Synthesis and pharmacological evaluation of analogs of indole-based cannabimimetic agents

Article abstract of DOI:10.1111/j.1747-0285.2009.00910.x

Aminoalkylindoles (AAIs), although structurally dissimilar from the classical cannabinoids, are known to be capable of binding to cannabinoid receptors and of evoking cannabinomimetic responses. With the aim of investigating the structure-activity relationships (SAR) for the binding of non-classical agonists to CB1 and CB2 cannabinoid receptors, we designed and synthesized a series of indole derivatives. The compounds were tested for their analgesic action by formalin test and compared to WIN 55212-2, an AAI acting to the cannabinoid receptors. In receptor binding assay, compound 5 showed affinity for the CB1 receptor comparable to WIN 55212-2.

Full text of DOI:10.1111/j.1747-0285.2009.00910.x

ª 2009 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2009.00910.x
Chem Biol Drug Des 2010; 75: 106–114
Research Article
Synthesis and Pharmacological Evaluation of
Analogs of Indole-Based Cannabimimetic Agents
Orazio Mazzoni1,†, Maria V. Diurno1,*,
These therapeutic applications claimed for cannabinoid receptor
agonists have triggered the synthesis of new compounds that are
able to modulate the activity of cannabinoid receptors (16–20). One
of the most common examples of non-classic type of cannabinoids
is pravadoline that, together with WIN 55212-2 (Chart 1), belongs
to the AAI class (21–23). These compounds show high affinity
Antonio M. di Bosco1, Ettore Novellino1,
Paolo Grieco1, Giovanni Esposito1, Alessia
Bertamino1, Antonio Calignano2 and
Roberto Russo2
1Dipartimento di Chimica Farmaceutica e Tossicologica, Via D.
Montesano 49 Universitꢀ di Napoli Federico II, Naples, Italy
2Dipartimento di Farmacologia Sperimentale, Via D. Montesano 49
Universitꢀ di Napoli Federico II, Naples, Italy
toward the cannabinoid receptors (24) and in addition, in several
pharmacological and behavioral assays, they are more active with
respect to the classical cannabinoid (CC) THC (D9-tetrahydrocannabi-
nol) acting as an agonist at cannabinoid receptors (25,26).
*Corresponding author: Prof Maria V. Diurno, diurno@unina.it
This manuscript is dedicated to Orazio Mazzoni.
Originally, AAIs analogs were synthesized as cyclo-oxygenase inhibi-
tors (27,28), but subsequently, they were also found to possess
antinociceptive activity. Unlike the opioids, their inhibitory effects
were not blocked by naloxone (29). More evidences have shown
that AAIs analogs of pravadoline, lacking the cyclooxygenase inhibi-
tory effect, endow cannabinomimetic response (27,30,31), by binding
at cannabinoid receptors and represent an important class of can-
nabinoid receptor agonists.
Aminoalkylindoles (AAIs), although structurally
dissimilar from the classical cannabinoids, are
known to be capable of binding to cannabinoid
receptors and of evoking cannabinomimetic
responses. With the aim of investigating the struc-
ture–activity relationships (SAR) for the binding of
non-classical agonists to CB1 and CB2 cannabi-
noid receptors, we designed and synthesized a ser-
ies of indole derivatives. The compounds were
tested for their analgesic action by formalin test
and compared to WIN 55212-2, an AAI acting to
the cannabinoid receptors. In receptor binding
assay, compound 5 showed affinity for the CB1
receptor comparable to WIN 55212-2.
The most potent AAIs analogs share some common features, such
as an indole ring system, a tertiary amine moiety, and a naphthoyl
or aroyl group. In contrast, the potent CCs are structures containing
a phenolic moiety, a cyclohexyl ring and, a hydrophobic side chain
(32,33). However, AAIs and CCs share a similar high lipophilicity.
Previously, have been reported two alternative binding conforma-
tions for WIN 55212-2, defined aroyl-up1 and aroyl-up2 (34), where
the naphthyl ring of this ligand is oriented within the receptor
upward with respect to the extracellular side. Aromatic–aromatic
interactions are important not only for the binding of WIN but also
for inducing receptor conformational change. It is possible that dif-
ferences in the nature of the ligand binding could contribute to
ligand-specific conformational changes in the receptor. In fact, it
was observed that AAI structural analogs devoid of the carbonyl
oxygen of the aroyl moiety showed an increased binding affinity
compared with the corresponding AAI compound. This finding would
suggest that the C3 carbonyl oxygen and its possible H-bonding
would not be important for AAI binding to the CB1 receptor.
Key words: aminoalkylindole analogs, analgesic agents, cannabimi-
metic agents, CB1 receptor, SAR study
Received 28 May 2009, revised 25 September 2009 and accepted for
publication 26 September 2009
Cannabinoids interact with cannabinoid receptors to produce a vari-
ety of potential beneficial therapeutic effects including attenuation
of nausea and vomiting in cancer chemotherapy, management of
glaucoma, the suppression of muscle spasticity ⁄ spasm associated
with multiple sclerosis (1), disorders associated with Alzheimer's
disease (2,3), and therapeutic effects of analgesia (4–6). Further-
more, CB1 receptor antagonists ⁄ inverse agonists have therapeutic
application as appetite suppressant (7,8); the rimonabant
(SR141716A, Acompliaꢀ, Sanofi-Aventis, Paris, France) (9) has been
approved in some countries for the treatment of obesity (10–13)
and in the management of schizophrenia (14,15), although recently
this compound has been withdrawn from clinical development.
Starting from the structure of pravadoline and WIN 55212-2, we
have synthesized a series of indole derivatives (General structures
A and B of Chart 1) that show the common features of AAIs.
These compounds, prepared by reaction of 1-morpholinoethyl-indole-
2,3-dione or 1-morpholinoethyl-indole-3-aldheyde, with aryl or naph-
thyl amine (Schemes 1 and 2) display the spatial and electronic
106

Indole-based Ligands. SAR Study on CB1 Receptor
A
A
O
O
R
OMe
X
X
N
N
H
CH3
CH3
Pravadoline
O
N
N
N
N
O
General Structure B
General Structure A
Chart 1: Design of Synthesized
compounds in relation to Pravado-
line and WIN55212-2.
WIN 55212-2
N
N
N
O
N
O
O
O
requirements for binding to the cannabinoid receptors: two aromatic
moieties, one of them represented by an indole nucleus which is
connected to a cyclic lipophilic group and a hydrogen acceptor
group, represented by an azomethinic or aminic nitrogen in our mol-
ecules.
group ranges from 1690 to 1710 ⁄ cm while the C=N stretching lies at
1640 ⁄ cm. The purity of compounds was checked by ascending TLC
on Merck's silica gel plates (0.25 mm) with fluorescent baking. NMR
measurements (data reported in d) were performed on a Varian
500 MHz spectrometer. The chemical shifts are referenced to CDCl3
solvent signals at d 7.26. Me4Si was used as internal reference.
We performed molecular modeling and molecular dynamic studies
on compounds 1–10 and on pravadoline and WIN 55212-2, taken
as reference compounds in this study. The Figure 1A–C shows the
3D structures of compound 5, obtained by a simulated annealing
method (see Experimental section) and the conformation of pravado-
line and WIN 55212-2, (Cambridge database)a. As showed in
Figure 1, compound 5, pravadoline, and WIN 55212-2 are well over-
lapped (D–F).
3-(Arylimino)-1-(2-morpholin-4-yl-ethyl)-
1,3-dihydro-indole-2-one (1 and 4) and their
5-substituted-derivatives (2, 3, 5 and 6). General
Procedure
To a mixture of indole-2,3-dione (or 5-substituted-indole-2,3-dione)
(10 mmol) and sodium hydride (15 mmol), in anhydrous Dimethylfor-
mamide (DMF) (20 mL) at room temperature, was added a solution of
N-(chloroethyl)morpholine hydrochloride (10 mmol) in anhydrous ace-
tonitrile. The reaction mixture, stirred for 8 h at reflux temperature,
kept at room temperature for 48 h and evaporated in vacuum yielded
the corresponding intermediate 1-(2-morpholin-4-yl-ethyl)-1H-indole-
2,3-dione (I) (or its 5-substituted derivatives (II and III)) as a brown
oil. Chromatography on silica gel using ethyl acetate: carbon tetra-
chloride (3:2, v:v) as eluent afforded compounds as colored powder.
Experimental Section
Chemistry
Melting points were determined by a Kofler apparatus and are
uncorrected. The elemental analysis (C, H, and N) of reported com-
pounds agrees with the calculated values and was within €0.4% of
theoretical values. Mass spectra (ESI) were obtained on an API 2000
spectrometer (Applied Biosystems, Foster City, CA, USA). The IR
spectra were taken on a Perkin-Elmer 1760-X IFT spectrophotometer
(Wellesley, MA, USA) in potassium bromide, and the amide carbonyl
I (or its 5-substituted derivatives II and III) was dissolved in abso-
lute ethanol, the appropriate aromatic amine was added to the
solution, and the mixture was stirred for 4 h at refluxing tempera-
R
O
R
O
O
N
H
NaH
DMF
O
N
Cl
N
N
O
O
I
R = H
II
R = Cl
III
R = CH3
Ar-NH2
EtOH
Ar
R
H
Cl CH3
R
N
Ar
O-CH3
1
2
5
3
6
O
N
4
N
Scheme 1: Synthesis of com-
O
pound 1–6.
Chem Biol Drug Des 2010; 75: 106–114
107

Mazzoni et al.
H
H
O
O
NaH
N
H
N
DMF
Cl
N
N
O
O
IV
R' NH2
EtOH
NaBH4
C / Pd
MeOH
R'
R'
N
H
4-OCH3-Ph
7
8
9
Naphth-1-yl
4-Cl-Ph-
4-CH3-Ph
N
10
N
Scheme 2: Synthesis of com-
pounds 7–10.
O
A
B
C
Figure 1: The 3D structures of
WIN 55212-2 (A), pravadoline (B)
obtained by Cambridge database,
and compound 5 (C) obtained by
molecular dynamics. The super-
imposition of WIN 55212-2 and
compound 5 (D), pravadoline and
compound 5 (E), and WIN 55212-2,
pravadoline, and compound 5 (F).
D
E
F
ture. After cooling, the mixture was evaporated in vacuo yielding
the crude product that was recrystallized from a mixture of ethyl
acetate–ethanol (2:1, v:v).
J = 10.0 Hz), 3.06 (2H, t; J = 7.1 Hz), 2.62 (2H, t; J = 7.1 Hz), 2.37
(4H, dt; J = 10.0 Hz); anal C14H15ClN2O3 C, H, N; Mass calcd.
294.73; MS (ESI+) m ⁄ z 295.70 (M + H+; 100%).
(I) 1-(2-Morpholin-4-yl-ethyl)-1H-indole-2,3-dione
Orange powder. Yield: 45%. Mp 172–4 ꢁC; 1H NMR 7.83 (1H, d;
J = 8.0 Hz), 7.79 (1H, d; J = 8.0 Hz), 7.52 (1H, t; J = 8.0 Hz), 7.19
(1H, t; J = 8.0 Hz), 3.65 (4H, dt; J = 10.0 Hz), 3.09 (2H, t;
J = 7.1 Hz), 2.60 (2H, t; J = 7.1 Hz), 2.36 (4H, dt; J = 10.0 Hz); anal
C14H16N2O3 C, H, N; Mass calcd. 260.29; MS (ESI+) m ⁄ z found
261.31 (M + H+; 100%).
(III) 5-Methyl-1-(2-morpholin-4-yl-ethyl)-1
H-indole-2,3-dione
Brown powder. Yield: 56%. Mp 130–2 ꢁC; 1H NMR 7.71 (1H, d;
J = 8.2 Hz), 7.59 (1H, s), 7.32 (1H, d; J = 8.2 Hz), 3.67 (4H, dt;
J = 10.0 Hz), 3.07 (2H, t; J = 7.1 Hz), 2.61 (2H, t; J = 7.1 Hz), 2.37
(4H, dt; J = 10.0 Hz), 2.35 (3H, s); anal C15H18N2O3 C, H, N; Mass
calcd. 274.32; MS (ESI+) m ⁄ z 275.31 (M + H+; 100%).
(II) 5-Chloro-1-(2-morpholin-4-yl-ethyl)-1
(1) 3-(4-Methoxy-phenylimino)-1-(2-morpholin-
4-yl-ethyl)-1,3-dihydro-indole-2-one
Yellow powder. Yield: 65%. Mp 174–75 ꢁC; 1H NMR 7.67 (1H, d;
J = 8.2 Hz), 7.60 (1H, d; J = 8.2 Hz), 7.27 (1H, t; J = 8.1 Hz), 7.20
H-indole-2,3-dione
Dark orange powder. Yield: 38%. Mp 120–1 ꢁC; H NMR 7.80 (1H,
1
s), 7.77 (1H, d; J = 8.1 Hz), 7.53 (1H, d; J = 8.1 Hz), 3.67 (4H, dt;
108
Chem Biol Drug Des 2010; 75: 106–114

Indole-based Ligands. SAR Study on CB1 Receptor
added solution of N-(chloroethyl)morpholine hydrochloride
(10 mmol). The reaction mixture, stirred for 12 h at reflux tempera-
ture, kept at room temperature 24 h and evaporated in vacuum,
yielded the corresponding 1-(2-morpholin-4-yl-ethyl)-1H-indole-3-car-
baldheyde (IV) as a brown oil. Chromatography on silica gel using
ethyl acetate: chloroform 2:1 v:v, as eluent afforded pure IV as an
orange powder.
(2H, d; J = 8.3 Hz), 7.03 (1H, t; J = 8.2 Hz), 6.80 (2H, d; J = 8.3 Hz),
3.73 (3H, s), 3.67 (4H, dt; J = 11.0 Hz), 3.06 (2H, t; J = 7.2 Hz), 2.62
(2H, t; J = 7.2 Hz), 2.37 (4H, dt; J = 11.0 Hz); anal C21H23N3O3 C, H,
N; Mass calcd. 365.43; MS (ESI+) m ⁄ z 366.42 (M + H+; 100%).
a
(2) 5-Chloro-3-(4-methoxy-phenylimino)-1-(2-
morpholin-4-yl-ethyl)-1,3-dihydro-indole-2-one
1
Orange powder. Yield: 62%. Mp 205–6 ꢁC; H NMR 7.61 (2H, d+s;
IV was dissolved in absolute ethanol, and appropriate arylamine
was added to the solution. The mixture was stirred for 4 h at
refluxing temperature. After cooling, the mixture was evaporated in
vacuum yielding the crude product. Owing to the instability of the
indole immine derivative, it was submitted to the reduction without
purification.
J = 8.2 Hz), 7.28 (1H, d; J = 8.1 Hz), 7.22 (2H, d; J = 8.3 Hz), 6.78
(2H, d; J = 8.3 Hz), 3.75 (3H, s), 3.68 (4H, dt; J = 11.0 Hz), 3.05 (2H,
t; J = 7.2 Hz), 2.60 (2H, t; J = 7.2 Hz), 2.37 (4H, dt; J = 11.0 Hz);
anal C21H21ClN3O3 C, H, N; Mass calcd. 398.86; MS (ESI+) m ⁄ z
399.78 (M + H+; 100%).
To a suspension of NaBH4 (10 mmol) and C ⁄ Pd (15 mmol) in etha-
nol ⁄ water (1:1 v:v) (30 mL), crude immine was added, and the
mixture was stirred for 3 h at room temperature. The resulting
mixture was treated with HCl 2N (20 mL), filtered, and evaporated
in vacuum. The resulting residue was collected, dissolved in chlo-
roform and washed with water (3 · 50 mL). Organic layer was
evaporated and residue purified by chromatography on silica gel
using ethyl acetate: n-hexane 1:1 as eluent yielded products a col-
ored oils.
(3) 5-Methyl-3-(4-methoxy-phenylimino)-1-(2-
morpholin-4-yl-ethyl)-1,3-dihydro-indole-2-one
Dark orange powder. Yield: 58%. Mp 140–1 ꢁC; H NMR 7.55 (1H,
d; J = 8.1 Hz), 7.40 (1H, s), 7.19 (2H, d; J = 8.3 Hz), 7.08 (1H, d;
J = 8.1 Hz), 6.81 (2H, d; J = 8.2 Hz), 3.70 (3H, s), 3.65 (4H, dt;
J = 11.0 Hz), 3.08 (2H, t; J = 7.0 Hz), 2.61 (2H, t; J = 7.0 Hz), 2.39
(4H, dt; J = 11.0 Hz); anal C22H24N3O3 C, H, N; Mass calcd. 378.44;
MS (ESI+) m ⁄ z 379.46 (M + H+; 100%).
1
(4) 3-(Naphthalen-1-ylimino)-1-(2-morpholin-4-
yl-ethyl)-1,3-dihydro-indole-2-one
Orange powder. Yield: 71%. Mp 178–80 ꢁC; H NMR 7.70 (3H, m),
7.67 (1H, d; J = 8.5 Hz), 7.60 (1H, d; J = 8.4 Hz), 7.30 (4H, m), 7.27
(1H, t; J = 8.4 Hz), 7.03 (1H, t; J = 8.5 Hz), 3.67 (4H, dt;
J = 11.0 Hz), 3.07 (2H, t; J = 7.2 Hz), 2.62 (2H, t; J = 7.2 Hz), 2.37
(4H, dt; J = 11.0 Hz); anal C24H22N3O2 C, H, N; Mass calcd. 384.45;
MS (ESI+) m ⁄ z 385.46 (M + H+; 100%).
(IV) 1-(2-Morpholin-4-yl-ethyl)-1H-indole-
3-carbaldheyde
Dark orange oil. Yield: 52%. 1H NMR 9.95 (1H, s), 8,27 (1H, d;
J = 7.8Hz), 7.79 (1H, s), 7.25 (3H, d+t+t), 4.24 (2H, t; J = 7.5 Hz), 3.65
(4H, m), 2.74 (2H, t; J = 7.5 Hz), 2.42 (4H, m); anal C15H18N2O2 C, H,
N; Mass calcd. 258.32; MS (ESI+) m ⁄ z 259.31 (M + H+; 100%).
1
(7) (4-Methoxy-phenyl)-[1-(2-morpholin-4-
yl-ethyl)-1H-indole-3-ylmethyl]-amine
1
(5) 5-Chloro-3-(naphthalen-1-ylimino)-1-(2-
Orange oil. Yield: 52%. H NMR 7.66 (1H, d; J = 7.6 Hz); 7.35 (1H,
morpholin-4-yl-ethyl)-1,3-dihydro-indole-2-one
Orange powder. Yield: 68%. Mp 130–31 ꢁC; H NMR 7.72 (3H, m),
7.63 (2H, d+s; J = 8.3 Hz), 7.28 (4H, m), 7.28 (1H, d; J = 8.3 Hz),
3.65 (4H, dt; J = 10.0 Hz), 3.09 (2H, t; J = 7.1 Hz), 2.60 (2H, t;
J = 7.1 Hz), 2.37 (4H, dt; J = 10.0 Hz); anal C24H21ClN3O2 C, H, N;
Mass calcd. 418.9; MS (ESI+) m ⁄ z 419.91 (M + H+; 100%).
d; J = 7.6Hz); 7.25 (1H, t; J = 7.6 Hz); 7.13 (1H, t; J = 7.6 Hz); 6.81
(2H, d; J = 8.4 Hz); 6.76 (2H, d; J = 8.8 Hz), 6.71 (1H, s), 4.42 (2H,
s), 4.22 (2H, t; J = 7.8 Hz), 3.76 (3H, s), 3.69 (4H, m), 2.74 (2H, t;
J = 7.8 Hz), 2.49 (4H, m); anal C22H27N3O2 C, H, N; Mass calcd.
365.47; MS (ESI+) m ⁄ z 366.42 (M + H+; 100%).
1
(8) [1-(2-Morpholin-4-yl-ethyl)-1H-indole-
(6) 5-Methyl-3-(naphthalen-1-ylimino)-1-(2-
morpholin-4-yl-ethyl)-1,3-dihydro-indole-2-one
Dark orange powder. Yield: 55%. Mp 188–89 ꢁC; H NMR 7.70 (3H,
m), 7.55 (1H, d; J = 8.3 Hz), 7.40 (1H, s), 7.31 (4H, m), 7.07 (1H, d;
J = 8.3 Hz), 3.67 (4H, dt; J = 10.0 Hz), 3.07 (2H, t; J = 7.1 Hz), 2.61
(2H, t; J = 7.1 Hz), 2.37 (4H, dt; J = 10.0 Hz); anal C25H24N3O2 C, H,
N; Mass calcd. 398.48; MS (ESI+) m ⁄ z 399.51 (M + H+; 100%).
3-ylmethyl]-naphthalen-1-yl-amine
Pale yellow oil. Yield: 64%. H NMR 7.81 (1H, d; J = 7.6 Hz), 7.74
1
1
(2H, m), 7.44 (3H, m), 7.30 (3H, m), 7.22 (1H, s), 7.15 (1H, t;
J = 7.6 Hz), 6.79 (1H, d; J = 8.0 Hz), 4.63 (2H, s), 4.25 (2H, t;
J = 7.8 Hz), 3.71 (4H, m), 2.77 (2H, t; J = 7.8 Hz), 2.51 (4H, m); anal
C25H27N3O C, H, N; Mass calcd. 385.50; MS (ESI+) m ⁄ z 386.51
(M + H+; 100%).
(4-Aryl)-[1-(2-morpholin-4-yl-ethyl)-1H-indole-
3-ylmethyl]-amine (7–10). General Procedure
To a mixture of indole-3-aldehyde (10 mmol) and sodium hydride
(30 mmol), in anhydrous DMF (20 mL) at room temperature, was
(9) (4-Chloro-phenyl)-[1-(2-morpholin-4-yl-ethyl)-
1H-indole-3-ylmethyl]-amine
1
Pale yellow oil. Yield: 48%. HNMR 7.62 (1H, d; J = 8.8 Hz); 7.39
(1H, d; J = 8,8 Hz); 7.23 (1H, t; J = 8.8 Hz); 7.12 (3H, m); 7.02 (1H,
Chem Biol Drug Des 2010; 75: 106–114
109

Mazzoni et al.
s; 6.49 (2H, d; J = 8.8 Hz); 4.41 (2H, s); 4.21 (2H, t; J = 7.8 Hz);
3.64 (4H, m); 2.75 (2H, t; J = 7.8 Hz); 2.44 (4H, m); anal
C21H24ClN3O C, H, N; Mass calcd. 369.89; MS (ESI+) m ⁄ z 370.89
(M + H+; 100%).
until tested. The experiments were conducted in accordance with
the Guide for Care and Use of Laboratory Animals published by the
National Institutes of Health and the ethical guidelines of the Inter-
national Association for the Study of Pain.
(10) [1-(2-morpholin-4-yl-ethyl)-1H-indole-
Statistical analysis
3-ylmethyl]-p-tolyl-amine
Orange oil. Yield: 64%. H NMR 7.62 (1H, d; J = 8.0 Hz) 7.35 (1H,
All data were expressed as a mean € SEM. In each case, the
cumulative licking-response was calculated for each mouse, and the
dose–response curves were expressed as the licking activity
(seconds) for the first 15 min and last 15–40 min. Response values
and its 95% confidence intervals were calculated using linear
regression analysis. The data were examined by analysis of vari-
ance (ANOVA) for multiple comparisons with a single control group.
When the analysis was restricted to two means, Student's test
(two-tailed) was used. Level of significance was set to 5% (p 0.05).
1
d; J = 8.0 Hz), 7.20 (1H, s), 7.1 (2H, t; J = 7.6 Hz), 7.01 (2H, d;
J = 8.0 Hz), 6.62 (2H, d; J = 8.0 Hz), 4.40 (2H, s), 4.18 (2H, t
J = 7.8 Hz), 3.65 (4H, m), 2.71 (2H, t; J = 7.8 Hz), 2.46 (4H, m), 2.22
(3H, s); anal C22H27N3O C, H, N; Mass calcd. 349.47; MS (ESI+) m ⁄ z
350.48 (M + H+; 100%).
Pharmacology
Paw formalin assay
Binding assays
The formalin test was performed in mice that had been individually
exposed to the observation chamber for 45 min before experiments.
For the formalin injection, 10 lL 5% formalin was administered into
the plantar surface of the right hind paw using a 30-gauge needle.
The animals were then placed in a clear Plexiglas cylinder
(20 · 30 cm) for observation. The pain behavior was quantified by
determining the amount of time (s) spent by mouse licking the
injected paw, over 40–60 min using 5-min bins. Two phases of spon-
taneous licking-behavior were observed after the formalin injection.
The interval from 0 to 15 min has been defined as Phase I, and the
interval from 15 to 40 min has been defined as Phase II (35).
CB1 competitive binding assays were performed in rat cerebellar
membranes using [3H]WIN 55212-2 (Perkin Elmer) as a radioligand.
Binding studies were performed at 25 ꢁC with 20 lg of receptor
protein in a 20 mM HEPES ⁄ 1 mM MgCl2 (pH = 7.4) buffer along
with [3H]WIN 55212-2 at a concentration of 100 nM. Final assay
volume was 500 lL, and the incubation time was 90 min. Receptor-
bound [3H]WIN 55212-2 was separated from free [3H]WIN 55212-2
by filtration. The filter paper was washed twice with 200 lL of
cold assay buffer and counted by liquid scintillation (46). The Ki val-
ues were calculated based on the Cheng–Prusoff equation:
Ki = IC50 ⁄ (1 + L ⁄ Kd).
Criteria for exclusion from the study included incomplete formalin
injection or excessive bleeding from the injection site. Time–
response data were presented as the mean € SEM of 5-min bins
over 40 min.
Molecular modeling
Molecular modeling and graphic manipulations were performed
using the INSIGHTII software package (Accelrys, San Diego, CA, USA)
running on Silicon Graphics Octane2 workstation and on Dell Preci-
sion 780, a dual-core Pentium D 3800 GHz, machine running Linux
Fedora 4 as operating system.
All the compounds, pravadoline, WIN 55212-2, and the antagonist
SR141716A were dissolved in dimethylsulfoxide (DMSO) and diluted
in saline containing DMSO 10% final concentrations. Ten microliters
of these solutions (containing 100 lg of compounds or controls)
was administered by intraplantar injection.
The 3D structures of analyzed compounds were constructed using
the module Builder of Insight program and then optimized using a
molecular dynamic simulation, calculations were performed by a
simulated annealing method (46–48), in vacuo, using the consistent-
valence force field (49,50) in INSIGHTII ⁄ DISCOVER software packages.
All tested compounds and references were given 30 min before for-
malin administration and were compared to saline injections (control
group). For the response analysis for the paw formalin assay, data
from Phase I and Phase II observations were considered separately. In
each case, the licking response was calculated for each mouse, and
the response expressed as percentage of control. Response values
and their 95% confidence intervals were calculated using linear
regression analysis (38). Displacement test of compounds by CB1 and
CB2 antagonists was performed by intravenous administration of SR
141716A and SR144528, 30 min before test compounds.
Results and Discussion
The antinociceptive and cannabinomimetic activities of compounds
1–10 were evaluated at peripheral level by formalin-induced pain
in the mouse (paw-licking test) (35,36). Paw-licking test is the
widely employed test to identify pharmacological properties of new
molecules.
Animals
The pain behavior is quantified by determining the amount of time
(in seconds) that the mouse spent licking the injected paw, over
40–60 min using 5-min bins. Two phases of spontaneous licking
behavior are observed after the formalin injection. The interval from
All the experiments were performed on male Swiss mice (20–25 g;
Harlan Italy), maintained on a 12-h light ⁄ dark cycle with food and
water available ad libitum. Mice were housed in groups of five
110
Chem Biol Drug Des 2010; 75: 106–114

Indole-based Ligands. SAR Study on CB1 Receptor
0 to 15 min has been defined as Phase I, and it is related to the
direct interaction with cannabinoid receptors, while the interval
from 15 to 40 min has been defined as Phase II, and it is related
to the production of inflammatory mediators (37).
biologic activity similar to AAIs (Table 2). The reversion of the antin-
ociceptive activity in the presence of antagonist suggests that the
analgesia could be related to the interaction with cannabinoid
receptors. It is interesting to note that the reversion of the activity
is obtained only by CB1 antagonist suggesting selectivity for the
tested compounds.
Compounds 1–10, WIN 55212-2, and pravadoline, used as refer-
ences, the CB1 antagonist SR141716A and the CB2 antagonist
SR144528 were given 30 min before formalin administration and
were compared to saline injections (control group). For the response
analysis of the formalin assay, data from Phase I and Phase II obser-
vations were considered separately. In each case, the licking
response was calculated for each mouse. The responses reported in
Table 1 are expressed as licking time and in parenthesis are reported
the percentage of inhibition of pain with respect to untreated control
(39). Compounds that gave more interesting results were subse-
quently screened to evaluate the analgesic reversion by contempo-
rary administration of CB1 and CB2 receptor antagonists.
Binding affinity at rat native CB1 receptor was performed in rat cer-
ebellar membranes, according to the procedure reported in experi-
mental section, using [3H]WIN 55212-2 (Perkin Elmer) as ligand. The
results reported in Table 3 show that indole derivatives exhibited
good affinities toward CB1 cannabinoid receptor. Compound 5
showed an affinity comparable to that of reference WIN 55212-2
(8.4 and 7.3 nM, respectively).
The compounds 7–10 displaying the substitution of the imino group
with a methyleneamino group showed a consistent reduction in the
analgesic activity in vitro. In particular, the compounds 7 and 9
resulted to be almost inactive, while compounds 8 and 10 showed
to endow a moderate analgesic activity (Table 1). The difference
between binding and in vivo values seems to suggest a partial
agonism or, more importantly, the interference of pharmacokinetic
factors for the compound 10. Further investigation is currently in
progress.
The maximal nociceptive response was observed during the first
15 min, with a subsequent drop of licking activity for the next 15–
40 min. Chloroisatin derivatives 2, 5, and the indole derivative 10
showed a licking time reduction values ranging between 35% and
45%, while the other compounds showed only a slight antinocicep-
tive effect in the Phase I. No activity (except for the compound 5)
was detected in the Phase II. The values of reduction of nociception
in the Phase I were comparable to pravadoline but less active when
compared to WIN 55212-2.
Figure 1 shows a graphical representation of the preferred con-
former of the molecule 5, as representative of the series, with
the three key pharmacophores, the aryl, morpholino, and the azo-
methinic nitrogen lone pair, aligned with the corresponding groups
of the references pravadoline and WIN 55212-2. The molecule of
compound 5 fits in with the reference compounds with a 0.4 ꢀ
average deviation of atoms, suggesting the possibility of interac-
tions with CB1 through the exploration of the same receptorial
spaces. In fact, although the formal substitution of the AAI car-
bonyl with an azomethinic group, the napthyl residues of 5, and
WIN well overlap each other, such as the indole and the alkylam-
inic moieties. The capability of 5 to interact with CB1 is consis-
tent with the hypothesis that the potential interactions of the AAI
carbonyl group with CB1 are unnecessary for both the binding
and the activation (34).
Differently to other compounds reported in literature (2,39–44)
among the indole 2,3-dione derivatives 1–6, the activity seems to
be affected by the electron-withdrawing nature of the substituent
on the indole moiety. In fact, the 5-chloro compounds 2 and 5 were
the most active in this study. Regarding the derivatives 7–10, shar-
ing the indole moiety, the activity seems to be affected by the
arylamino methylene moiety in position 3. The activity decreases in
the order 10 > 8>7 > 9 and seems to be related to the electron-
donating nature of the aminic aryl substituent. The activity of
compound 10 could be explained by an increase in the electronic
density on aminic nitrogen and consequently to generate a
hydrogen bond with the target.
Compounds 2, 5, 10, and the slightly active compound 6 were sub-
sequently screened in the presence of CB1 and CB2 antagonists to
evaluate receptorial displacement. The results seem to confirm a
Besides, the presence of the chlorine atom in position 5 of the
indole-2-one residue also seems to be important (compare 5 with 4
and 6, and 2 with 1 and 3). This could be related to the extension of
Table 1: Paw-licking time and, in parenthesis, the percentage of inhibition of pain induced by formalin with respect to untreated control
Compound
WIN
Pravadoline 55212-2 Formalin
1
2
3
4
5
6
7
8
9
10
Phase I 101 € 9
% inhib. (8)
71 € 7
(35)
96 € 8 104 € 9
(12) (5)
60 € 5
(45)
80 € 4
(26.5)
111 € 12
(0)
88 € 6
(19.8)
110 € 1
(0)
67 € 5
(39)
58 € 3
(48)
44 € 4
(60)
110 € 18
–
Phase II 162 € 10 160 € 12 165 € 2 163 € 11 122 € 9 164 € 13 163 € 16 164 € 12 141 € 2 165 € 14 120 € 9
% inhib. (0) (0) (0) (0) (25.6) (0) (0) (0) (15) (0) (27)
59 € 6
(65)
165 € 16
–
Groups of mice received formalin injection into the paw (5%–10 lL, 10 mice ⁄ group). The licking response was observed for each mouse and response expres-
sed as paw-licking time in second, and in parenthesis was reported percentage of reduction of pain with respect to control. Results were determined using lin-
ear regression analysis as described in the methods.
Chem Biol Drug Des 2010; 75: 106–114
111

Mazzoni et al.
Table 2: Pain reversion in paw-licking test obtained by contemporary administration of antagonist at CB1 and CB2 receptors
SR 141716A
Phase I
SR 144528
Phase I
Comp
Phase I
Phase II
Phase II
Phase II
2
71 € 1 (35)
60 € 1 (45)
80 € 1 (26.5)
67 € 2 (39)
58 € 1 (48)
44 € 2 (60)
110 € 18
160 € 2 (3)
122 € 3 (25.6)
164 € 1 (0)
165 € 1 (0)
120 € 2 (27)
59 € 3 (65)
165 € 16
107 € 2 (2)
104 € 1 (5)
107 € 2 (2)
106 € 1 (3)
108 € 2 (2)
107 € 2 (2)
(0)
(0)
(0)
(0)
(0)
71 € 2 (33)
75 € 2 (41)
83 € 2 (24)
66 € 2 (40)
59 € 2 (46)
49 € 1 (45)
(0)
5
123 € 2 (25)
(0)
(0)
112 € 2 (28)
60 € 2 (27)
6
10
Pravadoline
WIN 55212-2
Formalin
75 € 2 (50)
Groups of mice received formalin into either paw. The mice were treated intravenously with antagonists (2 mg ⁄ kg) 30 min before compounds administration.
The licking response was observed for each mouse and expressed also as % of reduction of pain with respect to control. The reported results were determined
using linear regression analysis as described in the methods.
Table 3: Binding affinity values at rat native CB1 receptor
Studi di Napoli ``Federico II''. The assistance of the staff is grate-
fully appreciated.
Ki (nM)
rCB1
Ki (nM)
Comp
Comp
rCB1
References
1
311 € 14.8
74.6 € 3.8
390 € 20.4
553 € 36.1
8.4 € 2.0
6
69.6 € 3.3
42.6 € 2.9
49.4 € 3.4
24.1 € 2.2
15.7 € 1.9
2
7
1. Pertwee G.R. (2002) Cannabinoids and multiple sclerosis. Phar-
macol Ther;95:165–174.
3
8
4
9
10
2. Benito C., Nunez E., Tolon R.M., Carrier E.J., Rabano A., Hillard
C.J., Romero J. (2003) Cannabinoid CB2 receptors and fatty acid
amide hydrolase are selectively overexpressed in neuritic plac-
que-associated glia in Alzheimer's disease brains. J Neurosci;
23:11136–11141.
5
WIN 55212-2
7.3 € 1.3
The reported results are expressed as the mean € SEM of at least three
independent experiments.
3. Volicer L., Stelly M., Morris J., McLaughlin J., Volicer B.J.
(1997) Effects of Dronabinol on anorexia and disturbed behavior
in patients with Alzheimer's disease. Int J Geriatr Psychiatry;
12:913–919.
the electron-withdrawing effect until the arylamino moiety through
the electronic conjugate system, and consequently, to the formation
of the necessary p-stacking interactions with CB1 receptor.
4. Rice A.S. (2001) Cannabinoids and pain. Curr Opin Investig
Drugs;2:399–414.
5. Di Carlo G., Izzo A.A. (2003) Cannabinoids for gastrointestinal
diseases: potential therapeutic applications. Expert Opin Investig
Drugs;12:39–49.
6. Porter A.C., Felder C.C. (2001) The endocannabinoid nervous sys-
tem: unique opportunities for therapeutic intervention. Pharmacol
Ther;90(1):45–60.
Conclusion
Although the activity of some compounds reported here are compa-
rable to pravadoline but less active when compared to WIN, these
indole derivatives represent an interesting starting point for further
studies in the field of cannabimimetic agents.
7. Dow R.L., Carpino P.A., Hadcock J.R., Black S.C., Iredale P.A.,
DaSilva-Jardine P., Shneider S.R., Paight E.S., Griffith D.A., Scott
D.O., O'Connor R.E., Nduaka C.I. (2009) Discovery of 2-(2-Chlor-
ophenyl)-3-(4-chlorophenyl)-7-(2,2-difluoropropyl)-6,7-dihydro-2H-
pyrazolo[3,4-f][1,4] oxazepin-8(5H)-one (PF-514273), a novel, bicy-
clic lactam-based cannabinoid-1 receptor antagonist for the
treatment of obesity. J Med Chem;52:652–2655.
In particular, the synthetic method developed to produce these
compounds could be used to perform libraries by combinatorial
approach to optimize the activity of these indole derivatives. In
fact, the results obtained indicate that it is possible to design
analogs that could be more effective on nociceptive response by
introducing appropriate structural modifications into indole moiety.
8. Hurst D., Umejiego U., Lynch D., Seltzman H., Hyatt S., Roche
M., McAllister S., Fleischer D., Kapur A., Abood M., Shi S.,
Jones J., Lewis D., Reggio P. (2006) Biarylpyrazole inverse agon-
ists at the cannabinoid CB1 receptor: importance of the C-3
carboxamide oxygen ⁄ Lysine3.28(192) Interaction. J Med Chem;
49:5969–5987.
In conclusion, the first results confirm the validity of our synthetic
method providing practical access to indole-based derivatives of
intense current interest in antinociceptive effect.
Acknowledgments
9. Thornton-Jones Z.D., Vickers S.P., Clifton P.G. (2005) SR141716A
reduces appetitive and consummatory responses for food. Psy-
chopharmacology;179:452–460.
The LC–MS and NMR spectral data were provided by Centro di Ric-
erca Interdipartimentale di Analisi Strumentale, Universitꢁ degli
112
Chem Biol Drug Des 2010; 75: 106–114

Indole-based Ligands. SAR Study on CB1 Receptor
10. Doggrell S.A. (2005) Will the new CB1 cannabinoid receptor
antagonist SR-147778 have advantages over rimonabant? Expert
Opin Investig Drugs;14:339–342.
11. Williamson E.M., Evans F.J. (2000) Cannabinoids in clinical prac-
tice. Drugs;60:1303–1314.
potent, enantioselective, (aminoalkyl)indole agonists of the can-
nabinoid receptor. J Med Chem;35:124–135.
24. Showalter V.M., Compton D.R., Martin B.R., Abood M.E. (1996)
Evaluation of binding in a transfected cell line expressing a
peripheral cannabinoid receptor (CB2); identification of cannabi-
noid receptor subtype selective agonists. J Pharmacol Exp
Ther;278:989–999.
25. Martin B.R., Compton D.R., Thomas B.F., Prescott W.R., Little
P.J., Razdaçn R.K., Johnson M.R., Melvin L.S., Mechoulam R.,
Ward S.J. (1991) Behavioral, biochemical, and molecular model-
ing evaluations of cannabinoid analogs. Pharmacol Biochem
Behav;40:471–478.
26. Compton D.R., Gold L.H., Ward S.J., Balster R.L., Martin B.R.
(1992) Aminoalkylindole analogs: cannabimimetic activity of a
class of compounds structurally distinct from D9-tetrahydrocan-
nabinol. J Pharmacol Exp Ther;263:1118–1126.
27. Bell M.R., D'Ambra T.E., Kumar V., Eissenstat M.A., Herrmann
J.L., Wetzel J.R. Jr, Rosi D. et al. (1991) Antinociceptive (amin-
oalkyl) indoles. J Med Chem;34:1099–1100.
12. De Vry J., Schreiber R., Eckel G., Jentzsch K.R. (2004) Behavioral
mechanisms underlying inhibition of food-maintained responding
by the cannabinoid receptor antagonist ⁄ inverse agonist
SR141716A. Eur J Pharmacol;483:55–63.
13. McLaughlin P.J., Winston K., Swezey L., Wisniecki A., Aberman
J., Tardif D.J., Betz A.J., Ishiwari K., Makriyannis A., Salamone
J.D. (2003) The cannabinoid CB1 antagonists SR 141716A and
AM 251 suppress food intake and food-reinforced behavior in a
variety of tasks in rats. Behav Pharmacol;14:583–588.
14. Gambi F., de Berardis D., Sepede G., Quartesan R., Calcagni E.,
Salerno R.M., Conti C.M.V., Ferro F.M. (2005) Cannabinoid recep-
tors and their relationships with neuropsychiatric disorders. Int J
Immunopathol Pharmacol;18:15–19.
15. Zavitsanou K., Garrick T., Huang X.F. (2004) Selective antagonist
[3H]SR141716A binding to cannabinoid CB1 receptors is
increased in the anterior cingulate cortex in schizophrenia. Prog
Neuropsychopharmacol Biol Psychiatry;28(2):355–360.
28. Pacheco M., Childers S.R., Arnold R., Casiano F., Ward S.J.
(1991) Amminoalkylindoles: action on specific G-protein-linked
receptors. J Pharmacol Exp Ther;257:170–183.
16. Huffman J.W. (2005) CB2 receptor ligands. Mini Rev Med
Chem;5:641–649.
29. Xie X.Q., Eissenstat M.A., Makriyannis A. (1995) Common can-
nabimimetic pharmacophoric requirements between aminoalkyl
indoles and classical cannabinoids. Life Sci;56:1963–1970.
30. Ward S.J., Mastriani D., Casiano F., Arnold R. (1990) Pravado-
line: profile in isolated tissue preparations. J Pharmacol Exp
Ther;255:1230–1239.
31. Devane W.A., Dysarz F.A. III, Johnson M.R., Melvin L.S., Howlett
A.C. (1988) Determination and characterization of a cannabinoid
receptor in rat brain. Mol Pharmacol;34:605–613.
32. Makriyannis A., Rapaka R.S. (1990) The molecular basis of can-
nabinoid activity. Life Sci;47:2173–2184.
33. Xie X., Han X., Chen J., Eissenstat M., Makriyannis A. (1999)
High resolution NMR and computer modeling studies of the can-
nabimimetic aminoalkylindole prototype, WJN55212-2. J Med
Chem;42:4021–4027.
34. Shim J.-Y., Howlett A.C. (2006) WIN55212-2 docking to the CB1
cannabinoid receptor and multiple pathways for conformational
induction. J. Chem. Inf. Model.;46:1286–1300.
35. Calignano A., La Rana G., Piomelli D. (2001) Antinociceptive
activity of the endogenous fatty acid amide, palmitylethanola-
mide. Eur J Pharmacol;419:191–198.
36. Kolesnikov Y., Cristea M., Osknam G., Torosjan A., Wilson R.
(2004) Evaluation of the tail formalin test in mice as a new
model to assess local analgesic effects. Brain Res;1029:217–
223.
37. Abbot F.V., Franklin K.B., Westbrook R.F. (1995) The formalin test:
scoring properties of the first and second phases of the pain
response in rats. Pain;60:91–102.
17. Deng H., Gifford A.N., Zvonok A.M., Cui G., Li X., Fan P., Des-
champs J.R., Flippen-Anderson J.L., Gatley S.J., Makriyannis A.
(2005) Potent cannabinergic indole analogues as radioiodinatable
brain imaging agents for the CB1 cannabinoid receptor. J Med
Chem;48:6386–6392.
18. Willis P.G., Pavlova O.A., Chefer S.I., Vaupel D.B., Mukhin A.G.,
Horti A.G. (2005) Synthesis and structure-activity relationship of
a novel series of aminoalkylindoles with potential for imaging
the neuronal cannabinoid receptor by positron emission tomog-
raphy. J Med Chem;48:5813–5822.
19. Page D., Yang H., Brown W., Walpole C., Fleurent M., Fyfe M.,
Gaudreault F., St-Onge S. (2007) New 1,2,3,4-tetrahydropyrrol-
o[3,4-b]indole derivatives as selective CB2 receptor agonists.
Bioorg Med Chem Lett;17:6183–6187.
20. Adam J., Cowley P.M., Kiyoi T., Morrison A.J., Mort C.J.W.
(2006) Recent progress in cannabinoid research. Prog Med
Chem;44:207–329.
21. Kai H., Morioka Y., Murashi T., Morita K., Shinonome S., Nakaz-
ato H., Kawamoto K. et al. (2007) 2-Arylimino-5,6-dihydro-4H-
1,3-thiazines as a new class of cannabinoid receꢂtor agonists.
Part 1: discovery of CB2 receptor selective compounds. Bioorg
Med Chem Lett;17:4030–4034.
22. Huffman J.W., Zengin G., Wu M.-J., Lu J., Hynd G., Bushell K.,
Thompson A.L.S., Bushell S., Tartal C., Hurst D.P., Reggio P.H.,
Selley D.E., Cassidy M.P., Wiley J.L., Martin B.R. (2005) Struc-
ture-activity relationships for 1-alkyl-3-(1-naphthoyl)indoles at
the cannabinoid CB1 and CB2 receptors: steric and electronic
effects of naphtoyl substituent. New highly selective CB2 recep-
tor agonists. Bioorg Med Chem;13:89–112.
38. Dubuisson D., Dennis S.G. (1977) The formalin test: a quantita-
tive study of the analgesic effects of morphine, meperidine and
brain stem stimulation in rats and cats. Pain;4:161–174.
39. Eissenstat M.A., Bell M.R., D'Ambra T.E., Alexander E.J., Daum
S.J., Ackerman J.H., Gruett M.D. et al. (1995) Aminoalkylindoles:
structure-activity relationships of novel cannabinoid mimetics.
J Med Chem;38:3094.
23. D'Ambra T.E., Estep K.G., Bell M.R., Eissenstat M.A., Josef K.A.,
Ward S.J., Haycock D.A., Baizman E.R., Casiano F.M., Beglin
N.C., Chippari S.M., Grego J.D., Kullnig R.K., Daley G.T. (1992)
Conformationally restrained analogues of pravadoline: nanomolar
Chem Biol Drug Des 2010; 75: 106–114
113

Mazzoni et al.
40. Diaz P., Xu J., Astruc-Diaz F., Pan H.M., Brown D.L., Naguib M.
(2008) Design and synthesis of a novel series of N-alkyl isatin
acylhydrazone derivatives that act as selective cannabinoid
receptor 2 agonists for the treatment of neuropathic pain. J
Med Chem;51:4932–4947.
41. Diaz P., Phatak S.S., Xu J., Astruc-Diaz F., Cavasotto C.N., Na-
guib M. (2009) 6-methoxy-N-alkyl isatin acylhydrazone deriva-
tives as a novel series of potent selective cannabinoid receptor
2 inverse agonists: design, synthesis, and binding mode predic-
tion. J Med Chem;52:433–444.
42. Huffman J.W., Padgett L.W. (2005) Recent developments in the
medicinal chemistry of cannabimimetic indoles, pyrroles and ind-
enes. Curr Med Chem;12:1395–1411.
43. Shim J.Y., Collantes E.R., Welsh W.J., Subramaniam B., Howlett
A.C., Eissenstat M.A., Ward S.J. (1998) Three-dimensional quan-
titative structure-activity relationship study of the cannabimimet-
ic (aminoalkyl)indoles using comparative molecular field analysis.
J Med Chem;41:4521–4532.
46. Brꢃnger A.T., Kuriyan J., Karplus M. (1987) Crystallographic R
factor refinement by molecular dynamics. Science;235:458–
460.
47. Nilges M., Clore G.M., Gronenborn A.M. (1988) Determination of
three-dimensional structures of proteins from interproton dis-
tance data by dynamical simulated annealing from a random
array of atoms. Circumventing problems associated with folding.
FEBS Lett;239:129–136.
48. Havel T.F. (1991) An evaluation of computational strategies for
use in the determination of protein structure from distance con-
straints obtained by NMR spectroscopy. Prog Biophys Mol
Biol;56:43–78.
49. Dauber-Osguthorpe P., Roberts V.A., Osguthorpe D.J., Wolff J.,
Genest M., Hagler A.T. (1988) Structure and energetics of ligand
binding to proteins: Escherichia coli dihydrofolate reductase-tri-
methoprim, a drug–receptor system. Proteins;4:31–47.
50. Allinger N.L., Yuh Y.H., Lii J.H. (1989) Molecular mechanics. The
MM3 force field for hydrocarbons. 1. J Am Chem Soc;111:
8551–8566.
44. Manera C., Tuccinardi T., Martinelli A. (2008) Indoles and related
compounds as cannabinoid ligands. Mini Rev Med Chem;8:370–
387.
Note
45. Tarzia G., Duranti A., Tontini A., Spadoni G., Mor M., Rivara S.,
Plazzi P.V., Kathuria S., Piomelli D. (2003) Synthesis and struc-
ture-activity relationships of a series of pyrrole cannabinoid
receptor agonists. Bioorg Med Chem;11:3965–3973.
awww.ccdc.cam.ac.uk (1994) Cambridge Structural Database System
Users Manual. Cambridge, UK: Cambridge Crystallographic Data
Centre.
114
Chem Biol Drug Des 2010; 75: 106–114