Design, synthesis, and pharmacological characterization of indol-3-ylacetamides, indol-3-yloxoacetamides, and indol-3-ylcarboxamides: Potent and selective CB2 cannabinoid receptor inverse agonists

Article abstract of DOI:10.1021/jm3003334

In our search for new cannabinoid receptor modulators, we describe herein the design and synthesis of three sets of indole-based ligands characterized by an acetamide, oxalylamide, or carboxamide chain, respectively. Most of the compounds showed affinity

Full text of DOI:10.1021/jm3003334

Article
pubs.acs.org/jmc
Design, Synthesis, and Pharmacological Characterization of Indol-3-
ylacetamides, Indol-3-yloxoacetamides, and Indol-3-ylcarboxamides:
Potent and Selective CB2 Cannabinoid Receptor Inverse Agonists
Serena Pasquini,† Claudia Mugnaini,† Alessia Ligresti,‡ Andrea Tafi,† Simone Brogi,† Chiara Falciani,†
Valentina Pedani,† Nicolo Pesco,† Francesca Guida,§ Livio Luongo,§ Katia Varani,∥ Pier Andrea Borea,∥
̀
Sabatino Maione,§ Vincenzo Di Marzo,*,‡ and Federico Corelli*,†
†Dipartimento Farmaco Chimico Tecnologico, Universita
̀
degli Studi di Siena, Via A. Moro, 53100 Siena, Italy
‡Endocannabinoid Research Group, Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Via dei Campi Flegrei 34,
80078 Pozzuoli (Napoli), Italy
§Endocannabinoid Research Group, Dipartimento di Medicina Sperimentale − Sezione di Farmacologia “ Donatelli”,
̀
Seconda Universita di Napoli, Via S. Maria di Costantinopoli 16, 80138 Napoli, Italy
∥Dipartimento di Medicina Clinica e Sperimentale, Sezione di Farmacologia, Universita
Via Fossato di Mortara 17-19, 44121 Ferrara, Italy
̀
degli Studi di Ferrara,
S
* Supporting Information
ABSTRACT: In our search for new cannabinoid receptor
modulators, we describe herein the design and synthesis of
three sets of indole-based ligands characterized by an
acetamide, oxalylamide, or carboxamide chain, respectively.
Most of the compounds showed affinity for CB2 receptors in
the nanomolar range, with Ki values spanning 3 orders of
magnitude (377−0.37 nM), and moderate to good selectivity
over CB1 receptors. Their in vitro functional activity as inverse
agonists was confirmed in vivo in the formalin test of acute
peripheral and inflammatory pain in mice, in which
compounds 10a and 11e proved to be able to reverse the effect of the CB2 selective agonist COR167.
atherosclerosis,10 myocardial infarction,11 stroke,12 gastro-
INTRODUCTION
■
intestinal inflammatory,13and autoimmune14 and neurodege-
nerative disorders15 to hepatic ischemia/reperfusion injury,16
inflammation17 and fibrosis,18 kidney19 and bone20 disorders,
and cancer.21
Cannabinoid receptors 1 and 2 (CB1 and CB2) are G protein-
coupled receptors that were characterized and cloned in the early
1990s.1,2 While CB1 receptors are expressed at high levels in the
central nervous system (CNS), CB2 receptors are primarily
found in the immune system, in tonsils, spleen, macrophages,
and lymphocytes (B-cells and natural killer cells),3 although there
is some evidence of their presence also in the CNS.4
Agonists of both cannabinoid receptor types produce strong
antinociceptive effects in animal models of chronic, neuro-
pathic, and inflammatory pain and are intensively investigated
as potential new antihyperalgesic and antiinflammatory agents.5
Activation of central CB1 receptors, rather than peripheral
CB1 or CB2 receptors, is thought to mediate the psycho-
tropic effects associated with nonselective agonists such as
Δ9-tetrahydrocannabinol (Δ9-THC).6 The occurrence of these
adverse effects limits the therapeutic usefulness of mixed
cannabinoid agonists that show high affinity for CB1 receptors.
Differences in receptor distribution and signal transduction
mechanisms7,8 are likely to account for the relative absence of
CNS side effects induced by CB2 agonists, which, in addition to
pain, are also being actively investigated for use in a multitude
of disparate diseases and pathological conditions,9 ranging from
The indole nucleus represents a “privileged structure” in
medicinal chemistry22 and a well-established scaffold for the
development of CB2 receptor ligands.23 Pioneering work at
the Sterling-Winthrop Research Institute, leading to the potent
but nonselective ligand 124 (Chart 1), was followed by the
development of CB2-selective ligands 2 by Huffmann et al.25
and 3 by Makriyannis and co-workers26. CB2-Selective agonists
as well as inverse agonists (exemplified by 427 and 5,28
respectively) were obtained by fine chemical modulation of
the prototypical structures, while a ground-breaking work by
Frost et al.23 demonstrated that indol-3-ylcycloalkyl ketones,
such as 6, lacking an aromatic acyl group, are new high-affinity
CB2 ligands.
In our research program on new cannabinoid receptor
modulators,29 we have described the rapid combinatorial
Received: March 9, 2012
© XXXX American Chemical Society
A
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Chart 1. Representative Indole-Based CB2 Ligands
synthesis, starting from 5-hydroxyindole-3-acetic acid, of a
library of N,O-dialkylated 5-hydroxy-3-indole-N-alkylaceta-
mides, some of which (e.g., 7, Chart 2) proved to be weak
Accordingly, the new compounds were designed through an
approach that, acknowledging the structure−affinity relationship
developed for quinolone-3-carboxamide CB2 ligands29a,f,i
(such as 8), entailed (i) the removal of the linker between
the aromatic rings of 7 in order to obtain 5-(hetero)aryl-
substituted indoles 9−11, more closely mimicking 8, and (ii)
either preserving the acetamide moiety (9) or replacing it with
oxalylamide (10) and carboxamide (11) side chains. We have
recently developed a pharmacophore based three-dimensional
quantitative structure−selectivity relationship model (3D-QSSR)
able to predict in a semiquantitative manner the selectivity index
(SI) of novel CB2 receptor ligands belonging to several
structural classes.29h Herein, we report the prediction of the SI
of three molecules, taken as representative samples of structural
classes 9, 10, and 11, by application of our selectivity model. The
outcome of this prediction was expected to possibly support the
approach we had chosen to design CB2-selective 5-(hetero)aryl-
substituted indoles and to provide a theoretical grounding to
rationalize the subsequent pharmacological results.
Chart 2. Prototypical Structures (7, 8) and Novel
5-(Hetero)arylindole-Based Acetamides (9), Oxoacetamides
(10), and Carboxamides (11)
The SI of prototypes 9j, 10b, and 11d (Table 1) was pre-
dicted by application of our 3D-QSSR model after a conforma-
tional search was performed on these derivatives and their
conformers were aligned onto the CB2 pharmacophore
CB2PHAM.29h The results of the in silico selectivity prediction
are shown in Table 1. All three derivatives 9j, 10b, and 11d
were predicted to be selective, even if at a different extent.
Compounds 10b and 11d were estimated to possess similar
selectivities (corresponding to an activity difference between
CB1 and CB2 receptors of at least 2 orders of magnitude) and
to be roughly 10 times more selective than 9j. Accordingly,
compounds 9j, 10b, and 11d were synthesized and tested.
Comparison between calculated and experimental SI values for
the three compounds confirmed the prediction ability of our
model and hence all the designed derivatives 9−11 (as well as
compounds 13 and 14) were checked by theoretical estimation
of their SI. As can be seen in Table 1, for all these compounds
the calculated SI values were larger than 10. On the basis of
these results, the preparation of the whole set of compounds
9−11 was confidently undertaken.
but selective CB2 ligands.30 Here we describe our investigations
aimed at identifying new indole-based compounds with
enhanced affinity and/or selectivity for the CB2 receptor.
RESULTS AND DISCUSSION
■
Compound Design and Selectivity Prediction. Struc-
tural modifications were planned to reduce the conformational
freedom of lead compound 7, thereby modifying receptor
selectivity and affinity according to a popular tactic in medicinal
chemistry.31 On the other hand, the choice of the substituents
to decorate the indole scaffold was suggested by results obtained
in our previous studies on quinolone cannabinoid ligands.
B
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Chemistry. The preparation of indole derivatives 9 was
accomplished by the route shown in Scheme 1, following synthetic
procedures already set up for 3-indole-N-alkylacetamide30 and
quinolone-3-carboxamide analogues.29a,f,i 5-Bromo-1H-indole-3-ace-
tic acid was converted into the corresponding amides 12a and 12b
(method A), which then underwent N-alkylation with the
appropriate alkyl halide under phase-transfer catalysis (PTC) con-
ditions (method B) to yield compounds 13a and 13b,c, respec-
tively. Suzuki coupling (method C) with furan-2-ylboronic acid pro-
vided the final compounds 9a−c. With the aim to enhance chemical
a
Table 1. CB1 and CB2 Receptor Affinity Values for Compounds 9a−k, 10a−e, 11a−g, 13b,c, and 14
C
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Table 1. continued
a
b
Data represent mean values for at least three separate experiments performed in duplicate and are expressed as Ki (nanomolar). Ad, adamantan-1-yl.
cCB1, human cannabinoid type 1 receptor. dCB2, human cannabinoid type 2 receptor. eFor both receptor binding assays, the new compounds were tested
by use of membranes from HEK cells transfected with either the CB1 or CB2 receptor and [3H]-(−)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-
4-(3-hydroxypropyl)-cyclohexanol ([3H]CP-55,940). fKi = “Eeuilibrium dissociation constant”, that is, the concentration of the competing ligand that will
g
bind to half the binding sites at equilibrium, in the absence of radioligand or other competitors. SI = selectivity index for CB2, calculated as Ki(CB1)/
Ki(CB2) ratio. hBinding affinities of reference compounds were evaluated in parallel with the test compounds under the same conditions. iCB2 reference
j
compound. CB1 reference compound.
a
Scheme 1. Synthesis of 2-(1H-Indol-3-yl)acetamides 9a−k
a
Reagents and conditions: (a) Method A: appropriate amine, EDC, HOBt, DCM, room temp. (b) Method B: alkyl halide, TBAB, 20% NaOH/
DCM, room temp. (c) Method C: arylboronic acid, Pd(OAc)2, PPh3, 2 M Na2CO3, DME, EtOH, MW, 150 °C, 10 min.
diversity at N1 position, amide 12b was first transformed with
phenylboronic acid to 14, which was then subjected to N-alkylation
with several alkyl halides to provide a set of 1-substituted 2-(5-
bromo-1H-indol-3-yl)-N-(adamantan-1-yl)acetamides (9d−k).
D
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a
Scheme 2. Synthesis of 2-(1H-Indol-3-yl)-2-oxoacetamides 10a−d
a
Reagents and conditions: (a) Method D: appropriate amine, TEA, DCM, room temp. (b) Method B: alkyl halide, TBAB, 20% NaOH/DCM,
room temp. (c) Method C: arylboronic acid, Pd(OAc)2, PPh3, 2 M Na2CO3, DME, EtOH, MW, 150 °C, 10 min.
Starting from 2-(5-bromo-1H-indol-3-yl)-2-oxoacetyl chlor-
ide32 (Scheme 2), the final oxalylamides 10a−d were
synthesized via amidation (method D) to 15, alkylation to
derivatives 16a,b, and final cross-coupling reaction to install the
5-(hetero)aryl substituent. To test a different synthetic route,
compound 10e (Scheme 3) was prepared by Suzuki arylation of
16c, in turn obtained through amidation of the N-alkylated
indol-3-oxalyl chloride 17, which could be prepared by
treatment of 18 with oxalyl chloride.
Scheme 3. Synthesis of 2-(1H-Indol-3-yl)-2-oxoacetamide
10e
a
5-Bromo-1H-indol-3-carboxylic acid and its 6-bromo isomer
(Scheme 4), prepared according to a literature protocol,33 were
converted into the amides 19a,b (method E), which were then
subjected to alkylation (to 20a,b,c), followed by Pd-catalyzed
arylation to afford the indol-3-yl-carboxamides 11a−g.
a
Reagents and conditions: (a) Method B: pentyl iodide, TBAB, 20%
NaOH/DCM, room temp. (b) Oxalyl chloride, Et2O, 0 °C. (c)
Method D: cyclohexylamine, TEA, DCM, room temp. (d) Method C:
phenylboronic acid, Pd(OAc)2, PPh3, 2 M Na2CO3, DME, EtOH,
MW, 150 °C, 10 min.
In Vitro Pharmacology and SAR. All the newly
synthesized indole derivatives were screened, in a competitive
binding experiment, for their affinity and selectivity toward the
human recombinant CB1 and CB2 receptors. The tested
compounds were evaluated in parallel with SR14452834 and
rimonabant35 as CB2 and CB1 reference ligands, respectively, as
previously described.29 The results, in terms of binding affinities
for the two receptors (Ki values), are reported in Table 1.
With the exception of compound 13b, which proved to be
devoid of affinity at both cannabinoid receptor types, all the
tested compounds showed affinity at CB2 receptor in the nano-
molar range, with Ki values spanning 3 orders of magnitude
(from 377 for 9a to 0.37 for 10a; Table 1).
indole derivatives. Thus, the direct connection of an aryl group
to the indole moiety, by removing the methylene bridge at
position 5 of 7 while retaining the acetamide chain at position
3, positively affected CB2 affinity, as compounds 9 and 14
showed Ki values 3−30 times lower than that of 7. Further
conformational restriction at the level of the amide side chain,
as in compounds 10 and 11, characterized by a carbonyl group
directly linked to the indole nucleus, caused a 16−1000-fold
enhancement in CB2 receptor affinity compared to the
prototype 7.
Our computational approach not only demonstrated
predictive significance but also allowed us to rationalize the
structure−affinity and structure−selectivity relationships
through its microscopic interpretation. In Figure 1 the super-
position of the 3D-QSSR model and compounds 9j, 10b, and
The structural optimization strategy, based on conforma-
tional restriction of the aryl substituent at position 5 and amide
side chain at position 3 of the prototypical compound 7, led to
a significant improvement in the binding profile of the new
E
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a
Scheme 4. Synthesis of (1H-Indol-3-yl)carboxamides 11a−g
a
Reagents and conditions: (a) Method E: 1-aminoadamantane, CDI, DMF, room temp to 120 °C. (b) Method B: alkyl halide, TBAB, 20% NaOH/
DCM, room temp. (c) Method C: arylboronic acid, Pd(OAc)2, PPh3, 2 M Na2CO3, DME, EtOH, MW, 150 °C, 10 min.
Figure 1. (Crossed stereoview) Superposition of the 3D-QSSR model with compounds 9j (yellow carbon atoms), 10b (green carbon atoms), and
11d (white carbon atoms).
11d (the three chosen representatives) is shown. The different
matching of both the CB2 pharmacophore and the QSSR
model by the three compounds is quite evident. The acetamide
moiety of 9j, in fact, causes a different orientation of the indole
moiety with respect to that of 10b and 11d, thereby markedly
affecting the orientation of the phenyl substituent at position 5
of the indole nucleus. The different three-dimensional arrange-
ment of crucial substituents of 9j, 10b, and 11d is likely to
account for the difference observed in their receptor affinity.
Moreover, the three sets of compounds elicited the following SI
values: 9, 5 ≤ SI ≤ 487; 10, 154 ≤ SI ≤ 932; 11, 26 ≤ SI ≤ 568.
Therefore, compounds 9 are the least selective while
compounds 10 are the most selective for CB2 receptor.
Inspection of Figure 1 can explain this selectivity trend. With
respect to 11d, in fact, compound 10b is able to locate its
amide carbonyl in the blue area representing increased CB2
selectivity, whereas 9j occupies the red area of decreased CB2
selectivity with the methylene group of the amide side chain.
Compounds bearing the acetamide chain on the indole ring
(general structures 9, 13, and 14) exhibited moderate to good
CB2 affinity. In this series, the replacement of the 1-adamantyl
group (as in 9b) by the cycloheptyl residue (compound 9a)
had a detrimental effect on the binding profile. The substituent
on the amide nitrogen matches a hydrophobic feature of our
CB2 pharmacophore model, and hence it is reasonable that
CB2 affinity may be modulated by the lipophilicity of this
substituent. This finding is in line with previous observations
on quinolone-3-carboxamide derivatives, where replacement of
the adamantyl group by less bulky/lipophilic moieties markedly
reduced CB2 receptor affinity,29a,f as a result of decreased
F
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Table 2. Effects of Compounds 9g, 10a−c, and 11b,e,f on cAMP Production in hCB2 CHO Cells in Comparison with Reference
a
Compound JWH133
increase in cAMP production (%)
concn
9g
10a
10b
55
70
10c
62
75
11b
51
68
11e
93
110 11
11f
JWH133
1 μM
67
88
6
9
138 12
163 15
4
6
5
7
5
6
9
106 10
135 14
−87
−94
8
9
10 μM
a
Data represent mean values for at least four independent experiments performed in duplicate.
hydrophobic interactions with lipophilic residues.36 The
insertion of an aromatic or heteroaromatic substituent at the
5 position of the indole ring improved receptor affinity
(compare 13b,c with compounds 9), although no substantial
difference was noticed among compounds bearing a phenyl or a
2-furyl group.
On the other hand, the N1 side chain exerts a modulatory
effect on affinity, depending on its length, the highest affinity
being demonstrated by compounds with chains of 3−4 carbon
atoms (9f−I).
is not completely surprising on the basis of what described in
the arylquinolone-3-carboxamide series, where CB2 selectivity
was lost by moving the aryl substituent from position 6 to
position 7, nevertheless it cannot be rationalized by resorting to
our selectivity model.
Functional Activity at CB2 Receptors in Vitro. The
effect of compounds 9g, 10a−c, and 11b,e,f on forskolin-
induced elevation of cyclic adenosine monophosphate (cAMP)
levels in Chinese hamster ovary (CHO) cells transfected with
human CB2 receptor was also assessed. Table 2 reports the
effect of the examined compounds at concentrations of 1 and
10 μM, expressed as percentage of the cAMP increase.
In our experimental conditions, a well-known CB2 agonist,
JWH-133, was able to inhibit cAMP production, whereas the
novel CB2 ligands were able to increase cAMP production with a
typical inverse agonist behavior. This finding was not completely
unexpected, because 6-arylquinolone-3-carboxamides behave
similarly. Thus, if one takes into consideration the structural
analogy between 6-arylquinolones and 5-arylindoles and grants
that both these heterocyclic systems simply act as scaffolds able
to correctly orient their substituents in the 3D space, it is not
surprising that compounds belonging to either class may exert
the same functional effects.
In Vivo Pharmacology. Finally, the in vivo activity of 10a
and 11e, that is, the most potent and CB2-selective indole
derivatives, was evaluated in the formalin test of acute peripheral
and inflammatory pain in mice. Formalin injection induces
a biphasic stereotypical nocifensive behavior. Nociceptive
responses are divided into an early, short-lasting first phase
(0−7 min), caused by a primary afferent discharge produced by
the stimulus, followed by a quiescent period and then a second,
prolonged phase (15−60 min) of tonic pain. Fifteen minutes
before injection of formalin, mice received intraperitoneal (ip)
administration of vehicle or either of the two compounds
(1 or 3 mg/kg), alone or in combination with the selective CB2
agonist N-(adamantan-1-yl)-6-isopropyl-4-oxo-1-pentyl-1,4-di-
hydroquinoline-3-carboxamide29a (hereafter referred to as
COR167) (1 mg/kg, ip), administered 5 min before the
compound.29e The results are presented in Figures 2 and 3.
When assayed in the formalin test, 10a and 11e exhibited a
behavior we have shown to be typical of CB2 inverse agonists
in several previous studies,29e,f,i with no effect on the first phase
of formalin nocifensive behavior and a weak but dose-related
effect on the second, inflammatory phase. Accordingly,
compound 10a, the least potent of the two compounds, at a
dose inactive per se (1 mg/kg), completely reversed the effect
of the CB2 agonist COR167; and compoind 11e, despite
having being tested at a dose still exerting some effect per se
(1 mg/kg), still counteracted the effect of COR167.
All the compounds in the oxalamide series (10a−e) are high-
affinity CB2 ligands, also endowed with remarkable selectivity
over CB1 receptor. Comparison between corresponding
acetamide and oxalamide derivatives 9j/10b, 9b/10c, and 9g/
10d clearly shows that higher CB2 selectivity of compounds 10,
with respect to derivatives of 9, is due to both greater affinity
for CB2 receptor and lower affinity for CB1 receptor. Such
an in vitro profile was determined by the replacement of a
methylene group by a carbonyl group at position 3 of the
indole ring, that in turn influences the steric and electronic
properties of the amide side chain. Thus, the different relative
orientation of the amide group and indole ring may help
discriminate between cannabinoid receptor subtypes, while a
more profitable H-bond networking between ligands 10 and
CB2 receptor is likely to account for enhanced CB2 affinity. In
fact, although all the tested compounds are able to match the
H-bond donor feature with a carbonyl oxygen when super-
imposed to the 3D-QSSR model, compounds of structure 10
possess another carbonyl group that may be involved in further
H-bonding interactions within the CB2 receptor binding
pocket, so as to enhance receptor affinity. Compound 10a, in
particular, showed Ki(CB2) = 0.37 nM. Although no definitive
explanation for such a notable affinity can be given, this resulted
in the most active compound among the 26 indole derivatives
assayed. Contrary to what was observed in the acetamide series,
substitution of a cyclohexyl group for the 1-adamantyl one did
not alter the binding properties (compare 10b with 10e).
Also, the compounds belonging to the carboxamide series
(11a−g) displayed very high affinity for CB2 receptor, with Ki
values ranging from 22.2 nM (11b) to 1.82 nM (11a), and in
most cases well compare with the CB2 reference standard
SR144528 (Ki = 5.4 nM). By comparison with the cor-
responding derivatives 10, it can be seen that differences in
Ki(CB2) values are within 1 order of magnitude. In particular,
the ratio between Ki(CB2) values for the couples 11a/10d,
11c/10c, 11d/10b, and 11e/10a are 0.07, 0.6, 0.9, and 6.7,
respectively. As a result, compounds 11 are substantially
equivalent to derivatives 10 in terms of affinity, though
endowed with somewhat lower receptor selectivity. It is
noteworthy that moving the aromatic substituent from the
5-position (11d) to the 6-position (11g) gave rise to a dramatic
increase in CB1 receptor affinity, leading to a compound
possessing very high affinity at both CB1 and CB2 receptors
and inverted receptor selectivity (SI < 1). Although this finding
In summary, three different series of 5-arylindole derivatives
namely, indol-3-ylacetamides 9, indol-3-yloxalamides 10, and
indol-3-ylcarboxamides 11were studied as CB2 receptor
ligands. Most of these compounds, particularly those belonging
to the second and third groups, elicited high affinity at CB2
G
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compare with other indole analogues previously described as
CB2 receptor agonists.23 However, compounds 9−11 differ in
terms of functional activity, in that they act as inverse agonists
in vitro and such an activity profile has been confirmed in in
vivo assays, where they proved to be able to reverse the effect
of the CB2 selective agonist COR167. These results are
reminiscent of those obtained with 6-(hetero)arylquinolone-3-
carboxamides (like 8) and seem to support our initial idea that
the quinolone and indole rings may act as interchangeable
scaffolds, which, though not identical in terms of physicochem-
ical properties, are able to orient in a similar way some crucial
pharmacophoric groups, thereby yielding compounds that exert
similar pharmacodynamic effects.
A number of reports have suggested that selective cannabinoid
CB2 inverse agonists may serve as novel immunomodulatory
agents in the treatment of a variety of acute and chronic
inflammatory disorders.29e,37,38 With this background, the new
compounds here described might represent an interesting
starting point for the development of potential antiinflammatory
agents with an innovative mechanism of action.
EXPERIMENTAL SECTION
■
Figure 2. Effect of compound 10a (1−3 mg/kg, ip), (top) alone or
(bottom) in combination, at the dose of 1 mg/kg, with COR167
(1 mg/kg, ip) in the formalin test in mice. The total time of the
nociceptive response was measured every 5 min. Results are mean
Chemistry. Reagents were purchased from commercial suppliers
and used without further purification. Anhydrous reactions were run
under positive pressure of dry N2. Merck silica gel 60 was used for
flash chromatography (23−400 mesh). IR spectra were recorded on a
Perkin−Elmer BX Fourier transform infrared (FT-IR) system with
▲
SEM (n = 8−10 for each group). ( , top) Statistically significant
◆
differences vs formalin; ( , bottom) statistically significant differences
CHCl3 as the solvent or a Nujol dispersion. H NMR and 13C NMR
1
■
(P < 0.05) vs formalin; ( , bottom) statistically significant differences
were recorded at 200 and 50 MHz, respectively, on a Bruker AC200F
spectrometer and at 400 and 100 MHz on a Bruker Advance DPX400.
Chemical shifts are reported relative to that of tetramethylsilane
at 0.00 ppm. Mass spectral (MS) data were obtained on an Agilent
1100 LC/MSD VL system (G1946C) with a 0.4 mL/min flow rate by
use of a binary solvent system of 95:5 methanol/water. UV detection
was monitored at 254 nm. Mass spectra were acquired in either
positive or negative mode scanning over the mass range 105−1500.
Melting points were determined on a Gallenkamp apparatus and are
uncorrected. Microwave irradiations were conducted with a CEM
Discover synthesis unit (CEM Corp., Matthews, NC). Elemental
analyses were performed on a Perkin-Elmer PE 2004 elemental
analyzer, and the data for C, H, and N are within 0.4% of the theoretical
values. The chemical purity of the target compounds was determined
under the following conditions: zorbax eclipse C8, MeOH/H2O
(MeOH 80−95, t = 20 min), 0.8 mL/min. The purity of each
compound was >96% in either analysis.
(P < 0.05) vs COR167. One-way analysis of variance followed by a
Tukey−Kramer multiple comparisons test was used for data analysis.
Synthesis of Amides 12a,b by Method A: General
Procedure. To a solution of 5-bromoindoleacetic acid (254 mg,
1 mmol) in dichloromethane (DCM; 15 mL) were added successively
HOBt (135 mg, 1 mmol), EDC (383 mg, 2 mmol), and the
appropriate amine (2 mmol). After the mixture was stirred at room
temperature for 2 h, solvent was evaporated and the residue was flash-
chromatographed on silica gel eluted with DCM/MeOH (95:5) to
give the pure product.
Example: 2-(5-Bromo-1H-indol-3-yl)-N-(adamantan-1-yl)-
acetamide (12b). Obtained in quantitative yield. White solid, mp
1
93−94 °C. H NMR (400 MHz, DMSO-d6) δ 10.94 (br s, 1H), 7.68
Figure 3. Effect of compound 11e (1−3 mg/kg, ip), (top) alone or
(bottom) in combination, at the dose of 1 mg/kg, with COR167 (1 mg/kg,
ip) in the formalin test in mice. The total time of the nociceptive
response was measured every 5 min. Results are mean SEM (n = 8−
(s, 1H), 7.39 (br s, 1H), 7.22 (d, J = 8.0 Hz, 1H), 7.11−7.06 (m, 2H),
3.23 (s, 2H), 1.90 (s, 3H), 1.83 (s, 6H), 1.52 (s, 6H). 13C NMR (100
MHz, CDCl3) δ 170.3, 135.1, 128.8, 125.2, 124.8, 121.4, 113.0, 109.2,
51.9, 41.5, 36.3, 34.6, 29.4. IR (Nujol) 1654 cm−1. MS (ESI) m/z 388
[M + H]+, 410 [M + Na]+. Anal. (C20H23BrN2O) C, H, N.
●
▲
10 for each group). ( , , top) Statistically significant differences
◆
●
■
(P < 0.05) vs formalin; (
,
, bottom) statistically significant
Synthesis of Compounds 9d−k, 13a−c, 16a,b, 18, and 20a−
c by Method B: General Procedure. A mixture of the appropriate
starting compound (0.9 mmol), alkyl halide (1.1 mmol), tetrabuty-
lammonium bromide (TBAB; 0.5 mmol), 20% aqueous sodium
hydroxide (10 mL), and DCM (15 mL) was stirred at room temp-
erature overnight. The reaction mixture was diluted with water and the
organic layer was extracted with DCM (3 × 10 mL). The combined
differences (P < 0.05) vs formalin; ( ) statistically significant differences
(P < 0.05) vs COR167. One-way analysis of variance followed by a
Tukey−Kramer multiple comparisons test was used for data analysis.
receptor and moderate to good selectivity over CB1 receptor.
As a whole, the new indole-based CB2 ligands favorably
H
dx.doi.org/10.1021/jm3003334 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
organic solution was washed with brine, dried over anhydrous sodium
sulfate, and evaporated to dryness. The crude residue was purified by
flash chromatography on silica gel, eluting with hexanes/ethyl acetate
(2:1) to provide the title compound.
2-(5-Bromo-1-pentyl-1H-indol-3-yl)-2-oxoacetyl chloride (17).
Oxalyl chloride (197 μL, 2.25 mmol) was added at 0 °C to a solution
of 5-bromo-1-pentyl-1H-indole (500 mg, 1.88 mmol) in dry Et2O.
After the solution was stirred at room temperature for 1 h, a further
amount of oxalyl chloride (164 μL, 1.88 mmol) was added and the
solution was maintained at room temperature for a further 20 min.
The solution was evaporated in vacuo and the yellow-orange residue
was washed with hexanes to give 402 mg (60%) of the title compound,
which was used in the next step without further purification.
Molecular Modeling. Three-dimensional structure modeling and
matching onto the previously developed atom-based 3D-QSSR model
were carried out on an IBM workstation with Linux operating system
running Maestro 8.0, MacroModel 9.5 and phase 2.5 programs
Example: 2-[[5-Bromo-1-(prop-2-en-1-yl)-1H-indol]-3-yl]-2-oxo-
N-(adamantan-1-yl)acetamide (16b). Prepared in 80% yield from 15.
1
Yellow solid, mp 150−152 °C. H NMR (400 MHz, CDCl3) δ 8.97
(d, J = 4.0 Hz, 1H), 8.55 (s, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.29 (s,
1H), 7.18 (d, J = 8.0 Hz, 1H), 6.00−5.91 (m, 1H), 5.28 (d, J =
12.0 Hz, 1H), 5.16 (d, J = 20.0 Hz, 1H), 4.72 (s, 2H), 2.10 (s, 9H),
1.72 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 181.1, 161.2, 141.6,
134.9, 131.3, 129.5, 126.7, 125.3, 119.2, 117.1, 111.7, 111.5 51.8,
49.8, 41.2, 36.3, 29.4. IR (Nujol) 1679, 1639 cm−1. MS (ESI) m/z 442
[M + H]+, 464 [M + Na]+. Anal. (C23H25BrN2O2) C, H, N.
(Schrodinger, LLC, New York). The use of phase, implemented in the
̈
Maestro modeling package, to generate a pharmacophore and a 3D-
QSSR model for cannabinoid receptor CB2 has been already described
elsewhere.29h The 3D structure of all the derivatives modeled in this
study was built in Maestro. Conformers of each molecule were
generated in MacroModel by use of the OPLS_2005 force field,
GB/SA water, and no cutoff for nonbonded interactions. Molecular
energy minimizations were performed via the PRCG method with
5000 maximum iterations and a numerical value of 0.001 as gradient
convergence threshold. The conformational searches were carried out
by application of the MCMM torsional sampling method, performing
automatic setup with 20 kJ/mol in the energy window for saving
structure and a 0.5 Å cutoff distance for redundant conformers. Each
SI value was predicted by application of our 3D-QSSR model after
alignment of the corresponding molecule to the CB2 pharmacophore.
In Vitro Functional Assay. CHO cells transfected with human
CB2 receptors (Perkin-Elmer Life and Analytical Sciences, Boston,
MA) were washed with phosphate-buffered saline and diluted trypsin
and centrifuged for 10 min at 200g. The pellet containing CHO cells
(1 × 106 cells/assay) was suspended in 0.5 mL of incubation mixture:
NaCl 150 mM, KCl 2.7 mM, NaH2PO4 0.37 mM, MgSO4 1 mM,
CaCl2 1 mM, Hepes 5 mM, MgCl2 10 mM, and glucose 5 mM, pH 7.4
at 37 °C. Then 0.5 mM 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidi-
none (Ro 20-1724) as phosphodiesterase inhibitor was added and
preincubated for 10 min in a shaking bath at 37 °C. The effects of test
compounds (9g, 10a−c, and 11b,e,f) and reference compound JWH-133
were studied in the presence of forskolin (1 μM). The reaction was
terminated by the addition of cold 6% trichloroacetic acid (TCA). The
TCA suspension was centrifuged at 2000g for 10 min at 4 °C and the
supernatant was extracted four times with water-saturated diethyl
ether. The final aqueous solution was tested for cAMP levels by
a competition protein binding assay. Samples of cAMP standard
(0−10 pmol) were added to each test tube containing the incuba-
tion buffer (Trizma base 0.1 M, aminophylline 8.0 mM, and 2
mercaptoethanol 6.0 mM, pH 7.4) and [3H]cAMP (specific activity 26
Ci/mmol; Perkin-Elmer Life and Analytical Sciences, Boston, MA).
The binding protein, previously prepared from beef adrenal glands,
was added to the samples previously incubated at 4 °C for 150 min,
and after the addition of charcoal thes samples were centrifuged at
2000g for 10 min. The clear supernatant was counted by using a Tri
Carb Packard 2810 TR scintillation counter.
In Vivo Antinociceptive Assay (Formalin Test). The exper-
imental procedures applied in the formalin test were approved by the
Animal Ethics Committee of the Second University of Naples. Animal
care was in compliance with the IASP and European Community
guidelines on the use and protection of animals in experimental
research (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 behavior.39
Nociceptive responses are divided into an early, short-lasting first
phase (0−7 min) caused by a primary afferent discharge produced by
the stimulus, followed by a quiescent period and then a second,
prolonged phase (15−60 min) of tonic pain. Mice received formalin
(1.25% in saline, 30 μL) in the dorsal surface of one side of the hind-
paw. Each mouse was randomly assigned to one of the experimental
groups (n = 8−10) and placed in a Plexiglas cage and allowed to move
freely for 15−20 min. A mirror was placed at a 45° angle under the
Synthesis of Compounds 9a−c, 10a−e, 11a−g, and 14 by
Method C: General Procedure. A 5-mL process vial was charged
with the appropriate bromoindole derivative (1 mmol), the appro-
priate boronic acid (5 mmol), Pd(OAc)2 (22.5 mg, 0.1 mmol), PPh3
(78.6 mg, 0.3 mmol), 2 M Na2CO3 (2 mL, 4 mmol), EtOH (1 mL),
and 1,2-dimethoxyethane (DME) (4 mL). The vessel was sealed under
air and exposed to microwave heating for 10 min at 150 °C. The
reaction mixture was thereafter cooled down to room temperature,
diluted with AcOEt, and filtered through a short plug of Celite. The
solution was washed with brine, dried over anhydrous sodium sulfate,
and evaporated. The residue was purified by flash chromatography on
silica gel, eluted with DCM/MeOH (95:5).
Example: 2-[[5-Phenyl-1-(prop-2-en-1-yl)-1H-indol]-3-yl]-2-oxo-
N-(adamantan-1-yl)acetamide (10d). Prepared from 16b in 62%
1
yield. White solid, mp 151−152 °C. H NMR (400 MHz, CDCl3) δ
9.06 (s, 1H), 8.70, (s, 1H), 7.71 (m, 2H), 7.57 (d, J = 8.0 Hz, 1H),
7.48−7.33 (m, 5H), 6.07−5.98 (s, 1H), 5.32 (d, J = 8.0 Hz, 1H), 5.23
(d, J = 16.0 Hz, 1H), 4.78 (d, J = 4.0 Hz, 2H), 2.14 (s, 9H), 1.75
(s, 6H). 13C NMR (100 MHz, CDCl3) δ 181.0, 161.7, 141.9, 141.9,
137.1, 135.9, 131.8, 128.9, 128.8, 127.8, 127.0, 123.6, 121.4, 119.3, 112.4,
110.8, 52.0, 50.1, 41.4, 36.5, 29.6. IR (Nujol) 1676, 1636 cm−1. MS (ESI)
m/z 439 [M + H]+, 461 [M + Na]+. Anal. (C29H30N2O2) C, H, N.
Synthesis of Oxalylamides 15 and 16c by Method D:
General Procedure. A solution of the appropriate 2-(5-bromo-1H-
indol-3-yl)-2-oxoacetyl chloride (1.8 mmol), primary amine (2.1 mmol),
and triethylamine (TEA) (2.6 mmol) in dry DCM (25 mL) was stirred at
0 °C for 30 min and then at room temperature overnight. The solution was
washed with 1 N HCl, a saturated solution of sodium bicarbonate, and
brine. After the solution was dried over anhydrous sodium sulfate, volatiles
were removed in vacuo and the residue was recrystallized from EtOH.
Example: 2-(5-Bromo-1H-indol-3-yl)-2-oxo-N-(adamantan-1-yl)-
acetamide (15). Yield 82%. White solid, mp >300 °C. 1H NMR (400
MHz, DMSO-d6) δ 12.37 (br s, 1H), 8.67 (s, 1H), 8.31 (s, 1H), 7.83 (s,
1H), 7.50 (d, J = 8.0 Hz, 1H), 7.39 (d, J = 8.0 Hz, 1H), 2.49 (s, 3H),
2.05 (s, 6H), 1.65 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 222.6,
205.3, 138.4, 127.1, 125.2, 51.6, 41.2, 36.3, 29.4. IR (Nujol) 1684, 1622
cm−1. MS (ESI) m/z 402 [M + H]+. Anal. (C20H21BrN2O2) C, H, N.
Synthesis of Carboxamides 19a,b by Method E: General
Procedure. A solution of the appropriate carboxylic acid (6.1 mmol)
and CDI (7.3 mmol) in N,N-dimethylformamide (DMF; 7 mL) was
stirred at room temperature for 45 min. To this yellowish solution, 1-
aminoadamantane (12.2 mmol) dissolved in DMF (10 mL) was added
and the reaction mixture was heated at 120 °C for 7 h. After cooling,
the solution was diluted with ethyl acetate and a saturated solution of
sodium bicarbonate. The organic layer was washed with brine (4 × 30 mL),
and the precipitate that formed was filtered off. The organic solution was
evaporated in vacuo and the crude solid residue was triturated with ethyl
acetate and hexanes.
Example: (5-Bromo-1H-indol-3-yl)-N-(adamantan-1-yl)-
carboxamide (19a). Yield 76%. White solid, mp > 280 °C (decomp).
1H NMR (400 MHz, DMSO-d6) δ 11.62 (br s, 1H), 8.26 (s, 1H), 8.12
(s, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.23 (d, J = 8.0 Hz, 1H), 7.10 (s,
1H), 2.49 (s, 6H), 2.08 (s, 9H). 13C NMR (100 MHz, CDCl3) δ
164.3, 135.2, 129.4, 128.8, 124.6, 123.9, 114.1, 113.4, 111.6, 51.5, 43.7,
41.9, 41.6, 36.7, 36.4, 36.0, 29.5, 29.2. IR (Nujol) 1621 cm−1. MS
(ESI) m/z 372 [M − H]−. Anal. (C19H21BrN2O) C, H, N.
I
dx.doi.org/10.1021/jm3003334 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
cage to allow full view of the hind-paws. Lifting, favoring, licking,
shaking, and flinching of the injected paw were recorded as nociceptive
responses. The duration of those mentioned noxious behaviors was
monitored by an observer blind to the experimental treatment for
periods of 0−10 min (early phase) and 20−60 min (late phase) after
functional characterization of brainstem cannabinoid CB2 receptors.
Science 2005, 310, 329−332. (c) Beltramo, M.; Bernardini, N.;
Bertorelli, R.; Campanella, M.; Nicolussi, E.; Fredduzzi, S.; Reggiani, A.
CB2 receptor-mediated antihyperalgesia: possible direct involvement
of neural mechanisms. Eur. J. Neurosci. 2006, 23, 1530−1538.
(5) Pertwee, R. G. Emerging strategies for exploiting cannabinoid
receptor agonists as medicines. Br. J. Pharmacol. 2009, 156, 397−411.
(6) Pertwee, R. G.; Thomas, A. Therapeutic applications for agents
that act at CB1 and CB2 receptors. In The Cannabinoid Receptors;
Reggio, P., Ed.; Humana Press: Totowa, NJ, 2009; pp 361−392.
(7) Di Marzo, V.; De Petrocellis, L. Plant, synthetic, and endogenous
cannabinoids in medicine. Annu. Rev. Med. 2006, 57, 553−574.
(8) Felder, C. C.; Joyce, K. E.; Briley, E. M.; Mansouri, J.; Mackie, K.;
Blond, O.; Lai, Y.; Ma, A. L.; Mitchell, R. L. Comparison of the
pharmacology and signal transduction of the human cannabinoid CB1
and CB2 receptors. Mol. Pharmacol. 1995, 48, 443−450.
formalin administration. Results are expressed as means
SEM.
Significant differences between groups were evaluated by using analysis
of variance followed by Dunnett’s test. The version of the formalin
test we applied is based on the fact that a correlational analysis showed
that no single behavioral measure can be a strong predictor of formalin
or drug concentrations on spontaneous behaviors.40 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).41 Groups of 8−10 animals per treatment were
used, with each animal being used for one treatment only. Mice
received intraperitoneal vehicle (20% dimethyl sulfoxide, DMSO, in
0.9% NaCl) or different doses of before-mentioned compounds.
(9) Pacher, P.; Mechoulam, R. Is lipid signaling through cannabinoid
2 receptors part of a protective system? Prog. Lipid Res. 2011, 50, 193−
211 and references cited therein.
ASSOCIATED CONTENT
* Supporting Information
(10) Montecucco, F.; Matias, I.; Lenglet, S.; Petrosino, S.; Burger, F.;
Pelli, G.; Braunersreuther, V.; Mach, F.; Steffens, S.; Di Marzo, V.
Regulation and possible role of endocannabinoids and related
mediators in hypercholesterolemic mice with atherosclerosis. Athero-
sclerosis 2009, 205, 433−441.
■
S
Analytical data for compounds 9a−k, 10a−c, 10e, 11a−g, 12a,
13a−c, 14, 16a, 16c, 18, 19a, 19b, and 20a−c. This material is
available free of charge via the Internet at http://pubs.acs.org.
(11) (a) Reinhard, W.; Stark, K.; Neureuther, K.; Sedlacek, K.;
Fischer, M.; Baessler, A.; Weber, S.; Kaess, B.; Wiedmann, S.;
Erdmann, J.; Lieb, W.; Jeron, A.; Riegger, G.; Hengstenberg, C.
Common polymorphisms in the cannabinoid CB2 receptor gene
(CNR2) are not associated with myocardial infarction and
cardiovascular risk factors. Int. J. Mol. Med. 2008, 22, 165−174.
(b) Zhang, M.; Adler, M. W.; Abood, M. E.; Ganea, D.; Jallo, J.; Tuma,
R. F. CB2 receptor activation attenuates microcirculatory dysfunction
during cerebral ischemic/reperfusion injury. Microvasc. Res. 2009, 78,
86−94.
AUTHOR INFORMATION
Corresponding Author
■
*(V.D.) phone +39 081 8675093, fax +39 081 8041770,
e-mail vdimarzo@icmib.na.cnr.it; (F.C.) phone +39 0577
234308, fax +39 0577 234333, e-mail corelli@unisi.it.
Notes
The authors declare no competing financial interest.
(12) (a) Murikinati, S.; Juttler, E.; Keinert, T.; Ridder, D. A.;
̈
ACKNOWLEDGMENTS
Authors from the University of Siena gratefully acknowledge
financial support from Siena Biotech S.p.A. We thank Fabrizio
Muhammad, S.; Waibler, Z.; Ledent, C.; Zimmer, A.; Kalinke, U.;
Schwaninger, M. Activation of cannabinoid 2 receptors protects
against cerebral ischemia by inhibiting neutrophil recruitment. FASEB
J. 2010, 24, 788−798. (b) Hillard, C. J. Role of cannabinoids and
endocannabinoids in cerebral ischemia. Curr. Pharm. Des. 2008, 14,
2347−2361.
■
Vincenzi (University of Ferrara) and Marco Allara
Pozzuoli) for technical assistance.
̀
(CNR,
(13) (a) Izzo, A. A.; Sharkey, K. A. Cannabinoids and the gut: new
developments and emerging concepts. Pharmacol. Ther. 2010, 126,
21−38. (b) Wright, K.; Rooney, N.; Feeney, M.; Tate, J.; Robertson,
D.; Welham, M.; Ward, S. Differential expression of cannabinoid
receptors in the human colon: cannabinoids promote epithelial wound
healing. Gastroenterology 2005, 129, 437−453. (c) Storr, M. A.;
Keenan, C. M.; Zhang, H.; Patel, K. D.; Makriyannis, A.; Sharkey, K. A.
Activation of the cannabinoid 2 receptor (CB2) protects against
experimental colitis. Inflamm. Bowel Dis. 2009, 15, 1678−1685.
(14) (a) Pandey, R.; Mousawy, K.; Nagarkatti, M.; Nagarkatti, P.
Endocannabinoids and immune regulation. Pharmacol. Res. 2009, 60,
85−92. (b) Hegde, V. L.; Hegde, S.; Cravatt, B. F.; Hofseth, L. J.;
Nagarkatti, M.; Nagarkatti, P. S. Attenuation of experimental
autoimmune hepatitis by exogenous and endogenous cannabinoids:
involvement of regulatory T cells. Mol. Pharmacol. 2008, 74, 20−33.
(15) (a) Bisogno, T.; Di Marzo, V. Cannabinoid receptors and
endocannabinoids: role in neuroinflammatory and neurodegenerative
disorders. CNS Neurol. Disord. Drug Targets 2010, 9, 564−573.
ABBREVIATIONS
■
CB1, cannabinoid type 1; CB2, cannabinoid type 2; CDI, 1,1′-
carbonyldiimidazole; COR167, N-(adamantan-1-yl)-6-isoprop-
yl-4-oxo-1-pentyl-1,4-dihydroquinoline-3-carboxamide; DME,
1,2-dimethoxyethane; EDC, N-(3-dimethylaminopropyl)-N′-
ethylcarbodiimide hydrochloride; HOBt, 1-hydroxybenzotria-
zole; MW, microwaves; SI, selectivity index; TBAB, tetrabutyl-
ammonium bromide; TEA, triethylamine
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