Investigations on the 4-Quinolone-3-carboxylic acid motif. 3. Synthesis, structure-affinity relationships, and pharmacological characterization of 6-substituted 4-quinolone-3-carboxamides as highly selective cannabinoid-2 receptor ligands

Article abstract of DOI:10.1021/jm100123x

A set of quinolone-3-carboxamides 2 bearing diverse substituents at position 1, 3, and 6 of the bicyclic nucleus was prepared. Except for six compounds exhibiting K_i > 100 nM, all the quinolone-3-carboxamides 2 proved to be high affinity CB2 li

Full text of DOI:10.1021/jm100123x

J. Med. Chem. 2010, 53, 5915–5928 5915
DOI: 10.1021/jm100123x
Investigations on the 4-Quinolone-3-carboxylic Acid Motif. 3. Synthesis, Structure-Affinity
Relationships, and Pharmacological Characterization of 6-Substituted 4-Quinolone-3-carboxamides
as Highly Selective Cannabinoid-2 Receptor Ligands
Serena Pasquini,† Alessia Ligresti,‡ Claudia Mugnaini,† Teresa Semeraro,† Lavinia Cicione,†,# Maria De Rosa,†
Francesca Guida,§ Livio Luongo,§ Maria De Chiaro,§ Maria Grazia Cascio,^ Daniele Bolognini, ,^ Pietro Marini,^
Roger Pertwee,^ Sabatino Maione,§ Vincenzo Di Marzo,*,‡ and Federico Corelli*,†
†Dipartimento Farmaco Chimico Tecnologico, Universitaꢀ degli Studi di Siena, Via A. Moro, 53100 Siena, Italy, ‡Endocannabinoid Research
Group, Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Via dei Campi Flegrei 34, 80078 Pozzuoli (Naples), Italy,
§Department of Experimental Medicine;Section of Pharmacology “L. Donatelli”, Second University of Naples, Via S. Maria di
Costantinopoli 16, 80138 Naples, Italy, DBSF, Pharmacology Section and Neuroscience Centre, University of Insubria, Busto Arsizio (Va),
Italy, and ^Institute of Medical Sciences University of Aberdeen, Aberdeen AB25 2ZD, Scotland, U.K. # Present address: Dipartimento di
ꢀ
Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, Universita del Piemonte Orientale “A. Avogadro”, Vercelli, Italy.
Received January 26, 2010
A set of quinolone-3-carboxamides 2 bearing diverse substituents at position 1, 3, and 6 of the bicyclic
nucleus was prepared. Except for six compounds exhibiting Ki >100 nM, all the quinolone-3-
carboxamides 2 proved to be high affinity CB2 ligands, with Ki values ranging from 73.2 to 0.7 nM
and selectivity [SI = Ki(CB1)/Ki(CB2)] varying from >14285 to 1.9, with only 2ah exhibiting a reverse
selectivity (SI<1). In the formalin test of peripheral acute and inflammatory pain in mice, 2ae showed
analgesic activity that was antagonized by a selective CB2 antagonist. By contrast, 2e was inactive per se
and antagonized the effect of a selective CB2 agonist. Finally, 2g and 2p exhibited CB2 inverse agonist-
like behavior in this in vivo test. However, two different functional assays carried out in vitro on 2e and
2g indicated for both compounds an overall inverse agonist activity at CB2 receptors.
Introduction
side the CNS and are most abundant in the immune system,
i.e. in tonsils, spleen, macrophages, and lymphocytes (B-cells
and natural killer cells).8
Cannabinoid receptors, their endogenous ligands, and pro-
teins responsible for endocannabinoid cellular uptake and
inactivation, i.e. the putative anandamide membrane trans-
porter (AMTa),1 and the fatty acid amide hydrolase (FAAH),2
or the monoacylglycerol lipase (MAGL)3 in the case of 2-AG,
constitute the “endocannabinoid (EC) system” and represent
potentially interesting targets4 for the development of new
therapeutic agents to be employed in many pathologies, such
as, for example, pain, loss of appetite in patients with AIDS,
obesity, chemotherapy-induced nausea and vomiting, immune
and inflammatory disorders, cardiovascular and gastrointest-
inal disorders, and neurodegenerative diseases.5
So far, two cannabinoid receptors (cannabinoid receptor 1
[CB1] and cannabinoid receptor 2 [CB2]), have been char-
acterized and cloned.6,7 CB1 receptors are expressed at high
levels in the central nervous system (CNS), whereas CB2
receptors are found predominantly, but not exclusively, out-
Agonists of both cannabinoid receptor subtypes produce
strong antinociceptive effects in animal models of chronic,
neuropathic, and inflammatory pain and are intensively
investigated as potential new analgesic and antiinflammatory
agents.9 Unfortunately, CB1/CB2 agonists are not devoid of
unwanted side effects (such as muscle weakness, palpitations,
dry mouth, disorientation, altered time perception, impair-
ment of memory, tremor, confusion, paranoia and hallucina-
tion, nausea and vomiting), many of which are thought to be
due to activation of central CB1 receptors rather than periph-
eral CB1 or CB2 receptors.10 The occurrence of these adverse
effects limits the therapeutic usefulness of mixed cannabinoid
agonists that show high affinity for CB1 receptors.
Differences in receptor distribution and signal transduction
mechanisms11,12 are likely to account for the relative absence
of CNS side effects induced by CB2 agonists. These consid-
erations suggest that novel pharmacotherapies selectively
targeting CB2 receptors may have considerable therapeutic
potential.13 Significant medicinal chemistry efforts have been
directed to the characterization of selective CB2 agonists
(Chart 1), leading to the identification of compounds eliciting
antinociceptive effects in models of acute pain, persistent
inflammatory pain, postoperative pain, cancer pain, and
neuropathic pain.14
*To whom correspondence should be addressed. For F.C.: phone,
þ39 0577 234308; fax, þ39 0577 234333; E-mail, corelli@unisi.it. For V.
D.: phone, þ39 081 8675093; fax, þ39 081 8041770; E-mail,
vdimarzo@icmib.na.cnr.it.
a Abbreviations: AMT, anandamide membrane transporter; FAAH,
fatty acid amide hydrolase; MAGL, monoacylglycerol lipase; EC,
endocannabinoid system; CB1, cannabinoid receptor type 1; CB2,
cannabinoid receptor type 2; HOBt, 1-hydroxybenzotriazole; HBTU,
O-benzotriazol-1-yl-N,N,N0,N0-tetramethyluronium hexafluorophosphate;
DIPEA, diisopropylethylamine; TEA, triethylamine; SI, selectivity
index.
In recent years a number of reports have suggested that
also CB2 inverse agonists/antagonists (Chart 1) may possess
r
2010 American Chemical Society
Published on Web 07/27/2010
pubs.acs.org/jmc

5916 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Pasquini et al.
Chart 1. Representative CB2-Selective Agonists (HU-308,40 JWH-133,27 AM1241,31 GW405833,41 and GW842166X42) and Inverse
Agonists/Antagonists (JTE-907,15b SR144528,15a AM630,26 and Sch.33643)
Chart 2. Lead Compound 1 and General Structure of 6-Sub-
stituted 4-Quinolone-3-carboxamide Derivatives 2
Scheme 1. Synthesis of 6-Substituted 4-Quinolone-3-carboxa-
mide Derivatives 2a,ba
antiinflammatory activity, being able to inhibit carrageenan-
induced paw edema in mice, to reduce leukocytes trafficking
and to impair the migration of cells toward cannabinoid
agonists.15 Taken together, these previous results show that
selective cannabinoid CB2 inverse agonists may serve as
novel immunomodulatory agents in the treatment of a variety
of acute and chronic inflammatory disorders. On the other
hand, CB2 agonists have also been proposed as useful ther-
apeutic agents against chronic (neuropathic) pain and inflam-
mation.16 Although it may appear puzzling that both agonists
and inverse agonists at a given receptor may cause anti-
inflammatory effects, this phenomenon has been widely de-
scribed for CB2 receptors and was ascribed to the capability
of these receptors to be coupled, under different conditions
and in a time- and site-selective way, to either recruitment
of immune cells or interference with the action of other
chemoattractants.4a,17
a Reagents and conditions: (i) pentyl iodide, K2CO3, DMF, 100 ꢀC, 4
h; (ii) 10% aq NaOH, reflux, 2 h; (iii) 1-aminoadamantane, HBTU,
HOBt, DIPEA, DMF, rt, 4 h.
Within a research program aimed at characterizing
novel cannabinoid ligands,18 we have recently described
a family of quinolone-3-carboxamides bearing diverse
substituents at different positions of the aromatic ring as
new CB2 ligands. The tested compounds exhibited high
CB2 affinity and selectivity over the CB1 receptor subtype;
compound 1 (Chart 2), in particular, exhibited >190-fold
selectivity over CB1 in the [3H]CP-55,940 binding assay
(hCB2/hCB1 Ki=6.3 nM/1220 nM). Moreover, this com-
pound showed analgesic activity in the formalin test of
acute peripheral and inflammatory pain in mice as a
result of selective CB2 agonistic activity.18a,19 On the basis

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 5917
of these promising results, we chose derivative 1 as the
prototype for the development of a set of 6-substituted
quinolone-3-carboxamides of general structure 2, where
the N-1 pentyl chain and adamantyl group of 1 were either
retained or replaced by other lipophilic groups, whereas a
number of different substituents in place of the 2-propyl
group at C-6 position were extensively investigated.20 Our
main objectives were: (i) to identify new compounds
endowed with increased CB2 affinity and/or selectivity,
(ii) to highlight the relationship between chemical struc-
ture and the in vitro/in vivo pharmacological profile of the
new compounds.
Scheme 4. Synthesis of 6-Substituted 4-Quinolone-3-carboxa-
mide Derivatives 2af-2ama
Scheme 2. Synthesis of 6-Substituted 4-Quinolone-3-carboxa-
mide Derivatives 2c,da
a Reagents and conditions: (i) amine, HBTU, HOBt, DIPEA, DMF,
rt, 8 h; (ii) alkyl halide, K2CO3, DMF, 100 ꢀC, 4 h; (iii) 2-furanboronic
acid, Pd(OAc)2, PPh3, 1 N Na2CO3, DME, EtOH, MW, 150 ꢀC, 10 min.
a Reagents and conditions: (i) Oxone, 1,4-dioxane, H2O, rt, 24 h.
Scheme 3. Synthesis of 6-Substituted 4-Quinolone-3-carboxamide Derivatives 2e-2aea
a Reagents and conditions: (i) (for 2e, 2 g-k, 2m, 2n, 2q, 2r, 2t, 2u): 7, aryl- or alkenylboronic acid, Pd(OAc)2, PPh3, 1 N Na2CO3, DME, EtOH, MW,
150 ꢀC, 10 min; (for 2f, 2l, 2o, 2p, 2s): 2a, aryl- or heteroarylboronic acid, Pd(OAc)2, PPh3, 1 N Na2CO3, DME, EtOH, MW, 150 ꢀC, 10 min. (ii) (for 2v):
2a, phenylacetylene, i-Pr2NH, PdCl2(PPh3)2, toluene, reflux, 2 h; (for 2w): (1) 2a, (trimethylsilyl)acetylene, i-Pr2NH, PdCl2(PPh3)2, toluene, reflux, 2 h;
(2) K2CO3, MeOH, THF, rt, 30 min. (iii) 2a, styrene, Pd(OAc)2, TEA, PPh3, DMF, reflux, 3 h. (iv) 2a, imidazole or pyrrole, Cu(OAc)2, DBU, DMSO,
130 ꢀC, 10 min. (v) H2, Pd/C, EtOH, 4 h.

5918 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Pasquini et al.
Table 1. CB1 and CB2 Receptor Affinity Values for Compounds 2a-2ama
CB1b,d
Kif (nM)
CB2c,d
Ki (nM)
compd
R1
R2
R3
SIe
2a
iodo
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-adamantyl
1-fenchyl
1-pentyl
1-pentyl
1-pentyl
1-pentyl
1-pentyl
1-pentyl
1-pentyl
1-pentyl
1-pentyl
1-pentyl
1-pentyl
1-pentyl
1-pentyl
1-pentyl
1-pentyl
1-pentyl
1-pentyl
1-pentyl
1-pentyl
1-pentyl
1-pentyl
1-pentyl
1-pentyl
1-pentyl
1-pentyl
1-pentyl
1-pentyl
1-pentyl
1-pentyl
1-pentyl
1-pentyl
propargyl
allyl
2080
>10000
460
20.6
3881
73.2
65.9
0.7
101
>3
2b
2,4-dichlorobenzyl
phenylsulfinyl
phenylsulfonyl
2-furyl
2cg
2d
6
>10000
>10000
>10000
>10000
>10000
>10000
>10000
NTh
>152
>14285
>4348
>2381
>1205
>909
>625
2e
2f
2g
2-thienyl
2.3
4.2
4-hydroxyphenyl
1-cyclohexenyl
4-chlorophenyl
2,4-difluorophenyl
3-hexen-3-yl
5-chloro-2-thienyl
2-methoxypyridin-5-yl
4-isopropoxyphenyl
3,5-dimethylisoxazol-4-yl
4-cyanophenyl
3,5-dimethyl-4-methoxyphenyl
6-methoxy-2-naphthyl
1-isobutylpyrazol-4-yl
E-3,3-dimethylbuten-1-yl
4-biphenyl
2h
2i
8.3
11.0
16.0
NT
44.8
59.3
NT
125.7
41.9
21.5
52.6
51.0
71.6
181
343.0
8.8
2j
2k
2l
>10000
>10000
NT
>223
>169
2m
2n
2o
2p
>10000
>10000
510
>80
>239
24
2q
2r
360
245.7
7
2s
5
2t
2u
>10000
>10000
>10000
>10000
>10000
29.7
>140
>55
>29
>1136
>153
42
2v
2-phenylethynyl
ethynyl
styryl
2w
2x
2y
65.4
0.7
pyrrol-1-yl
2z
imidazol-1-yl
cyclohexyl
3-hexyl
>10000
>10000
NT
8.0
7.3
>1250
>1370
2aa
2ab
2ac
2ad
2ae
2af
2ag
2ah
2ai
2aj
2ak
2al
NT
NT
16.9
56.6
49.8
NT
5.0
3,3-dimethylbutyl
ethyl
NT
900
53
>177
64
2-phenylethyl
bromo
bromo
>10000
3210
NT
bromo
bromo
1-pentyl
1-pentyl
1-pentyl
allyl
2.8
1900
0.6
5
1-piperidinyl
1-hexyl
400
900
59.0
4.4
bromo
2-furyl
1700
2
1-adamantyl
1-adamantyl
1-fenchyl
>10000
>10000
480
>169
2273
200
2-furyl
2-furyl
1-buten-4-yl
1-pentyl
2am
1i
SR144528i,j
Rimonabanti,k
2.4
6.3
1220
>2820
12.0
194
>522
0.015
5.4
790
a Data represent mean values for at least three separate experiments performed in duplicate and are expressed as Ki (nM). b CB1: human cannabinoid
type 1 receptor. c CB2: human cannabinoid type 2 receptor. d For both receptor binding assays, the new compounds were tested using membranes from
HEK cells transfected with either the CB1 or CB2 receptor and [3H]-(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)-phenyl]-trans-4-(3-hydroxy-propyl)-
cyclohexanol ([3H]CP-55,940). e SI: selectivity index for CB2, calculated as Ki(CB1)/Ki(CB2) ratio. f Ki: “Equilibrium dissociation constant”, that is, the
concentration of the competing ligand that will bind to half the binding sites at equilibrium in the absence of radioligand or other competitors. g Tested as
the racemate. h NT, not tested because of solubility problems. i The binding affinities of reference compounds were evaluated in parallel with compounds
2 under the same conditions. j CB2 reference compound. k CB1 reference compound.
Chemistry
Most of the final compounds 2 (namely, 2e-2ae) were
obtained through metal-catalyzed cross-coupling reactions
starting from either the bromo derivative 718a or the corre-
sponding iodo derivative 2a (Scheme 3). In particular, all the
Sonogashira, Heck, and Ullman-type reactions (conditions ii,
iii, and iv in Scheme 3) were performed on precursor 2a in
order to obtain the expected products in satisfactory yield,
whereas in the case of the Suzuki reaction (condition i), the
choice between 2a and 7 as the reaction substrate basically
depended upon the nature of the boronic acid to be used; thus,
aryl- and alkenylboronic acids gave good results also with the
less reactive bromo derivative 7, while the cross-coupling
reaction using heteroarylboronic acids, with the only excep-
tion of 2-furylboronic acid, was best carried out on the iodo
derivative 2a. Compounds 2h, 2k, 2t, 2w, and 2x were
The synthesis of the new compounds 2 was accomplished as
depicted in Schemes 1-4. 4-Oxo-1,2-dihydroquinolin-3-car-
boxylic acid ethyl esters 3a21 and 3b22 (Scheme 1) were
alkylated with pentyl iodide in the presence of potassium
carbonate to give derivatives 4a and 4b, respectively. Alkaline
hydrolysis of the ester function to 5a and 5b was followed by
coupling (HBTU, HOBt, DIPEA) with 1-aminoadamantane
to afford the final compounds 2a and 2b in an overall yield of
45 and 55%, respectively.
Oxidation of the sulfide 618a (Scheme 2) by means of
oxone in 1,4-dioxane/water gave a mixture of the corres-
ponding sulfoxide 2c (7% yield) and sulfone 2d (50% yield),
which were separated by column chromatography on
silica gel.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 5919
Figure 1. Effect of intraperitoneal (ip) administration of vehicle (20% DMSO in saline), compound 2e (1 or 3 mg/kg) alone, or compound 2e
(1 mg/kg ip) in combination with JWH-133 (3 mg/kg ip), a selective CB2 receptor agonist, on the nociceptive behavior (seconds) induced by
subcutaneous formalin (1.25%) injection into the hind paw of mice (n = 8-10). Open columns represent the early phase and filled columns the
late phase of formalin-evoked nociception. Formalin was injected 15 min after vehicle or drug treatments. Asterisks indicate significant
differences (P<0.05) vs vehicle and circles vs compound 2e (1 mg/kg ip).
subjected to catalytic hydrogenation to yield the correspond-
ing saturated derivatives 2aa-2ae.
minutes before injection of formalin, mice received intraper-
itoneal (ip) administration of vehicle or compound 2e, 2g, 2p,
2aa, and 2ae (1 or 3 mg/kg), alone or in combination with
either the selective CB2 antagonist, 6-iodo-2-methyl-1-[2-(4-
morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)metha-
none26 (AM630, Chart 1) (1 mg/kg, ip) or the selective CB2
agonist 3-(10,10-dimethylbutyl)-1-deoxy-Δ8-THC27 (JWH-
133, Chart 1) (3 mg/kg, ip), administered 5 min before the
compound. The results are presented in Figures 1-5. Finally,
the effect of compounds 2e and 2g on forskolin-induced
elevation of cAMP levels in CHOcells transfectedwithhuman
CB2 and on [35S]GTPγS binding to membranes from these
cells as well as from the mouse spleen (which expresses
selectively CB2 receptors) were also assessed. The effect of
2e as a potential antagonist against CP-55,940 agonist effects
on forskolin-induced elevation of cAMP levels and [35S]-
GTPγS binding in these assays was also evaluated.
Finally, with the aim of exploring the effects on receptor
affinity of enhanced chemical diversity within this family of
4-oxoquinoline-3-carboxamides, diverse substituents were in-
troduced at either the N-1 position or carboxamide group.
Accordingly, 6-bromo-4-oxo-1,4-dihydroquinoline-3-carbo-
xylic acid (8)18a (Scheme 4) by reaction with 1-aminoadaman-
tane and rac-1-fenchylamine23 was converted into the N-(1-
adamantyl)amide 9a and N-(1-fenchyl)amide 9b, which were
in turn alkylated at the N-1 position; in particular, the
adamantylamide 9a was treated with propargyl, allyl, and
1-buten-4-yl halides to afford the corresponding derivatives
2af, 2ag, and 10,18a while the fenchylamide 9b was alkylated
with n-pentyl iodide to give 2ah. Conversely, compounds 2ai
and 2aj were obtained by amidation with 1-aminopiperidine
and 1-aminohexane, respectively, of the acid 11, in turn
prepared according to a general procedure for the synthesis
of 4-quinolone-3-carboxylic acids (see Supporting Infor-
mation). Suzukireactionwith2-furylboronic acidon6-bromo
derivatives 2ag, 10, and 2ah provided the corresponding 6-(2-
furanyl) derivatives 2ak, 2al, and 2am.
Results and Discussion
In Vitro Pharmacology and SAR. Out of the 39 synthesized
quinolones 2, compounds 2k, 2n, 2ab, 2ac, 2ag could not be
tested in the cannabinoid receptor binding assay because of
their very low solubility in DMSO/water. With the exception
of six compounds (2b, 2o, 2u, 2v, 2ai, 2aj) exhibiting Ki >
100 nM (Table 1), all the remaining 28 quinolone carbox-
amides of the 2 series proved to be high affinity CB2 ligands,
with Ki values ranging from 73.2 nM (2c) to 0.7 nM (2e and
2y). Under the conditions of our assay, the CB2-selective
reference ligand SR14452815a showed Ki = 5.4 nM. With
regard to the CB1 affinity, Ki values spanned 3 orders of
magnitude in the range from >10000 nM (22 out of the
34 tested compounds) to 245.7 nM (2s), with rimonabant
(SR141716A)24 used as the CB1-selective reference ligand,
displaying Ki = 12.0 nM. Because of the high variability of
affinity for both CB1 and CB2 receptors, the CB2 selectivity
index (SI), calculated as Ki(CB1)/Ki(CB2) ratio, for tested
compounds also varied widely, from >14285 to 1.9. Only in
Pharmacology
The binding affinities (Ki values) of compounds 2 for hu-
man recombinant CB1 and CB2 receptors are reported in
Table 1. The tested compounds were evaluated in parallel with
SR14452815a (Chart 1) and rimonabant24 as reference CB2
and CB1 ligands, respectively, as previously described.25
The functional activity of compounds 2e, 2g, 2p, 2aa, and
2ae was assessed using the formalin test of acute peripheral
and inflammatory pain in mice. Formalin injection induces
a biphasic stereotypical nocifensive behavior. Nociceptive
responses are divided into an early, short lasting first phase
(0-7 min) caused by a primary afferent discharge produced
by the stimulus, followed by a quiescent period, and then a
second, prolonged phase (15-60 min) of tonic pain. Fifteen

5920 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Pasquini et al.
Figure 2. Effect of intraperitoneal (ip) administration of vehicle (20% DMSO in saline), compound 2ae (1 or 3 mg/kg) alone, or compound 2ae
(3 mg/kg) in combination with AM630 (1 mg/kg), a selective CB2 antagonist, on the nociceptive behavior (seconds) induced by subcutaneous
formalin (1.25%) injection into the hind paw of mice (n = 8-10). Open columns represent the early phase and filled columns the late phase
of formalin-evoked nociception. Formalin was injected 15 min after vehicle or drug treatments. Asterisks indicate significant differences
(P<0.05) vs vehicle and circles vs compound 2ae (3 mg/kg).
Figure 3. Effect of intraperitoneal (ip) administration of vehicle (20% DMSO in saline), compound 2aa (1 or 3 mg/kg), JWH-133 (1 or 3 mg/
kg), compound 2aa (3 mg/kg) in combination with JWH-133 (1 mg/kg), or compound 2aa (1 mg/kg) in combination with JWH-133 (3 mg/kg)
on the nociceptive behavior (seconds) induced by subcutaneous formalin (1.25%) injection into the hind paw of mice (n = 8-10). Open
columns represent the early phase and filled columns the late phase of formalin-evoked nociception. Formalin was injected 15 min after vehicle
or drug treatments. Asterisks indicate significant differences (P < 0.05) vs vehicle and circles vs JWH-133 (1 and 3 mg/kg).
one case the SI was lower than 1, with compound 2ah
[Ki(CB1) = 2.8, Ki(CB2) = 5.0] exhibiting a reverse selecti-
vity. Derivatives 2e-h, 2w, 2z, 2aa, and 2al all showed CB1
and CB2 affinities >10000 nM and <10 nM, respectively
and hence a SI>1000. To our knowledge, compound 2e is the
CB2 ligand endowed with the highest affinity (Ki = 0.7 nM)
and selectivity (SI>14285) described so far in the literature
and compares favorably with structurally different com-
pounds claimed in recent patent applications, such as for in-
stance a benzimidazole derivative showing SI>1300028 and
an indole derivative with Ki = 0.24 nM.29,30
A chain of appropriate length and lipophilicity at position
1 of the quinolone nucleus is required for best CB2 affinity/
selectivity. In fact, replacement of the pentyl group of 2e with
a four-carbon atom chain (1-buten-4-yl in compound 2al)
and then with a three-carbon atom chain (allyl in compound
2ak) progressively reduced CB2 affinity by approximately 1
order of magnitude. Similarly, the insertion of a propargyl
chain at the N-1 position (2af) decreased both CB2 and CB1
affinities by more than 3 times with respect to compound 718a
bearing a n-pentyl moiety.
The nature of the 3-carboxamido substituent also mark-
edly affects both receptor affinity and selectivity. Thus,
compounds 2ah and 2am, where the adamantyl group has
been replaced with the fenchyl moiety characteristic of
SR144528, retained basically the same CB2 affinity as the
corresponding adamantyl derivatives 7 [Ki(CB2) = 14.3 nM]18a
and 2e, respectively, while exhibiting dramatically improved
affinity for the CB1 receptor, that increased from 996 nM (for 7)
to 2.8 nM (for 2ah) and from >10000 nM (for 2e) to 480 nM
(for 2am). These changes in CB1 affinity strongly influenced
receptor selectivity, leading to the much less CB2-selective
ligand 2am or even to a compound (2ah) showing reversed
selectivity. On the other hand, N-1-piperidinyl and N-1-hexyl
amides 2ai and 2aj proved to be substantially devoid of affinity
toward both receptor subtypes.
The most striking results were obtained by inserting sub-
stituents at position 6 of the quinolone scaffold, particularly

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 5921
Figure 4. Effect of intraperitoneal (ip) administration of vehicle (20% DMSO in saline), compound 2g (1 or 3 mg/kg), JWH-133 (1 or 3 mg/kg),
compound 2g (3 mg/kg) in combination with JWH-133 (1 mg/kg), or compound 2g (1 mg/kg) in combination with JWH-133 (3 mg/kg) on the
nociceptive behavior (seconds) induced by subcutaneous formalin (1.25%) injection into the hind paw of mice (n = 8-10). Open columns
represent the early phase and filled columns the late phase of formalin-evoked nociception. Formalin was injected 15 min after vehicle or drug
treatments. Asterisks indicate significant differences (P<0.05) vs vehicle and circles vs JWH-133 (1 and 3 mg/kg).
Figure 5. Effect of intraperitoneal (ip) administration of vehicle (20% DMSO in saline), compound 2p (1 or 3 mg/kg), JWH-133 (3 mg/kg), or
compound 2p (3 mg/kg) in combination with JWH-133 (3 mg/kg), on the nociceptive behavior (seconds) induced by subcutaneous formalin
(1.25%) injection into the hind paw of mice (n = 8-10). Open columns represent the early phase and filled columns the late phase of formalin-
evoked nociception. Formalin was injected 15 min after vehicle or drug treatments. Asterisks indicate significant differences (P<0.05) vs
vehicle and circles vs JWH-133 (3 mg/kg).
aryl and heteroaryl groups. Quinolones 2e-g, 2i, 2j, 2l, 2m,
2p, and 2z, all characterized by this type of substitution,
displayed high CB2 affinity and selectivity, although more
sterically demanding aromatic substituents, as in com-
pounds 2o, 2q-s, and 2u, negatively affected either CB2
affinity or selectivity. No definitive explanation can yet be
given for the particular biochemical profile of the 6-pyrrolyl
derivative 2y, which seems to be an outlier within this series,
showing the same CB2 potency as 2e, but greatly reduced
selectivity. The replacement of the 6-bromo substituent with
the 2-furyl group favorably influenced the whole biochem-
ical profile even in compounds, such as 2al and 2am, with a
diverse substitution pattern at N-1 and carboxamide nitro-
gen. It is interesting to note how the conversion of 2ah into
2am was able not only to maintain very high CB2 affinity,
but also to restore the CB2 selectivity, despite the CB1-
directing effect of the fenchyl group on the amide.
less active compounds were generally obtained. Thus, the
6-dichlorobenzyl derivative 2b was actually inactive, and
further spacing out of the aromatic rings by either unsatu-
rated or saturated spacers resulted in compounds possessing
moderate (2x, 2ae) or low (2v) CB2 affinity. It is worth noting
that 2ae showed lower CB2 affinity than the corresponding
6-ethyl derivative (2ad) or 6-phenyl derivative,18a but the
presence of the aromatic ring in the 6-substituent probably
prevented a favorable interaction with the CB1 receptor,
thus giving rise to a more selective ligand than 2ad.
Also alkyl, alkenyl, and alkynyl substituents at the 6 posi-
tion are well tolerated, as demonstrated by the CB2 affinity
and selectivity values of compounds 2h, 2t, 2w, 2aa, and 2ad.
However, the risk exists to get too lipophilic compounds, the
biological testing of which becomes problematic (as for 2k,
2ab, 2ac).
Oxidation of the 6-phenylthio analogue 618a to the sulf-
oxide 2c and sulfone 2d caused a descrease in CB2 affinity by
approximately 20 times. Considering the modest affinity/
When the aromatic ring at the position 6 was moved away
from the quinolone nucleus by means of different spacers,

5922 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Pasquini et al.
Figure 6. Dose-dependent effects (means ( SE) (A) of CP-55,940 (n = 2) and of 2e and 2g (n = 3) on forskolin-induced stimulation of cyclic
AMP production by CHO-hCB2 cells, (B) of CP-55,940, 2e and 2g on [35S]GTPγS binding to mouse spleen membranes (n = 4-12), and (C) of
2e on [35S]GTPγS binding to CHO-hCB2 cell membranes (n = 4).
selectivity profile of 2c as well as the low yield in its prepara-
tion, no attempt was made to separate the racemate in order
to test the individual enantiomers. Finally, the 6-iodo deri-
vative 2a elicited receptor affinity and selectivity comparable
to the 6-bromo analogue 7, and in particular it showed 4-fold
reduced CB2 affinity but 10-fold enhanced receptor selec-
tivity when compared to its 6-unsubstituted counterpart.18a
In Vivo Pharmacology. Compounds with high to moderate
affinity (Ki ranging from 0.7 to 56.6 nM) and selectivity (SI
ranging from >177 to >14286) for CB2, i.e. 2e, 2g, 2p, 2aa
and 2ae, were selected to be tested in the formalin test of
acute (both phases) and inflammatory (second phase) pain
(Figures 1-5). Compound 2e (Figure 1), possibly the highest
affinity and most selective CB2 ligand developed to date (see
above), behaved as a potent CB2 neutral antagonist in this
test because, unlike CB2 inverse agonists,31 it did not reduce
the second phase of the nocifensive response to formalin but
fully antagonized, already at the dose of 1 mg/kg, the effect of
even a high dose (3 mg/kg) of the CB2-selective ligand JWH-
133 on both phases of the formalin response. Given the lack
of CB2-neutral antagonists in the scientific literature, this
CB2-selective neutral antagonism-like behavior of 2e might
provide an unprecedented pharmacological tool for in vivo
studies of CB2 receptor biological function. By contrast,
compound 2ae (Figure 2) behaved as a CB2 agonist,
although weaker than JWH-133 because it counteracted
both phases of the nocifensive response to formalin but only
at the highest dose tested (3 mg/kg), and this effect was
antagonized by the CB2 receptor inverse agonist AM630 at a
per se inactive dose (1 mg/kg). Importantly, the CB2 affinity
of 2ae (Ki = 56.6 nM) was significantly lower than that (Ki =
3.4 nM) previously reported for JWH-133,27 in agreement
with its lower efficacy in vivo. Compound 2aa (Figure 3), like
2e, was also inactive in the formalin test, but it fully antag-
onized only the effect of a low dose of JWH-133 (1 mg/kg) at
the highest dose tested (3 mg/kg). Therefore, also 2aa might
be considered as a neutral CB2-selective antagonist, albeit
less efficacious than 2e, in agreement with its lower affinity
for CB2 receptors as compared to 2e (Ki = 7.3 nM vs
0.7 nM). Unlike 2e, 2ae, and 2aa, the effects of compounds
2g and 2p in the formalin test were more open to interpreta-
tion. Compound 2g (Figure 4) significantly inhibited only the
second phase of the nocifensive response to formalin only at
the highest dose tested (3 mg/kg). This, in view of the high
affinity (Ki = 4.2 nM) and selectivity (SI > 2381) of this
compound for CB2, is the behavior previously reported for
high affinity CB2 inverse agonists.32 Accordingly, the effect
of 2g (3 mg/kg) was partly antagonized by a low dose of

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 5923
Figure 7. (A) Inhibitory effect of CP-55,940 on forskolin-induced stimulation of cyclic AMP production by CHO-hCB2 cells in the presence
of 20 nM 2e or of its vehicle, DMSO (means ( SE, n = 4). Stimulatory effect of CP-55,940 on [35S]GTPγS binding to mouse spleen membranes
(B) or CHO-hCB2 cell membranes (C) in the presence of 20 nM 2e or of its vehicle, DMSO (means ( SE, n = 4).
JWH-133 (1 mg/kg). However, a per se inactive dose of 2g (1
mg/kg) was not capable of fully antagonizing the antinoci-
fensive effect of JWH-133 (3 mg/kg) although it rendered the
effect of the latter nonstatistically significant. This observa-
tion, given the high affinity of 2g for CB2, is not completely
in agreement with it behaving as an inverse agonist.32 Indeed,
the pharmacological behavior of 2g is also compatible with
that of a potent, albeit partial, CB2 agonist because, as
mentioned above, it obstructed the effect of a low, but still
active, dose of JWH-133. Finally, compound 2p (Figure 5),
endowed with lower affinity (Ki = 41.9 nM) and selectivity
(SI > 177) for CB2 receptor than 2g, appeared also less
efficacious than the latter compound at reducing the second
phase of the nocifensive response to formalin at the highest
dose tested (3 mg/kg). This behavior might be due to 2p
acting again as an inverse agonist, as suggested by the fact
that this compound and JWH-133 obstructed each other,
rather than to its activity as a potential dual CB1/CB2
agonist at high doses, which would have resulted in an
inhibition of both the first and second phase of the nocifen-
sive response to formalin.
Functional Activity at CB2 Receptors in Vitro. The above
hypotheses on the possible agonist, neutral antagonist-, and
inverse agonist-like behaviors of the five compounds exam-
ined here in the formalin test needed to be confirmed, at least
to some extent, by appropriate in vitro assays of CB2
receptor functional activity. Therefore, of the five com-
pounds, we selected the ones with highest affinity on CB2,
i.e. 2e and 2g, and tested them in two assays of CB2-mediated
functional activity. These two compounds were also selected
because, for the former, we needed to confirm the unprece-
dented behavior as a neutral antagonist in the formalin test
and, for the latter, we needed to distinguish between the two
possibilities (inverse agonism vs partial agonism) suggested
by this in vivo assay (see above). Both compounds behaved
as inverse agonists in the forskolin-induced cAMP elevation
assay carried out in CHO cells overexpressing the human
recombinant CB2 receptor (Figure 6A), although the effect
of 2e in this assay was only seen at concentrations higher than
100 nM, i.e. more than 140-fold higher than its Ki. Both
compounds also acted as inverse agonists in the [35S]GTPγS
binding assay carried out with mouse spleen membranes

5924 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Pasquini et al.
(Figure 6B), which preferentially express CB2 receptors over
CB1. In this case, compound 2e was more potent but
significantly less efficacious than 2g, with an Emax of only
13.9%. Therefore, given its activity in vivo, we thought that
2e, at concentrations <100 nM, could behave as a neutral
antagonist, and tested it against CP-55,940-induced inhibi-
tion of forskolin-induced cAMP elevation in CHO cells
overexpressing the human recombinant CB2 receptors or
stimulation of [35S]GTPγS binding in mouse spleen mem-
branes. Surprisingly, in neither of these assays, 2e, at con-
centrations up to 20 nM, could antagonize the effect of CP-
55,940 (Figure 7), the reported CB2 receptor affinity of
which is similar to that observed here for 2e. Thus, while
the in vitro assays performed here confirmed for 2g its
behavior as inverse agonist, observed in the formalin test,
they failed to confirm the very promising neutral antagonist
activity of 2e suggested by the in vivo data. A possible
explanation for this discrepancy is that this compound might
act, in vivo, rather than as a competitive antagonist, as a
“functional” antagonist instead, for example by blocking
effects that are downstream of CB2 receptors, and hence
counteracting only some effects (e.g., analgesia) of certain
agonists (e.g., JWH133) rather than other effects (e.g.,
inhibition of adenylate cyclase) by other agonists (e.g., CP-
55,940). Indeed, it is known that different agonists can
induce the trafficking of their G-protein-coupled receptors
toward different G-proteins and biological effects. It is also
possible that 2e is transformed in vivo to a metabolite that
instead does act as a neutral competitive antagonist at CB2
receptors. Indeed, unpublished in vitro data from our labo-
ratories indicate that this compound is rapidly oxidized by
cytochrome P450 enzymes (after 1 h incubation with
CYP3A4, only 4% of compound 2e remains unaltered).
Finally, a possibly much simpler explanation is that 2e
behaves in vitro predominantly as an inverse agonist at
CB2 receptors, and that the putative neutral antagonism
observed with 2e in vivo might be observed also in vitro but
only at concentrations that are too low to counteract the
agonist effects of a potent agonist such as CP-55,940. Indeed,
we did find here that 2e behaves as an inverse agonist at
concentrations >40 nM also in a [35S]GTPγS binding assay
carried out with CHO cells overexpressing the human re-
combinant CB2 receptor (Figure 6C), in which, again, a per
se inactive concentration of the compound could not coun-
teract the stimulatory effect of CP-55,940 (Figure 7C).
at least in vivo, behaved as agonists, inverse agonists or, most
importantly, neutral antagonists. This would have been a
quite significant finding, since the CB2 “antagonists” most
widely used so far (such as SR144528 and AM630) all behave
indeed as inverse agonists,5b able to reverse the constitutive
activity of the receptor, whereas neutral antagonists have
affinity but no efficacy for their receptor. Selected compounds
were assayed in the formalin test of acute peripheral and
inflammatory pain in mice by ip administration. In previous
studies, compounds acting as CB2 agonists exhibited in this
test analgesic activity, whereas CB2 antagonists antagonized
the antinociceptive responses of agonists and inverse agonists
also caused antinocifensive effects at the highest dose tested.33
In the present study, data obtained in this animal model of
inflammatory pain allowed us to conclude that 6-substituted
4-quinolone-3-carboxamides agonists may possess CB2 re-
ceptor-mediated analgesic activity in vivo and show favorable
drug-safety profile. These data suggest that the compound
with highest CB2 affinity obtained in this study, i.e 2e, might
behave as a neutral antagonist in vivo, whereas others, such as
2g, might act as inverse agonists. However, two different
functional assays carried out in vitro could only confirm the
latter hypothesis, thus opening the way to further future
studies aimed at understanding how the very high affinity of
2e for CB2, and its weak efficacy and/or potency as an inverse
agonist at this receptor in vitro, result in the antagonism of a
CB2-mediated effect only in vivo. A different behavior of CB2
ligands infunctional assayscarried outinvitro orinvivoisnot
unusual. Indeed, the concept of “protean agonists”, defined as
those compounds the functional efficacies of which in various
assay systems may depend on the local levels of receptor
constitutive activities, and for which, therefore, the efficacies
observed in vitro may not necessarily predict in vivo
activities,34 was applied to explain the pharmacological activ-
ity of certain CB2 receptor-selective ligands as early as 2006.35
Importantly, a recent study showed that such different beha-
viors in functional tests do not seem to affect the final
outcome in assays of antihyperalgesic activity,36 exactly as
observed here, possibly because, as emphasized by many
authors,4a,17,44 both agonists and inverse agonists at CB2
may inhibit inflammation via different cellular mecha-
nism of actions. Finally, further investigations will be also
directed to enhance both water solubility and metabolic
stability of the most promising CB2 ligands within this series
of compounds.
Experimental Section
Conclusions
Chemistry. Reagents were purchased from commercial sup-
pliers and used without further purification. Anhydrous reac-
tions were run under (a positive pressure) dry N2. Merck silica
gel 60 was used for flash chromatography (23-400 mesh). IR
spectra were recorded on a Perkin-Elmer BX FT-IR system
using CHCl3 as the solvent or a Nujol dispersion. 1H NMR and
13C NMR were recorded at 200 and 50 MHz respectively on a
Bruker AC200F spectrometer and at 400 and 100 MHz on a
Bruker Advance DPX400. Chemical shifts are reported relative
to tetramethylsilane at 0.00 ppm. Mass spectral (MS) data were
obtained using Agilent 1100 LC/MSD VL system (G1946C)
with a 0.4 mL/min flow rate using a binary solvent system of 95:5
methanol/water. UV detection was monitored at 254 nm. Mass
spectra were acquired either in positive or in negative mode
scanning over the mass range of 105-1500. Melting points were
determined on a Gallenkamp apparatus and are uncorrected.
Microwave irradiations were conducted using a CEM Dis-
cover Synthesis Unit (CEM Corp., Matthews, NC). Elemental
Within a research program aimed at characterizing novel
cannabinoid ligands, we describe herein new 6-substituted
4-quinolone-3-carboxamides possessing high affinity for the
human CB2 receptor at nanomolar or subnanomolar concen-
tration and selectivity index values as high as >14000. Thus,
some of these compounds proved to be very potent toward the
human CB2 receptor, while no affinity for the human CB1
receptor could be measured at all. It should be considered that
the issue of receptor selectivity has a major relevance in the
cannabinoid area, since none of the CB2-selective agonists
that have been developed to date are completely CB2-
specific.5b Thus they are all expected to display CB2 selectivity
only within a finite dose range and to target CB1 receptors as
well when administered at a dose that lies above this range.
As far as the functional activity is concerned, among the
new compounds it was possible to identify compounds that,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 5925
analyses were performed on a Perkin-Elmer PE 2004 elemental
analyzer and the data for C, H, and N are within 0.4% of the
theoretical values. The chemical purity of the target compounds
was determined using the following conditions: an Agilent 1100
series LC/MSD with a Lichrocart 125-4 Lichrospher 100 RP-18
(4.6 mm ꢀ 100 mm, 5 μm) reversed phase column; method: 86%
(v/v) of MeOH in H2O, isocratic, flow rate of 1 mL/min, UV
detector, 254 nm. The purity of each compound was g95% in
either analysis.
Synthesis of Compounds 4a,b and 2af-2ah by N-Alkylation
Reaction. General Procedure. To a solution of the appropriate
substrate 3a,b or 9a,b (1 mmol) in DMF (1 mL) were added
K2CO3 (0.39 g, 2.8 mmol) and the alkyl halide (2.8 mmol). The
reaction mixture was heated at 100 ꢀC for 4 h, then diluted with
water (10 mL) and extracted with CH2Cl2 (3 ꢀ 3 mL). The
organic layer was dried over Na2SO4 and evaporated to dryness.
The crude residue was recrystallized from EtOH to provide the
title compound.
Example. 6-Bromo-4-oxo-1-pentyl-N-(1,3,3-trimethylbicyclo-
[2.2.1]heptan-2-yl)-1,4-dihydroquinoline-3-carboxamide (2ah).
Prepared in 88% yield starting from 9b; white solid; mp
159-160 ꢀC. 1H NMR (200 MHz, CDCl3): δ 10.01 (d, J =
9.1 Hz, 1H), 8.71 (s, 1H), 8.63 (d, J = 1.9 Hz, 1H), 7.72 (d, J1=
1.9, J2 = 8.9 Hz, 1H), 7.34 (d, J = 8.9 Hz, 1H), 4.15 (t, J = 6.9
Hz, 2H), 3.80 (d, J = 9.1 Hz, 1H), 1.82-1.58 (m, 6H), 1.51-1.17
(m, 7H), 1.10 (s, 3H), 1.04 (s, 3H), 0.87-0.81 (m, 6H). 13C NMR
(100 MHz, CDCl3): δ 175.54, 165.03, 147.78, 137.78, 135.64,
130.20, 129.38, 118.88, 117.66, 112.68, 64.17, 54.39, 48.80, 48.35,
42.70, 39.57, 31.02, 28.70, 27.76, 26.07, 22.20, 21.71, 19.78,
13.80. MS (ESI): m/z: 474 [M þ H]þ. IR (CHCl3): ν 1607,
1658 cm-1. Anal. (C25H33BrN2O2) C, H, N.
CH2Cl2. The organic layers were washed with brine, dried over
anhydrous Na2SO4, and evaporated to dryness. The crude
residue was purified by chromatography using light petroleum
ether/AcOEt (2:1) as eluent to provide compounds 2c (5 mg, 7%
yield) as a colorless oil and 2d (32 mg, 50% yield) as a white solid.
N-(Adamantan-1-yl)-4-oxo-1-pentyl-6-(phenylsulfinyl)-1,4-di-
hydroquinoline-3-carboxamide (2c). Colorless oil. 1H NMR (200
MHz, CDCl3): δ 9.69 (s, 1H), 8.78 (d, J = 1.9 Hz; 1H), 8.50 (s,
1H), 8.10 (dd, J1 = 1.9, J2 = 8.6 Hz, 1H), 7.90-7.55 (m, 6H),
4.20 (t, J = 7.2 Hz, 2H), 2.14-2.09 (m, 10H), 1.90-1.81 (m,
2H), 1.80-1.71 (m, 5H), 1.37 (m, 4H), 0.92-0.86 (m, 3H). MS
(ESI): m/z 517 [M þ 1]þ, 539 [M þ Na]þ. IR (CHCl3): ν 1601,
1653 cm-1. Anal. (C31H36N2O3S) C, H, N.
N-(Adamantan-1-yl)-4-oxo-1-pentyl-6-(phenylsulfonyl)-1,4-
dihydroquinoline-3-carboxamide (2d). White solid; mp 133-
1
135 ꢀC. H NMR (200 MHz, CDCl3): δ 9.60 (s, 1H), 8.89 (d,
J = 2.4 Hz, 1H), 8.67 (s, 1H), 8.15 (dd, J1 = 2.4, J2 = 8.8 Hz, 1H),
7.91 (d, J = 6.8 Hz, 2H), 7.56 (d, J = 8.8 Hz, 1H), 7.50-7.43
(m, 3H), 4.16 (t, J = 6.8 Hz, 2H), 2.11-2.09 (m, 9H), 1.79-1.65
(m, 8H), 1.35-1.31 (m, 4H), 0.82-0.77 (m, 3H). MS (ESI): m/z:
533 [M þ H]þ. IR (CHCl3): ν 1601, 1655 cm-1. Anal. (C31H36-
N2O4S) C, H, N.
Synthesis of Compounds 2e-2u and 2ak-2am by Suzuki
Reaction. General Procedure. To a solution of the appropriate
bromoquinolone 7 or iodoquinolone 2a (1 mmol) in DME (4 mL),
Pd(OAc)2 (0.1 mmol), PPh3 (0.3 mmol), the appropriate boronic
acid (0.5 mmol), EtOH (1 mL), and 1 N Na2CO3 (2 mL) were
added. The mixture was irradiated with microwaves at 150 ꢀC
for 10 min, then filtered through a plug of Celite. The filtrate was
washed with H2O, brine, dried over anhydrous Na2SO4, and
evaporated to dryness. The solid residue was purified by flash
chromatography using CH2Cl2/MeOH (98:2) as eluent.
Example. N-(Adamantan-1-yl)-6-(furan-2-yl)-4-oxo-1-pentyl-
1,4-dihydroquinoline-3-carboxamide (2e). Prepared from 7 in
Synthesis of Compounds 5a and 5b by Basic Hydrolysis.
General Procedure. A suspension of the appropriate ester 4a,b
(1 mmol) in 10% aq NaOH (7 mL) was refluxed for 3 h. After
cooling at room temperature, the reaction mixture was acidified
using conc HCl. The solid precipitated was collected by filtra-
tion and washed with water and petroleum ether. The solid
obtained was recrystallized from EtOH to afford the pure acid.
Example. 6-Iodo-4-oxo-1-pentyl-1,4-dihydroquinoline-3-car-
boxylic Acid (5a). Prepared from compound 4a in 65% yield;
1
81% yield; light-brown solid; mp 180-181 ꢀC. H NMR (200
MHz, CDCl3): δ 9.88 (s, 1H), 8.65-8.61 (m, 2H), 7.93-7.89 (m,
1H), 7.44-7.40 (m, 2H), 6.73-6.72 (m, 1H), 6.43-6.42 (m, 1H),
4.14 (t, J = 7.6 Hz, 2H), 2.15 (s, 6H), 2.06 (s, 3H), 1.80-1.74 (m,
2H), 1.66-1.61 (m, 6H), 1.31-1.28 (m, 4H), 0.83-0.80 (m, 3H).
MS (ESI): m/z: 459 [M þ H]þ. IR (CHCl3): ν 1601, 1653 cm-1
.
1
Anal. (C29H34N2O3) C, H, N.
beige solid; mp 186-187 ꢀC. H NMR (200 MHz, CDCl3): δ
Synthesis of Compounds 2v and 2w by Sonogashira Reaction.
General Procedure. To a solution of 2a (518 mg, 1 mmol) in
toluene (7 mL) under N2 were added successively CuI (38 mg, 0.2
mmol), i-Pr2NH (7 mL), the appropriate alkyne (2.2 mmol), and
PdCl2(PPh3)2 (70.2 mg, 0.1 mmol). The reaction mixture was
refluxed for 2 h and volatiles were removed under reduced
pressure. The crude residue was purified by flash chromatogra-
phy using CH2Cl2/MeOH (99:1) as eluent to give 2v or the
trimethylsilyl derivative of 2w.
N-(Adamantan-1-yl)-4-oxo-1-pentyl-6-(2-phenylethynyl)-1,4-
dihydroquinoline-3-carboxamide (2v). Yield: 72%; yellow solid;
mp 199-200 ꢀC. 1H NMR (200 MHz, CDCl3): δ 9.86 (s, 1H),
8.68 (s, 1H), 8.63 (d, J = 1.4 Hz, 1H), 7.35-7.31 (m, 3H), 4.17 (t,
J = 7.3 Hz, 2H), 2.16 (s, 6H), 2.09 (s, 3H), 1.85-1.78 (m, 2H),
1.75-1.70 (m, 6H), 1.35-1.32 (m, 4H), 0.91-0.85 (m, 3H). MS
(ESI): m/z: 493 [M þ H]þ, 515 [M þ Na]þ. IR (CHCl3): ν 1601,
1653 cm-1. Anal. (C33H36N2O2) C, H, N.
14.64 (s, 1H), 8.82 (s, 1H), 8.72 (s, 1H), 8.06 (d, J = 8.8 Hz, 1H),
7.34 (d, J = 8.8 Hz, 1H), 4.27 (t, J = 7.2 Hz, 2H), 1.98-1.79 (m,
2H), 1.36-1.34 (m, 4H), 1.00-0.80 (m, 3H). MS (ESI): m/z:
386 [M þ H]þ. IR (CHCl3): ν 1722 cm-1. Anal. (C15H16INO3)
C, H, N.
Synthesis of Compounds 2a,b, 9a,b, and 2ai,aj by Amidation
Reaction. General Procedure. The appropriate carboxylic acid
5a,b, 8, or 11 (2 mmol) was dissolved in DMF (5 mL). HOBt (270
mg, 2 mmol), HBTU (1.72 g, 4 mmol), DIPEA (152 μL, 3 mmol),
and the amine (2.4 mmol) were added to the solution and the
reaction mixture was stirred at room temperature for 30 min.
Further DIPEA (152 μL, 3 mmol) was thereafter added, and the
reaction mixture was stirred at room temperature for further 4 h.
The reaction mixture was poured into ice and the solid pre-
cipitated was collected by filtration and washed with water and
petroleum ether. Recrystallization from EtOH gave the pure
amide.
N-(Adamantan-1-yl)-6-ethynyl-4-oxo-1-pentyl-1,4-dihydro-
quinoline-3-carboxamide (2w). The trimethylsilyl derivative ob-
tained by the Sonogashira reaction (40 mg, 43% yield) was
dissolved in MeOH/THF (1:1, 10 mL) and K2CO3 (110 mg, 0.8
mmol) was added. The reaction mixture was stirred at room
temperature for 30 min and evaporated to dryness. The crude
residue was taken up in CH2Cl2 and washed with water and
brine. The organic layer was evaporated to dryness to afford
Example. 6-Bromo-4-oxo-1-pentyl-N-(piperidin-1-yl)-1,4-di-
hydroquinoline-3-carboxamide (2ai). Prepared in 88% yield from
11; white solid; mp 195-196 ꢀC. 1H NMR (200 MHz, CDCl3): δ
10.06 (s, 1H), 8.61 (d, J = 1.6 Hz, 1H), 8.43 (s, 1H), 7.64 (dd, J1
= 1.6, J2 = 7.0 Hz, 1H), 7.28 (d, J = 7.0 Hz, 1H), 4.11, (t, J =
6.8 Hz, 2H), 2.77-2.65 (m, 4H), 1.72-1.62 (m, 6H), 1.44-1.28
(m, 6H), 0.76 (t, J = 6.8 Hz, 3H). MS (ESI): m/z: 421 [M þ H]þ.
IR (CHCl3): ν 1601, 1655 cm-1. Anal. (C20H26BrN3O2) C, H, N.
Synthesis of Compounds 2c and 2d. To a solution of oxone (240
mg, 0.39 mmol) in H2O (3 mL), a solution of 6 (65 mg, 0.13
mmol) in 1,4-dioxane (2 mL) was added. The reaction mixture
was stirred at room temperature for 20 h and extracted with
1
compound 2w (32 mg, 96% yield) as a colorless oil. H NMR
(200 MHz, CDCl3): δ 9.81, (s, 1H), 8.69 (s, 1H), 8.61 (d, J = 1.9
Hz, 1H), 7.74 (dd, J1 = 1.9 Hz, J2 = 7.0 Hz, 1H), 7.42 (d, J = 7.0
Hz, 1H), 7.42 (d, J = 8.9 Hz, 1H), 4.17 (t, J = 7.4 Hz, 2H), 3.13

5926 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Pasquini et al.
(s, 1H), 2.15 (s, 6H), 2.09 (s, 3H), 1.85-1.70 (m, 2H), 1.63-1.61
(m, 6H), 1.36-1.33 (m, 4H), 0.91-0.88 (m, 3H). MS (ESI): m/z:
417 [M þ H]þ, 439 [M þ Na]þ. IR (CHCl3): ν 1599, 1660, 3303
cm-1. Anal. (C27H32N2O2) C, H, N.
second, prolonged phase (15-60 min) of tonic pain. Mice
received formalin (1.25% in saline, 30 μL) in the dorsal surface
of one side of the hind-paw. Each mouse was randomly assigned
to one of the experimental groups (n = 8-10) and placed in a
Plexiglas cage and allowed to move freely for 15-20 min. A
mirror was placed at a 45ꢀ angle under the cage to allow full view
of the hind-paws. Lifting, favoring, licking, shaking, and flinch-
ing of the injected paw were recorded as nociceptive responses.
The duration of those mentioned noxious behaviors were mon-
itored by an observer blind to the experimental treatment for
periods of 0-10 min (early phase) and 20-60 min (late phase)
after formalin administration. Results are expressed as means (
SEM. Significant differences between groups were evaluated by
using analysis of variance followed by the Dunnett’s test. The
version of the formalin test we applied is based on the fact that a
correlational analysis showed that no single behavioral measure
can be a strong predictor of formalin or drug concentrations on
spontaneous behaviors.37 Consistently, we considered that a
simple sum of time spent licking plus elevating the paw, or the
weighted pain score, is in fact superior to any single (lifting,
favoring, licking, shaking, and flinching) measure (r ranging
from 0.75 to 0.86).38 Treatments: groups of 8-10 animals per
treatment were used with each animal being used for one
treatment only. Mice received intraperitoneal vehicle (20%
DMSO in 0.9% NaCl) or different doses of before mentioned
compounds.
(E)-N-(Adamantan-1-yl)-4-oxo-1-pentyl-6-styryl-1,4-dihydro-
quinoline-3-carboxamide (2x). A suspension of compound 2a
(100 mg, 0.19 mmol), styrene (65 μL, 0.57 mmol), Pd(OAc)2 (42
mg, 0.19 mmol), TEA (26 μL, 0.19 mmol),and PPh3 (20 mg,
0.076 mmol) in DMF (1 mL) was refluxed under N2 for 3 h. The
reaction mixture was poured into ice-water and extracted with
CH2Cl2. The organic layer was washed with water, then brine,
dried over anhydrous Na2SO4, and evaporated to dryness. The
crude residue was purified by flash chromatography using
CH2Cl2/MeOH (99:1) as eluent to give the title compound 2x.
Yield: 40%; yellow oil. 1H NMR (200 MHz, CDCl3): δ 9.96 (s,
1H), 8.69 (s, 1H), 8.8.62-8.61 (m, 1H), 7.87-7.83 (m, 1H),
7.54-7.40 (m, 3H), 7.37-7.31 (m, 3H), 7.26 (d, J = 5.4 Hz, 1H),
4.20 (t, J = 7.4 Hz, 2H), 2.17 (s, 6H), 2.10 (s, 3H), 1.88-1.72 (m,
2H), 1.61-1.60 (m, 6H), 1.37-1.35 (m, 4H), 0.90 (t, J = 6.8 Hz,
3H). MS (ESI): m/z: 495 [M þ H]1. IR (CHCl3): ν 1600, 1655
cm-1. Anal. (C33H38N2O2) C, H, N.
Synthesis of Compounds 2y and 2z by Ullman-Type Reaction.
General Procedure. To a solution of imidazole or pyrrole (1.5
mmol) in dry DMSO (0.2 mL), Cu(OAc)2 (182 mg, 1 mmol), 2a
(518 mg, 1 mmol), and DBU (300 μL, 2 mmol) were added under
N2. The reaction mixture was irradiated with microwaves at
130 ꢀC for 10 min and then diluted with MeOH/AcOEt and
filtered through a plug of Celite. The filtrate was washed with
water, brine, dried over anhydrous Na2SO4, and evaporated to
dryness. The solid residue was purified by flash chromatography
using CH2Cl2/MeOH (98:2) as eluent.
Example. N-(Adamantan-1-yl)-6-(1H-imidazol-1-yl)-4-oxo-1-
pentyl-1,4-dihydroquinoline-3-carboxamide (2z). Yield: 47%;
light-brown solid; mp 282-283 ꢀC. 1H NMR (200 MHz,
CDCl3): δ 9.76, (s, 1H), 8.74 (s, 1H), 8.52 (s, 2H), 7.75-7.70
(m, 2H), 7.64-7.60 (m, 2H), 4.23 (t, J = 7.2 Hz, 2H), 2.14 (s,
6H), 2.09 (s, 3H), 1.99-1.69 (m, 2H), 1.65-1.63 (m, 6H),
1.37-1.33 (m, 4H), 0.92-0.85 (m, 3H). MS (ESI): m/z: 459 [M
þ H]þ, 481 [MþNa]þ. IR (CHCl3): ν 1601, 1654 cm-1. Anal.
(C28H34N4O2) C, H, N.
Synthesis of Compounds 2aa-2ae by Hydrogenation Reaction.
General Procedure. To a previously degassed solution of the
appropriate starting material (1 mmol) in EtOH (5-10 mL) was
added 10% Pd/C (0.1 mmol). The reaction mixture was stirred
under H2 (1 atm) at room temperature for 4 h and then was
filtered through a plug of Celite and evaporated to dryness.
Example. N-(Adamantan-1-yl)-6-ethyl-4-oxo-1-pentyl-1,4-di-
hydroquinoline-3-carboxamide (2ad). Yield: 100%; colorless oil.
1H NMR (200 MHz, CDCl3): δ 10.01 (s, 1H), 8.70 (s, 1H), 8.32
(d, J = 1.7 Hz, 1H), 7.54 (dd, J1 = 1.7 Hz, J2 = 7.2 Hz, 1H), 7.41
(d, J = 7.2 Hz, 1H), 4.18 (t, J = 7.4 Hz, 2H), 2.77 (d, J = 7.6 Hz,
2H)q, 2.15 (s, 6H), 2.08 (s, 3H), 1.89-1.82 (m, 2H), 1.76-1.70
(m, 6H), 1.35-1.31 (m, 7H), 0.88 (t, J = 6.9 Hz, 3H). MS (ESI):
m/z: 421 [M þ H]þ. IR (CHCl3): ν 1601, 1656 cm-1. Anal.
(C27H36N2O2) C, H, N.
In Vitro Functional Assays. CHO Cells. Chinese hamster
ovary (CHO) cells transfected with human cannabinoid CB2
receptors39 were maintained in Dulbecco’s modified Eagles’s
medium (DMEM) nutrient mixture F-12 HAM, supplemented
with 1 mM L-glutamine, 10% fetal bovine serum, 0.6% peni-
cillin-streptomycin, and G418 (400 μg/mL). Cells were main-
tained at 37 ꢀC and 5% CO2 in their media and passaged twice a
week using nonenzymatic cell dissociation solution.
Membrane Preparation. Binding assays with [35S]GTPγS
were performed with membranes obtained from CHO-hCB2
cells39 or mouse spleens.40 The hCB2 transfected cells were
removed from flasks by scraping and then frozen as a pellet at
-20 ꢀC until required. Spleen tissue was obtained from adult
male C57BL/6J mice weighing 25-40 g and maintained on a 12/
12 h light/dark cycle with free access to food and water. Spleens
were cut into several pieces and placed in a Choi lysis buffer
(Tris-HCl 20 mM, sucrose 0.32 M, EDTA 0.2 mM, EGTA
0.5 mM, pH 7.5) containing Roche protease inhibitor cocktail
(1:40 v/v; Roche Diagnostics, Mannheim, Germany) and PMSF
(150 μM) and then homogenized. The homogenate was centri-
fuged at 500g for 2 min and the resultant supernatant was
recentrifuged at 16000g for 20 min. The harvested membranes
were resuspended in TME buffer (50 mM Tris-HCl; EDTA
1.0 mM; MgCl2 3.0 mM; pH 7.4) and stored at -80 ꢀC for no
more than 1 month. Protein assays were performed using a Bio-
Rad Dc kit (Hercules, CA).
Cyclic AMP Assay. Assays were performed using HitHunter
cyclic AMP assay kits according to the vendor’s protocol.
Briefly, CHO cells expressing hCB2 receptors were detached
using cell dissociation buffer, counted, and seeded at 2 ꢀ 104
cells per well in 100 μL of complete medium onto white 96-well
plates. They were then incubated at 37 ꢀC with 5% CO2 for
approximately 24 h before running the experiment. The assays
and the drug dilutions were performed using a 1:1 mixture of
DMEM and Ham’s F12 medium without phenol red (DMEM/
F12 Media), containing 10 μM of rolipram and forskolin.
Before running the assay, the medium was discarded and cells
were washed once with DMEM/F12 Media. Cells were then
treated with the compounds under investigation (30 μL per well)
and incubated for 30 min at 37 ꢀC with 5% CO2. Finally, a cyclic
AMP standard curve was constructed, after which the appro-
priate mixture of kit components was added, as described by the
manufacturer (DiscoveRx). Plates were incubated overnight at
room temperature in the dark. Chemiluminescent signals were
Binding Assays. CB1 and CB2 receptor binding assays were
performed exactly as described previously,18 using membranes
of cells overexpressing the human recombinant CB1 or CB2
receptors.
Formalin Test. The experimental procedures applied in the
formalin test were approved by the Animal Ethics Committee of
the Second University of Naples. Animal care was in compliance
with the IASP and European Community guidelines on the use
and protection of animals in experimental research (EC L358/1
18/12/86). All efforts were made to minimize animal suffering
and to reduce the number of animals used. Formalin injection
induces a biphasic stereotypical nocifensive behavior.33 Noci-
ceptive responses are divided into an early, short-lasting first
phase (0-7 min) caused by a primary afferent discharge pro-
duced by the stimulus, followed by a quiescent period and then a

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 5927
detected using a Synergy HT Multi-Mode Microplate Reader
(BioTek, Winooski, VT).
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and in vivo pharmacological characterization of JTE-907, a novel
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Jackson, J. V.; Fan, X.; Kung, T. T.; Gonsiorek, W.; Schwarz, M. A.;
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J. A. Cannabinoid receptor type 2 activation induces a microglial
anti-inflammatory phenotype and reduces migration via MKP
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Tamburo, S.; Giordano, C.; Gatta, L.; Scafuro, M. A.; Rossi, F. S.;
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1-(20,40-Dichlorophenyl)-6-methyl-N-cyclohexylamine-1,4-
[35S]GTPγS Binding Assay. This assay was performed as
described previously.40 It was carried out with GTPγS binding
buffer (50 mM Tris-HCl, 100 mM NaCl, 0.1% BSA) in the
presence of [35S]GTPγS and GDP, in a final volume of 500 μL.
The GTPγS binding buffer also contained 50 mM Tris-Base,
5 mM MgCl2, 1 mM dithiothreitol and 1 mM EDTA (CHO cell
membrane experiments) or 3 mM MgCl2 and 0.2 mM EGTA
(spleen membrane experiments). Binding was initiated by the
addition of [35S]GTPγS to the wells. Nonspecific binding was
measured in the presence of 30 μM GTPγS. Compound(s) under
investigation were incubated in the assay for 60 min at 30 ꢀC.
The reaction was terminated by a rapid vacuum filtration
method using Tris-binding buffer, and the radioactivity was
quantified by liquid scintillation spectrometry. In all the [35S]-
GTPγS-binding assays we used 0.1 nM [35S]GTPγS, 30 μM
GDP and 40 μg (spleen membranes) or 50 μg (cell membranes)
protein per well. Additionally, mouse spleen membranes were
preincubated for 30 min at 30 ꢀC with 0.5 U/mL adenosine
deaminase (200 U/mL) to remove any endogenous adenosine.
Materials. Rolipram was supplied by Sigma-Aldrich (Poole,
Dorset, UK) and forskolin and CP-55,940 {(-)-3-[2-hydroxy-
4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-
1-ol} by Tocris (Bristol, UK). The HitHunter cyclic AMP assay
kit was purchased from DiscoveRx (Fremont, CA).
Acknowledgment. The authors from the University of
Siena gratefully acknowledge financial support from Siena
Biotech SpA. The authors from CNR, Pozzuoli, are very
dihydroindeno[1,2-c]pyrazole-3-carboxamide, a novel CB2 agonist,
alleviates neuropathic pain through functional microglial changes in
mice. Neurobiol. Dis. 2010, 37, 177–185. (d) Racz, I.; Nadal, X.;
ꢀ
grateful to Marco Allara for technical assistance.
~
Alferink, J.; Banos, J. E.; Rehnelt, J.; Martín, M.; Pintado, B.; Gutierrez-
Supporting Information Available: Additional synthetic, spec-
tral, and analytical data. This material is available free of charge
via the Internet at http://pubs.acs.org.
Adan, A.; Sanguino, E.; Manzanares, J.; Zimmer, A.; Maldonado, R.
Crucial role of CB(2) cannabinoid receptor in the regulation of central
immune responses during neuropathic pain. J. Neurosci. 2008, 28,
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(17) Miller, A. M.; Stella, N. CB2 receptor-mediated migration of
immune cells: it can go either way. Br. J. Pharmacol. 2008, 153,
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