Investigations on the 4-quinolone-3-carboxylic acid motif. 4. Identification of new potent and selective ligands for the cannabinoid type 2 receptor with diverse substitution patterns and antihyperalgesic effects in mice

Article abstract of DOI:10.1021/jm200476p

Experimental evidence suggests that selective CB2 receptor modulators may provide access to antihyperalgesic agents devoid of psychotropic effects. Taking advantage of previous findings on structure-activity/selectivity relationships for a class of 4-quin

Full text of DOI:10.1021/jm200476p

ARTICLE
pubs.acs.org/jmc
Investigations on the 4-Quinolone-3-carboxylic Acid Motif. 4.
Identification of New Potent and Selective Ligands for the
Cannabinoid Type 2 Receptor with Diverse Substitution Patterns
and Antihyperalgesic Effects in Mice
Serena Pasquini,† Maria De Rosa,† Valentina Pedani,† Claudia Mugnaini,† Francesca Guida,‡ Livio Luongo,‡
Maria De Chiaro,‡ Sabatino Maione,‡ Stefania Dragoni,§ Maria Frosini,§ Alessia Ligresti,||
Vincenzo Di Marzo,|| and Federico Corelli*,†
†Dipartimento Farmaco Chimico Tecnologico, Universitꢀa degli Studi di Siena, Via A. Moro, 53100 Siena, Italy
‡Department of Experimental Medicine—Section of Pharmacology “L. Donatelli”, Second University of Naples,
Via S. Maria di Costantinopoli 16, 80138 Naples, Italy
§Dipartimento di Neuroscienze, Sezione di Farmacologia, Universitꢀa 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
S Supporting Information
b
ABSTRACT: Experimental evidence suggests that selective CB2 receptor
modulators may provide access to antihyperalgesic agents devoid of psychotropic
effects. Taking advantage of previous findings on structureꢀactivity/selectivity
relationships for a class of 4-quinolone-3-carboxamides, further structural
modifications of the heterocyclic scaffold were explored, leading to the discovery
of the 8-methoxy derivative 4a endowed with the highest affinity and selectivity
ever reported for a CB2 ligand. The compound, evaluated in vivo in the formalin
test, behaved as an inverse agonist by reducing at a dose of 6 mg/kg the second phase of the formalin-induced nocifensive response
in mice.
’ INTRODUCTION
have been reported with regard to its effects on bone resorption
and osteoclast function. In fact, in one study carried out in the
mouse, it has been demonstrated that CB2-selective antagonist/
inverse agonist AM6307 (Chart 1) inhibited osteoclast formation
and activity in vitro, whereas the CB2-selective agonists
JWH1338 and HU3089 stimulated osteoclast formation.10 How-
ever, a more recent study performed in human cells has shown
that AM630 instead enhances osteoclast activity.11 Ofek and co-
workers have reported that activation of CB2 stimulates osteo-
blast proliferation and bone marrow derived colony-forming
units osteoblastic and that selective and nonselective CB2
agonists are mitogenic in MC3T3 E1 and newborn mouse
calvarial osteoblastic cultures.12
Very recently we have described a family of 6-substituted-
4-quinolone-3-carboxamide derivatives displaying high CB2 recep-
tor affinity and selectivity over CB1 receptor. Some of these
compounds, such as 113 and 214 (Chart 2), were demonstrated to
act as agonists or inverse agonists, respectively, in functional
activity assays, depending on the aliphatic or aromatic nature
of the 6-substituent. On the other hand, a three-dimensional
Research in medicinal chemistry during the last years has led
to the identification of a spectrum of compounds with different
affinities and selectivities for cannabinoid type 1 (CB1) and
type 2 (CB2) receptors, compounds that have been essential in
characterizing the role of cannabinoid receptors in the body.1
Differences in receptor distribution and signal transduction
mechanisms2 are likely to account for the relative absence of
CNS side effects induced by CB2 ligands. These considerations
suggest that novel pharmacotherapies selectively targeting CB2
receptors may have considerable therapeutic potential.3 Accord-
ingly, significant medicinal chemistry efforts have been directed
to the characterization of selective CB2 agonists (Chart 1),
leading to the identification of compounds eliciting antinocicep-
tive effects in models of acute pain, persistent inflammatory pain,
postoperative pain, cancer pain, and neuropathic pain.4 In recent
years, a number of reports have suggested that CB2 inverse
agonists/antagonists (Chart 1) may possess antiinflammatory
activity, being able to inhibit carrageenan-induced paw edema in
mice, to reduce leukocytes trafficking, and to impair the migra-
tion of cells toward cannabinoid agonists.5 The CB2 receptor is
also an emerging target in osteoporosis disease modification,6 as
it has been reported to regulate bone mass, but conflicting results
Received: April 20, 2011
Published: June 27, 2011
r
2011 American Chemical Society
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Chart 1. Representative CB2-Selective Agonists (HU-308,
JWH-133) and Inverse Agonists/Antagonists (SR144528,
AM630)
Chart 2. Lead Compounds 1ꢀ3 and General Structure of
Variously Substituted 4-Quinolone-3-carboxamide Deriva-
tives 4ꢀ8
quantitative structureꢀselectivity relationship (3D-QSSAR) analysis
performed on a large set of known CB1 and CB2 ligands resulted in
the rational design of the 8-methyl-4-quinolone-3-carboxamide 3 as a
high affinity and very selective CB2 receptor ligand.15
Starting from these achievements, we first synthesized some
8-substituted-4-quinolone-3-carboxamide derivatives 4aꢀc,e
and subsequently 6,8-disubstituted quinolones 4d,fꢀg to explore
the possibility of obtaining a more pronounced effect in terms of
CB2 affinity and selectivity through double substitution. Then,
treasuring the indications delivered by the molecular modeling
studies, compounds 6a and 6b, exhibiting an aliphatic chain at
position 2 and a free NH group at position 1, were investigated,
together with the tricyclic derivative 5. These structural mod-
ifications were expected to improve the hydrophilic/hydropho-
bic balance of the compounds and to provide useful information
on the role of the aliphatic chain in the receptors binding. Finally,
6-substituted-4-hydroxy-2-quinolone-3-carboxamides 7a and 7b
(pKa < 5)16 were prepared by considering the possibility of con-
verting them into the corresponding sodium salts. For comparison
purposes, the 2-quinolone and 2-alkoxyquinoline derivatives 7c and
8 were also synthesized.
The 1,4-oxazino[2,3,4-ij]quinoline 5 was synthesized in a
different way, according to a modified GroheꢀHeitzer procedure
(Scheme 2).13 Treatment of 2,3-difluorobenzoyl chloride with
3-dimethylaminopropenoic acid ethyl ester led to the enamino
ketone 12, which was subsequently reacted with 2-amino-1-
butanol to yield the quinolone ester 14 via the intermediate 13.
Taking advantage of the ease of displacement of the fluorine
substituent, the tricyclic acid 15 was obtained by treating 14 with
potassium fluoride/potassium carbonate17 in DMF at 150 °C,
followed by alkaline hydrolysis of the ester group. The product
15 so obtained was subjected to coupling with 1-aminoadaman-
tane to deliver the amide 5 in 8% overall yield (from 2,3-
difluorobenzoyl chloride).
Both 2-substituted 4-quinolones 6a,b and 4-hydroxy-2-quino-
lones 7a,b were synthesized from 5-bromoisatoic anhydride
(Scheme 3), which was condensed with either methyl
3-oxoheptanoate18 to give 16 or diethyl malonate,16 after alkyla-
tion to 17, to yield the 4-hydroxy-2-quinolone derivative 18.
Compound 16 was converted into the corresponding adamanta-
nyl amide 6a by the hydrolysis/amidation sequence, while the
amide 7a was obtained in quantitative yield by direct aminolysis
of 6b with 1-aminoadamantane in refluxing toluene. Compounds
6a and 7a in turn provided the 6-(2-furanyl)-quinolones 6b and
’ CHEMISTRY
The synthesis of the final compounds 4ꢀ8 was accomplished
as depicted in Schemes 1ꢀ4. Commercially available anilines 9aꢀd
and 2-(1-pentyloxy)aniline (9e) (see Supporting Information)
were converted into the corresponding N-(adamantan-1-yl)-1,4-
dihydro-4-oxoquinoline-3-carboxamide derivatives 10aꢀe in
7ꢀ37% overall yield according to synthetic procedures pre-
viously set up for similar compounds (Scheme 1).13,14 Inter-
mediates 10aꢀd were reacted with 1-iodopentane in the
presence of potassium carbonate to provide the final quinolones
4aꢀd in a yield of 57ꢀ74% (23% for 4b). Compound 10c was
subjected to Suzuki cross-coupling reaction with phenylboronic
acid to give 11, which proved to be resistant to N-alkylation using
1-iodopentane. Therefore, the final derivative 4e was prepared
from 4c via Suzuki reaction. Analogously, compounds 4f and 4g
were obtained from 4d using phenylboronic acid and 2-furan-
boronic acid, respectively.
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Scheme 1. Synthesis of 4-Quinolone-3-carboxamide Deriva-
Scheme 2. Synthesis of 2H-[1,4]-Oxazino[2,3,4-ij]quino-
tives 4aꢀg and 11a
line-6-carboxamide Derivative 5a
a Reagents and conditions: (i) ethyl 3-dimethylaminoacrylate, Et3N,
toluene, 100 °C, 4 h; (ii) (1) 2-aminobutanol, EtOH, Et2O, rt, 2 h, (2)
K2CO3, DMF, 90 °C, 3 h; (iii) (1) KF, K2CO3, DMF, 150 °C, 3 h, (2)
10% NaOH, reflux, 1 h; (iv) 1-aminoadamantane, HOBt, HBTU,
DIPEA, DMF, rt, 3 h.
Cytotoxicity assays were also performed to establish the
effect of selected quinolone-3-carboxamides on cell proliferation
in vitro, using human hepatoblastoma (Hep-G2) cells via
MTT assay.
Furthermore, in order to exclude the possibility that these
molecules could indirectly activate CB1 and CB2 receptors,
through inhibition of endocannabinoid inactivation by fatty acid
amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL),
four compounds, i.e. 4a, 4g (the ligands endowed with highest
affinity and selectivity for CB2), 5, and 7a (the ligand endowed
with the lowest CB2 affinity), were selected for enzymatic assays,
for which rat brain FAAH and COS-7 cell MAGL activities were
used.22
Finally, the in vivo activity of the same compounds was
evaluated in the formalin test of acute peripheral and inflamma-
tory pain in mice. Formalin injection induces a biphasic
stereotypical nocifensive behavior. Nociceptive responses are
divided into an early, short-lasting first phase (0ꢀ7 min) caused
by a primary afferent discharge produced by the stimulus,
followed by a quiescent period and then a second, prolonged
phase (15ꢀ60 min) of tonic pain. Fifteen min before injection
of formalin, mice received intraperitoneal (ip) administration
of vehicle or of either of the four compounds (1, 3, or 6 mg/kg),
alone or in combination with either the selective CB2 antago-
nist, 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-
yl](4-methoxyphenyl)methanone (AM630) (1 mg/kg, ip),
administered 5 min before the compound.23 The results are
presented in Figures 1ꢀ4.
a Reagents and conditions: (i) pentyl iodide, K2CO3, DMF, 90 °C, 4 h;
(ii) arylboronic acid, Pd(OAc)2, PPh3, 2 M Na2CO3, DME, EtOH, MW,
150 °C, 10 min.
7b, respectively, through Pd-catalyzed reaction with 2-furan-
boronic acid.
Finally, 2-oxo-1,2-dihydroquinoline-3-carboxylic acid (19)19
was converted into the N-adamantyl amide 20 (Scheme 4),
which on treatment with 1-iodopentane and potassium carbo-
nate afforded compounds 7c and 8 in 53% and 33% yield,
respectively, after chromatographic separation.
’ PHARMACOLOGY
With the exception of compounds 4c, 4e, 8, and 10c, which
proved to be insoluble under the test conditions, all the
newly synthesized quinolones were screened, in a competitive
binding experiment, for their affinity and selectivity toward
the human recombinant CB1 and CB2 receptors. The tested
compounds were evaluated in parallel with SR14452820 and
rimonabant21 as CB2 and CB1 reference ligands, respectively, as
previously described.13,14 The results, in terms of binding
affinities for the two receptors (Ki values), are reported in
Table 1.
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Scheme 3. Synthesis of Quinolone-3-carboxamide Deriva-
tives 6a,b and 7a,ba
Scheme 4. Synthesis of Compounds 7c and 8a
a Reagents and conditions: (i) 1-aminoadamantane, HOBt, HBTU,
DIPEA, DMF, rt, 3 h; (ii) pentyl iodide, K2CO3, DMF, 90 °C, 4 h.
positions 6 and 8 of the quinolone nucleus was not beneficial.
In fact, starting from the 8-methoxy derivative 4a, eliciting excellent
values of affinity (Ki 0.6 nM) and selectivity (SI > 16666) toward the
CB2 receptor, the introduction of a bromine atom (compound 4d)
or a (hetero)aromatic moiety (compounds 4f and 4g) led to a loss of
CB2 affinity.
1-Unsubstituted compounds 6a and 10a showed the lowest
receptor affinity within the series in line with a previuos report
describing the importance of the N1-nitrogen substitution for
quinolones to bind at the CB2 receptor.24 However, when
additional lipophilic substituents were introduced in other posi-
tions of the bicyclic scaffold, moderate (6b, 11) to significant
(10e) CB2 affinity was restored, although in the latter case
associated with poor selectivity.
Reducing the conformational freedom of the N1-pentyl chain,
as in compound 5, did not abolish the binding capability,
although 5 exhibited 10-fold lower CB2 receptor affinity and
100-fold lower selectivity compared to the lead compound 2.
Isomeric 2-quinolone derivatives 7a, 7b, and 7c all proved to
be potent CB2 ligands, with 7c displaying highest CB2 affinity
among the tested compounds, including the reference com-
pound 2, although with markedly reduced selectivity. These
findings, which are in agreement with the results recently
reported for structurally related 1-substituted-1,8-naphthyridin-
4(1H)-on-3-carboxamide derivatives,25 deserve further attention
as they represent a good starting point for the development of a
novel class of CB2 ligand based on the 2-quinolone scaffold.
Furthermore, we show here that at least four of the novel
compounds, i.e. 4a, 4g, 5, and 7a, are devoid of any potential
“indirect” agonist activity at cannabinoid receptors, exerted by
prolonging the lifespan of endocannabinoids because these com-
pounds did not inhibit anandamide or 2-AG degradation by FAAH
or MAGL, respectively, at up to a 10 μM concentration.
Cytotoxicity Evaluation. Cytotoxicity assays were performed
to establish the effect of selected quinolone-3-carboxamides on
cell proliferation in vitro. Because many drugs are cleared
from the body through the liver, this organ is a good choice for
target organ toxicity assessment. Consequently, the cytotoxicity
of compounds 3, 4a, 4d, 4f, 4g, 5, 6b, 7a, 7b, 10e, and 11
was evaluated toward human hepatoblastoma (Hep-G2) cells
via the MTT assay. The compounds were tested at a single
a Reagents and conditions: (i) methyl 3-oxoheptanoate, NaOH,
1,4-dioxane, reflux, 48 h; (ii) (1) 10% NaOH, reflux, 1 h, (2) 1-aminoada-
mantane, HOBt, HBTU, DIPEA, DMF, rt, 30 h; (iii) furanboronic acid,
Pd(OAc)2, PPh3, 2 M Na2CO3, DME, EtOH, MW, 150 °C, 10 min; (iv)
1. pentyl iodide, DIPEA, DMF, 80 °C, 18 h, (2) diethyl malonate,
sodium tert-butoxide, DMF, 100 °C, 2 h; (v) 1-aminoadamantane,
toluene, reflux, 1 h.
’ RESULTS AND DISCUSSION
In Vitro Pharmacology and SAR. All the tested compounds
showed moderate to very high affinity toward the CB2 receptor,
with Ki values in the range 430ꢀ0.2 nM, except for 6a and 10a,
which proved to be completely inactive on both receptors
subtypes. Only compounds 4b, 7b, 7c, and 10e displayed
significant CB1 affinity. According to this binding profile, the
selectivity index (SI), calculated as Ki(CB1)/Ki(CB2) ratio, for
tested compounds varied widely, going from >16666 (for 4a) to
4.3 (for 10e).
In the series of 8-substituted quinolones, the best results in
terms of both CB2 affinity and selectivity were obtained with the
introduction of electron-donating groups, such as methyl
(compound 3)15 or methoxy group (compound 4a), while
electron-withdrawing groups, such as nitro, were responsible for
significant CB1 affinity as well, resulting in a loss of CB2 selectivity
(compound 4b). Unexpectedly, the double substitution at
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Table 1. CB1 and CB2 Receptor Affinity Values for Compounds 4ꢀ7, 10, and 11a
a Data represent mean values for at least three separate experiments performed in duplicate and are expressed as Ki (nM). b Ad = adamantan-1-yl. c CB1: human
cannabinoid type 1 receptor. d CB2: human cannabinoid type 2 receptor. e 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). 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 SI: selectivity index for CB2, calculated as Ki(CB1)/Ki(CB2) ratio. h The binding affinities of
reference compounds were evaluated in parallel with the test compounds under the same conditions. i CB2 reference compound. j CB1 reference compound.
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Figure 2. Effect of 4g (1ꢀ3 mg/kg, ip) alone (A) or in combination, at
the dose of 3 mg/kg, with AM630 (1 mg/kg, ip) (B) in the formalin test
in mice. The total time of the nociceptive response was measured every 5
min and expressed as the total time of the nociceptive responses in min.
Results are mean ( SEM (n = 8ꢀ10 for each group). In (A), filled
symbols denote statistically significant differences vs formalin. In (B),
filled symbols denote statistically significant differences (P < 0.05) vs 4g.
One-way analysis of variance followed by a TukeyꢀKramer multiple
comparisons test was used for data analysis.
Figure 1. Effect of 4a (3ꢀ6 mg/kg, ip) alone (A) or in combination, at
the dose of 6 mg/kg, with AM630 (1 mg/kg, ip) (B) in the formalin test
in mice. The total time of the nociceptive response was measured every 5
min and expressed as the total time of the nociceptive responses in min.
Results are mean ( SEM (n = 8ꢀ10 for each group). In (A), filled
symbols denote statistically significant differences vs formalin. In (B),
filled symbols denote statistically significant differences (P < 0.05) vs 4a.
One-way analysis of variance followed by a TukeyꢀKramer multiple
comparisons test was used for data analysis.
reduced by AM630 (Figure 2B), thus indicating that this
compound might indeed act as a potent and selective CB2
agonist. Conversely, the effects of 5 were only partly attenuated
by AM630 (Figure 3B), thus suggesting for this compound a
potential dual action at CB2 and other targets (including CB1).
Finally, compound 7a also exhibited antihyperalgesic effects
against both the first and second phase of the formalin response,
although only at the highest dose tested of 3 mg/kg (Figure 4A).
These effects were strongly antagonized by AM630 (Figure 4B),
thus suggesting also for this compound a selective CB2 agonist-
like activity, although less potent than that observed with 4g, in
agreement with the relative affinities of the two compounds for
CB2, measured here (Ki = 8.5 and 29.4 nM, respectively, see
Table 1). In conclusion, these in vivo data, while confirming that
4-quinolone-3-carboxamide CB2 ligands produce antihyperalge-
sic effects in vivo, suggest for compounds 4a, 4g, 5, and 7a
different tentative functional activities as a: selective CB2 inverse
agonist, potent and selective CB2 agonist, weaker but still
selective CB2 agonist, and nonselective CB2 agonist, respec-
tively. Clearly, appropriate in vitro functional assays will have to
be carried out in order to confirm these predictions.
concentration of 1 μM, i.e. about 1ꢀ3 orders of magnitude
higher than their Ki toward CB2 receptors. Results show that all
the derivatives exhibit very low or no cytotoxicity, the cell
viability being always above 95% after a 72 h treatment (see
Supporting Information).
In Vivo Pharmacology. Compounds 4a, 4g, 5, and 7a
exhibited antinociceptive activity in the formalin test in mice.
The compound with highest affinity and selectivity for CB2, 4a,
was surprisingly the less efficacious in this test, a dose of 6 mg/kg
being necessary in order to observe a reduction of the second
(and, to a much lesser extent, the first) phase of the formalin-
induced nocifensive response (Figure 1A). This might suggest
that 4a might behave as an inverse agonist, rather than an agonist,
at CB2 receptors. Indeed, CB2 inverse agonists exert activity in
the formalin test, particularly on the second phase, only at
relatively high doses.13,23 This behavior differs from that of
potent CB2 agonists, which reduce both phases of the formalin
response at doses usually proportional to their corresponding Ki
values. In agreement with its potential activity as inverse agonist,
the effect of 4a was not significantly reversed by the CB2
antagonist AM630 (Figure 1B). The two compounds with
similar affinity for CB2 receptors, 4g and 5, exhibited different
efficacies in the formalin test, the former being very potent (with
maximal effect being reached already at the 1 mg/kg dose) and
efficacious also on the first phase of the nocifensive response
(Figure 2A), the latter instead showing activity only at the higher
tested dose (3 mg/kg) (Figure 3A). The effect of 4g was strongly
’ CONCLUSIONS
In our continuing effort to explore structureꢀaffinity relation-
ship for quinolones binding at cannabinoid receptors, we have
described herein the synthesis and pharmacological evaluation of
new compounds characterized by diverse substitution patterns of
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Figure 4. Effect of 7a (1ꢀ3 mg/kg, ip) alone (A) or in combination, at
the dose of 3 mg/kg, with AM630 (1 mg/kg, ip) (B) in the formalin test
in mice. The total time of the nociceptive response was measured every 5
min and expressed as the total time of the nociceptive responses in min.
Results are mean ( SEM (n = 8ꢀ10 for each group). In (A), filled
symbols denote statistically significant differences vs formalin. In (B),
filled symbols denote statistically significant differences (P < 0.05) vs 7a.
One-way analysis of variance followed by a TukeyꢀKramer multiple
comparisons test was used for data analysis.
Figure 3. Effect of 5 (1ꢀ3 mg/kg, ip) alone (A) or in combination, at
the dose of 3 mg/kg, with AM630 (1 mg/kg, ip) (B) in the formalin test
in mice. The total time of the nociceptive response was measured every 5
min and expressed as the total time of the nociceptive responses in min.
Results are mean ( SEM (n = 8ꢀ10 for each group). In Figure (A), filled
symbols denote statistically significant differences vs formalin. In (B),
filled symbols denote statistically significant differences (P < 0.05) vs 5.
One-way analysis of variance followed by a TukeyꢀKramer multiple
comparisons test was used for data analysis.
’ EXPERIMENTAL SECTION
the bicyclic core. In particular, 8-substituted 4-quinolone-3-
carboxamides possessing high affinity for the human CB2
receptor at nanomolar or subnanomolar concentration and
selectivity index values as high as >16000 were identified. 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. Because of the relevance of the
selectivity issue in the cannabinoid area,26 these results seem to
hold some promise with regard to the possibility that truly
selective CB2 ligands can be developed. Moreover, other com-
pounds belonging to structurally related chemical classes, namely
2-quinolones (compound 7c) and 4-hydroxy-2-quinolones
(compound 7b), demonstrated high CB2 affinity although
accompanied with less receptor selectivity. However, it is worth
reminding that 4a possesses CB2 affinity and selectivity approxi-
mately 8-fold and 1800-fold, respectively, higher than the 8-un-
substituted parent compound.13 Therefore, considering the
crucial effect frequently exerted by appropriate substituents at
positions 6 and/or 8 of quinolones in increasing CB2 receptor
affinity and selectivity, compound 7c emerges as a specially
interesting candidate for further structural optimization.
When assayed in vivo in the formalin test of analgesia in mice,
compounds 4a, 4g, 5, and 7a elicited antihyperalgesic effects, 4g
resulting more potent than the other tested compounds. On the
basis of the results obtained in the formalin assay in the absence and
in the presence of the CB2 antagonist AM630, a functional profile of
CB2 agonist (inverse agonist for 4a) could be inferred for these
compounds. In any case, the analgesic effects should be ascribed to
direct activity at CB2 receptor, as no inhibitory activity of FAAH and
MAGL by the test compounds could be demonstrated.
Chemistry. Reagents were purchased from commercial suppliers
and used without further purification. Anhydrous reactions were run
under a positive pressure of dry N2. Merck Silica Gel 60 was used for
flash chromatography (23ꢀ400 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 105ꢀ1500. Melting points were determined on a Gallenkamp
apparatus and are uncorrected. Microwave irradiations were conducted
using a CEM Discover Synthesis Unit (CEM Corporation, Matthews,
NC). 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. 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 Quinolones 4aꢀd by Alkylation Reaction.
General Procedure. A mixture of the appropriate quinolone derivative
10aꢀd (1 mmol), 1-iodopentane (0.4 mL, 2.8 mmol), and solid K2CO3
(386 mg, 2.8 mmol) in dry DMF (1 mL) was heated under N2
atmosphere at 90 °C for 4 h and then poured into an iceꢀwater mixture.
The precipitated N-alkylquinolone was extracted with DCM and
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purified by flash chromatography on silica gel eluting with DCM/
MeOH (97:3).
Example: N-(Adamantan-1-yl)-3-ethyl-3,7-dihydro-7-oxo-2H-[1,4]-
oxazino[2,3,4-ij]quinoline-6-carboxamide (5). Prepared in 54% yield
from 15 as a light-brown solid; mp 218ꢀ219 °C. 1H NMR (200 MHz,
CDCl3): δ 10.61 (s, 1H), 8.62 (s, 1H), 8.02ꢀ8.00 (m, 1H), 7.34ꢀ7.30
(m, 1H), 7.20ꢀ7.15 (m, 1H), 4.51 (d, J = 12 Hz, 1H), 4.29ꢀ4.25
(m, 1H), 4.19 (t, J = 9.2 Hz, 1H), 2.16ꢀ2.11 (m, 6H), 2.09ꢀ1.83 (m,
3H), 1.76ꢀ1.70 (m, 8H), 1.03 (t, J = 8.0 Hz, 3H). 13C NMR (100 MHz,
CDCl3): δ 177.0, 158.8, 144.1, 142.2, 133.9, 127.0, 122.2, 121.3, 118.8,
111.8, 68.9, 63.7, 40.9, 37.9, 36.6, 25.8, 21.0, 11.9. IR (CHCl3): ν 1654,
1728 cmꢀ1. MS (ESI): m/z 393 [M + H]+, 415 [M + Na]+. Anal.
(C24H28N2O3) C, H, N.
Methyl 6-Bromo-2-butyl-1,4-dihydro-4-oxoquinoline-3-
carboxylate (16). Solid NaOH (25 mg, 0.6 mmol) and methyl
3-oxoheptanoate (978 μL, 6 mmol) were added to a solution of
5-bromoisatoic anhydride (1 g, 4 mmol) in 1,4-dioxane (6 mL). The
mixture was refluxed for 48 h, then cooled to room temperature and
concentrated under reduced pressure. The residue was taken up into
water and AcOEt, and the organic layer was dried and evaporated.
Purification by column chromatography on silica gel (3:1 AcOEt/PE)
furnished 16 (660 mg, 47%) as a white solid; mp 222ꢀ223 °C. 1H NMR
(200 MHz, DMSO-d6) δ 11.08 (bs, 1H), 8.26 (d, J = 2.4 Hz 1H), 7.73
(dd, J1 = 2.4 Hz, J2= 8.7 Hz, 1H), 7.52 (d, J = 8.7 Hz, 1H), 3.77 (s, 3H),
2.71 (t, J = 7.8 Hz, 2H), 1.74ꢀ1.62 (m, 2H), 1.42ꢀ1.26 (m, 2H), 0.87
(t, J = 7.3 Hz, 3H). IR (CHCl3): ν 1634, 1720 cmꢀ1. MS (ESI): m/z 339
[M + H]+. Anal. (C15H16BrNO3) C, H, N.
N-(Adamantan-1-yl)-6-bromo-2-butyl-1,4-dihydro-4-oxo-
quinoline-3-carboxamide (6a). Prepared in 25% overall yield from
16 by ester hydrolysis followed by amidation reaction according to the
procedures described for the preparation of 15 and 5, respectively. White
solid; mp 172ꢀ173 °C. 1H NMR (400 MHz, CDCl3): δ 10.40 (bs, 1H),
9.30 (s, 1H), 8.38 (d, J = 2.0 Hz, 1H), 7.58 (dd, J1 = 2.0, J2 = 8.8 Hz, 1H),
7.34 (d, J = 8.8 Hz, 1H), 3.65 (q, J = 7.2 Hz, 1H), 3.04 (t, J = 8.0 Hz, 2H),
2.10 (s, 5H), 2.02 (s, 2H), 1.63ꢀ1.55 (m, 5H), 1.54ꢀ1.48 (m, 2H),
1.20ꢀ1.12 (m, 4H), 0.66 (t, J = 8.0 Hz, 3H). 13C NMR (100 MHz,
CDCl3): δ 176.2, 165.6, 158.6, 137.0, 135.6, 129,0, 126.9, 119.4, 118.4,
113.6, 58.7, 52.3, 42.0, 36.7, 33.9, 31.8, 29.7, 22.8, 18.6, 13.9. IR (Nujol):
ν 1616, 1650 cmꢀ1. MS (ESI): m/z 458 [M + H]+. Anal. (C24H29BrN2O2)
C, H, N.
Ethyl 6-Bromo-1,2-dihydro-4-hydroxy-2-oxo-1-pentyl-
quinoline-3-carboxylate (18). A solution of 5-bromoisatoic anhy-
dride (400 mg, 1.65 mmol), 1-iodopentane (239 μL, 1.82 mmol), and
DIPEA (575 μL, 3.3 mmol) in DMF (5 mL) was heated to 80 °C for 18 h
while monitoring the progress of the reaction by TLC (98:2 DCM/
MeOH). Diethyl malonate (500 μL, 3.3 mmol) and a solution of sodium
tert-butoxide in DMF, prepared by adding NaH (87 mg, 3.6 mmol) to
dry tert-butanol (315 μL, 3.3 mmol) in DMF (1.5 mL), were added.
After heating to 100 °C for 2 h, the reaction mixture was poured into ice
and 37% HCl was added until pH 2 is reached. Extraction with DCM,
followed by the usual workup of the organic phase, and purification by
silica gel chromatography (99:1 DCM/MeOH) of the solid residue gave
18 (240 mg, 40% overall yield from 5-bromoisatoic anhydride); mp
84ꢀ85 °C (EtOH). 1H NMR (200 MHz, CDCl3): δ 17.01 (s, 1H), 8.32
(d, J = 2.0Hz, 1H),7.70(dd,J1 =2,0Hz, J2 =8.8Hz,1H),7.18(d,J=8.8Hz,
1H), 4.21 (t, J = 7.0 Hz, 2H), 4.15 (t, J = 7.2 Hz, 2H), 1.74ꢀ1.62 (m, 2H),
1.42ꢀ1.02 (m, 7H), 0.90 (t, J= 7.3 Hz, 3H). 13CNMR(50MHz, CDCl3):δ
172.5, 170.5, 158.8, 139.4, 137.0, 128.2, 115.9, 114.6, 98.5, 62.6, 42.5, 29.1,
27.1, 22.5, 14.2, 14.0. IR (CHCl3): ν 1640, 1721 cmꢀ1. MS (ESI): m/z 383
[M + H]+. Anal. (C17H20BrNO4) C, H, N.
Example: N-(Adamantan-1-yl)-8-methoxy-4-oxo-1-pentyl-1,4-di-
hydroquinoline-3-carboxamide (4a). Prepared in 64% yield from
10a. White solid; mp 153ꢀ154 °C (EtOH). 1H NMR (200 MHz,
CDCl3): δ 9.91 (s, 1H), 8.54 (s, 1H), 8.10ꢀ8.05 (m, 1H), 7.34ꢀ7.26
(m, 1H), 7.10ꢀ7.05 (m, 1H), 4.43 (t, J = 7.5 Hz, 2H), 3.90 (s, 3H),
2.08ꢀ1.99 (m, 12H), 1.80ꢀ1.59 (m, 5H), 1.32ꢀ1.19 (m, 4H), 0.83
(t, J = 6.5 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 175.9, 163.6, 149.9,
149.6, 130.5, 130.2, 125.0, 119.1, 114.1, 112.3, 59.8, 56.2, 51.5, 41.8,
36.5, 31.1, 29.5, 28.6, 22.2, 13.9. IR (CHCl3): ν 1655 cmꢀ1. MS (ESI):
m/z 423 [M + H]+. Anal. (C26H34N2O3) C, H, N.
Synthesis of Quinolones 4eꢀg, 6b, 7b, and 11 by Suzuki
Coupling. General Procedure. A 5 mL process vial was charged with
the appropriate bromo derivative 4c, 4d, 6a, 7a, or 10c (1 mmol), the
appropriate boronic acid (5 mmol), Pd(OAc)2 (22.5 mg, 0.1 mmol),
PPh3 (78.6 mg, 0.3 mmol), 2 M Na2CO3 (2 mL, 4 mmol), EtOH
(1 mL), and DME (4 mL). The vessel was sealed under air and exposed
to microwave heating for 10 min at 150 ̊C. The reaction mixture was
thereafter cooled down to room temperature, diluted with AcOEt, and
filtered through a short plug of Celite. The solution was washed with
brine, dried over anhydrous sodium sulfate, and evaporated. The residue
was purified by flash chromatography on silica gel.
Example: N-(Adamantan-1-yl)-6-(furan-2-yl)-8-methoxy-4-oxo-1-
pentyl-1,4-dihydroquinoline-3-carboxamide (4g). Prepared in 97%
yield from 4d. White solid; mp 202ꢀ203 °C (EtOH). 1H NMR
(200 MHz, CDCl3): δ 9.50 (s, 1H), 8.58 (s, 1H), 8.38 (d, J = 1.4 Hz,
1H), 7.49ꢀ7.45 (m, 2H), 6.81 (d, J = 3.3 Hz, 1H), 6.51ꢀ6.49 (m, 1H),
4.50 (t, J = 7.6 Hz, 2H), 4.02 (s, 3H), 2.16ꢀ2.09 (m, 9H), 1.79ꢀ1.70 (m,
8H), 1.34ꢀ1.30 (m, 4H), 0.88 (t, J = 6.5 Hz, 3H). 13C NMR (100 MHz,
CDCl3): δ 175.9, 163.8, 152.6, 150.5, 149.6, 142.6, 128.0, 113.9, 112.1,
109.6, 106.5, 59.9, 56.42, 51.7, 41.8, 36.8, 31.2, 29.6, 28.6, 22.3, 13.9. IR
(Nujol): ν 1501, 1556, 1666, 3112 cm1. MS (ESI): m/z 489 [M + H]+,
511 [M + Na]+. Anal. (C30H36N2O4) C, H, N.
3-Ethyl-3,7-dihydro-7-oxo-2H-[1,4]oxazino[2,3,4-ij]quino-
line-6-carboxylic Acid (15). KF (88 mg, 1.5 mmol) and K2CO3
(415 mg, 3 mmol) were added to a solution of 14 (307 mg, 1 mmol, for
its preparation see Supporting Information) in dry DMF (2.7 mL), and
the mixture was heated to 150 °C for 3 h under a nitrogen atmosphere.
After cooling, the reaction is diluted with water and extracted with
DCM (3 ꢁ 5 mL). The organic phase was washed with brine, dried,
and evaporated to afford a residue, which was hydrolyzed with 10%
NaOH (33 mL) by refluxing for 1 h. After cooling to rt, 37% HCl was
added to pH 2 and the mixture was extracted with DCM (3 ꢁ 10 mL).
After usual workup of the organic solution, a residue was obtained,
which was filtered through a short column of silica gel (9:1 DCM/
MeOH) to give the pure compound 15 (142 mg, 55%) as a white
solid; mp 209ꢀ212 °C. 1H NMR (200 MHz, CDCl3): δ 14.53
(s, 1H), 8.73 (s, 1H), 8.00ꢀ7.95 (m, 1H), 7.47ꢀ7.29 (m, 2H), 4.57ꢀ4.50
(m, 1H), 4.39ꢀ4.32 (m, 2H), 2.00ꢀ1.77 (m, 2H), 1.04 (t, J = 7.1 Hz, 3H).
IR (CHCl3): ν 1616, 1718 cmꢀ1. MS (ESI): m/z 260 [M + H]+, 282
[M + Na]+. Anal. (C14H13NO4) C, H, N.
Synthesis of Compounds 5, 6a, and 20 by Amidation
Reaction. General Procedure. The appropriate carboxylic acid
(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 1-aminoada-
mantane (360 mg, 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 precipitated was collected by filtration
and washed with water and petroleum ether. Recrystallization from
EtOH gave the pure amide.
N-(Adamantan-1-yl)-6-bromo-1,2-dihydro-4-hydroxy-2-
oxo-1-pentylquinoline-3-carboxamide (7a). A solution of 18
(500 mg, 1.4 mmol) and 1-aminoadamantane (420 mg, 27.6 mmol) in
toluene (100 mL) was refluxed with azeotropical removal of water
during 1 h. Solvent was evaporated and the residue was filtered through a
short pad of silica gel (DCM as eluent) to give the title compound 7a
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Journal of Medicinal Chemistry
ARTICLE
(630 mg, 100%) as a white solid; mp 191ꢀ192 °C. 1H NMR (200 MHz,
CDCl3): δ 17.68 (s, 1H), 8.30 (d, J = 2.1 Hz, 1H), 7.68 (dd, J1 = 2.1 Hz,
J2 = 8.7 Hz, 1H), 7.15 (d, J = 8.7 Hz, 1H), 4.14 (t, J = 7.1 Hz, 2H), 2.13
(s, 9H), 1.70ꢀ1.56 (m, 8H), 1.42ꢀ1.36 (m, 4H), 0.90 (t, J = 7.2 Hz, 3H).
13C (100 MHz, CDCl3): δ 171.3, 170.3, 163.1, 152.3, 143.6, 138.3, 136.0,
128.0, 115.9, 115.0, 112,5, 97.8, 72.6, 52.6, 45.9, 42.3, 41.4, 36.6, 36.3, 36.2,
29.7, 29.4, 29.0, 27.3, 13.9. IR (CHCl3): ν 1632, 1724 cmꢀ1 MS (ESI): m/z
488 [M + H]+. Anal. (C25H31BrN2O3) C, H, N.
Binding Assays. CB1 and CB2 receptor binding assays were
performed exactly as described previously,13,14 using membranes of
cells overexpressing the human recombinant CB1 or CB2 receptors.
Enzymatic Assays. The inhibitory activity of varying concentra-
tions (0.1, 1, 5, and 10 μM) of the compounds on the hydrolysis of
[14C]-anandamide and [14C]-2-arachidonoyl-glycerol by rat brain
FAAH-like and COS-7 cell MAGL-like enzymatic activities was assessed
as described previously.22
filtered MTT stock solution (5 mg/mL in PBS, pH 7.4) and incubated
for additional 4 h at 37 °C to allow the yellow dye to be transformed
into blue crystals. Then, the MTT solution was carefully aspirated,
and formazan was extracted from the cells with 200 μL/well of
DMSO. The optical density was measured by 96-well ELISA plate
reader at a wavelength of 540 nm. Cell viability (%) was calculated as
([A]test/[A]control) ꢁ 100. Wells containing cell culture medium without
test compounds were used as control. Data are reported as mean ( SEM
(n = 6).
’ ASSOCIATED CONTENT
S
Supporting Information. Additional synthetic, spectro-
b
scopic/analytical, and biological data. This material is available
free of charge via the Internet at http://pubs.acs.org.
Formalin Test. The experimental procedures applied in the for-
malin 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.27 Nociceptive responses are divided into an early, short-lasting
first phase (0ꢀ7 min) caused by a primary afferent discharge produced
by the stimulus, followed by a quiescent period and then a second,
prolonged phase (15ꢀ60 min) of tonic pain. Mice received formalin
(1.25% in saline, 30 μL) in the dorsal surface of one side of the hindpaw.
Each mouse was randomly assigned to one of the experimental groups
(n = 8ꢀ10) and placed in a Plexiglas cage and allowed to move freely for
15ꢀ20 min. A mirror was placed at a 45° angle under the cage to allow
full view of the hindpaws. Lifting, favoring, licking, shaking, and flinching
of the injected paw were recorded as nociceptive responses. The
duration of those mentioned noxious behaviors were monitored by an
observer blind to the experimental treatment for periods of 0ꢀ10 min
(early phase) and 20ꢀ60 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.28 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).29 Treatments: groups
of 8ꢀ10 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.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: +39 0577 234308. Fax: +39 0577 234333. E-mail:
corelli@unisi.it.
’ ACKNOWLEDGMENT
The authors from the University of Siena gratefully acknowl-
edge financial support from Siena Biotech SpA and from the
University of Siena (PAR Progetti 2006). The authors from
CNR, Pozzuoli, are very grateful to Marco Allarꢀa for technical
assistance.
’ ABBREVIATIONS USED
CB1, cannabinoid type-1; CB2, cannabinoid type-2; DIPEA,
diisopropylethylamine; 3D-QSSAR, three-dimensional quantitative
structureꢀselectivity relationship; DCM, dichloromethane; DME,
1,2-dimethoxyethane; DMF, dimethylformamide; FAAH, fatty acid
amide hydrolase; MAGL, monoacylglycerol lipase; HOBt, 1-hydro-
xybenzotriazole; HBTU, O-benzotriazol-1-yl-N,N,N0,N0-tetra-
methyluronium hexafluorophosphate; SI, selectivity index; TEA,
triethylamine
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