Rational design and synthesis of an orally active indolopyridone as a novel conformationally constrained cannabinoid ligand possessing antiinflammatory properties

Article abstract of DOI:10.1021/jm020329q

A series of unique indazoles and pyridoindolones have been rationally designed and synthesized as novel classes of cannabinoid ligands based on a proposed bioactive amide conformation. This has led to the discovery of the novel indolopyridone 3a as a conf

Full text of DOI:10.1021/jm020329q

2110
J . Med. Chem. 2003, 46, 2110-2116
Ra tion a l Design a n d Syn th esis of a n Or a lly Active In d olop yr id on e a s a Novel
Con for m a tion a lly Con str a in ed Ca n n a bin oid Liga n d P ossessin g
An tiin fla m m a tor y P r op er ties
Stephen T. Wrobleski,*,† Ping Chen,† J ohn Hynes, J r.,† Shuqun Lin,† Derek J . Norris,† Chennagiri R. Pandit,†
Steven Spergel,† Hong Wu,† J ohn S. Tokarski,‡ Xiaorong Chen,§ Kathleen M. Gillooly,§ Peter A. Kiener,§
Kim W. McIntyre,§ Vina Patil-koota,§ David J . Shuster,§ Lori A. Turk,§ Guchen Yang,§ and Katerina Leftheris†
Departments of Discovery Chemistry, Structural Biology and Modeling, and Immunology, Inflammation,
and Pulmonary Discovery, Bristol-Myers Squibb, P.O. Box 4000, Princeton, New J ersey 08543-4000
Received J uly 26, 2002
A series of unique indazoles and pyridoindolones have been rationally designed and synthesized
as novel classes of cannabinoid ligands based on a proposed bioactive amide conformation.
This has led to the discovery of the novel indolopyridone 3a as a conformationally constrained
cannabinoid ligand that displays high affinity for the CB2 receptor (Ki(CB2) ) 1.0 nM) and
possesses antiinflammatory properties when administered orally in an in vivo murine
inflammation model.
In tr od u ction
of THC, as well as other cannabinoids, were predomi-
nantly mediated through the CB1 receptor whereas
the immunomodulatory or antiinflammatory effects
were mediated through the peripheral CB2 receptor
subtype. More recent studies have suggested a broader
tissue distribution of CB1 receptors,5 and several re-
ports have provided evidence for antiinflammatory
effects mediated through the CB1 as well as CB2
receptors.6
The most widely known cannabinoid, ∆9-tetrahydro-
cannabinol (∆9-THC, 1), is considered to be the primary
In an effort to understand the role of cannabinoids
and their receptors in biological responses, a significant
amount of research effort has been focused on the
identification of THC analogues as well as nonclassical
or synthetic cannabinoids.7 One of the most widely
studied examples of the nonclassical type of cannab-
inoids is WIN-55212 (2), which belongs to the ami-
noalkylindole (AAI) class of compounds originally re-
ported by Sterling-Winthrop in 1992.8 This compound
has been extensively investigated because of its high
affinity toward the cannabinoid receptors (Ki(CB1) ) 1.9
nM; Ki(CB2) ) 0.3 nM)9 and has played an important
role in the identification and characterization of the
cannabinoid receptors and their associated functions.
In addition, 2 is more active than the classical cannab-
inoid THC in several pharmacological and behavioral
assays10 and acts as an agonist toward cannabinoid
receptors, resulting in a significant reduction of li-
popolysaccharide (LPS) induced proinflammatory cy-
tokine production (TNF-R and IL-1) in mice.6b,c Fur-
thermore, a recent report has demonstrated the ability
of 2 to inhibit LPS-induced TNF-R production in human
peripheral blood monocytes (PBMCs).6a
psychoactive constituent of marijuana and is known to
produce a variety of potentially beneficial therapeutic
effects including analgesia, antiemesis, anticonvulsive,
immunomodulation, and lowered intraocular pressure.1
Unfortunately, the medical use of THC has been limited,
as well as controversial, because of its often undesir-
able central nervous system (CNS) effects and poten-
tially addictive properties. In an effort to identify the
receptors responsible for cannabinoid activity, two can-
nabinoid receptor subtypes, CB1 and CB2 belonging to
the GPCR superfamily of 7-transmembrane receptors,
were first cloned in 1990 and 1993, respectively.2,3 Early
studies found the CB1 receptors to be highly expressed
in the tissues of the central nervous system, while the
CB2 receptors were detected mainly in peripheral
tissues, particularly on the surface of immune cells.4
As a result, it was widely believed that the CNS effects
As part of our efforts to identify novel CB2 agonists
as antiinflammatory agents, we have recently identified
substituted indazoles and conformationally constrained
indolopyridones as novel classes of cannabinoid ligands.
This report describes the rational design and synthesis
of these compounds leading to the identification of the
novel indolopyridone 3a as a potent cannabinoid ligand.
In addition, the in vivo evaluation of 3a as an antiin-
* To whom correspondence should be addressed. Phone: 609-252-
4873. Fax: 609-252-6601. E-mail: stephen.wrobleski@bms.com.
† Department of Discovery Chemistry.
‡ Department of Structural Biology and Modeling.
§ Department of Immunology, Inflammation, and Pulmonary Dis-
covery.
10.1021/jm020329q CCC: $25.00 © 2003 American Chemical Society
Published on Web 04/26/2003

Design and Synthesis of Indolopyridone
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11 2111
Ta ble 1. Effect of Indole C2 Substitution on Binding Affinity
Ta ble 2. Calculated Effect of C2 Substitution on Amide
Conformations
Ki (nM)
compd
R
CB2
CB1
4a
4b
4c
4d
H
11 ( 3
29 ( 6
110 ( 23
6%a
245 ( 52
8 ( 2
ndb
a
compd
R
∆E (kcal/mol)
methyl
ethyl
n-propyl
4a
4b
4c
4d
H
+0.6
+2.9
+3.5
+3.8
ndb
methyl
ethyl
n-propyl
% inhibition at 100 nM ligand concentration. b Not determined.
a
a
(Energy of amide conformer I) - (energy of amide conformer
II).
flammatory agent using a murine model of inflamma-
tion is reported.
energy conformations about the C3-carboxamide bond
depicted as amide conformer I and amide conformer II
in Table 2. Both conformers maintain the amide group
near the plane and in conjugation with the indole ring
system. Furthermore, the DFT energy calculations
suggest that the rotomer populations of the C3 amide
group are most likely influenced by the size of the C2
group as illustrated in Table 2. More specifically, the
calculated energy difference (∆E) between conformers
I and II indicate that conformer II is favored over
conformer I in the range 0.6-3.8 kcal/mol for analogues
4a -d . We rationalized that larger C2 substituents
would destabilize conformer I relative to II because of
unfavorable interactions between the C2 substituent
and the amide nitrogen substituents. Interestingly,
comparison of the CB2 binding affinities of 4a -d listed
in Table 1 with their respective amide conformational
preferences as calculated in Table 2 suggests a trend of
decreasing CB2 binding affinity with an increasing
tendency to favor amide conformer II over conformer I.
These results suggested to us that compounds that more
strongly favor the amide conformer I may provide
increased CB2 binding affinity. To further test this
hypothesis, and in an attempt to identify compounds
with increased cell potency relative to the indole ana-
logues, chemotypes that would be expected to favor or
adopt this amide orientation were pursued.
Design
Previously, we have reported substituted indole C3
carboxamides bearing a (1S)-endo-fenchyl group as
potent cannabinoid ligands.11 Interestingly, analysis of
the effects of C2 substitution on CB1 and CB2 binding
affinity in this series revealed a strong preference for a
small substituent at the C2 position as illustrated in
Table 1. Only a hydrogen or methyl substituent was
tolerated in obtaining good CB2 affinity as observed for
compounds 4a and 4b. Increasing the size of the C2
substituent to ethyl as in 4c or to n-propyl as in 4d
resulted in a significant decrease of CB2 binding affin-
ity. Importantly, a similar trend of increasing size of
the indole C2 substituent leading to decreased CB
binding affinity has also been reported in the SAR
analysis of 2 as well as other aminoalkylindole cannab-
inoid ligands.8,11
Despite having potent CB2 binding affinities, com-
pounds 4a and 4b displayed only modest CB2 agonist
activity as measured by the in vitro efficacy in inhibition
of TNF-R release in human peripheral blood mono-
nuclear cells (IC50 ) 13 and 33 µM, respectively) relative
to 2 in this assay (IC50 ) 6 µM). In addition, the most
potent compound 4a , when administered orally up to a
dose of 50 mg/kg, failed to show efficacy in a murine
model of acute inflammation where LPS-induced TNF-R
production was measured.11 In light of these results,
alternative chemotypes relative to the indole nucleus
were pursued in an effort to identify, in vivo, orally
active CB2 ligands.
On the basis of DFT energy calculations performed
identically to the calculations on the indole series, the
proposed indazole 5a was predicted to strongly favor
The observed trend of a decrease in CB binding
affinity in the indole series with an increase in the size
of the C2 substituent was taken into consideration in
the design of other potential chemotypes. It was ratio-
nalized that larger C2 substituents in the indole series
may lead to decreased binding affinity by directly
creating unfavorable contacts with receptor and/or
indirectly by influencing the adjacent C3 amide rotomer
population and position of the pendent fenchyl group.
To investigate the influence of the C2 substituent on
the C3 amide rotomer population, molecular modeling
calculations in the form of torsional energy scans about
the C3-carboxamide bond were performed on analogues
4a -d . These calculations suggest two local minimum
amide conformer I over conformer II by >4 kcal/mol.
We rationalize that favorable stereoelectronic interac-
tions between the indazole N-2 nitrogen and the amide
hydrogen should stabilize conformer I relative to II. In
addition, unfavorable stereoelectronic interactions be-
tween the N-2 nitrogen and the C3 carbonyl oxygen

2112 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11
Wrobleski et al.
Sch em e 1a
Sch em e 2a
a
Conditions: (a) (1) 2.2 equiv of nBuLi, THF, -30 to 0 °C and
then DMF, (2) 3 N aqueous HCl, 55 °C, 85%.
Ta ble 3. Selected Indazole Carboxamides 5a -d
a
Conditions: (a) Boc anhydride, THF, reflux, 80%; (b) nBuLi,
THF, -40 to -10 °C and then CO2, 80%; (c) EDCI, HOBt, Me2NH,
DMF, room temp, 78%; (d) NaNO2, aqueous AcOH, 82%; (e) NaH,
4-(2-chloroethyl)morpholine hydrochloride, DMF, 45 °C, 72%; (f)
aqueous NaOH, EtOH, 80 °C, 80%; (g) R1R2NH, EDCI, HOBt,
(iPr)2NEt, DMF, room temp, 66-87%.
TNF-R
Ki(CB2)
(nM)
(PBMC)
Ki(CB1)
(nM)
R
IC50 (µM)b
5a HN[(1S)-fenchyl]
2.0 ( 0.5
30
20
43
36
24 ( 10
162 ( 60
ndc
would be expected to destabilize the alternative con-
former II in the indazole case. In addition to 5a , the
novel indolopyridone 3a was designed as a constrained
analogue that would restrict the carboxamide to the
desired orientation present in conformer I.
5b HN[R-(2-pyridyl)benzyl]a 146 ( 79
5c HN[(2)-methoxybenzyl]
77 ( 20
5d N[(N-2-propyl)(2-chloro- 69 ( 26
ndc
6-fluorobenzyl]
a
b
Racemic. n ) 1. c Not determined.
tion was measured. For selected compounds, CB1 recep-
tor affinities were also determined by displacement of
radiolabeled 2 from the CB1 receptor.
Ch em istr y
Since the 7-methoxy substituent and the morpholi-
noethyl groups were previously found to be the optimal
substitutions in the indole carboxamide series, the
chemistry was initially designed to maintain these
pharmacophores in the preparation of the indazole and
indolopyridone chemotypes. Appropriately substituted
indazoles were prepared from commercially available
2-methoxy-6-methylaniline (6) as outlined in Scheme 1.
Protection of the aniline to afford 714 followed by
carboxylation and amide bond formation provided dim-
ethylamide 9. Sodium nitrite mediated cyclization15 of
9 afforded indazole 10 that underwent a selective N-1
alkylation employing sodium hydride and commercially
available 4-(2-chloroethyl)morpholine to provide 11.
Hydrolysis of the dimethylamide followed by amide bond
formation with a variety of primary and secondary
amines afforded the desired indazole products 5.
The conformationally constrained indolopyridone ana-
logues were easily synthesized from the previously
prepared11 2-methyl-3-carboxamide indoles employing
an efficient formylation and cyclization sequence16 as
illustrated by the synthesis of derivative 3a in Scheme
2.
In d a zole Ser ies. By use of the previously outlined
synthesis, four indazoles 5a -d were identified as having
appreciable CB2 binding affinity (IC50 < 200 nM).
Analogous to the indole series, the (1S)-fenchylamide
analogue 5a provided the highest CB2 and CB1 binding
affinity within the indazole series (Ki(CB2) ) 2.0 ( 0.5
nM; Ki(CB1) ) 24 ( 10 nM). In addition, indazole 5a
showed an increase in CB2 binding affinity relative to
the most potent indole fenchylamide 4a (Ki(CB2) ) 11
( 3 nM; Ki(CB1) ) 245 ( 52 nM) in the CB2 and CB1
assays. These initial results appeared to support our
hypothesis that compounds that favor the amide con-
former I based on molecular modeling may provide
increased affinity for the CB2 receptor (vide supra).
Unfortunately, despite having excellent CB2 and CB1
binding affinity, indazoles 5a -d showed minimal ago-
nist activity at the CB2 receptor, resulting in only
moderate cell potency against TNF-R production in the
PBMC cell assay (IC50 ) 20-43 µM) as listed in Table
3. In light of these results, the indazole analogues were
abandoned from further in vivo studies and attention
was focused on evaluating the conformationally con-
strained indolopyridone derivatives.
In d olop yr id on e Ser ies. Three out of four of the
amide analogues that were prepared in the indazole
series were synthesized and evaluated in the corre-
sponding indolopyridone series as listed in Table 4. As
in the previous indole and indazole cases, the (1S)-
fenchyl analogue 3a was the most potent CB2 ligand
in the indolopyridone series (Ki(CB2) ) 1.0 ( 0.2 nM).
In addition, indolopyridone 3a was the most potent CB2
ligand compared to any of the indole or indazole deriva-
tives prepared to date. Interestingly, indolopyridone 3b
Resu lts a n d Discu ssion
All compounds were initially screened for CB2 binding
affinity by displacement of radiolabeled 2. Compounds
at 100 nM concentration that gave >60% inhibition of
2 in binding to the CB2 receptor were subsequently
screened for in vitro inhibition of TNF-R release in
human peripheral blood mononuclear cells (PBMCs).
Compounds that were active in the PBMC assay were
then evaluated for oral activity in a murine model of
acute inflammation where LPS-induced TNF-R produc-

Design and Synthesis of Indolopyridone
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11 2113
Ta ble 4. Selected Substituted Indolopyridones 3a -c
TNF-R
(PBMC)
Ki(CB2)
Ki(CB1)
R
(nM)
IC50 (µM)
(nM)
F igu r e 2. In vivo activity of CB ligands dosed orally in
inhibiting LPS-induced TNF-R release in mice (n ) 8 animals/
group).
3a (1S)-fenchyl
1.0 ( 0.2
67 ( 23
8 ( 4b
7 ( 7b
ndd
16 ( 4
3700 ( 1000
3b (2)-methoxybenzyl
3c R-(2-pyridyl)benzyla 13%c
ndd
tantly, this work had led to the identification of indol-
opyridone 3a as a potent and orally active cannabinoid
ligand that possesses antiinflammatory properties in
vivo when administered in a murine inflammation
model.
a
b
d
Racemic. n ) 4. c % inhibition at 500 µM. Not determined.
Exp er im en ta l Section
Ch em istr y. Proton magnetic resonance (1H NMR) spectra
were recorded on a Bruker ARX-400 spectrometer and are
reported in ppm relative to the reference solvent of the sample
in which they were run. HPLC and LC-MS analyses were
conducted using a Shimadzu LC-10AS liquid chromatograph
and a SPD UV-vis detector at 220 nm with the MS detection
performed with a Micromass Platform LC spectrometer. HPLC
analyses were performed using the following conditions: Bal-
listic YMC S5 ODS 4.6 mm × 50 mm column with a binary
solvent system where solvent A is 10% methanol, 90% water,
and 0.2% phosphoric acid and solvent B is 90% methanol, 10%
water, and 0.2% phosphoric acid; flow rate ) 4 mL/min; linear
gradient time ) 4 min; start %B ) 0, final %B ) 100. LC-
MS analyses were performed using the following conditions:
Waters Xterra 5 µm, 4.6 mm × 30 mm column with a binary
solvent system where solvent A is 10% methanol, 90% water,
and 0.1% trifluoroacetic acid and solvent B is 90% methanol,
10% water, and 0.1% trifluoroacetic acid; flow rate ) 4 mL/
min; linear gradient time ) 2 min; start %B ) 0, final %B )
100. Melting points were measured on a Mel-Temp 3.0 melting
point apparatus and are uncorrected. All reagents were
purchased from commercial sources unless otherwise noted.
Preparation of (1S)-endo-fenchylamine was performed accord-
ing to the previously reported procedures.17 All reagents were
used without further purification unless otherwise noted. All
reactions were performed under an argon atmosphere. Reac-
tions in aqueous media were run under an ambient atmo-
sphere unless otherwise noted.
[3-Meth oxy-2-(ter t-bu tyloxyca r bon yla m in o)]p h en yla c-
etic Acid (8). To 73 mL (73 mmol) of a 1 M solution of di-
tert-butyl dicarbonate in THF at room temperature was added
10 g (73 mmol) of 3-methoxy-2-(tert-butyloxycarbonylamino)-
toluene, and the resulting solution was refluxed for 24 h. The
mixture was cooled to room temperature and concentrated on
a rotary evaporator. The resulting viscous oil was dissolved
in methylene chloride (125 mL), and the solution was succes-
sively washed with 6% aqueous citric acid (2 × 30 mL), water
(2 × 30 mL), and brine (30 mL) and then dried over anhydrous
sodium sulfate, filtered, and concentrated in vacuo to afford
the crude product as a viscous oil. The resulting oil was
dissolved in a minimal amount of warm methylene chloride
(∼30 mL), and hexanes were added (∼125 mL). The cloudy
mixture was cooled to 0 °C for 1 h. The crystalline product
was collected by filtration and dried in vacuo to afford 13.9 g
(80%) of 8 as a white solid: mp 161.3-162.4 °C. 1H NMR (400
MHz, MeOH-d4): δ 7.19 (t, J ) 8.2 Hz, 1H), 6.93 (d, J ) 8.3
Hz, 1H), 6.89 (d, J ) 7.7 Hz, 1H), 3.82 (s, 3H), 3.61 (s, 2H),
1.48 (br s, 9H). HPLC: tR ) 2.41 min, 99.2% purity. HRMS
(EI) m/z calcd for C14H18NO5 [M - H]+: 280.1185. Found:
280.1174.
F igu r e 1. The iv dose-response of 3a in LPS-induced TNF-R
release in mice (n ) 8 animals/group).
(Ki(CB2) ) 67 ( 23 nM) containing the 2-methoxyben-
zylamide was similar in potency toward the CB2 recep-
tor compared to its indazole counterpart 5c (Ki(CB2) )
77 ( 20 nM). In contrast, the indolopyridone 3c con-
taining the R-(2-pyridyl)benzyl substituent appeared
less active at the CB2 receptor (13% inhibition at 500
µM) compared to indazole 5b (Ki(CB2) ) 146 ( 79 nM).
Most gratifyingly, however, indolopyridone 3a showed
increased cell activity (IC50 ) 8 µM) relative to the most
potent indole and indazoles 4a and 5a (IC50 ) 13 and
30 µM, respectively). In addition, the 2-methoxybenzy-
lamide 3b also gave an increase in cell activity (IC50
)
7 µM) relative to its indazole counterpart 5c (IC50 ) 43
µM) despite having similar CB2 binding affinities. After
identification of indolopyridone 3a as the most potent
CB2 ligand with significant cell activity, 3a was evalu-
ated in an in vivo model.
Indolopyridone 3a exhibited a dose-dependent inhibi-
tion of LPS-stimulated TNF-R release when dosed
intravenously, having an ED50 between 0.66 and 2 mg/
kg as illustrated in Figure 1. In addition, 3a was also
orally efficacious resulting in a 61% reduction of serum
TNF-R levels when dosed at 50 mg/kg as shown in
Figure 2. It is noteworthy to mention that 2 did not show
any efficacy in this murine inflammation model when
administered orally.
In conclusion, molecular modeling calculations and
conformation-activity relationships within a class of
indole-derived cannabinoid ligands have suggested the
involvement of the amide conformer I as a bioactive
conformation at the CB2 receptor. This hypothesis has
been used to rationally design novel, conformationally
restricted indazole and indolopyridone ligands having
an increased affinity for the CB2 receptor. Most impor-
[3-Meth oxy-2-(ter t-bu tyloxyca r bon yla m in o)]p h en yla c-
etic Acid Dim eth yla ceta m id e (9). To a solution of 8.32 g

2114 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11
Wrobleski et al.
(29.6 mmol) of 8, 8.51 g (44.4 mmol) of 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide hydrochloride, 4.80 g (35.5 mmol)
of 1-hydroxybenzotriazole, and 9.66 g (118 mmol) of dimethy-
lamine hydrochloride in 90 mL of dimethylformamide at room
temperature was added 25.8 mL (148 mmol) of diisopropyl-
ethylamine, and the resulting solution was stirred for 48 h.
The reaction mixture was concentrated in vacuo, and the
resulting oil was dissolved in 350 mL of dichloromethane and
washed with aqueous 1 N sodium hydroxide (3 × 125 mL),
6% aqueous citric acid solution (3 × 100 mL), water (100 mL),
and brine (100 mL). After the mixture was dried over anhy-
drous sodium sulfate, the resulting solution was decanted and
concentrated on a rotary evaporator to afford a reddish-orange
oil as the crude product. This material was dissolved in diethyl
ether (ca. 100 mL) and reconcentrated on the rotary evapora-
tor, yielding a yellow solid that was subsequently triturated
with two 35-mL portions of hexanes to remove any residual
dimethylformamide. The resulting solid was dried in vacuo to
deliver 7.09 g (78%) of 9 as a yellow solid: mp 111.8-113.0
°C. 1H NMR (400 MHz, MeOH-d4): δ 7.20 (t, J ) 8.0 Hz, 1H),
6.93 (d, J ) 8.4 Hz, 1H), 6.79 (d, J ) 7.7 Hz, 1H), 3.82 (s, 3H),
3.71 (s, 2H), 2.99 (s, 3H), 2.95 (s, 3H), 1.47 (br s, 9H). HPLC:
tR ) 2.46 min, 99.0% purity. HRMS (EI) m/z calcd for
sodium hydroxide. The aqueous solution was concentrated on
a rotary evaporator, and the remaining solid was azeotroped
with toluene, dissolved in methylene chloride (ca. 10 mL), dried
over anhydrous sodium sulfate, filtered, and concentrated in
vacuo to afford 79 mg (80%) of a white solid as the sodium
carboxylate salt. This compound was used directly in the next
step without further purification.
To the sodium carboxylate salt (79 mg, 0.23 mmol) were
successively added 65 mg (0.34 mmol) of EDCI, 38 mg (0.28
mmol) of HOBt, 0.6 mL of DMF, 0.16 mL (0.92 mmol) of
diisopropylethylamine, and 53 mg (28 mmol) of (1S)-endo-
fenchylamine.17 The resulting mixture was stirred at 70 °C
for 16 h and then cooled to ambient temperature, and 20 mL
of ethyl acetate was added. The mixture was successively
washed with saturated aqueous sodium bicarbonate (3 × 5
mL), water (2 × 5 mL), and brine (5 mL), and the organic
solution was dried over anhydrous sodium sulfate, filtered, and
concentrated in vacuo to afford the crude product. Purification
by flash chromatography on silica gel using 5% methanol in
ethyl acetate as the eluant afforded 91 mg (87%) of 5a as a
1
white solid. H NMR (400 MHz, MeOH-d4): δ 7.79 (d, J ) 8.2
Hz, 1H), 7.25 (t, J ) 8.0 Hz, 1H), 7.00 (d, J ) 7.6 Hz, 1H),
4.87-4.85 (m, 2H), 4.07 (s, 1H), 4.06 (overlapping s, 3H), 3.83-
3.78 (m, 6H), 3.63 (br s, 2H), 3.30 (br s, 2H), 1.83-1.76 (m,
3H), 1.60-1.54 (m, 2H), 1.35-1.22 (m, 2H), 1.19 (s, 3H), 1.12
(s, 3H), 0.09 (s, 3H). HPLC: tR ) 2.92 min, 98.9% purity. LC-
MS: tR ) 1.80 min, [M + H]+ ) 441.5. HRMS (EI) m/z calcd
for C25H37N4O3 [M + H]+: 441.2866. Found: 441.2885.
C
16H25N2O4 [M + H]+: 309.1814. Found: 309.1812.
7-Meth oxy-3-d im eth yla m id oin d a zole (10). To a stirring
solution of 1.27 g (4.12 mmol) of 9 in 4% aqueous acetic acid
at 95 °C was slowly added an aqueous solution of 0.85 g (12.4
mmol) of sodium nitrite in 1.4 mL of water over 2 h. After the
addition was complete, HPLC analysis showed nearly complete
consumption of the substrate. The reaction mixture was cooled
to room temperature and concentrated on a rotary evaporator,
and the resulting solid was suspended in approximately 30
mL of water. The product was collected by vacuum filtration,
washed with water (20 mL), and then dried in vacuo to afford
7-Meth oxy-N-[p h en yl(2-p yr id in yl)m eth yl]-1-[2-(4-m or -
p h olin yl)eth yl]-1H-in d a zole-3-ca r boxa m id e (5b). 5b was
prepared as described for 5a to obtain 26 mg (66%) of 5b as a
1
yellow solid. H NMR (400 MHz, MeOH-d4): δ 8.58 (dd, J )
4.3, 0.9 Hz, 1H), 7.79 (td, J ) 7.7, 1.7 Hz, 1H), 7.72 (d, J ) 8.3
Hz, 1H), 7.50 (d, J ) 7.9 Hz, 1H), 7.41 (d, J ) 8.6 Hz, 2H),
7.34-7.30 (m, 3H), 7.26-7.22 (m, 1H), 7.14 (t, J ) 7.9 Hz, 1H),
6.87 (d, J ) 7.6 Hz, 1H), 6.38 (s, 1H), 4.85 (t, J ) 6.9 Hz, 2H),
3.99 (s, 3H), 3.62 (t, J ) 4.6 Hz, 4H), 2.88 (t, J ) 6.9 Hz, 2H),
2.54 (t, J ) 4.4 Hz, 4H). HPLC: tR ) 2.07 min, 99.0% purity.
LC-MS: tR ) 1.35 min, [M + H]+ ) 472.8. HRMS (EI) m/z
calcd for C27H30N5O3 [M + H]+: 472.2349. Found: 472.2343.
1
0.74 g (82%) of 10 as a yellow solid: mp 224.5-226.2 °C. H
NMR (400 MHz, MeOH-d4): δ 7.47 (t, J ) 8.5 Hz, 1H), 7.13
(t, J ) 7.8 Hz, 1H), 6.85 (d, J ) 7.6 Hz, 1H), 4.01 (s, 3H), 3.36
(s, 3H), 3.18 (s, 3H). HPLC: tR ) 2.10 min, 99.1% purity.
HRMS (EI) m/z calcd for C11H14N3O2 [M + H]+: 220.1086.
Found: 220.1080.
7-Meth oxy-N-[(2-m eth oxyp h en yl)m eth yl]-1-[2-(4-m or -
p h olin yl)eth yl]-1H-in d a zole-3-ca r boxa m id e (5c). 5c was
prepared as described for 5a to obtain 18 mg (77%) of 5c as
an off-white solid. 1H NMR (400 MHz, MeOH-d4): δ 7.76 (d, J
) 8.1 Hz, 1H), 7.29 (d, J ) 7.4 Hz, 1H), 7.25 (td, J ) 8.1, 1.5
Hz, 1H), 7.15 (t, J ) 7.9 Hz, 1H), 6.97 (d, J ) 8.1 Hz, 1H),
6.92-6.86 (m, 2H), 4.81 (t, J ) 6.9 Hz, 2H), 4.60 (s, 2H), 3.99
(s, 3H), 3.89 (s, 3H), 3.61 (t, J ) 4.7 Hz, 4H), 2.86 (t, J ) 6.9
Hz, 2H), 2.51 (t, J ) 4.4 Hz, 4H). HPLC: tR ) 2.27 min, 98.4%
purity. LC-MS: tR ) 1.48 min, [M + H]+ ) 425.3. HRMS (EI)
m/z calcd for C23H29N4O4 [M + H]+: 425.2189. Found: 425.2203.
N-[(2-Ch lor o-6-flu or op h en yl)m eth yl]-7-m eth oxy-N-(1-
m et h ylet h yl)-1-[2-(4-m or p h olin yl)et h yl]-1H -in d a zole-3-
ca r boxa m id e (5d ). 5d was prepared as described for 5a to
obtain 21 mg (75%) of 5d as a pale-yellow solid. 1H NMR (400
MHz, MeOH-d4): δ 7.51-7.49 (m, 1H), 7.28-7.22 (m, 2H), 7.14
(d, J ) 7.9 Hz, 1H), 7.12-7.03 (m, 1H), 6.89 (d, J ) 7.6 Hz,
1H), 5.32 (br s, 2H), 4.79 (br s, 2H), 4.00 (s, 3H), 3.60-3.51
(m, 5H), 2.88-2.80 (m, 2H), 2.45 (br s, 4H), 1.25-1.13 (m, 6H).
HPLC: tR ) 2.79 min, 98.8% purity. LC-MS: tR ) 1.74 min,
[M + H]+ ) 489.3. HRMS (EI) m/z calcd for C25H31ClFN4O3
[M + H]+: 489.2069. Found: 489.2071.
2,5-Dih yd r o-6-m et h oxy-5-[2-(4-m or p h olin yl)et h yl]-2-
[(1S,2S)-1,3,3-tr im eth ylbicyclo[2.2.1]h ep ta n -2-yl]-1H-p y-
r id o[4,3-b]in d ol-1-on e (3a ). To a solution of 4b11 (0.20 g, 0.44
mmol) in 4 mL of anhydrous THF at -30 °C was added 0.88
mL (1.3 mmol) of a 1.5 M solution of nBuLi in hexanes, and
the resulting solution was warmed to room temperature over
45 min. The solution was cooled to -30 °C, DMF (0.14 mL,
1.8 mmol) was added, and the mixture was stirred at -30 °C
for 15 min and then at room temperature for 1 h. The reaction
mixture was transferred via cannula into 6 mL of a well-stirred
10% aqueous HCl solution that had been deoxygenated by
bubbling argon through the solution for approximately 10 min
7-Meth oxy-N,N-d im eth yl-1-[2-(4-m or p h olin yl)eth yl)]-
1H-in d a zole-3-ca r boxa m id e (11). To a room-temperature
solution of 0.549 g (2.50 mmol) of 10 and 0.75 g (5.00 mmol)
of 4-(2-chloroethyl)morpholine in 5 mL of anhydrous dimeth-
ylformamide was added 0.20 g (5.0 mmol) of 60% sodium
hydride dispersion in two portions over 10 min. The reaction
mixture was stirred for 14 h, and then an additional 0.75 g
(5.0 mmol) of 4-(2-chloroethyl)morpholine was added followed
by heating to 40 °C for an additional 2 h. The reaction mixture
was cooled to room temperature, and 10 mL of water was
slowly added. The mixture was extracted with ethyl acetate
(4 × 30 mL), and the combined extracts were washed with
water (3 × 7 mL) and brine (7 mL), then dried over anhydrous
sodium sulfate, decanted, and concentrated in vacuo to afford
a yellow liquid that partially solidified upon standing. This
material was triturated with three 20-mL portions of hexanes
and the remaining white solid was dried in vacuo to afford
1
0.60 g (72%) of 11: mp 109.5-110.3 °C. H NMR (400 MHz,
MeOH-d4): δ 7.49 (d, J ) 8.2 Hz, 1H), 7.14 (t, J ) 7.8 Hz,
1H), 6.89 (d, J ) 7.6 Hz, 1H), 4.82 (t, J ) 6.7 Hz, 2H), 4.00 (s,
3H), 3.62-3.60 (m, 4H), 3.37 (s, 3H), 3.17 (s, 3H), 2.87(t, J )
6.7 Hz, 2H), 2.53-2.51 (m, 4H). HPLC: tR ) 1.46 min, 99.4%
purity. HRMS (EI) m/z calcd for C17H25N4O3 [M + H]+:
333.1927. Found: 333.1914.
7-Meth oxy-1-[2-(4-m or p h olin yl)eth yl]-N-[(1S,2S)-1,3,3-
tr im eth ylbicyclo[2.2.1]h ep ta n -2-yl)-1H-in d a zol-3-ca r box-
a m id e (5a ). To 100 mg (0.30 mmol) of 11 was added 0.5 mL
of 3 N aqueous sodium hydroxide and 0.5 mL of ethanol, and
the resulting solution was heated at 80 °C for 16 h and then
cooled to room temperature and concentrated. The residue was
dissolved in water (5 mL) and adjusted to pH 7 by addition of
1 N aqueous HCl and then was reconcentrated. The resulting
residue was redissolved in water (5 mL) and made basic
(greater than pH 10) by adding a few drops of 1 N aqueous

Design and Synthesis of Indolopyridone
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11 2115
prior to the addition. The mixture was heated to 55 °C for 17
h, cooled to room temperature, and made basic by a dropwise
addition of 3 N aqueous KOH to a pH of approximately 10.
The mixture was extracted with methylene chloride (3 × 20
mL), washed with water (20 mL) and brine (10 mL), dried over
sodium sulfate, and concentrated in vacuo to afford the crude
product as a yellow semisolid. The crude product was purified
by flash chromatography on silica gel using a gradient elution
of 80% ethyl acetate in hexanes initially and then 100% ethyl
acetate to elute the product. Concentration of the desired
fractions in vacuo provided 0.17 g (85%) of 3a as an off-white
solid: mp 164.4-165.4 °C. 1H NMR (400 MHz, CDCl3): δ 8.09
(d, J ) 7.8 Hz, 1H), 7.65 (d, J ) 7.7 Hz, 1H), 7.22 (app t, J )
8.0 Hz, 1H), 6.86 (d, J ) 8.0 Hz, 1H), 6.40 (d, J ) 7.7 Hz, 1H),
5.29 (s, 1H), 4.62 (t, J ) 6.9 Hz, 2H), 3.99 (s, 3H), 3.72-3.70
(m, 4H), 2.78 (t, J ) 7.4 Hz, 2H), 2.57-2.54 (m, 4H), 2.06-
1.96 (m, 3H), 1.78-1.60 (m, 1H), 1.39-1.34 (m, 3H), 1.34
(overlapping s, 3H), 1.09 (s, 3H), 0.95 (s, 3H). HPLC: tR ) 2.80
min, 99.5% purity. LC-MS: tR ) 1.77 min, [M + H]+ ) 464.5.
HRMS (EI) m/z calcd for C28H38N3O3 [M + H]+: 464.2913.
Found: 464.2914. Anal. (C28H37N3O3) C, H, N.
with a methyl group on N1 instead of the morpholinoethyl
group to increase the efficiency of calculations. Because the
morpholinoethyl group can adopt similar conformations for all
the analogues and does not affect the conformation about the
C3-carboxamide bond, it was felt that this was a reasonable
approximation. The conformation of the C2 substituent was
that conformation found to give the lowest energy in the Monte
Carlo simulations.
CB2 a n d CB1 Assa y P r otocol. The following assay has
been carried out using the human cannabinoid receptor
expressed in Chinese hamster ovary (CHO) cells. Radioactive
tracer label (WIN 55,212-2 mesylate [5,7-3H] for CB2, CP55,-
940 for CB1) and test compounds are incubated together
in a 96-well tissue culture plate. All reagents are dissolved
or resuspended in binding buffer (10 mM HEPES, pH 7.4,
1 mM EDTA, 5 mM MgCl2, 0.3% BSA). The reaction is
initiated by the addition of membranes (50 mg) from CHO-
K1 cells expressing either CB1 or CB2. The plates are
incubated 2 h with shaking at room temperature, and the
reaction mixture is harvested on a Wallac Filtermat B with
seven wash cycles using wash buffer (10 mM HEPES, pH 7.4,
0.1% BSA). The filter is counted in a Betaplate scintillation
counter to ascertain the cannabinoid inhibitory activity of the
test compound (activity inversely proportional to the amount
of labeled WIN55,212-2 incorporated). Routinely the radiolabel
was used at a concentration of 10 nM, but the exact concentra-
tion of reagents and the amount of label can be varied as
needed.
2,5-Dih yd r o-6-m et h oxy-2-(2-m et h oxyp h en yl)-5-[2-(4-
m or p h olin yl)eth yl]-1H-p yr id o[4,3-b]in d ol-1-on e (3b). 3b
was prepared as described for 3a to obtain 38 mg of 3b as a
yellow solid. 1H NMR (400 MHz, CDCl3): δ 8.64 (d, J ) 5.0
Hz, 1H), 8.05 (d, J ) 8.0 Hz, 1H), 7.80 (s, 1H), 7.69 (t, J ) 7.7
Hz, 1H), 7.54 (d, J ) 7.6 Hz, 1H), 7.42 (d, J ) 7.9 Hz, 1H),
7.36-7.30 (m, 3H), 7.28-7.18 (m, 4H), 6.83 (d, J ) 7.9 Hz,
1H), 6.43 (d, J ) 7.6 Hz, 1H), 4.59 (app t, J ) 6.8 Hz, 2H),
3.96 (s, 3H), 3.70-3.67 (m, 4H), 2.73 (app t, J ) 6.8 Hz, 2H),
2.53-2.50 (m, 4H). HPLC: tR ) 1.91 min, 98.6% purity. LC-
MS: tR ) 1.93 min, [M + H]+ ) 495.1. HRMS (EI) m/z calcd
for C30H31N4O3 [M + H]+: 495.2396. Found: 495.2381.
2,5-Dih yd r o-6-m et h oxy-5-[2-(4-m or p h olin yl)et h yl]-2-
[p h e n yl(2-p yr id in yl)m e t h yl]-1H -p yr id o[4,3-b]in d ol-1-
on e (3c). 3c was prepared as described for 3a to obtain 68
mg (55%) of 3c as a light-orange solid. 1H NMR (400 MHz,
CDCl3): δ 8.08 (d, J ) 7.9 Hz, 1H), 7.43 (d, J ) 7.5 Hz, 1H),
7.31 (d, J ) 7.9 Hz, 1H), 7.26-7.19 (m, 4H), 6.92-6.88 (m,
2H), 6.84 (d, J ) 7.9 Hz, 1H), 6.42 (d, J ) 7.5 Hz, 1H), 4.60
(app t, J ) 7.0 Hz, 2H), 3.97 (s, 3H), 3.88 (s, 3H), 3.69 (app t,
J ) 4.4 Hz, 4H), 2.74 (t, J ) 7.9 Hz, 2 H), 2.53 (t, J ) 4.4 Hz,
4H). HPLC: tR ) 2.91 min, 98.1% purity. LC-MS: tR ) 1.84
min, [M + H]+ ) 448.2. HRMS (EI) m/z calcd for C26H30N3O4
[M + H]+: 448.2236. Found: 448.2236.
P BMC Assa y P r otocol. Human PBMCs were isolated from
whole blood collected from healthy donors. Blood was diluted
into RPMI 1640 (Life Technologies) containing 2.5 mM EDTA
(Life Technologies) and 10 µg/mL polymyxin (Sigma), and then
it was underlaid with ficoll (Accurate Scientific Co.) and
centrifuged at 600g for 25 min. The interface is collected, and
cells are washed twice and resuspended in RPMI, 10% FBS.
Cells are then distributed (200 µL/well) into 96-well tissue
culture treated plates (Falcon) at 1 × 106 cells/mL in RPMI,
10% FBS. Test compounds were added to appropriate wells
and incubated with cells for 30 min. Cells were then stimulated
by the addition of lipopolysaccharide (LPS, BioWhittaker), with
a final concentration of 25 ng/mL, and incubated for 6 h at 37
°C. The cell supernatants were removed and assayed for
TNF-R by ELISA (R&D Systems).
In h ibition of TNF -r Relea se in Mice. BALB/c female
mice, 6-8 weeks of age, were obtained from Harlan labora-
tories and maintained ad libitum on water and standard
rodent chow (Harlan Teklad). Mice were acclimated to ambient
conditions for at least 1 week prior to use. For intravenous
(iv) dosing, the compounds were prepared in a solution of 10%
propylene glycol in phosphate-buffered saline. Lipopolysac-
charide (LPS, E. coli O111:B4; Sigma) was added to the dosing
solution at a final concentration of 10 µg/mL. The mixture was
injected in a total volume of 0.1 mL per mouse. Blood samples
were obtained 60 min after the compound + LPS injection.
For oral dosing, the compounds were prepared in a solution
of 50% propylene glycol in water and a dosing volume of 0.2
mL per mouse was administered by gavage 10 min prior to
LPS injection (0.1 mL of LPS suspended at 10 µg/mL in PBS,
administered iv). Blood samples were obtained 60 min after
LPS injection. Serum was separated from clotted blood samples
by centrifugation (5 min, 5000g, room temperature) and
analyzed for levels of TNF-R by ELISA assay (R&D Systems)
according to the manufacturer’s directions. Results are shown
as the mean ( SD of n ) 8 mice per treatment group. All
procedures involving animals were reviewed and approved by
the Institutional Animal Care and Use Committee.
Molecu la r Mod elin g. The modeling was performed on an
SGI Octane workstation. The starting coordinates of the
structures were generated using CONCORD. The conforma-
tional profiles of the compounds were examined by a 5000-
step Monte Carlo (MC) conformational search using Batchmin
as implemented in Macromodel18 (version 7.0) using the Merck
molecular mechanics force field19 (MMFF) and the GB/SA
water continuum model. The trial conformation from each MC
step was subjected to conjugate gradient minimization using
the Polak-Ribiere first derivative method until a derivative
convergence criterion of 0.01 kcal mol-1 Å-1 was reached or
until a maximum of 400 iterations had been performed. The
resulting unique conformations within a 20 kcal/mol energy
window (a conformation was deemed unique if it met a heavy
atom root-mean-square criterion of >0.25 Å compared to an
existing conformer) were further subjected to 500 steps of
conjugate gradient minimization. The pool of conformations
of each compound, within an energy window of 10 kcal/mol,
was reduced by means of cluster analysis (Xcluster20 option
implemented in Macromodel). The unique conformations were
subjected to 30 steps of DFT energy optimizations as imple-
mented in J aguar21 (version 4.0) at B3LYP/G-31G**, and a
single-point energy was calculated for the resulting geometries
using B3LYP/cc-PVTZ(-f)+. The grid torsional energy scans
about the C3-carboxamide bond were performed from 0° to
330° in 30° increments using the same energy optimization
protocol as for the unique set of Monte Carlo conformations.
The scans were performed on the core part of the molecules
Ack n ow led gm en t. We gratefully acknowledge Dr.
Mark Witmer for his assistance in providing experi-
mental data for this manuscript and Dr. Kyoung S. Kim
for his helpful discussions.

2116 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11
Wrobleski et al.
(10) (a) Martin, B. R.; Compton, D. R.; Thomas, B. F.; Prescott, W.
R. Little, P. J .; Razdan, R. K.; J ohnson, M. R.; Melvin, L. S.;
Mechoulam, R.; Ward, S. J . Behavioral, Biochemical, and
Molecular Modeling Evaluations of Cannabinoid Analogs. Phar-
macol. Biochem. Behav. 1991, 40, 471-478. (b) Compton, D. R.;
Gold, L. H.; Ward, S. J .; Balster, R. L.; Martin, B. R. Aminoalky-
lindole Analogs: Cannabimimetic Activity of a Class of Com-
pounds Structurally Distinct from ∆9-Tetrahydrocannabinol. J .
Pharmacol. Exp. Ther. 1992, 263, 1118-1126.
(11) Hynes, J ., J r.; Leftheris, K.; Wu, H.; Pandit, C.; Chen, P.; Norris,
D. J .; Chen, B.-C.; Zhao, R.; Kiener, P. A.; Chen, X.; Turk, L. A.;
Patil-Koota, V.; Gillooly, K. M.; Shuster, D. J .; McIntyre, K. W.
C-3 Amido-Indole Cannabinoid Receptor Modulators. Bioorg.
Med. Chem. Lett. 2002, 12, 2399-2402.
(12) Eissenstat, M. A.; Bell, M. R.; D’Ambra, T. E.; Alexander, E. J .;
Daum, S. J .; Ackerman, J . A.; Gruett, M. D.; Kumar, V.; Estep,
K. G.; Olefirowicz, E. M.; Wetzel, J . R.; Alexander, M. D.;
Weaver, J . D., III; Haycock, D. A.; Luttinger, D. A.; Casiano, F.
M.; Chippari, S. M.; Kuster, J . E.; Stevenson, J . I.; Ward, S. E.
Aminoalkylindoles: Structure-Activity Relationships of Novel
Cannabinoid Mimetics. J . Med. Chem. 1995, 38, 3094-3105.
(13) A similar syn conformation of structurally related WIN-55212-2
cannabinoid ligand has also been implicated as the bioactive
conformation. Reggio, P. H.; Basu-Dutt, S.; Barnett-Norris, J .;
Castro, M. T.; Hurst, D. P.; Seltzman, H. H.; Roche, M. J .;
Gilliam, A. F.; Thomas, B. F.; Stevenson, L. A.; Pertwee, R. G.;
Abood, M. E. The Bioactive Conformation of Aminoalkylindoles
at the Cannabinoid CB1 and CB2 Receptors: Insights Gained
from (E)- and (Z)-Naphthylidene Indenes. J . Med. Chem. 1998,
41, 5177-5187.
(14) Clark, R. D.; Muchowski, J . M.; Fisher, L. E.; Flippin, L. A.;
Repke, D. B.; Souchet, M. Preparation of Indoles and Oxindoles
from N-(tert-Butoxycarbonyl)-2-alkylanilines. Synthesis 1991, 10,
871-878.
(15) Yoshida, T.; Matsuura, N.; Yamamoto, K.; Doi, M.; Shimada,
K.; Morie, T.; Kato, S. Practical Synthesis of 1H-Indazole-3-
carboxylic Acid and Its Derivatives. Heterocycles 1996, 43, 2710-
2712.
(16) Experimental conditions were modified from the following. Clark,
R. D.; Miller, A. B.; Berger, J .; Repke, D. B.; Weinhardt, K. K.;
Kowalczyk, B. A.; Eglen, R. M.; Bonhaus, D. W.; Lee, C.-H.;
Michel, A. D.; Smith, W. L.; Wong, E. H. F. 2-(Quinuclidin-3-
yl)pyrido[4,3-b]indol-1-ones and Isoquinolin-1-ones. Potent Con-
formationally Restricted 5-HT3 Receptor Antagonists. J . Med.
Chem. 1993, 36, 2645-2657.
(17) Suchocki, J . A.; Everette, L. M.; Martin, T. J .; George, C.; Martin,
B. R. Synthesis of 2-exo- and 2-endo-Mecamylamine Analogues.
Structure-Activity Relationships for Nicotinic Antagonism in
the Central Nervous System. J . Med. Chem. 1991, 34, 1003-
1010.
Refer en ces
(1) (a) Porter, A. C.; Felder, C. C. The Endocannabinoid Nervous
System: Unique Opportunities for Therapeutic Intervention.
Pharmacol. Ther. 2001, 90, 45-60. (b) Williamson, E. M.; Evans,
F. J . Cannabinoids in Clinical Practice. Drugs 2000, 60 (6),
1303-1314. (c) Hollister, L. E. Health Aspects of Cannabis.
Pharmacol. Rev. 1986, 38, 1-20.
(2) Matsuda, L. A.; Lolait, S. J .; Brownstein, M. J .; Young, A. C.;
Bonner, T. I. Structure of a Cannabinoid Receptor and Func-
tional Expression of the Cloned cDNA. Nature 1990, 346, 561-
564.
(3) Munro, S.; Thomas, K. L.; Abu-Shaar, M. Molecular Character-
ization of a Peripheral Receptor for Cannabinoids. Nature 1993,
365, 61-65.
(4) For a comprehensive review, see the following. Pertwee, R. G.
Pharmacology of Cannabinoid CB1 and CB2 Receptors. Phar-
macol. Ther. 1997, 74, 129-180.
(5) Wagner, J . A.; Varga, K.; Ellis, E. F.; Rzigalinski, B. A.; Martin,
B. R.; Kunos, G. Activation of Peripheral Cannabinoid Receptors
in Haemorrhagic Shock. Nature 1997, 390, 518-521.
(6) (a) Germain, N.; Boichot, E.; Advenier, C.; Berdyshev, E. V.;
Lagente, V. Effect of the Cannabinoid Receptor Ligand, WIN
55,212-2 on the Superoxide Anion and TNF-R Production by
Human Mononuclear Cells. Int. Immunopharmacol. 2002, 2,
537-543. (b) Smith, S. R.; Terminelli, C.; Denhardt, G. Modula-
tion of Cytokine Responses in Corynebacterium Parvum-Primed
Endotoxemic Mice by Centrally Administered Cannabinoid
Ligands. Eur. J . Pharmacol. 2001, 425, 73-83. (c) Smith, S. R.;
Terminelli, C.; Denhardt, G. Effects of Cannabinoid Receptor
Agonist and Antagonist Ligands on Production of Inflammatory
Cytokines and Anti-inflammatory Interleukin-10 in Endotoxemic
Mice. J . Pharmacol. Exp. Ther. 2000, 293, 136-150. (d) J agger,
S. I.; Sellaturay, S.; Rice, A. S. The Endogenous Cannabinoid
Anandamide, but Not the CB2 Ligand Palmitoylethanolamide,
Prevents the Viscero-Visceral Hyper-Reflexia Associated with
Inflammation of the Rat Urinary Bladder. Neurosci. Lett. 1998,
253, 123-126. (e) Richardson, J . D.; Kilo, S.; Hargreaves, K. M.
Cannabinoids Reduce Hyperalgesia and Inflammation via In-
teraction with Peripheral CB1 Receptors. Pain 1998, 75, 111-
119. (f) Berdyshev, E.; Boichet, E.; Corbel, M.; Germain, N.;
Lagente, V. Effects of Cannabinoid Receptor Ligands on LPS-
Induced Pulmonary Inflammation in Mice. Life Sci. 1998, 63,
125-129.
(7) For recent reviews, see the following. (a) Di Marzo, V.; De
Petrocellis, L.; Bisogno, T. Endocannabinoids Part I: Molecular
Basis of Endocannabinoid Formation, Action and Inactivation
and Development of Selective Inhibitors. Emerging Ther. Targets
2001, 5 (2), 241-265. (b) Pertwee, R. G. Cannabinoid Receptor
Ligands: Clinical and Neuropharmacological Considerations,
Relevant to Future Drug Discovery and Development. Exp. Opin.
Invest. Drugs 2000, 9 (7), 1553-1571.
(8) D’Ambra, T. E.; Estep, K. G.; Bell, M. R.; Eissenstat, M. A.; J osef,
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. Conformationally Restrained Analogs of Pravado-
line: Nanomolar Potent, Enantioselective, (Aminoalkyl)indole
Agonists of the Cannabinoid Receptor. J . Med. Chem. 1992, 35,
124-135.
(18) Mohamadi, F.; Richards, N. G. J .; Guida, W. C.; Liskamp, R.;
Lipton, M.; Caufield, C.; Chang, G.; Hendrickson, T.; Still, W.
C. MacroModelsAn Integrated Software System for Modeling
Organic and Bioorganic Molecules Using Molecular Mechanics.
J . Comput. Chem. 1990, 11, 440.
(19) Halgren, T. A. Merck Molecular Force Field. I. Basis, Form,
Scope, Parameterization, and Performance of MMFF94. J .
Comput. Chem. 1996, 17, 490.
(9) Showalter, V. M.; Compton, D. R.; Martin, B. R.; Abood, M. E.
Evaluation of Binding in a Transfected Cell Line Expressing a
Peripheral Cannabinoid Receptor (CB2): Identification of Can-
nabinoid Receptor Subtype Selective Ligands. J . Pharmacol.
Exp. Ther. 1996, 278, 989-999.
(20) Shenkin, P. S.; McDonald, D. Q. Cluster Analysis of Molecular
Conformations. J . Comput. Chem. 1994, 15, 899.
(21) J aguar, version 4.0; Schrodinger, Inc.: Portland, OR, 1991-2000.
J M020329Q