4-oxo-1,4-dihydropyridines as selective CB2 cannabinoid receptor ligands: Structural insights into the design of a novel inverse agonist series

Article abstract of DOI:10.1021/jm100286k

Growing evidence shows that CB₂ receptor is an attractive therapeutic target. Starting from a series of 4-oxo-1,4-dihydroquinoline-3- carboxamide as selective CB₂ agonists, we describe here the medicinal chemistry approach leading to

Full text of DOI:10.1021/jm100286k

7918 J. Med. Chem. 2010, 53, 7918–7931
DOI: 10.1021/jm100286k
4-Oxo-1,4-dihydropyridines as Selective CB2 Cannabinoid Receptor Ligands: Structural Insights
into the Design of a Novel Inverse Agonist Series
Jamal El Bakali,†,^ Giulio G. Muccioli,‡,#,^ Nicolas Renault,§ Delphine Pradal,† Mathilde Body-Malapel, Madjid Djouina,
‡
Laurie Hamtiaux, Virginie Andrzejak, Pierre Desreumaux, Philippe Chavatte, Didier M. Lambert,* and Regis Millet*
†
§
,‡
,†
ꢀ
†Universiteꢀ Lille-Nord de France, Institut de Chimie Pharmaceutique Albert Lespagnol, EA 2692, IFR 114, 3 Rue du Professeur Laguesse,
BP 83, 59006 Lille Cedex, France, ‡Uniteꢀ de Chimie Pharmaceutique et de Radiopharmacie, Louvain Drug Research Institute,
Universiteꢀ Catholique de Louvain, 73 Avenue E. Mounier UCL-CMFA (7340), B-1200 Bruxelles, Belgium, §Universiteꢀ Lille-Nord de France,
Faculteꢀde Pharmacie, Laboratoire de Chimie Theꢀrapeutique, EA 1643, IFR 114, 3 Rue du Professeur Laguesse, BP 83, 59006 Lille Cedex, France,
and Physiopathologie des Maladies Inflammatoires de l’Intestin, Universiteꢀ Lille-Nord de France, Amphi J & K, U795, IFR 114,
Boulevard du Professeur Leclercq, 59045 Lille Cedex, France. ^ Both authors contributed equally to this work.
# Present address: Bioanalysis and Pharmacology of Bioactive Lipids Lab, Louvain Drug Research Institute, Universiteꢀ Catholique de Louvain,
72 Avenue E. Mounier UCL-CHAM (7230), B-1200 Bruxelles, Belgium
Received March 4, 2010
Growing evidence shows that CB2 receptor is an attractive therapeutic target. Starting from a series of
4-oxo-1,4-dihydroquinoline-3-carboxamide as selective CB2 agonists, we describe here the medicinal
chemistry approach leading to the development of CB2 receptor inverse agonists with a 4-oxo-1,4-
dihydropyridine scaffold. The compounds reported here show high affinity and potency at the CB2
receptor while showing only modest affinity for the centrally expressed CB1 cannabinoid receptor.
Further, we found that the functionality of this series is controlled by its C-6 substituent because
agonists bear a methyl or a tert-butyl group and inverse agonists, a phenyl or 4-chlorophenyl group,
respectively. Finally, in silico studies suggest that the C-6 substituent could modulate the conformation
of W6.48 known to be critical in GPCR activation.
Introduction
(CNSa) side effects. Because these undesirable effects are
thought to be CB1 receptor-mediated,5 the main strategy to
avoid them is to develop CB2 selective ligands.6 The high ex-
pression of CB2 receptor in immune tissues and cells, both in
periphery2,7 and in the CNS8,9 as well as the enhancement of
its expression following inflammatory insults,10,11 suggests
that CB2 receptor selective ligands might be effective in
modulating inflammation. These observations were con-
firmed by the lack of immunomodulation induced by canna-
binoids in CB2 knockout mice.12 The CB2 receptor exerts a
critical role in the immune system by modulating cytokines
release13,14 and immune cells migration.15-17 Besides, recent
studies have emphasized the major role of CB2 receptors in
pathologies where an inflammatory component is involved,
including Alzheimer disease,18 inflammatory bowel disease,19-21
or multiple sclerosis.22,23 Other studies indicate that CB2
receptors could be involved in alleviating pain24 and could
provide protection from bone loss.25
This therapeutic potential has prompted the development
of several CB2 receptor selective ligands, either as agonists or
as antagonists/inverse agonists (Chart 1). Among the selective
agonists, classical cannabinoids and aminoalkylindoles have
been extensively studied.26-28 (6aR,10aR)-3-(1,1-Dimethyl-
butyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,
d]pyran (1, JWH-133) was shown to decrease experimental
colitis induced by oil of mustard and dextran sulfate sodium in
vivo,19 whereas (1-(2-morpholin-4-yl-ethyl)-1H-indol-3-yl)-
(2,2,3,3-tetramethylcyclopropyl) methanone (2, A-796260)
has demonstrated analgesic activity in models of inflammatory,
postoperative, neuropathic, and osteoarthritic pain.24 More
The CB2 receptor belongs to the class A of G protein-
coupled receptors superfamily. It is one of the components of
the endocannabinoid system, which is a physiological system
composedof cannabinoidreceptors, CB1 and CB2, their endo-
genous ligands, named endocannabinoids, and the biotrans-
formation enzymes involved in the synthesis, degradation,
and cellular uptake of these endocannabinoids.1-3 This sys-
tem plays a key role in numerous biological processes and is
involved in maintaining homeostasis. Cannabinoids exhibit
pharmacological effects in a large spectrum of diseases and
disorders.4 Thus, in the past years, investigations were aimed
at designing new synthetic molecules that target cannabinoid
receptors. One of the main challenges for such compounds is
to be as much as possible devoid of central nervous system
*To whom correspondence should be addressed. For R.M.: phone,
ꢀ
þ33-3-2096-4374; fax, þ33-3-2096-4906; E-mail, regis.millet@univ-lille2.fr.
For D.M.L.: phone, þ32-2-764-7347; fax, þ32-2-764-7363; E-mail,
didier.lambert@uclouvain.be.
a Abbreviations: CNS, central nervous system; CHO, Chinese ham-
ster ovary; hCB1, human CB1 cannabinoid receptor; hCB2, human CB2
cannabinoid receptor; Ki, inhibition constant; EC50, half-maximal
effective concentration; [35S]-GTPγS, guanosine-50-O-(3-[35S]thio)-
triphosphate); Emax, maximum efficacy; TM, transmembrane domain;
EL, extracellular loop; rmsd, root-mean-square deviation; LHMDS,
lithium hexamethyldisilazane; DMF, N,N-dimethylformamide; DMS,
dimethylsulfate; EtOAc, ethyl acetate; EtOH, ethanol; AcOH, acetic
acid; DMSO, dimethylsulfoxide; DIPEA, N,N-diisopropylethylamine;
HOBt, 1-hydroxybenzotriazole; HBTU, 2-(1H-benzotriazol-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate; DPPA, diphenyl-
phosphoryl azide; t-BuOK, potassium tert-butoxide; t-BuOH, tert-butyl
alcohol; rt, room temperature; TLC, thick-layer chromatography
r
pubs.acs.org/jmc
Published on Web 10/27/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 7919
Chart 1. Structure of Representatives CB2 Selective Ligands (1)
JWH-133, (2) A-796260, (3) CBS-0550, (4) GW842166X, (5)
SR144528, (6) JTE907, (7) Sch225336, (8) ALICB122
Chart 2. General Structure of the Tested Compounds 11,
17-41, and 43
However, it is essential to better understand the pharma-
cology of CB2 receptor ligands because, for instance, anti-
inflammatory properties have been described for both ago-
nists and inverse agonists. Similar trends were observed con-
cerning studies on bone physiology. Indeed, some authors
suggested that blocking the CB2 receptor protects from bone
loss in ovariectomized mice37,38 and others showed the same
effect using the highly selective CB2 receptor agonist {4-
[4-(1,1-dimethylheptyl)-2,6-dimethoxy-phenyl]-6,6-dimethyl-
bicyclo[3.1.1]hept-2-en-2-yl}-methanol (HU-308, structure
not shown).25,39
In this context, our groups previously described the synthe-
sis, pharmacological characterization, and structure-activity
relationships of selective CB2 ligands based on a 4-oxo-1,4-
dihydroquinoline-3-carboxamide scaffold (e.g., 8).40,41 The
structure-activity relationship studies highlighted a signifi-
cant correlation between affinity and/or selectivity toward
the CB2 receptor and structural features such as an aliphatic
moiety, especially an adamantyl substituent, on the C-3 car-
boxamide group as well as a n-pentyl chain in N-1 position.
Concerning the functionality of these ligands, most of the
compounds behaved as selective CB2 agonists and, more
surprisingly, small changes in the position of the substituents
around the heterocycle resulted in modifications of the com-
pounds functionality.41
In light of these considerations, we sought to find new CB2
selective ligands. Our efforts to identify such compounds
allowed us to describe novel series of 4-oxo-1,4-dihydropyr-
idines and 4-thioxo-1,4-dihydropyridines (general structure
shown in Chart 2) that were found to be potent and selective
CB2 receptor ligands. We also report here the identification of
a key substituent on the 4-oxo-1,4-dihydropyridine scaffold
responsible for a functionality switch within this series of
compounds.
recently, other compounds have been disclosed such as the
iminopyrazole N-(5-tert-butyl-2-cyclopropylmethyl-1-methyl-
1,2-dihydropyrazol-3-ylidene)-2-fluoro-3-(trifluoromethyl)-
benzamide (3, CBS-0550)29 and the pyrimidine derivative
2-[(2,4-dichlorophenyl)amino]-N-[(tetrahydro-2H-pyran-4-yl)-
methyl]-4-(trifluoromethyl)-5-pyrimidinecarboxamide (4,
GW842166X),30 which was chosen as a clinical candidate for the
treatment of inflammatory pain. As for selective antagonists/
inverse agonists, much fewer compounds have beendescribed.
The first to be discovered and the most widely used is the 1,5-
diarylpyrazole, 5-(4-chloro-3-methylphenyl)-1-[(4-methylph-
enyl)methyl]-N-[(1S,4R,6S)-1,5,5-trimethyl-6-bicyclo[2.2.1]-
heptanyl]pyrazole-3-carboxamide (5, SR144528).31 This com-
pound as well as the 2-quinolone derivative N-(benzo[1,3]-
dioxol-5-ylmethyl)-7-methoxy-2-oxo-8-pentyloxy-1,2-dihydro-
quinoline-3-carboxamide (6, JTE907) received attention due
to their anti-inflammatory properties.32 More recently, a new
class of CB2 selective inverse agonists based on a triaryl bis-
sulfone scaffold has been described.33-35 This class is repre-
sented by N-[1(S)-[4-[[4-methoxy-2-[(4-methoxyphenyl)sulfo-
nyl]phenyl]-sulfonyl]phenyl]ethyl]methanesulfonamide (7,
Sch225336), which was shown to block the recruitment of
leucocytes in vivo.36 Taken together, the available data strongly
support CB2 ligands as modulators of inflammation.
Chemistry
The synthesis of N3-(1-adamantyl)-6-methyl-1-pentyl-4-
oxo-1,4-dihydropyridine-3-carboxamide (compound 11), out-
lined in Scheme 1, was performed using a methodology adapted
from the literature.42,43 The commercially available 4-hydroxy-
6-methyl-2-pyrone was reacted with N,N-dimethylformamide
dimethyl acetal in mild conditions to give 9, which, when
treated with n-pentylamine under alkaline conditions followed
by acidification gave the carboxylic acid 10. Finally, amidation
was accomplished with 1-aminoadamantane hydrochloride
under peptide coupling conditions to give the target amide 11.
The synthesis of the 6-tert-butyl, 6-phenyl or 6-(4-chloro-
phenyl) substituted 4-oxo-1,4-dihydropyridine-3-carboxamides
and 4-thioxo-1,4-dihydropyridine-3-carboxamides 17-36 is

7920 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
El Bakali et al.
Scheme 1a
a Reagents and conditions: (i) DMF-DMA, dioxane, rt, 4 h, 63%; (ii) t-BuOK, n-pentylamine, EtOH, reflux, 12 h, 73%; (iii) (a) HOBt, HBTU,
DIPEA, DMF, rt, 3 h, (b) 1-aminoadamantane hydrochloride, rt, 12 h, 57%.
Scheme 2a
a Reagents and conditions: (i) DMF/DMS, Et3N, CH2Cl2, rt, 2 h, 79%; (ii) (a) LHMDS, RCOCl, THF, -78 ꢀC, 15 min, (b) 3N HCl, Et2O, rt, 45 min,
35-67%; (iii) R0-NH2, AcOH/EtOH 1:3 reflux, 1-4 h, 45-77%; (iv) (when 14d) P4S10, pyridine, reflux, 12 h, 90%; (v) 10% NaOH/EtOH v/v, reflux, 6 h,
66-96%; (vi) (a) HOBt, HBTU, DIPEA, DMF, rt, 3 h, (b) R00-NH2, rt, 12 h, 28-74%.
described in Scheme 2. Structures of intermediate compounds
13-16 as well as target amides 17-40 are summarized in
Table 1. The 4-oxo-1,4-dihydropyridine scaffold was prepared
from ethyl acetoacetate as previously described.44 Enaminone
12 was obtained by reaction of ethyl acetoacetate with the N,
N-dimethylformamide/dimethylsulfate adduct (DMF/DMS)
in combination with triethylamine. Subsequent deproton-
ation of 12 with lithium hexamethyldisilazane (LHMDS) in the
presence of the appropriate acyl chloride at -70 ꢀC followed
by acidification at room temperature led directly to pyran-4-
ones 13a-g. Aminolysis in acidic conditions afforded 4-oxo-
1,4-dihydropyridines 14a-k in acceptable yields. The ana-
logue 4-thioxo-1,4-dihydropyridine15was synthesized invery
good yields from the corresponding 4-oxo-1,4-dihydropyri-
dine (14d) by a thionation reaction using phosphorus penta-
sulfide as reagent. After saponification of the ethyl ester
functions of compounds 14a-k and 15 (sodium hydroxide),
the resulting carboxylic acids 16a-l were engaged in an
amidation reactionwiththe appropriate aminesunder peptide
coupling conditions to afford target amide compounds 17-40.
Carboxylic acid 16d was also converted to the carbamate 41
(Boc protected amine) via a Curtius rearrangement using
diphenylphosphoryl azide and potassium tert-butoxide as
reagents and tert-butanol as solvent (Scheme 3). Treatment
of 41 with hydrochloric acid in isopropyl alcohol followed by
neutralization led to amine 42. The latter reacted with cyclo-
hexyl carboxylic acid under peptide coupling conditions to
afford the “reverse amide” 43.
and human CB1 cannabinoid receptor (hCB1), respectively, as
previously described.45 Membranes from Chinese hamster
ovary (CHO) cells expressing either the hCB1 or the hCB2
cannabinoid receptor were used in these experiments. All
compounds were first screened at 10 μM concentration for
their affinity toward both cannabinoid receptors. The inhibi-
tion constant (Ki) valueswerethen determinedfor compounds
exhibiting a specific displacement superior to 60% either for
hCB1 or the hCB2 (Table 2), and the selectivity index (CB2 vs
CB1) was calculated whenever possible. We also investigated
their functionality at the CB2 receptor using a guanosine-50-
O-(3-[35S]thio)triphosphate ([35S]-GTPγS) binding assay and
hCB2-CHO cells membranes, as previously described.46 This
assay constitutes a functional measure of the interaction of the
receptor and the G-protein, the first step in activation of the
G-protein coupled receptors. In this system, neutral antago-
nists do not affect [35S]-GTPγS binding, while agonists and
inverse agonists respectively increase and decrease nucleotide
binding. The functionality of reference cannabinoid agonists
1, WIN-55,212-2 and CP-55,940 as well as the inverse agonist
5 were determined. Half-maximal effective concentration
(EC50) and maximum efficacy (Emax) values of reference and
original compounds are summarized in Table 3.
Structure-Affinity/Activity Relationships
The starting point of our investigation was the lead com-
pound 8, which was described by our groups as a potent
selective CB2 agonist (Ki=16.4 nM). In the present work, we
removed the condensed benzene ring of 4-oxo-1,4-dihydro-
quinoline-3-carboxamide derivatives resulting in the 4-oxo-
1,4-dihydropyridine core. A set of four compounds was first
synthesized on the basis of our previously reported work (11, 17,
25, and 32).40 These compounds possessed some features in
common, like the N-1 pentyl chain and a carboxamidoadamantyl
Pharmacology
The affinities of the newly synthesized compounds 11,
17-41, and 43 were determined by a competitive radioligand
displacement assay using [3H]-CP55,940 and [3H]-SR141716A
as radioligands for human CB2 cannabinoid receptor (hCB2)

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 7921
Table 1. Structures of the Newly Synthesized Compounds 13-40
compd
R
R0
R00
X
Y
Z
13a
13b
13c
13d
13e
13f
13g
14a
14b
14c
14d
14e
14f
14g
14h
14i
14j
14k
15
tert-butyl
phenyl
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
S
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
O
O
O
O
O
O
O
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
4-chlorophenyl
3-chlorophenyl
2-chlorophenyl
4-methylphenyl
4-methoxyphenyl
tert-butyl
pentyl
ethyl
butyl
phenyl
phenyl
phenyl
phenyl
phenyl
pentyl
hexyl
phenyl
pentyl
pentyl
pentyl
pentyl
pentyl
pentyl
pentyl
ethyl
4-chlorophenyl
3-chlorophenyl
2-chlorophenyl
4-methylphenyl
4-methoxyphenyl
phenyl
16a
16b
16c
16d
16e
16f
16g
16h
16i
16j
16k
16l
17
tert-butyl
phenyl
O
O
O
O
O
O
O
O
O
O
O
S
phenyl
phenyl
butyl
pentyl
hexyl
phenyl
phenyl
phenyl
pentyl
pentyl
pentyl
pentyl
pentyl
pentyl
pentyl
pentyl
ethyl
4-chlorophenyl
3-chlorophenyl
2-chlorophenyl
4-methylphenyl
4-methoxyphenyl
phenyl
tert-butyl
tert-butyl
1-adamantyl
cyclohexyl
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
S
18
19
phenyl
phenyl
(R,S)-1-(adamantyl)ethyl
(R,S)-1-(adamantyl)ethyl
(R,S)-1-(adamantyl)ethyl
1-(adamantyl)methyl
1-(3,5-dimethyl)adamantyl
1-adamantyl
20
21
pentyl
phenyl
pentyl
pentyl
butyl
phenyl
phenyl
22
23
phenyl
phenyl
24
25
phenyl
phenyl
pentyl
hexyl
1-adamantyl
1-adamantyl
cyclohexyl
26
27
phenyl
phenyl
pentyl
pentyl
pentyl
pentyl
pentyl
pentyl
pentyl
pentyl
pentyl
pentyl
pentyl
pentyl
pentyl
pentyl
28
29
(R)-1-(1,2,3,4-tetrahydronaphthyl)
(S)-1-(1,2,3,4-tetrahydronaphthyl)
piperidin-1-yl
phenyl
phenyl
phenyl
30
31
3-(trifluoromethyl)phenyl
1-adamantyl
cyclohexyl
32
33
4-chlorophenyl
4-chlorophenyl
4-chlorophenyl
3-chlorophenyl
2-chlorophenyl
4-methylphenyl
4-methoxyphenyl
phenyl
34
35
3-(trifluoromethyl)phenyl
1-adamantyl
1-adamantyl
36
37
1-adamantyl
1-adamantyl
38
39
1-adamantyl
cyclohexyl
40
phenyl
S
moiety at position 3, but differed by their substituents at posi-
tion 6 of the heterocycle (i.e., methyl, tert-butyl, phenyl, and
4-chlorophenyl). All of these 6-substituted analogues were
selective, displaying high affinity at the CB2 receptor and low
or no affinity at CB1 receptor. Compound 11, with a methyl at
position 6, exhibits a Ki value at CB2 receptor (20 nM) of the
same magnitude as the starting compound 8 and was found to
be highly selective (Ki at CB1 >3000). A similar result was

7922 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
El Bakali et al.
Scheme 3a
a Reagents and conditions: (i) DPPA, t-BuOK, t-BuOH, reflux, 12 h, 38%; (ii) (a) 5N HCl, isopropyl alcohol, rt, 14 h, (b) 10% NaOH, 95%; (iii) (a)
cyclohexyl carboxylic acid, HOBt, HBTU, DIPEA, DMF, rt, 3 h, (b) 38, rt, 12 h, 38%.
Table 2. Affinities (Ki values) of Compounds 11, 17-41, 43, and
Reference Compounds (5, WIN-55,212-2, CP-55,940, and 1) towards
hCB1 and hCB2 Cannabinoid Receptorsa
Table 3. Potency (EC50) and Maximal Stimulation (Emax) of Selected
Compounds and Reference Ligands on hCB2 Cannabinoid Receptora
[
35S]-GTPγS (hCB2)
Emax (%)b
binding affinity Ki (nM)
compd
EC50 (nM)
selectivity ratio CB2
versus CB1
11
17
18
20
22
23
24
25
26
27
28
29
30
31
32
33
34
39
40
41
43
5
5.5 ( 1.1
12.2 ( 2.5
7.8 ( 3.5
6.5 ( 1.5
10.9 ( 2.8
5.8 ( 1.1
7.4 ( 0.9
3.2 ( 1.6
8.7 ( 0.9
1.9 ( 0.5
1.9 ( 0.4
60 ( 16
148 ( 2c
212 ( 3d
135 ( 3c
46 ( 3e
compd
hCB1
>3000
hCB2
20 ( 3
29 ( 3
11
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
43
5
>150
>103
>103
>7
>3000
>3000
>3000
368 ( 84
>3000
626 ( 92
454 ( 55
>1000
592 ( 97
596 ( 75
929 ( 131
131 ( 20
>1000
>3000
>3000
>1000
>1000
>1000
51.2 ( 6
34.4 ( 6
134 ( 19
384 ( 56
>1000
>1000
>3000
>3000
ND
37 ( 1e
29 ( 3
41 ( 2e
414 ( 61
14.3 ( 2.1
>3000
40 ( 2e
26
39 ( 2e
33 ( e
23.3 ( 1.9
6.6 ( 0.7
18.4 ( 1.3
4.0 ( 0.4
13.5 ( 0.9
10.6 ( 1.1
10.1 ( 1.1
369 ( 41
92 ( 7
25.8 ( 3
79 ( 7
78 ( 9
>1000
27
69
62 ( 2e
42 ( 2e
>54
148
48 ( 3e
26 ( 6
14 ( 4
23 ( 6
22 ( 4
48 ( 2e
44
88
42 ( 4 e
42 ( 2 e
13
>3
39 ( 2 e
158 ( 42
17 ( 2
27 ( 3
164 ( 28
25 ( 3
1.8 ( 0.9
24.6 ( 1.6
6.1 ( 2.1
145.6 ( 3
42 ( 3 e
>33
>116
>13
>13
30 ( 1 e
45 ( 1 e
26 ( 3 e
43 ( 2 e
21.6 ( 2.7 e
207.1 ( 10.1 d
230.5 ( 13.7 d
201.4 ( 7.5 d
1.8 ( 0.2
0.7 ( 0.1
13.2 ( 1.6
91.1 ( 14
18.8 ( 2.3
48 ( 4
209 ( 33
51 ( 4
51.7 ( 4.8
9.1 ( 0.8
15.4 ( 1.4
20.3 ( 2.6
29
48
WIN-55,212-2
CP-55,940
1
10
4
>53
>21
>14
>59
a The results are expressed as mean ( SEM of at least four experi-
ments performed in duplicate. b Basal constitutive activity of the recep-
c
tor has been set at a value of 100%. Emax values between 100% and
200%indicated that the compound behaves as a partial agonist. d Emax
values around 200% indicated that the compound behaves as a full
agonist. e Emax values under 100% indicated that the compound behaves
as an inverse agonist.
WIN-55,212-2
CP-55,940
1
ND
ND
ND
(EC50 = 12.2 nM and Emax = 212%). More surprisingly,
introducing a phenyl or a 4-chlorophenyl group in place of
methyl or tert-butyl shifted the functionality from agonist to
inverse agonist because both compounds 25 and 32 decreased
[35S]-GTPγS binding with Emax values of 39% and 42%,
respectively. Because the functionality switch observed within
this series of compounds is an interesting feature, we decided
to investigate whether the change in functionality we observed
when replacing the C-6 tert-butyl substituent with a phenyl or
4-chlorophenyl substituent was dependent on the phenyl
substituent. Therefore we synthesized four additional com-
pounds characterized by differently substituted phenyls
(35-38) and evaluated their affinity and functionality. These
compounds show a similar affinity for the CB2 receptor when
compared to 25, although they are marginally less selective
(Table 2). When looking at their functionality, they too
behave as inverse agonists decreasing [35S]-GTPγS binding
(Emax values of 75 ( 2%, 88 ( 1%, and 75 ( 5% for 35, 36,
a The Ki values were obtained from nonlinear analysis of competition
curves using using [3H]-SR141716A and [3H]-CP-55,940 as radioligands
for hCB1 and hCB2 cannabinoid receptors, respectively, and are ex-
pressed as mean ( SEM of at least four experiments performed in
duplicate.
obtained for compound 17 bearing a tert-butyl group. Repla-
cing the methyl or tert-butyl groups by a phenyl resulted in a
5-fold enhancement of the affinity (e.g., 25 with a Ki value of
4 nM), while selectivity was notaltered. Introducing a chlorine
atom in the para position of the phenyl ring resulted in a
decrease in both selectivity and CB2 affinity (compare com-
pounds 25 and 32). When considering their functionality, we
noticed that compound 11 dose-dependently increased the
[35S]-GTPγSbindingupto148%withanEC50 value of 5.5 nM,
which means that this compound behaves as a partial agonist,
whereas compound 17 behaves as a potent full agonist increas-
ing [35S]-GTPγS binding to the same extent as CP-55,940

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 7923
Figure 1. Sequence alignment between the model human CB2 receptor and the crystal template sequences. This JalView64 graphical
representation shows the alignment between the whole CB2 model sequence and the partial sequence (without N-terminal, C-terminal, and
IL3 regions) of the X-ray β2-adrenergic receptor (PDB 2RH1). The identical conserved amino acids and relevant CB2 features Ser4.53, Ser4.57,
and Tyr5.58 are respectively displayed as blue and red overlined residues. The TM domains deduced from crytstal template are annotated in the
“TM pred” section.
and 37, respectively), although the 4-methoxyphenyl substi-
tuted 4-oxo-1,4-dihydropyridine behaves as a partial agonist
(Emax = 148 ( 4%). Taken together, these observations
suggest that the C-6 substituent of the 4-oxo-1,4-dihydropyr-
idine ring appears to be crucial for the control of functionality
at CB2 receptor and also modulates affinity, efficacy, and
selectivity.
Because derivative 25 combines a good affinity with the
highest selectivity, we decided to retain the C-6 phenyl moiety
and to develop a series of CB2 inverse agonists with various
N-1 and 3-carboxamido substituents. Therefore, the replace-
ment of the n-pentyl chain by n-ethyl, n-butyl, or n-hexyl
groups resulted in moderate to strong reduction of the affinity
(see compounds 19, 24, 26) while introducing a phenyl
(compound 21) completely abolished the CB2 affinity. Taken
together, these data, in accordance with our previous work,40
clearly emphasize that the affinity toward the CB2 receptor is
very sensitive to N-1 substituent, with n-pentyl chain being the
preferred one.
Next, we investigated the modification of the amide substitu-
ent because it seems to affect affinity toward both cannabi-
noid receptors. It was shown for the 4-oxo-1,4-dihydroquino-
line-3-carboxamide derivatives that bulky aliphatic substituents
(especially adamantyl) are favorable for CB2 affinity and
selectivity,40,41 and compounds 11, 17, and 25 confirmed this
observation. Enhancement of adamantyl lipophilicity by in-
troducing two methyl groups in position 3 and 5 of the
adamantyl ring (compound 23) improved neither affinity nor
efficacy at CB2 receptor but enhanced CB1 receptor affinity.
When the adamantyl moiety was positioned further away
from the 4-oxo-1,4-dihydropyridine core (by a methylene
link) leading to compound 22, affinity at CB2 decreased by
6-fold. However, when a methyl group was added on the
methylene link, the affinity for both cannabinoid receptors
was increased (compare 22 and 20). When a phenyl group was
placed at position 6 of the 4-oxo-1,4-dihydropyridine ring, the
replacement of the adamantyl moiety by a less bulky aliphatic
group like cyclohexyl induced a slight decrease in affinity
(compare 25 and 27). However, no effect was observed when a
tert-butyl or a 4-chlorophenyl was placed at position 6
(compare for instance 17 and 18 or 32 and 33).
pounds 25 and 31, which exhibit Ki values of 4 and 26 nM,
respectively.
By analogy with 4-oxo-1,4-dihydroquinoline-3-carboxa-
mide derivatives, we introduced a chiral center on the carbox-
amido function in order to assess the effect of stereoselectivity
on the affinity, selectivity, and functionality of our inverse
agonist series. Therefore, we prepared two compounds char-
acterized by the 1-(1,2,3,4-tetrahydronaphthyl) moiety, 28
represents the (R) enantiomer and 29 the (S) enantiomer.
The eutomer (compound 28) of this novel series exhibited
more than 30-fold higher affinityfor the CB2 receptor than the
distomer (compound 29). In accordance with our earlier
work, a stereoselectivity is observed with the (R) enantiomer
exhibiting a better affinity and efficacy than the (S) enantio-
mer. Furthermore, compound 28 as well as compound 27 are
the most potent compounds of our series, with EC50 values of
1.9 nM.
Asexpected, the 3-carboxamido substituent is animportant
parameter for the affinity, efficacy, and selectivity. However,
and opposite to what we found for the C-6 position, this
modification did not affect the functionality because, regard-
less the carboxamido substituent, all the compounds retained
their respective functionality.
When looking at the amide link of 27, we found that the
reverse amide 43 has 5-fold lower affinity as compared to the
amide analogue. In a similar manner, the carbamate inter-
mediate 41, which can be regarded as an analogue of com-
pound 43 wherein its cyclohexyl moiety is replaced by a tert-
butoxy group, displayed a lower affinity with a Ki value of 209
nM at CB2 and no affinity at CB1.
We also introduced a thioketone in place of the carbonyl of
4-oxo-1,4-dihydropyridine core, leading to compounds 39
and 40. Albeit this frequent substitution has already been
proved to affect the affinity or functionality of some canna-
binoid ligands,47,41 we found here that this modification
resulted in a 5-fold reduction of the affinity and had no effect
on the functionality.
In Silico Insights from an Inactive State Model of the CB2
Receptor
The inactive state of the human CB2 apo-receptor was built
from the homologous crystal template of the human β2-
adrenergic receptor (Figure 1), as specified in the Experimen-
tal Section. Thus, the modeled CB2 receptor should be in its
inactive state because the human β2-adrenergic receptor
template used was cocrystallized with a high-affinity inverse
agonist.48-51
Next, we prepared compound 30, with a piperidyl group on
the amide moiety, which showed a Ki value of 92 nM at CB2
receptor and was found inactive at CB1. Introducing an
aromatic moiety, especially the 3-(trifluoromethyl)phenyl
group, in place of adamantyl caused a reduction in affinity
and efficacy at CB2. This is illustrated for instance by com-

7924 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
El Bakali et al.
As illustrated in Figure 2, the resulting ligand binding site is
restricted by transmembrane domain (TM) III, IV, V, VI, and
VII, as well as extracellular loops (EL) 2 and 3. The resulting
docking poses of both compounds 17 and 25 revealed a
consensual binding mode, as the five best ones were super-
imposed within a 1.5 A root-mean-square deviation (rmsd)
(Figure 3). Both compounds 17 and 25 are anchored by
hydrogen bonds with Tyr5.39 (Tyr190) and Phe183 backbone
of EL2. The pentyl chains spread out in a lipophilic pocket
definedbyPhe91, Phe94, Phe106, Ile110, Val113, and Leu182.
The adamantyl groups fit in the extracellular region particu-
larly EL2, whereas the C-6 tert-butyl or phenyl substituents of
compounds 17 and 25, respectively, orient toward the bottom
of the pocket defined by an aromatic cage including Phe3.36
(Phe117), Trp5.43 (Trp194), and Trp6.48 (Trp258). Trp6.48 is
included in the CWXP pattern of helix 6 and is strictly
conserved among the class A GPCRs. Trp6.48 and adjacent
side chains have been shown to undergo conformational
transitions as a “rotamer toggle switch” during the activation
of rhodopsin52-54 and β2-adrenergic receptors.55 This “rota-
mer toggle switch” has also been shown to be critical for the
activation of the CB1 receptor with specific contacts between
Phe3.36 and Trp6.48 in the inactive state, which are broken
during the activation, leading to a χ1 rotamer switch (F3.36
trans(180ꢀ)/W6.48 gþ(-60ꢀ)) f (F3.36 gþ, W6.48 trans).56
As observed in the CB2 receptor bound to the compound 25
(Figure 3A), Phe3.36 and Trp6.48 form a π-π face-to-face
interaction, whereas the C-6 phenyl substituent of compound
25 establishes edge-to-face aromatic interactions with
Trp6.48. During the MD simulation, the placement of both
ligands was examined by monitoring the distance between the
C-6 substituents of the ligands and the side chain of Trp6.48
(Figure 4). Taking into account the distance between the
closest carbon atoms in the minimized complex at t=0, the
simulation shows that the distance implying the C-6 phenyl
group of compound 25 tends to converge toward a distance of
4 A, whereas the distance between the C-6 tert-butyl substi-
tuent of compound 17 and the side chain of Trp6.48 does not
converge and varies between 5 and 8 A. The preservation of
the aromatic cluster between the C-6 substituent of compound
25 and Trp6.48 is also depicted by monitoring the receptor-
ligand binding energy during the MD simulation (Figure 4).
Indeed, the binding energy of the complex with compound 25
ranges from -65 to -55 kcal/mol and is thus more stabilizing
Figure 2. Homology model of the CB2 apo-receptor. A global
overview of the complex model is represented where the binding
site is displayed as a solvent-accessible surface colored from blue for
polar regions to brown for hydrophobic regions.
Figure 3. Ligand-bound states of the CB2 receptor with compounds 25 (A) and 17 (B). The best docking poses for each ligand are displayed as
lines, whereas the ligand pose carried out from energy minimization is illustrated as sticks. The hydrogen bonds figure as yellow dashed lines.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 7925
we demonstrate that substituents at N-1 and C-3 position
are crucial for affinity but not for functionality. Using a β2-
adrenergic receptor-based CB2 receptor model, we suggest
that the phenyl at C-6 confers the inverse agonist profile by
blocking the χ1 torsion of Trp6.48 side chain in its inactive
conformation. Overall, the data presented here show that the
4-oxo-1,4-dihydropyridine ring is a highly effective scaffold
for the design of new CB2 receptor ligands. Moreover, the
novel selective CB2 ligands reported here will be useful tools
for characterizing the functions of CB2 receptor.
Experimental Section
Chemistry. All commercial reagents and solvents were used
without further purification. Analytical thin-layer chromatog-
raphy was performed on precoated Kieselgel 60F254 plates
(Merck); the spots were located by UV (254 and 366 nm) and/
or with iodine. Silica gel 60 230-400 mesh purchased from
Merck was used for column chromatography. Alumina silica gel
(neutral) 150 mesh purchased from Sigma Aldrich was used for
column chromatography for purification of compound 13c.
Preparative thick-layer chromatography (TLC) was performed
using silica gel from Merck, the compounds were extracted from
the silica using CHCl3/MeOH (8:2, v/v). All melting points were
€
determined with a Buchi 535 capillary apparatus and remain
uncorrected. H NMR spectra were obtained using a Bruker
1
€
300 MHz spectrometer, chemical shifts (δ) are expressed in
ppm relative to tetramethylsilane used as an internal standard,
J values are in hertz, and the splitting patterns are designated as
follows: s, singlet; d, doublet; t, triplet; m, multiplet. All com-
pounds were analyzed by HPLC-MS on a HPLC combined with
a Surveyor MSQ (Thermo Electron) equipped with an APCI-
source. All tested compounds showed a purity of >96% in
APCIþ mode.
3-(Dimethylaminomethylene)-6-methyl-4-oxo-2-pyrone 9 was
prepared according to a procedure already described.42,43 Com-
pound 9 crystallizes in a mixture of toluene/cyclohexane (2:8, v/v).
The ethyl 2-[(dimethylamino)methylene]-3-oxobutanoate 12,
pyran-4-ones 13a (ethyl 6-tert-butyl-4-oxo-4H-pyran-3-carboxylate)
and 13b (ethyl 4-oxo-6-phenyl-4H-pyran-3-carboxylate) and
pyridin-4-one 14f (ethyl 4-oxo-1,6-diphenyl-1,4-dihydropyri-
dine-3-carboxylate) were obtained using the procedures
previously described44 with minor modifications. In our case,
pyran-4-ones 13a-g were unstable on silica gel and could not be
purified even by flash chromatography. We found that pyran-4-
ones 13a-b and 13d-g crystallize in diethyl ether at low
temperature under reduced pressure.
Figure 4. Analysis of molecular dynamics simulations. Binding
energy between 17 and 25 with the CB2 receptor was plotted (A)
as well as the distance between the less buried carbon atom of
Trp258 (W258) side chain and the more buried carbon atom of the
ligand (B).
than the complex, including compound 17, for which the
binding energy ranges from -55 to -45 kcal/mol.
6-Methyl-4-oxo-1-pentyl-1,4-dihydropyridine-3-carboxylic Acid
(10). To a stirred solution of compound 9 (0.2 g; 1.1 mmol) in
anhydrous ethanol (EtOH) (10 mL) were added under nitrogen
atmosphere, potassium tert-butoxide (0.17 g; 1.7 mmol), and
n-pentylamine (0.25 mL; 2.2 mmol). This mixture was refluxed
under nitrogen for 12 h. EtOH was removed under reduced
pressure and the residue dissolved in water. The aqueous phase
was washed with ethyl acetate (EtOAc), and the resulting
solution was carefully acidified with a 1 N HCl solution to pH
2 and extracted with EtOAc. The combined organic extracts
were washed with H2O, dried over MgSO4, and the solvent
removed under reduced pressure to afford pure 10 as a white
solid (0.18 g, 73%); mp 126 ꢀC. 1H NMR (CDCl3) δ 16.61
(s, 1H), 8.43 (s, 1H), 6.58 (s, 1H), 3.97 (t, 2H, J=7.9 Hz), 2.45
(s, 3H), 1.80 (m, 2H), 1.38 (m, 4H), 0.93 (t, 3H, J=6.8 Hz).
LC-MS (APCIþ) m/z 224.2 (MHþ).
Even though the duration of the simulation is not long
enough to observe a conformational switch of Trp6.48 χ1
torsion from the g(þ) (-60ꢀ) toward its trans conformation
(180ꢀ), these results suggest that the phenyl substituent at the
C-6 position of compound 25 confers the inverse agonist
profile by stabilizing the χ1 torsion of Trp6.48 side chain in
its inactive g(þ) conformation and thus could prevent its
transition toward the trans conformation thought to be
essential for the receptor activation.
Conclusion
We have synthesized a series of selective CB2 receptor
ligands based on a 4-oxo-1,4-dihydropyridine scaffold. Bind-
ing assays showed that the nature of substituents around the
heterocycle strongly impacts on both affinity and function-
ality. We have identified the C-6 substituent as crucial in
controlling the functionality of this series of compounds
because replacing an alkyl group by a phenyl group switched
the functionality from agonist to inverse agonist. Conversely,
General Procedure for the Preparation of Ethyl 6-Aryl-4-oxo-
4H-pyran-3-carboxylate (13c-13g). These compounds were
obtained using the same methodology as already described.44
Ethyl 6-(4-Chlorophenyl)-4-oxo-4H-pyran-3-carboxylate (13c).
Compound 13c was purified by neutral silica-alumina gel
chromatography (dichloromethane/ethyl acetate 1:1, v/v); beige

7926 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
El Bakali et al.
solid (52%); mp 155 ꢀC. 1H NMR (CDCl3) δ 8.58 (s, 1H), 7.7
(d, 2H, J = 9.1 Hz), 7.5 (d, 2H, J = 9.1 Hz), 6.84 (s, 1H), 4.40
(q, 2H, J = 7.0 Hz), 1.41 (t, 3H, J = 7.0 Hz). LC-MS (APCIþ)
m/z 279.4 (MHþ).
Ethyl6-(3-Chlorophenyl)-4-oxo-4H-pyran-3-carboxylate(13d).
Beige solid (57%); mp 118 ꢀC. 1H NMR (CDCl3) δ 8.59 (s, 1H),
7.76 (s, 1H), 7.65 (d, 2H, J = 7.6 Hz), 7.48 (m, 2H), 6.86 (s, 1H),
4.40 (q, 2H, J = 7.0 Hz), 1.41 (t, 3H, J = 7.0 Hz). LC-MS
(APCIþ) m/z 279.0 (MHþ).
Ethyl 6-(4-Chlorophenyl)-4-oxo-1-pentyl-1,4-dihydropyridine-
3-carboxylate (14g). Purification by silica gel chromatography
(dichloromethane/methanol 9:1, v/v); yellow oil (75%). 1H NMR
(CDCl3) δ 8.22 (s, 1H), 7.50 (d, 2H, J = 8.8 Hz), 7.30 (d, 2H, J =
8.8 Hz), 6.40 (s, 1H), 4.40 (q, 2H, J = 7.1 Hz), 3.72 (t, 2H, J =
7.7 Hz), 1.55 (m, 2H), 1.40 (t, 3H, J = 7.1 Hz), 1.18 (m, 4H), 0.82
(t, 3H, J = 7.0 Hz). LC-MS (APCIþ) m/z 348.2 (MHþ).
Ethyl 6-(3-Chlorophenyl)-4-oxo-1-pentyl-1,4-dihydropyridine-
3-carboxylate (14h). Purification by silica gel chromatography
(dichloromethane/methanol 9:1, v/v); yellow oil (63%). 1H NMR
(CDCl3) δ 8.22 (s, 1H), 7.48 (m, 2H), 7.34 (s, 1H), 7.24 (d, 2H, J =
7.6 Hz), 6.41 (s, 1H), 4.40 (q, 2H, J = 7.1 Hz), 3.72 (t, 2H, J =
7.7 Hz), 1.55 (m, 2H), 1.40 (t, 3H, J = 7.1 Hz), 1.13 (m, 4H), 0.82
(t, 3H, J = 7.0 Hz). LC-MS (APCIþ) m/z 348.1 (MHþ).
Ethyl 6-(2-Chlorophenyl)-4-oxo-1-pentyl-1,4-dihydropyridine-
3-carboxylate (14i). Purification by silica gel chromatography
(dichloromethane/methanol 9:1, v/v); yellow oil (45%). 1H
NMR (CDCl3) δ 8.10 (s, 1H), 7.17 (m, 4H), 6.12 (s, 1H), 4.10
(q, 2H, J = 7.1 Hz), 3.78 (t, 2H, J = 7.7 Hz), 1.32 (m, 2H), 1.12
(t, 3H, J = 7.1 Hz), 0.86 (m, 4H), 0.51 (t, 3H, J = 7.0 Hz). LC-
MS (APCIþ) m/z 348.1 (MHþ).
Ethyl 6-(4-Methylphenyl)-4-oxo-1-pentyl-1,4-dihydropyridine-
3-carboxylate (14j). Purification by silica gel chromatography
(dichloromethane/methanol 9:1, v/v); yellow oil (75%). 1H
NMR (CDCl3) δ 8.02 (s, 1H), 6.98 (q, 2H, J = 6.9 Hz), 6.10
(s, 1H), 4.06 (q, 2H, J = 7.1 Hz), 3.57 (t, 2H, J = 7.4 Hz), 2.11
(s, 3H), 1.27 (m, 2H), 1.07 (t, 3H, J = 7.1 Hz), 0.82 (m, 4H), 0.48
(t, 3H, J = 6.9 Hz). LC-MS (APCIþ) m/z 328.2 (MHþ).
Ethyl 6-(4-Methoxyphenyl)-4-oxo-1-pentyl-1,4-dihydropyridine-
3-carboxylate (14k). Purification by silica gel chromatography
(dichloromethane/methanol 9:1, v/v); yellow oil (57%). 1H
NMR (CDCl3) δ 8.15 (s, 1H), 7.20 (d, 2H, J = 8.8 Hz), 6.90
(d, 2H, J = 8.8 Hz), 6.31 (s, 1H), 4.29 (q, 2H, J = 7.1 Hz), 3.72
(t, 2H, J = 7.6 Hz), 3.79 (s, 3H), 1.52 (m, 2H), 1.34 (t, 3H, J =
7.1 Hz), 1.06 (m, 4H), 0.72 (t, 3H, J = 7.0 Hz). LC-MS (APCIþ)
m/z 344.2 (MHþ).
Ethyl 6-(2-Chlorophenyl)-4-oxo-4H-pyran-3-carboxylate (13e).
1
Beige solid (35%). H NMR (CDCl3) δ 8.39 (s, 1H), 7.21 (m,
4H), 6.49 (s, 1H), 4.12 (q, 2H, J = 7.0 Hz), 1.12 (t, 3H,
J = 7.0 Hz). LC-MS (APCIþ) m/z 279.1 (MHþ).
Ethyl 6-(4-Methylphenyl)-4-oxo-4H-pyran-3-carboxylate (13f).
Beige solid (63%); mp 96 ꢀC. 1H NMR (CDCl3) δ 8.57 (s, 1H),
7.67 (d, 2H, J = 8.2 Hz), 7.32 (d, 2H, J = 8.2 Hz), 6.83 (s, 1H),
4.40 (q, 2H, J = 7.1 Hz), 1.41 (t, 3H, J = 7.1 Hz). LC-MS
(APCIþ) m/z 259.1 (MHþ).
Ethyl 6-(4-Methoxyphenyl)-4-oxo-4H-pyran-3-carboxylate (13g).
Yellow solid (57%); mp 134 ꢀC. 1H NMR (CDCl3) δ 8.56
(s, 1H), 7.70 (d, 2H, J = 9.0 Hz), 7.0 (d, 2H, J = 9.0 Hz), 6.77
(s, 1H), 4.40 (q, 2H, J = 7.0 Hz), 3.88 (s, 3H) 1.39 (t, 3H,
J = 7.0 Hz). LC-MS (APCIþ) m/z 275.1 (MHþ).
Ethyl 6-tert-Butyl-4-oxo-1-pentyl-1,4-dihydropyridine-3-car-
boxylate (14a). A solution of pyrone ester (13a) (3 g, 13.4 mmol),
n-pentylamine (3.1 mL, 26.7 mmol) in EtOH (60 mL) and acetic
acid (AcOH) (40 mL) was refluxed for 4 h. The mixture was
cooled to room temperature and the solvents were distilled off to
leave a brown oil. Water was added and the product was
extracted with CH2Cl2. The crude oil was chromatographed
on silica gel (dichloromethane/methanol 98:2) to give a rust
colored oil (2.24 g, 57%); 1H NMR (CDCl3) δ 8.12 (s, 1H), 6.55
(s, 1H), 4.38 (q, 2H, J = 7.1 Hz), 4.02 (t, 2H, J = 8.3 Hz), 1.81
(m, 2H), 1.40-1.34 (m, 16H), 0.94 (t, 3H, J = 6.7 Hz). LC-MS
(APCIþ) m/z 294.4 (MHþ).
General Procedure for the Preparation of Ethyl 1-Alkyl-6-aryl-
4-oxo-1,4-dihydropyridine-3-carboxylate (14b-e, 14g-k). To a
solution of pyrone in EtOH/AcOH (3:1, v/v) was added, slowly,
the appropriate alkylamine or aniline (2 equiv) at 5 ꢀC. The
mixture was refluxed for 1 h. After cooling to room temperature,
solvents were evaporated and the residue partitioned in
H2O-CHCl3. The organic phase was washed both with water
and brine, dried, and evaporated to leave an oil. The latter was
chromatographed on silica gel to afford pure product.
Ethyl 1-Ethyl-4-oxo-6-phenyl-1,4-dihydropyridine-3-carboxy-
late (14b). Purification by silica gel chromatography (dichloro-
methane/ethyl acetate 1:1); beige solid (77%); mp 159 ꢀC.
1H NMR (CDCl3) δ 8.41 (s, 1H), 7.50 (m, 5H), 6.07 (s, 1H), 4.22
(q, 2H, J = 6.9 Hz), 3.81 (q, 2H, J = 7.4 Hz), 1.26 (t, 3H, J = 6.9
Hz), 1.04 (t, 3H, J = 7.4 Hz). LC-MS (APCIþ) m/z 272.1 (MHþ).
Ethyl 1-Butyl-4-oxo-6-phenyl-1,4-dihydropyridine-3-carboxy-
late (14c). Purification by silica gel chromatography (dichloro-
methane/ethyl acetate 1:1, v/v); brown oil (74%). 1H NMR
(CDCl3) δ 8.22 (s, 1H), 7.48 (m, 3H) 7.32 (m, 2H), 6.42 (s, 1H),
4.39 (q, 2H, J = 7.2 Hz), 3.74 (t, 2H, J = 7.6 Hz), 1.52 (m, 2H),
1.38 (t, 3H, J = 7.2 Hz), 1.12 (m, 2H), 0.75 (t, 3H, J = 7.4 Hz).
LC-MS (APCIþ) m/z 300.2 (MHþ).
Ethyl 1-Pentyl-6-phenyl-4-thioxo-1,4-dihydropyridine-3-carbo-
xylate (15). A mixture of 14d (2.6 g, 8.3 mmol) and phosphorus
pentasulfide (3.7 g, 16.6 mmol) was refluxed for 12 h in pyridine
(80 mL). After cooling to room temperature, the solvent was
removed under reduced pressure and the residue was partitioned
in H2O-EtOAc. The organic layer was washed with brine,
dried, and concentrated under reduced pressure to leave a
rust-colored oil. The latter was purified by silica gel chroma-
tography (cyclohexane/ethyl acetate 9:1, v/v) to give an orange
oil (2.46 g, 90%). 1H NMR (DMSO-d6) δ 8.22 (s, 1H), 7.54 (m,
5H), 7.06 (s, 1H), 4.26 (q, 2H, J = 7.0 Hz), 3.85 (t, 2H, J = 7.6
Hz), 1.45 (m, 2H), 1.28 (t, 3H, J = 7.0 Hz), 1.03-0.98 (m, 4H),
0.70 (t, 3H, J = 6.7 Hz). LC-MS (APCIþ) m/z 330.8 (MHþ).
General Procedure for the Preparation of Carboxylic Acids
(16a-h). Esters 14a-g and 15 were dissolved in ethanol and 2.5
N NaOH (v/v). The mixture was refluxed for 6 h. After cooling
to room temperature, EtOH was removed under reduced pres-
sure and the residue was dissolved in water and washed with
EtOAc. The aqueous phase was acidified (1 N HCl, pH 2) and
extracted with EtOAc. The combined organic extracts were
washed with water and brine, dried over MgSO4, and concen-
trated under reduced pressure to afford essentially pure car-
boxylic acids 16a-h.
Ethyl 4-Oxo-1-pentyl-6-phenyl-1,4-dihydropyridine-3-carbox-
ylate (14d). Purification by silica gel chromatography (dichloro-
methane/ethyl acetate 1:1, v/v); brown oil (71%). 1H NMR
(DMSO-d6) δ 8.40 (s, 1H), 7.51 (m, 5H), 6.08 (s, 1H), 4.22 (q, 2H,
J = 7.0 Hz), 3.81 (t, 2H, J = 7.6 Hz), 1.38 (m, 2H), 1.26 (t, 3H,
J = 7.0 Hz), 1.02 (m, 4H), 0.69 (t, 3H, J = 7.0 Hz). LC-MS
(APCIþ) m/z 314.2 (MHþ).
6-tert-Butyl-4-oxo-1-pentyl-1,4-dihydropyridine-3-carboxylic
Acid (16a). Beige powder (77%); mp 89 ꢀC. 1H NMR (DMSO-
d6) δ 11.97 (s, 1H), 8.66 (s, 1H), 6.68 (s, 1H), 4.31 (t, 2H, J = 8.2
Hz), 1.73 (m, 2H), 1.41 (s, 9H), 1.32 (m, 4H), 0.87 (t, 3H, J = 6.7
Hz). LC-MS (APCIþ) m/z 266.3 (MHþ).
Ethyl 4-Oxo-1-hexyl-6-phenyl-1,4-dihydropyridine-3-carbox-
ylate (14e). Purification by silica gel chromatography (dichloro-
methane/ethyl acetate 1:1, v/v); brown oil (68%). 1H NMR
(CDCl3) δ 8.22 (s, 1H), 7.41 (m, 5H), 6.08 (s, 1H), 4.19 (q, 2H,
J = 6.9 Hz), 3.93 (t, 2H, J = 7.3 Hz), 1.26-1.02 (m, 11H), 0.73
(t, 3H, J = 7.1 Hz). LC-MS (APCIþ) m/z 328.2 (MHþ).
1-Ethyl-4-oxo-6-phenyl-1,4-dihydropyridine-3-carboxylic Acid
(16b). Beige powder (96%); mp 108 ꢀC. 1HNMR(CDCl3) δ12.56 (s,
1H), 8.51 (s, 1H), 7.54 (m, 5H), 6.09 (s, 1H), 4.02 (q, 2H, J = 7.4 Hz),
1.17 (t, 3H, J = 7.4 Hz). LC-MS (APCIþ) m/z 244.2 (MHþ).
1-Butyl-4-oxo-6-phenyl-1,4-dihydropyridine-3-carboxylic Acid
(16c). White powder (92%); mp 121 ꢀC. 1H NMR (DMSO-d6)

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 7927
δ 16.35 (s, 1H), 8.87 (s, 1H), 7.56 (m, 5H), 6.63 (s, 1H), 4.03
(t, 2H, J = 7.6 Hz), 1.43 (m, 2H),1.03 (m, 2H), 0.63 (t, 3H, J =
7.4 Hz). LC-MS (APCIþ) m/z 272.1 (MHþ).
N3-(1-Adamantyl)-6-methyl-1-pentyl-4-oxo-1,4-dihydropyr-
idine-3-carboxamide (11). Beige solid (57%); mp 195 ꢀC.
1H NMR (CDCl3) δ 10.15 (s, 1H), 8.38 (s, 1H), 6.38 (s, 1H),
3.86 (t, 2H, J = 7.8 Hz), 2.35 (s, 3H), 2.14 (m, 9H), 1.71
(m, 8H), 1.34 (m, 4H), 0.92 (t, 3H, J = 6.9 Hz). LC-MS (APCIþ)
m/z 357.4 (MHþ).
N3-(1-Adamantyl)-6-tert-butyl-1-pentyl-4-oxo-1,4-dihydro-
pyridine-3-carboxamide (17). Beige solid (49%); mp 72 ꢀC.
1H NMR (CDCl3) δ 10.07 (s, 1H), 8.39 (s, 1H), 6.58 (s, 1H),
4.06 (t, 2H, J = 8.3 Hz), 2.14 (m, 9H), 1.71 (m, 8H), 1.42 (s, 9H),
1.36 (m, 4H), 0.93 (t, 3H, J = 6.6 Hz). LC-MS (APCIþ) m/z
399.3 (MHþ).
N3-(Cyclohexyl)-6-tert-butyl-1-pentyl-4-oxo-1,4-dihydropyri-
dine-3-carboxamide (18). Beige solid (53%); mp 91 ꢀC. 1H NMR
(CDCl3) δ 10.2 (d, 1H, J = 7.3 Hz), 8.40 (s, 1H), 6.58 (s, 1H),
4.08 (t, 2H, J = 8.3 Hz), 3.96 (m, 1H), 1.94-1.73 (m, 7H),
1.43-1.37 (m, 18H), 0.93 (t, 3H, J = 6.6 Hz). LC-MS (APCIþ)
m/z 347.2 (MHþ).
(R,S)-N3-(1-(1-Adamantyl)ethyl)-1-ethyl-4-oxo-6-phenyl-1,4-
dihydropyridine-3-carboxamide (19). Beige solid (54%); mp 224 ꢀC.
1H NMR (CDCl3) δ 10.41 (d, 1H, J = 9.1 Hz), 8.58 (s, 1H),
7.53 (m, 3H), 7.35 (m, 2H), 6.47 (s, 1H), 3.87 (m, 3H), 2.00
(m, 3H), 1.64 (m, 12H), 1.25 (t, 3H, J = 7.3 Hz), 1.13 (d, 3H, J =
7.0 Hz). LC-MS (APCIþ) m/z 405.1 (MHþ).
(R,S)-N3-(1-(1-Adamantyl)ethyl)-4-oxo-1-pentyl-6-phenyl-1,4-
dihydropyridine-3-carboxamide (20). White solid (52%); mp 169 ꢀC.
1H NMR (CDCl3) δ 10.41 (d, 1H, J = 9.1 Hz), 8.55 (s, 1H), 7.52
(m, 3H), 7.33 (m, 2H), 6.47 (s, 1H), 3.91 (m, 1H), 3.79 (t, 2H, J =
7.7 Hz), 2.00 (m, 3H), 1.64 (m, 14H), 1.15 (m, 7H), 0.79 (t, 3H, J =
6.9 Hz). LC-MS (APCIþ) m/z 447.4 (MHþ).
(R,S)-N3-(1-(1-Adamantyl)ethyl)-1,6-diphenyl-4-oxo-1,4-di-
hydropyridine-3-carboxamide (21).White solid (47%); mp >250 ꢀC.
1H NMR (CDCl3) δ 10.35 (d, 1H, J = 9.1 Hz), 8.70 (s, 1H), 7.32
(m, 6H), 7.10 (m, 4H), 6.86 (s, 1H), 3.90 (m, 1H), 2.00 (m, 3H), 1.69
(m, 12H), 1.17 (d, 3H, J = 6.7 Hz). LC-MS (APCIþ) m/z 453.4
(MHþ).
4-Oxo-1-pentyl-6-phenyl-1,4-dihydropyridine-3-carboxylic Acid
(16d). Purification by silica gel chromatography (dichloro-
methane/ethyl acetate 1:1, v/v); white powder (88%); mp 115 ꢀC.
1H NMR (CDCl3) δ 13.5 (s, 1H), 8.4 (s, 1H), 7.48 (m, 5H), 6.08
(s, 1H), 3.81 (t, 2H, J = 7.1 Hz), 1.38 (m, 2H), 1.02-0.97 (m, 4H),
0.69 (t, 3H, J = 7.0 Hz). LC-MS (APCIþ) m/z 286.1 (MHþ).
1-Hexyl-4-oxo-6-phenyl-1,4-dihydropyridine-3-carboxylic Acid
(16e). Purification by silica gel chromatography (dichloromet-
hane/ethyl acetate 1:1, v/v); beige oil (86%). 1H NMR (DMSO-
d6) δ 16.35 (s, 1H), 8.88 (s, 1H), 7.56 (m, 5H), 6.63 (s, 1H), 4.02
(t, 2H, J = 7.6 Hz), 1.43-0.85 (m, 8H), 0.73 (t, 3H, J = 7.2 Hz).
LC-MS (APCIþ) m/z 300.1 (MHþ).
4-Oxo-1,6-diphenyl-1,4-dihydropyridine-3-carboxylic Acid (16f).
1
White powder (94%); mp 182 ꢀC. H NMR (CDCl3) δ 13.20
(s, 1H), 8.76 (s, 1H), 7.42-7.21 (m, 10H), 6.82 (s, 1H). LC-MS
(APCIþ) m/z 292.3 (MHþ).
6-(4-Chlorophenyl)-4-oxo-1-pentyl-1,4-dihydropyridine-3-car-
boxylic Acid (16g). White powder (86%); mp 184 ꢀC. 1H NMR
(CDCl3) δ 13.88 (s, 1H), 8.58 (s, 1H), 7.7 (d, 2H, J = 9.1 Hz), 7.5
(d, 2H, J = 9.1 Hz), 6.84 (s, 1H), 4.01 (t, 2H, J = 7.0 Hz), 1.52
(m, 2H), 1.26 (m, 4H), 1.02 (t, 3H, J = 6.9 Hz). LC-MS (APCIþ)
m/z 320.5 (MHþ).
6-(3-Chlorophenyl)-4-oxo-1-pentyl-1,4-dihydropyridine-3-car-
1
boxylic Acid (16h). Beige powder (70%); mp 82 ꢀC. H NMR
(MeOD-d4) δ 8.80 (s, 1H), 7.61 (m, 3H), 7.50 (d, 2H, J = 7.3 Hz),
6.69 (s, 1H), 4.03 (t, 2H, J = 7.0 Hz), 1.63 (m, 2H), 1.15 (m, 4H),
0.81 (t, 3H, J = 6.9 Hz). LC-MS (APCIþ) m/z 320.0 (MHþ).
6-(2-Chlorophenyl)-4-oxo-1-pentyl-1,4-dihydropyridine-3-car-
1
boxylic Acid (16i). Yellow solid (66%); mp 153 ꢀC. H NMR
(MeOD-d4) δ 8.84 (s, 1H), 7.47 (m, 4H), 6.67 (s, 1H), 4.03 (t, 2H,
J = 7.0 Hz), 1.61 (m, 2H), 1.12 (m, 4H), 0.78 (t, 3H, J = 6.9 Hz).
LC-MS (APCIþ) m/z 320.1 (MHþ).
6-(4-Methylphenyl)-4-oxo-1-pentyl-1,4-dihydropyridine-3-
carboxylic Acid (16j). Yellow solid (81%); mp 68 ꢀC. 1H NMR
(MeOD-d4) δ 8.77 (s, 1H), 7.39 (q, 4H, J = 6.9 Hz), 6.59 (s, 1H),
4.07 (t, 2H, J = 7.0 Hz), 2.42 (s, 3H), 1.59 (m, 2H), 1.10 (m, 4H),
0.77 (t, 3H, J = 6.9 Hz). LC-MS (APCIþ) m/z 300.1 (MHþ).
6-(4-Methoxyphenyl)-4-oxo-1-pentyl-1,4-dihydropyridine-3-
carboxylic Acid (16k). White solid (85%); mp 73 ꢀC. 1H NMR
(MeOD-d4) δ 8.76 (s, 1H), 7.45 (d, 2H, J = 8.1 Hz), 7.10 (d, 2H,
J = 8.1 Hz), 6.61 (s, 1H), 4.09 (t, 2H, J = 7.0 Hz), 3.87 (s, 3H),
1.60 (m, 2H), 1.13 (m, 4H), 0.77 (t, 3H, J = 6.9 Hz). LC-MS
(APCIþ) m/z 316.1 (MHþ).
1-Pentyl-6-phenyl-4-thioxo-1,4-dihydropyridine-3-carboxylic
Acid (16l). Purification by silica gel chromatography (dichloro-
methane/ethyl acetate 1:1, v/v); orange powder (84%); mp 108 ꢀC.
1H NMR (DMSO-d6) δ 13.75 (s, 1H), 9.00 (s, 1H), 7.59 (m, 5H),
7.40 (s, 1H), 4.10 (t, 2H, J = 8.4 Hz), 1.49 (m, 2H), 1.02 (m, 4H),
0.69 (t, 3H, J = 6.7 Hz). LC-MS (APCIþ) m/z 302.1 (MHþ).
General Procedure for the Preparation of Carboxamides (11 and
17-40). To a solution of carboxylic acid 10 and 16a-l in dry DMF
were added N,N-diisopropylethylamine (DIPEA) (3 equiv) and
the coupling agents 1-hydroxybenzotriazole (HOBt) (0.5 equiv),
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-
phosphate (HBTU) (1.5 equiv). The resulting mixture was stirred
at room temperature until thin-layer chromatography showed the
starting material to be consumed (ca. 3 h). The appropriate amine
(1.5 equiv) was then added, and the solution was stirred at room
temperature for 12 h. The solvent was removed under reduced
pressure and the residue taken up in water and extracted with
CH2Cl2. The organic phase was washed with saturated aqueous
NaHCO3 solution, with 1 N aqueous HCl, and water. The organic
extract was dried over MgSO4 and concentrated in vacuo to a
brown oil. The crude material was purified by TLC using the
appropriate eluent (dichloromethane/methanol 9:1, v/v) and re-
crystallized in heptane or acetonitrile (except for compound 30) to
afford the titled compounds (11 and 17-40).
N3-((1-Adamantyl)methyl)-4-oxo-1-pentyl-6-phenyl-1,4-dihy-
dropyridine-3-carboxamide (22). White solid (57%); mp 154 ꢀC.
1H NMR (CDCl3) δ 10.39 (m, 1H), 8.56 (s, 1H), 7.51 (m, 3H),
7.35 (m, 2H), 6.47 (s, 1H), 3.79 (t, 2H, J = 7.7 Hz), 3.16 (d, 2H,
J = 6.1 Hz), 1.99 (m, 3H), 1.69-1.61 (m, 14H), 1.12 (m, 4H),
0.78 (t, 3H, J = 6.9 Hz). LC-MS (APCIþ) m/z 433.3 (MHþ).
N3-(1-(3,5-Dimethyl)adamantyl)-4-oxo-1-pentyl-6-phenyl-1,4-
dihydropyridine-3-carboxamide (23). White solid (62%); mp
1
133 ꢀC. H NMR (CDCl3) δ 10.21 (s, 1H), 8.5 (s, 1H), 7.5-
7.35 (m, 5H), 6.45 (s, 1H), 3.77 (t, 2H, J = 7.1 Hz), 2.00-1.18
(m, 19H), 0.88 (s, 6H), 0.79 (t, 3H, J = 7.0 Hz). LC-MS (APCIþ)
m/z 447.3 (MHþ).
N3-(1-Adamantyl)-1-butyl-4-oxo-6-phenyl-1,4-dihydropyridine-
1
3-carboxamide (24). White solid (30%); mp 146 ꢀC. H NMR
(DMSO-d6) δ 10.19 (s, 1H), 8.53 (s, 1H), 7.53 (m, 5H), 6.25 (s,
1H), 3.90 (t, 2H, J = 7.4 Hz), 2.03 (m, 9H), 1.66 (m, 6H), 1.40
(m, 2H), 1.04 (m, 2H), 0.65 (t, 3H, J = 7.3 Hz). LC-MS (APCIþ)
m/z 405.2 (MHþ).
N3-(1-Adamantyl)-4-oxo-1-pentyl-6-phenyl-1,4-dihydropyri-
dine-3-carboxamide (25). Beige solid (55%); mp 145 ꢀC. 1H
NMR (CDCl3) δ 10.17 (s, 1H), 8.51 (s, 1H), 7.51 (m, 3H), 7.34
(m, 2H), 6.45 (s, 1H), 3.77 (t, 2H, J = 7.7 Hz), 2.16 (m, 9H),
1.72-1.58 (m, 8H), 1.12 (m, 4H), 0.81 (t, 3H, J = 6.9 Hz). LC-
MS (APCIþ) m/z 419.3 (MHþ).
N3-(1-Adamantyl)-1-hexyl-4-oxo-6-phenyl-1,4-dihydropyri-
dine-3-carboxamide (26). White solid (32%); mp 167 ꢀC. 1H
NMR (DMSO-d6) δ 10.19 (s, 1H), 8.53 (s, 1H), 7.54 (m, 5H),
6.25 (s, 1H), 3.90 (t, 2H, J = 7.6 Hz), 2.03 (m, 9H), 1.66 (m, 6H),
1.41 (m, 2H), 1.00 (m, 6H), 0.74 (t, 3H, J = 7.0 Hz). LC-MS
(APCIþ) m/z 433.3 (MHþ).
N3-(Cyclohexyl)-4-oxo-1-pentyl-6-phenyl-1,4-dihydropyridine-
1
3-carboxamide (27). White solid (64%); mp 120 ꢀC. H NMR
(CDCl3) δ 10.31 (d, 1H, J = 7.6 Hz), 8.54 (s, 1H), 7.52 (m, 3H),
7.35 (m, 2H), 6.46 (s, 1H), 4.00 (m, 1H), 3.80 (t, 2H, J = 7.7 Hz),

7928 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
El Bakali et al.
1.98-1.12 (m, 16H), 0.79 (t, 3H, J = 6.9 Hz). LC-MS (APCIþ)
m/z 366.2 (MHþ).
J = 8.7 Hz), 7.01 (d, 2H, J = 8.7 Hz), 6.43 (s, 1H), 3.88 (s, 3H) 3.79
(t, 2H, J = 7.6 Hz), 2.16 (m, 9H), 1.70 (m, 8H), 1.14
(m, 4H), 0.80 (t, 3H, J = 6.8 Hz). LC-MS (APCIþ) m/z 449.3
(MHþ).
N3-(1-Adamantyl)-1-pentyl-6-phenyl-4-thioxo-1,4-dihydro-
pyridine-3-carboxamide (39). Yellow solid (56%); mp 163 ꢀC. 1H
NMR (CDCl3) δ 11.41 (s, 1H), 8.75 (s, 1H), 7.60 (s, 1H), 7.54 (m,
3H), 7.34 (m, 2H), 3.88 (t, 2H, J = 7.7 Hz), 2.22 (m, 9H),
1.75-1.56 (m, 8H), 1.13 (m, 4H), 0.79 (t, 3H, J = 7.0 Hz). LC-
MS (APCIþ) m/z 435.3 (MHþ).
(R)-4-Oxo-1-pentyl-6-phenyl-N3-(1-(1,2,3,4-tetrahydronapht-
hyl))-1,4-dihydropyridine-3-carboxamide (28). To obtain com-
pound 28, the pure enantiomer, (R)-1,2,3,4-tetrahydro-1-
1
naphthylamine was used. White solid (60%); mp 139 ꢀC. H
NMR (CDCl3) δ 10.66 (d, 1H, J = 8.2 Hz), 8.61 (s, 1H), 7.52 (m,
3H), 7.35 (m, 3H), 7.13 (m, 3H), 6.44 (s, 1H), 5.45 (m, 1H), 3.81
(t, 2H, J = 7.7 Hz), 2.86 (m, 2H), 1.97 (m, 2H), 1.57 (m, 4H),
1.44-1.28 (m, 4H), 0.8 (t, 3H, J = 7.0 Hz). LC-MS (APCIþ) m/z
415.3 (MHþ).
N3-(Cyclohexyl)-1-pentyl-6-phenyl-4-thioxo-1,4-dihydropyri-
1
dine-3-carboxamide (40). Yellow solid (59%); mp 119 ꢀC. H
(S)-4-Oxo-1-pentyl-6-phenyl-N3-(1-(1,2,3,4-tetrahydronaphthyl))-
1,4-dihydropyridine-3-carboxamide (29). To obtain compound
29, the pure enantiomer, (S)-1,2,3,4-tetrahydro-1-naphthyl-
amine was used. White solid (52%); mp 139 ꢀC. 1H NMR (CDCl3)
and LC-MS (APCIþ) (see compound 28).
NMR (CDCl3) δ 11.53 (d, 1H, J = 7.3 Hz), 8.76 (s, 1H), 7.60 (s,
1H), 7.53 (m, 3H), 7.35 (m, 2H), 4.09 (m, 1H), 3.91 (t, 2H, J =
7.7 Hz), 1.97 (m, 2H), 1.80-1.59 (m, 10H), 1.13 (m, 4H), 0.82 (t,
3H, J = 6.8 Hz). LC-MS (APCIþ) m/z 383.3 (MHþ).
tert-Butyl 4-Oxo-1-pentyl-6-phenyl-1,4-dihydropyridin-3-yl
carbamate (41). To a solution of carboxylic acid 16d (0.4 g, 1.4
mmol) in tert-butyl alcohol (20 mL) were added under nitrogen
atmosphere, potassium tert-butoxide (0.17 g, 1.7 mmol) and
diphenylphosphoryl azide (0.36 mL, 1.7 mmol). The mixture
was refluxed for 12 h, cooled to room temperature, and diluted
with EtOAc. The organic phase was washed with saturated
aqueous NaHCO3 solution and brine, dried over MgSO4, and
concentrated under reduced pressure to a yellowish oil. The latter
was purified by silica gel chromatography (dichloromethane/
methanol 95:5, v/v) to afford pure 41 (0.17 g, 38%) as a yellow
oil. 1H NMR (DMSO-d6) δ 8.39 (s, 1H), 7.74 (s, 1H), 7.56-7.43
(m, 5H), 6.12 (s, 1H), 3.81 (t, 2H, J = 7.6 Hz), 1.48 (m, 11H),
1.03 (m, 4H), 0.69 (t, 3H, J = 6.8 Hz). LC-MS (APCIþ) m/z
357.1 (MHþ).
4-Oxo-1-pentyl-6-phenyl-N3-(piperidin-1-yl)-1,4-dihydropyri-
dine-3-carboxamide (30). White powder (40%); mp 122 ꢀC. 1H
NMR (CDCl3) δ 11.17 (s, 1H), 8.56 (s, 1H), 7.53 (m, 3H), 7.33
(m, 2H), 6.48 (s, 1H), 3.81 (t, 2H, J = 7.6 Hz), 2.92 (t, 4H, J =
5.1 Hz), 1.77-1.48 (m, 8H), 1.12 (m, 4H), 0.79 (t, 3H, J = 6.9 Hz).
LC-MS (APCIþ) m/z 368.3 (MHþ).
4-Oxo-1-pentyl-6-phenyl-N3-(3(trifluoromethyl)phenyl)-1,4-
dihydropyridine-3-carboxamide (31). White solid (74%); mp 125 ꢀC.
1H NMR (CDCl3) δ 12.80 (s, 1H), 8.64 (s, 1H), 8.18 (s, 1H), 7.89
(d, 1H, J = 7.6 Hz), 7.54-7.37 (m, 7H), 6.56 (s, 1H), 3.86 (t, 2H,
J = 7.7 Hz), 1.64 (m, 2H), 1.14 (m, 4H), 0.8 (t, 3H, J = 6.7 Hz).
LC-MS (APCIþ) m/z 429.6 (MHþ).
N3-(1-Adamantyl)-6-(4-chlorophenyl)-1-pentyl-4-oxo-1,4-di-
hydropyridine-3-carboxamide (32). White powder (28%); mp
190 ꢀC. 1H NMR (DMSO-d6) δ 10.17 (s, 1H), 8.53 (s, 1H),
7.61 (d, 2H, J = 8.7 Hz), 7.55 (d, 2H, J = 8.7 Hz), 6.27 (s, 1H),
3.9 (t, 2H, J = 7.6 Hz), 2.03 (m, 9H), 1.66 (m, 6H), 1.42 (m, 2H),
1.05 (m, 4H), 0.71 (t, 3H, J = 6.8 Hz). LC-MS (APCIþ) m/z
453.3 (MHþ).
6-(4-Chlorophenyl)-N3-cyclohexyl-4-oxo-1-pentyl-1,4-dihydro-
pyridine-3-carboxamide (33). White powder (32%); mp 157 ꢀC.
1H NMR (CDCl3) δ 10.31 (d, 1H, J = 7.9 Hz), 8.58 (s, 1H), 7.61
(d, 2H, J = 8.7 Hz), 7.55 (d, 2H, J = 8.7 Hz), 6.29 (s, 1H), 3.92 (t,
2H, J = 7.6 Hz), 3.82 (m, 1H) 1.83-1.31 (m, 12H), 1.03 (m, 4H),
0.71 (t, 3H, J = 6.8 Hz). LC-MS (APCIþ) m/z 401.3 (MHþ).
6-(4-Chlorophenyl)-4-oxo-1-pentyl-N3-(3(trifluoromethyl)phenyl)-
1,4-dihydropyridine-3-carboxamide (34). White powder (38%);
mp 100 ꢀC. 1H NMR (CDCl3) δ 13.03 (s, 1H), 8.80 (s, 1H), 8.29
(s, 1H), 7.78 (d, 2H, J = 8.1 Hz), 7.63 (m, 3H), 7.45 (d, 2H, J =
7.6 Hz), 6.46 (s, 1H), 3.99 (t, 2H, J = 7.4 Hz), 1.45 (m, 2H), 1.06
(m, 4H), 0.72 (t, 3H, J = 6.8 Hz). LC-MS (APCIþ) m/z 463.2
(MHþ).
5-Amino-1-pentyl-2-phenyl-1H-pyridin-4-one (42). To a solu-
tion of 5N hydrochloric acid in isopropyl alcohol (20 mL) was
added the carbamate 41 (0.1 g, 0.28 mmol). The mixture was
stirred at room temperature for 14 h and then concentrated
under reduced pressure. The resulting solid was solubilized in
water and washed with Et2O. The aqueous phase was alkali-
nized with 10% NaOH and extracted with EtOAc. The organic
phase was dried over MgSO4 and the solvent removed under
reduced pressure to afford pure 42 as a white solid (0.068 g,
1
95%); mp 98 ꢀC. H NMR (DMSO-d6) δ 7.48-7.38 (m, 5H),
7.19 (s, 1H), 5.82 (s, 1H), 4.67 (s, 2H), 3.67 (t, 2H, J = 7.3 Hz),
1.46 (m, 2H), 1.04 (m, 4H), 0.72 (t, 3H, J = 6.7 Hz). LC-MS
(APCIþ) m/z 257.1 (MHþ).
N-(4-Oxo-1-pentyl-6-phenyl-1,4-dihydropyridin-3-yl)cyclo-
hexanecarboxamide (43). Compound 43 was obtained using the
same procedure described for amides 11 and 17-40. Purifica-
tion by silica gel chromatography (ethyl acetate/cyclohexane
1
N3-(1-Adamantyl)-6-(3-chlorophenyl)-1-pentyl-4-oxo-1,4-di-
hydropyridine-3-carboxamide (35). Beige solid (62%); mp 178 ꢀC.
1H NMR (CDCl3) δ 10.10 (s, 1H), 8.50 (s, 1H), 7.49 (m, 2H), 7.34
(s, 1H), 7.24 (d, 2H, J = 7.6 Hz), 6.42 (s, 1H), 3.75 (t, 2H, J =
7.6 Hz), 2.15 (m, 9H), 1.70 (m, 8H), 1.13 (m, 4H), 0.80 (t, 3H, J =
6.8 Hz). LC-MS (APCIþ) m/z 453.2 (MHþ).
N3-(1-Adamantyl)-6-(2-chlorophenyl)-1-pentyl-4-oxo-1,4-di-
hydropyridine-3-carboxamide (36). White solid (58%); mp 166
ꢀC. 1H NMR (CDCl3) δ 10.15 (s, 1H), 8.54 (s, 1H), 7.50 (m, 4H),
6.42 (s, 1H), 3.66 (t, 2H, J = 7.6 Hz), 2.16 (m, 9H), 1.70 (m, 8H),
1.11 (m, 4H), 0.79 (t, 3H, J = 6.8 Hz). LC-MS (APCIþ) m/z
453.2 (MHþ).
1:1); white solid (38%); mp 179 ꢀC. H NMR (CDCl3) δ 8.94
(s, 1H), 8.57 (s, 1H), 7.48 (m, 3H), 7.33 (m, 2H), 6.37 (s, 1H), 3.72
(t, 2H, J = 7.9 Hz), 2.37 (m, 1H), 2.00-1.11 (m, 16H), 0.91 (t,
3H, J = 7.0 Hz). LC-MS (APCIþ) m/z 367.3 (MHþ).
Pharmacology. hCB1 and hCB2 membranes of CHO cells were
purchased from PerkinElmer. Fatty acid free bovine serum
albumin (BSA) was purchased from Sigma Chemical Co. (St.
Louis, MO). WIN-55,212-2 was purchased from RBI (Natick,
MA), HU-210 and CP-55,940 from Tocris (Bristol, UK), and
SR141716A and 5 were kindly donated by Sanofi Recherche
(Montpellier, France). [3H]-SR141716A (52 Ci/mol) was from
Amersham (Roosendaal, The Netherlands), [3H]-CP-55,940
(101 Ci/mol) was from NEN Life Science (Zaventem, Belgium),
and HU-210 was from Tocris (Bristol, UK). Glass fiber filters
were purchased from Whatman (Maidstone, UK), while Aqua-
luma was from PerkinElmer (Schaesberg, The Netherlands). [35S]-
GTPγS (1173 Ci/mmol) was from Amersham (Roosendaal, The
Netherlands).
N3-(1-Adamantyl)-6-(4-methylphenyl)-1-pentyl-4-oxo-1,4-di-
hydropyridine-3-carboxamide (37). White solid (73%); mp
1
210 ꢀC. H NMR (CDCl3) δ 10.19 (s, 1H), 8.50 (s, 1H), 7.30
(d, 2H, J = 8.4 Hz), 7.22 (d, 2H, J = 8.1 Hz), 6.43 (s, 1H), 3.78
(t, 2H, J = 7.6 Hz), 2.43 (s, 3H) 2.16 (m, 9H), 1.72 (m, 6H), 1.58
(m, 2H), 1.13 (m, 4H), 0.79 (t, 3H, J = 6.8 Hz). LC-MS (APCIþ)
m/z 433.3 (MHþ).
Competition Binding Assay. Stock solutions of the com-
pounds were prepared in DMSO and further diluted (100ꢀ)
with the binding buffer to the desired concentration. Final
DMSO concentrations in the assay were less than 0.1%. The
N3-(1-Adamantyl)-6-(4-methoxyphenyl)-1-pentyl-4-oxo-1,4-
dihydropyridine-3-carboxamide (38). White solid (73%); mp 144 ꢀC.
1H NMR (CDCl3) δ 10.19 (s, 1H), 8.50 (s, 1H), 7.24 (d, 2H,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 7929
competitive binding experiments were performed as described
earlier.57 Briefly [3H]-SR141716A (1 nM) or [3H]-CP-55,940
(1nM) as radioligands for the hCB1 and the hCB2 cannabinoid
receptor, respectively, were added to 40 μg of membranes
resuspended in 0.5 mL (final volume) binding buffer (50 mM
Tris-HCl, 3 mM MgCl2, 1 mM EDTA, 0.5% bovine serum
albumine, pH 7.4). After 1 h at 30 ꢀC, the incubation was
stopped and the solutions were rapidly filtered through 0.5%
PEI pretreated GF/B glass fiber filters on a M-48T Brandell cell
harvester and washed twice with 5 mL of ice-cold binding buffer
without serum albumin. The radioactivity on the filters was
measured using a Pharmacia Wallac 1410 β-counter using 10
mL of Aqualuma, after 10 s shaking and 3 h resting. Assays were
performed at least in triplicate. The nonspecific binding was
determined in the presence of 10 μM HU-210.
very variable among GPCRs and known to be critical for ligand
binding.63-65 The compact structural folding of the template
loop was conserved by adjusting the 10 gap deletions in the
N-terminal moiety of the loop. Contrary to the unstructured
C-terminal moiety of EL2 region in the β2-adrenergic receptor,
the N-terminal segment is structured as an R-helix and could
keep reliable CR-CR restraints editing deletion gaps in this
region (see Figure 1). The intracellular lopp (IL) 3 region was
also fully automatically generated as previously described be-
cause this loop is lacking in the crystal template, whereas the
N-terminal (Met1-Leu28) and C-terminal (His316-Cys360)
overhangs that are not aligned to the template sequence were
not modeled. The homology process was carried out on the basis
of the input sequence alignment as well as spatial restraints
applied for the highly potential disulfide bridge between Cys174
and Cys179.
[35S]-GTPγS Assays. The binding experiments were per-
formed at 30 ꢀC in tubes containing 40 μg protein in 0.5 mL
(final volume) binding buffer (50 mM Tris-HCl, 3 mM MgCl2,
1 mM EDTA, 100 mM NaCl, 0.1% bovine serum albumin, pH
7.4) supplemented with 20 μM GDP. The assay was initiated by
the addition of [35S]-GTPγS (0.05 nM, final concentration).
After 1 h, the incubations were terminated by the addition of
5 mL of ice-cold washing buffer (50 mM Tris-HCl, 3 mM MgCl2,
1 mM EDTA, 100 mM NaCl). The suspension was immediately
filtered through GF/B filters using a 48 well Brandell cell
harvester and washed twice with the same ice-cold buffer. The
radioactivity on the filters was counted as mentioned above.
Assays were performed in triplicate. The nonspecific binding
was measured in the presence of 100 μM Gpp(NH)p. Results
were expressed as EC50 (nM) and Emax (%). Basal constitutive
activity of the receptor has been set at a value of 100%; reported
Emax values above 100% indicated that the compound behaves
as an agonist (either partial or full), values under 100% indi-
cated inverse agonist properties.
An energy minimization of hydrogens and side chains was
then performed using the CHARMM forcefield66 and proces-
sing 5000 steps with the steepest descent algorithm converging to
a 0.1 kcal mol-1 A-1 gradient, followed by 20000 steps with the
3
3
conjugate gradient, converging to a 0.001 kcal mol-1 A-1
3
3
gradient. Graphical inspection of models was performed using
the Sybyl 6.9.2 software (Tripos Associates, Inc., 1699 South
Hanley Road, St. Louis, MO 63144).
Modeling Ligand-Bound States of the CB2 Receptor Model
with Both Compounds 17 and 25. After the identification of the
putative binding site using the MOLCAD program of Sybyl
6.9.2, both compounds 17 and 25 were therein submitted dock-
ing routines using GOLDv4.1.1 software67 in a 8 A sphere
including the entire solvent-accessible surface. For each ligand,
30 preferential conformations were calculated and ranked fol-
lowing the goldscore fitness scoring function. An early termina-
tion was allowed as three top-ranked solutions were within a 1.5
A RMSd. Once the most relevant docking solution was chosen
for each ligand, both receptor-ligand complexes underwent a
CHARMM energy minimization as previously described in
order to discard steric clashes. Finally, both complexes were
submitted to a 10 ns molecular dynamics (MD) CHARMM
simulation applying harmonic restraints on the TM bundle
backbone and a distance-dependent dielectrics arguing for an
implicit representation of solvent in outer regions. During the
MD simulation, the binding energy was monitored in each
Data Analysis. IC50 and EC50 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-Prus-
off equation: Ki = IC50/(1 þ L/Kd). Statistical signification of
[35S]-GTPγS assay results was assessed using a one-way ANO-
VA followed by a Dunett post-test.
Homology Modeling of the Inactive Human CB2 Apo-Recep-
tor. Sequence homology rates are very comparable between each
GPCR crystal and CB2 sequences (44%, 46%, 44%, and 46%
in conserved pattern and 40%, 37%, 37%, and 39% in whole
sequences in bovine rhodopsin, turkey β1-adrenergic receptor,
human β2-adrenergic receptor, and A2A adenosine receptor,
respectively). However, the two serine residues 4.53 (161) and
4.57 (165), known as critical for the binding of 5,58 were
simultaneously conserved only in the sequences of the β-adre-
nergic receptors. The human β2-adrenergic receptor (PDB 2RH1)
was selected as the crystal template for homology modeling
because it has the advantage of conserving the tyrosine residue
in position 5.58 (209) of the critical TM V instead of an alanine
residue in the sequence of the turkey β1-adrenergic receptor.
Indeed, CB1 and CB2 receptors lack the highly conserved residue
Pro5.50 in the class A GPCRs. Thus the second most highly
conserved residue, Tyr5.58, has been described as the new reference
amino acid in TM V concerning CB1 and CB2 receptors.59,60
The input sequence alignment for homology modeling by
MODELER software61 was automatically generated by
ClustalW,62 taking care to shift gaps out from the transmem-
brane regions. Then the insertion/deletion gaps were manually
adjusted in loop regions, which are generated without any
homology restraint by a simulated annealing procedure in-
cluded in the MODELER program. Thus the one-residue gaps
in IL1 (insertion), EL1 (deletion), and EL3 (insertion) were
moved to the center of the loops so that no bulky or misfolded
peptide is introduced in regions flanking transmembrane he-
lices. Further attention was given to the EL2 region, which is
receptor-ligand complex following the formula: ΔGbinding
ΔGcomplex - ΔGligand - ΔGprotein
=
.
Acknowledgment. This work was financially supported by
a grant from the Nord-Pas-de-Calais Regional Council and
University of Lille 2.
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