Investigations on the 4-quinolone-3-carboxylic acid motif. 2. Synthesis and structure-activity relationship of potent and selective cannabinoid-2 receptor agonists endowed with analgesic activity in vivo

Article abstract of DOI:10.1021/jm800552f

Quinolone-3-carboxamides 11 bearing at position 5, 6, 7, or 8 diverse substituents such as halides, alkyl, aryl, alkoxy, and aryloxy groups differing in their steric/electronic properties, were prepared. The new compounds were tested in vitro for CB1 and

Full text of DOI:10.1021/jm800552f

J. Med. Chem. 2008, 51, 5075–5084
5075
Investigations on the 4-Quinolone-3-carboxylic Acid Motif. 2. Synthesis and Structure-Activity
Relationship of Potent and Selective Cannabinoid-2 Receptor Agonists Endowed with Analgesic
Activity in Vivo
Serena Pasquini,† Lorenzo Botta,† Teresa Semeraro,† Claudia Mugnaini,† Alessia Ligresti,‡ Enza Palazzo,§ Sabatino Maione,§
Vincenzo Di Marzo,‡ and Federico Corelli*,†
Dipartimento Farmaco Chimico Tecnologico, UniVersita` 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, and
Department of Experimental Medicine, Section of Pharmacology “L. Donatelli”, Second UniVersity of Naples, Via S. Maria di Costantinopoli
16, 80138 Naples, Italy
ReceiVed May 13, 2008
Quinolone-3-carboxamides 11 bearing at position 5, 6, 7, or 8 diverse substituents such as halides,
alkyl, aryl, alkoxy, and aryloxy groups differing in their steric/electronic properties, were prepared.
The new compounds were tested in vitro for CB1 and CB2 receptor affinity in comparison with the
reference compounds rimonabant and SR144528. The tested compounds exhibited CB2 affinity in the
range from 55.9 to 0.8 nM and CB1 affinity in the range from >10 000 to 5.3 nM, with selectivity
indeces [Ki(CB1)/Ki(CB2)] varying from >2666.6 to 1.23. On the basis of the structure-selectivity
relationship developed, the presence of a substituent at C6/C8 or C7 well accounts for the high or low
CB2 selectivity, respectively. Compound 11c, characterized by high CB2 affinity and selectivity, showed
analgesic activity in the formalin test of acute peripheral and inflammatory pain in mice as a result of
selective CB2 agonistic activity.
Introduction
exclusively expressed by microglia, blockade of its activation
may be attained by CB2 agonists with no overt psychoac-
tivity. All these findings may set the basis for the use of
CB2 agonists as a therapeutic approach for the treatment of
multiple sclerosis and the prevention of neurodegenerative
disorders, such as Alzheimer’s disease. CB2 activation can
also produce analgesic effects devoid of psychotropic activity,
and importantly, the CB2-specific ligand 3 (GW842166X)16
is under clinical development for the treatment of inflam-
matory pain. Other representatives of CB2 selective agonists
are, for example, compounds 4 (HU308), 5 (AM1241), 6
(GW405833), and 7 (JWH-015), while the pyrazole derivative
8 (SR144528) is a CB2 selective antagonist/inverse agonist.2
Very recently, 1,8-naphthyridine-4(1H)-on-3-carboxamide
derivatives,17 such as 9, and 4-oxo-1,4-dihydroquinoline-3-
carboxamide derivatives18 (for example, 10) have been described
as structurally diverse ligands endowed with high affinity and
selectivity toward CB2 (Chart 2). Our interest in the quinolone
chemistry19 led us to choose compound 10 as the prototype for
the development of a new family of CB2 selective agonists.
This compound is characterized by a 1-adamantyl substituent
on the carboxamide nitrogen and a n-pentyl substituent at the
N-1 position. It was demonstrated that whatever the carboxamide
substituent, the n-pentyl group is the preferred N-1 side chain.18
However, the effects on CB2 affinity and/or selectivity of
modifications on the benzene portion of the quinolone ring have
not been explored in great detail so far, as only few examples
of such modified quinolone carboxamides have been recently
reported.17,20 In light of these considerations and following our
previous findings on CB1/CB2 ligands,21 we decided to
investigate the effect on cannabinoid receptor activity/selectivity
of decoration of the quinolone nucleus with various substituents,
such as halides, alkyl, aryl, alkoxy, and aryloxy groups differing
in their respective steric/electronic properties as well as their
position on the condensed benzene ring. The N-1 pentyl chain
The progress achieved over the past 15 years in under-
standing the action mechanisms of cannabinoids has revived
the therapeutic interest in these substances.1 On the basis of
several controlled clinical trials, it is possible to affirm that
cannabinoids possess interesting therapeutic potential in a
number of pathological conditions, such as pain, immuno-
suppression, peripheral vascular disease, appetite enhance-
ment or suppression, and locomotor disorders.2 Currently,
cannabinoid type 1 receptor (CB1) antagonists/inverse ago-
nists are evaluated for obesity, metabolic disorders, smoking
cessation, and alcohol abuse.3,4 Rimonabant (1) (Chart 1) is
a potent and selective antagonist for the CB1 that was
launched in Europe in 2006 by Sanofi-Aventis for the
treatment of obesity and associated risk factors.5,6 On the
other hand, selective cannabinoid type 2 receptor (CB2)
ligands may be used to treat pain,7 inflammation,8 osteoporo-
sis,9 CB2-expressing malignant gliomas,10 tumors of immune
origin,11 and immunological disorders such as multiple
sclerosis.12 Among the different therapeutic strategies de-
signed to interfere with the deleterious processes involved
in the pathology of multiple sclerosis, the use of cannabinoid
compounds is currently one of the most promising ones.13,14
Growing literature describes a beneficial role of cannabinoids,
and more specifically CB2 selective agonists such as 2 (JWH-
133),2 in Alzheimer’s disease, the most common form of
dementia, by blocking ꢀ-amyloid peptide-induced activation
of microglial cells.15 Since CB2 appears to be almost
†
Universita` degli Studi di Siena.
Consiglio Nazionale delle Ricerche.
Second University of Naples.
‡
§
Dedicated with respect and gratitude to Professor Marino Artico on
the occasion of his retirement.
* To whom correspondence should be addressed. Phone: +39 0577
234308. Fax: +39 0577 234333. E-mail: corelli@unisi.it.
10.1021/jm800552f CCC: $40.75
2008 American Chemical Society
Published on Web 08/05/2008

5076 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Chart 1. CB1 and CB2 Receptors Selective Ligands
Pasquini et al.
Chart 2. CB2 Selective Quinolones
some chemical diversity, a parallel synthesis approach was
performed using a carousel reaction station. Six commercially
available or easily accessible anilines23 were condensed with
diethyl ethoxymethylenemalonate (EMME)a according to the
Gould-Jacobs reaction.24 The intermediate enaminoesters were
directly cyclized in refluxing diphenyl ether, and the quinolone
esters were hydrolyzed by adding 10% aqueous NaOH to the
reaction mixture and by heating at reflux temperature for 4 h.
Acids were precipitated by adding HCl to the reaction mixture
and collected by filtration. In this way it was possible to prepare
six 4-oxo-1,4-dihydroquinoline-3-carboxylic acids (12a-f) in
a single step in good overall yield (23-40%) (Scheme 1, Table
1).
Acids 12a-f were subjected to coupling reaction with
1-aminoadamantane using 1-hydroxybenzotriazole (HOBt) and
O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluoro-
phosphate (HBTU) as coupling reagents in the presence of
diisopropylethylamine (DIPEA) in DMF to give the correspond-
ing amides, which in turn were directly reacted with the
appropriate alkyl halide and potassium carbonate in the same
solvent. The final compounds 11b-h were obtained in a
satisfactory overall yield of 32-50%, whereas 11a was obtained
in low yield (7%), probably because of some steric hindrance
toward the approaching electrophile exerted by the methyl
substituent at position 8.
and adamantyl group were either retained or replaced with other
lipophilic groups, such as prop-1-en-3-yl, but-1-en-4-yl, pent-
1-en-5-yl, or (R)-1-phenylethyl, to obtain the new quinolon-
ecarboxamides of general structure 11.22
Other final compounds 11i-m were prepared by a linear,
step-by-step synthesis (Scheme 2). Thus, esters 13a25 and 13b26
a Abbreviations: EMME, diethyl ethoxymethylenemalonate; HOBt, 1-hy-
droxybenzotriazole; HBTU, O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluro-
nium hexafluorophosphate; DIPEA, diisopropylethylamine; SI, selectivity
index.
Chemistry
The synthesis of the target compounds 11a-v is highlighted
in Schemes 1-4. Initially, in order to achieve in a fast way

4-Quinolone-3-carboxylic Acid Motif
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 5077
Scheme 1a
a For R1-R5, see Table 1. Reagents and conditions: (i) diethyl ethoxymethylenemalonate, 120 °C, 4 h; (ii) diphenyl ether, reflux, 16 h; (iii) 10% NaOH,
reflux, 4 h, then HCl; (iv) 1-aminoadamantane, HOBt, HBTU, DIPEA, DMF, room temp, 20 h; (v) alkyl halide, KI, K2CO3, DMF, 90 °C, 20 h.
Scheme 2a
a For R1-R5, see Table 1. Reagents and conditions: (i) see (v) in Scheme 1; (ii) 10% NaOH, reflux, 2 h, then HCl; (iii) see (iv) in Scheme 1; (iv) ethyl
3-dimethylaminoacrylate, Et3N, toluene, 100 °C, 4 h; (v) 1-pentylamine, K2CO3, DMF, 100 °C, 2 h; (vi) 6 N HCl, reflux, 7 h.
were first N-alkylated to 14a-c, which were then hydrolyzed
to the corresponding acids 15a-c. Finally, coupling reaction
of 15a-c with 1-aminoadamantane under the usual conditions
provided amides 11i-k.
Taking advantage of the ease of displacement of their
fluorine substituent, amides 11l,m were reacted with different
nucleophiles to enhance the chemical diversity within this
family of 4-oxoquinoline-3-carboxamides. Accordingly, when
treated with pyrrolidine or sodium ethoxide, 11m afforded
11n and 11o in 38% and 61% yield, respectively (Scheme
3). Similarly, amide 11l yielded the alkoxy derivatives 11p
(60%) and 11q (50%) by reaction with sodium methoxide
or sodium ethoxide, respectively. Displacement of fluorine
by thiophenol converted 11l into 11r, which in turn was
oxidized using Oxone in 1,4-dioxane/water to give an
approximately 1:1 mixture of the corresponding sulfone 11s
and sulfoxide 11t, which were separated by column chro-
matography on silica gel.
7-Fluoro-4-oxo-1-(1-pentyl)-1,4-dihydroquinoline-3-carboxy-
lic acid (15d) was synthesized in a different way, according to
a modified Grohe-Heitzer procedure.27 Treatment of 2,4-
difluorobenzoyl chloride with 3-dimethylaminopropenoic acid
ethyl ester led to the enamino ketone 16 that was subsequently
reacted with n-pentylamine to yield the quinolone ester 17.
Because of the high reactivity toward nucleophiles exhibited
by the fluorine atom at position 7 (para to the carbonyl group),
the ester 17 could not be subjected to hydrolysis under basic
conditions but was conveniently hydrolyzed using 6 N HCl.
The product 15d so obtained was transformed into amides 11l,m
by coupling with 1-aminoadamantane and (R)-1-phenylethy-
lamine.
Finally, the bromoamide 11g was further modified through
Suzuki coupling using phenylboronic acid and trans-(4-
methoxyphenyl)vinylboronic acid under microwave irradia-

5078 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Pasquini et al.
Scheme 3a
a Reagents and conditions: (i) pyrrolidine or thiophenol, K2CO3, DMF, 100 °C, 6 h; (ii) MeONa or EtONa, THF, 50 °C, 16 h; (iii) Oxone, 1,4-dioxane,
H2O, room temp, 24 h.
tion.28 Compounds 11u and 11v were obtained in reasonable
yield (30 and 45%, respectively) (Scheme 4).
min) of tonic pain. Fifteen minutes before injection of formalin,
mice received intraperitoneal (ip) administration of vehicle or
11c (1 or 3 mg/kg), alone or in combination with the selective
CB2 antagonist, 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-
indol-3-yl](4-methoxyphenyl)methanone (AM630) (3 mg/kg,
ip), administered 5 min before the compound. The results are
presented in Figure 1.
Biology
The binding affinities (Ki values) of compounds 11 for human
recombinant CB1 and CB2 receptors are reported in Table 2.
Tested compounds were evaluated in parallel with 8 (Chart 1)
and 1 as CB2 and CB1 reference compounds, respectively, as
previously described.29
Results and Discussion
The functional activity of compound 11c 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
Out of the 22 synthesized quinolone carboxamides 11,
compounds 11p and 11q elicited solubility issues that prevented
their biological testing as DMSO/water solution in the cannab-
inoid receptor binding assay, while amides 11a-o,r-v proved
to be high affinity CB2 ligands, with Ki values ranging from
55.9 nM (11v) to 0.8 nM (11l) (Table 2). Under the conditions
of our assay, compound 10, taken as a representative of

4-Quinolone-3-carboxylic Acid Motif
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 5079
Scheme 4a
compound 10 (SI ) 9.0), all belong to the group of the less
selective ligands. The effect on selectivity of C5 or C8
substitution cannot be yet defined, since the C5 or C8 substituted
compounds 11a, 11i, and 11j also present substituents at other
positions on the benzene ring. On the basis of these observations,
the possibility for a substituent at the C6/C8 positions on the
quinolone ring of 11, regardless of its electronic properties, to
occupy defined regions of the space inside CB2 receptor well
accounts for the ability of the given ligand to selectively bind
to this receptor subtype. That does not mean that substitution
at C7 position is detrimental for CB2 affinity, since some of
the 7-substituted quinolone carboxamides, such as 11l, 11s, 11r,
and 11t, are among those with the highest affinity at CB2
receptor. This finding is in line with those reported by Stern et
al.,18 who demonstrated that the introduction of a chlorine atom
at position 7 of the quinoline template has a favorable effect
on CB2 affinity. On the other hand, moving the substituent from
C6 to C7 markedly enhances also CB1 affinity, thus giving rise
to compounds endowed with lower selectivity (compare, for
instance, 11h and 11r). Such a result is difficult to be compared
with Manera’s findings, regarding the increased or decreased
CB2 selectivity for their 7-chloro or 7-methyl substituted
quinolones, respectively. However, it is considered that com-
pounds of general structure 11 here described differ from those
reported by Manera et al.17 (such as 9), being characterized by
different substituents at positions 1 and 3 of the bicyclic nucleus.
When tested for functional activity, compound 11c, one of
the most potent and CB2-selective ligands in binding assays,
dose-dependently inhibited the second phase of the formalin-
induced nocifensive response in mice (Figure 1A). The effect
was maximal with the 3 mg/kg dose, the action of which was
significantly attenuated by the CB2 antagonist AM630 (1 mg/
kg) (Figure 1B). These findings suggest that 11c is able to exert
analgesic activity in vivo by acting as a CB2 agonist.
a Reagents and conditions: (i) phenylboronic acid or trans-2-(4-meth-
oxyphenyl)vinylboronic acid, Pd(OAc)2, Ph3P, Na2CO3, DME, EtOH,
microwave, 150 °C, 10 min.
quinolone CB2 agonist, showed Ki ) 4.7 nM instead of the
reported value of 16.4 nM,18 while the CB2-selective reference
ligand 8 showed Ki ) 5.4 nM. With regard to the CB1 affinity,
Ki values spanned 4 orders of magnitude in the range from
>10000 nM (11a,j,u) to 5.3 nM (11r), with 1 (used as the CB1-
selective reference ligand) displaying Ki ) 12.0 nM. As a
consequence 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 compounds 11a-o,r-v also
varied widely, from >2666.6 to 1.2. In no case was the SI value
lower than 1; that is, no compound exhibited a reversed
selectivity (higher affinity for CB1 than for CB2).
The nature of the 3-carboxamido substituent seems to affect
both receptor affinity and selectivity, since the highly lipophilic
adamantylamido substituent confers higher affinity and selectiv-
ity for the CB2 receptor with respect to its 1-phenylethylamido
counterpart (compare, for instance, 11l and 11m) and the two
completely unselective compounds 11m and 11o are both
1-phenylethylamido derivatives. Coming to the effects of the
N1 alkyl chain, affinity increased in the order 1-butenyl <
1-pentyl < 1-pentenyl, while no substantial effect on selectivity
was noticed (compare 11d, 11g, and 11f). While this result could
be expected on the basis of the findings of Stern et al.,18 no
definitive explanation for the better affinity and selectivity profile
of the N-allyl derivative 11j, with respect to the corresponding
N-pentyl analogue 11i, can yet be given.
To best discuss the effects of substitution on the condensed
benzene ring, the issues of CB2 affinity and selectivity should
be distinguished. Thus, several substituents with different steric
and electronic properties are compatible with high CB2 affinity,
since seven compounds (11l, 11s, 11r, 11h, 11k, 11t, and 11u)
displayed a Ki value lower than that of the unsubstituted
compound 10, and even the less active compound 11v was still
a nanomolar affinity ligand with a Ki value of 55.9 nM. On the
other hand, a preliminary structure-selectivity relationship
emerged, with CB2 selectivity depending on the substitution
pattern on the aromatic ring. In fact, when the quinolone
carboxamides are ranked in order of decreasing SI value, it can
be seen that the 12 more selective derivatives 11u, 11j, 11a,
11b, 11c, 11h, 11d, 11f, 11g, 11v, 11e, and 11i (2666.6 g SI
g 39.9) are all substituted at least at C6 or C8, followed by
11k (SI ) 38.8) bearing substituents at both C6 and C7, whereas
the compounds 11l, 11n, 11t, 11s, 11r, 11m, and 11o (38.1 g
SI g 1.2) substituted only at C7, along with the unsubstituted
Conclusions
The results of our preliminary screening program on substi-
tuted quinolone-3-carboxamides led to the identification of
cannabinoid receptor ligands characterized by high affinity and
selectivity. In in vitro assays, some of the new compounds
exhibited affinity and selectivity values for the CB2 subtype
that are even higher than those of the CB2-specific ligand 8,
used as the reference compound. Such a biochemical profile is
strongly dependent upon the presence of a substituent at the
C6 position of the quinolone ring, while the nature of the
substituent itself is far less crucial. The activity shown by
derivative 11c in the formalin-induced nocifensive response in
mice suggests that quinolone-3-carboxamides possess agonist
properties at the CB2 receptor and might be considered as
potential analgesic agents. On the other hand, ligands with high
CB1 affinity, though substantially devoid of receptor selectivity,
were identified as well, possessing a C7-substituent on the
quinolone nucleus. Also in this case, some of the compounds
showed CB1 affinity superior to that of 1, used in our tests as
the CB1 reference standard.
The structure-affinity/selectivity relationships emerging from
our study suggest that the quinolone carboxamide scaffold can
be optimized through selected chemical modifications aimed at
designing either CB1 or CB2 selective ligands.
Experimental Section
Chemistry. Reagents were purchased from commercial suppliers
and used without further purification. Anhydrous reactions were
run under (a positive pressure) dry N2. Merck silica gel 60 was

5080 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Table 1. Structure of the New Compounds 11-15
Pasquini et al.
compd
R1
R2
R3
R4
R5
R6
11a
11b
11c
11d
11e
11f
11g
11h
11i
H
H
H
H
H
H
H
H
MeO
MeO
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
MeO
H
MeO
MeO
H
MeO
MeO
H
H
Cl
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
MeO
MeO
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
1-pentyl
1-pentyl
1-pentyl
but-1-en-4-yl
1-pentyl
pent-1-en-5-yl
1-pentyl
1-pentyl
1-pentyl
allyl
1-pentyl
1-pentyl
1-pentyl
1-pentyl
1-pentyl
1-pentyl
1-pentyl
1-pentyl
1-pentyl
1-pentyl
1-pentyl
1-pentyl
H
1-adamantylamino
1-adamantylamino
1-adamantylamino
1-adamantylamino
1-adamantylamino
1-adamantylamino
1-adamantylamino
1-adamantylamino
1-adamantylamino
1-adamantylamino
1-adamantylamino
1-adamantylamino
(R)-1-phenylethylamino
(R)-1-phenylethylamino
(R)-1-phenylethylamino
1-adamantylamino
1-adamantylamino
1-adamantylamino
1-adamantylamino
1-adamantylamino
1-adamantylamino
1-adamantylamino
OH
OH
OH
OH
OH
OH
OEt
OEt
OEt
OEt
OEt
OH
OH
OH
OH
CF3
i-Pr
Br
3-Cl-4-F-PhO
Br
Br
PhS
H
H
F
H
H
H
H
H
H
H
11j
11k
11l
pyrrol-1-yl
F
F
pyrrolidin-1-yl
EtO
MeO
EtO
PhS
PhSO2
PhSO
H
H
Cl
H
H
11m
11n
11o
11p
11q
11r
11s
11t
11u
11v
12a
12b
12c
12d
12e
12f
13a
13b
14a
14b
14c
15a
15b
15c
15d
H
H
Ph
(E)-4-MeO-styryl
H
CF3
i-Pr
H
H
H
H
H
H
H
Br
H
H
H
H
3-Cl-4-F-PhO
H
H
PhS
H
F
H
H
F
H
H
F
MeO
H
MeO
MeO
H
MeO
MeO
H
pyrrol-1-yl
H
H
pyrrol-1-yl
H
H
1-pentyl
allyl
1-pentyl
1-pentyl
allyl
pyrrol-1-yl
F
1-pentyl
1-pentyl
H
H
H
used for flash chromatography (23-400 mesh). 1H NMR and 13
C
Example. 4-Oxo-6-(trifluoromethyl)-1,4-dihydroquinoline-3-car-
boxylic Acid (12b). 12b was prepared by parallel synthesis from
4-(trifluoromethyl)aniline in 25% yield. White solid; mp >280 °C
NMR were recorded at 200 and 50 MHz, respectively, on a Brucker
AC200F spectrometer and at 400 and 100 MHz on a Brucker
Advance DPX400. Chemical shifts are reported relative to tetram-
ethylsilane at 0.00 ppm. Mass spectral (MS) data were obtained
using an 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 mode or in negative mode scanning over
the mass range of 105-1500. Melting points were determined on
a Gallenkamp apparatus and are uncorrected. Elemental 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. Microwave irradiations were conducted using a CEM
Discover Synthesis Unit (CEM Corp., Matthews, NC).
1
(dec). H NMR (200 MHz, DMSO-d6): δ 17.09 (s, 1H), 8.83 (s,
1H), 8.44 (s, 1H), 7.81-7.75 (m, 2H). MS (ESI): m/z 258 [M +
H]+, 280 [M + Na]+. IR (nujol): ν 3406, 1643, 1619 cm-1. Anal.
(C11H6F3NO3) C, H, N.
Parallel Synthesis of Amides 11a-h. Acids 12a-f (1 mmol) and
DMF (3 mL) were placed in different vessels of a Syncore reactor
under a nitrogen atmosphere. HOBt (1 mmol), HBTU (2 mmol),
DIPEA (1.5 mmol), and 1-aminoadamantane (1.2 mmol) were
added to the solutions, and the reaction mixtures were stirred at
room temperature for 30 min. Further DIPEA (1.5 mmol) was
thereafter added, and the reaction mixtures were stirred at room
temperature for 20 h. K2CO3 (5 mmol), the appropriate alkyl halide
(5 mmol), and KI (5 mmol) were added to every mixture, which
was heated at 90 °C for 20 h. Each reaction mixture was poured
into ice and extracted with AcOEt. The organic layers were washed
with brine, dried over anhydrous Na2SO4, and evaporated to dryness.
The crude residues were purified by chromatography using light
petroleum ether/AcOEt (5:1 to 1:1) to afford the pure amides.
Example. N-(Adamant-1-yl)-7-chloro-8-methyl-4-oxo-1-pentyl-
1,4-dihydroquinoline-3-carboxamide (11a). 11a was prepared by
parallel synthesis from 12a in 7% yield. Light-yellow oil. 1H NMR
(200 MHz, CDCl3): δ 9.29 (s, 1H), 8.51 (s, 1H), 7.88 (d, J ) 9.0
Hz, 1H), 7.51 (d, J ) 9.0 Hz, 1H), 4.12 (t, J ) 6.8 Hz, 2H), 2.8 (s,
3H), 2.15-1.96 (m, 8H), 1.92-1.89 (m, 2H), 1.73-1.63 (m, 7H),
Parallel Synthesis of Acid Derivatives 12a-f. Six anilines (1
mmol) were added into six round-bottom flasks of a carousel
reactor, and diethyl ethoxymethylemalonate (1 mmol) was added.
The reaction mixtures were heated at 120 °C for 4 h and cooled to
room temperature. Ph2O (1 mL) was added to each vessel, and the
reaction mixtures were heated at reflux temperature for 16 h. After
the mixture was cooled to room temperature, 10% aqueous NaOH
was added to each flask and the reaction mixtures were refluxed
for 4 h. After the mixture was cooled, concentrated HCl was added
to the reaction mixtures, allowing the precipitation of the five acid
derivatives, which were collected by filtration, washed with water,
then petroleum ether, and recrystallized from DMF.

4-Quinolone-3-carboxylic Acid Motif
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 5081
Table 2. CB1 and CB2 Receptor Affinity Values for Compounds
11a-va
18 h and then poured into ice-water. The resulting N-alkylqui-
nolone was collected by filtration and recrystallized from DMF or
extracted in CH2Cl2 and purified by flash chromatography on silica
gel eluting with CH2Cl2/MeOH (98:2).
Example. Ethyl 5,8-Dimethoxy-4-oxo-1-pentyl-1,4-dihydroquin-
oline-3-carboxylate (14a). 14a was prepared in 41% yield from 13a.
White solid, mp 121-122 °C. 1H NMR (200 MHz, CDCl3): δ 8.17
(s, 1H), 7.05 (d, J ) 8.9 Hz, 1H), 6.76 (d, J ) 8.9 Hz, 1H),
4.38-4.28 (m, 4H), 3.87 (s, 3H), 3.85 (s, 3H), 1.77-1.63 (m, 2H),
1.35 (t, J ) 7.0 Hz, 3H), 1.28-1.20 (m, 4H), 0.86 (t, J ) 6.2 Hz,
3H). MS (ESI): m/z 348 [M + H]+, 370 [M + Na]+. IR (nujol):
ν 1656, 1615 cm-1. Anal. (C19H25NO5) C, H, N.
Synthesis of 7-Substituted-4-oxo-1,4-dihydroquinoline-3-car-
boxamides 11n and 11r. General Procedure. K2CO3 (3 mmol) and
pyrrolidine (7.6 mmol) or thiophenol (3 mmol) were added to a
solution of 11m or 11l (1 mmol), respectively, in DMF (8 mL)
under a nitrogen atmosphere. The reaction mixture was heated at
100 °C for 6 h, cooled to room temperature, and poured into water.
The solid precipitate was collected by filtration, washed with light
petroleum ether, and recrystallized from EtOH.
Example. N-(Adamant-1-yl)-4-oxo-1-pentyl-7-(phenylthio)-1,4-
dihydroquinoline-3-carboxamide (11r). 11r was prepared from 11l
and thiophenol in 58% yield. White solid; mp 164.5 °C. 1H NMR
(200 MHz, CDCl3): δ 9.86 (s, 1H), 8.60 (s, 1H), 8.33 (d, J ) 8.6
Hz, 1H), 7.57-7.52 (m, 2H), 7.50-7.41 (m, 3H), 7.24-7.23 (m,
1H), 7.19 (s, 1H), 3.92 (t, J ) 7.7 Hz, 2H), 2.14-2.08 (m, 8H),
1.76-1.57 (m, 9H), 1.32-1.10 (m, 4H), 0.86 (t, J ) 6.8 Hz, 3H).
MS (ESI): m/z 501 [M + H]+, 523 [M + Na]+. IR (nujol): ν 1659,
1602 cm-1. Anal. (C31H36N2O2S) C, H, N.
Synthesis of 7-Substituted-4-oxo-1,4-dihydroquinoline-3-car-
boxamides 11o-q. General Procedure. A solution of 11l or 11m
(0.14 mmol) in dry THF (3 mL) was added to a solution of freshly
prepared MeONa or EtONa (0.28 mmol) under a nitrogen atmo-
sphere. The reaction mixture was heated at 50 °C for 16 h and
neutralized using 1 N HCl. The mixture was concentrated under
reduced pressure and extracted with CH2Cl2. The organic layer was
washed with brine, dried over Na2SO4, and evaporated to dryness.
The crude residue was purified by chromatography using light
petroleum ether:AcOEt (1:1) as eluent.
Example. (R)-7-Ethoxy-4-oxo-1-pentyl-N-(1-phenylethyl)-1,4-di-
hydroquinoline-3-carboxamide (11o). 11o was prepared from 11m
and EtONa in 61% yield. White solid; mp 128.2 °C (from EtOH).
1H NMR (400 MHz CDCl3): δ 10.50 (d, J ) 6.8 Hz, 1H), 8.61 (s,
1H), 8.37 (d, J ) 9.1 Hz, 1H), 7.36-7.14 (m, 5H), 7.0-6.97 (m,
1H), 6.76 (s, 1H), 5.24 (t, J ) 7.0 Hz, 1H), 4.12-4.05 (m, 4H),
1.80-1.73 (m, 2H), 1.53 (d, J ) 6.8 Hz, 3H), 1.42 (t, J ) 6.8 Hz,
3H), 1.31-1.27 (m, 4H), 0.87-0.78 (m, 3H). MS (ESI): m/z 407
[M + H]+, 429 [M + Na]+. IR (nujol): ν 1667, 1603 cm-1. Anal.
(C25H30N2O3) C, H, N.
Synthesis of Compounds 11s and 11t. To a solution of Oxone
(1.1 g, 1.8 mmol) in H2O (2 mL), a solution of 3r (28 mg, 0.06
mmol) in 1,4-dioxane (2 mL) was added. The reaction mixture was
stirred at room temperature for 20 h and extracted with 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 11s (9.5 mg, 33% yield) and 11t (11
mg, 37% yield) as white solids.
N-(Adamant-1-yl)-4-oxo-1-pentyl-7-(phenylsulfonyl)-1,4-dihyd-
roquinoline-3-carboxamide (11s). Mp 215.6 °C. 1H NMR (200
MHz, CDCl3): δ 9.36 (s, 1H), 8.78 (s, 1H), 8.60 (d, J ) 8.7 Hz,
1H), 8.17 (s, 1H), 7.99-7.96 (m, 2H), 7.82 (d, J ) 8.7 Hz, 1H),
7.57-7.54 (m, 3H), 4.26 (t, J ) 7.5 Hz, 2H), 2.14-2.02 (m, 10H),
1.88-1.81 (m, 2H), 1.80-1.70 (m, 5H), 1.37 (m, 4H), 0.92-0.84
(m, 3H). MS (ESI): m/z 555 [M + Na]+. IR (CHCl3): ν 1716, 1659
1598 cm-1. Anal. (C31H36N2O4S) C, H, N.
f
Ki (nM)
compd
CB1b,d
CB2c,d
SIe
11a
11b
11c
11d
11e
11f
11g
11h
11i
>10000
2520
1220
2680
739
696
996
640
2100
>10000
139.8
30.5
25.5
11.6
6.3
23.8
16.1
9.4
14.3
3.4
52.6
9.2
>392.7
216.7
194.3
112.7
46.0
74.4
69.4
189.3
39.9
11j
11k
11l
>1091.7
38.8
38.1
3.6
0.8
11m
11n
11o
11p
11q
11r
11s
11t
11u
11v
10h
8h,i
14.3
160
10.7
17.1
24.2
NTg
NTg
1.6
0.9
3.5
3.8
55.9
4.7
1.3
9.4
1.2
29.7
NTg
NTg
5.3
6.1
28.0
>10000
2630
42.1
>2820
12.0
3.3
6.8
8.0
>2666.6
47.0
9.0
5.4
790
>522.2
0.015
1h,j
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-
hydroxypropyl)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 NT: not tested because of solubility problems. h The
binding affinities of reference compounds were evaluated in parallel with
compounds11 under the same conditions. i SR144528, CB2 reference
compound. j Rimonabant, CB1 reference compound.
1.53-1.35 (m, 4H), 0.96 (t, J ) 6.9 Hz, 3H). MS (ESI): m/z 441
[M + H]+, 464 [M + Na]+. Anal. (C26H33ClN2O2) C, H, N.
Synthesis of 4-Oxo-1,4-dihydroquinoline-3-carboxamides 11i-m
by Amidation Reaction. General Procedure. To a solution of the
appropriate acid derivative 15a-d (1 mmol) in DMF (5 mL), HBTU
(2 mmol) was added followed by HOBt (1 mmol), DIPEA (1.5
mmol), and 1-aminoadamantane or (R)-1-phenylethylamine (1.2
mmol). The mixture was stirred at room temperature under N2 for
30 min, and further DIPEA (1.5 mmol) was added. The reaction
mixture was stirred at room temperature for 1 h, poured into water,
and extracted with CH2Cl2. The organic layer was washed with
brine, dried over anhydrous Na2SO4, and evaporated. The crude
residue was recrystallized from EtOH, with the exception of 11k,
which was triturated with diethyl ether.
Example. N-(Adamant-1-yl)-7-fluoro-4-oxo-1-pentyl-1,4-dihyd-
roquinoline-3-carboxamide (11l). 11l was prepared from 15d and
1-aminoadamantane in 76% yield. Pink solid; mp 197.5-200.5 °C.
1H NMR (400 MHz, CDCl3): δ 9.76 (s, 1H), 8.65 (s, 1H),
8.49-8.42 (m, 1H), 7.15-7.07 (m, 2H), 4.09 (t, J ) 7.5 Hz, 2H),
2.11-2.05 (m, 10H), 1.97-1.78 (m, 2H), 1.74-1.66 (m, 5H),
1.33-1.19 (m, 4H), 0.85 (t, J ) 6.7 Hz, 3H). MS (ESI): m/z 411
[M + H]+, 433 [M + Na]+. IR (CHCl3): 1659, 1605 cm-1. Anal.
(C25H31FN2O2) C, H, N.
Synthesis of 1-Alkyl-4-oxo-1,4-dihydroquinoline Derivatives
14a-c by Alkylation Reaction. General Procedure. To a mixture
of the quinolone derivative 13a,b (1 mmol) and K2CO3 (2.8 mmol)
in dry DMF (2 mL) under a nitrogen atmosphere, the appropriate
alkyl iodide (2.8 mmol) or alkyl bromide along with KI (2.8 mmol
each) was added. The reaction mixture was heated at 90 °C for
N-(Adamant-1-yl)-4-oxo-1-pentyl-7-(phenylsulfinyl)-1,4-dihyd-
roquinoline-3-carboxamide (11t). Mp 102-103 °C. 1H NMR (200
MHz, CDCl3): δ 9.73 (s, 1H), 8.78 (s, 1H), 8.50 (d, J ) 8.1 Hz,
1H), 8.02 (s, 1H), 7.68-7.34 (m, 6H), 4.27 (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

5082 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Pasquini et al.
Figure 1. (A) Analgesic effect of 11c in the formalin test in mice. Each point represents the mean ( standard error of the mean (SEM) of 8-10
animals per group. Asterisks denote values that were significantly different from vehicle (P < 0.05), as assessed using two-way ANOVA followed
by the Bonferroni’s test. (B) Effect of the CB2 antagonist 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-y l](4-methoxyphenyl)methanone
(AM630) on the analgesic effect of 11c. Asterisks denote values that were significantly different from vehicle (P < 0.05), whereas circles denote
values that were significantly different from 11c only, as assessed using two-way ANOVA followed by the Bonferroni’s test.
(m, 4H), 0.92-0.86 (m, 3H). MS (ESI): m/z 517 [M + 1]+, 539
[M + Na]+. IR (CHCl3): ν 1722, 1660, 1598 cm-1. Anal.
(C31H36N2O3S) C, H, N.
7-Fluoro-4-oxo-1-pentyl-1,4-dihydroquinoline-3-carboxylic Acid
(15d). A suspension of 17 (305 mg, 1 mmol) in EtOH (3.5 mL)
and 6 N HCl (3.5 mL) was refluxed for 7 h. After the mixture was
cooled to room temperature, the solid precipitate was filtered and
recrystallized from EtOH to furnish 15d (260 mg, 94%) as a light-
Synthesis of Compounds 11u and 11v by Suzuki Reaction.
General Procedure. To a solution of 11g (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 as eluent.
1
pink solid; mp 148.3 °C. H NMR (200 MHz, CDCl3): δ 14.7 (b,
1H), 8.73 (s, 1H), 8.61-8.53 (dd, J1 ) 6.6 Hz, J2 ) 8.8 Hz, 1H),
7.33-7.21 (m, 2H), 4.23 (t, J ) 7.3 Hz, 2H), 1.95-1.88 (m, 2H),
1.55-1.39 (m, 4H), 0.96-0.89 (m, 3H). MS (ESI): m/z 278 [M +
H]+, 300 [M + Na]+. IR (CHCl3): ν 1722, 1615 cm-1. Anal.
(C15H16FNO3) C, H, N.
Ethyl 2-(2,4-Difluorobenzoyl)-3-(dimethylamino)acrylate (16).
To a solution of 2,4-difluorobenzoyl chloride (2.8 mL, 22.7 mmol)
and Et3N (4.8 mL, 34.1 mmol) in toluene, ethyl (dimethylami-
no)acrylate (3.2 g, 22.7 mmol) was added, and the reaction mixture
was heated at 90 °C for 4 h. The solvent was removed at reduced
pressure, and the crude solid was purified by chromatography using
AcOEt/light petroleum ether (1:1 to 2:1) to afford 16 (4.29 g, 67%
yield) as a yellow solid, which was recrystallized from EtOH. Mp
>280 °C (dec). 1H NMR (200 MHz CDCl3): δ 7.73 (s, 1H),
7.66-7.55 (m, 1H), 6.92-6.82 (m, 1H), 6.79-6.69 (m, 1H), 3.97
(q, J ) 7.1 Hz, 2H), 3.05 (b, 6H), 0.94 (t, J ) 7.1 Hz, 3H). MS
(ESI): m/z 284 [M + H]+, 306 [M + Na]+. IR (CHCl3): ν 1725,
1612, 1234, 1220 cm-1. Anal. (C14H15F2NO3) C, H, N.
Example. N-(Adamant-1-yl)-4-oxo-1-pentyl-6-phenyl-1,4-dihy-
droquinoline-3-carboxamide (11u). 11u was prepared from 11g and
phenylboronic acid in 45% yield. Yellow oil. 1H NMR (200 MHz,
CDCl3): δ 9.97 (s, 1H), 8.76-8.74 (m, 2H), 7.96 (dd, J1 ) 1.8 Hz,
J2 ) 8.8 Hz, 1H), 7.72-7.67 (m, 2H), 7.57 (d, J ) 8.9 Hz, 1H),
7.51-7.32 (m, 3H), 4.23 (t, J ) 7.4 Hz, 2H), 2.18-2.10 (m, 8H),
1.95-1.78 (m, 2H), 1.72-1.62 (m, 7H), 1.40-1.27 (m, 4H), 0.91
(t, J ) 6.8 Hz, 3H). MS (ESI): m/z 469 [M + H]+, 491 [M +
Na]+. IR (CHCl3): ν 3248, 1657, 1601 cm-1. Anal. (C31H36N2O2)
C, H, N.
Synthesis of 1-Alkyl-4-oxo-1,4-dihydroquinoline-3-carboxylic
Acids 15a-c by Basic Hydrolysis. General Procedure. A suspen-
sion of ester 14a-c (1 mmol) in 10% aqueous NaOH (10 mL)
was refluxed for 2 h (until the suspension became a solution). After
cooling at room temperature, the reaction mixture was acidified
using concentrated HCl. The resulting precipitate was filtered and
washed with water to give the corresponding quinolone-3-carboxylic
acid, which was recrystallized from EtOH.
Ethyl 7-Fluoro-4-oxo-1-pentyl-1,4-dihydroquinoline-3-carboxy-
late (17). n-Pentylamine (1.7 mL, 15.2 mmol) was added dropwise
to a solution of 16 (4.29 g, 15.2 mmol) in Et2O (160 mL) and EtOH
(69 mL). The reaction mixture was stirred at room temperature for
25 min, and the solvent was removed under reduced pressure. The
residue was washed with 0.1 N HCl, dried under anhydrous Na2SO4,
and evaporated to dryness. DMF (20 mL) and K2CO3 (3.5 g, 25.6
mmol) were added to the solid residue, and the mixture was heated
at 90 °C for 1 h, then poured into ice-water. The precipitated solid
was collected by filtration and recrystallized from EtOH to give
Example. 5,8-Dimethoxy-4-oxo-1-pentyl-1,4-dihydroquinoline-
3-carboxylic Acid (15a). 15a was prepared from 14a in 86% yield.
White solid; mp 209.7 °C. 1H NMR (200 MHz, DMSO-d6): δ 15.73
(s, 1H), 8.59 (s, 1H), 7.40 (d, J ) 9.0 Hz, 1H), 6.97 (d, J ) 9.0
Hz, 1H), 4.54 (t, J ) 7.5 Hz, 2H), 3.87 (s, 3H), 3.78 (s, 3H),
1.68-1.50 (m, 2H), 1.03 (m, 4H), 0.81 (t, J ) 6.8 Hz, 3H). MS
(ESI): m/z 320 [M + H]+, 342 [M + Na]+. IR (nujol): ν 3378,
1728, 1630 cm-1. Anal. (C17H21NO5) C, H, N.
1
17 (3.88 g, 89% yield) as a yellow solid; mp 104-106.3 °C. H
NMR (200 MHz, CDCl3): δ 8.57-8.49 (m, 1H), 8.43 (s,1H),
7.16-7.04 (m, 2H), 4.39 (q, J ) 7.2 Hz, 2H), 4.08 (t, J ) 7.5 Hz,
2H), 1.90-1.80 (m, 2H), 1.43-1.34 (m, 7H), 0.92 (t, J ) 6.6 Hz,

4-Quinolone-3-carboxylic Acid Motif
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 5083
3H). MS (ESI): m/z 306 [M + H]+, 328 [M + Na]+. IR (CHCl3):
ν 1726, 1689 cm-1. Anal. (C17H20FNO3) C, H, N.
(6) Marketing authorization of European Medicines Agency (EMEA) valid
throughout the European Union: June 19, 2006. European Public
Assessment Report (EPAR). Acomplia. Protocol EMEA/H/C/666.
(7) Malan, T. P., Jr.; Ibrahim, M. M.; Lai, J.; Vanderah, T. W.;
Makriyannis, A.; Porreca, F. CB2 cannabinoid receptor agonists: pain
relief without psychoactive effects. Curr. Opin. Pharmacol. 2003, 3,
62–67.
(8) Iwamura, H.; Suzuki, H.; Ueda, Y.; Kaya, T.; Inaba, T. In vitro and
in vivo pharmacological characterization of JTE-907, a novel selective
ligand for cannabinoid CB2 receptor. J. Pharmacol. Exp. Ther. 2001,
296, 420–425.
(9) Ofek, O.; Karsak, M.; Leclerc, N.; Fogel, M.; Frenkel, B.; Wright,
K.; Tam, J.; Attar-Namdar, M.; Kram, V.; Shohami, E.; Mechoulam,
R.; Zimmer, A.; Bab, I. Peripheral cannabinoid receptor, CB2, regulates
bone mass. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 696–701.
(10) Sanchez, C.; de Ceballos, M. L.; Go´mez del Pulgar, T.; Rueda, D.;
Corbacho, C.; Velasco, G.; Galve-Roperh, I.; Huffman, J. W.; Ramon
y Cajal, S.; Guzman, M. Inhibition of glioma growth in vivo by
selective activation of the CB2 cannabinoid receptor. Cancer Res. 2001,
61, 5784–5789.
CB1 and CB2 Receptors Binding Assays. The new compounds
were evaluated in CB1 and CB2 receptors binding assays using
membranes from HEK cells transfected with either the CB1 or CB2
receptor and [3H]-(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phe-
nyl]-trans-4-(3-hydroxypropyl)cyclohexanol ([3H]CP-55,940; Kd )
0.18 nM for CB1R and Kd ) 0.31 nM for CB2R) as the high affinity
ligand, as described by the manufacturer (Perkin-Elmer, Italy).29
Stock solutions of compounds were daily prepared in DMSO with
final DMSO concentration less than 0.1%. Displacement curves
were generated by incubating drugs with [3H]CP-55,940 (0.14 nM
for CB1R and 0.084 nM for CB2R binding assay). In all cases, Ki
values were calculated by applying the Cheng-Prusoff equation30
to the IC50 values (obtained by GraphPad) for the displacement of
the bound radioligand by increasing concentrations of the test
compounds.
Formalin Test in Mice. Mice received formalin (1.25%) 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 45° angle under the cage to
allow full view of the hind paws. Lifting, favoring, licking, shaking,
and flinching of the injected paw were recorded as nociceptive
responses. Fifteen minutes before injection of formalin, mice
received intraperitoneal vehicle (10% DMSO in 0.9% NaCl, 50
µL) or 11c (1 or 3 mg/kg, ip) in the same volume solution, alone
or in combination with the selective CB2 antagonist, AM630 (3
mg/kg, ip), administered 5 min before the compound. The total
time of the nociceptive response was measured every 5 min and
expressed as the total time of the nociceptive responses in min
(mean ( SEM). Recording of nociceptive behavior commenced
immediately after formalin injection and was continued for 60 min.
(11) McKallip, R. J.; Lombard, C.; Fisher, M.; Martin, B. R.; Ryu, S.;
Grant, S.; Nagarkatti, P. S.; Nagarkatti, M. Targeting CB2 cannabinoid
receptors as a novel therapy to treat malignant lymphoblastic disease.
Blood 2002, 100, 627–634.
(12) Pertwee, R. G. Cannabinoids and multiple sclerosis. Pharmacol. Ther.
2002, 95, 165–174.
(13) Docagne, F.; Mun˜eto´n, V.; Clemente, D.; Ali, C.; Lor´ıa, F.; Correa,
F.; Hernango´mez, M.; Mestre, L.; Vivien, D.; Guaza, C. Excitotoxicity
in a chronic model of multiple sclerosis: Neuroprotection effects of
cannabinoids through CB1 and CB2 receptor activation. Mol. Cell.
Neurosci. 2007, 34, 551–561.
(14) Ortega-Gutierrez, S.; Molina-Holgado, E.; Are´valo-Mart´ın, A.; Correa,
F.; Viso, A.; Lopez-Rodriguez, M. L.; Di Marzo, V.; Guaza, C.
Activation of the endocannabinoid system as therapeutic approach in
a murine model of multiple sclerosis. FASEB J. 2005, 19, 1338–1340.
(15) Ram´ırez, B. G.; Bla´zquez, C.; Go´mez del Pulgar, T.; Guzma´n, M.; de
Ceballos, M. L. Prevention of Alzheimer’s disease pathology by
cannabinoids: neuroprotection mediated by blockade of microglial
activation. J. Neurosci. 2005, 25, 1904–1913.
Acknowledgment. We thank Dr. Maria Paola Castelli,
Dipartimento di Neuroscienze, University of Cagliari, Italy, for
helpful discussions. Authors from the CNR of Pozzuoli thank
Marco Allara` for technical assistance. Authors from the
University of Siena thank the Ministero dell’Universita` e della
Ricerca (PRIN 2006, Prot. n 2006030948_002) for financial
support.
(16) Giblin, G. M. P.; O’Shaughnessy, C. T.; Naylor, A.; Mitchell, W. L.;
Eatherton, A. J.; Slingsby, B. P.; Rawlings, D. A.; Goldsmith, P.;
Brown, A. J.; Haslam, C. P.; Clayton, N. M.; Wilson, A. W.; Chessell,
I. P.; Wittington, A. R.; Green, R. Discovery of 2-[(2,4-Dichlorophe-
nyl)amino]-N-[(tetrahydro-2H pyran-4-yl)methyl]-4-(trifluoromethyl)-
5-pyrimidinecarboxamide, a selective CB2 receptor agonist for the
treatment of inflammatory pain. J. Med. Chem. 2007, 50, 2597–2600.
(17) (a) Manera, C.; Benetti, V.; Castelli, M. P.; Cavallini, T.; Lazzaretti,
S.; Pibiri, F.; Saccomanni, G.; Tuccinardi, T.; Vannacci, A.; Martinelli,
A.; Ferrarini, P. L. Design, synthesis, and biological evaluation of
new 1,8-naphtyridin-4(1H)-on-3-carboxamide and quinolin-4-(1H)-
on-3-carboxamide derivatives as CB2 selective agonists. J. Med. Chem.
2006, 49, 5947–5957. (b) Manera, C.; Cascio, M. G.; Benetti, V.;
Allara`, M.; Tuccinardi, T.; Martinelli, A.; Saccomanni, G.; Vivoli,
E.; Ghelardini, C.; Di Marzo, V.; Ferrarini, P. L. New 1,8-naphthiridine
and quinoline derivatives as CB2 selective agonists. Bioorg. Med.
Chem. Lett. 2007, 17, 6505–6510.
Supporting Information Available: Synthetic and spectral data
for compounds 11b-k,m,n,p,q,v, 12a,c-f, 14b,c, 15b,c and
elemental analyses for compounds 11-17. This material is available
free of charge via the Internet at http://pubs.acs.org.
References
(18) Stern, E.; Muccioli, G.; Millet, R.; Goossens, J. F.; Farce, A.; Chavatte,
P.; Poupaert, J. H.; Lambert, D. M.; Depreux, P.; He´nichart, J. P. Novel
4-oxo-1,4-dihydroquinoline-3-carboxamide derivatives as new CB2
cannabinoid receptors agonists: synthesis, pharmacological properties
and molecular modelling. J. Med. Chem. 2006, 49, 70–79.
(19) Massa, S.; Corelli, F.; Mai, A.; Artico, M.; Panico, S.; Simonetti, N.
Research on antibacterial and antifungal agents. XI. New antibacterial
quinolones related to pirfloxacin. Farmaco 1989, 44, 779–793.
(20) Stern, E.; Muccioli, G.; Bosier, B.; Hamtiaux, L.; Millet, R.; Poupaert,
J. H.; He´nichart, J. P.; Depreux, P.; Goossens, J. F.; Lambert, D. M.
Pharmacomodulations around the 4-oxo-1,4-dihydroquinoline-3-car-
boxamides, a class of potent CB2-selective cannabinoid receptor
ligands: consequences in receptor affinity and functionality. J. Med.
Chem. 2007, 50, 5471–5484.
(21) Silvestri, R.; Cascio, M. G.; La Regina, G.; Piscitelli, F.; Lavecchia,
A.; Brizzi, A.; Pasquini, S.; Botta, M.; Novellino, E.; Di Marzo, V.;
Corelli, F. Synthesis, hCB1 and hCB2 cannabinoid receptor affinity
and molecular modeling of substituted 1-phenyl-5-(1H-pyrrol-1-yl)-
1H-pyrazole-3-carboxamides, as potent pyrrole bioisosteres of rimona-
bant and SR144528. J. Med. Chem. 2008, 51, 1560–1576.
(22) This work was presented in part at the meeting Frontiers in CNS and
Oncology Medicinal Chemistry, Siena, Italy, October 7-9, 2007; P75.
(23) For the preparation of 4-(3-chloro-4-fluorophenoxy)aniline and 4-(phe-
nylthio)aniline, see the following: Fotsch, C.; Sonnenberg, J. D.; Chen,
N.; Hale, C.; Karbon, W.; Norman, M. H. Synthesis and structure-
activity relationships of trisustituted phenyl urea derivatives as
(1) (a) Ben Amar, M. Cannabinoids in medicine: a review of their
therapeutic potential. J. Ethnopharmacol. 2006, 105, 1–25. (b)
Piomelli, D. The endocannabinoid system: a drug discovery perspec-
tive. Curr. Opin. InVest. Drugs 2005, 6, 672–679. (c) Di Marzo, V.;
Bifulco, M.; De Petrocellis, L. The endocannabinoid system and its
therapeutic exploitation. Nat. ReV. Drug DiscoVery 2004, 3, 771–784.
(2) Lambert, D. M.; Fowler, C. J. The endocannabinoid system: drug
targets, lead compounds, and potential therapeutic applications. J. Med.
Chem. 2005, 48, 5059–5087.
(3) (a) Lange, J. H. M.; Kruse, C. G. Medicinal chemistry strategies to
CB1 cannabinoid receptor antagonists. Drug DiscoVery Today 2005,
10, 693–702. (b) Barth, F. CB1 cannabinoid receptor antagonist. Annu.
Rep. Med. Chem. 2005, 40, 103–118. (c) Muccioli, G. G.; Lambert,
D. M. Latest advances in cannabinoid receptor antagonists and inverse
agonists. Expert Opin. Ther. Pat. 2006, 16, 1405–1423.
(4) Tucci, S. A.; Halford, J. C. G.; Harrold, J. A.; Kirkham, T. C.
Therapeutic potential of targeting the endocannabinoids; implications
for the treatment of obesity, metabolic syndrome, drug abuse and
smoking cessation. Curr. Med. Chem. 2006, 13, 2669–2680.
(5) (a) Rinaldi-Carmona, M.; Barth, F.; He´aulme, M.; Shire, D.; Calandra,
B.; Congy, C.; Martinez, S.; Maruani, J.; Ne´liat, G.; Caput, D.; Ferrara,
P.; Soubrie´, P.; Brelie`re, J. C.; Le Fur, G. SR141716A, a potent and
selective antagonist of the brain cannabinoid receptor. FEBS Lett. 1994,
350, 240–244. (b) Sorbera, L. A.; Castaner, J.; Silvestre, J. S.
Rimonabant hydrochloride. Drugs Future 2005, 30, 128–137.

5084 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Pasquini et al.
neuropeptide Y5 receptor antagonists. J. Med. Chem. 2001, 44, 2344–
2356.
(24) Gould, R. G., Jr.; Jacobs, W. A. The synthesis of certain substituted
quinolones and 5,6-benzoquinolines. J. Am. Chem. Soc. 1939, 61,
2890–2895.
(25) Link, V. H.; Bernauer, K.; Englert, G. Abko¨mmlinge der 3-chinolin-
carbonsa¨ure mit sauerstoffsubstitution in stellung 4, 5 und 8: synthese,
reaktionen, NMR.-studien. HelV. Chim. Acta 1982, 65, 2645–2667.
(26) Stefancich, G.; Artico, M.; Corelli, F.; Massa, S.; Panico, S.; Simonetti,
N. 1-Ethyl-6-fluoro-1,4-dihydro-4-oxo-7-(1H-pyrrol-1-yl)quinoline-3-
carboxylic acid, a new fluorinated compound of oxacin family with
high broad-spectrum antibacterial activities. Farmaco, Ed. Sci. 1985,
40, 237–243.
antibacterial evaluation of 6-amino-8-methylquinolones. J. Med. Chem.
1996, 39, 436–445.
(28) Ax, A.; Schaal, W.; Vrang, L.; Samuelsson, B.; Halberg, A.; Karle´n,
A. Cyclic sulfamide HIV-1 protease inhibitors, with sidechains
spanning from P2/P2′ to P1/P1′. Bioorg. Med. Chem. 2005, 13, 755–
764.
(29) Brizzi, A.; Brizzi, V.; Cascio, M. G.; Bisogno, T.; Siriani, R.; Di
Marzo, V. Design, synthesis, and binding studies of new potent ligands
of cannabinoid receptors. J. Med. Chem. 2005, 48, 7343–7350.
(30) Cheng, Y.; Prusoff, W. H. Relationship between the inhibition constant
(Ki) and the concentration of inhibitor which causes 50% inhibition
(I50) of an enzymatic reaction. Biochem. Pharmacol. 1973, 22,
3099–3108.
(27) (a) Grohe, R.; Heitzer, H. Cycloaracylation of enamines. I. Synthesis
of 4-quinolone-3-carboxylic acids. Liebigs Ann. Chem 1987, 1, 29–
37. (b) Cecchetti, V.; Fravolini, A.; Lorenzini, M. C.; Tabarrini, C.;
Terni, P.; Xin, T. Studies on 6-aminoquinolones: synthesis and
JM800552F