Investigations on the 4-Quinolone-3-Carboxylic Acid Motif Part5: Modulation of the Physicochemical Profile of a Set of Potent and Selective Cannabinoid-2 Receptor Ligands through a Bioisosteric Approach

Article abstract of DOI:10.1002/cmdc.201100573

Three heterocyclic systems were selected as potential bioisosteres of the amide linker for a series of 1,6-disubstituted-4-quinolone-3-carboxamides, which are potent and selective CB2 ligands that exhibit poor water solubility, with the aim of improving their physicochemical profile and also of clarifying properties of importance for amide bond mimicry. Among the newly synthesized compounds, a 1,2,3-triazole derivative (1-(adamantan-1-yl)-4-[6-(furan-2-yl)-1,4-dihydro-4-oxo-1-pentylquinolin-3-yl]-1H-1,2,3-triazole) emerged as the most promising in terms of both physicochemical and pharmacodynamic properties. When assayed in vitro, this derivative exhibited inverse agonist activity, whereas, in the formalin test in mice, it produced analgesic effects antagonized by a well-established inverse agonist. Metabolic studies allowed the identification of a side chain hydroxylated derivative as its only metabolite, which, in its racemic form, still showed appreciable CB2 selectivity, but was 150-fold less potent than the parent compound.

Full text of DOI:10.1002/cmdc.201100573

MED
DOI: 10.1002/cmdc.201100573
Investigations on the 4-Quinolone-3-Carboxylic Acid Motif
Part 5: Modulation of the Physicochemical Profile of a Set
of Potent and Selective Cannabinoid-2 Receptor Ligands
through a Bioisosteric Approach**
Claudia Mugnaini,*[a] Stefania Nocerino,[a] Valentina Pedani,[a] Serena Pasquini,[a]
Andrea Tafi,[a] Maria De Chiaro,[d] Luca Bellucci,[a] Massimo Valoti,[b] Francesca Guida,[d]
Livio Luongo,[d] Stefania Dragoni,[b] Alessia Ligresti,[c] Avraham Rosenberg,[e]
Daniele Bolognini,[f] Maria Grazia Cascio,[f] Roger G. Pertwee,[f] Ruin Moaddel,[e]
Sabatino Maione,[d] Vincenzo Di Marzo,[c] and Federico Corelli*[a]
Three heterocyclic systems were selected as potential bio-
isosteres of the amide linker for a series of 1,6-disubstituted-4-
quinolone-3-carboxamides, which are potent and selective CB2
ligands that exhibit poor water solubility, with the aim of im-
proving their physicochemical profile and also of clarifying
properties of importance for amide bond mimicry. Among
the newly synthesized compounds, a 1,2,3-triazole derivative
(1-(adamantan-1-yl)-4-[6-(furan-2-yl)-1,4-dihydro-4-oxo-1-pentyl-
quinolin-3-yl]-1H-1,2,3-triazole) emerged as the most promising
in terms of both physicochemical and pharmacodynamic prop-
erties. When assayed in vitro, this derivative exhibited inverse
agonist activity, whereas, in the formalin test in mice, it pro-
duced analgesic effects antagonized by a well-established in-
verse agonist. Metabolic studies allowed the identification of
a side chain hydroxylated derivative as its only metabolite,
which, in its racemic form, still showed appreciable CB2 selec-
tivity, but was 150-fold less potent than the parent compound.
Introduction
The pharmacological effects of cannabinoids are mediated by
two distinct receptors, CB1 and CB2.[1,2] These are seven-trans-
membrane receptors that belong to the rhodopsin-like family
(class A) of G protein-coupled receptors (GPCRs) and are in-
volved in a wide variety of multiple intracellular signal trans-
duction pathways.[3]
It has been demonstrated that CB2-selective agonists display
antinociceptive activity in well-validated models of acute pain,
persistent inflammatory pain, post-operative pain, cancer pain
and neuropathic pain.[7–9] Other potential therapeutic indica-
[a] Dr. C. Mugnaini, S. Nocerino, V. Pedani, Dr. S. Pasquini, Prof. A. Tafi,
Dr. L. Bellucci, Prof. F. Corelli
CB1 receptors are expressed predominantly in the central
nervous system (CNS), with particularly high levels in the cere-
bellum, hippocampus and basal ganglia, but are also ex-
pressed, albeit at much lower levels, in the peripheral nervous
system, as well as in cells of the immune system, in the heart,
vascular tissues, and the testis. In contrast, the expression of
the CB2 receptor is more restricted, limited primarily to
immune and hematopoietic cells.[4,5]
Dipartimento Farmaco Chimico Tecnologico, Universitꢀ degli Studi di Siena
Via De Gasperi 2, 53100 Siena (Italy)
E-mail: claudia.mugnaini@unisi.it
federico.corelli@unisi.it
[b] Prof. M. Valoti, Dr. S. Dragoni
Dipartimento di Neuroscienze, Sezione di Farmacologia, Universitꢀ degli
Studi di Siena
Via De Gasperi 2, 53100 Siena (Italy)
[c] Dr. A. Ligresti, Dr. V. Di Marzo
Endocannabinoid Research Group, Istituto di Chimica Biomolecolare
Consiglio Nazionale delle Ricerche (CNR)
Via Campi Flegrei 34, 80078 Pozzuoli, Napoli (Italy)
The human CB2 receptor shows only 44% total homology
with the human CB1 receptor. Despite this low homology,
most plant-derived, endogenous, and classical synthetic canna-
binoids have similar affinities for both receptors. This limits the
therapeutic utility of nonselective, brain-penetrable cannabi-
noid agonists due to their known psychotropic effects via the
CB1 receptor. Given the predominant peripheral distribution of
the CB2 receptor and considering the differences in signal
transduction mechanisms between the two receptors, it has
been hypothesized that selective activation of the CB2 recep-
tor should circumvent the undesired psychoactive effects typi-
cally induced by activation of the CB1 receptor. A selective CB2
agonist is, therefore, therapeutically desirable.[6]
[d] M. De Chiaro, Dr. F. Guida, Dr. L. Luongo, Prof. S. Maione
Dipartimento di Medicina Sperimentale, Seconda Universitꢀ di Napoli
Via S. Maria di Costantinopoli 16, 80138 Napoli (Italy)
[e] Dr. A. Rosenberg, Dr. R. Moaddel
NIH Biomedical Research Center, National Institute on Aging
251 Bayview Boulevard, Baltimore, MD 21224 (USA)
[f] Dr. D. Bolognini, Dr. M. G. Cascio, Prof. R. G. Pertwee
Institute of Medical Sciences, University of Aberdeen
Foresterhill, Aberdeen AB25 2ZD (UK)
[**] For parts 1–4, see References [22–25], respectively.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201100573.
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tions for CB2-selective agonists include multiple sclerosis,[10]
amyotrophic lateral sclerosis,[11] Huntington’s disease,[12]
stroke,[13] atherosclerosis,[14] gastrointestinal inflammation
states,[15] chronic liver diseases,[15] bone disorders[16] and
cancer.[17–20] On the other hand, a number of reports suggest
that selective CB2 inverse agonists/antagonists could serve as
immunomodulatory agents in the treatment of a variety of
acute and chronic inflammatory disorders.[21]
molecules.[26] It has also become clear that low solubility can
affect biological assays by reducing the concentration of the
compound at the target site, thus resulting in erroneous struc-
ture–activity relationship analysis.[27] Keeping these concepts in
mind and starting from lead compounds 3 and 4 (Figure 1),
which have solubility issues that make biological evaluation
difficult,[24,25] we aimed to improve their solubility through
a bioisosteric approach[28] to give high-affinity cannabinoid re-
ceptor ligands endowed with good selectivity for the type 2 re-
ceptor and characterized by an acceptable physicochemical
profile.
During the course of our investigations into the 4-quino-
lone-3-carboxylic acid motif,[22–25] we recently identified and re-
ported a series of 6-substituted 4-quinolone-3-carboxamides as
highly selective CB2 receptor ligands, with Ki values in the low
nanomolar or sub-nanomolar range and selectivity index
values as high as >14000.[23,24] Some of the derivatives, such
as compound 1,[23] characterized by an agonist profile, proved
to have an antinociceptive effect in vivo, while compound 2[24]
showed inverse agonist-like activity (Figure 1).
To this end, we took into consideration three different
heterocyclic systems to be used as amide surrogates in com-
pound 4, speculating that replacing the amide moiety with
suitable five-membered heterocyclic rings could improve solu-
bility. In particular, based on previous findings described in the
literature, 1,2,3-triazole[29] and 1,3,4-oxadiazole[30] ring systems
(compounds 5 and 6, respectively; Figure 1) were selected as
amide replacements owing to their favorable logP values. De-
spite its higher logP value, 1,2,4-oxadiazole-containing com-
pound 7 was synthesized as well due to the good pharmacoki-
netic properties shown by 1,2,4-oxadiazole-based CB2 ago-
nists.[31] Moreover, in order to study the effect of bioisosteric
replacement on the affinity and selectivity of our compounds,
Despite their interesting pharmacodynamic profiles, the
compounds of this series suffer from low solubility which has,
in some cases, hampered their biological evaluation. Balancing
lipophilicity (necessary to reach the target receptor) and water
solubility (necessary for biological testing and acceptable bio-
availability) is therefore a challenging endeavor for the struc-
tural optimization of CB2 ligands that are inherently lipophilic
Figure 1. Structures of prototypical 4-quinolone-3-carboxamides 1–4, 14, and 17 and 4-quinolone-3-carboxamide bioisosteres 5–13 and 15–16.
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4-quinolones 8–10 and 11–13 (Figure 1) were pre-
pared as analogues of 14[23] and 2,[24] respectively,
which exhibited both high CB2 affinity and selectivity
in binding assays. The results obtained for the newly
synthesized bioisosteres in terms of solubility and af-
finity/selectivity were finally used to guide the design
of the 2-quinolone 15 and the 2-alkoxyquinoline 16
(Figure 1), 1,2,3-triazole analogues of compounds 17
and 3, respectively.[25]
Metabolic studies were carried out on compound
11 with the aim of investigating its metabolic stabili-
ty and, possibly, identifying its principal metabolites.
In general, drug metabolism reactions convert lipo-
philic compounds into more hydrophilic products so
that the body can remove these xenobiotics more
easily. Therefore, an active metabolite can serve as
a modified lead compound around which new struc-
ture–activity relationships can be investigated with
the aim of designing second-generation molecules
endowed with an improved solubility profile. Also, in
Scheme 1. Synthesis of compounds 5, 8, 11. Reagents and conditions: a) BMICl, H2O,
microwave, 2408C, sealed tube; b) 1. HMT, TFA, microwave, 1208C, sealed tube; 2. H2O;
c) 1. dimethyl-1-diazo-2-oxopropylphosphonate, K2CO3, MeOH/THF (1:1); 2. AdN3, CuI, mi-
crowave, 1008C, sealed tube; d) RB(OH)2, Pd(OAc)2, PPh3, Na2CO3 (1n), DME, EtOH, micro-
wave, 1508C.
cases where inactive metabolites are formed, appropriate
structural modification could decrease the drug metabolism,
resulting in improved drug exposure.[32–34] For unequivocal
identification, the major metabolites predicted in silico for
compound 11 were also synthesized. This allowed us to use
these compounds as analytical standards in the HPLC and MS/
MS studies and as authentic samples for biological testing.
Cyclization of acylhydrazide 22 to 1,3,4-oxadiazolyl derivative 9
was successfully accomplished using phosphorus oxychloride
at 1108C.[38] Suzuki coupling between 6-bromo derivative 9
and the appropriate boronic acid afforded 6-isopropoxyphenyl
derivative 6 and 6-furanyl derivative 12.
Preparation of 1,2,4-oxadiazole derivatives 7, 10 and 13
started from compound 18, which was reacted overnight with
1-adamantanecarboxamidoxime[39] to give O-acylamidoxime in-
termediate 23 (Scheme 3).
Results and Discussion
Irradiation of the reaction mixture with microwave at 1508C
for 10 min gave 6-bromo derivative 10, which was in turn con-
verted to 6-isopropoxyphenyl derivative 7 and 6-furanyl deriva-
tive 13 by Suzuki reaction.[40] For the synthesis of 1,2,3-triazole
derivatives 15 and 16, easily accessible 3-bromo-quinolin-
2(1H)-one (24)[41] was reacted with pentyl iodide to give a mix-
ture of N-alkylated quinolinone 25 and 2-alkoxyquinoline 26 in
Chemistry
1,2,3-Triazolyl derivatives 5, 8 and 11 were prepared starting
from carboxylic acid 18n[24] which was first decarboxylated by
microwave irradiation to give the 3-unsubstituted quinolone
19 (Scheme 1). The reaction was carried out at 2408C using 1-
butyl-3-methyl-imidazolium chloride (BMIC) as the
solvent and a stoichiometric amount of water.[35] Iso-
lation of 19 was easily accomplished by simple ex-
traction followed by rapid filtration through silica gel.
Treatment of 19 with hexamethylenetetramine (HMT)
in trifluoroacetic acid (TFA) followed by hydrolysis of
the intermediate imine gave C-3 formylated deriva-
tive 20 in almost quantitative yield.[36] The 1,2,3-tria-
zole moiety (compound 8) was obtained by elabora-
tion of the formyl group through a two-step, one-pot
procedure consisting of a Bestmann–Ohira homolo-
gation followed by microwave-assisted, copper-cata-
lyzed azide–alkyne 1,3-dipolar cycloaddition.[37] Final-
ly, 6-isopropoxyphenyl derivative 5 and 6-furanyl de-
rivative 11 were prepared from 6-bromoquinolone 8
through a Suzuki coupling.
1,3,4-Oxadiazolyl derivatives 6, 9 and 12 were pre-
pared from intermediate 21,[24] where the ester group
was converted into acylhydrazide by treatment with
Scheme 2. Synthesis of compounds 6, 9, 12. Reagents and conditions: a) NH2NH2·H2O,
EtOH, reflux; b) 1-adamantanecarbonyl chloride, DMAP, Et3N, CH2Cl2, 08C!RT; c) POCl3,
hydrazine followed by acylation with 1-adamantane-
carbonyl chloride to give compound 22 (Scheme 2). 110 8C; d) RB(OH)2, Pd(OAc)2, PPh3, Na2CO3 (1n), DME, EtOH, microwave, 1508C.
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compounds. Indeed, in order to synthesize com-
pound 29, a predicted possible metabolite of 11 (see
below), we had to change the synthetic approach
(Scheme 5). 6-Bromo 3-iodo-quinolone[42] was first
protected at the NH group using ethyl acrylate, then
a regioselective Sonogashira coupling with 33 gave
protected alkyne 30. Removal of the trimethylsilyl
(TMS) group to give compound 34, followed by
a Huisgen reaction, afforded derivative 31 bearing
the appropriate substitution at position 1 of the
1,2,3-triazole ring. Finally, through a Suzuki reaction,
we obtained target compound 29 by simultaneous
insertion of a furyl moiety at position 6 and deprotec-
tion at N1. N-Substituted derivative 32 was obtained
by alkylation with (5-iodopentan-2-yloxy)(tert-butyl)-
dimethylsilane[43] followed by deprotection of the hy-
droxy group.
Lipophilicity and solubility studies
Scheme 3. Synthesis of compounds 7, 10, 13. Reagents and conditions: a) 1-Adamantane-
carboxamidoxime, HBTU, DIPEA, DMF; b) microwave, 1508C, 10 min; c) RB(OH)2,
Pd(OAc)2, PPh3, Na2CO3 (1n), DME, EtOH, microwave, 1508C.
LogP values for amides 2, 4 and 14 and newly syn-
thesized bioisosteres 5–7, 8–10 and 11–13 were both
calculated in silico using ChemDraw Ultra 8.0 and ex-
a 2.5/1 ratio (Scheme 4). After separation by flash chromatogra-
phy, compounds 25 and 26 were transformed into the corre-
sponding alkynyl derivatives (27 and 28) through Sonogashira
reaction with trimethylsilylacetylene (TMSA) followed by depro-
tection. Elaboration of the triple bond into a 1,2,3-triazole
moiety afforded compounds 15 and 16 in 70% and 37% yield,
respectively.
perimentally determined by HPLC (Table 1). Indeed, HPLC pro-
vides an easy and reliable way to determine the partition prop-
erties of a compound based on its chromatographic retention
time, which directly relates to the compound’s distribution be-
tween the aqueous/organic mobile phase and the standard re-
versed-phase stationary phase.[44] In order to cover a wide
range of lipophilicities, several measurements at different con-
centrations of organic solvent in the mobile phase are needed
for each compound. Then, by comparing the retention times
in the different organic phase concentrations, the organic sol-
vent concentration can be extrapolated to zero. Anal-
ysis of the data reveals that, despite minor differen-
ces between the calculated and experimental logP
values, compounds with a 1,2,3-triazole and 1,3,4-ox-
adiazole nucleus are characterized by the lowest
logP values, and in all cases, these values are lower
than those of the corresponding amides (c.f., com-
pounds 2, 11 and 12; Table 1).
The chemical synthesis of drug metabolites can sometimes
be rather challenging, requiring the complete revision of the
synthetic pathway set up for the preparation of the parent
Solubility studies were carried out on 6-furyl deriv-
atives 11–13 together with amide derivative 2 fol-
lowing a kinetic approach, a methodology becoming
increasingly popular since DMSO stock solution has
been established as a de facto standard for com-
pound storage and distribution to different assays.[45]
We started with a 10mm stock solution of the com-
pound in DMSO, which was diluted with water to get
a 5% DMSO environment. After an 18h incubation
period, the precipitate was filtered off and the con-
centration of the compound was determined by
HPLC.[46] Solubility values (given in parentheses) were
in accordance with calculated logP values and re-
Scheme 4. Synthesis of compounds 15 and 16. Reagents and conditions: a) Pentyl iodide,
K2CO3, 18-crown-6, CH3CN, 908C; b) 1. TMSA, CuI, Pd(Ph3)2Cl2, DIPEA, dioxane, microwave,
1208C, sealed tube; 2. K2CO3, MeOH, THF; c) AdN3, CuSO4, sodium ascorbate, microwave,
1508C, sealed tube, DMF.
vealed the following solubility trend: 11
(132 mgmLꢀ1) >12 (128 mgmLꢀ1) >2 (75 mgmLꢀ1) >
13 (7 mgmLꢀ1).
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C. Mugnaini, F. Corelli et al.
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The bioisosteric substitution
of the amide moiety with
a
1,2,3-triazole or 1,3,4-oxadi-
azole nucleus resulted in a signifi-
cant improvement in terms of
reduced lipophilicity and in-
creased water solubility of the
molecules. Although the logP
and solubility values for 1,2,4-
oxadiazole derivatives were not
promising, all three synthesized
compounds 7, 10 and 13 exhib-
ited adequate solubility under
the conditions used for the bio-
logical assay, thus ensuring the
possibility to evaluate the affinity
and selectivity of these new CB2
ligands.
Affinity studies
Scheme 5. Synthesis of compounds 29 and 32. Reagents and conditions: a) Ethyl acrylate, TRITON B, DMF, 908C;
b) TMSA, CuI, Pd(Ph3)2Cl2, DIPEA, THF; c) KF, EtOH; d) AdN3, CuSO4, sodium ascorbate, tBuOH/H2O (1:1); e) 2-furyl
boronic acid, Pd(OAc)2, PPh3, Na2CO3 (1n), DME, EtOH, microwave, 1508C. f) 1. (5-iodopentan-2-yloxy)(tert-butyl)-
dimethylsilane, K2CO3, DMF, 908C;. 2. TBAF, THF.
We recently developed an online
screening method for CB1 and
CB2 ligands, where cellular
membrane fragments of a chron-
ic myelogenous leukemia cell line, KU-812, were immobilized
on the surface of an open tubular (OT) capillary to create
a CB1/CB2-OT column.[47] Compounds 7, 9–11, 13, selected
as representatives of the structural variability around the qui-
nolone scaffold, were submitted to a preliminary test using
the OT column to rank them as strong/moderate/weak/no
affinity ligands for the CB2 receptor. [3H]-Win-55,1212 was
used as a marker ligand for the CB2 receptor at a concentra-
tion of 0.25 nm.[47] With the exception of compound 7 which
showed very little displacement, all tested compounds were
able to displace the marker with high efficacy suggesting
a potentially high CB2 affinity for all of the evaluated ligands.
This is consistent with the Ki values determined by competi-
tive binding experiments (see below).
Table 1. Calculated and experimental lipophilicity (logP) data and pharmaco-
logical data against cannabinoid type 1 (CB1) and type 2 (CB2) receptors of
compounds 2, 4, 5, 7–16, 29, 32.
Compd
logP[a]
Ki[b] [nm]
S.I.[c]
Calcd Exp
CB1
CB2
2[24]
4
5
7
8
9
10
11
12
13
14[23]
15
16
29
32
6.41 6.40 >10000
0.7ꢁ0.2 >14285
7.78 8.08
NT
NT
–
>6
6.92 7.78 >10000
8.33 7.71 >10000
5.15 5.83 >10000
5.53 5.15 >10000
1680.0ꢁ24.5
>10000
–
32.2ꢁ1.2
250.0ꢁ8.6
2.7ꢁ0.8
>284
>40
76
>8620
>1543
82
6.56 5.77
5.55 5.18 >10000
5.93 5.26 >10000
207.5ꢁ5.5
1.2ꢁ0.2
6.5ꢁ2.2
6.96 6.38
6.01 6.44
33.0ꢁ0.6
0.4ꢁ0.1
996. 0ꢁ12.1
14.3ꢁ2.1
11.1ꢁ3.6
12.6ꢁ4.3
69
29
271
–
5.81
6.65
3.39
3.35
–
–
–
–
–
–
–
321.9ꢁ2.1
3414.6ꢁ9.8
In vitro pharmacology
>10000
>10000
>10000
>2820
190.0ꢁ10.0
5.4ꢁ1.3
>52
>522
In order to assess their affinity and selectivity towards the
human recombinant CB1 and CB2 receptors and, also, to fur-
ther validate our analytical methodology, all the synthesized
compounds were screened in a competitive binding experi-
ment in parallel with SR144528[48] and rimonabant[49] as refer-
ence CB2 and CB1 ligands, respectively, as previously de-
scribed.[23] The results, in terms of binding affinities (Ki) for
the two receptors, are reported in Table 1.
SR144528[d]
Rimonabant[d]
–
12.0ꢁ8.3
790.0ꢁ11.2
0.015
[a] LogP values were calculated (calcd) as clogP using the software Chem-
Draw Ultra 8.0 (CambridgeSoft Corp.; Cambridge, MA, USA) or experimentally
(exp) determined by HPLC (see Experimental Section for details). [b] Equilibri-
um dissociation constant (Ki) is the concentration of the competing ligand
that binds 50% of the binding sites at equilibrium in the absence of a radioli-
gand or other competitors. Data represent the mean ꢁSD of at least three
separate experiments performed in duplicate. For both receptor binding
assays, 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-dime-
thylheptyl)-phenyl]-trans-4-(3-hydroxy-propyl)-cyclohexanol ([3H]CP-55,940).
NT indicates that the compound was not tested because of solubility issues.
[c] Selectivity index (SI) for CB2 is the ratio of Ki (CB1)/Ki (CB2). [d] The binding
affinities of reference compounds SR144528 (CB2) and rimonabant (CB1)
were evaluated in parallel with the test compounds under the same condi-
tions.
With the exception of the 6-isopropoxyphenyl derivatives
5 and 7 (Ki (CB2)=1680 nm and >10000 nm, respectively),
all of the newly synthesized bioisosteres showed high affini-
ty for the CB2 receptor with Ki values ranging from 250 nm
(9) to 0.4 nm (13). Keeping the substituent at the 6-position
fixed and analyzing the effect of the bioisosteric substitution,
the best results in terms of CB2 affinity were obtained with
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the 1,2,4-oxadiazole derivatives, while 1,2,3-triazole analogues
proved to be the most selective, with selectivity index (SI)
values, the ratio of Ki (CB1)/Ki (CB2), up to 8620 (11). The strong
affinity of the 1,2,4-oxadiazole derivatives for both receptors
subtypes resulted in a significant loss of selectivity (in all cases,
SI<100). When considering the affinity and selectivity, com-
pound 11 emerged as the most promising with a Ki value
against CB2 of 1.2 nm and a SI value of >8620. In addition, the
increased solubility of compound 11 indicates that the inser-
tion of a furyl ring at the 6-position of the quinolone scaffold
combined with a 1,2,3-triazole bioisosteric substitution ensures
the best results in terms of physicochemical and pharmacody-
namic properties in the series.
The bioisosteric substitution of the amide bond with a 1,2,3-
triazole was also promising in the case of insoluble 2-alkoxy-
quinoline 3. Indeed, its bioisostere 16 showed excellent CB2 af-
finity and good selectivity (Ki (CB2)=12.6 nm; SI=272), thus
representing a potential new lead for the development of
a novel class of selective CB2 ligands. Interestingly, all the pre-
liminary data obtained in our online screening with OT col-
umns were in line with those obtained in the in vitro pharma-
cological assays, confirming the validity of our analytical meth-
odology for the prescreening of libraries of potential cannabi-
noid ligands.
35
!
^
Figure 2. The effect of compounds 11 ( ) and 12 ( ) on [ S]GTPgS binding
to hCB2-CHO cell membranes (n=8). Each symbol represents the mean
change in [35S]GTPgS binding ꢁSEM.
In order to gain some insight into the functional activity of
some of the novel compounds synthesized here, compounds
11 and 12 were tested for their effect on [35S]GTPgS binding to
hCB2-CHO cell membranes.[50,51] Both compounds were found
to elicit inverse agonist effects (Figure 2) with potencies reflect-
ing their affinities for human CB2 (Tables 1 and 2).
segments of compounds 11–13 by methyl groups. The five-
membered ring models are depicted in Table 3 and differ in
the number and disposition of the heteroatoms that are able
to act as hydrogen-bond acceptors. To quantify the relative
strength of the hydrogen-bond acceptor atoms, a total of
eight complexes were generated by placing one water mole-
cule in front of each heteroatom to form a hydrogen bond. In-
teraction energies of the complexes were analyzed according
Table 2. The EC50 and Emax values of compounds 11, 12 and CP55940 for
stimulation of [35S]GTPgS binding to hCB2-CHO cell membranes.
Compd
Emax[a] [%]
EC50[a] [ nm]
n[b]
Table 3. Interaction energies (DE) for complexes between models a–
c and a water molecule.
CP55940
11
12
84.7 (78.3/91.1)
1.8 (0.9/3.3)
29.3 (16.5/52.2)
57.6 (33.2/102.8)
4
8
8
ꢀ52 (ꢀ57.2/ꢀ46.4)
ꢀ46.6 (ꢀ52.5/ꢀ40.6)
Compd
System[a]
Complex[b]
W:O1 (a)
DE[c] [kcalmolꢀ1
]
[a] The 95% confidence limits are shown in parentheses. [b] n represents
the number of data values.
12
ꢀ2.03
W:N3 (a)
W:N4 (a)
ꢀ6.12
ꢀ6.12
Computational studies
The relative balance of the intermolecular interactions plays
a significant role in the binding site stabilization, affecting re-
ceptor affinity and selectivity.[52] Analysis of the strength and
nature of such competitive, noncovalent interactions, thus rep-
resents an important aspect to rationalize the experimental
data obtained from biological assays. In particular, we were in-
terested in giving a possible explanation for the peculiar be-
havior of 1,2,4-oxadiazole derivative 13, which, in contrast to
the other bioisosteric counterparts 11 and 12 endowed with
high affinity and excellent CB2 selectivity, exhibited high affini-
ty for both CB1 and CB2 receptors.
13
11
W:O1 (b)
ꢀ3.68
W:N2 (b)
W:N4 (b)
ꢀ4.77
ꢀ5.08
W:N2 (c)
W:N3 (c)
ꢀ5.75
ꢀ5.95
[a] Schematic representation of the molecular models. [b] Complexes are
designated as W:X (m) where W stands for “water” molecule, “X” for the
heteroatoms and “m” for molecule model. [c] Interaction energies be-
tween molecule and water.
For this purpose, simplified five-membered ring molecular
models were created replacing the adamantyl and quinolone
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925

C. Mugnaini, F. Corelli et al.
MED
to the Kitaura–Morokuma (KM)[53–55] scheme and the energy
decomposition analysis (EDA) are reported in Table 3. The KM
analysis is in agreement with the general observation that a ni-
trogen atom in an unsaturated system is a much stronger hy-
drogen-bond acceptor than an oxygen atom.[56,57] Indeed, the
W:O1 (a) complex shows a lower interaction energy value (E=
ꢀ2.03 kcalmolꢀ1), whereas W:N3 (a) and W:N4 (a) possess the
highest interaction energy values (E=ꢀ6.12 kcalmolꢀ1) with
the relative balance of the hydrogen-bonding interactions
amounting to more than 4 kcalmolꢀ1 in favor of nitrogen
atoms. Thus, it is improbable that the oxygen atom acts as
a hydrogen-bond acceptor in this system. The W:O1 (b) com-
plex shows an interaction energy value of ꢀ3.68 kcalmolꢀ1,
whereas W:N4 (a) and W:N4 (b) show similar values of
ꢀ4.38 kcalmolꢀ1 and ꢀ4.69 kcalmolꢀ1, respectively. Due to the
smaller differences in the interaction energy, the oxygen atom
of model (b) can effectively compete as a hydrogen-bond ac-
ceptor with nitrogen atoms. Thus, compound 13 can exist as
two possible conformers, namely 13a and 13b in which O1
and N4, respectively, act as hydrogen-bond acceptors
(Figure 3). In these two conformations, the adamantyl group
In vivo pharmacology
The in vivo activity of compounds 11 and 12 was evaluated in
the formalin test of acute peripheral and inflammatory pain in
mice. Formalin injection induces a biphasic, stereotypical, noci-
fensive behavior. Nociceptive responses are divided into an
early, brief first phase (0–7 min) caused by a primary afferent
discharge produced by the stimulus, followed by a quiescent
period, and then a second, prolonged phase (15–60 min) of
tonic pain. Prior to injection of formalin (15 min), mice received
intraperitoneal (ip) administration of vehicle or test com-
pounds (1 or 3 mgkgꢀ1) alone or in combination with the se-
lective CB2 antagonist, 6-iodo-2-methyl-1-[2-(4-morpholinyl)-
ethyl]-1H-indol-3-yl](4-methoxyphenyl)methanone (AM630) ad-
ministered at a dose of 1 mgkgꢀ1 (ip) 5 min before dosing
with the test compound.[58] The results obtained for com-
pounds 11 and 12 are presented in Figure 4.
Both ligands exhibited antinociceptive activity in the formal-
in test. The dose of 3mgkgꢀ1 induced a significant reduction
of the second phase—and to a much lesser extent, the first
phase as well, although only for 11—of formalin-induced noci-
fensive behavior (Figure 4a,c). Pretreatment with AM630
(1mgkgꢀ1) completely abolished the antinociceptive effects ex-
erted by compound 11 (Figure 4b), while it did not induce any
change on the effect mediated by compound 12 (Figure 4d).
Based on our previous studies on quinolones as CB li-
gands,[23,24,25,58] these data suggest that compound 11 acts as
a direct CB2 agonist (in partial disagreement with the results
of the [35S]GTPgS binding assay), and that compound 12 acts
as a possible CB2 inverse agonist (as also suggested by the
[35S]GTPgS binding assay). Different outcomes from in vitro and
in vivo functional data have also been reported previously, par-
ticularly for CB2 ligands, and have been explained with a po-
tential behavior as protean agonists for those compounds be-
having as inverse agonists/antagonists in vitro and as agonists
in vivo, possibly depending on constitutive activity of CB2 re-
ceptors.[59–61] We believe that the protean agonist behavior of
CB2 ligands acting as inverse agonists in vitro and as agonist
in vivo, might be explained in part by the different endocanna-
binoid signaling that is likely to be present in vitro or in vivo.
For example, the formation of heterodimers between CB2 re-
ceptors and other receptors in “real” cells and in vivo, or the
occurrence of higher endocannabinoid levels, could dramati-
cally alter the pharmacology of the ligands. Furthermore, al-
though CB2 can inhibit the release of inflammatory mediators,
thus in principle producing anti-inflammatory effects, this re-
ceptor has also been reported to promote migration of some
inflammatory cells, which might produce either anti-inflamma-
tory or pro-inflammatory actions, depending on where the re-
ceptor is activated in vivo.[62] This could have influenced the re-
sponse we observed in vivo in the second phase of the formal-
in test, which does have an inflammatory component.
Figure 3. Alternative conformations for compound 12 and 13. Arrows indi-
cate hydrogen-bond acceptor sites.
would arrange in different spatial regions, thus possibly allow-
ing profitable interactions with both cannabinoid receptors.
The W:N2 (c) and W:N3 (c) models show intermediate interac-
tion energies and are able to form hydrogen-bond interactions
assuming the same spatial conformation of compound 12. The
computational study performed on compounds 11–13 gave an
insight into structural aspects differentiating compound 13
with respect to the other bioisosteres 11 and 12. However, it
should not be assumed that these results provide the defini-
tive explanation for the affinity of 13 towards the CB1 recep-
tor.
Metabolic studies
Among all the test compounds, quinolone 11, possessing the
most interesting pharmacodynamic, functional and solubility
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Potent and Selective Cannabinoid-2 Receptor Ligands
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profile, was selected for further investigation with the aim of
characterizing its cytochrome P450 (CYP)-dependent metabo-
lism in human liver microsomal preparations.[63] Prediction of
possible metabolites of 11 by using the software MetaSite
3.1.2[64] resulted in the identification of two molecules, namely
N-dealkylated derivative 29 and side chain hydroxylated deriv-
ative 32, which were also synthesized in our laboratories. In
parallel, in vitro metabolic studies were carried out on com-
pound 11 in the presence of human liver microsomes. Prelimi-
nary studies showed that the amount of product formed in-
creased linearly with time up to 60min and that the initial
rates were proportional to the amount of microsomal protein
added up to 1mgmLꢀ1 (data not shown). Kinetic analysis of
compound 11 metabolism was performed at 60min incubation
time and in the presence of 0.5mg of microsomal proteins. At
all of the concentrations tested, only one peak was observed
in the HPLC-MS analysis of the reaction mixture at approxi-
mately 4.3min retention time, which was identified by compar-
ison with an authentic sample of compound 32 (Figure 5).
Analysis of the mass spectra of the two compounds showed
the presence of peaks with identical molecular weights and
the same fragmentation profile (Figure 6); both compounds
showed peaks at 499 and 537 m/z corresponding to [M+H]+
and [M+K]+, respectively. These data, together with the evi-
dence that compound 32 and the CYP-dependent metabolite
of compound 11 presented an overlapping HPLC profile, indi-
Figure 4. Results of the formalin test in mice. Effect of a) compound 11 (i.p.)
ꢀ1
ꢀ1
&
*
alone: 1.25% formalin ( ); 1 mgkg compound 11 ( ); 3 mgkg com-
~
pound 11 ( ). Filled symbols denote statistically significant values versus for-
malin. Effect of b) a combination of compound 11 and AM630 (i.p.): 1.25%
ꢀ1
formalin ( ); 3 mgkg compound 11 ( ); AM630 (1 mgkgꢀ1)+11
~
&
(3 mgkgꢀ1) ( ). Statistically significant results (P<0.05) versus formalin (
~
)
^
^
and compound 11 ( ). Effect of c) compound 12 (i.p.) alone: 1.25% formalin
ꢀ1
ꢀ1
~
&
*
( ); 1 mgkg compound 12 ( ); 3 mgkg compound 13 ( ). Filled sym-
bols denote statistically significant values versus formalin. Effect of d) a com-
ꢀ1
&
bination of compound 12 and AM630 (i.p.): 1.25% formalin ( ); 3 mgkg
compound 12 ( ); AM630 (1 mgkgꢀ1)+12 (3 mgkgꢀ1) ( ). Statistically sig-
~
^
~
^
nificant results (P<0.05) versus formalin ( ) and compound 12 ( ). The
total time of the nociceptive response (y-axis) was measured every 5 min
(x-axis). Results are mean ꢁSEM (n=8–10 for each group). One-way analysis
of variance followed by a Turkey–Kramer multiple comparisons test was
used for data analysis.
Figure 5. HPLC-MS analysis of compound 32 and the CYP-dependent metab-
olite of compound 11. a) HPLC (total ion current) profile of the reaction mix-
ture obtained by incubating compound 11 with human microsomal prepara-
tions. b) Chromatogram of the peak at 499 m/z present in the human micro-
somal incubation mixture. c) Chromatogram of the peak at 499 m/z of au-
thentic compound 32.
ChemMedChem 2012, 7, 920 – 934
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927

C. Mugnaini, F. Corelli et al.
MED
amides allowed us to improve the solubility of the starting
compounds, obtaining new molecules still endowed with very
high affinity for the CB2 receptor. Among all the active com-
pounds, only derivative 13 exhibited high-affinity for both re-
ceptor subtypes, resulting in a loss of CB2 selectivity. Theoreti-
cal studies were used to attempt to rationalize this peculiar be-
havior, which highlighted the crucial role played by the hydro-
gen-bonding properties of the particular heteroaromatic ring
used as the amide mimic. Both compounds 11 and 12 be-
haved as inverse agonists in the [35S]GTPgS binding assay and,
when evaluated in the formalin test of acute peripheral and in-
flammatory pain in mice, exhibited antinociceptive activity by
inducing a significant reduction of the second phase of the for-
malin-induced nocifensive behavior, an effect that, with other
quinolones, including parent compound 2,[24] was previously
ascribed to either agonism or inverse agonism of the CB2 re-
ceptor. However, whilst pretreatment with AM630 did not pro-
duce any change in the effect induced by 1,3,4-oxadiazole 12,
indicating that this compound is a CB2 inverse agonist, the an-
algesic activity of 1,2,3-triazole derivative 11 was reversed by
the selective CB2 antagonist AM630, thus suggesting that
compound 11 is a full CB2 agonist in vivo and a potential CB2
protean agonist.[59–61]
Figure 6. MS spectra of a) authentic compound 32 and b) CYP-dependent
metabolite of compound 11.
In conclusion, our results highlight the possibility of modu-
lating the physicochemical properties of cannabinoid ligands
through a simple bioisosteric approach without significant loss
of receptor affinity. However, caution should be used, on the
basis of differences in aromatic, electrostatic and hydrogen
bonding properties of different heteroaromatic ring systems
used as amide surrogates, when considering the issue of selec-
tivity and functional activity of the new designed compounds.
cate that the CYP-dependent metabolism of 11 promoted the
formation of the C4-alkyl side chain hydroxylated derivative.
The interaction of 11 with CYPs followed a hyperbolic, Mi-
chealis–Menten, dependence. The apparent Km values of CYP
for hydroxylated derivative formation was 5.21ꢁ0.60 mm sug-
gesting high affinity of CYP towards the compound. However,
this high affinity was accompanied by a low formation rate
(Vmax =0.16ꢁ0.01 nmolminꢀ1 mgꢀ1 protein, which resulted in
a low intrinsic clearance value, as indicated by the Vmax/Km
value (0.030 minꢀ1). With the aim of identifying the CYPs in-
volved in the metabolic process of compound 11, an inhibition
study was also performed indicating that CYP3A4 is the major
isoform involved, while CYP1A and CYP2A are only partially im-
plicated in this process (for further details, see the Supporting
Information).
Compounds 29 and 32 were finally evaluated in the [3H]CP-
55,940 binding assay (Table 1). As expected, compound 29,
lacking the alkyl chain, showed no affinity for either receptor
subtype, while compound 32, the actual metabolite of com-
pound 11, maintained an appreciable CB2 selectivity (SI>52)
but lost receptor affinity (Ki =190 nm vs Ki =1.2 nm for com-
pound 11), thus strongly reducing the interest in using this
molecule as a lead compound for future developments. Stud-
ies directed at preparing the two enantiomers of compound
32 are ongoing in our laboratories.
Experimental Section
Abbreviations
3-Aminopropyltriethoxysilane (APTS); 2-(1H-benzotriazole-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU); 1-butyl-
3-methyl-imidazolium chloride (BMIC); 3-[(3-cholamidopropyl)di-
methylammonio]-1-propanesulfonate (CHAPS); N,N-diisopropyl-
ethylamine (DIPEA); N,N-dimethylformamide (DMF); 1,2 dime-
thyoxyethane (DME); ethylenediaminetetraacetic acid (EDTA); gua-
nosine diphosphate (GDP); hexamethylenetetramine (HMT); petro-
lium ether (PE); phenylmethylsulfonyl fluoride (PMSF); tetra-n-buty-
lammonium fluoride (TBAF); tetrahydrofuran (THF); trifluoroacetic
acid (TFA); N,N,N-trimethyl-1-phenylmethanaminium hydroxide
(TRITON B); trimethylsilylacetylene (TMSA).
Chemistry
General: Reagents were purchased from commercial suppliers and
used without further purification. Anhydrous reactions were run
under a positive pressure of dry N2. Silica gel 60 (Merck) was used
for flash chromatography (23–400 mesh). Infrared (IR) spectra were
recorded on a PerkinElmer BX FT-IR system using CHCl3 as the sol-
1
vent. H NMR and 13C NMR were recorded at 200 MHz and 50 MHz,
Conclusions
respectively, on a Brucker AC200F spectrometer, and at 400 MHz
and 100 MHz, respectively, on a Brucker Advance DPX400. Chemi-
cal shifts (d) are reported relative to tetramethylsilane (TMS), set to
The bioisosteric substitution of the amide moiety with three
different heterocyclic rings on a set of 4-quinolone-3-carboxy-
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Potent and Selective Cannabinoid-2 Receptor Ligands
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d=0.00 ppm. Mass spectrometry (MS) was performed on an Agi-
lent 1100 LC/MSD VL system (G1946C) with a flow rate of
0.4 mLminꢀ1 using a binary solvent system of 95:5 MeOH/H2O. Ul-
traviolet (UV) detection was monitored at 254 nm. Mass spectra
were acquired either in the positive or negative scanning mode
over a mass range of 105–1500. Melting points (mp) were deter-
mined on a Gallenkamp apparatus and are uncorrected. Microwave
irradiations were conducted using a Discover Synthesis unit (CEM
Corp., Matthews, NC, USA). All the reactions were performed in
sealed tube maintaining constant temperature and variable wat-
tage. Elemental analyses were performed on a PerkinElmer PE 2004
elemental analyzer and the data for C, H and N are within 0.4% of
the theoretical values.
The solution was cooled to 08C and treated with Et3N (356 mL,
2.56 mmol), DMAP (312 mg, 2.56 mmol) and 1-adamantanecarbon-
yl chloride (507 mg, 2.56 mmol). The reaction mixture was stirred
for 2 h at RT, and then concentrated in vacuo. The resultant solid
(100 mg) was dissolved in POCl3 (6 mL, 65.5 mmol) and heated at
110 8C for 5 h. After removal of POCl3 by distillation, the residue
was dissolved in CH2Cl2 (10 mL). The organic phase was washed
with H2O and brine (10 mL), then dried (anhyd Na2SO4), filtered
and concentrated in vacuo to give pure compound 9 as a yellow
1
solid (70 mg, 72%): mp: 241–2438C; H NMR (200 MHz, CDCl3): d=
0.87 (t, J=6.3 Hz, 3H), 1.34–1.35 (m, 4H), 1.77–2.11 (m, 15H), 4.17
(t, J=7.4 Hz, 2H), 7.34 (d, J=8.8 Hz, 1H), 7.74 (dd, J1 =1.8 Hz, J2 =
8.9 Hz, 1H), 8.48 (s, 1H), 8.62 ppm (d, J=1.8 Hz, 1H); 13C NMR
(100 MHz, CDCl3): d=13.8, 22.2, 28.1, 28.6, 34.3, 36.6, 40.4, 54.3,
107.4, 117.7, 119.3, 129.8, 130.7, 135.8, 137.6, 146.3, 171.8, 172.8,
176.7 ppm; IR (CHCl3): n˜ =1632, 1594 cmꢀ1; MS (ESI): m/z (%): 518
(100) [M+Na]+, 520 (62) [M+Na]+; Anal. calcd for C26H30BrN3O2: C
62.90, H 6.09, N 8.46, found: C 62.79, H 6.10, N 8.43.
6-Bromo-1-pentylquinolin-4(1H)-one (19):
A mixture of 18
(150 mg, 0.44 mmol), 1-butyl-3-methyl-imidazolium chloride
(477 mg, 2.73 mmol) and H2O (53 mL, 2,96 mmol) was irradiated by
microwave at 1408C for 10 min (two cycles). The reaction mixture
was then diluted with EtOAc (5 mL) and washed with brine (5 mL).
The organic layer was dried (anhyd Na2SO4), filtered and concen-
trated in vacuo. Purification by flash chromatography (EtOAc) gave
3-(Adamantan-1-yl)-5-(6-bromo-1,4-dihydro-4-oxo-1-pentylqui-
nolin-3-yl)-1,2,4-oxadiadole (10):
A solution of 18 (68 mg,
1
19 as a white solid (107 mg, 82%): mp: 1898C; H NMR (200 MHz,
0.2 mmol), HBTU (79 mg, 0.2 mmol), DIPEA (82 mL, 0.47 mmol) and
1-adamantanecarboxamidoxime (46 mg, 0.24 mmol) in DMF
(2.4 mL) was stirred overnight at RT and then irradiated by micro-
wave at 1508C for 10 min. The reaction mixture was poured into
H2O (10 mL) and extracted with CH2Cl2 (5ꢁ7 mL). The organic
phase was washed with HCl (1n), H2O (15 mL) and brine (10 mL),
then dried (anhyd Na2SO4), filtered and concentrated in vacuo. Pu-
rification by flash chromatography (EtOAc, PE, 1:1) gave 9 as
a white solid (80 mg, 80%): mp: 187–1888C; 1H NMR (400 MHz,
CDCl3): d=0.95 (t, J=7.2 Hz, 3H), 1.42–1.43 (m, 4H), 1.82–2.10 (m,
15H), 4.23 (t, J=7.2 Hz, 2H), 7.37 (d, J=9.0 Hz, 1H), 7.79 (dd, J1 =
2.0 Hz, J2 =9.1 Hz, 1H), 8.55 (s, 1H), 8.67 ppm (d, J=2.0 Hz, 1H);
13C NMR (100 MHz, CDCl3): d=13.8, 22.2, 27.8, 28.7, 34.4, 36.3, 40.0,
54.2, 106.9, 117.8, 118.9, 129.4, 130.3, 135.63, 137.7, 144.9, 162.0,
171.9, 173.0 ppm; IR (CHCl3): n˜ =1636 cmꢀ1; MS (ESI): m/z (%): 496
(75) [M+H]+, 498 (82) [M+H]+, 518 (60) [M+Na]+, 520 (100) [M+
Na]+; Anal. calcd for C26H30BrN3O2: C 62.90, H 6.09, N 8.46, found: C
62.78, H 6.10, N 8.48.
CDCl3): d=0.9 (t, J=6.8 Hz, 3H), 1.40 (m, 4H), 1.90 (t, J=7.2 Hz,
2H), 4.27 (t, J=7.2 Hz, 2H), 7.48 (d, J=9.2 Hz, 1H), 7.90 (dd, J1 =
2.3 Hz, J2 =8.2 Hz, 1H), 8.47 (s, 1H), 8.72 ppm (s, 1H); IR (CHCl3):
n˜ =1725, 1615 cmꢀ1; MS (ESI): m/z (%): 294 (100) [M+H]+, 296 (98)
[M+H]+, 316 (78) [M+Na]+, 318 (97) [M+Na]+; Anal. calcd for
C14H16BrNO: C 57.16, H 5.48, N 4.76, found: C 57.32, H 5.47, N 4.75.
1-(Adamantan-1-yl)-4-(6-bromo-1,4-dihydro-4-oxo-1-pentylqui-
nolin-3-yl)-1H-1,2,3-triazole (8):
A solution of 19 (430 mg,
1.27 mmol) and hexamethylenetetramine (178 mg, 1.27 mmol) in
TFA (1.8 mL, 24.2 mmol) was irradiated by microwave at 1208C for
15 min. Further hexamethylenetetramine (178 mg, 1.27 mmol) was
added, and the reaction mixture was irradiated for an additional
5 min. The reaction was treated with H2O (5 mL) and stirred for
10 min at RT, then neutralized with saturated aq Na2CO3 and ex-
tracted with CH2Cl2 (3ꢁ5 mL). The organic phase was washed with
brine (5 mL), dried (anhyd Na2SO4), filtered and concentrated in
vacuo. The crude (200 mg), dimethyl-1-diazo-2-oxopropylphospho-
nate (357 mg, 1.86 mmol) and K2CO3 (387 mg, 2.80 mmol) were
suspended in MeOH/THF (1:1, 20 mL) and stirred overnight at RT.
After addition of CuI (23 mg, 0.12 mmol) and 1-adamantly azide
(110 mg, 0.62 mmol), the reaction mixture was irradiated by micro-
wave at 1008C for 13 min. The solvent was removed in vacuo, and
the residue was dissolved in EtOAc (15 mL). The organic phase was
washed with saturated aq NH4Cl, H2O and brine (15 mL), dried
(anhyd Na2SO4), filtered and concentrated in vacuo. Purification by
flash chromatography (EtOAc/PE, 1:1) gave 8 as a white solid
General procedure for the synthesis of compounds 5–7, 11–13:
A mixture of the appropriate 6-bromo derivative (1 equiv), DME,
Pd(OAc)2 (0.1 equiv), Ph3P (0.3 equiv), EtOH, Na2CO3 (1n) and the
appropriate boronic acid (5 equiv) was irradiated by microwave at
1508C for 5 min. The reaction was then filtered and diluted with
EtOAc. The organic mixture was washed with brine, dried (anhyd
Na2SO4), filtered and concentrated in vacuo. Purification by flash
chromatography afforded the target compounds.
1
1-(Adamantan-1-yl)-4-[6-(4-(2-propyloxy)phenyl]-1,4-dihydro-4-
oxo-1-pentylquinolin-3-yl]-1H-1,2,3-triazole (5): yellow solid
(86%): mp: 83–848C; 1H NMR (200 MHz, CDCl3): d=0.89 (t, J=
7.0 Hz, 3H), 1.18–1.37 (m, 10H), 1.78–2.30 (m, 15H), 4.26 (t, J=
7.0 Hz, 2H), 4.52–4.65 (m, 1H), 6.97 (d, J=8.6 Hz, 1H), 7.52 (d, J=
8.9 Hz, 1H), 7.62 (d, J=8.6 Hz, 2H), 7.86 (d, J=8.9 Hz, 1H), 8.73–
8.75 (m, 2H), 8.84 ppm (s, 1H); 13C NMR (100 MHz, CDCl3): d=13.9,
22.1, 22.3, 28.8, 29.0, 29.5, 29.7, 36.0, 43.0, 53.9, 59.5, 70.0, 112.5,
116.1, 116.3, 119.4, 124.2, 127.4, 128.1, 130.3, 131.9, 136.4, 137.5,
140.3, 140.8, 157.8, 174.4 ppm; IR (CHCl3): n˜ =2927, 1632 cmꢀ1; MS
(ESI): m/z (%): 551 (41) [M+H]+, 573 (100) [M+Na]+; Anal. calcd
for C35H42N4O2: C 76.33, H 7.69, N 10.17, found: C 76.59, H 7.66, N
10.14.
(86 mg, 28%): H NMR (200 MHz, CDCl3): d=0.87 (t, J=6.7 Hz, 3H),
1.13–1.48 (m, 4H), 1.77–2.27 (m, 15H), 4.2 (t, J=6.7 Hz, 2H), 7.34
(d, J=9.2 Hz, 1H), 7.70 (d, J=9.2 Hz, 1H), 8.64–8.66 (m, 2H),
8.79 ppm (s, 1H); 13C NMR (100 MHz, CDCl3): d=13.9. 22.3, 28.8,
29.5, 36.0, 43.0, 53.9, 59.5, 113.1, 117.5, 117.6, 119.5, 128.4, 129.9,
134.7, 137.4, 140.3, 140.6, 173.0 ppm; MS (ESI): m/z (%): 495 (100)
[M+H]+, 497 (97) [M+H]+, 517 (32) [M+Na]+, 519 (38) [M+Na]+;
Anal. calcd for C26H31BrN4O: C 63.03, H 6.31, N 11.31, found: C
62.89, H 6.31, N 11.33.
2-(Adamantan-1-yl)-5-(6-bromo-1,4-dihydro-4-oxo-1-pentylqui-
nolin-3-yl)-1,3,4-oxadiadole (9):
A solution of 21 (800 mg,
2.18 mmol) in EtOH (4 mL) was added dropwise to a refluxing solu-
tion of N2H4·H2O (1 mL, 21.8 mmol) in EtOH (3 mL). After 1 h, the
reaction mixture was filtered then concentrated. The residue was
washed with Et2O (10 mL), dried and redissolved in CH2Cl2 (33 mL).
2-(Adamantan-1-yl)-5-[6-(4-(2-propyloxy)phenyl]-1,4-dihydro-4-
oxo-1-pentylquinolin-3-yl]-1,3,4-oxadiadole (6): yellow solid
ChemMedChem 2012, 7, 920 – 934
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929

C. Mugnaini, F. Corelli et al.
MED
1
(41%): H NMR (400 MHz, CDCl3): d=1.24 (t, J=8.0 Hz, 3H), 1.34–
1.38 (m, 4H), 1.81–2.19 (m, 15H), 4.23 (t, J=8.0 Hz, 2H), 4.58–4.64
(m, 1H), 6.98 (d, J=12.0 Hz, 2H), 7.46–7.69-(m, 3H), 7.93 (d, J=
8.0 Hz, 1H), 8.55 (s, 1H), 8.77 ppm (s,1H); 13C NMR (100 MHz,
CDCl3): d=13.9, 22.1, 22.3, 27.8, 28.8, 29.7, 34.4, 36.4, 40.0, 54.1,
70.0, 116.3, 124.7, 128.1, 128.4, 128.6, 131.0, 131.3, 131.9, 132.0,
132.1, 137.6, 144.4, 158.1, 162.6, 172.3, 173.4 ppm; MS (ESI): m/z
(%): 552 (73) [M+H]+, 574 (100) [M+Na]+; Anal. calcd for
C35H41N3O3: C 76.19, H 7.49, N 7.62, found: C 76.10, H 7.51, N 7.59.
was dissolved in CH2Cl2 (50 mL). The organic layer was washed
with H2O (50 mL) and brine (50 mL), dried (anhyd Na2SO4), filtered
and concentrated in vacuo. Purification by flash chromatography
(PE/EtOAc, 98:2) gave 25 as a red oil (839 mg, 65%) and 26 as an
orange oil (336 mg, 26%). Compound 25: 1H NMR (200 MHz,
CDCl3): d=0.84–0.91 (m, 3H), 1.27–1.47 (m, 4H), 1.64–1.76 (m, 2H),
4.23–4.31 (m, 2H), 7.15–7.32 (m, 2H), 7.45–7.59 (m, 2H), 8.06 ppm
(s, 1H); 13C NMR (100 MHz, CDCl3): d=14.0, 22.4, 27.1, 29.1, 44.1,
114.4, 117.6, 120.8, 122.5, 128.3, 130.8, 138.5, 140.5, 158.1 ppm; IR
(CHCl3): n˜ =1647 cmꢀ1; MS (ESI): m/z (%): 294 (15) [M+H]+, 296
(14) [M+H]+, 316 (80) [M+Na]+, 318 (100) [M+Na]+; Anal. calcd
for C14H16BrNO: C 57.16; H 5.48; N 4.76, found: C 57.24; H 5.46; N
4.75. Compound 26: 1H NMR (200 MHz, CDCl3): d=0.95 (t, J=
7.0 Hz, 3H), 1.25–1.58 (m, 4H), 1.80–1.95 (m, 2H), 4.50 (t, J=6.6 Hz,
2H), 7.24–7.39 (m, 1H), 7.57–7.63 (m, 2H), 7.78–7.82 (m, 1H),
8.20 ppm (s, 1H); 13C NMR (100 MHz, CDCl3): d=14.1, 22.5, 28.3,
28.5, 67.4, 108.44, 124.6, 126.0, 126.5, 127.1, 130.7, 140.6, 145.3,
157.5 ppm; IR (CHCl3): n˜ =3020 cmꢀ1; MS (ESI): m/z (%): 294 (100)
[M+H]+, 296 (92) [M+H]+; Anal. calcd for C14H16BrNO: C 57.16, H
5.48, N 4.76, found: C 57.13, H 5.50, N 4.75.
3-(Adamantan-1-yl)-5-[6-(4-(2-propyloxy)phenyl]-1,4-dihydro-4-
oxo-1-pentylquinolin-3-yl]-1,2,4-oxadiadole (7): yellow solid
(58%): mp: 215–2168C; H NMR (200 MHz, CDCl3): d=0.87–0.94 (m,
1
3H). 1.19–1.48 (m, 10H), 1.78–2.07 (m, 15H), 4.2 (t, J=7.3 Hz, 2H),
4.52–4.61 (m, 1H), 6.95 (d, J=8.7 Hz, 2H), 7.48 (d, J=8.8 Hz, 1H)
7.58 (d, J=8.7 Hz, 2H), 7.86 (dd, J1 =2.1 Hz, J2 =8.8 Hz, 1H), 8.49 (s,
1H), 8.71 ppm (d, J=2.1 Hz, 1H); 13C NMR (100 MHz, CDCl3): d=
13.9, 22.1, 22.3, 28.1, 28.7, 36.6, 40.4, 47.9, 54.2, 70.0, 116.3, 116.4,
125.0, 128.2, 128.7, 131.1, 131.3, 137.4, 137.8, 145.8, 153.2, 158.1,
173.2 ppm; IR (CHCl3): n˜ =1640, 1614 cmꢀ1; MS (ESI): m/z (%): 552
(100) [M+H]+, 574 (93) [M+Na]+; Anal. calcd for C35H41N3O3: C
76.19, H 7.49, N 7.62, found: C 76.32, H 7.47, N 7.61.
1-(Adamantan-1-yl)-4-(1-pentylquinolin-2(1H)-one)-1H-1,2,3-tri-
azole (15): A suspension of 25 (247 mg, 0.841 mmol), TMSA
(291 mL, 1.85 mmol), CuI (32 mg, 0.17 mmol), Pd(PPh3)2Cl2 (59 mg,
0.084 mmol) and DIPEA (16 mL) in 1,4-dioxane (16 mL) was irradiat-
ed by microwave at 1208C for 10 min. The reaction mixture was fil-
tered through Celite, concentrated in vacuo. The residue was redis-
solved in CH2Cl2 (20 mL) and treated with HCl (1n; 20 mL). The or-
ganic phase was washed with H2O (20 mL) and brine (20 mL), and
dried (anhyd Na2SO4), filtered and concentrated in vacuo. The resi-
due was dissolved in MeOH/THF (1:1, 30 mL), treated with K2CO3
(1.1 g), and stirred for 30 min at RT. After removal of the solvents,
the residue was dissolved in EtOAc (20 mL), and the organic phase
was washed with H2O (20 mL) and brine (20 mL), and dried (anhyd
Na2SO4), filtered and concentrated in vacuo. The crude material
(85 mg), AdN3 (69 mg, 0.39 mmol), CuSO4 (5.6 mg, 0.035 mmol) and
sodium ascorbate (6.9 mg, 0.035 mmol) were dissolved in DMF
(2 mL), and the solution was irradiated by microwave for 10 min at
1508C. The reaction mixture was poured into H2O (10 mL) and ex-
tracted with CH2Cl2 (3ꢁ10 mL). The organic phase was washed
with brine (20 mL), dried (anhyd Na2SO4), filtered and concentrated
in vacuo. Purification by flash chromatography (PE/EtOAc, 4:1)
1-(Adamantan-1-yl)-4-[6-(furan-2-yl)-1,4-dihydro-4-oxo-1-pentyl-
quinolin-3-yl]-1H-1,2,3-triazole (11): yellow solid (94%): mp: 80–
818C; 1H NMR (200 MHz, CDCl3): d=0,88 (t, J=6.6 Hz, 3H), 1.18–
1.37 (m, 4H), 1.78–2.29 (m, 15H), 4.22 (t, J=6.6 Hz, 2H), 6.48 (d, J=
3.6 Hz, 1H), 6.77 (d, J=3.6 Hz, 1H), 7.45–7.50 (m, 2H), 7.96 (d, J=
8.9 Hz, 1H), 8.72–8.78 ppm (m, 3H); 13C NMR (50 MHz, CDCl3): d=
13.9, 22.3, 28.8, 28.9, 29.5, 36.0, 43.0, 53.9, 59.5, 105.8, 111.9, 112.6,
116.1, 119.5, 121.9, 126.8, 127.2, 127.5, 137.6, 140.3, 140.6, 142.5,
153.0, 174.2 ppm; IR (CHCl3): n˜ =1577, 1461 cmꢀ1; MS (ESI): m/z
(%): 483 (100) [M+H]+, 505 (75) [M+Na]+; Anal. calcd for
C30H34N4O2: C 74.66, H 7.10, N 11.61, found: C 74.70, H 7.08, N
11.59.
2-(Adamantan-1-yl)-5-[6-(furan-2-yl)-1,4-dihydro-4-oxo-1-pentyl-
quinolin-3-yl]-1H-1,3,4-oxadiadole (12): yellow solid (42%): mp:
118–1208C; 1H NMR (200 MHz, CDCl3): d=0.89 (t, J=6.7 Hz, 3H),
1.34–1.38 (m, 4H), 1.78–2.15 (m, 15H), 4.18 (t, J=7.3, 2H), 6.50 (d,
J=1.9 Hz, 1H), 6.78 (d, J=3.3 Hz, 1H), 7.45–7.49 (m, 2H), 7.99 (dd,
J1 =1.8 Hz, J2 =8.9 Hz, 1H), 8.46 (s, 1H), 8.77 ppm (d, J=1.8 Hz,
1H); 13C NMR (100 MHz, CDCl3): d=13.9, 22.2, 28.1, 28.7, 29.7, 36.4,
36.6, 39.3, 40.4, 54.3, 106.6, 106.9, 112.0, 116.4, 122.5, 126.0, 128.2,
128.7, 137.6, 142.8, 145.8, 152.4, 173.0, 173.1, 176.6 ppm; IR
(CHCl3): n˜ =1637, 1615 cmꢀ1; MS (ESI): m/z (%): 484 (62) [M+H]+,
506 (100) [M+Na]+; Anal. calcd for C30H33N3O3: C 74.51, H 6.88, N
8.69, found: C 74.38, H 6.90, N 8.70.
1
gave 15 as a pale yellow solid (103 mg, 70%): mp: 1698C; H NMR
(200 MHz, CDCl3): d=0.89–0.95 (m, 3H), 1.39–1.52 (m, 4H), 1.62–
1.87 (m, 9H), 2.17–2.34 (m, 8H), 4.30–4.39 (m, 2H), 7.21–7.29 (m,
1H), 7.34–7.39 (m, 1H), 7.52–7.60 (m, 1H), 7.70–7.74 (m, 1H), 8.63
(s, 1H), 8.79 ppm (s, 1H); 13C NMR (100 MHz, CDCl3): d=14.0, 22.5,
27.3, 29.3, 29.5, 36.0, 43.0, 59.6, 114.0, 121.0, 122.3, 129.5, 130.2,
133.9, 138.2, 141.3, 160.1 ppm; IR (CHCl3): n˜ =1618 cmꢀ1; MS (ESI):
m/z (%): 417 (100) [M+H]+, 439 (12) [M+Na]+; Anal. calcd for
C26H32N4O: C 74.97, H 7.74, N 13.45, found: C 75.21, H 7.71, N
13.42.
3-(Adamantan-1-yl)-5-[6-(furan-2-yl)-1,4-dihydro-4-oxo-1-pentyl-
quinolin-3-yl]-1H-1,2,4-oxadiadole (13): white solid (65%): mp:
1
234–2368C, H NMR (400 MHz, CDCl3): d=0.88–0.93 (m, 3H), 1.16–
1.25 (m, 4H), 1.71–2.10 (m, 15H), 4.21 (t, J=8.0 Hz, 2H), 6.50 (s,
1H), 7.27 (s, 1H), 7.46 (s, 1H), 7.50 (s, 1H), 7.95 (d, J=8.0 Hz, 1H),
8.47 (s, 1H), 8.73 ppm (s, 1H); 13C NMR (100 MHz, CDCl3): d=13.9,
22.2, 27.8, 29.7, 36.4, 40.0, 54.1, 106.4, 112.1, 116.4, 122.2, 127.9,
128.1, 128.4, 137.8, 142.8, 144.4, 152.5, 162.5, 173.0, 173.2 ppm; IR
(CHCl3): n˜ =1640, 1614 cmꢀ1; MS (ESI): m/z (%): 484 (100) [M+H]+,
506 (36) [M+Na]+; Anal. calcd for C30H33N3O3: C 74.51, H 6.88, N
8.69, found: C 74.64, H 6.86, N 8.71.
1-(Adamantan-1-yl)-4-[2-(pentyloxy)quinoline]-1H-1,2,3-triazole
(16): Prepared from 26 according to the procedure used for the
preparation of 15. pale yellow solid (37%): mp: 1708C; 1H NMR
(200 MHz, CDCl3): d=0.83–0.92 (m, 3H), 1.43–1.62 (m, 4H), 1.75–
2.08 (m, 9H), 2.23–2.42 (m, 8H), 4.60 (t, J=6.7 Hz, 2H), 7.33–7.41
(m, 1H), 7.55–7.63 (m, 1H), 7.79–7.84 (m, 2H), 8.21 (s, 1H),
9.00 ppm (s, 1H); 13C NMR (100 MHz, CDCl3): d=14.4, 22.9, 29.1,
29.7, 36.3, 43.3, 59.7, 66.7, 116.0, 120.3, 124.5, 125.5, 127.0, 128.2,
129.5, 135.0, 141.3, 145.8, 158.5 ppm; IR (CHCl3): n˜ =3018 cmꢀ1; MS
(ESI): m/z (%): 417 (100) [M+H]+, 439 (19) [M+Na]+; Anal. calcd
3-Bromo-1-pentylquinolin-2(1H)-one (25) and 3-bromo-2-(penty-
loxy)quinoline (26): A suspension of 24 (1.00 g, 4.46 mmol), K2CO3
(1.72 g, 12.48 mmol), 18-crown-6 (1.2 mg, 0.0045 mmol) and pentyl
iodide (1.65 mL, 12.48 mmol) in CH3CN (35 mL) was heated over-
night at 908C. CH3CN was then removed in vacuo, and the residue
930
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ChemMedChem 2012, 7, 920 – 934

Potent and Selective Cannabinoid-2 Receptor Ligands
MED
for C26H32N4O: C 74.97, H 7.74, N 13.45, found: C 74.82, H 7.76, N
13.40.
0.14 mmol) and AdN3 (27 mg, 0.15 mmol) in tBuOH (1.5 mL) was
treated with a solution of CuSO4 (2.2 mg, 0.014 mmol) and sodium
ascorbate (2.7 mg, 0.014 mmol) in H2O (1.5 mL). The reaction mix-
ture was stirred under N2 for 3 d at RT, then poured into H2O
(10 mL) and extracted with CH2Cl2 (3ꢁ10 mL). The organic extracts
were washed with saturated aq NH4OH (20 mL) and brine (20 mL),
dried (anhyd Na2SO4), filtered and concentrated in vacuo. Purifica-
tion by column chromatography (CH2Cl2/MeOH, 98:2) afforded 31
Ethyl 3-(6-bromo-3-iodo-4-oxoquinolin-1(4H)-yl)propanoate (33):
A solution of 6-bromo-3-iodoquinolone (1.30 g, 3.7 mmol) in DMF
(32 mL) was treated with TRITON B (0.8 mL) and ethyl acrylate
(4 mL, 37.1 mmol). The reaction mixture was stirred at 908C for 5 h,
then cooled and poured into H2O (300 mL). The precipitate was
isolated by filtration, washed with H2O and Et2O, and dried in
vacuo to give 33 as a white solid (1.25 g, 75%), which was used in
1
as a white solid (40 mg, 58%): mp: 208–2098C; H NMR (400 MHz,
CDCl3): d=1.19–1.24 (m, 3H), 1.76 (s, 6H), 2.28 (s, 9H), 2.88 (t, J=
7.0 Hz, 2H), 4.13 (dq, J1 =7.0 Hz, J2 =1.0 Hz, 2H), 4.55 (t, J=7.0 Hz,
2H), 7.39 (d, J=9.0 Hz), 7.73 (d, J=9.0 Hz), 8.63 (s, 1H), 8.67 (s,
1H), 8.89 ppm (s, 1H);13C NMR (100 MHz, CDCl3): d=14.1, 29.5,
33.7, 36.0, 43.0, 48.9, 59.5, 61.5, 113.6, 117.1, 117.8, 119.5, 128.3,
130.1, 135.0, 137.1, 140.1, 140.7, 169.3, 173.1 ppm; IR (CHCl3): n˜ =
1732, 1630 cmꢀ1; MS (ESI): m/z (%): 525 (100) [M+H]+, 527 (82)
[M+1]+; Anal. calcd for C26H29BrN4O3: C 59.43, H 5.56, N 10.66,
found: C 59.22, H 5.57, N 10.69.
1
the next step without further purification. mp: 182–1848C; H NMR
(200 MHz, CDCl3): d=1.22 (t, J=6.9, 3H), 2.83 (t, J=6.22, 2H), 4.14
(q, J=6.9, 2H), 4.41 (t, J=6.22, 2H), 7.26 (d, J=9.1 Hz, 1H), 7.73
(dd, J1 =2.3 Hz, J2 =9.1 Hz, 1H), 8.18 (s, 1H), 8.54 ppm (d, J=
2.3 Hz, 1H); 13C NMR (100 MHz, CDCl3): d=14.1, 33.5, 48.5, 61.6,
116.8, 118.2, 124.9, 130.7, 135.4, 137.5, 148.4, 170.0, 172.9 ppm; IR
(CHCl3): n˜ =1731, 1618 cmꢀ1; MS (ESI): m/z (%): 452 (90) [M+2H]2+
;
Anal. calcd for C14H13BrINO3: C 37.36, H 2.91, N 3.11, found: C 37.44,
H 2.91, N 3.10.
3-(1-Adamantyl-1H-1,2,3-triazol-4-yl)-6-(furan-2-yl)quinolin-
Ethyl 3-(6-bromo-4-oxo-3-((trimethylsylil)ethynyl)quinolin-1(4H)-
yl)propanoate (30): A solution of 33 (100 mg, 0.22 mmol) in dry
THF (8 mL) was treated with DIPEA (384 mL, 2.2 mmol), CuI (4.2 mg,
0.022 mmol), and PdCl2(PPh3)2 (15.4 mg, 0.022 mmol). The solution
was degassed at RT for 10 min. During this time, a solution of
TMSA (48 mL, 0.34 mmol) in anhyd THF (3 mL) was also degassed
and added dropwise to the first solution. The reaction mixture was
stirred at RT for 3 h, then filtered through a pad of Celite. The filter
cake was rinsed with EtOAc (10 mL), and the filtrate was washed
with saturated aq NH4Cl (10 mL) and brine (10 mL), dried (anhyd
Na2SO4), filtered and concentrated in vacuo. Purification by flash
column chromatography (EtOAc/PE, 1:1) gave target product 30 as
a beige solid (90 mg, 97%): mp: 144–1458C; 1H NMR (200 MHz,
CDCl3): d=0.23 (s, 9H), 1.23 (t, J=7.2 Hz, 3H), 2.82 (t, J=6.6 Hz,
2H), 4.15 (q, J=7.2 Hz, 2H), 4.40 (t, J=6.6 Hz, 2H), 7.25 (d, J=
8.9 Hz, 1H), 7.71 (dd, J1 =2.1 Hz, J2 =8.9 Hz, 1H), 7.94 (s, 1H),
8.55 ppm (d, J=2.1 Hz, 1H); 13C NMR (100 MHz, CDCl3): d=0.1,
14.1, 33.4, 48.9, 61.5, 98.4, 98.5, 106.8, 117.2, 118.2, 127.6, 129.9,
135.4, 137.1, 147.5, 170.02, 174.9 ppm; IR (CHCl3): n˜ =2359, 1734,
1622 cmꢀ1; MS (ESI): m/z (%): 420 (100) [M+H]+, 422 (85) [M+H]+,
442 (90) [M+Na]+, 444 (87) [M+Na]+; Anal. calcd for
C19H22BrNO3Si: C 54.29 H 5.28, N 3.33, found: C 54.40, H 5.26, N
3.34.
4(1H)-one (29):
A mixture of 31 (20 mg, 0.038 mmol), DME
(776 mL), Pd(OAc)2 (0.8 mg, 0.0038 mmol), Ph3P (3 mg, 0.011 mmol),
EtOH (164 mL), Na2CO3 (1n, 100 mL) and 2-furanboronic acid
(6.7 mg, 0.057 mmol) was irradiated by microwave at 1508C for
5 min. The reaction mixture was then filtered, diluted with EtOAc
(10 mL), was washed with brine (5 mL), dried (anhyd Na2SO4), fil-
tered and concentrated in vacuo. Purification by analytical TLC
(CH2Cl2/MeOH, 95:5) gave 29 as a white solid (10 mg, 63%):
1H NMR (200 MHz, CDCl3/CD3OD): d=1.73 (s, 6H), 2.22 (s, 9H),
6.41–6.43 (m, 1H), 6.71 (d, J=3.2 Hz, 1H), 7.40–7.45 (m, 2H), 7.87
(dd, J1 =1.4 Hz, J2 =8.8 Hz, 1H), 8.56 (s, 1H), 8.60 (s, 1H), 8.64 ppm
(s, 1H); 13C NMR (100 MHz, CDCl3/CD3OD): d=18.6, 29.5, 35.9, 42.9,
59.8, 105.6, 111.9, 118.7, 120.5, 127.1, 127.6, 135.9, 142.4, 153.2,
163.8, 175.4 ppm; MS (ESI): m/z (%): 413 (100) [M+H]+; Anal. calcd
for C25H24N4O2: C 72.80, H 5.86, N 13.58, found: C 73.01, H 5.87, N
13.53.
3-(1-Adamantyl-1H-1,2,3-triazol-4-yl)-6-(furan-2-yl)-1-(4-hydroxy-
pentyl)quinolin-4(1H)-one (32): 5-(Iodopentan-2-yloxy)(tert-butyl)-
dimethylsilane (88 mg, 0.268 mmol) was added to a suspension of
29 (40 mg, 0.096 mmol) and K2CO3 (37 mg, 0.096 mmol) in DMF
(120 mL). The reaction mixture was stirred at 908C for 6 h, then
cooled to RT and treated with H2O (10 mL). The aqueous mixture
was extracted with CH2Cl2 (3ꢁ10 mL), and the combined organic
extracts were washed with brine (20 mL), dried (anhyd Na2SO4), fil-
tered and concentrated in vacuo. Purification by analytical TLC
(CH2Cl2/MeOH, 98:2) gave the alkylated intermediate as a yellow oil
(26 mg). The intermediate was dissolved in THF (2 mL), and the so-
lution was treated with TBAF (1m in THF, 54 mL, 0.054 mmol). Two
further additions of TBAF (30 mL, 0.03 mmol) were added after 30
and 60 min. The reaction mixture was stirred overnight at RT, then
concentrated in vacuo, and diluted with CH2Cl2 (20 mL). The organ-
ic phase was then washed with H2O (15 mL) and brine (15 mL),
dried (anhyd Na2SO4), filtered and concentrated in vacuo. Purifica-
tion by analytical TLC (CH2Cl2/MeOH, 95:5) gave 32 as a white solid
(3.2 mg, 16%): 1H NMR (200 MHz, CDCl3): d=1.19–1.61 (m, 5H),
1.79 (s, 6H), 1.99–2.10 (m, 2H), 2.29 (s, 9H), 3.82–3.91 (m, 2H),
4.28–4.35 (m, 1H), 6.50–6.51 (m, 1H), 6.78 (d, J=3.1 Hz, 1H), 7.51
(m, 1H), 7.56 (s, 1H), 7.95–8.00 (m, 1H), 8.74 (s, 1H), 8.78 (d, J=
1.6 Hz, 1H), 8.84 ppm (s, 1H); 13C NMR (100 MHz, CDCl3): d=23.6,
25.1, 25.8, 29.0, 29.5, 35.9, 36.1, 42.9, 59.8, 105.6, 111.9, 118.7, 120.5,
127.1, 127.6, 135.9, 142.4, 154.2, 175.4 ppm; MS (ESI): m/z (%): 499
(100) [M+H]+, 500 (35) [M+2H]2+; Anal. calcd for C30H34N4O3: C
72.26, H 6.87, N 11.24, found: C 72.15, H 6.88, N 11.20.
Ethyl 3-(6-bromo-3-ethynyl-4-oxoquinolin-1(4H)-yl)propanoate
(34): KF (28 mg, 0.48 mmol) was added to a solution of 30
(100 mg, 0.24 mmol) in dry EtOH (7 mL). The reaction mixture was
stirred at RT for 2 h, then poured onto ice and stirred for a further
15 min. The aqueous mixture was extracted with CH2Cl2 (3ꢁ
20 mL), and the organic extracts were washed with brine (50 mL),
dried (anhyd Na2SO4), filtered and concentrated in vacuo. Purifica-
tion by column chromatography (EtOAc/PE, 1:1 then 2:1) gave 34
1
as a white solid (35 mg, 42%): mp: 157–1608C; H NMR (200 MHz,
CDCl3): d=1.22 (t, J=7.0 Hz, 3H), 2.83 (t, J=6.4 Hz, 2H), 3.23 (s,
1H), 4.12 (q, J=7.0 Hz, 2H), 4.42 (t, J=6.4 Hz, 2H), 7.25 (d, J=
8.9 Hz, 1H), 7.71 (dd, J1 =2.1 Hz, J2 =8.9 Hz, 1H), 7.97 (s, 1H),
8.52 ppm (d, J=2.0 Hz, 1H); 13C NMR (100 MHz, CDCl3): d=14.1,
33.3, 48.9, 61.6, 81.3, 106.8, 117.0, 118.3, 127.1, 130.1, 135.6, 137.2,
147.6, 170.1, 179.1 ppm; IR (CHCl3): n˜ =3310, 2361, 1731,
1621 cmꢀ1; MS (ESI): m/z (%): 348 (100) [M+H]+, 350 (98) [M+1]+,
370 (92) [M+Na]+, 372 (93) [M+Na]+; Anal. calcd for C16H14BrNO3:
C 55.19, H 4.05, N 4.02, found: C 55.04, H 4.06, N 4.03.
Ethyl 3-(6-bromo-3-(1-adamantyl-1H-1,2,3-triazol-4-yl)-4-oxoqui-
nolin-1(4H)-yl)propanoate (31):
A suspension of 34 (50 mg,
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using an online scintillation detector (IN/US system, b-ram Model 3,
Tampa, FL, USA) with a dwell time of 1 s, using Laura Lite 3 soft-
ware and a 50 mL flow cell with the scintillation flow rate set at
150 mLminꢀ1. A series of compounds (7, 9, 10, 11 and 12) were
screened on the KU-812 open tubular columns. A 2 mL frontal
sample containing [3H]Win-55,1212 (0.25 nm) with a mixture of
10 nm of four compounds was carried out as previously de-
scribed.[47]
LogP determination
Experiments were performed according to the procedure described
by Dreassi et al.[65] HPLC analysis was performed on an Agilent
1100 series liquid chromatography/mass selective detector (LC/
MSD) (Agilent Technologies, Palo Alto, CA, USA) with a Lichrocart
125–4 Lichrospher 100 RP-18 column (4.6 mmꢁ100 mm, 5 mm);
method: 86% (v/v) of MeOH in H2O, isocratic, flow rate=
1 mLminꢀ1, UV detector: 254 nm. The Agilent 1100 series LC/MSD
system constituted of a vacuum solvent degassing unit, a binary
high-pressure gradient pump, a 1100 series UV detector, and
a 1100 MSD model VL bench-top single-quadrupole mass spec-
trometer, with orthogonal atmospheric pressure ionization with
electrospray (API-ES). Nitrogen was used as a nebulizing and
drying gas. Parameters were set as follows: pressure of the nebuliz-
ing gas: 40 psi; flow of the drying gas: 9 Lminꢀ1; capillary voltage:
3000 V; fragmentor voltage: 70 V; vaporization temperature:
3508C.
Binding assays
CB1 and CB2 receptor binding assays were performed exactly as
described previously,[23,24] using membranes of cells overexpressing
the human recombinant CB1 or CB2 receptors.
[35S]GTPgS binding assay: For binding experiments, [35S]GTPgS
(1250 Cimmolꢀ1) was obtained from PerkinElmer Life Sciences Inc.
(Boston, MA, USA), GTPgS was purchased from Roche Diagnostic
(Indianapolis, IN, USA) and GDP was obtained from Sigma–Aldrich.
(ꢀ)-cis-3-[2-Hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hy-
droxypropyl)cyclohexanol (CP55940) was purchased from Tocris
(Bristol, UK). Chinese hamster ovary (CHO) cells transfected with
human cannabinoid CB2 receptors (hCB2-CHO cells) were main-
tained in Dulbecco’s modified Eagles’s medium nutrient mixture
F-12 HAM, supplemented with l-glutamine (1 mm), 10% fetal
bovine serum, penicillin–streptomycin (0.6%) and G418
(400 mgmLꢀ1). Cells were maintained at 378C with 5% CO2 in their
media and passaged twice a week using nonenzymatic cell dissoci-
ation solution.
Solubility determination
Experiments were performed according to the procedure described
by Dreassi et al.[66] using the same HPLC conditions described
above.
Column preparation: The CB1/CB2-OT column was synthesized
using a previously described protocol.[47] Briefly, KU812 cells (107)
were homogenized in Tris buffer (10 mL, 50 mm, pH 7.5) containing
NaCl (100 mm), MgCl2 (5 mm), CaCl2 (1 mm), benzamidine (100 mm),
pepstatin A (4 mm), aprotinin (100 mg), leupeptin (100 mg), and
PMSF (1 mg). The homogenate was centrifuged at 700 g for 5 min,
and the supernatant was subsequently collected and centrifuged
at 100000 g for 30 min at 48C. The resulting pellet was suspended
in Tris buffer (2 mL, 50 mm, pH 7.5) containing CHAPS (2% w/v),
NaCl (100 mm), MgCl2 (5 mm), CaCl2 (1 mm), benzamidine (100 mm),
aprotinin (100 mg), leupeptin (100 mg), PMSF (1 mg), pepstatin A
(4 mm), glycerol (10%), and the resulting mixture was rotated with
shaking at 150 rpm for 18 h at 48C. The mixture was then centri-
fuged at 100000 g for 25 min, and the supernatant was used for
immobilization onto the open tubular capillary. The open tubular
capillary (25 cmꢁ100 mm i.d.) was primed with NaOH (1m) for
20 min, rinsed with water, and dried at 958C for 1 h. A 10% aque-
ous solution of APTS was passed through the capillary for 5 min
followed by a 30 min incubation at 958C in an oven, and the pro-
cess was repeated. After 18 h, a 1% aqueous solution of gluteral-
dehyde was passed through the capillary for 1 h followed by
water, and a solution of avidin (25 mm, 5 mg in 2 mL water) was
recycled through the capillary for 5 min. Both tips of the capillary
were submerged in the avidin solution for 7 days at 48C. Then,
a solution of biotin-X (3.5 mm, 5 mg in DMSO/water, 3:1 v/v) was
run through the capillary for 1 h. The solution containing the solu-
bilized KU812 membranes was recycled through the column for
30 min. The open tubular capillary was then dialysed against Tris
buffer (50 mm, pH 7.4) containing NaCl (100 mm), MgCl2 (5 mm),
CaCl2 (1 mm), and EDTA (1 mm) and rotated with shaking overnight
at 48C. The following day, solubilized KU812 membranes were
again passed through the capillary for 30 min, and the dialysis was
repeated.
Binding assays with [35S]GTPgS were performed with membranes
obtained from hCB2-CHO cells.[51] The hCB2 transfected cells were
removed from the flask by scraping, centrifuged at 2500 rpm and
then frozen as a pellet at ꢀ208C until required. Before use in the
[35S]GTPgS binding assay, cells were defrosted and diluted in GTPgS
binding buffer. Protein assays were performed using a Bio-Rad DC
kit (Bio-Rad, Hercules, CA, USA).
[35S]GTPgS binding assay: This assay was performed as described
previously.[50] Briefly, assays were conducted in GTPgS binding
buffer (50 mm Tris-HCl, 50 mm Tris-Base, 5 mm MgCl2, 1 mm EDTA,
100 mm NaCl, 1 mm dithiothreitol, 0.1% bovine serum albumin in
the presence of [35S]GTPgS (0.1 nm) and GDP (30 mm) in a final
volume of 500 mL. Binding was initiated by the addition of hCB2-
CHO cell membranes (20 mg protein per well). Nonspecific binding
was measured in the presence of GTPgS (30 mm). Compounds
under investigation were incubated in the assay for 60 min at
308C. The reaction was terminated by a rapid vacuum filtration
method using Tris-binding buffer, and the radioactivity was quanti-
fied by liquid scintillation spectrometry.
Formalin test: The experimental procedures applied in the formalin
test were approved by the Animal Ethics Committee of the Second
University of Naples (Italy). Animal care was in compliance with the
International Association for the Study of Pain (IASP) and European
Community guidelines on the use and protection of animals in ex-
perimental research (E.C. 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 be-
havior.[67] Nociceptive responses are divided into an early, brief first
phase (0–7 min) caused by a primary afferent discharge produced
by the stimulus, followed by a quiescent period, and then
a second, prolonged phase (15–60 min) of tonic pain. Mice re-
ceived formalin (1.25% in saline, 30 mL) in the dorsal surface of one
Chromatographic system and screening: The CB1/CB2-OT column
was connected to a LC-10AD isocratic HPLC pump (Shimadzu, Co-
lumbia, MD, USA). The mobile phase consisted of ammonium ace-
tate (10 mm, pH 7.4)/MeOH (90:10, v/v) delivered at a rate of
50 mLminꢀ1 at room temperature. Detection was accomplished
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Potent and Selective Cannabinoid-2 Receptor Ligands
MED
side of the hind paw. Each mouse was randomly assigned to one
of the experimental groups (n=8–10) and placed in a Plexiglas
cage and allowed to move freely for 15–20 min. A mirror was
placed at a 458 angle under the cage to allow full view of the
hind-paws. Lifting, favoring, licking, shaking and flinching of the in-
jected paw were recorded as nociceptive responses. The duration
of those mentioned noxious behaviors were monitored by an ob-
server blind to the experimental treatment for periods of 0–10 min
(early phase) and 20–60 min (late phase) after formalin administra-
tion. Groups of 8–10 animals per treatment group were used with
each animal being used for one treatment only. Mice received vehi-
cle (20% DMSO in 0.9% NaCl) or different doses of test com-
pounds by intraperitoneal administration.
80% for the metabolite. The sensitivity limit of the method, taken
as the ratio between the compound peak height and the back-
ground noise height ꢂ10, was 2 pmol of injected material. The re-
lationship between analyte concentration and peak area ratio
versus the peak area of the internal standard was linear up to
300 mm. The metabolite was identified by HPLC-MS analysis. Analy-
ses were performed using ESI in the positive mode: detector volt-
age: 1650 V; drying gas pressure: 25.0 psi; desolvation tempera-
ture: 200.08C; nebulizing gas pressure: 45.0 psi; needle voltage:
5000 V; shield voltage: 600 V; capillary voltage: 65 V. Nitrogen was
used as both the nebulizer and drying gas. Mass spectra (MS) were
acquired over the scan range of 50–1500 m/z. Chromatographic
analysis was carried out using isocratic elution (eluent A: acetoni-
trile; eluent B: 0.05% aqueous formic acid; 80:20 v/v) and a flow
rate of 1.0 mLminꢀ1. After elution, an aliquot of the eluent (200 mL)
was directed to the MSD unit for spectral analysis. The MS spec-
trum of the metabolite was compared with the spectrum obtained
from the authentic compound.
Results are expressed as the mean ꢁ standard error of the mean
(SEM). Significant differences between groups were evaluated by
using analysis of variance followed by the Dunnett’s test. The ver-
sion of the formalin test we applied is based on the fact that a cor-
relational analysis showed that no single behavioral measure can
be a strong predictor of formalin or drug concentrations on spon-
taneous behaviors.[61] 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.[68]
Supporting Information
Equations used in logP determination and the details of the inhibi-
tion studies of metabolism of compound 11 are available as Sup-
porting Information (http://dx.doi.org/10.1002/cmdc.201100573).
Computational studies
Acknowledgements
The simplified compound models were obtained replacing ada-
mantyl and “quinolone” segments of compounds 11–13 by methyl
groups. A total of eight complexes were then generated placing
one water molecule in front of each heteroatom to form a hydro-
gen bond. Geometries of the complexes between the five-mem-
bered ring and water were optimized at the HF/6–311G**+ + level
of theory, using ab initio quantum chemistry program GAMESS.[69]
Total interaction energies were decomposed via the Kitaura–Moro-
kuma scheme[53] as implemented in the GAMESS program.[69]
A.L. and V.D.M. thank Mr. Marco Allarꢀ (CNR, Pozzuoli, Italy) for
technical assistance. R.M. and A.R. thank the Intramural Research
Program of the US National Institute on Aging for financial sup-
port. D.B., M.G.C. and R.G.P. thank the US National Institutes of
Health (NIH) for funding (grant no.: DA-03672).
Keywords: bioisosteres
·
cannabinoids
·
quinolones
·
receptors · structure–activity relationships
Metabolic studies of compound 11 in vitro
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Received: December 5, 2011
Revised: January 24, 2012
Published online on March 2, 2012
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ChemMedChem 2012, 7, 920 – 934