 ( 2.6b
15.8 ( 1.4b
9.1 ( 0.8b
15.4 ( 1.4b
4
5c
N.D.
N.D.
a The Ki values were obtained from nonlinear analysis of competition curves using [3H]-CP-55,940 as radioligand. Data are mean ( SEM of three to four
experiments performed in duplicate. b From ref 19. c CP-55,940.
These observations open the way for further molecular
pharmacology research in terms of which key amino acid
residues are involved in the binding and the efficacy of these
compounds in the cannabinoid CB2 receptor.
of potent and selective agonist or antagonist/inverse agonist of
the CB2 cannabinoid receptor.
Experimental Section
Chemistry. All commercial reagents and solvents were used
without further purification. Analytical thin layer chromatography
was performed on precoated Kieselgel 60F254 plates (Merck); the
spots were located by UV (254 and 366 nm) and with iodine; Rf
values are given for guidance. Silica gel 60 230-400 mesh
purchased from Merck was used for column chromatography.
Preparative thick-layer chromatography (PTLC) was performed
using silica gel from Merck, the compounds were extracted from
silica gel by the following solvent system: CH2Cl2/MeOH 7:3 (v/
v). All melting points were measured with a Bu¨chi 535 capillary
apparatus and remain uncorrected. 1H NMR spectra were obtained
using a Bru¨cker 300 MHz spectrometer, chemical shifts (δ) were
expressed in ppm relative to the tetramethylsilane peak used as
internal standard, J values are in Hertz, and the splitting patterns
were designated as follows: s, singlet; d, doublet; t, triplet; m,
multiplet. IR spectra were determined with a Bru¨cker Vector 22
spectrometer on a germanium crystal. APCI+ (atmospheric pressure
Conclusion
The present study allowed us to extend the structure-affinity
relationship studies of the 4-oxo-1,4-dihydroquinolines, a class
of potent and selective CB2 cannabinoid receptor ligands.
Reversing the amide bond or introducing an additional nitrogen
in the quinoline nucleus, leading to [1,6]-naphthyridine or [1,8]-
naphthyridine, did not elicit major changes in the affinity of
the compounds. However, the consequences of such modifica-
tions deeply affected the functionality of these ligands. Indeed,
small changes in the compound structure, like the replacement
of the 4-oxo-dihydroquinoline by a 4-thioxo-1,4-dihydroquino-
line or the introduction of a chlorine atom, resulted in the switch
of the functionality from agonist to antagonist or inverse agonist.
This information will be very useful in the future development
of this class of derivatives, allowing for the targeted synthesis

5478 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
Stern et al.
Table 2. Structure and Binding Data of Compounds 52-62, 67, 68, 71, and 76 on hCB1 and hCB2 Cannabinoid Receptorsa
% of displacement
Ki (nM)
cmpd
R′
hCB1-R
<20
59.6 ( 5.1
23.8 ( 13.1
42.4 ( 0.9
56.3 ( 6.6
<20
88.2 ( 5.1
91.6 ( 6.0
22.4 ( 3.8
60.4 ( 1.6
59.1 ( 4.3
26.3 ( 5.6
30.0 ( 11.8
50.0 ( 11.5
26.1 ( 8.6
hCB2-R
hCB2-R
52
53
54
55
59
60
56
61
62
57
58
67
68
71
76
6-chloro
7-chloro
8-chloro
6-chloro
6-chloro
6-chloro
7-chloro
7-chloro
7-chloro
6-chloro
6-chloro
[1,5]-naphthyridine
[1,6]-naphthyridine
[1,8]-naphthyridine
cinnoline
1-(3,5-dimethyl)adamantyl
1-(3,5-dimethyl)adamantyl
1-(3,5-dimethyl)adamantyl
(()-1-(adamantyl)ethyl
86.7 ( 0.2
105.8 ( 9.0
98.7 ( 2.6
81.8 ( 13.0
87.7 ( 7.7
97.2 ( 0.5
102.1 ( 2.8
102.4 ( 1.3
92.9 ( 6.5
98.2 ( 4.7
92.8 ( 2.4
93.4 ( 7.0
95.0 ( 2.1
101.8 ( 1.4
94.6 ( 0.4
54.6 ( 8.2
5.3 ( 0.7
27.4 ( 9.1
222 ( 32
235 ( 77
144 ( 45
41.1 ( 3.4
26.1 ( 3.5
265 ( 66
506 ( 88
121 ( 5.6
259 ( 22
30.9 ( 3.9
23.5 ( 1.8
93.0 ( 13.0
(-)-1-(adamantyl)ethyl
(+)-1-(adamantyl)ethyl
(()-1-(adamantyl)ethyl
(-)-1-(adamantyl)ethyl
(+)-1-(adamantyl)ethyl
(()-1-(1,2,3,4-tetrahydronaphthyl)
(+)-1-(1,2,3,4-tetrahydronaphthyl)
1-(3,5-dimethyl)adamantyl
1-(3,5-dimethyl)adamantyl
1-(3,5-dimethyl)adamantyl
1-(3,5-dimethyl)adamantyl
a The Ki values were obtained from nonlinear analysis of competition curves using [3H]-CP-55,940 as radioligand. Data are mean ( SEM of three to four
experiments performed in duplicate.
Table 3. Structure and Binding Data of Compounds 79a and 80-98 on hCB1 and hCB2 Cannabinoid Receptorsa
% of displacement
Ki (nM)
cmpd
R
Ar
hCB1-R
<10
hCB2-R
hCB2-R
79a
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
H
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
1-naphthyl
2-naphthyl
phenyl
31.0 ( 4.3
77.7 ( 5.7
97.0 ( 3.8
89.5 ( 0.8
100.2 ( 4.2
111.6 ( 0.3
96.5 ( 6.6
93.7 ( 4.3
56.8 ( 16.0
80.4 ( 0.4
78.5 ( 3.2
46.1 ( 2.4
74.3 ( 2.6
94.5 ( 5.4
<10
N.D.
butyl
pentyl
hexyl
benzyl
33.0 ( 3.5
55.8 ( 4.3
35.8 ( 3.2
47.1 ( 2.3
70.8 ( 11.7
73.8 ( 6.3
79.8 ( 2.8
29.3 ( 5.2
31.3 ( 0.2
28.7 ( 3.3
<10
1550 ( 529
154 ( 18
582 ( 60
656 ( 19
225 ( 85
1120 ( 222
966 ( 148
N.D.
4-fluorobenzyl
4-chlorobenzyl
4-bromobenzyl
2-phenylethyl
3-phenylpropyl
2-cyclohexylethyl
2-(morpholin-4-yl)ethyl
pentyl
>2000
1670 ( 256
N.D.
35.7 ( 4.5
49.5 ( 5.1
<10
1288 ( 92
446 ( 80
N.D.
pentyl
butyl
pentyl
hexyl
pentyl
pentyl
pentyl
4-methoxyphenyl
4-methoxyphenyl
4-methoxyphenyl
3,4-methylenedioxyphenyl
2-(6-methoxy)naphthyl
9-anthracenyl
<20
46.9 ( 6.4
68.8 ( 2.0
70.2 ( 8.0
44.1 ( 4.0
76.5 ( 3.1
N.D.
39.9 ( 5.9
27.2 ( 5.4
<20
>2000
>2000
N.D.
52.9 ( 3.2
1500 ( 223
a The Ki values were obtained from nonlinear analysis of competition curves using [3H]-CP-55,940 as radioligand. Data are mean ( SEM of three to four
experiments performed in duplicate.
chemical ionization) mass spectra were obtained on an LC-MS
system Thermo Electron Surveyor MSQ. Optical rotations ([R]D)
were measured on a Perkin-Elmer 343 polarimeter. Specific
rotations are given as deg/dm, and the concentration values are
reported as g/mL of the specified solvent and were recorded at
25 °C. Elemental analyses were performed by the “Service Central
d’Analyses” at the CNRS, Vernaison (France). Chiral separations
were carried out using a gradient Waters 600E metering pump
model equipped with a Waters 996 photodiode array spectropho-
tometer. Chromatographic data were collected and processed on a
computer running with Millennium 2010. The column eluate was
monitored at 220 and 230 nm. The sample loop was 20 µL
(Rheodyne 7125 injector). Chiral chromatography was carried out
on a Chiralpak AD-H (Tris-3,5-dimethylphenylcarbamate; 250 mm

CB2-SelectiVe Cannabinoid Receptor Ligands
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22 5479
Table 4. [35S]-GTPγS Binding Stimulation Assays of Selected
Compounds and Reference Compounds for the hCB2 Cannabinoid
Receptorsa
N3-(1-(3,5-Dimethyl)adamantyl)-4-oxo-1-(4-bromobenzyl)-
1,4-dihydroquinoline-3-carboxamide (12). Compound 12 was
purified by TLC (dichloromethane/methanol 99:1), white solid (280
1
mg, 60%); mp; IR; H NMR (CDCl3); LC-MS (APCI+) m/z 520
[35S]-GTPγS
specific binding
(control ) 100%)
[35S]-GTPγS
specific binding
(control ) 100%)
(MH+). Anal. (C29H31BrN2O2) C, H, N.
cmpd
cmpd
N3-(1-(3,5-Dimethyl)adamantyl)-4-oxo-1-(2-phenylethyl)-1,4-
dihydroquinoline-3-carboxamide (13). Compound 13 was purified
by TLC (dichloromethane/methanol 98:2), white solid (102 mg,
25%); mp; IR; 1H NMR (CDCl3); LC-MS (APCI+) m/z 455 (MH+).
Anal. (C30H34N2O2) C, H, N.
10
13
14
16
19
22
23
24
25
29
30
32
35
38
39
40
47
52
53
157.1 ( 4.7**
140.6 ( 8.1**
98.8 ( 3.56
54
55
59
60
56
61
62
57
58
67
68
71
76
1
187.4 ( 4.0**
97.3 ( 4.3
86.1 ( 2.3**
76.7 ( 2.5**
56.3 ( 6.1**
53.5 ( 3.2**
42.7 ( 2.6**
98.5 ( 4.3
141.0 ( 4.9**
182.9 ( 6.7**
155.0 ( 3.25**
155.6 ( 3.8**
174.6 ( 3.5**
172.5 ( 1.9**
128.2 ( 4.5**
27.1 ( 3.2**
185.9 ( 9.0**
26.5 ( 2.9**
119.7 ( 10.1
183.3 ( 7.4**
139.4 ( 4.9**
65.6 ( 1.8**
49.6 ( 1.8**
51.6 ( 0.8**
N3-(1-(3,5-Dimethyl)adamantyl)-4-oxo-1-(3-phenylpropyl)-
1,4-dihydroquinoline-3-carboxamide (14). Compound 14 was
purified by TLC (dichloromethane/methanol 98:2), white solid (147
1
mg, 35%); mp; IR; H NMR (CDCl3); LC-MS (APCI+) m/z 469
123.7 ( 2.7**
170.4 ( 3.4**
135.6 ( 3.4**
123.5 ( 2.8**
109.3 ( 3.1
21.6 ( 2.7**
201.4 ( 7.5**
130.7 ( 1.8**
207.1 ( 10.1**
230.5 ( 13.7**
(MH+). Anal. (C31H36N2O2) C, H, N.
N3-(1-(3,5-Dimethyl)adamantyl)-4-oxo-1-(2-cyclohexylethyl)-
1,4-dihydroquinoline-3-carboxamide (15). Compound 15 was
purified by TLC (cyclohexane/ethyl acetate 7:3), white solid (248
1
mg, 60%); mp; IR; H NMR (CDCl3); LC-MS (APCI+) m/z 461
(MH+). Anal. (C30H41N2O2) C, H, N.
2
3
4
5
N3-(1-(3,5-Dimethyl)adamantyl)-4-oxo-1-(2-(morpholin-4-yl)-
ethyl)-1,4-dihydroquinoline-3-carboxamide Hydrochloride (16).
Compound 16 was purified by TLC (dichloromethane/methanol 98:
2), white solid (217 mg, 65%); mp; IR; 1H NMR (CDCl3); LC-MS
(APCI+) m/z 464 (MH+). Anal. (C28H38ClN3O3) C, H, N.
N3-(1-(2-Phenylethyl))-4-oxo-1-(2-(morpholin-4-yl)ethyl)-1,4-
dihydroquinoline-3-carboxamide Hydrochloride (17). Compound
17 was purified by TLC (cyclohexane/ethyl acetate 6:4), white solid
a Results are expressed as the percentages of stimulation of [35S]-GTPγS
binding (basal value set at 100%) obtained for a concentration of ligands
of 10 µM. Data are the mean ( SEM of three experiments performed in
duplicate. Statistical significance assessed by one-way ANOVA followed
by a Dunett post-test (*P < 0.05 and **P < 0.01).
1
(178 mg, 45%); mp; IR; H NMR (CDCl3); LC-MS (APCI+) m/z
406 (MH+). Anal. (C24H28ClN3O3) C, H, N, Cl.
× 4.6 mm i.d.; 10 µm; Daicel Chemical Industries, Baker, France).
The mobile phase consisting of hexane/propan-2-ol (9:1, v/v) was
degassed with a Waters inline degasser apparatus and delivered at
a flow rate of 1.0 mL‚min-1. All the separations were carried out
at 20 °C. The peak of the solvent front was considered to be equal
to the dead time (t0) and was about 3.70 min. For preliminary
studies, compounds were dissolved in propan-2-ol at a concentration
of 0.50 mmol‚L-1 (concentration 100%) and passed through a 0.45
µm membrane filter prior to loading the column.
Procedures for the preparation of 2-phenylaminomethylenema-
lonic acid diethyl ester (6) and 4-oxo-1,4-dihydroquinoline-3-
carboxylic acid ethyl ester (7) have been previously described.19
The procedures for the synthesis of 1-alkyl-4-oxo-1,4-dihydro-
quinoline-3-carboxylic acid ethyl ester (8a-i) and 1-alkyl-4-oxo-
1,4-dihydroquinoline-3-carboxylic acid (9a-i), as well as their
characterization, are described in the Supporting Information.
General Procedure for the Preparation of N3-Aryl-1-alkyl-
4-oxo-1,4-dihydroquinoline-3-carboxamide (10-25). To a solu-
tion of PybrOP (1.5 mmol) in 3 mL of dry DMF were added at
room temperature compounds 9a-i and diisopropylethylamine (3.0
mmol). The preswollen resin (0.75 g) in dry DMF was treated with
the above mixture at room temperature for 3 h, after which time
the resin was washed three times with dry DMF and three times
with dichloromethane. The same activation procedure was repeated
once. The appropriate amine (0.67 mmol) dissolved in dry DMF
was reacted with the polymer-bound activated ester for 24 h at room
temperature. The supernatant was then separated from the resin by
filtration and the polymer beads were washed three times with dry
DMF and three times with dichloromethane. The combined
solutions were concentrated and the residue was purified either by
crystallization or preparative TLC.
(-)-N3-(1-(1-Phenylethyl))-4-oxo-1-(2-(morpholin-4-yl)ethyl)-
1,4-dihydroquinoline-3-carboxamide Hydrochloride (18). Com-
pound 18 was purified by TLC eluting from dichloromethane/
methanool 95:5, white solid (262 mg, 66%); mp; [R]25 ) -75°
D
(c ) 0.01, CH2Cl2); IR; H NMR (CDCl3); LC-MS (APCI+) m/z
1
406 (MH+). Anal. (C24H28ClN3O3) C, H, N, Cl.
(+)-N3-(1-(1,2,3,4-Tetrahydronaphthyl))-4-oxo-1-pentyl-1,4-
dihydroquinoline-3-carboxamide (19). Compound 19 was purified
by TLC (cyclohexane/ethyl acetate 7:3), white oil (209 mg, 60%);
1
[R]25 ) +1.8° (c ) 0.01, CH2Cl2); IR; H NMR (CDCl3); LC-
D
MS (APCI+) m/z 389 (MH+). Anal. (C25H28N2O2) C, H, N.
(-)-N3-(1-(1,2,3,4-Tetrahydronaphthyl))-4-oxo-1-pentyl-1,4-
dihydroquinoline-3-carboxamide (20). Compound 20 was purified
by TLC (cyclohexane/ethyl acetate 7:3), white oil (216 mg, 62%);
1
[R]25 ) -1.8° (c ) 0.01, CH2Cl2); IR; H NMR (CDCl3); LC-
D
MS (APCI+) m/z 389 (MH+). Anal. (C25H28N2O2) C, H, N.
N3-(1-Adamantyl)-4-oxo-1-(2-(morpholin-4-yl)ethyl)-1,4-di-
hydroquinoline-3-carboxamide Hydrochloride (21). Compound
21 was purified by TLC (dichloromethane/methanol 98:2), white
solid (156 mg, 40%); mp; IR; 1H NMR (CDCl3); LC-MS (APCI+)
m/z 436 (MH+). Anal. (C26H33N3O3) C, H, N.
N3-((1-Adamantyl)methyl)-4-oxo-1-pentyl-1,4-dihydroquino-
line-3-carboxamide (22). Compound 22 was purified by TLC
(cyclohexane/ethyl acetate 7:3), white solid (201 mg, 55%); mp;
1
IR; H NMR (CDCl3); LC-MS (APCI+) m/z 407 (MH+). Anal.
(C26H34N2O2) C, H, N.
(()-N3-(1-(1-Adamantyl)ethyl)-4-oxo-1-pentyl-1,4-dihydro-
quinoline-3-carboxamide (23). Compound 23 was purified by TLC
(cyclohexane/ethyl acetate 7:3), white solid (208 mg, 55%); mp;
1
[R]25 ) +0° (c ) 0.01, CH2Cl2); IR; H NMR (CDCl3); LC-MS
D
(APCI+) m/z 422 (MH+). Anal. (C27H36N2O2) C, H, N.
(-)-N3-(1-(1-Adamantyl)ethyl)-4-oxo-1-pentyl-1,4-dihydro-
quinoline-3-carboxamide (24). Compound 24 was prepared by
chiral preparative HPLC (stationary phase: Chiralpak AD (20 µm);
mobile phase: n-hexane/propan-2-ol, 90/10; separation yield: 94%),
white solid (245 mg); mp; [R]25D ) -93° (c ) 0.01, CH2Cl2); IR;
1H NMR (CDCl3); LC-MS (APCI+) m/z 422 (MH+). Anal.
(C27H36N2O2) C, H, N.
N3-(1-(3,5-Dimethyl)adamantyl)-4-oxo-1-(4-fluorobenzyl)-1,4-
dihydroquinoline-3-carboxamide (10). Compound 10 was purified
by TLC (dichloromethane/methyl alcohol 99:1), white solid (185
1
mg, 45%); mp; IR; H NMR (CDCl3); LC-MS (APCI+) m/z 459
(MH+). Anal. (C29H31FN2O2) C, H, N.
N3-(1-(3,5-Dimethyl)adamantyl)-4-oxo-1-(4-chlorobenzyl)-1,4-
dihydroquinoline-3-carboxamide (11). Compound 11 was purified
by TLC (dichloromethane/methanol 99:1), white solid (235 mg,
55%); mp; IR; 1H NMR (CDCl3); LC-MS (APCI+) m/z 476 (MH+).
Anal. (C29H31ClN2O2) C, H, N.
(+)-N3-(1-(1-Adamantyl)ethyl)-4-oxo-1-pentyl-1,4-dihydro-
quinoline-3-carboxamide (25). Compound 25 was prepared by
chiral preparative HPLC (stationary phase: Chiralpak AD (20 µm);

5480 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
Stern et al.
mobile phase: n-hexane/propan-2-ol, 90/10; separation yield: 91%),
white solid (235 mg); mp; [R]25D ) +93° (c ) 0.01, CH2Cl2); IR;
1H NMR (CDCl3); LC-MS (APCI+) m/z 422 (MH+). Anal.
(C27H36N2O2) C, H, N.
Synthesis and Characterization of 26, 27a,b, and 28a,b. The
synthesis and characterization of 26, 27a,b, and 28a,b are described
in the Supporting Information.
residue was dissolved in ethyl acetate (50 mL) and washed with
saturated aqueous sodium hydrogenocarbonate (2 × 50 mL), water
(2 × 50 mL), and brine (2 × 50 mL). The organic layer was
separated and dried over anhydrous magnesium sulfate. The
concentrate was purified by flash chromatography (dichloromethane/
methanol 98:2, v/v) to afford the corresponding carboxamide
derivatives 38-40.
General Procedure for the Preparation of N3-Aryl-1-alkyl-
4-oxo-1,4-dihydroquinoline-3-carboxamide (29-35). To a solu-
tion of PybrOP (1.5 mmol) in 3 mL of dry DMF were added at
room temperature compounds 28a,b and diisopropylethylamine
(3.0 mmol). The preswollen resin (0.75 g) in dry DMF was
treated with the above mixture at room temperature for 3 h, and
after this time, the resin was washed three times with dry DMF
and three times with dichloromethane. The same activation
procedure was repeated a second time. The appropriate amine
(0.67 mmol) dissolved in dry DMF was reacted with the poly-
mer-bound activated ester for 24 h at room temperature. The
supernatant was then separated from the resin by filtration, and
the polymer beads were washed three times with dry DMF and
three times with dichloromethane. The combined solutions were
concentrated and the residue was purified either by crystallization
or preparative TLC.
Adamantane-1-carboxylic Acid (4-Oxo-1-pentyl-1,4-dihyd-
roquinolin-3-yl)amide (38). White solid (686 mg, 40%); mp; IR;
1H NMR (CDCl3); LC-MS (APCI+) m/z 393 (MH+). Anal.
(C25H32N2O2) C, H, N.
N-(4-Oxo-1-pentyl-1,4-dihydroquinolin-3-yl)-3-phenyl-propi-
onamide (39). White solid (727 mg, 40%); mp; IR; 1H NMR
(CDCl3); LC-MS (APCI+) m/z 363 (MH+). Anal. (C23H26N2O2) C,
H, N.
Naphthalene-1-carboxylic Acid (4-Oxo-1-pentyl-1,4-dihyd-
roquinolin-3-yl)amide (40). White solid (1.07 g, 56%); mp; IR;
1H NMR (CDCl3); LC-MS (APCI+) m/z 385 (MH+). Anal.
(C25H24N2O2) C, H, N.
General Procedure for the Preparation of 3-(Substituted-
aminomethyl)-1-pentyl-1H-quinolin-4-one (42-44). A mixture
of aldehyde 41 (0.30 g, 1.23 mmol) in dry methanol (30 mL) was
stirred at room temperature under nitrogen in the presence of 3 Å
molecular sieve. Selected amine (1.85 mmol) and triethylamine
(0.70 mL, 4.93 mmol) were added, and the resulting mixture was
stirred overnight at 50 °C under nitrogen. Then sodium cyanoboro-
hydride (0.08 g, 1.35 mmol) was added, and the stirring was
continued at 50 °C for 24 h. The molecular sieve was eliminated
by filtration, and the solvent was removed by evaporation under
reduced pressure. The residue was dissolved in ethyl acetate (50
mL) and washed with saturated aqueous sodium hydrogenocar-
bonate (2 × 50 mL), water (2 × 50 mL), and brine (2 × 50 mL).
The organic layer was separated and dried over anhydrous
magnesium sulfate. The concentrate was purified by flash-chro-
matography, using specified eluent, to afford the corresponding
amines, which were isolated as hydrochloride except for compound
(42).
N3-(1-(3,5-Dimethyl)adamantyl)-2-methyl-4-oxo-1-pentyl-1,4-
dihydroquinoline-3-carboxamide (29). Compound 29 was purified
by TLC (cyclohexane/ethyl acetate 6:4), yellow oil (140 mg, 36%);
1
IR; H NMR (CDCl3); LC-MS (APCI+) m/z 435 (MH+). Anal.
(C28H38N2O2) C, H, N.
N3-(1-(3,5-Dimethyl)adamantyl)-4-oxo-2-phenyl-1-pentyl-1,4-
dihydroquinoline-3-carboxamide (30). Compound 30 was purified
by TLC (dichloromethane/methanol 98:2), white solid (134 mg,
30%); mp; IR; 1H NMR (CDCl3); LC-MS (APCI+) m/z 497 (MH+).
Anal. (C33H40N2O2) C, H, N.
N3-(2-Phenylethyl)-2-methyl-4-oxo-1-pentyl-1,4-dihydroquin-
oline-3-carboxamide (31). Compound 31 was purified by TLC
1
(cyclohexane/ethyl acetate 4:6), white oil (250 mg, 74%); IR; H
NMR (CDCl3); LC-MS (APCI+) m/z 377 (MH+). Anal. (C24H28N2O2)
C, H, N.
3-((1-Adamantyl)aminomethyl)-1-pentyl-1H-quinolin-4-one (42).
Compound 42 was purified by chromatography (dichloromethane/
(-)-N3-(1-Phenylethyl)-2-methyl-4-oxo-1-pentyl-1,4-dihydro-
1
quinoline-3-carboxamide (32). Compound 32 was purified by TLC
methanol 9:1, v/v), white solid (163 mg, 35%); mp; IR; H NMR
(cyclohexane/ethyl acetate 6:4), white oil (170 mg, 50%); [R]25
(DMSO-d6); LC-MS (APCI+) m/z 379 (MH+). Anal. (C25H34N2O)
C, H, N.
D
) -90° (c ) 0.01, CH2Cl2); IR; 1H NMR (CDCl3); LC-MS
(APCI+) m/z 377 (MH+). Anal. (C24H28N2O2) C, H, N.
1-Pentyl-(3-phenylethylaminomethyl)-1H-quinolin-4-one Hy-
drochloride (43). Compound 43 was purified by chromatography
(dichloromethane/methanol 9:1, v/v), white solid (151 mg, 32%);
(-)-N3-(1-(2-Naphthyl)ethyl)-2-methyl-4-oxo-1-pentyl-1,4-di-
hydroquinoline-3-carboxamide (33). Compound 33 was purified
1
by TLC (cyclohexane/ethyl acetate 4:6), white oil (130 mg, 34%);
mp; IR; H NMR (DMSO-d6); LC-MS (APCI+) m/z 349 (MH+).
1
[R]25 ) -148° (c ) 0.01, CH2Cl2); IR; H NMR (CDCl3); LC-
Anal. (C23H29ClN2O) C, H, N.
D
MS (APCI+) m/z 427 (MH+). Anal. (C28H30N2O2) C, H, N.
(-)-N3-(1-(1-Naphthyl)ethyl)-2-methyl-4-oxo-1-pentyl-1,4-di-
hydroquinoline-3-carboxamide (34). Compound 34 was purified
(()1-Pentyl-(3-(1,2,3,4-tetrahydronaphthyl)aminomethyl)-1H-
quinolin-4-one Hydrochloride (44). Compound 44 was purified
by chromatography (dichloromethane/methanol 9:1, v/v), white
solid (167 mg, 33%); mp; IR; 1H NMR (DMSO-d6); LC-MS
(APCI+) m/z 375 (MH+). Anal. (C25H30ClN2O) C, H, N.
1-Pentyl-4-thioxo-1,4-dihydroquinoline-3-carboxylic Acid
Ethyl Ester (45). A mixture of 8a (2.00 g, 6.96 mmol) and
phosphorus pentasulfide (3.09 g, 13.92 mmol) was refluxed for 12
h in pyridine (40 mL). After cooling, the solvent was removed under
reduced pressure and the residue was taken up in water and then
extracted with ethyl acetate. The organic layer was dried over
magnesium sulfate, concentrated under reduced pressure, and finally
purified by flash chromatography (cyclohexane/ethyl acetate 9:1)
by TLC (cyclohexane/ethyl acetate 4:6), white oil (130 mg, 34%);
1
[R]25 ) -175° (c ) 0.01, CH2Cl2); IR; H NMR (CDCl3); LC-
D
MS (APCI+) m/z 427 (MH+). Anal. (C28H30N2O2) C, H, N.
N3-(1-Adamantyl)-4-oxo-1-pentyl-2-phenyl-1,4-dihydroquino-
line-3-carboxamide (35). Compound 35 was purified by TLC
(dichloromethane/methanol 98:2), white solid (130 mg, 34%); mp;
IR; H NMR (CDCl3); LC-MS (APCI+) m/z 469 (MH+). Anal.
1
(C31H36N2O2) C, H, N.
Synthesis and Characterization of Intermediates 36, 37, and
41. The synthesis and characterization of intermediates 36, 37, and
41 are described in the Supporting Information.
1
to provide 1.98 g (94%) of compound 45 as a yellow oil; IR; H
NMR (CDCl3).
General Procedure for the Preparation of N-(4-Oxo-1-pentyl-
1,4-dihydroquinolin-3-yl)-aryl-carboxamide (38-40). A mixture
of selected carboxylic acid (5.00 mmol) and thionyl chloride (10
mL) was refluxed for 2 h. Excess of thionyl chloride was evaporated
under reduced pressure, and dry toluene (20 mL) was added
(solution A). A mixture of amine 37 (7.5 mmol) and ethyldiisoproyl
amine (7.5 mmol) in dry toluene (30 mL) under nitrogen was cooled
at 0 °C. A solution of freshly prepared acyl chloride (solution A)
was added dropwise. The mixture was stirred overnight at room
temperature and then concentrated under reduced pressure. The
1-Pentyl-4-thioxo-1,4-dihydroquinoline-3-carboxylic Acid (46).
A mixture of 45 (2.00 g, 6.59 mmol) and lithium hydroxide (1.10
g, 26.36 mmol) was stirred (RT, N2) in a mixture of tetrahydrofuran/
water 50:50 (100 mL). Tetrahydrofuran was removed under reduced
pressure. The solution was adjusted to pH 4 with aqueous 10%
hydrochloric acid. The resulting precipitate was collected by
filtration, washed with water, and recrystallized from diisopropyl
ether to afford 1.50 g (83%) of carboxylic acid 46 as a yellow
1
solid; mp; IR; H NMR (DMSO-d6).

CB2-SelectiVe Cannabinoid Receptor Ligands
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22 5481
N3-(1-(3,5-Dimethyl)adamantyl)-1-pentyl-4-thioxo-1,4-dihy-
droquinoline-3-carboxamide (47). This compound was obtained
using the same methodology previously described for 1-pentyl-4-
oxo-1,4-dihydroquinoline-3-carboxamide (10-25). Purified by TLC
(cyclohexane/ethyl acetate 8:2), white solid (235 mg, 60%); mp;
NMR (CDCl3); LC-MS (APCI+) m/z 423 (MH+). Anal. (C25H27-
ClN2O2) C, H, N.
(+)-N3-(1-(1,2,3,4-Tetrahydronaphthyl))-6-chloro-4-oxo-1-
pentyl-1,4-dihydroquinoline-3-carboxamide (58). Purified by
TLC (cyclohexane/ethyl acetate 7:3), white solid (232 mg, 82%);
1
IR; H NMR (CDCl3); LC-MS (APCI+) m/z 437 (MH+). Anal.
1
[R]25 ) +2.0° (c ) 0.01, CH2Cl2); mp; IR; H NMR (CDCl3);
D
LC-MS (APCI+) m/z 423 (MH+). Anal. (C25H27ClN2O2) C, H, N.
Synthesis and Characterization of Intermediates 63a,b, 64a,b,
65a,b, 66a,b, 69, and 70. The synthesis and characterization of
intermediates 63a,b, 64a,b, 65a,b, 66a,b, 69, and 70 are described
in the Supporting Information.
(C27H36N2OS) C, H, N.
Synthesis and Characterization of Intermediates 48a-c, 49a-
c, 50a-c, and 51a-c. The synthesis and characterization of
intermediates 48a-c, 49a-c, 50a-c, and 51a-c are described in
the Supporting Information.
General Procedure for the Preparation of N3-Aryl-1-alkyl-
4-oxo-1,4-dihydronaphthyridine-3-carboxamide (67, 68, and 71).
These compounds were obtained using the same methodology
previously described for 1-pentyl-4-oxo-1,4-dihydroquinoline-3-
carboxamide (10-25).
General Procedure for the Preparation of N3-Aryl-1-alkyl-
4-oxo-1,4-dihydroquinoline-3-carboxamide (52-58). These com-
pounds were obtained using the same methodology previously
described for 1-pentyl-4-oxo-1,4-dihydroquinoline-3-carboxamide
(10-25).
N3-(1-(3,5-Dimethyl)adamantyl)-4-oxo-1-pentyl-1,4-dihydro-
[1,5]-naphthyridine-3-carboxamide (67). Purified by TLC (dichlo-
romethane), white solid (40 mg, 25%); mp; IR; 1H NMR (CDCl3);
LC-MS (APCI+) m/z 422 (MH+). Anal. (C26H35N3O2) C, H, N.
N3-(1-(3,5-Dimethyl)adamantyl)-4-oxo-1-pentyl-1,4-dihydro-
[1,6]-naphthyridine-3-carboxamide (68). Compound 68 was puri-
fied by TLC (dichloromethane), white solid (47 mg, 30%); mp;
N3-(1-(3,5-Dimethyl)adamantyl)-6-chloro-4-oxo-1-pentyl-1,4-
dihydroquinoline-3-carboxamide (52). Compound 52 was purified
by TLC (cyclohexane/ethyl acetate 7:3), white solid (167 mg, 55%);
mp; IR; 1H NMR (CDCl3); LC-MS (APCI+) m/z 456 (MH+). Anal.
(C27H35ClN2O2) C, H, N.
N3-(1-(3,5-Dimethyl)adamantyl)-7-chloro-4-oxo-1-pentyl-1,4-
dihydroquinoline-3-carboxamide (53). Compound 53 was purified
by TLC (cyclohexane/ethyl acetate 7:3), white solid (219 mg, 72%);
mp; IR; 1H NMR (CDCl3); LC-MS (APCI+) m/z 456 (MH+). Anal.
(C27H35ClN2O2) C, H, N.
N3-(1-(3,5-Dimethyl)adamantyl)-8-chloro-4-oxo-1-pentyl-1,4-
dihydroquinoline-3-carboxamide (54). Compound 54 was purified
by TLC (cyclohexane/ethyl acetate 7:3), white solid (225 mg, 74%);
mp; IR; 1H NMR (CDCl3); LC-MS (APCI+) m/z 456 (MH+). Anal.
(C27H35ClN2O2) C, H, N.
1
IR; H NMR (CDCl3); LC-MS (APCI+) m/z 422 (MH+). Anal.
(C26H35N3O2) C, H, N.
N3-(1-(3,5-Dimethyl)adamantyl)-4-oxo-1-pentyl-1,4-dihydro-
[1,8]-naphthyridine-3-carboxamide (71). Compound 71 was puri-
fied by TLC (dichloromethane), white solid (63 mg, 40%); mp;
1
IR; H NMR (CDCl3); LC-MS (APCI+) m/z 422 (MH+). Anal.
(C26H35N3O2) C, H, N.
Diethyl 2-(Phenylhydrazono)malonate (72). Aniline (9.78 mL,
107.37 mmol) was added to aqueous HCl 37% (23.30 mL). The
resulting aniline hydrochloride solution was cooled to -10 °C. Then
a solution of sodium nitrite (7.58 g, 109.88 mmol) in water (30
mL) was added keeping the reaction temperature below 0 °C.
Because the reaction was exothermic, the addition has to proceed
very slowly under rigorous cooling. The resulting orange solution
was added to a solution consisting of sodium acetate and diethyl
malonate. The latter solution was freshly prepared by dissolution
of sodium acetate (20.19 g, 246.25 mmol) in water (40 mL). Then
diethyl malonate (16.68 mL, 109.88 mmol) was dissolved in ethanol
(215 mL). Both solutions were combined and brought to 0 °C,
which is accompanied by some precipitations. To the resulting slurry
stirred at 0 °C was slowly added the previously prepared cold
solution of phenyl diazonium chloride. After this addition, the
reaction mixture was allowed to reach room temperature and stirred
for 5 h. After keeping the mixture at -13 °C overnight, a white
inorganic solid and the crude product (dark red oil) precipitated.
The solid was removed by filtration. The filtrate was evaporated
to give a red, viscous oil, which was taken up in ethyl acetate and
extracted twice with water. The combined organic layers were dried
over anhydrous magnesium sulfate, evaporated under reduce
pressure, and finally purified by flash chromatography (dichlo-
romethane) the afford 26.95 g (95%) of compound 72 as an orange
(()-N3-(1-(1-Adamantyl)ethyl)-6-chloro-4-oxo-1-pentyl-1,4-
dihydroquinoline-3-carboxamide (55). Compound 55 was purified
by TLC (cyclohexane/ethyl acetate 7:3), white solid (140 mg, 46%);
mp; [R]25D ) +0° (c ) 0.01, CH2Cl2); IR; 1H NMR (CDCl3); LC-
MS (APCI+) m/z 456 (MH+). Anal. (C27H35ClN2O2) C, H, N.
(-)-N3-(1-(1-Adamantyl)ethyl)-6-chloro-4-oxo-1-pentyl-1,4-
dihydroquinoline-3-carboxamide (59). Compound 59 was pre-
pared by chiral preparative HPLC (stationary phase: Chiralpak AD
(20 µm); mobile phase: n-hexane/propan-2-ol, 92/8, v/v; separation
yield: 94%), white solid (235 mg); mp; [R]25D ) -101° (c ) 0.01,
CH2Cl2); IR; 1H NMR (CDCl3); LC-MS (APCI+) m/z 422 (MH+).
(+)-N3-(1-(1-Adamantyl)ethyl)-6-chloro-4-oxo-1-pentyl-1,4-
dihydroquinoline-3-carboxamide (60). Prepared by chiral prepara-
tive HPLC (stationary phase: Chiralpak AD (20 µm); mobile
phase: n-hexane/propan-2-ol, 92/8, v/v; separation yield: 91%),
white solid (227 mg); mp; [R]25 ) +101° (c ) 0.01, CH2Cl2);
D
1
IR; H NMR (CDCl3); LC-MS (APCI+) m/z 422 (MH+).
(()-N3-(1-(1-Adamantyl)ethyl)-7-chloro-4-oxo-1-pentyl-1,4-
dihydroquinoline-3-carboxamide (56). Compound 56 was purified
by TLC (cyclohexane/ethyl acetate 7:3), white solid (164 mg, 54%);
mp; [R]25D ) +0° (c ) 0.01, CH2Cl2); IR; 1H NMR (CDCl3); LC-
MS (APCI+) m/z 456 (MH+). Anal. (C27H35ClN2O2) C, H, N.
(-)-N3-(1-(1-Adamantyl)ethyl)-7-chloro-4-oxo-1-pentyl-1,4-
dihydroquinoline-3-carboxamide (61). Prepared by chiral prepara-
tive HPLC (stationary phase: Chiralpak AD (20 µm); mobile
phase: n-hexane/propan-2-ol, 90/10, v/v; separation yield: 96%),
white solid (240 mg); mp; [R]25D ) -95° (c ) 0.01, CH2Cl2); IR;
1H NMR (CDCl3); LC-MS (APCI+) m/z 422 (MH+).
1
oil: IR; H NMR (CDCl3).
2-(Phenylhydrazono)malonic Acid (73). To diethyl 2-(phenyl-
hydrazono)malonate 72 (5.00 g, 18.92 mmol) dissolved in refluxing
ethyl alcohol (95%, 20 mL) was added dropwise aqueous sodium
hydroxide (2 N, 21 mL). The resulting mixture was heated to reflux
for 30 min, then allowed to reach room temperature and evaporated
under reduced pressure. The resulting concentrate was precipitated
into aqueous HCl (10%). The resulting precipitate was collected
by filtration, washed with water, and dried in vacuo over P2O5 to
(+)-N3-(1-(1-Adamantyl)ethyl)-7-chloro-4-oxo-1-pentyl-1,4-
dihydroquinoline-3-carboxamide (62). Compound 62 was pre-
pared by chiral preparative HPLC (stationary phase: Chiralpak AD
(20 µm); mobile phase: n-hexane/propan-2-ol, 90/10, v/v; separa-
1
afford 3.19 g (81%) of 73 as a yellow solid: mp; IR; H NMR
tion yield: 93%), white solid (232 mg); mp; [R]25 ) +95° (c )
(DMSO-d6).
D
1
0.01, CH2Cl2); IR; H NMR (CDCl3); LC-MS (APCI+) m/z 422
4-Oxo-1,4-dihydrocinnoline-3-carboxylic Acid (74). To 2-(phe-
nylhydrazono)malonic acid 73 (2.00 g, 9.61 mmol) suspended in
1,2-dichlorobenzene (20 mL) was added dropwise a solution of
thionyl chloride (2 mL) in 1,2-dichlorobenzene (10 mL). The
resulting mixture was heated to 70 °C for 5 h. The excess of thionyl
chloride was distilled off under ambient pressure. To this solution
(MH+).
(()-N3-(1-(1,2,3,4-Tetrahydronaphthyl))-6-chloro-4-oxo-1-
pentyl-1,4-dihydroquinoline-3-carboxamide (57). Compound 57
was purified by TLC (cyclohexane/ethyl acetate 7:3), white solid
1
(226 mg, 80%); [R]25 ) +0° (c ) 0.01, CH2Cl2); mp; IR; H
D

5482 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
Stern et al.
was added a solution of titanium tetrachloride (2 mL) in 1,2-
dichlorobenzene (30 mL) within 15 min. The reaction suspension
was stirred at 90 °C for 14 h. Subsequently, the excess of titanium
tetrachloride and then the 1,2-dichlorobenzene were evaporated
under reduced pressure. The resulting brown solid was extracted
several times with small portions of boiling hot aqueous sodium
hydroxide (4 M, total volume: 30 mL). The resulting suspension
was passed through cellite. The product precipitated into concen-
trated HCl (37%, 150 mL) while being agitated. The resulting solid
was filtered off and then dried under reduced pressure over P2O5,
romethane/methanol 98:2, v/v), white solid (1267 mg, 65%); mp;
1
IR; H NMR (CDCl3); LC-MS (APCI+) m/z 424 (MH+). Anal.
(C27H18ClNO2) C, H, N.
1-(4-Bromobenzyl)-3-(naphthalene-1-carbonyl)-1,4-dihydro-
quinolin-4-one (86). Compound 86 was purified by TLC (dichlo-
romethane/methanol 98:2, v/v), white solid (1400 mg, 65%); mp;
1
IR; H NMR (CDCl3); LC-MS (APCI+) m/z 469 (MH+). Anal.
(C27H18BrNO2) C, H, N.
3-(Naphthalene-1-carbonyl)-1-phenylethyl-1,4-dihydroquino-
lin-4-one (87). Compound 87 was purified by TLC (dichlo-
romethane/methanol 98:2, v/v), white solid (371 mg, 30%); mp;
1
offering 1.19 g (65%) of 74 as a brown solid: mp; IR; H NMR
(DMSO-d6).
1
IR; H NMR (CDCl3); LC-MS (APCI+) m/z 404 (MH+). Anal.
(C28H21NO2) C, H, N.
N3-(1-(3,5-Dimethyl)adamantyl)-4-oxo-1,4-dihydrocinnoline-
3-carboxamide (75). To a stirred solution of carboxylic acid 74
(0.50 g, 2.63 mmol) in dry DMF (30 mL) was added diisopropy-
lethyl amine (1.83 mL, 10.52 mmol). The resulting solution was
stirred at room temperature for 10 min before adding HBTU (1.49
g, 3.94 mmol) and stirring for 3 more hours. 1-Amino-3,5-
dimethyladamantane (0.85 g, 3.94 mmol) was then added, and the
solution was stirred for 24 h. DMF was evaporated under reduced
pressure, and the residue was dissolved in ethyl acetate and
successively washed with aqueous saturated sodium bicarbonate,
water, and brine. The organic phase was dried over anhydrous
magnesium sulfate, evaporated, and finally purified by flash
chromatography using dichloromethane/methanol 98:2 (v/v) as
eluent, yielding 0.63 g (68%) of 75 as an orange solid: mp; IR; 1H
NMR (DMSO-d6).
3-(Naphthalene-1-carbonyl)-1-phenylpropyl-1,4-dihydroquin-
olin-4-one (88). Compound 88 was purified by TLC (dichlo-
romethane/methanol 98:2, v/v), white solid (691 mg, 36%); mp;
1
IR; H NMR (CDCl3); LC-MS (APCI+) m/z 418 (MH+). Anal.
(C29H23NO2) C, H, N.
3-(Naphthalene-1-carbonyl)-1-(2-(cyclohexyl)ethyl)-1,4-dihy-
droquinolin-4-one (89). Compound 89 was purified by TLC
(dichloromethane/methanol 98:2, v/v), white solid (376 mg, 20%);
mp; IR; 1H NMR (CDCl3); LC-MS (APCI+) m/z 410 (MH+). Anal.
(C28H27NO2) C, H, N.
3-(Naphthalene-1-carbonyl)-1-(2-(morpholin-4-yl)ethyl)-1,4-
dihydroquinolin-4-one Hydrochloride (90). Compound 90 was
purified by TLC (dichloromethane/methanol 97:3, v/v), white solid
(681 mg, 33%); mp; IR; H NMR (CDCl3); LC-MS (APCI+) m/z
1
413 (MH+). Anal. (C26H25ClN2O3) C, H, N.
N3-(1-(3,5-Dimethyl)adamantyl)-4-oxo-1-pentyl-1,4-dihydro-
cinnoline-3-carboxamide (76). This compound was obtained
using the same methodology previously described for 1-pentyl-
4-oxo-1,4-dihydroquinoline-3-carboxylic acid ethyl esters
(8a-i), purified by TLC, eluting from cyclohexane/ethyl acetate
3-(Naphthalene-2-carbonyl)-1-pentyl-1,4-dihydroquinolin-4-
one (91). Compound 91 was purified by TLC (cyclohexane/ethyl
acetate 7:3, v/v) ,white oil (681 mg, 40%); 1H NMR (CDCl3); LC-
MS (APCI+) m/z 370 (MH+). Anal. (C25H23NO2) C, H, N.
3-Benzoyl-1-pentyl-1,4-dihydroquinolin-4-one (92). Compound
92 was purified by TLC (dichloromethane/methanol 95:5, v/v),
white oil (631 mg, 43%); IR; 1H NMR (CDCl3); LC-MS (APCI+)
m/z 320 (MH+). Anal. (C21H21NO2) C, H, N.
1
6:4 (v/v), white solid (775 mg, 40%); mp 121-122 °C; IR; H
NMR (CDCl3); LC-MS (APCI+) m/z 422 (MH+). Anal. (C26H35N3O2)
C, H, N.
Synthesis and Characterization of 77a-g, 78a-g, and 79a-
g. The synthesis and characterization of 77a-g, 78a-g, and 79a-g
are reported in the Supporting Information.
General Procedure for the Preparation of N-Alkyl-3-aroyl-
1,4-dihydroquinolin-4-one (80-98). These compounds were ob-
tained using the same methodology previously described for
1-pentyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid ethyl esters
(8a-i).
1-Butyl-3-(naphthalene-1-carbonyl)-1,4-dihydroquinolin-4-
one (80). Compound 80 was purified by TLC (dichloromethane/
methanol 98:2, v/v), white solid (490 mg, 30%); mp; IR; 1H NMR
(CDCl3); LC-MS (APCI+) m/z 356 (MH+). Anal. (C24H21NO2) C,
H, N.
1-Butyl-3-(4-methoxybenzoyl)-1,4-dihydroquinolin-4-one (93).
Compound 93 was purified by TLC (dichloromethane/methanol 98:
1
2, v/v), white oil (416 mg, 27%); IR; H NMR (CDCl3); LC-MS
(APCI+) m/z 336 (MH+). Anal. (C21H21NO3) C, H, N.
3-(4-Methoxybenzoyl)-1-pentyl-1,4-dihydroquinolin-4-one (94).
Compound 94 was purified by TLC (dichloromethane/methanol 98:
1
2, v/v), white oil (562 mg, 35%); IR; H NMR (CDCl3); LC-MS
(APCI+) m/z 350 (MH+). Anal. (C22H23NO3) C, H, N.
1-Hexyl-3-(4-methoxybenzoyl)-1,4-dihydroquinolin-4-one (95).
Compound 95 was purified by TLC (dichloromethane/methanol 98:
1
2, v/v), white oil (769 mg, 46%); IR; H NMR (CDCl3); LC-MS
(APCI+) m/z 364 (MH+). Anal. (C23H25NO3) C, H, N.
3-(Benzo[1,3]dioxole-5-carbonyl)-1-pentyl-1,4-dihydroquino-
lin-4-one (96). Compound 96 was purified by TLC (dichlo-
3-(Naphthalene-1-carbonyl)-1-pentyl-1,4-dihydroquinolin-4-
one (81). Compound 81 was purified by TLC (cyclohexane/ethyl
1
romethane/methanol 95:5, v/v), white oil (417 mg, 25%); IR; H
1
acetate 7:3, v/v), white solid (1155 mg, 68%); mp; IR; H NMR
NMR (CDCl3); LC-MS (APCI+) m/z 364 (MH+). Anal. (C22H21-
NO4) C, H, N.
(CDCl3); LC-MS (APCI+) m/z 370 (MH+). Anal. (C25H23NO2) C,
H, N.
3-((6-Methoxy)naphthalene-2-carbonyl)-1-pentyl-1,4-dihyd-
roquinolin-4-one (97). Compound 97 was purified by TLC
(dichloromethane/methanol 98:2, v/v), white oil (459 mg, 25%);
1-Hexyl-3-(naphthalene-1-carbonyl)-1,4-dihydroquinolin-4-
one (82). Compound 82 was purified by TLC (dichloromethane/
methanol 98:2, v/v), white solid (917 mg, 52%); mp; IR; 1H NMR
(CDCl3); LC-MS (APCI+) m/z 384 (MH+). Anal. (C26H25NO2) C,
H, N.
1-Benzyl-3-(naphthalene-1-carbonyl)-1,4-dihydroquinolin-4-
one (83). Compound 83 was purified by TLC (dichloromethane/
methanol 98:2, v/v), white solid (358 mg, 20%); mp; IR; 1H NMR
(CDCl3); LC-MS (APCI+) m/z 390 (MH+). Anal. (C27H19NO2) C,
H, N.
1
IR; H NMR (CDCl3); LC-MS (APCI+) m/z 400 (MH+). Anal.
(C26H25NO3) C, H, N.
3-(Anthracene-9-carbonyl)-1-pentyl-1,4-dihydroquinolin-4-
one (98). Compound 98 was purified by TLC (dichloromethane/
methanol 98:2, v/v), yellow solid (482 mg, 25%); mp; IR; 1H NMR
(CDCl3); LC-MS (APCI+) m/z 420 (MH+). Anal. (C29H25NO2) C,
H, N.
Pharmacology. Fatty acid free bovine serum albumin (BSA)
was purchased from Sigma Chemical Co. (St. Louis, MO).
Compound 4 was purchased from RBI (Natick, MA), 2, HU-210,
and CP-55,940 (5) were acquired from Tocris (Bristol, U.K.). SR-
141716A and 1 were kindly donated by Sanofi Recherche (Mont-
pellier, France).
1-(4-Fluorobenzyl)-3-(naphthalene-1-carbonyl)-1,4-dihydro-
quinolin-4-one (84). Compound 84 was purified by TLC (dichlo-
romethane/methanol 98:2, v/v), white solid (1105 mg, 59%); mp;
1
IR; H NMR (CDCl3); LC-MS (APCI+) m/z 408 (MH+). Anal.
(C27H18FNO2) C, H, N.
1-(4-Chlorobenzyl)-3-(naphthalene-1-carbonyl)-1,4-dihydro-
quinolin-4-one (85). Compound 85 was purified by TLC (dichlo-
Cell Culture and Preparation of hCB1- or hCB2-Transfected
CHO Cell Membranes. CHO cells stably transfected with the

CB2-SelectiVe Cannabinoid Receptor Ligands
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22 5483
(8) Pacher, P.; Batkai, S.; Kunos, G. The endocannabinoid system as an
emerging target of pharmacotherapy. Pharmacol. ReV. 2006, 58,
389-462.
cDNA sequences encoding either the human CB1 or the human
CB2 cannabinoid receptors were kindly provided by Dr. M.
Detheux and Dr. P. Nokin, respectively (Euroscreen s.a., Gosselies,
Belgium). Cell cultures and membrane preparation were reported
previously.19
Competition Binding Assay. [3H]-SR-141716A (52 Ci/mol) was
purchased from Amersham (Roosendaal, The Netherlands) and
[3H]-CP-55,940 (101 Ci/mol) from NEN Life Science (Zaventem,
Belgium). Binding assay procedure was previously reported,19 under
those conditions, using [3H]-SR-141716A, the Bmax value was 57
pmoles/mg protein and the Kd value was 1.13 ( 0.13 nM for the
hCB1 cannabinoid receptor, and using [3H]-CP-55,940, the Bmax
value was 194 pmoles/mg protein and the Kd value was 4.3 ( 0.13
nM for the hCB2 cannabinoid receptor. The results are expressed
as mean ( SEM of at least three experiments performed in
duplicate.
[35S]GTPγS Assay. [35S]-GTPγS (1173 Ci/mmol) was obtained
from Amersham (Roosendaal, The Netherlands). The experiments
were performed as previously described.19 Gpp(NH)p 100 µM
was used to determine the nonspecific binding. The results are
expressed as mean ( SEM of at least three experiments performed
in duplicate and are reported for a concentration of ligand of
10 µM.
Data Analysis. IC50 values were determined by nonlinear
regression analysis performed using the GraphPad prism 4.0
program (GraphPad Software, San Diego). The Ki values were
calculated from the IC50, based on the Cheng-Prusoff equation:
Ki ) IC50/(1 + L/Kd). The statistical significance of [35S]-GTPγS
assay results was assessed using a one-way ANOVA followed by
a Dunett post-test.
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Acknowledgment. Dr. Laurence Goossens is acknowledged
for her contribution to the chiral preparative HPLC separation
of 24, 25, and 59-62. GGM is a postdoctoral researcher from
the National Fund for Scientific Research (FNRS, Belgium).
This work was partially supported by Charcot Foundation
(Bruxelles, Belgium) and by the National Fund for Scientific
Research (FNRS, Cre´dit aux chercheurs. Belgium).
Supporting Information Available: Elemental analysis of
compounds 10-25, 29-35, 38-40, 42-44, 47, 52-58, 67, 68,
71, 76, and 80-98, spectroscopic data and melting point for all
compounds, and procedures for the synthesis of intermediates.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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JM070387H