Investigations on the 4-quinolone-3-carboxylic acid motif. 6. Synthesis and pharmacological evaluation of 7-substituted quinolone-3-carboxamide derivatives as high affinity ligands for cannabinoid receptors

Article abstract of DOI:10.1016/j.ejmech.2012.09.035

Within our studies on structure-activity relationships of 4-quinolone-3-carboxamides as cannabinoid ligands, a new series of compounds characterized by a fluoro or phenylthio group at 7-position and different substituents at N1 and carboxamide nitrogen were synthesized and evaluated for their binding ability to cannabinoid type 1 (CB1) and type 2 (CB2) receptors. Most of the compounds showed affinity for one or both cannabinoid receptors at nanomolar concentration, with K_i(CB1) and K_i(CB2) values ranging from 2.45 to >10,000 nM and from 0.09 to 957 nM, respectively. The N-(3,4-dichlorobenzyl)amide derivatives 27 and 40 displayed relatively low affinity, but high selectivity towards the CB1 receptor. Compounds 4 and 40, a CB2 and a CB1 ligand, respectively, behaved as partial agonists in the [ ³⁵S]GTPγS assay. They showed very low permeability through (MDCK-MDR1) cells and might, therefore, represent possible lead structures for further optimization in the search for cannabinoid ligands unable to cross the blood-brain barrier.

Full text of DOI:10.1016/j.ejmech.2012.09.035

European Journal of Medicinal Chemistry 58 (2012) 30e43
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Investigations on the 4-quinolone-3-carboxylic acid motif. 6. Synthesis and
pharmacological evaluation of 7-substituted quinolone-3-carboxamide
derivatives as high affinity ligands for cannabinoid receptors
Serena Pasquini a, Maria De Rosa a, Alessia Ligresti b, Claudia Mugnaini a, Antonella Brizzi a,
Nicola P. Caradonna c, Maria Grazia Cascio d, Daniele Bolognini d, Roger G. Pertwee d, Vincenzo Di Marzo b,
,
Federico Corelli a
*
a Dipartimento Farmaco Chimico Tecnologico, Università degli Studi di Siena, Via A. Moro, 53100 Siena, Italy
b Endocannabinoid Research Group, Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Via dei Campi Flegrei 34, 80078 Pozzuoli (Napoli), Italy
c Siena Biotech SpA, Strada del Petriccio e Belriguardo 35, 53100 Siena, Italy
d Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, Scotland, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Within our studies on structureeactivity relationships of 4-quinolone-3-carboxamides as cannabinoid
ligands, a new series of compounds characterized by a fluoro or phenylthio group at 7-position and
different substituents at N1 and carboxamide nitrogen were synthesized and evaluated for their binding
ability to cannabinoid type 1 (CB1) and type 2 (CB2) receptors. Most of the compounds showed affinity
for one or both cannabinoid receptors at nanomolar concentration, with Ki(CB1) and Ki(CB2) values
ranging from 2.45 to >10,000 nM and from 0.09 to 957 nM, respectively. The N-(3,4-dichlorobenzyl)
amide derivatives 27 and 40 displayed relatively low affinity, but high selectivity towards the CB1
receptor. Compounds 4 and 40, a CB2 and a CB1 ligand, respectively, behaved as partial agonists in the
Received 23 July 2012
Received in revised form
21 September 2012
Accepted 25 September 2012
Available online 3 October 2012
Keywords:
Synthesis
Quinolones
[
35S]GTP
gS assay. They showed very low permeability through (MDCK-MDR1) cells and might, therefore,
represent possible lead structures for further optimization in the search for cannabinoid ligands unable
to cross the bloodebrain barrier.
Cannabinoid ligands
Partial agonists
Peripheral ligands
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
a pharmacological rodent model about 10 years ago [2] and the
subsequent approval of rimonabant as an antiobesity drug in
The endocannabinoid system is involved in various pathological
conditions, such as pain, peripheral vascular diseases, appetite
enhancement/suppression, immune and inflammatory disorders,
cardiovascular and gastrointestinal disorders, and neurodegenera-
tive diseases [1]. Since the identification of the two cannabinoid
receptors CB1 and CB2, a wide spectrum of compounds with
different receptors affinity and selectivity have been reported. The
observed antiobesity effect of rimonabant (SR141716A) in
Europe under the registered name AcompliaÒ has led to a surge in
the search for novel CB1 antagonists/inverse agonists as new
therapeutic agents for the treatment of obesity and other metabolic
disorders, such as dyslipidemia, and type 2 diabetes [3]. Despite the
withdrawal of Acomplia for safety reasons (see below), CB1
receptor antagonists still have promise in other therapeutic areas,
including smoking and alcohol addiction as well as cognitive
impairment [4]. Additionally, selective/non selective agonists of
both cannabinoid receptor subtypes produce strong anti-
nociceptive effects in animal model of chronic, neuropathic, and
inflammatory pain and are intensively investigated as potential
new analgesic and anti-inflammatory agents [5]. Several studies
have suggested that also CB2 receptor antagonists/inverse agonists
may possess anti-inflammatory activity and could be useful as
immunomodulatory agents in the treatment of a variety of acute
and chronic disorders [6]. Moreover, a beneficial effect of CB2
agonists in various neurological disorders [7], including mouse
Abbreviations: BBB, bloodebrain barrier; CB1, cannabinoid type-1; CB2,
cannabinoid type-2; CHO, chinese hamster ovary cell; CNS, central nervous system;
DIPEA, diisopropylethylamine; DME, 1,2-dimethoxyethane; DMF, N,N-dime-
thylformamide;
HBTU,
O-benzotriazol-1-yl-N,N,N0,N0-tetramethyluronium
hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; MDCK-mdr1, MadineDarby
canine kidney cells transfected with the human MDR1 gene; MW, microwaves;
[
35S]GTP S, [35S]guanosine-50-O-(3-thiotriphosphate); SI, selectivity index.
g
* Corresponding author.
E-mail address: corelli@unisi.it (F. Corelli).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.09.035

S. Pasquini et al. / European Journal of Medicinal Chemistry 58 (2012) 30e43
31
focal ischaemia and reperfusion brain injury, has been demon-
strated [8].
Unfortunately, compounds targeting the endocannabinoid
O
O
O
COOH
i
N
H
system are not devoid of unwanted side effects (such as muscle
weakness, palpitations, dry mouth, disorientation, altered time
perception, impairment of memory, tremor, confusion, paranoia
and hallucination, nausea and vomiting), many of which are
thought to be due to activation of central CB1 receptors rather than
peripheral CB1 or CB2 receptors [9]. The European Medicines
Agency (EMEA) has recommended the suspension of marketing
authorization for rimonabant as of 23 October 2008 owing, among
other reasons, to an approximate doubling of the risk of psychiatric
disorders, including anxiety, depression, and suicidal ideation, in
obese or overweight patients taking Acomplia compared to those
taking placebo. As a result, the development of compounds selec-
tively targeting CB2 receptor or peripherally acting on CB1 receptor
may represent in principle a strategy towards safer cannabinoid
drugs [10].
Recently, we have described a large family of 4-quinolone-3-
carboxamide derivatives endowed with high CB2 receptor affinity
and selectivity over CB1 receptor and characterized by different
functional profiles [11e13]. On the other hand, ligands with high
CB1 affinity, even if devoid of receptor selectivity, were identified as
well, possessing a C7-substituent on the 4-quinolone nucleus
(compounds 1 and 2, Chart 1) [11]. The structureeactivity rela-
tionships (SAR) emerging from our previous studies suggested that
the 4-quinolone-3-carboxamide scaffold could be optimized
through selected chemical modifications in order to obtain either
CB1 or CB2 receptor ligands with improved affinity and/or selec-
tivity. At least in one instance, we also showed that the replacement
of the 1-adamantyl group by a fenchyl moiety can greatly enhance
CB1 affinity [12].
F
N
H
F
N
H
26
25
ii
O
O
O
O
iii
N
H
N
H
F
N
R
PhS
N
R
3: R= methyl
4: R= ethyl
5: R= 1-propyl
6: R= 1-butyl
7: R= 1-pentyl
8: R= 3-methylbenzyl
9: R= 4-acetoxy-1-butyl
14: R= methyl
15: R= ethyl
16: R= 1-propyl
17: R= 1-butyl
18: R= 1-pentyl
19: R= 3-methylbenzyl
20: R= 4-acetoxy-1-butyl
10: R= 2-(N,N-dimethylamino)ethyl
11: R= 2-methoxyethyl
12: R= ethenyl
21: R= 2-(N,N-dimethylamino)ethyl
22: R= 2-methoxyethyl
iv
iv
23: R= 4-phenylthio-1-butyl
24: R= 4-hydroxy-1-butyl
13: R= 4-hydroxy-1-butyl
Scheme 1. Synthesis of fenchylamides 3e24. Reagents and conditions: (i) (1R,2S)-
(ꢀ)-fenchylamine, HBTU, HOBt, DIPEA, DMF, rt, 4 h; (ii) appropriate alkyl iodide, K2CO3,
DMF, MW, 100 ꢁC, 5 min; (iii) PhSH, K2CO3, MW, 150 ꢁC, 15 min; (iv) 6 N HCl, EtOH,
100 ꢁC, 2 h.
substituents were expected to increase potency and hydrophilicity,
respectively.
On the basis of these findings and considering the limited
number of C7-substituted 4-quinolone-3-carboxamides reported
so far in the literature as cannabinoid receptors ligands [11,14,15],
we decided to investigate in detail the cannabinoid receptors
affinity/selectivity profile of C7-substituted 4-quinolones to see if
the combination of substitution at this particular position with
appropriate modifications of the residues on the carboxamide and
N1 nitrogen atoms, could drive CB1 receptor selectivity. Thus,
taking 1 and 2 as prototypical structures, we designed new 4-
quinolones (Chart 1), bearing a fluoro or a phenylthio substituent
at C7 position, while different groups, such as 2-fenchyl, 3,4-
dichlorobenzyl, 1-piperidinyl, isopropyl, methoxy, dimethylamino,
(rac)-1-hydroxy-2-butyl, (S)-pirrolidine-2-carbony1, 2-(morpho-
lin-4-yl)-ethyl, benzyloxy, were introduced on the carboxamide
nitrogen. The N1 pentyl chain was retained or replaced with other
moieties, such as lower alkyl chains and 3-methylbenzyl, 2-
2. Chemistry
Initially,
a series of 4-quinolone-3-fenchylamides (3e24)
bearing a fluoro or a phenylthio group at C7 position and various
alkyl chain at N1 position were prepared (Scheme 1). The fenchy-
lamide 25 was obtained by coupling reaction between compound
26 [16] and (1R,2S)-(ꢀ)-fenchylamine, prepared according to
a literature procedure [17]. The final 7-fluoro-substituted products
3e11 were prepared from 25 by alkylation reaction using the
appropriate alkyl halides. Under the basic conditions used for the
alkylation reaction, compound 10 underwent a concurrent
b-
elimination of dimethylamine to give 12 as a by-product. Finally,
basic hydrolysis of 9 yielded compound 13. The 7-phenylthio
derivatives 14e22 were prepared from the corresponding 7-
fluoro derivatives through nucleophilic displacement using thio-
phenol under microwave (MW) irradiation. When this reaction was
applied to substrate 9, compound 23 was isolated as a result of the
nucleophilic displacement of both the 7-fluoro group and the
acetoxy moiety on the side chain at N1 position. Ester hydrolysis of
20 afforded 24.
methoxyethyl,
4-hydroxybutyl,
2-N,N-dimethylaminoethyl
groups. The substituents at the nitrogen atoms were chosen with
the aim to modulate both the pharmacological and physicochem-
ical profile of the compounds, as lipophilic groups or more polar
O
O
O
N
O
R2
N
H
6
7
N
H
R
R3
N
R1
8
R= 6-alkyl group: CB2 selective agonists
New Quinolones
3-24, 27, 30-40, 44, and 50
R= 6-(heteroaryl group): CB2 selective inverse agonists
R= 8-alkoxy group: CB2 selective inverse agonists
R= 1: 7-F; 2: 7-PhS group: unselective ligands
For R1-R3, seeTable 1 and Table 2
Chart 1. Prototypical structures (left) and novel 7-substituted analogues (right).

32
S. Pasquini et al. / European Journal of Medicinal Chemistry 58 (2012) 30e43
Cl
3. Results and discussion
Cl
3.1. In vitro pharmacology and SAR
O
O
O
i
COOH
The binding affinities (Ki values) of compounds 3e25, 27, and
30e40 for human recombinant CB1 and CB2 receptors are reported
in Tables 1 and 2. The tested compounds were evaluated in parallel
with lead compounds 1 and 2 as well as SR144528 [20] and
rimonabant [21] as reference CB2 and CB1 ligands, respectively, as
previously described [22]. The effects of compounds 4 and 40 on
N
H
Br
N
H
Br
N
H
28
29
Cl
Cl
ii
[
35S]guanosine-50-O-(3-thiotriphosphate) ([35S]GTP
gS) binding to
Cl
Cl
membranes from chinese hamster ovary cells (CHO cells) trans-
fected with human CB1 or CB2 receptors as well as from the mouse
O
N
O
O
O
iii
N
N
H
H
Br
N
Table 1
Me
Me
CB1 and CB2 receptor affinity values for compounds 3e8, 10e19, 21e25.a,b
27
30
O
O
Scheme 2. Synthesis of 3,4-dichlorobenzylamides 27 and 30. Reagents and conditions:
(i) 3,4-dichlorobenzylamine, HBTU, HOBt, DIPEA, DMF, rt, 20 h; (ii) 1-iodopentane,
K2CO3, DMF, 90 ꢁC, 1.5 h; (iii) phenylboronic acid, Pd(OAc)2, PPh3, 1 M Na2CO3, DME,
EtOH, MW, 150 ꢁC, 5 min.
N
H
X
N
R1
Compound 27 was prepared starting from 28 [18] (Scheme 2),
through
a reaction sequence entailing amidation with 3,4-
Compd
X
R1
ClogPc CB1d,e
CB2g,e
SIh
dichlorobenzylamine to give 29, followed by N-alkylation with
pentyl iodide to provide 30. Suzuki coupling of 30 with phenyl-
boronic acid led to 27.
Kif (nM) Ki (nM)
3
4
5
6
7
8
10
F
F
F
F
F
F
F
Methyl
Ethyl
1-Propyl
1-Butyl
1-Pentyl
3-Methylbenzyl
2-(N,N-Dimethyl)
aminoethyl
2-Methoxyethyl
1-Ethenyl
3.79
4.12
4.61
5.03
5.45
5.76
3.79
>10000 93.9
106
131
57
17
17
959.7
193.0
14.4
4.0
>10000 31.5
178.7
7.3
2.9
0.8
0.3
A
second series of 1-pentyl-7-phenylthio-4-quinolone-3-
carboxamide derivatives 31e40 was then prepared as reported in
Scheme 3. Treatment of 41 [19] with thiophenol in the presence of
potassium carbonate under MW irradiation allowed, at the same
time, the introduction of the phenylthio group at C7 by displace-
ment of the fluorine atom and the removal of the ester moiety at C3
position to afford the 3-carboxy-7-(phenylthio)-4-quinolone 42,
which was then reacted with various amines to give the final
compounds 31e40.
317
22
7.9
11
12
13
14
15
16
17
18
19
21
F
F
F
3.63
4.12
3.83
5.73
6.07
6.56
6.97
7.39
7.70
5.73
296.8
3572.6
398.5
1866.9
181.3
11.0
4.7
2.45
669.3
28.8
5.5
54
235
26
117
42
31
31
27.2
5
15.2
15.2
16.0
4.4
0.4
0.1
0.09
139.1
1.3
4-Hydroxy-1-butyl
PhS Methyl
PhS Ethyl
PhS 1-Propyl
PhS 1-Butyl
PhS 1-Pentyl
O
O
N
PhS 3-Methylbenzyl
PhS 2-(N,N-Dimethyl)
aminoethyl
PhS 2-Methoxyethyl
PhS 4-Phenylthio-1-butyl 8.52
23
COOEt
COOH
i
22
23
24
25
1
2
5.58
23.8
1.1
22
>1545
15
>10
38
3.3
F
N
PhS
>10000 6.5
38.6 2.5
>10000 957.1
30.5
5.3
Me
Me
PhS 4-Hydroxy-1-butyl
F
5.77
3.00
4.44
6.38
7.77
6.28
H
41
42
0.8
1.6
5.4
790
ii
SR144528i
Rimonabantj
>2820
12.0
>522
0.015
a
Data represent mean values for at least three separate experiments performed
O
O
in duplicate and are expressed as Ki (nM).
31: R = isopropyl
32: R = N,N-dimethylamino
33: R = ( )-1-hydroxy-2-butyl
34: R = (S)-1-pyrrolidinyl-2-carboxamide
35: R = 3-quinuclidinyl
R
b
The binding affinities of reference compounds were evaluated in parallel with
N
H
test compounds under the same conditions.
c
PhS
N
Calculated with ChemBioDraw Ultra 11.0.
CB1: human cannabinoid type 1 receptor.
For both receptor binding assays, the new compounds were tested using
d
Me
e
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-55940).
36: R = 1-piperidinyl
37: R = N-benzyloxy
38: R = 2-(4-morpholinyl)ethyl
39: R = methoxy
40: R = 3,4-dichlorobenzyl
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
CB2: human cannabinoid type 2 receptor.
SI: selectivity index for CB2, calculated as Ki(CB1)/Ki(CB2) ratio.
CB2 reference compound.
CB1 reference compound.
h
Scheme 3. Synthesis of 7-phenylthio derivatives 31e40 bearing different amide
groups. Reagents and conditions: (i) PhSH, K2CO3, DMF, MW, 150 ꢁC, 15 min; (ii)
appropriate amine, HBTU, HOBt, DIPEA, DMF, rt, 18 h.
i
j

S. Pasquini et al. / European Journal of Medicinal Chemistry 58 (2012) 30e43
33
Table 2
CB1 and CB2 receptor affinity values for compounds 27 and 30e40.a,b
O
N
O
R1
R2
Compd
R1
R2
ClogPc
CB1d,e
CB2g,e
Ki (nM)
SIh
Kif (nM)
Cl
Cl
Cl
Cl
27
Phenyl
6.97
390
>10000
0.04
H
N
30
Br
6.13
660.0
130.0
5
H
N
31
32
PhS
PhS
5.21
4.79
20.9
86.6
14.8
77.5
1.4
1.1
N
H
N
N
OH
33
34
PhS
PhS
4.84
3.87
58.2
19.9
2.9
6.5
N
H
CONH
N
2
1490.0
230.0
N
35
PhS
4.74
151.1
89.1
1.7
N
H
36
37
PhS
PhS
5.34
6.48
28.9
12.9
13.3
19.3
2.2
0.7
N
O
N
H
N
H
O
38
39
PhS
PhS
4.15
4.75
63.2
490
118.7
340
0.5
1.4
N
N
H
O
N
H
Cl
Cl
40
44
PhS
7.40
3.71
420
>10000
0.04
H
N
F
>10000
>10000
e
N
N
H
(continued on next page)

34
S. Pasquini et al. / European Journal of Medicinal Chemistry 58 (2012) 30e43
Table 2 (continued )
Compd
R1
R2
ClogPc
CB1d,e
CB2g,e
Ki (nM)
SIh
Kif (nM)
50
PhS
5.65
>10000
>10000
e
N
N
H
1
2
4.44
6.38
7.77
6.28
30.5
5.3
>2820
12.0
0.8
1.6
5.4
38
3.3
>522
0.015
SR144528i
Rimonabantj
790
a
Data represent mean values for at least three separate experiments performed in duplicate and are expressed as Ki (nM).
The binding affinities of reference compounds were evaluated in parallel with test compounds under the same conditions.
Calculated with ChemBioDraw Ultra 11.0.
b
c
d
e
CB1: human cannabinoid type 1 receptor.
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-55940).
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
CB2: human cannabinoid type 2 receptor.
SI: selectivity index for CB2, calculated as Ki(CB1)/Ki(CB2) ratio.
CB2 reference compound.
CB1 reference compound.
h
i
j
spleen (which expresses selectively CB2 receptors) were also
assessed.
On the other hand, the insertion of more polar chains, pos-
sessing -systems (8, 12, 19), H-bond acceptor (11, 22) or donor (13,
p
All the 7-substituted 4-quinolone-3-fenchylamides reported in
Table 1 showed affinity for one or both cannabinoid receptors at
nanomolar concentration, with Ki(CB1) and Ki(CB2) values
ranging from 2.45 to >10,000 nM and from 0.09 to 957 nM,
respectively. Accordingly, the values of selectivity index
[SI ¼ Ki(CB1)/Ki(CB2)] varied between 5 and >1545. As a result, all
these compounds can be considered CB2 receptor ligands
endowed with moderate to very high CB2 selectivity, since no
compound elicited higher affinity for CB1 receptor than for CB2
receptor. Several compounds compared well in terms of affinity
with rimonabant and SR144528, taken as CB1 and CB2 reference
ligands, respectively.
The replacement of the adamantyl group by the fenchyl moiety
on the amide nitrogen led to a significant improvement in binding
ability (compare 7 and 18 with the lead compounds 1 and 2,
respectively), thereby confirming not only the general hypothesis
that the amide substituent plays a crucial role in determining
receptor affinity and selectivity, but also that the fenchyl group is
somewhat superior to the adamantyl one in this regard. This effect
may be related to hydrophobicity, as 7 and 18 display ClogP values
higher than those of 1 and 2, respectively, by approximately 1
unit.
24) groups, or a protonatable amino group (10, 21) slightly reduced
binding affinity, but in some cases caused an improvement in CB2
selectivity. Therefore, one might argue that the pocket accommo-
dating the N1 chain better tolerates more polar/less hydrophobic
groups in the CB2 receptor compared to the CB1 receptor. In
particular, it is worth noting that some polar chains, such as 2-
methoxyethyl and 2-N,N-dimethylaminoethyl chains, at N1 posi-
tion in 7-fluoro derivatives (compounds 11 and 10, respectively)
were able to significantly reduce lipophilicity, leading to
compounds with ClogP values lower than 4.0, with only modest
decrease in CB2 receptor affinity (one order of magnitude or less).
In the 7-phenylthio series, the insertion of these chains at N1
position did not affect CB2 affinity, but only slightly reduced affinity
for the CB1 receptor (compare 21 and 22 with 2).
Finally, the very good affinity and selectivity for CB2 receptors
shown by compound 23 leads us to infer that the presence of
a bulky residue at N1 side chain might help the ligand discriminate
between the two cannabinoid receptor types.
Among this first series of compounds, 18, bearing the N1-pentyl
chain and 7-phenylthio substituent, exhibited the highest affinity
values for both receptors, and is one of the highest affinity canna-
binoid ligands ever described. Accordingly,
a second set of
Direct comparison of 7-fluoro and 7-phenylthio derivatives
unequivocally demonstrated that, all the other substituents being
the same, 7-phenylthio compounds exhibited higher affinity for
both receptors, whilst selectivity met with only marginal fluctua-
tions between the compounds couples. These findings suggest that
the binding sites of either receptor can take in even relatively bulky
substituents, not only at position 6 as already reported [11,12], but
also at position 7.
Within both series (7-fluoro and 7-phenylthio derivatives), the
introduction at N1 position of progressively longer alkyl chains (3e
7 and 14e18) led to increasingly more potent ligands at both
receptors. However, CB1 affinity rose more than CB2 affinity,
resulting in a decrease of CB2 selectivity. Conversely, the presence
of the 3-methylbenzyl group in 8 restored the CB2 selectivity, in
good agreement with previous findings [15], whilst in compound
19 receptor affinity and selectivity were substantially lost, likely as
a result of the presence in the molecule of several bulky groups that
hamper drug interaction with both receptor types.
compounds (31e40) was designed in a way to be characterized by
the same substitution patterns at C7 and N1 as 18, but differing for
the residues on the amide nitrogen (Table 2). Such a structural
modification generally produced substantially unselective ligands
(SI for compounds 31e39 varied between 6.5 and 0.5) possessing
good affinity for CB1 receptor (Ki in the range 1490e12.9 nM) and/
or CB2 receptor (Ki in the range >10,000e13.3 nM). To our satis-
faction, compound 40 emerged as a CB1 ligand showing significant
affinity (420 nM) and selectivity over CB2 receptor (SI ¼ 0.04).
When a 7-phenyl group was substituted for the 7-phenylthio
moiety of 40, compound 27 was obtained with a completely
superimposable binding profile. Noteworthy, the structural modi-
fications of the amide group that reduced lipophilicity proved to be
unfavourable in terms of receptor affinity and selectivity. Thus,
although the affinity values for some of the compounds (e.g. 31, 33,
36, 37) were not negligible per se, the drop in CB1 and CB2 affinity
compared to 18 was approximately 5e25 and 150e200 times,
respectively.

S. Pasquini et al. / European Journal of Medicinal Chemistry 58 (2012) 30e43
35
Table 3
120
100
80
60
40
20
0
CP 55940
EC50 (nM) and Emax (%) values with 95% confidence limits shown in brackets for
compound 40
compounds 4, 40 and CP-55940 in [35S]GTP
gS assay performed in hCB1 or hCB2 CHO
cell membranes.
Compd
Cells
EC50 (nM)
(95% CI)
Emax (%)
(95% CI)
45.0a
(38.4 & 51.5)
100
n
4
hCB2-CHO
hCB2-CHO
hCB1-CHO
hCB1-CHO
17.2
(4.3 & 69.7)
7.4
(1.6 & 34.1)
790.6
(289.7 & 2158)
0.7
10
10
12
12
CP-55940
40
(79.9 & 130.1)
55.8a
(45.4 & 66.2)
100
(91.4 & 112.0)
CP-55940
(0.3 & 1.6)
a
Results are expressed as percentage of the maximum response to CP-55940
which it induced at a concentration of ꢀ7.
3.2. Functional activity at CB1 and CB2 receptors in vitro
-20
Compounds 4 and 40, taken as representatives of relatively
potent/selective CB2 and CB1 ligands, respectively, were subject to
further in vitro pharmacological evaluation. Thus, with the aim of
investigating whether these two compounds were able to activate
or antagonize cannabinoid CB1 (for compound 40) or CB2 (for
-11 -10 -9 -8 -7 -6 -5 -4
Log M
Fig. 2. Mean log concentrationeresponse curve of compound 40 and CP-55940. Each
symbol represents the mean percentage change in binding of [35S]GTP
gS to hCB1-CHO
compound 4) receptors, [35S]GTP
gS binding assays were performed
cell membranes ꢂ SEM (n ¼ 12).
using membranes obtained from CHO cells stably transfected with
hCB1 or hCB2 receptors, according to previously described proce-
dures [23a]. The results of this functional assay are reported in
Table 3. We found that compound 4, at concentrations ranging from
A tiered screening approach was used on metabolic evaluation of
compounds. Primary screening was performed to assess the
stability towards cytochrome P450 3A4 (CYP3A4), deeming this
isoform responsible for the majority of known drug oxidation
reactions and being the most abundant P450 enzyme in human
liver and small intestine. Both compounds underwent a CYP3A4-
dependent metabolism, though in a different way. In particular,
while compound 4 was completely depleted, compound 40 showed
a remaining percentage of 25% after 1 h incubation in the presence
of recombinant hCYP3A4. The percentage of remaining compound
was used to classify compounds as stable or unstable with a cut-off
threshold of 20%. This threshold was selected by reference to
typical CYP3A4 substrates, using human in vivo pharmacokinetics
as starting point. In order to assess whether this different behaviour
towards hCYP3A4 was also reflected towards CYP450-dependent
metabolism (Phase I metabolism), human and rat liver micro-
somes were used to estimate the metabolic stability of the
compounds. Both compounds exhibited very high in vitro intrinsic
clearance (Clint) in both microsomal species, levelling the differ-
ences towards CYP3A4, indicating the involvement of other iso-
forms in the metabolism of compound 40. Based on our previous
1 nM up to 10
membranes (Table 3). Similarly, compound 40 at concentrations
ranging from 1 nM up to 30 S binding to
M, stimulated [35S]GTP
m gS binding to hCB2-CHO cell
M, stimulated [35S]GTP
m
g
hCB1-CHO cell membranes (Table 3). Both compounds 4 and 40
showed Emax values significantly lower that that of the full CB1/CB2
agonist, CP-55940, thus behaving as partial agonists at CB2 and CB1
receptors, respectively (Figs. 1 and 2).
3.3. ADME properties evaluation
Next, selected ADME properties of compounds 4 and 40 were
evaluated (Table 4), according to previously reported protocols [24].
120
CP 55940
compound 4
100
80
60
40
20
0
Table 4
Selected ADME properties of compounds 4 and 40.
4
40
Metabolic stability (%)a
Plasma stability at 3 h (%)b
Clint (human)c
1
92
25
100
>700
>700
m
m
L/min/mg
L/min/mg
>700
>700
m
m
L/min/mg
L/min/mg
Clint (rat)c
MDCK-MDR1 ABd
MDCK-MDR1 BA/ABe
0.17ꢃ10ꢀ6 cm/s
0.14ꢃ10ꢀ6 cm/s
1.0
0.4
a
Metabolic stability was determined as percentage remaining after incubation
for 1 h with recombinant hCYP3A4.
Plasma stability was determined as percentage remaining after incubation for
3 h with fresh rat plasma.
-20
b
-11 -10 -9 -8 -7 -6 -5 -4
Log M
c
In vitro intrinsic clearance from human and rat liver microsomal stability assay.
d
MS-based quantification of apical
M across contiguous monolayers of MDCK-MDR1 cells.
Ratio of (basolateral / apical) to (apical / basolateral) transfer rate of a test
compound at 10 M across contiguous monolayers of MDCK-MDR1 cells.
/ basolateral transfer rate of a test
Fig. 1. Mean log concentrationeresponse curve of compound 4 and CP-55940. Each
compound at 10 m
e
symbol represents the mean percentage change in binding of [35S]GTP
gS to hCB2-CHO
cell membranes ꢂ SEM (n ¼ 10).
m

36
S. Pasquini et al. / European Journal of Medicinal Chemistry 58 (2012) 30e43
findings with other 4-quinolone derivatives, the high metabolic
instability displayed by compounds 4 and 40 might be related to
the presence of the aliphatic chain at N1 [25]. However, further
in vitro studies, such as the identification of the main metabolic
pathways, would be required to provide feedback for chemistry
strategy refinement and overcome metabolic issues in the subse-
quent phase of lead optimization. Despite the presence of structural
alerts, both compounds showed high stability in plasma, as the
percentage of compound remaining after incubation for 3 h with
fresh rat plasma was 92% and 100% for 4 and 40, respectively.
Subsequently, compounds 4 and 40 were tested in Madine
Darby canine kidney cells transfected with the human mdr1 gene
(MDCK-MDR1), a model of bloodebrain barrier (BBB) penetration,
which is widely used to predict in vivo permeability of compounds
[26], including CNS penetration of CB1 antagonists [10a]. Although
the compounds are not substrates for P-gp, as they presented low
efflux ratios, their extremely low cell permeability (0.14e
0.17 10ꢀ6 cm/s) suggests poor probability of rapid BBB penetration.
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 105e1500. Melting points were
determined on a Gallenkamp apparatus and are uncorrected.
Microwave irradiations were conducted using a CEM Discover
Synthesis Unit (CEM Corp., Matthews, NC). Elemental analyses
were performed on a PerkineElmer 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 ꢃ 100 mm, 5
mm)
reversed phase column; method: 86% (v/v) of MeOH in H2O, iso-
cratic, flow rate of 1 mL/min, UV detector, 254 nm. The purity of
each compound was ꢄ95% in either analysis.
4. Conclusions
5.2. General procedure for the amidation reaction
In conclusion, two sets of 7-substituted 4-quinolone-3-
carboxamide derivatives were synthesized as cannabinoid ligands
possibly selective for either CB1 or CB2 receptor with the aim of
expanding the SAR within this family of compounds. The results of
in vitro evaluation of the new compounds, in terms of receptor
affinity and selectivity, demonstrated our working hypothesis, that
is, chemical modulation of the 4-quinolone scaffold can be
exploited to design potent and/or selective CB1 and CB2 receptor
ligands. In this respect, the replacement of the adamantyl moiety
with the fenchyl one on the carboxamide nitrogen generally
enhanced ligand potency, with marginal effects on receptor selec-
tivity. Conversely, when the 3,4-dichlorobenzyl group was
substituted for the adamantyl one, a marked increase in CB1
selectivity was observed, though at the expense of potency, and
compounds 27 and 40 emerged as the first, to our knowledge, CB1
selective ligands in the 4-quinolone-3-carboxamide family.
Chemical modulation of the N1 position by the insertion of more
polar chains allowed to reduce the high lipophilicity of the hit
compounds 1 and 2 to a more acceptable level (ClogP < 4) with no
dramatic loss in both affinity and selectivity values. This finding
might help design new cannabimimetic 4-quinolones possessing
more drug-like properties, an issue that so far has been only
partially addressed.
To a solution of the appropriate acid derivative (1 mmol) in dry
DMF (5 mL) O-benzotriazol-1-yl-N,N,N0,N0-tetramethyluronium
hexafluorophosphate (HBTU, 2 mmol) was added followed by
1-hydroxybenzotriazole (HOBt, 1 mmol), diisopropylethylamine
(DIPEA, 1.5 mmol) and the appropriate amine (1.2 mmol). The
mixture was stirred under N2 atmosphere at room temperature for
30 min and DIPEA (1.5 mmol) was added again. The reaction
mixture was stirred at room temperature for 18 h, poured into
water and extracted with dichloromethane. The organic layer was
washed with brine, dried over anhydrous Na2SO4 and evaporated to
dryness. The crude product, unless otherwise indicated, was
recrystallized from EtOH.
5.2.1. N-(1,3,3-Trimethylbicyclo[2.2.1]heptan-2-yl)-7-fluoro-4-oxo-
1,4-dihydroquinoline-3-carboxamide (25)
96% yield. White solid. Mp 279e280 ꢁC from EtOAc. 1H NMR
(400 MHz, CDCl3):
d
10.68 (d, J ¼ 9.3 Hz, 1H), 8.77 (d, J ¼ 6.0 Hz, 1H),
8.44e8.41 (m, 1H), 7.30e7.28 (m, 1H), 7.19 (m, 1H), 7.14e7.09 (m,
1H), 3.86 (d, J ¼ 9.5 Hz, 1H), 1.78e1.62 (m, 4H), 1.49e1.43 (m, 1H),
1.24e1.19 (m, 2H), 1.17 (s, 3H), 1.01 (s, 3H), 0.85 (s, 3H). 13C NMR
(100 MHz, CDCl3):
d 177.0, 166.6, 143.5, 140.8, 129.4, 123.5, 114.5,
111.6, 104.4, 104.2, 64.3, 48.9, 48.2, 47.7, 39.7, 31.2, 27.7, 26.0, 21.8,
19.8. MS (ESI): m/z 365 [M þ Na]þ (100), 343 [M þ H]þ (70). IR
The very low permeability elicited by compound 40 across the
MDCK-MDR1 cells does not guarantee per se reduced CNS exposure
in the absence of active asymmetric efflux by P-gp at BBB level.
Being not a substrate for P-gp, 40 is expected to reach the CNS once
a steady state between plasma and brain is established. Bearing
these considerations in mind, compound 40 can constitute
a possible starting point for further optimization en route to
develop CB1 receptor ligands with low CNS penetration and hence
low liability to psychotropic side effects.
(nujol):
n
2921, 1631, 1462 cmꢀ1. [
a
]
20: ꢀ70.44ꢁ (dichloromethane).
D
Anal. Calcd. for C20H23FN2O2: C, 70.15; H, 6.77; N, 8.18. Found: C,
70.32; H, 6.68; N, 8.32.
5.2.2. N-(3,4-Dichlorobenzyl)-7-bromo-4-oxo-1,4-dihydroquinoline-
3-carboxamide (29)
60% yield. Pink solid. Mp 185e186 ꢁC. 1H NMR (200 MHz,
DMSO):
d
12.71 (s, 1H), 10.31 (t, J ¼ 6.0 Hz, 1H), 8.77 (s, 1H), 8.13 (d,
J ¼ 8.7 Hz, 1H), 7.91e7.87 (m, 1H), 7.63e7.54 (m, 3H), 7.32e7.28 (m,
1H), 4.52 (d, J ¼ 6.0 Hz, 2H). MS (ESI): m/z 449 [M þ Na]þ (100). IR
5. Experimental
(nujol): n . Anal. Calcd. for
3390, 2929, 1647, 1622 cmꢀ1
C17H11BrCl2N2O2: C, 47.92; H, 2.60; N, 6.57. Found: C, 47.71; H, 2.52;
5.1. General
N, 6.76.
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 used for flash
chromatography (23e400 mesh). IR spectra were recorded on
a PerkineElmer 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 or at 400
5.2.3. N-Isopropyl-4-oxo-1-pentyl-7-(phenylthio)-1,4-
dihydroquinoline-3-carboxamide (31)
75% yield. Light yellow solid. Mp 158e159 ꢁC. 1H NMR (200 MHz,
CDCl3):
d
9.92 (d, J ¼ 7.5 Hz, 1H), 8.63 (s, 1H), 8.32 (d, J ¼ 8.7 Hz, 1H),
7.58e7.42 (m, 5H), 7.30e7.20 (m, 1H), 7.05e6.96 (m, 1H), 4.35e4.17
(m, 1H), 3.94 (t, J ¼ 7.6 Hz, 2H), 1.71e1.60 (m, 2H), 1.26e1.16 (m,
10H), 0.86 (t, J ¼ 6.7 Hz, 3H). 13C NMR (100 MHz, CDCl3):
d 176.1,

S. Pasquini et al. / European Journal of Medicinal Chemistry 58 (2012) 30e43
37
163.9,147.5,145.7,139.4,134.7,131.1,129.9,129.4,127.6,125.5,123.9,
(m, 5H), 7.25e7.20 (m, 1H), 6.99e6.95 (m, 1H), 3.94 (t, J ¼ 8.8 Hz,
2H), 2.89e2.84 (m, 4H), 1.75e1.63 (m, 6H), 1.45e1.43 (m, 2H), 1.25e
1.15 (m, 4H), 0.86 (t, J ¼ 4.6 Hz, 3H). 13C NMR (100 MHz, CDCl3):
112.9, 112.1, 54.1, 41.0, 28.5, 22.8, 22.1, 18.7, 13.8. MS (ESI): m/z 447
[M þ K]þ (60), 431 [M þ Na]þ (80), 409 [M þ H]þ (50). IR (CHCl3):
n
3019, 2360, 1652 cmꢀ1. Anal. Calcd. for C24H28N2O2S: C, 70.55; H,
6.91; N, 6.86. Found: C, 70.81; H, 6.78; N, 6.59.
d 175.7, 162.2, 147.6, 145.9, 139.3, 134.7, 131.0, 129.9, 129.4, 127.7,
125.4, 123.9, 112.9, 112.0, 56.9, 54.1, 28.5, 25.0, 23.5, 22.1, 13.8. MS
(ESI): m/z 472 [M þ Na]þ (50), 450 [M þ H]þ (100). IR (CHCl3):
n
5.2.4. N0,N0-Dimethyl-4-oxo-1-pentyl-7-(phenylthio)-1,4-
dihydroquinoline-3-carbohydrazide (32)
3019, 2360, 1663, 1223 cmꢀ1. Anal. Calcd. for C26H31N3O2S: C, 69.46;
H, 6.95; N, 9.35. Found: C, 69.64; H, 7.05; N, 9.06.
81% yield. White solid. Mp 173e174 ꢁC. 1H NMR (200 MHz,
CDCl3):
d
10.91 (s,1H), 8.66 (s,1H), 8.38e8.29 (m,1H), 7.59e7.43 (m,
5.2.9. N-(Benzyloxy)-4-oxo-1-pentyl-7-(phenylthio)-1,4-
dihydroquinoline-3-carboxamide (37)
5H), 7.26e7.20 (m, 1H), 6.97 (d, J ¼ 4.3 Hz, 1H), 3.94 (t, J ¼ 7.8 Hz,
2H), 2.77 (s, 6H), 1.70e1.64 (m, 2H), 1.27e1.15 (m, 4H), 0.87 (t,
31% yield. White solid. Mp 164e165 ꢁC. 1H NMR (200 MHz,
J ¼ 6.7 Hz, 3H). 13C NMR (100 MHz, CDCl3):
d
175.7, 162.8, 147.7,
CDCl3): d 12.25 (s,1H), 8.69 (s, 1H), 8.37e8.31 (m,1H), 7.57e7.22 (m,
134.8, 134.5, 130.1, 130.0, 129.9, 129.5, 129.3, 127.6, 123.9, 113.1,
11H), 6.96 (s, 1H), 5.03 (s, 2H), 3.98 (t, J ¼ 7.6 Hz, 2H), 1.72e1.57 (m,
112.8, 54.2, 47.9, 28.5, 28.5, 22.1, 13.8. MS (ESI): m/z 432 [M þ Na]þ
2H), 1.28e1.16 (m, 4H), 0.87 (t, J ¼ 6.7 Hz, 3H). 13C NMR (100 MHz,
(20), 410 [M þ H]þ (100). IR (CHCl3):
n
3019, 2360, 1606, cmꢀ1. Anal.
CDCl3): d 175.1, 163.3, 147.3, 146.2, 139.3, 135.6, 134.7, 131.0, 130.0,
Calcd. for C23H27N3O2S: C, 67.45; H, 6.65; N, 10.26. Found: C, 67.71;
H, 6.58; N, 10.01.
129.5, 128.9, 128.5, 127.8, 125.1, 124.1, 112.9, 111.2, 78.5, 63.8, 54.3,
30.8, 28.5, 22.1,13.8. MS (ESI): m/z 511 [M þ K]þ (20), 495 [M þ Na]þ
(40), 473 [M þ H]þ (100). IR (CHCl3):
n 3019, 2360, 1667, 1606,
5.2.5. N-(1-Hydroxybutan-2-yl)-4-oxo-1-pentyl-7-(phenylthio)-
1,4-dihydroquinoline-3-carboxamide (33)
1473 cmꢀ1. Anal. Calcd. for C28H28N2O3S: C, 71.16; H, 5.97; N, 5.93.
Found: C, 71.34; H, 6.05; N, 5.76.
65% yield. White solid. Mp 165e166 ꢁC. 1H NMR (200 MHz,
CDCl3):
d
10.23 (d, J ¼ 6.4 Hz, 1H), 8.62 (s, 1H), 8.31 (d, J ¼ 8.7 Hz,
5.2.10. N-[2-(Morpholin-4-yl)ethyl]-4-oxo-1-pentyl-7-
(phenylthio)-1,4-dihydroquinoline-3-carboxamide (38)
55% yield. Yellow solid. Mp 138e139 ꢁC. 1H NMR (200 MHz,
1H), 7.56e7.44 (m, 5H), 7.25e7.21 (m, 1H), 6.96 (d, J ¼ 1.4 Hz, 1H),
3.98e3.90 (t, J ¼ 7.6 Hz, 2H), 3.80e3.60 (m, 4H), 1.74e1.61 (m, 4H),
1.31e1.14 (m, 4H), 1.05 (t, J ¼ 7.4 Hz, 3H), 0.87 (t, J ¼ 6.8 Hz, 3H). 13
C
CDCl3):
d
10.12 (t, J ¼ 4.8 Hz,1H), 8.58 (s, 1H), 8.29 (d, J ¼ 8.6 Hz, 1H),
NMR (100 MHz, CDCl3):
d
176.0, 166.3, 147.6, 146.0, 139.4, 134.7,
7.54e7.48 (m, 5H), 7.22e7.17 (m,1H), 6.93 (m,1H), 3.96 (t, J ¼ 7.4 Hz,
131.0, 130.0, 129.5, 127.6, 125.3, 123.9, 112.9, 111.5, 66.8, 54.5, 54.3,
4H), 3.72 (t, J ¼ 4.8 Hz, 2H), 3.56 (t, J ¼ 6.0 Hz, 2H), 2.67e2.54 (m,
28.5, 24.3, 22.1, 13.8, 10.7. MS (ESI): m/z 461 [M þ Na]þ (100). IR
6H), 1.69e1.54 (m, 2H), 1.37e1.11 (m, 4H), 0.84 (t, J ¼ 6.7 Hz, 3H). 13
C
(CHCl3):
n
3019, 2400, 2360, 1652, 1221 cmꢀ1. Anal. Calcd. for
NMR (100 MHz, CDCl3): d 176.0, 165.0, 147.5, 145.7, 139.4, 134.6,
C
6.76.
25H30N2O3S: C, 68.46; H, 6.89; N, 6.39. Found: C, 68.24; H, 6.99; N,
131.1, 129.4, 127.8, 127.4, 125.6, 123.9, 113.0, 112.0, 66.9, 57.7, 54.1,
53.8, 53.6, 36.2, 29.6, 22.1, 13.8. MS (ESI): m/z 502 [M þ Na]þ (20),
480 [M þ H]þ (100). IR (CHCl3):
n
2927,1652,1461 cmꢀ1. Anal. Calcd.
5.2.6. (S)-1-[4-Oxo-1-pentyl-7-(phenylthio)-1,4-dihydroquinoline-
3-carbonyl]pyrrolidine-2-carboxamide (34)
for C27H33N3O3S: C, 67.61; H, 6.93; N, 8.76. Found: C, 67.34; H, 6.75;
N, 8.54.
93% yield. White solid. Mp 89e91 ꢁC. 1H NMR (200 MHz, CDCl3):
d
8.25 (d, J ¼ 8.4 Hz,1H), 8.13 (bs,1H), 7.93 (s,1H), 7.65e7.47 (m, 5H),
5.2.11. N-Methoxy-4-oxo-1-pentyl-7-(phenylthio)-1,4-
dihydroquinoline-3-carboxamide (39)
7.30e7.26 (m,1H), 7.00e6.96 (m,1H), 5.42 (bs,1H), 4.80 (t, J ¼ 6.2 Hz,
1H), 3.92e3.29 (m, 4H), 3.38e3.31 (m, 1H), 2.24 (t, J ¼ 8.0 Hz, 2H),
2.17e1.72 (m, 2H), 1.99e1.72 (m, 1H), 1.31e1.14 (m, 4H), 0.87 (t,
67% yield. Colourless viscous liquid. 1H NMR (200 MHz, CDCl3):
d
12.00 (s, 1H), 8.70 (s, 1H), 8.20e8.30 (m, 1H), 7.52e7.44 (m, 5H),
J ¼ 6.7 Hz, 3H).13C NMR (100 MHz, CDCl3):
d
174.7,173.9,166.2,146.0,
7.26e7.20 (m, 1H), 6.96 (m, 1H), 3.96 (t, J ¼ 7.7 Hz, 2H), 3.86 (s, 3H),
143.8,139.7,134.6,131.1,130.0,129.4,127.4, 124.8,123.8,118.8,112.9,
1.72e1.50 (m, 2H), 1.28e1.14 (m, 4H), 0.87 (t, J ¼ 6.8 Hz, 3H). 13C
59.8, 53.9, 47.8, 29.7, 28.5, 28.4, 24.4, 22.1, 13.8. MS (ESI): m/z 486
NMR (100 MHz, CDCl3): d 177.9, 167.3, 148.5, 148.4, 147.5, 136.9,
[M þ Na]þ (100), 464 [M þ H]þ (80). IR (CHCl3):
n
3019, 2400, 1633,
135.4, 135.1, 130.4, 130.2, 130.1, 129.8, 128.0, 113.0, 64.8, 55.1, 54.6,
1221 cmꢀ1. Anal. Calcd. for C26H29N3O3S: C, 67.36; H, 6.31; N, 9.06.
Found: C, 67.54; H, 6.45; N, 8.88.
28.8, 22.4, 14.1. MS (ESI): m/z 397 [M þ H]þ (80). IR (CHCl3):
n 2937,
1642 cmꢀ1. Anal. Calcd. for C22H24N2O3S: C, 66.64; H, 6.10; N, 7.07.
Found: C, 66.26; H, 6.45; N, 7.44.
5.2.7. N-(Quinuclidin-3-yl)-4-oxo-1-pentyl-7-(phenylthio)-1,4-
dihydroquinoline-3-carboxamide (35)
5.2.12. N-(3,4-Dichlorobenzyl)-4-oxo-1-pentyl-7-(phenylthio)-1,4-
dihydroquinoline-3-carboxamide (40)
62% yield. White solid. Mp 204e206 ꢁC. 1H NMR (200 MHz,
CDCl3):
d
10.63 (d, J ¼ 6.1 Hz, 1H), 8.69 (s, 1H), 8.30 (d, J ¼ 8.9 Hz,
30% yield. White solid. Mp 144e146 ꢁC. 1H NMR (200 MHz,
1H), 7.66e7.53 (m, 5H), 7.45e7.27 (m, 2H), 4.55e4.48 (m, 1H), 4.28
(t, J ¼ 7.5 Hz, 2H), 4.13e4.01 (m, 1H), 3.76e3.42 (m, 5H), 2.41e2.00
(m, 5H), 1.75 (t, J ¼ 7.6 Hz, 2H), 1.27e1.22 (m, 4H), 0.86 (t, J ¼ 6.5 Hz,
CDCl3):
d
10.47 (t, J ¼ 5.9 Hz,1H), 8.64 (s,1H), 8.32 (d, J ¼ 8.1 Hz,1H),
7.58e7.46 (m, 5H), 7.35e7.20 (m, 4H), 7.16 (d, J ¼ 1.7 Hz,1H), 4.57 (d,
J ¼ 5.9 Hz, 2H), 3.95 (t, J ¼ 7.7 Hz, 2H),1.72e1.61 (m, 2H),1.31e1.14 (m,
3H). 13C NMR (100 MHz, CDCl3):
d
175.9, 165.3, 147.6, 147.1, 139.8,
4H), 0.87 (t, J ¼ 6.8 Hz, 3H).13C NMR (100 MHz, CDCl3):
d 176.1,165.2,
130.0, 129.7, 127.3, 124.3, 112.8, 110.6, 103.3, 100.2, 91.7, 54.5, 49.5,
147.7,146.1,139.4,139.4,134.7,132.9,132.4,131.0,130.4,130.0,129.5,
129.5,127.7,126.9,125.5,124.0,113.0,111.6, 71.8, 54.3, 42.1, 28.5, 22.1;
13.8. MS (ESI): m/z 548 [M þ Na]þ (20), 526 [M þ H]þ (10). IR (CHCl3):
49.1, 46.9, 46.3, 43.7, 28.5, 24.1, 22.1, 21.7, 17.7, 13.7. MS (ESI): m/z
498 [M þ Na]þ (30), 476 [M þ H]þ (100). IR (CHCl3):
n 1645,
1559 cmꢀ1. Anal. Calcd. for C28H33N3O2S: C, 70.70; H, 6.99; N, 8.83.
Found: C, 70.34; H, 6.80; N, 8.66.
n
3019, 2360, 1659, 1531 cmꢀ1. Anal. Calcd. for C28H26Cl2N2O2S: C,
64.00; H, 4.99; N, 5.33. Found: C, 64.34; H, 4.85; N, 5.14.
5.2.8. N-(Piperidin-1-yl)-4-oxo-1-pentyl-7-(phenylthio)-1,4-
dihydroquinoline-3-carboxamide (36)
5.3. General procedure for the alkylation reaction
71% yield. White solid. Mp 145e146 ꢁC. 1H NMR (200 MHz,
To a mixture of the appropriate 4-quinolone derivative (1 mmol)
and potassium carbonate (2.8 mmol) in dry DMF the appropriate
CDCl3):
d
10.81 (s,1H), 8.64 (s,1H), 8.36 (d, J ¼ 6.7 Hz,1H), 7.58e7.46

38
S. Pasquini et al. / European Journal of Medicinal Chemistry 58 (2012) 30e43
alkyl iodide (1.5 mmol) was added under N2 atmosphere. The
reaction mixture was heated at 90 ꢁC for 18 h, unless otherwise
stated. After cooling to room temperature, the mixture was poured
into ice-water. The resulting N-alkyl 4-quinolone was collected by
filtration and recrystallized from the appropriate solvent or
extracted with dichloromethane and purified by flash column
chromatography eluting with dichloromethane/methanol (98:2).
8.53e8.51 (m,1H), 7.19e7.07 (m, 2H), 4.09 (t, J ¼ 7.7 Hz, 2H), 3.78 (d,
J ¼ 9.2 Hz, 1H), 1.83e1.80 (m, 2H), 1.72e1.55 (m, 4H), 1.45e1.31 (m,
5H), 1.22e1.19 (m, 2H), 1.17 (s, 3H), 1.14 (s, 3H), 0.89e0.81 (m, 6H).
13C NMR (100 MHz, CDCl3):
d 176.1, 165.1, 148.1, 130.6, 130.5, 113.7,
113.5, 112.6, 102.2, 101.9, 64.1, 54.4, 48.7, 48.3, 42.7, 39.5, 31.0, 28.7,
28.5, 27.7, 25.7, 22.2, 21.7, 19.8, 13.6. MS (ESI): m/z 435 [M þ Na]þ
(80), 413 [M þ H]þ (100). IR (nujol):
n
1633 cmꢀ1. [
a
]
20: þ3.77ꢁ
D
(dichloromethane). Anal. Calcd. for C25H33FN2O2: C, 72.79; H, 8.06;
N, 6.79. Found: C, 72.53; H, 8.21; N, 6.52.
5.3.1. N-(1,3,3-Trimethylbicyclo[2.2.1]heptan-2-yl)-7-fluoro-1-
methyl-4-oxo-1,4-dihydroquinoline-3-carboxamide (3)
88% yield. White solid. Mp 217e219 ꢁC from EtOH. 1H NMR
5.3.6. N-(1,3,3-Trimethylbicyclo[2.2.1]heptan-2-yl)-1-(3-methyl)
benzyl-7-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxamide (8)
86% yield. Grey solid. Mp 252e254 ꢁC from EtOH. 1H NMR
(200 MHz, CDCl3):
d
10.04 (d, J ¼ 8.6 Hz, 1H), 8.71 (s, 1H), 8.52e8.46
(m,1H), 7.19e7.07 (m, 2H), 3.82 (s, 3H), 3.78 (d, J ¼ 8.6 Hz,1H),1.74e
1.53 (m, 4H), 1.46e1.39 (m, 1H), 1.24e1.19 (m, 2H), 1.17 (s, 3H), 1.14
(400 MHz, CDCl3):
d
10.18 (d, J ¼ 10.0 Hz, 1H), 8.95 (s, 1H),
(s, 3H), 0.88 (s, 3H). 13C NMR (100 MHz, CDCl3):
d
166.6, 148.9, 141.4,
8.60e8.56 (m, 1H), 7.27e7.23 (m, 1H), 7.17e7.13 (m, 2H), 7.09e
7.06 (m, 1H), 6.97 (m, 2H), 5.36 (s, 2H), 3.89 (d, J ¼ 10.0 Hz,
1H), 2.32 (s, 3H), 1.80e1.67 (m, 4H), 1.51e1.50 (m, 1H), 1.28e1.18
(m, 2H), 1.21 (s, 3H), 1.18 (s, 3H), 0.99 (s, 3H). 13C NMR
130.3, 130.3, 113.9, 113.6, 112.7, 102.3, 102.0, 64.1, 48.7, 48.3, 42.7,
41.4, 39.5, 31.0, 27.7, 26.1, 21.7, 19.7. MS (ESI): m/z 379 [M þ Na]þ
(100), 357 [M þ H]þ (40). IR (CHCl3):
n .
2965, 1633 cmꢀ1
[
a]
20: þ7.08ꢁ (dichloromethane). Anal. Calcd. for C21H25FN2O2: C,
(100 MHz, CDCl3): d 176.4, 166.4, 165.0, 163.9, 149.0, 139.3, 133.7,
D
70.76; H, 7.07; N, 7.86. Found: C, 70.44; H, 7.12; N, 7.68.
130.3, 130.2, 126.7, 123.5, 113.9, 113.6, 112.8, 103.2, 103.0, 64.1,
57.9, 48.8, 48.4, 42.8, 39.5, 31.1, 27.8, 26.1, 21.7, 21.3, 19.8. MS
5.3.2. N-(1,3,3-Trimethylbiciclo[2.2.1]heptan-2-yl)-1-ethyl-7-
fluoro-4-oxo-1,4-dihydroquinoline-3-carboxamide (4)
(ESI): m/z 469 [M þ Na]þ (100), 447 [M þ H]þ (40). IR (nujol):
n
1659, 1601 cmꢀ1. [
a
]
20: þ13.71ꢁ (dichloromethane). Anal. Calcd.
D
86% yield. White solid. Mp 241e242 ꢁC from EtOH. 1H NMR
for C28H31FN2O2: C, 75.31; H, 7.00; N, 6.27. Found: C, 75.13; H,
7.18; N, 6.45.
(400 MHz, CDCl3):
d
10.09 (d, J ¼ 7.9 Hz, 1H), 8.75 (s, 1H), 8.54e8.50
(m, 1H), 7.17e7.10 (m, 2H), 4.19 (q, J ¼ 7.3 Hz, 2H), 3.80 (d,
J ¼ 7.9 Hz,1H),1.75e1.55 (m, 5H),1.47 (t, J ¼ 7.3 Hz, 3H),1.20e1.19 (m,
2H), 1.17 (s, 3H), 1.15 (s, 3H), 0.89 (s, 3H). 13C NMR (100 MHz, CDCl3):
5.3.7. 4-[3-(1,3,3-Trimethylbicyclo[2.2.1]heptan)-2-ylcarbamoyl]-
7-fluoro-4-oxo-1,4-dihydroquinolin-1-yl]butyl acetate (9)
67% yield. White solid. Mp 183e185 ꢁC from EtOH. 1H NMR
d
176.2, 165.1, 147.6, 130.6, 130.5, 113.7, 113.5, 112.8, 102.1, 101.8, 64.1,
49.1, 48.7, 48.3, 42.7, 39.5, 31.0, 27.7, 26.0, 21.7,19.7,14.3. MS (ESI): m/z
409 [Mþ K]þ (10), 393 [Mþ Na]þ (100), 371 [Mþ H]þ (20). IR(CHCl3):
(400 MHz, CDCl3):
d
10.05 (d, J ¼ 8.4 Hz, 1H), 8.69 (s, 1H), 8.51e8.47
(m, 1H), 7.19e7.07 (m, 2H), 4.14 (t, J ¼ 7.5 Hz, 2H), 4.45e3.98 (m,
4H), 3.78 (d, J ¼ 8.4 Hz,1H), 3.58 (t, J ¼ 6.3 Hz,1H),1.96 (s, 3H),1.94e
1.86 (m, 2H), 1.70e1.52 (m, 4H), 1.44e1.36 (m, 1H), 1.20e1.19 (m,
1H), 1.07 (s, 3H), 1.00 (s, 3H), 0.78 (s, 3H). MS (ESI): m/z 479
n
2963, 1662 cmꢀ1. [
a]
D
20: ꢀ5.22ꢁ (dichloromethane). Anal. Calcd. for
C22H27FN2O2:C,71.33;H,7.35;N,7.56.Found:C,71.55;H, 7.26;N,7.81.
5.3.3. N-(1,3,3-Trimethylbicyclo[2.2.1]heptan-2-yl)-7-fluoro-4-oxo-
1-propyl-1,4-dihydroquinoline-3-carboxamide (5)
[M þ Na]þ (100), 457 [M þ H]þ (60). IR (nujol):
n 1737, 1669,
1605 cmꢀ1. [
a
]
20: ꢀ22.29ꢁ (dichloromethane). Anal. Calcd. for
D
82% yield. White solid. Mp 217e219 ꢁC from DMF. 1H NMR
C26H33FN2O4: C, 68.40; H, 7.29; N, 6.14. Found: C, 68.12; H, 7.43; N,
(400 MHz, CDCl3):
d
10.16 (m, 1H), 8.78 (s, 1H), 8.60e8.56 (m, 1H),
6.53.
7.27e7.15 (m, 2H), 4.15 (t, J ¼ 6.9 Hz, 2H), 3.86 (d, J ¼ 9.0 Hz, 1H),
1.97e1.91 (m, 2H), 1.79e1.65 (m, 5H), 1.50e1.49 (m, 1H), 1.26e1.24
(m, 1H), 1.17 (s, 3H), 1.14 (s, 3H), 1.04 (t, J ¼ 7.5 Hz, 3H), 0.96 (s, 3H).
5.3.8. N-(1,3,3-Trimethylbicyclo[2.2.1]heptan-2-yl)-1-[2-
(dimethylamino)ethyl]-7-fluoro-4-oxo-1,4-dihydroquinoline-3-
carboxamide (10)
13C NMR (100 MHz, CDCl3):
d 176.1, 164.0, 148.2, 130.5, 130.4, 113.7,
113.4, 112.5, 102.2, 101.9, 64.1, 55.8, 48.7, 48.3, 42.7, 39.5, 31.2, 27.7,
53% yield. Mp 171e173 ꢁC from EtOH. 1H NMR (400 MHz, CDCl3):
26.0, 22.1, 21.7, 19.7, 11.0. MS (ESI): m/z 407 [M þ Na]þ (60), 385
d
10.13 (d, J ¼ 9.1 Hz, 1H), 8.79 (s, 1H), 8.60e8.57 (m, 1H), 7.22e7.18
[M þ H]þ (100). IR (nujol):
n
1661, 1606 cmꢀ1. [
a
]
20: ꢀ3.84ꢁ
(m, 2H), 4.30 (t, J ¼ 6.6 Hz, 2H), 3.84 (d, J ¼ 8.8 Hz, 1H), 2.79 (t,
J ¼ 6.4 Hz, 2H), 2.35 (s, 6H), 1.82e1.63 (m, 4H), 1.53e1.47 (m, 1H),
1.30e1.20 (m, 2H), 1.19 (s, 3H), 1.15 (s, 3H), 0.96 (s, 3H). 13C NMR
D
(dichloromethane). Anal. Calcd. for C23H29FN2O2: C, 71.85; H, 7.60;
N, 7.29. Found: C, 71.64; H, 7.51; N, 7.43.
(100 MHz, CDCl3):
d 176.2, 165.1, 148.6, 130.7, 130.6, 113.8, 113.6,
5.3.4. N-(1,3,3-Trimethylbiciclo[2.2.1]heptan-2-yl)-1-butyl-7-
fluoro-4-oxo-1,4-dihydroquinoline-3-carboxamide (6)
112.3, 102.2, 101.9, 64.2, 57.1, 52.0, 48.7, 48.3, 45.6, 42.7, 39.5, 31.0,
21.7, 26.1, 21.7, 19.8. MS (ESI): m/z 436 [M þ Na]þ (80), 414 [M þ H]þ
78% yield. White solid. Mp 166e168 ꢁC from EtOH. 1H NMR
(100). IR (nujol):
methane). Anal. Calcd. for C24H32FN3O2: C, 69.71; H, 7.80; N, 10.16.
Found: C, 69.42; H, 7.77; N, 10.38.
n
2965, 1661 cmꢀ1. [
a
]
20: þ16.18ꢁ (dichloro-
D
(400 MHz, CDCl3):
d
10.18 (m, 1H), 8.80 (s, 1H), 8.61e8.58 (m, 1H),
7.21e7.16 (m, 2H), 3.97 (t, J ¼ 6.7 Hz, 2H), 3.87 (d, J ¼ 8.8 Hz,1H),1.93e
1.66 (m, 6H), 1.54e1.42 (m, 3H), 1.28e1.22 (m, 2H), 1.19 (s, 3H), 1.14 (s,
3H), 1.01 (t, J ¼ 7.5 Hz, 3H), 0.97 (s, 3H). 13C NMR (100 MHz, CDCl3):
5.3.9. N-(1,3,3-Trimethylbicyclo[2.2.1]heptan-2-yl)-7-fluoro-1-(2-
methoxyethyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (11)
67% yield. Mp 185e187 ꢁC from cyclohexane. 1H NMR (400 MHz,
d
176.1, 165.1, 148.1, 130.6, 130.5, 124.7, 113.7, 113.4, 102.2, 101.9, 64.1,
54.1, 48.7, 48.3, 42.7, 39.5, 31.3, 30.8, 27.7, 26.0, 21.7,19.9,19.7,13.5. MS
(ESI): m/z 421 [M þ Na]þ (70), 399 [M þ H]þ (100). IR (nujol):
n
1661,
CDCl3):
d
10.14 (d, J ¼ 8.8 Hz, 1H), 8.80 (s, 1H), 8.60e8.56 (m, 1H),
20
1607 cmꢀ1
C
.
[
a
]
:
ꢀ3.88ꢁ (dichloromethane). Anal. Calcd. for
7.27e7.17 (m, 2H), 4.35 (t, J ¼ 5.1 Hz, 2H), 3.87 (d, J ¼ 8.8 Hz, 1H),
3.78 (t, J ¼ 5.1 Hz, 2H), 3.30 (s, 3H), 1.82e1.65 (m, 4H), 1.54e1.46 (m,
1H),1.39e1.27 (m, 2H),1.19 (s, 3H), 1.17 (s, 3H), 0.88 (s, 3H). 13C NMR
D
24H31FN2O2: C, 72.33;H, 7.84;N,7.15. Found:C, 72.55;H, 7.71; N, 6.93.
5.3.5. N-(1,3,3-Trimethylbicyclo[2.2.1]heptan-2-yl)-7-fluoro-4-oxo-
1-pentyl-1,4-dihydroquinoline-3-carboxamide (7)
(100 MHz, CDCl3):
112.7, 102.5, 102.2, 69.7, 64.1, 59.2, 53.6, 48.7, 48.3, 42.7, 39.5, 31.0,
d 176.3, 165.1, 148.8, 130.4, 130.3, 113.7, 113.4,
79% yield. White solid. Mp 181e183 ꢁC from cyclohexane. 1H
27.8, 26.1, 21.7, 19.8. MS (ESI): m/z 423 [M
þ
Na]þ (100),
NMR (400 MHz, CDCl3):
d
10.07 (d, J ¼ 8.7 Hz, 1H), 8.71 (s, 1H),
400 [M þ H]þ (95). IR (nujol):
n
1662 cmꢀ1. [
a
]
20: ꢀ19.19ꢁ
D

S. Pasquini et al. / European Journal of Medicinal Chemistry 58 (2012) 30e43
39
(dichloromethane). Anal. Calcd. for C23H29FN2O3: C, 68.98; H, 7.30;
5.4.3. N-(1,3,3-Trimethylbicyclo[2.2.1]heptan-2-yl)-4-oxo-7-
N, 6.99. Found: C, 68.73; H, 7.17; N, 6.75.
(phenylthio)-1-propyl-1,4-dihydroquinoline-3-carboxamide (16)
77% yield. White solid. Mp 162e164 ꢁC. 1H NMR (400 MHz,
5.3.10. N-(1,3,3-Trimethylbicyclo[2.2.1]heptan-2-yl)-7-fluoro-4-
oxo-1-vinyl-1,4-dihydroquinoline-3-carboxamide (12)
10% yield, as a by-product from the reaction between 25 and 1-
chloro-2-(N,N-dimethyl)ethanamine hydrochloride. White solid. Mp
CDCl3): d 10.30 (m, 1H), 8.77 (s, 1H), 8.42e8.40 (m, 1H), 7.58e7.48
(m, 5H), 7.27 (m, 1H), 7.02 (m, 1H), 3.96 (m, 2H), 3.87 (d,
J ¼ 8.8 Hz, 1H), 1.79e1.70 (m, 6H), 1.55e1.50 (m, 1H), 1.27e1.22 (m,
2H), 1.19 (s, 3H), 1.10 (s, 3H), 0.99e0.90 (m, 6H). 13C NMR (100 MHz,
187e189 ꢁC.1H NMR (200 MHz, CDCl3):
d
10.05 (d, J ¼ 8.8 Hz,1H), 8.94
CDCl3): d 176.4, 165.6, 147.6, 145.6, 139.4, 134.5, 131.3, 129.9, 129.4,
(s, 1H), 8.58e8.50 (m, 1H), 7.20e7.03 (m, 3H), 5.73 (dd, J1 ¼ 14.8 Hz,
J2 ¼ 1.6 Hz, 1H), 5.56e5.51 (m, 1H), 3.85 (d, J ¼ 8.8 Hz, 1H), 1.82e1.46
(m, 5H), 1.29e1.22 (m, 2H), 1.15 (s, 3H), 1.10 (s, 3H), 0.86 (s, 3H). MS
127.8, 125.5,123.97,113.1,112.3, 64.1, 55.6, 48.7, 48.3, 42.7, 39.5, 31.0,
27.8, 26.1, 22.0, 21.7, 19.8, 10.9. MS (ESI): m/z 497 [M þ Na]þ (60),
475 [M þ H]þ (100). IR (CHCl3):
n .
2917, 1655, 1600 cmꢀ1
(ESI): m/z 391 [M þ Na]þ (80), 369 [M þ H]þ (100). IR (nujol):
n
2951,
[a
]
20: ꢀ16.03ꢁ (dichloromethane). Anal. Calcd. for C29H34N2O2S: C,
D
1655, 1608 cmꢀ1. [
a]
D
20: þ46.67ꢁ (dichloromethane). Anal. Calcd. for
73.38; H, 7.22; N, 5.90. Found: C, 73.11; H, 7.13; N, 5.76.
C22H25FN2O2:C,71.72;H, 6.84;N,7.60. Found:C,71.45;H, 7.45;N, 7.83.
5.4.4. N-(1,3,3-Trimethylbicyclo[2.2.1]heptan-2-yl)-1-butyl-4-oxo-
7-(phenylthio)-1,4-dihydroquinoline-3-carboxamide (17)
5.3.11. N-(3,4-Dichlorobenzyl)-7-bromo-4-oxo-1-pentyl-1,4-
dihydroquinoline-3-carboxamide (30)
78% yield. White solid. Mp 166e168 ꢁC. 1H NMR (400 MHz,
40% yield. White solid. Mp 139e140 ꢁC from EtOH. 1H NMR
CDCl3): d 10.23 (m, 1H), 8.69 (s, 1H), 8.42e8.40 (m, 1H), 7.58e7.46
(200 MHz, DMSO):
d
10.45e10.37 (m, 1H), 8.73 (s, 1H), 8.36 (d,
(m, 5H), 7.28e7.25 (m, 1H), 7.01 (m, 1H), 3.97 (t, J ¼ 8.0 Hz, 2H),
3.86 (d, J ¼ 8.0 Hz, 1H), 1.78e1.64 (m, 6H), 1.50e1.49 (m, 1H), 1.29e
1.24 (m, 4H), 1.17 (s, 3H), 1.10 (s, 3H), 0.98e0.87 (m, 6H). 13C NMR
J¼ 8.7 Hz,1H), 7.7 (m,1H), 7.61e7.57 (m, 1H), 7.44 (d, J¼ 1.5 Hz, 1H), 7.37
(d, J¼ 8.3 Hz,1H), 7.19 (dd, J1 ¼8.0 Hz, J2 ¼1.7 Hz,1H), 4.59 (d, J¼ 5.6 Hz,
2H), 4.16 (t, J ¼ 7.3 Hz, 2H),1.92e1.85 (m, 2H), 1.56e1.39 (m, 4H), 0.91 (t,
J¼ 6.7 Hz, 3H). MS (ESI): m/z518 [M þ Na]þ (100), 497 [M þ H]þ (40). IR
(100 MHz, CDCl3):
d 176.3, 165.1, 147.5, 145.6, 139.4, 134.5, 131.3,
129.9, 129.39, 127.8, 125.6, 123.9, 113.1, 112.9, 64.1, 53.9, 48.7, 48.3,
(CHCl3):
n
3019, 1660, 1601, cmꢀ1. Anal. Calcd. for C22H21BrCl2N2O2: C,
42.7, 39.5, 31.0, 30.7, 27.8, 26.1, 21.7, 19.8, 13.8. MS (ESI): m/z 511
53.25; H, 4.27; N, 5.65. Found: C, 53.41; H, 4.18; N, 5.36.
[M þ Na]þ (60), 489 [M þ H]þ (100). IR (CHCl3):
n .
2920, 1665 cmꢀ1
[a
]
20: ꢀ1.62ꢁ (dichloromethane). Anal. Calcd. for C30H36N2O2S: C,
D
5.4. General procedure for the nucleophilic substitution reaction
73.73; H, 7.43; N, 5.73. Found: C, 73.51; H, 7.35; N, 5.90.
To a solution of appropriate 7-fluoro-4-quinolone derivative
(1 mmol) in dry DMF (1 mL) potassium carbonate (3 mmol) and
thiophenol (6 mmol) were added under N2 atmosphere and the
reaction mixture was heated using a microwave reactor at 150 ꢁC
for 15 min. The mixture was poured into water and extracted in
ethyl acetate. The organic layer was washed with brine, dried over
anhydrous Na2SO4 and evaporated to dryness. The crude product
was purified by flash column chromatography using ethyl acetate/
petroleum ether (3:7) as eluent.
5.4.5. N-(1,3,3-Trimethylbicyclo[2.2.1]heptan-2-yl)-4-oxo-1-pentyl-
7-(phenylthio)-1,4-dihydroquinoline-3-carboxamide (18)
74% yield. White solid. Mp 148e150 ꢁC from n-hexane. 1H NMR
(400 MHz, CDCl3):
d 10.18 (m, 1H), 8.65 (s, 1H), 8.35e8.31 (m, 1H),
7.50e7.39 (m, 5H), 7.21e7.20 (m, 1H), 6.94 (m, 1H), 3.90e3.88 (m,
2H), 3.79 (d, J ¼ 9.0 Hz, 1H), 1.71e1.59 (m, 6H), 1.45e1.39 (m, 1H),
1.24e1.19 (m, 6H), 1.17 (s, 3H), 1.15 (s, 3H), 0.84e0.83 (m, 6H). 13
C
NMR (100 MHz, CDCl3): d 176.3,165.4,147.5,145.6,139.5,134.6,131.3,
129.9,129.4,127.8,125.6,123.9,113.1,112.3, 64.1, 54.2, 48.7, 48.3, 42.7,
39.5, 31.0, 28.6, 27.8, 26.1, 22.1, 21.7, 19.8, 13.8. MS (ESI): m/z 525
5.4.1. N-(1,3,3-Trimethylbicyclo[2.2.1]heptan-2-yl)-1-methyl-4-
oxo-7-(phenylthio)-1,4-dihydroquinoline-3-carboxamide (14)
88% yield. White solid. Mp 217e219 ꢁC from EtOH. 1H NMR
[M þ Na]þ (100), 503 [M þ H]þ (60). IR (CHCl3):
n .
2924, 1659 cmꢀ1
[a
]
20: ꢀ12.79ꢁ (dichloromethane). Anal. Calcd. for C31H38N2O2S: C,
D
74.06; H, 7.62; N, 5.57. Found: C, 74.45; H, 7.76; N, 5.46.
(400 MHz, CDCl3):
d
10.14 (bs, 1H), 8.66 (s, 1H), 8.32 (d, J ¼ 7.7 Hz,
1H), 7.49e7.37 (m, 5H), 7.19e7.11 (m, 2H), 3.79 (d, J ¼ 9.3 Hz, 1H),
3.69 (s, 3H), 1.71e1.58 (m 5H), 1.46e1.38 (m, 1H), 1.22e1.20 (m, 1H),
1.17 (s, 3H), 1.13 (s, 3H), 0.80 (s, 3H). 13C NMR (100 MHz, CDCl3):
5.4.6. N-(1,3,3-Trimethylbicyclo[2.2.1]heptan-2-yl)-1-(3-methyl)
benzyl-4-oxo-7-(phenylthio)-1,4-dihydroquinoline-3-carboxamide (19)
74% yield. White solid. Mp 185e187 ꢁC from EtOH. 1H NMR
d
176.5, 165.3, 148.3, 145.4, 140.3, 134.1, 131.5, 129.9, 129.2, 127.7,
(400 MHz, CDCl3): d 10.29 (m, 1H), 8.88 (s, 1H), 8.41e8.39 (m, 1H),
125.4, 124.5, 113.6, 112.5, 64.1, 48.7, 48.4, 42.7, 41.2, 39.6, 31.7, 27.8,
7.45e7.36 (m, 5H), 7.27e7.23 (m,1H), 7.17e7.09 (m, 2H), 6.96 (m,1H),
6.76 (m, 1H), 6.66e6.64 (m, 1H), 5.14 (s, 2H), 3.89 (d, J ¼ 8.0 Hz, 1H),
2.30 (s, 3H), 1.84e1.68 (m, 4H), 1.54e1.48 (m,1H), 1.28e1.23 (m, 2H),
1.19 (s, 3H), 1.16 (s, 3H), 0.91 (s, 3H). 13C NMR (100 MHz, CDCl3):
26.1, 21.7, 19.8. MS (ESI): m/z 485 [M þ K]þ (20), 469 [M þ Na]þ
(100), 447 [M þ H]þ (40). IR (CHCl3):
n
2915, 1657 cm1 [
a
]
20: þ5.54ꢁ
D
(dichloromethane). Anal. Calcd. for C27H30N2O2S: C, 72.61; H, 6.71;
N, 6.27. Found: C, 72.34; H, 6.88; N, 6.47.
d
176.4, 165.4, 163.9, 148.4, 145.9, 139.8, 139.0, 134.4, 134.0, 130.7,
129.9, 129.3, 129.2, 129.0, 127.6, 126.5, 123.9, 123.3, 113.7, 103.2, 64.1,
5.4.2. N-(1,3,3-Trimethylbicyclo[2.2.1]heptan-2-yl)-1-ethyl-4-oxo-
7-(phenylthio)-1,4-dihydroquinoline-3-carboxamide (15)
57.7, 48.8, 48.3, 42.7, 39.6, 31.0, 27.8, 26.1, 21.8, 21.4,19.8. MS (ESI): m/z
559[Mþ Na]þ (50), 537[Mþ H]þ (100). IR(CHCl3):
n .
1654,1600cmꢀ1
70% yield. White solid. Mp 186e187 ꢁC. 1H NMR (400 MHz,
[
a
]
20: þ17.80ꢁ (dichloromethane). Anal. Calcd. for C34H36N2O2S: C,
D
CDCl3):
d
10.16 (d, J ¼ 8.2 Hz,1H), 8.68 (s,1H), 8.34 (d, J ¼ 8.6 Hz,1H),
76.08; H, 6.76; N, 5.22. Found: C, 76.31; H, 6.63; N, 5.07.
7.49e7.47 (m, 5H), 7.19e7.06 (m, 2H), 4.04e4.03 (m, 2H), 3.79 (d,
J ¼ 8.2 Hz,1H),1.83e1.58 (m, 4H),1.45e1.38 (m,1H),1.31 (t, J ¼ 7.2 Hz,
3H), 1.22e1.19 (m, 2H), 1.17 (s, 3H), 1.15 (s, 3H), 0.88 (s, 3H). 13C NMR
5.4.7. 4-[3-(1,3,3-Trimethylbicyclo[2.2.1]heptan-2-yl)carbamoyl]-
4-oxo-7-(phenylthio)-1,4-dihydroquinolin-1-yl]butyl acetate (20)
31% yield. White solid. Mp 78e80 ꢁC. 1H NMR (400 MHz, CDCl3):
(100 MHz, CDCl3):
d 176.3, 165.4, 147.2, 145.4, 139.2, 134.2, 132.4,
129.9, 129.3, 127.9, 125.7, 124.2, 113.4, 112.6, 64.1, 48.9, 48.7, 48.3,
d
10.14 (d, J ¼ 9.8 Hz, 1H), 8.64 (s, 1H), 8.36e8.35 (m, 1H), 7.52e7.51
42.7, 39.5, 31.0, 27.8, 26.1, 21.7,19.3,14.2. MS (ESI): m/z 483 [M þ Na]þ
(m, 5H), 7.20e7.19 (m, 1H), 6.90 (m, 1H), 3.81e3.79 (m, 2H), 3.78 (d,
J ¼ 9.0 Hz, 1H), 2.01 (s, 3H), 1.85e1.40 (m, 11H), 1.26e1.16 (m, 2H),
1.15 (s, 3H), 1.05 (s, 3H), 0.85 (s, 3H). MS (ESI): m/z 569 [M þ Na]þ
(100), 461 [M þ H]þ (60). IR (CHCl3):
n .
2981, 1655, 1600 cmꢀ1
[
a]
20: þ14.49ꢁ (dichloromethane). Anal. Calcd. for C28H32N2O2S: C,
D
73.91; H, 7.00; N, 6.08. Found: C, 73.63; H, 7.14; N, 6.87.
(100), 547 [M þ H]þ (80). IR (CHCl3):
n .
1655, 1602 cmꢀ1

40
S. Pasquini et al. / European Journal of Medicinal Chemistry 58 (2012) 30e43
[
a
]
20: þ20.00ꢁ (dichloromethane). Anal. Calcd. for C32H38N2O4S: C,
evaporated. The residue was filtered off and washed with water then
with diethyl ether and petroleum ether, to give the hydrolysis
products.
D
70.30; H, 7.01; N, 5.12. Found: C, 70.52; H, 6.90; N, 5.38.
5.4.8. N-(1,3,3-Trimethylbicyclo[2.2.1]heptan-2-yl)-1-[2-
(dimethylamino)ethyl]-4-oxo-7-(phenylthio)-1,4-dihydroquinoline-
3-carboxamide (21)
5.5.1. N-(1,3,3-Trimethylbicyclo[2.2.1]heptan-2-yl)-7-fluoro-1-(4-
hydroxybutyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (13)
93% yield. White solid. Mp 173e175 ꢁC. 1H NMR (400 MHz,
79%yield. White solid. Mp175e177 ꢁC.1HNMR(400 MHz, CDCl3):
d
10.17(d, J ¼ 8.9Hz,1H), 8.71 (s,1H), 8.40 (d, J¼ 8.6Hz,1H), 7.57e7.44
CDCl3):
d
10.14 (d, J ¼ 7.5 Hz, 1H), 8.76 (s, 1H), 8.55e8.50 (m, 1H),
(m, 5H), 7.26e7.23 (m, 2H), 4.23e4.20 (m, 2H), 3.84 (d, J ¼ 8.9 Hz,1H),
2.80e2.70 (m, 2H), 2.31 (s, 6H),1.78e1.61 (m, 4H),1.53e1.46 (m,1H),
1.26e1.21 (m, 2H), 1.19 (s, 3H), 1.10 (s, 3H), 0.95 (s, 3H). 13C NMR
7.22e7.12 (m, 2H), 4.24 (t, J ¼ 7.0 Hz, 2H), 3.80 (d, J ¼ 9.3 Hz, 1H),
3.70 (t, J ¼ 5.8 Hz, 2H), 2.02e1.96 (m, 2H), 1.73e1.61 (m, 6H),
1.47e1.41 (m, 1H), 1.21e1.19 (m, 3H), 1.16 (s, 3H), 1.11 (s, 3H), 0.82
(100 MHz, CDCl3):
d
176.3, 165.3, 148.0, 145.6, 139.6, 134.3, 131.4,
(s, 3H). 13C NMR (100 MHz, CDCl3):
d 173.5, 166.6, 148.2, 130.6,
129.9,129.3,127.9,125.5,124.1, 113.1, 112.4, 64.1, 57.2, 51.7, 48.7, 48.3,
130.5, 124.7, 113.8, 113.6, 102.4, 102.1, 64.2, 61.8, 54.2, 48.8, 48.3,
45.5, 42.7, 39.5, 31.0, 27.7, 26.0, 21.7,19.8. MS (ESI): m/z 526 [M þ Na]þ
42.7, 39.6, 31.0, 28.9, 27.8, 26.1, 25.8, 21.7, 19.8. MS (ESI): m/z 437
(80), 504 [M þ H]þ (100). IR (nujol):
n
1661,1601 cmꢀ1. [
a]
20: ꢀ16.90ꢁ
[M þ Na]þ (100), 415 [M þ H]þ (20). IR (nujol):
n 2950,
D
(dichloromethane). Anal. Calcd. for C30H37N3O2S: C, 71.54; H, 7.40; N,
8.34. Found: C, 71.31; H, 7.32; N, 8.51.
1656 cmꢀ1. [
a
]
20: ꢀ16.47ꢁ (dichloromethane). Anal. Calcd. for
D
C
24H31FN2O3: C, 69.54; H, 7.54; N, 6.76. Found: C, 69.53; H, 7.13;
N, 6.59.
5.4.9. N-(1,3,3-Trimethylbicyclo[2.2.1]heptan-2-yl)-1-(2-
methoxyethyl)-4-oxo-7-(phenylthio)-1,4-dihydroquinoline-3-
carboxamide (22)
5.5.2. N-(1,3,3-Trimethylbicyclo[2.2.1]heptan-2-yl)-1-(4-
hydroxybutyl)-4-oxo-7-(phenylthio)-1,4-dihydroquinoline-3-
carboxamide (24)
87% yield. White solid. Mp 147e148 ꢁC. 1H NMR (400 MHz,
CDCl3):
d
10.23 (m, 1H), 8.71 (s, 1H), 8.35 (d, J ¼ 8.4 Hz, 1H), 7.52e
66% yield. Yellow solid. Mp 94e96 ꢁC. 1H NMR (400 MHz,
7.33 (m, 5H), 7.21e7.05 (m, 2H), 4.20e4.07 (m, 2H), 3.90e3.80 (m,
1H), 3.70e3.59 (m, 2H), 2.01e1.50 (m, 8H), 1.49 (m, 2H), 1.21e1.19
(m, 2H), 1.18 (s, 3H), 1.14 (s, 3H), 0.82 (s, 3H). 13C NMR (100 MHz,
CDCl3):
d
10.23 (m, 1H), 8.71 (s, 1H), 8.35 (d, J ¼ 8.0 Hz, 1H), 7.52e
7.33 (m, 5H), 7.21e7.05 (m, 2H), 4.20e4.07 (m, 2H), 3.90e3.80 (m,
1H), 3.70e3.59 (m, 2H), 2.02e1.50 (m, 8H), 1.49e1.47 (m, 2H), 1.21e
1.19 (m, 2H), 1.18 (s, 3H), 1.14 (s, 3H), 0.82 (s, 3H). 13C NMR
CDCl3):
d 176.4, 165.3, 147.5, 145.7, 139.4, 134.5, 131.4, 129.9, 129.3,
127.8, 125.7, 124.1, 113.3, 112.3, 64.0, 62.0, 54.0, 48.5, 48.3, 42.7, 39.7,
(100 MHz, CDCl3): d 176.4, 165.3, 147.5, 145.7, 139.4, 134.5, 131.4,
129.9, 129.3, 127.8, 125.7, 124.1, 113.3, 112.3, 64.0, 62.0, 54.0, 48.9,
31.1, 28.9, 27.8, 26.1, 25.8, 21.7, 19.8. MS (ESI): m/z 513 [M þ Na]þ
(70), 491 [M
þ
H]þ (100). IR (nujol):
n
2979, 1606 cmꢀ1
.
48.3, 42.7, 39.7, 31.1, 28.9, 27.8, 26.1, 25.8, 21.7, 19.8. MS (ESI): m/z
[
a]
20: þ0.96ꢁ (dichloromethane). Anal. Calcd. for C30H36N2O3S: C,
527 [M þ Na]þ (30), 505 [M þ H]þ (100). IR (nujol):
n 2979,
D
71.40; H, 7.19; N, 5.55. Found: C, 71.19; H, 7.22; N, 5.36.
1655 cmꢀ1. [
a
]
20: þ5.43ꢁ (dichloromethane). Anal. Calcd. for
D
C30H36N2O3S: C, 71.40; H, 7.19; N, 5.55. Found: C, 71.69; H, 7.32; N,
5.4.10. N-(1,3,3-Trimethylbicyclo[2.2.1]heptan-2-yl)-4-oxo-7-
(phenylthio)-1-[4-(phenylthio)butyl]- 1,4-dihydroquinoline-3-
carboxamide (23)
5.20.
5.6. Synthesis of N-(3,4-dichlorobenzyl)-4-oxo-1-pentyl-7-phenyl-
Obtained in 46% yield as a by-product in the reaction between 9
1,4-dihydroquinoline-3-carboxamide (27)
and thiophenol. Colourless oil.1H NMR (200 MHz, CDCl3):
d 10.21 (m,
1H), 8.64 (s,1H), 8.36e8.34 (m,1H), 7.52e7.39 (m, 5H), 7.28e7.15 (m,
6H), 6.97 (m,1H), 3.96 (t, J ¼ 7.1 Hz, 2H), 3.80 (d, J ¼ 10.0 Hz,1H), 2.81
(t, J ¼ 8.0 Hz, 2H), 1.80e1.44 (m, 9H), 1.22e1.19 (m, 2H), 1.16 (s, 3H),
To a solution of the bromide 30 (80 mg, 0.16 mmol) in 1,2-
dimethoxyethane (DME, 0.65 mL), palladium(II) diacetate
(3.6 mg, 0.016 mmol), triphenylphosphine (12.75 mg
0.048 mmol), phenylboronic acid (99 mg 0.81 mmol), EtOH
(0.16 mL) and 1 M aq. Na2CO3 (0.32 mL) were added and the
reaction mixture was irradiated using a microwave apparatus at
150 ꢁC for 5 min. The mixture was then filtered through a plug of
Celite, the filtrate washed with brine, dried over anhydrous
Na2SO4 and evaporated to dryness. The solid residue was puri-
fied by flash column chromatography using dichloromethane/
methanol (99:1) as eluent, to give 27 as a white solid. 87% yield.
1.05 (s, 3H), 0.86 (s, 3H). 13C NMR (100 MHz, CDCl3):
d 176.3, 165.7,
147.5,145.7,139.3,135.4,134.5,131.3,130.0,129.8,129.4,129.0,127.9,
126.4, 125.6, 124.1, 113.1, 64.1, 53.6, 48.7, 48.3, 42.7, 39.5, 33.3, 31.0,
27.8, 27.6, 26.1, 25.7, 21.7, 19.8. MS (ESI): m/z 631 [M þ Cl]ꢀ (30), 595
[Mꢀ H]ꢀ (40). IR (CHCl3):
n
2955,1652,1601,1555 cmꢀ1. [
a
]
20: ꢀ5.49ꢁ
D
(dichloromethane). Anal. Calcd. for C36H40N2O2S2: C, 72.45; H, 6.76;
N, 4.69. Found: C, 72.72; H, 6.98; N, 4.33.
5.4.11. 4-Oxo-1-pentyl-7-(phenylthio)-1,4-dihydroquinoline-3-
carboxylic acid (42)
Mp 174e176 ꢁC. 1H NMR (200 MHz, CDCl3):
d 10.54 (m, 1H), 8.81
(s, 1H), 8.56 (d, J ¼ 8.2 Hz, 1H), 8.24 (d, J ¼ 7.1 Hz, 1H), 7.76e7.19
(m, 9H), 4.61 (d, J ¼ 6.0 Hz, 2H), 4.3 (t, J ¼ 7.4 Hz, 2H), 1.98e1.94
(m, 2H), 1.56e1.23 (m, 4H), 0.94e0.73 (m, 3H). 13C NMR
Obtained from 41 in 88% yield. White solid. Mp 174e176 ꢁC. 1H
NMR (200 MHz, CDCl3):
d
8.61 (s, 1H), 8.35 (d, J ¼ 8.6 Hz, 1H), 7.61e
7.47 (m, 5H), 7.30 (dd, J1 ¼ 8.6 Hz, J2 ¼ 1.7 Hz, 1H), 6.98 (m, 1H), 3.98
(t, J ¼ 7.8 Hz, 2H), 1.70e1.62 (m, 2H), 1.32e1.14 (m, 4H), 0.88 (t,
J ¼ 7.8 Hz, 3H). MS (ESI): m/z 390 [M þ Na]þ (70), 368 [M þ H]þ
(100 MHz, CDCl3): d 176.5, 165.4, 148.1, 146.1, 139.7, 139.4, 135.6,
132.7, 130.4, 130.1, 129.6, 129.2, 128.7, 128.0, 127.9, 127.5, 127.0,
126.8, 124.6, 114.1, 111.5, 54.3, 42.2, 28.7, 22.2, 13.8. MS (ESI): m/z
532 [M þ K]þ (30), 516 [M þ Na]þ (100), 494 [M þ H]þ (60). IR
(100). IR (CHCl3):
n
3019, 2360, 1718, 1610 cmꢀ1. Anal. Calcd. for
C
21H21NO3S: C, 68.64; H, 5.76; N, 3.81. Found: C, 68.34; H, 5.65; N,
(CHCl3): n
1657, 1603 cmꢀ1. Anal. Calcd. for C28H26Cl2N2O2: C,
3.64.
68.16; H, 5.31; N, 5.68. Found: C, 68.23; H, 5.45; N, 5.73.
5.5. General procedure for the hydrolysis reaction
5.7. Radioligand binding assays
To a suspension of the appropriate ester (1 mmol) in EtOH (4 mL)
6 N aq. HCl (4 mL) was added and the mixture was heated at 100 ꢁC
for 18 h. After cooling to room temperature the solvent was
CB1 and CB2 receptor binding assays were performed exactly as
described previously [9,22], using membranes of cells over-
expressing the human recombinant CB1 or CB2 receptors.

S. Pasquini et al. / European Journal of Medicinal Chemistry 58 (2012) 30e43
41
5.7.1. CHO cells
5.8.2. In vitro intrinsic clearance
Chinese hamster ovary cells (CHO) stably transfected with cDNA
encoding human cannabinoid CB1 or CB2 receptors were main-
tained at 37 ꢁC and 5% CO2 in Dulbecco’s modified Eagle’s medium
Test compounds were incubated separately at 1
m
M concentra-
tion in 100 mM phosphate buffer (pH 7.4) and 1 mM EDTA with
0.2 mg/mL of human or rat liver microsomes. The enzymatic
reaction was initiated by addition of a NADPH regenerating system
nutrient mixture F-12 HAM supplemented with 1 mM
10% foetal bovine serum and 0.6% penicillin-streptomycin together
with G418 (600 g/mL). These cells were passaged twice a week
L-glutamine,
(final concentrations: 2 mM b-nicotinamide adenine dinucleotide
m
phosphate (NADP) þ 10 mM glucose-6 phosphate (G6P) þ 0.4 U/ml
glucose-6-phosphate dehydrogenase (G6PDH)). Reactions were
terminated at regular time intervals (0e5e10e20e40 min) by
adding an equal volume of cold acetonitrile. All incubations were
performed in duplicate. Verapamil as positive control for the assay
was incubated in parallel under the same conditions. Samples were
analyzed by UPLC/TOF-MS. Semi-quantitative data were automat-
ically produced using the OpenLynx software. Calculations
Substrate depletion data (peak area at different time points) were
fitted to a monoexponential decay model (Eq. (1)), with a 1/y
weighting, C(t) ¼ C0 eꢀkt where C0 was the substrate concentration
in the incubation media at time 0 and k was the terminal rate
constant. Goodness of fit was assessed by the Akaike information
criterion (AIC) comparing to the biexponential fit. For analysis was
used GraphPad Prism 4.03 software. Under the assumption that the
using a non-enzymatic cell dissociation solution.
5.7.2. Membrane preparation
[
35S]GTP
gS binding assays were performed with hCB1- or hCB2-
CHO cell membranes [22]. Transfected cells were removed from
flasks byscraping and then frozen as a pellet at ꢀ20 ꢁC until required.
Before use in the radioligand binding assays, cells were defrosted,
diluted in Tris-buffer (50 mM TriseHCl and 50 mM Tris-base) and
homogenized with a 1 mL handheld homogenizer. Protein assays
were performed using a Bio-Rad Dc kit (Hercules, CA, USA).
5.7.3. [35S]GTP
These assays were carried out [23b] with GTP
gS binding assays
gS binding buffer
(50 mM TriseHCl, 50 mM Tris-base, 5 mM MgCl2, 1 mM EDTA,
100 mM NaCl, 1 mM dithiothreitol, 0.1% BSA) in the presence of
concentration of 1 mM is far below the Km (MichaeliseMenten
[
35S]GTP
g
S and GDP, in a final volume of 500
initiated by the addition of [35S]GTP
S to the wells. Nonspecific
binding was measured in the presence of 30 M GTP S. The drugs
m
L. Binding was
constant) of the main metabolic transformation of test compound
and, then, under linearity conditions respect to protein concen-
tration, the in vitro CLint was calculated by dividing the elimination
constant (k ¼ minꢀ1) for the Microsomal Protein Concentration
g
m
g
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
(PMS), expressed in mg/mL, to obtain CLint in units of mL/min/mg
protein Classification bands used for categorizing compounds into
low, medium or high clearance were in vitro Clint values of <1.8
(low), 1.8e49.4 (medium) and >49.4 (high) for human, and 3.4
(low), 3.4e92.4 (medium) and >92.4 (high) for rat.
spectrometry. In all the [35S]GTP
gS binding assays, we used 0.1 nM
[
35S]GTP
gS, 30 mM GDP and a protein concentration of 50 mg per
well. Compounds were stored at ꢀ20 ꢁC as 10 mM solutions in
DMSO.
5.8.3. Plasma stability
5.7.4. Analysis of the data
Fresh plasma (no more than 2e4 h from a blood draw), spiked
Values have been expressed as means and variability as SEM or
with a 250 mM solution of test item dissolved in acetonitrile 12%,
as 95% confidence limits. Net agonist stimulated
[
35S]GTP
gS
were divided in 3 aliquots (one for each time point) in a 96-well
plate (in duplicate) and incubated at 37 ꢁC under gentle agitation
for up to 3 h. At time 0 and afterwards at the prefixed time points of
30 min and 3 h, two aliquots were transferred and 3 volumes of
acetonitrile were added for protein precipitation, followed by
centrifugation (4000 rpm, 15 min, 4 ꢁC) and dilution with 0.1%
HCOOH. Analysis by LCeESIþeMSMS using a fast gradient were
performed using an API4000 triple quadrupole mass spectrometer
(Applied Biosystems) in the multiple reaction monitoring mode
(MRM). Results, as average of two replicates, were obtained by
comparing the peak area at the different time points (30 min and
3 h) with that at time 0 and were reported as percentage remain-
ing: Percent remaining ¼ AreaSTM/Areatime 0 ꢃ 100 (where
AreaSTM is the MS peak area of the sample at the specific time
points of 30 min and 3 h and Areatime 0 is the MS area of the
sample at time zero). Compounds were defined as stable, moder-
ately unstable, or unstable based on the Percent Remaining: 80e
100 stable, 50e80 moderately unstable, <50 unstable.
binding values were calculated by subtracting basal binding values
(obtained in the absence of agonist) from agonist-stimulated values
(obtained in the presence of agonist) as detailed elsewhere [22].
Values for EC50, maximal effect (Emax) and SEM or 95% confidence
limits of these values have been calculated by nonlinear regression
analysis using the equation for a sigmoid concentrationeresponse
curve (GraphPad Prism).
5.8. Evaluation of ADME properties
5.8.1. Metabolic stability assay
Compounds in 10 mM DMSO solution were added to an
incubation mixture in a 96-well microplate containing 20 pmol/
mL of hCYP3A4 (0.1e0.2 mg/mL protein) in 100 mM phosphate
buffer (pH 7.4) and 1 mM EDTA. The mixture was split in two
aliquots: one receiving
concentrations: mM
a
b
NADPH regenerating system (final
-nicotinamide adenine dinucleotide
2
phosphate (NADP) þ 10 mM glucose-6 phosphate (G6P) þ 0.4 U/
mL glucose-6-phosphate dehydrogenase (G6PDH)), the other an
equal amount of phosphate buffer. The final substrate concen-
5.8.4. MDCK-MDR1 cellular permeability
MadineDarby Canine Kidney stably transfected with human
mdr1 (obtained by Dott. Piet Borst, The Netherlands Cancer Insti-
tute, Amsterdam, Netherlands) were maintained in tissue culture
flasks in DMEM with Glutamax, Penicillin (100 U/mL), Strepto-
tration was 1 mM along with 0.25% of organic solvent. Incubation
proceeded for 1 h at 37 ꢁC and was stopped by addition of cold
acetonitrile to precipitate proteins. Metabolic stability was given
as the percent remaining following incubation with cofactor
(NADPH) with reference to the incubation mixture without
NADPH: % remaining ¼ Area NADPH ꢃ 100/Area ctrl where Area
ctrl was the MS peak area of the sample solution without NADPH
and Area NADPH was the MS area of the sample solution with
NADPH. The % CV obtained was typically within 10%.
mycin (100
mg/mL), 10% FBS. Five days before the permeability
experiment, the cells were split and placed on permeable cell
culture inserts (24-well Millipore) at a density of 250000 cells/well.
Transepithelial electrical resistance (TEER) was measured for each
well before incubation using an EVOMX instrument (WPI) to
ensure that the monolayer was confluent and the tight junctions

42
S. Pasquini et al. / European Journal of Medicinal Chemistry 58 (2012) 30e43
ꢃ cm2 was considered suitable for experi-
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in duplicate to the donor chamber and buffer to the acceptor
chamber (alternatively apical and basolateral) and incubated for 2 h
at 37 ꢁC under gentle agitation. Standards with a high (antipyrin)
and low (cimetidine) permeability and P-gp substrate (digoxin)
were incubated in the same plate under the same conditions. An
aliquot (100 mL) from each well (both apical and basolateral) at time
0 and 120 min was filtered and analyzed by UPLC/TOF-MS.
Following incubation, the cell monolayer was washed and incu-
bated with Lucifer Yellow, a fluorescent probe with low perme-
ability, to verify monolayer integrity after incubation. The UPLC
separation was performed using a C-18 column (Acquity UPLC BEH
C18, 1.7
mm, 2.1 ꢃ 50 mm, Waters). Samples were analyzed using an
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Acknowledgements
(b) E. Stern, G. Muccioli, B. Bosier, L. Hamitiaux, R. Millet, J.H. Poupaert,
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Authors wish to thank Dr. Elisa Turlizzi (Siena Biotech SpA,
Siena, Italy) for helpful discussions and Mr Marco Allarà (CNR,
Pozzuoli) and Mrs Lesley A. Stevenson (University of Aberdeen) for
technical support.
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2012.09.035.
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