Indole-2-carboxamides as allosteric modulators of the cannabinoid CB 1 receptor

Article abstract of DOI:10.1021/jm201485c

We synthesized new N-phenylethyl-1H-indole-2-carboxamides as the first SAR study of allosteric modulators of the CB₁ receptor. The presence of the carboxamide functionality was required in order to obtain a stimulatory effect. The maximum stimulatory activity on CB₁ was exerted by carboxamides 13 (EC₅₀ = 50 nM) and 21 (EC₅₀ = 90 nM) bearing a dimethylamino or piperidinyl group, respectively, at position 4 of the phenethyl moiety and a chlorine atom at position 5 of the indole.

Full text of DOI:10.1021/jm201485c

Brief Article
pubs.acs.org/jmc
Indole-2-carboxamides as Allosteric Modulators of the Cannabinoid
CB1 Receptor
Francesco Piscitelli,† Alessia Ligresti,‡ Giuseppe La Regina,† Antonio Coluccia,† Ludovica Morera,†
Marco Allara,‡ Ettore Novellino,§ Vincenzo Di Marzo,*,‡ and Romano Silvestri*,†
̀
†Istituto PasteurFondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Universita
̀
di Roma,
Piazzale Aldo Moro 5, I-00185 Roma, Italy
‡Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Via Campi Flegrei 34, Comprensorio Olivetti, I-80078
Pozzuoli, Napoli, Italy
§Dipartimento di Chimica Farmaceutica e Tossicologica, Universita
Napoli, Italy
̀
di Napoli Federico II, Via Domenico Montesano 49, I-80131,
S
* Supporting Information
ABSTRACT: We synthesized new N-phenylethyl-1H-indole-2-carboxamides as the first SAR study of allosteric modulators of
the CB1 receptor. The presence of the carboxamide functionality was required in order to obtain a stimulatory effect. The
maximum stimulatory activity on CB1 was exerted by carboxamides 13 (EC50 = 50 nM) and 21 (EC50 = 90 nM) bearing a
dimethylamino or piperidinyl group, respectively, at position 4 of the phenethyl moiety and a chlorine atom at position 5 of the
indole.
G-protein-coupled receptors show allosteric binding sites for
endogenous and/or synthetic ligands.21 The first evidence of an
allosteric binding site at the cannabinoid CB1 receptor was
reported by Price et al in 2005.22 To our knowledge, this site
was recognized by means of three small molecules, Org 27569
(1), Org 29647 (2), and Org 27759 (3), which showed
different efficacy versus affinity to the orthosteric site; later, the
phenylurea derivative PSNCBAM-1 (4) was shown to exhibit
similar pharmacological profile. In particular, these small
molecules proved to be allosteric enhancers of agonist binding
and affinity and allosteric inhibitors of agonist signaling
efficacy23,24 (Chart 1S, Supporting Information (SI)).
The identification of the CB1 allosteric binding site prompted
the search for other allosteric ligands as new therapeutic tools.
Allosteric enhancers of the CB1 receptor may lead to useful
agents without the CNS side effects that are characteristic of
direct receptor agonism because they would be inactive per se
and potentiate the effects of endogenous ligands, which are
more “site- and time-selective”.22 However, apart from the
recently reported parametrization of 1,25 neither crystallo-
graphic/modeling nor structure−activity relationship (SAR)
studies with these small molecules have been published so far.
Thus, this novel as well as original and potentially safer avenue
of therapeutic development has not yet been exhaustively
explored. Here, we report the first synthesis of indole-2-
carboxamides 8−26 structurally correlated to 1. Our results
show that new potent positive CB1 allosteric modulators may
be obtained by chemical modification of the N-phenylethyl-1H-
indole-2-carboxamide scaffold (Table 1).
INTRODUCTION
■
Cannabinoid (CB) receptors belong to the endocannabinoid
system that encompasses three components: (i) CB1 and CB2
G-protein-coupled receptors,1 (ii) endocannabinoids (i.e.,
endogenous CB receptor ligands), and (iii) proteins, enzymes,
and carriers involved in endocannabinoid formation and
inactivation.2 The endocannabinoid system is thought to be
involved in an ever-growing number of physiological processes
and pathologies. Metabolic and eating disorders,3 cancer,4
pain,5 neurodegeneration,6 immunosuppression,7 locomotion,8
and liver disease9 have demonstrated a strong correlation with
the endocannabinoid system. Thus, the endocannabinoid
system may provide excellent options for the development of
new drugs.10
CB1 receptors are found predominantly at neuronal terminals
where they act as modulators of neurotransmitter release. CB1
receptor antagonists/inverse agonists have therapeutic potential
in the treatment of a variety of pathological conditions2a
including obesity,11 Parkinson’s disease,12 metabolic disorders,
smoking cessation, and drug abuse.13,14 On the other hand,
selective CB2 agonists may be used to treat pain and
inflammation,15 osteoporosis,16 CB2-receptor-expressing malig-
nant gliomas,17 tumors of immune origin,18 and multiple
sclerosis.19
Monod20 described the term allosteric as the capability of
ligands to affect positively or negatively the biological activity of
enzymes and receptors by binding to sites (the allosteric sites)
that are separate from the substrate/ligand-binding site.
Interaction of an allosteric ligand induces conformational
changes in the protein which affect the binding of the
substrate/ligand into its orthosteric site, thereby fine-tuning
its biological activity.
Received: November 10, 2011
© XXXX American Chemical Society
A
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Table 1. Structure and Action of Compounds 5−26 and Reference Compound 1 as Stimulators or Inhibitors of [3H]CP55940
a
Binding to Human Recombinant Cannabinoid Receptors
a
EC50 or IC50 (means: n = 2; 8: n = 4) were determined by nonlinear or linear regression analysis, respectively, of enhancement or inhibition of
b
radioligand binding by increasing compound concentrations. BS: % of binding stimulation at the maximum concentration tested (10 μM; 25 μM for
17). BI: % of binding inhibition at the maximum concentration tested (10 μM). Weak radioligand binding inhibitor. Weak radioligand binding
c
d
e
stimulator.
CHEMISTRY
RESULTS AND DISCUSSION
■
■
The synthesis of new compounds 5−26 is depicted in Scheme
1. Compounds 1, 8−10, 14−17, 19−22, and 26 were
synthesized by the coupling reaction of the appropriate
indole-2-carboxylic acid 27−31 with the corresponding amine
32−38 in the presence of the BOP reagent and triethylamine in
anhydrous DMF. Compounds 5−7 were obtained by reaction
of 27 with alcohols 39−41 in the presence of DMAP and DCI
in anhydrous dichloromethane. Tin(II) chloride reduction of
the nitro group of 10 in ethyl acetate furnished 11, which upon
treatment with dimethyl sulfate in the presence of potassium
carbonate in acetone gave a mixture of the mono- (12) and
dimethylamino (13) derivatives, separated by column chroma-
tography. In a similar manner were obtained 23−25 starting
from 22. Acetylindole 17 was transformed into the correspond-
ing ethanol derivative 18 by sodium borohydride reduction in
aqueous tetrahydrofuran. The synthesis of the new synthetic
intermediates 27−31 and 32−41 is described in Schemes 1S−
4S of Supporting Information.
The potential properties of compounds 1 and 26 as positive
allosteric modulators of the binding of orthosteric ligands to the
CB1 receptor were evaluated by measuring the stimulation of
the specific binding of the nonselective CB1/CB2 receptor
agonist [3H]CP55940 (42) to cell membranes overexpressing
the human recombinant CB1 (Table 1). As a control, we also
carried out studies with preparations of cell membranes
overexpressing the human recombinant CB2 receptor. Com-
pounds 10, 12−15, and 19−21 were found to be enhancers of
the binding of 42 to the CB1 receptor, two of which (13 and
21) were particularly potent as indicated by their nanomolar
EC50 values, whereas 8, 9, 11, 16, the racemate 18, 22, 25, and
26 enhanced 42 binding only weakly. In contrast, 5−7 inhibited
the binding of 42 to the CB1 receptor. Clearly, in the case of
inhibition of 42, our assay does not allow to distinguish
between orthosteric and allosteric ligands. Only 8 and 19
showed very weak activity as enhancers of 42 binding to the
CB2 receptor. Compound 10 was devoid of any binding activity
B
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a b
,
In summary, the carboxamide at position 2 of the indole is
required in order to obtain the stimulatory effect. Both the
chlorine atom (R1) and alkyl group (R2) at the positions 5 and
3 of the indole, respectively, were useful, but not necessary, for
the stimulation of 42−CB1 binding. The piperidin-yl, nitro, and
dimethylamino groups at the para position of the phenylethyl
moiety (R3) were optimal for a potent stimulation. These
results show that the N-phenylethyl-1H-indole-2-carboxamide
scaffold may be properly decorated to obtain potent positive
allosteric ligands of the CB1 receptor. However, and this is a
limitation of the present study, whether these compounds
behave as functional positive modulators, as suggested by their
chemical similarity to 1, remains to be established by using ad
hoc in vitro and in vivo pharmacological studies. We also aim at
investigating in depth the chemical and physical properties
correlated to the stimulation of the CB1 receptor with the
synthesis of new derivatives.
Scheme 1. Synthesis of Compounds 1 and 5−26
a
1, 5−26: (see Table 1). 27: R1 = Cl, R2 = Et. 28: R1 = Cl, R2 =
COMe. 29: R1 = Cl, R2 = Me. 30: R1 = H, R2 = Et. 31: R1 = Cl, R2 =
H. 32: X = NH, R3 = NO2. 33: X = NH, R3 = piperidin-1-yl. 34: X =
NH, R3 = pyrrolidin-1-yl. 35: X = NH, R3 = 4-methylpiperazin-1-yl.
36: X = NH, R3 = Me. 37: X = NH, R3 = Cl. 38: X = NH, R3 = F. 39:
X = O, R3 = piperidin-1-yl. 40: X = O, R3 = NO2. 41: X = O, R3 =
CONCLUSION
■
We synthesized new N-phenylethyl-1H-indole-2-carboxamides
as the first SAR study of allosteric modulators of the CB1
receptor. These carboxamides were found to be selective
positive modulators of the human recombinant CB1 receptor.
The stimulatory activity on CB1 receptors required the
presence of the 2-carboxamide functionality as esters 5 and 7
was demonstrated to instead inhibit binding of 42 to CB1.
These results show that new potent CB1 stimulators may be
obtained by chemical modification of the N-phenylethyl-1H-
indole-2-carboxamide scaffold and represent the first attempt at
investigating the SARs of CB1 allosteric ligands using a
medicinal chemistry approach. SAR and chemicophysical
studies through the synthesis of new derivatives, and functional
studies, are now needed.
b
NMe2. Reagents and reaction conditions: (a) BOP, Et3N, DMF, rt,
16 h; (b) DCI, DMAP, CH2Cl2, rt, 12 h, for 5; (c) NaBH4, aqueous
THF, reflux, 2 h; (d) SnCl2·2H2O, AcOEt, 60 °C, 4 h; (e) Me2SO4,
K2CO3, acetone, rt, 14 h.
at CB2 receptors, and all other compounds not only lacked
stimulating binding activity but weakly inhibited the binding of
the radioligand. Also, in this case, our assay does not allow to
distinguish between orthosteric and allosteric ligands.
The presence of the carboxamide functionality at the position
2 of the indole emerged as being crucial for the enhancement of
the binding of 42 to the CB1 receptor. Replacement of the
carboxamide of 1 for an ester group (5−7) resulted in a
dramatic drop of stimulation with the concomitant appearance
of inhibitory activity. However, such a feature is necessary but
not sufficient per se to confer stimulatory properties to the
compounds investigated here because 17, 23, and 24 also did
not enhance 42 binding. Replacement of the piperidin-1-yl
group of 1 with the smaller pyrrolidin-1-yl (8) or the more
polar 4-methylpiperazin-1-yl (9) group, or the chlorine atom of
15 with a fluorine (16) led to weaker enhancement of the CB1
binding activity at all test concentrations. The stimulatory 42-
binding activity observed was fully restored by introduction of a
nitro group (10) in the place of the piperidin-1-yl moiety (R3),
suggesting that, at position 4 of the phenyl moiety, both steric
and electronic factors are involved in the observed activities.
The corresponding amino derivative 11 resulted in a weaker
stimulation of 42−CB1 receptor binding. The methyl analogue
(12) of 11 was comparable to 10 as binding stimulator. Finally,
the dimethylamino derivative 13 of 12 yielded the most potent
allosteric ligand within the present series (EC50 = 50 nM).
Neither acetyl (17) nor alcohol (18) produced compounds
capable of efficaciously stimulating 42−CB1 receptor binding.
The 3-methylindole 19 substantially retained the stimulatory
activity of the parent compound 1. Among 21−26 bearing a
hydrogen atom at position 3 of the indole, only compound 21
(EC50 = 90 nM) exhibited greater affinity than 1 as a stimulator
of 42−CB1 receptor binding. The indole 20 was less effective as
compared to 1 as a stimulator of 42−CB1 receptor binding.
These results have shown that the N-phenylethyl-1H-indole-2-
carboxamide represents an excellent scaffold for the design of
positive allosteric modulators of the human CB1 receptor.
EXPERIMENTAL SECTION
■
Chemistry. Combustion analysis was used as a method for
establishing compound purity. Purity of the tested compounds was
≥95%. See SI for details.
General Procedure for the Synthesis of 8−10, 14−17, 19−
22, and 1. Example: 5-Chloro-3-ethyl-N-(2-(4-(pyrrolidin-1-
yl)phenyl)ethyl)-1H-indole-2-carboxamide (8). A mixture of 27
(0.1 g, 0.44 mmol), 34 (0.13 g, 0.66 mmol), BOP (0.29 g, 0.66 mmol),
and triethylamine (0.28 g, 0.36 mL, 2.64 mmol) in anhydrous N,N-
dimethylformamide (5 mL) was stirred at room temperature for 16 h.
The reaction mixture was diluted with water and extracted with ethyl
acetate. The combined organic extracts were washed with brine, dried,
and evaporated. The residue was purified by silica gel column
chromatography (ethyl acetate−n-hexane 1:2 as eluent) to provide 8.
1
Yield 66%, mp 230−234 °C (from ethanol). H NMR (DMSO-d6): δ
11.73 (s, 1H), 8.32 (t, J = 5.2 Hz, 1H), 8.07 (d, J = 1.8 Hz, 1H), 7.82
(d, J = 8.7 Hz, 1H), 7.60 (dd, J = 8.6, 1.9 Hz, 1H), 7.48 (d, J = 8.4 Hz,
2H), 6.91 (d, J = 8.4 Hz, 2H), 3.94−3.81 (m, 2H), 3.66−3.55 (m,
4H), 3.39 (q, J = 7.4 Hz, 2H), 3.16 (t, J = 7.3 Hz, 2H), 2.40−2.31 (m,
4H), 1.55 ppm (t, J = 7.4 Hz, 3H). MS (ESI): m/z: 596 [M + H]+. IR:
ν 3315, 1595 cm−1. Anal. (C23H26ClN3O (395.93)) C, H, N, Cl.
Chemicophysical characterization, preparative, and combustion
analysis data of 9, 10, 14−17, 19−22, 26, and 1 are reported in SI.
General Procedure for the Synthesis of 5−7. Example: 2-(4-
(Piperidin-1-yl)phenyl)ethyl 5-chloro-3-ethyl-1H-indole-2-car-
boxylate (5). A mixture of 27 (0.076 g, 0.34 mmol), 39 (0.07 g,
0.34 mmol), and DMAP (0.004 g, 0.034 mmol) in anhydrous
dichloromethane (5 mL) was cooled to 0 °C on an ice−water bath.
DCI (0.07 g, 0.34 mmol) was added, and the mixture was stirred at
room temperature for 16 h. The solid was filtered off, and the solvent
was evaporated. The residue was purified by silica gel column
chromatography (chloroform as eluent) to provide 5 as a white solid.
C
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Yield 57%, mp 110−115 °C (from aqueous ethanol). 1H NMR
(DMSO-d6): δ 11.64 (s, 1H), 7.72 (d, J = 1.7 Hz, 1H), 7.43 (d, J = 8.7
Hz, 1H), 7.25 (dd, J = 8.7, 2.0 Hz, 1H), 7.16 (d, J = 8.7 Hz, 2H), 6.87
(d, J = 8.6 Hz, 2H), 4.48 (t, J = 6.9 Hz, 2H), 3.12−3.02 (m, 4H),
3.02−2.89 (m, 4H), 1.66−1.56 (m, 4H), 1.56−1.45 (m, 2H), 1.10
ppm (t, J = 7.4 Hz, 3H). MS (ESI): m/z: 411 [M + H]+. IR: ν 3313,
1677 cm−1. Anal. (C24H27ClN2O2 (410.94)) C, H, N, Cl.
Chemicophysical characterization, preparative, and combustion
analysis data of 5−7 are reported in SI.
N-(2-(4-Aminophenyl)ethyl)-5-chloro-3-ethyl-1H-indole-2-
carboxamide (11). Tin chloride dihydrate (0.45 g, 2.0 mmol) was
added to a solution of 10 (0.15 g, 0.43 mmol) in ethyl acetate (5.4
mL). The reaction mixture was heated to 60 °C for 3 h. After cooling,
the reaction mixture was treated with a saturated solution of sodium
bicarbonate, and the precipitate was filtered off. The aqueous layer was
extracted with ethyl acetate. The combined organic layers were washed
with brine, dried, and evaporated. The residue was purified by silica gel
column chromatography (ethyl acetate−n-hexane 1:1 as eluent) to
provide 11 as white solid. Yield 75%, mp 180−184 °C (from ethanol).
1H NMR (DMSO-d6): δ 11.35 (s, 1H), 7.93 (s, 1H), 7.65 (s, 1H),
ASSOCIATED CONTENT
* Supporting Information
Structures of 1−4, synthesis of 27−31 and 32−41, elemental
analysis of 5−26. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*For R.S.: phone, +39 06 4991 3800; fax, +39 06 4969 3268; E-
mail, romano.silvestri@uniroma1.it. V.D.M.: phone, +39 081
867 5193; fax, +39 081 804 1770; E-mail, vdimarzo@icmib.na.
cnr.it.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
A.C. thanks Istituto PasteurFondazione Cenci Bolognetti for
his Borsa di Studio per Ricerche in Italia. We thank Roberto
Cirilli, ISS, Roma, for the optical analysis of 18.
7.40 (d, J = 8.8 Hz, 1H), 7.18 (d, J = 8.4 Hz, 1H), 6.92 (d, J = 7.7 Hz,
2H), 6.51 (d, J = 8.0 Hz, 2H), 4.87 (s, 2H), 3.44 (m, 2H), 2.97 (q, J =
7.9 Hz, 2H), 2.69 (t, J = 7.3 Hz, 2H), 1.13 ppm (t, J = 7.4 Hz, 3H).
MS (ESI): m/z: 342 [M + H]+. IR: ν 3422, 3346, 3285, 1619 cm−1.
Anal. (C19H20ClN3O (341.83)) C, H, N, Cl.
ABBREVIATIONS USED
■
CB, cannabinoid; CB1, CB receptor type 1; CB2, CB receptor
type 2; SAR, structure−activity relationship; BOP, benzotria-
zole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluoro-
phosphate; DMAP, N,N-dimethyl-4-aminopyridine; DCI,
N,N′-dicyclohexylcarbodiimide; Xphos, 2-dicyclohexylphosphi-
no-2′,4′,6′-triisopropylbiphenyl; BS, percent of binding stim-
ulation at the maximum concentration tested is also reported;
BI, percent of binding inhibition at the maximum concentration
tested is also reported
5-Chloro-3-ethyl-N-(2-(4-methylaminophenyl)ethyl)-1H-in-
dole-2-carboxamide (12) and 5-chloro-N-(2-(4-
dimethylaminophenyl)ethyl)-3-ethyl-1H-indole-2-carboxa-
mide (13). A mixture of 11 (0.070 g, 0.20 mmol), potassium
carbonate (0.070 g, 0.51 mmol), and dimethylsulfate (0.025 g, 0.019
mL, 0.20 mmol) in anhydrous acetone (5 mL) was stirred at room
temperature for 16 h. The reaction mixture was diluted with water and
extracted with ethyl acetate. The combined organic layers were washed
with brine, dried, and evaporated. The residue was purified by silica gel
column chromatography (ethyl acetate−n-hexane 1:2 as eluent) as
eluent to give 13 as white solid. Yield 29%, mp 150−154 °C (from
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ethanol). H NMR (DMSO-d6): δ 11.32 (s, 1H), 7.93 (t, J = 5.4 Hz,
1H), 7.65 (d, J = 1.9 Hz, 1H), 7.40 (d, J = 8.7 Hz, 1H), 7.19 (dd, J =
8.7, 2.0 Hz, 1H), 7.09 (d, J = 8.6 Hz, 2H), 6.69 (d, J = 8.7 Hz, 2H),
3.55−3.41 (m, 2H), 2.97 (q, J = 7.4 Hz, 2H), 2.85 (s, 6H), 2.75 (t, J =
7.5 Hz, 2H), 1.13 ppm (t, J = 7.5 Hz, 3H). MS (ESI): m/z: 370 [M +
H]+. IR: ν 3438, 3251, 1630 cm−1. Anal. (C21H24ClN3O (369.89)) C,
H, N, Cl. Further elution with the same eluent furnished 12 as white
solid. Yield 31%, mp 110−114 °C (from ethanol). 1H NMR (DMSO-
d6): δ 11.32 (s, 1H), 7.91 (t, J = 5.4 Hz, 1H), 7.65 (d, J = 1.7 Hz, 1H),
7.40 (d, J = 8.7 Hz, 1H), 7.19 (dd, J = 8.7, 2.0 Hz, 1H), 6.99 (d, J = 8.3
Hz, 2H), 6.49 (d, J = 8.4 Hz, 2H), 5.43 (q, J = 5.3 Hz, 1H), 3.51−3.41
(m, 2H), 2.96 (q, J = 7.4 Hz, 2H), 2.71 (t, J = 7.4 Hz, 2H), 2.65 (d, J =
5.1 Hz, 3H), 1.13 ppm (t, J = 7.5 Hz, 3H). MS (ESI): m/z: 356 [M +
H]+. IR: ν 3279, 1634 cm−1. Anal. (C20H22ClN3O (355.86)) C, H, N,
Cl.
5-Chloro-3-(1-hydroxyethyl)-N-(2-(4-(piperidin-1-yl)phenyl)-
ethyl)-1H-indole-2-carboxamide (18). A solution of 17 (0.03 g,
0.06 mmol) and sodium borohydride (0.023 g, 0.06 mmol) in
tetrahydrofuran (2 mL) and water (0.09 mL) was heated to reflux
temperature for 2 h. After cooling, the reaction mixture was diluted
with water and extracted with ethyl acetate. The combined organic
layers were washed with brine, dried, and evaporated. The residue was
purified by a short silica gel column chromatography (ethyl acetate−n-
hexane 1:1 as eluent) to provide 18 as a white solid. Yield 92%, mp
1
132−134 °C (from ethyl acetate−hexane). H NMR (DMSO-d6): δ
11.54 (s, 1H), 9.41 (t, J = 5.4 Hz, 1H), 7.78 (d, J = 1.6 Hz, 1H), 7.41
(d, J = 8.7 Hz, 1H), 7.18 (dd, J = 8.7, 1.9 Hz, 1H), 7.11 (d, J = 8.1 Hz,
2H), 6.86 (d, J = 7.7 Hz, 2H), 6.18 (s, 1H), 5.46−5.33 (m, 1H), 3.64−
3.42 (m, 2H), 3.16−3.01 (m, 4H), 2.77 (t, J = 7.2 Hz, 2H), 1.71−1.56
(m, 4H), 1.56−1.47 (m, 2H), 1.37 ppm (d, J = 6.5 Hz, 3H). MS
(ESI): m/z: 426 [M + H]+. IR: ν 3232, 1635 cm−1. Anal.
(C24H28ClN3O2 (425.95)) C, H, N, Cl.
D
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