Identification and hit-to-lead optimization of a novel class of CB1 antagonists

Article abstract of DOI:10.1016/j.bmcl.2010.07.091

The discovery, synthesis and preliminary structure-activity relationships (SARs) of a novel class of CB1 antagonists is described. Initial optimization of benzimidazole-based screening hit 4 led to the identification of 'inverted' indole-based lead compound 18c with improved properties versus compound 4 including reduced A log P, improved microsomal stability and improved aqueous solubility. Compound 18c demonstrates in vivo CB1 antagonist efficacy (CB1 agonist induced hypothermia model) and is orally bioavailable in rat.

Full text of DOI:10.1016/j.bmcl.2010.07.091

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5449–5453
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification and hit-to-lead optimization of a novel class of CB1 antagonists
a
a
a
a
Jeffrey J. Letourneau a, , Patrick Jokiel , John Olson , Christopher M. Riviello , Koc-Kan Ho ,
Lihong McAleer a, Jingchun Yang a, Robert N. Swanson a, James Baker b, Phillip Cowley b, Darren Edwards b,
Nick Ward b, Michael H. J. Ohlmeyer a, Maria L. Webb a
*
a Ligand Pharmaceuticals, Inc., 3000 Eastpark Boulevard, Cranbury, NJ 08512, USA
b MSD, Newhouse, Lanarkshire ML1 5SH, Scotland, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The discovery, synthesis and preliminary structure–activity relationships (SARs) of a novel class of CB1
antagonists is described. Initial optimization of benzimidazole-based screening hit 4 led to the identifi-
cation of ‘inverted’ indole-based lead compound 18c with improved properties versus compound 4
including reduced A log P, improved microsomal stability and improved aqueous solubility. Compound
18c demonstrates in vivo CB1 antagonist efficacy (CB1 agonist induced hypothermia model) and is orally
bioavailable in rat.
Received 25 June 2010
Revised 20 July 2010
Accepted 21 July 2010
Available online 25 July 2010
Keywords:
Cannabinoid
CB1
Ó 2010 Elsevier Ltd. All rights reserved.
Antagonist
GPCR
Obesity
Benzimidazole
Indole
Inverted indole
A large number of CB1 antagonists and inverse agonists have
been reported in the literature as potential therapeutic agents for
the treatment of obesity.1 In fact, the archetypical CB1 inverse ago-
nist Rimonabant was approved in Europe for this indication,
although its marketing was recently discontinued by the European
Medicines Agency (EMEA) due to an increased risk of psychiatric
disorders.2 Many CB1 antagonists reported to date are similar in
structure to Rimonabant, sharing the common structural motif of
two adjacent phenyl groups (usually chloro-substituted) on a five
or six-membered heterocyclic scaffold. Most of these CB1 antago-
nists are highly lipophilic, with calculated log P’s >5 (i.e., Rimona-
bant A log P = 7.02).3
a novel series of CB1 antagonists which are structurally distinct
from most known ligands.
In a program aimed at the identification of a novel and structur-
ally distinct class of CB1 antagonists, high-throughput screening
(HTS) of a diverse set of combinatorial screening libraries, prepared
using ECLiPS™ technology,5 was undertaken. Multiple active struc-
tures, or hits, were identified in this HTS campaign. Among these
was compound 4 (Fig. 1) which was chosen as a starting point
for a HtL program, with the task of evaluating this hit class in terms
A-ring
Recent reports of efforts to reduce the log P –aimed at the
improvement of pharmaceutical properties of CB1 antagonists–
have appeared in the literature.4 Some of these efforts have met
with limited success in that improvements in lowering log P have
resulted in lower in vitro4a,4b and/or in vivo4a potency versus the
initial, highly lipophilic lead molecule. Herein is described a similar
effort to improve physicochemical properties (including reduction
of log P) during the course of hit-to-lead (HtL) optimization around
O
O
1
6
N
N
N
H
2
5
F
Cl
B-ring
O
4
N
N
N
N
H
H
Cl
X
N
N
N
Ar
Cl
O
Rimonabant
X = CH; 8a,b
X = N; 8c,d
* Corresponding author. Tel.: +1 609 452 3748; fax +1 609 452 3671.
E-mail address: jletourneau@ligand.com (J.J. Letourneau).
In 2008, Pharmacopeia, Inc. was acquired by Ligand Pharmaceuticals, Inc.
Figure 1. Structures of Rimonabant, and benzimidazole-based CB1 antagonists 4
and 8a–d.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.091

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J. J. Letourneau et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5449–5453
of both target independent (e.g., physicochemical properties) and
target dependent (e.g., potency) properties.6 The main objective
of this HtL program was to deliver a reasonably potent lead com-
pound (IC50 <100 nM) with appropriate drug-like physicochemical
properties7 to serve as the basis for further lead optimization ef-
forts. Importantly, we sought to minimize log P (A log P <5) and
The compounds in Tables 1 and 2 were evaluated for CB1 antag-
onistic activity in a luciferase-based CB1 reporter assay.8,9 In addi-
tion, metabolic stability was determined in both human liver
microsome (HLM) and mouse liver microsome (MLM) assays.10
The optimization effort around benzimidazole 4 initially entailed
exploration of SAR around both the A and B-rings of the biaryl moi-
ety and around the C(6) amide functionality. This effort gave rise to
a series of retroamides exemplified by structures 8a,b (A-ring =
phenyl) and 8c,d (A-ring = 2,6-disubstituted pyridyl) (see Table 1).
Interestingly, compound 8c was not only 5-fold more potent
than compound 4, but it also had modestly improved microsomal
stability. The salient SAR of compounds 8a–d and related analogs
regarding potency are the following: (1) meta-substitution of the
A-ring in the biaryl moiety is highly preferred; (2) substitution at
the 2-position of the benzimidazole scaffold is not tolerated; (3)
amido substitution at C(6) versus C(5) is preferred with sterically
bulky amides (e.g., pivalylamide) being optimal; (4) a secondary
amide is preferred over tertiary amide (i.e., N-H>N-Me); and, (5)
ortho and/or meta-substitution of the B-ring is preferred versus
para-substitution, with alkoxy substituents preferred versus alkyl,
halo, etc.
achieve modest aqueous solubility (>10 lg/mL).
The compounds appearing in Tables 1 and 2 were synthesized
via the general schemes depicted in Schemes 1–3. As illustrated
in Scheme 1, benzimidazole 4 was prepared in four steps from 3-
fluoro-4-nitrobenzoic acid. Benzimidazoles 8a–d, which contain a
retroamide at C(6), were also prepared from the same starting
material with a key step being a Curtius rearrangement of com-
pound 1 to give intermediate 5. Boc-deprotection followed by acyl-
ation gave intermediate 6. Finally, an SNAr2 reaction of
intermediate 6 with either 3-bromobenzylamine or (6-bromopyri-
din-2-yl)methanamine, to give intermediates 7a or 7b, followed by
Suzuki coupling with the appropriate arylboronic acids gave com-
pounds 8a–d. Indoles 12a,b and 13a–d were prepared in four steps
from commercially available 6-nitroindole 9 (Scheme 2). Lastly,
‘inverted’ indoles 17a,b and 18a–d were prepared from 5-nitroin-
dole 14 with the key steps being reaction of 14 with either 3-bro-
mobenzyaldehyde or 2-bromo-5-formylpyridine followed by
reduction with triethylsilane in the presence of trifluoroacetic acid
to give intermediates 15a,b (Scheme 3).
One concern which arose about these, and related, benzimid-
azole-based structures was the lack of microsomal stability.
Although compounds 8a and 8c represented a modest improve-
ment over compound 4, we sought further improvement in this re-
Table 1
In vitro potency, A log P values, and microsomal stability of benzimidazoles 4 and 8a–d, indoles 12a,b and ‘inverted’ indoles 17a,b
H
N
H
N
X
N
N
N
Ar
Ar
O
O
8a-d
12a,b
Ar
H
N
O
N
17a,b
Compds
X
Ar
IC50
(
l
M)a
A log P
HLM/MLM (% rem. @ 0.5 h)
2/0
4
0.104 ( .007)
5.63
O
O
O
*
*
8a
8b
CH
CH
N
0.064 ( .003)
5.09
5.51
4.58
5.00
5.31
5.73
5.70
6.12
35/2
0.269 ( .122)
0.021 ( .006)
0.073 ( .001)
0.051 ( .018)
0.058 ( .023)
0.037 ( .002)
0.045 ( .001)
ND
F
O
O
O
*
*
8c
28/17
14/1
8d
N
F
O
O
O
*
*
12a
12b
17a
17b
72/65
51/58
52/29
70/72
F
O
O
O
*
*
F
a
Values are means of at least two experiments, standard deviation is given in parentheses.

J. J. Letourneau et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5449–5453
5451
Table 2
In vitro potency, A log P values, microsomal stability and aqueous solubility of indoles 13a–d and ‘inverted’ indoles 18a–d
H
N
H
N
N
N
R
R
N
Ar
Ar
O
O
N
13a-d
18a-d
Compds
R
Ar
IC50
(
l
M)a
A log P
HLM/MLM (% rem. @ 0.5 h)
62/52
aq sol (
3.2
lg/mL)
O
HO
*
*
O
*
13a
0.007 ( .002)
0.045 ( .014)
0.003 ( .001)
0.023 ( .010)
0.007 ( .001)
0.027 ( .010)
0.005 ( .001)
0.019 ( .006)
3.71
O
HO
*
13b
13c
13d
18a
18b
18c
18d
4.13
3.42
3.84
3.90
4.32
3.60
4.02
45/54
51/53
35/44
56/16
47/5
11.7
<1
F
HO
HO
O
O
*
*
O
*
*
17.9
<1
F
O
O
HO
HO
*
*
O
*
*
2.0
F
HO
HO
O
O
*
*
O
*
*
56/36
41/14
14.9
10.6
F
a
Values are means of at least two experiments, standard deviation is given in parentheses.
O
O
O
O
Br
a
b,c
O
d,e
F
F
NH
N
H
HO
N
N
N
H
N
H
NO2
NO2
NH2
F
3
1
2
4
F
F
f
H
H
N
O
H
N
g,h
F
b,c,d
H
e
F
X
N
X
N
N
Br
N
N
N
O
Ar
O
NO2
O
NO2
O
5
6
7a; X = CH
8a-d
7b; X = N
Scheme 1. Reagents and conditions: (a) i-butylchloroformate, NMM, THF, 0 °C, 90 s; 3-fluorobenzylamine; (b) 3-bromobenzylamine or (6-bromopyridin-2-yl)methanamine,
DIPEA, ACN, 40 °C; (c) Fe, EtOH/H2O/AcOH, reflux; (d) triethylorthoformate, TsOHÁH2O (cat.); (e) Ar-B(OH)2, PS-PPh3-Pd, 1 N K2CO3 (aq), EtOH, microwave @ 120 °C, 5 min; (f)
DPPA, TEA, t-BuOH, dioxane, reflux; (g) 50% TFA/DCM; (h) trimethylacetylchloride, TEA, DCM.
gard. Part of our strategy for further improvements in properties,
including microsomal stability, was to employ the concept of ‘scaf-
fold-hopping’. It was discovered that replacement of the benzimid-
azole scaffold with either indole (structures 12a,b) or ‘inverted’
indole (structures 17a,b) led to improvement in microsomal stabil-
ity, while maintaining good potency.
into the C(6) amido substituent of both indoles and ‘inverted’ in-
doles was examined (Table 2). It had been demonstrated in our
earlier HtL efforts that the steric bulk of the pivalylamide substitu-
ent at C(6) was important for potency. Therefore, we chose to
incorporate either 3-hydroxy-3-methylbutanamide or 3-hydroxy-
2,2-dimethyl-propionamide substituents at C(6) because of their
similar steric demand relative to the pivalylamide substituent con-
tained in potent analogs 12a,b and 17a,b. To minimize c log P, a
2,6-disubstituted pyridyl ring, which was demonstrated to be an
adequate surrogate for phenyl in the benzimidazole series (e.g.,
compare 8a to 8c in Table 1), was incorporated at the A-ring posi-
tion. Both 4-benzo[1,3]dioxolyl and 2-methoxy-4-fluoro phenyl
While these indoles and ‘inverted’ indoles demonstrated ade-
quate potency and microsomal stability they lacked aqueous solu-
bility (i.e., measured aqueous solubility was <1 l
g/mL)11 and were
more lipophilic than desired (i.e., A log P’s >5). In an attempt to
lower c log P and improve aqueous solubility, while maintaining
adequate potency, the introduction of polar hydroxyl functionality

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J. J. Letourneau et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5449–5453
Table 3
H
N
b,c
a
O2N
Effect of compound 18c and Rimonabant on WIN 55,212-2 induced hypothermia in
mice
X
O2N
N
Br
Dose (lmol/kg, sc)
Percent ( sem) reversal of WIN 55,212-2
9
10a; X = CH
10b; X = N
(10
l
mol/kg, sc) induced hypothermia
18c
Rimonabant
H
N
d
0.01
0.1
0.3
1
—
—
—
—
—
—
À0.2 4.6
13.8 5.7
14.7 14.9
60.2 15.3
116.7 3.8
113.4 4.8
N
Ar
O
X
H2N
N
Ar
12a,b
3
e
11a; X = CH
11b; X = N
10
30
100
H
N
46.8 10.7
92.5 2.4
100.4 2.2
—
N
R
N
Ar
O
ED50 (95% Cl)
ꢀ30
0.8 (0.5–1.5)
13a-d
Scheme 2. Reagents and conditions: (a) 1-bromo-3-(bromomethyl)benzene or
2-bromo-6-(bromomethyl)pyridine, K2CO3, acetone, reflux; (b) Ar-B(OH)2, 1 N
Na2CO3 (aq), Pd(PPh3)4, dioxane, reflux; (c) raney Ni, H2 (1 atm), THF; (d) trimethy-
lacetylchloride, TEA, DCM; (e) 3-hydroxy-3-methylbutyric acid or 3-hydroxy-2,2-
dimethylpropanoic acid, DIPEA, HATU, DCM.
Table 4
Pharmacokinetic parameters for 18c in male Wistar rats at 5 mpk po and 1 mpk iva
AUCpo
1292 ng h/mL
0–7.5 h
Fpo (%)
CLiv (mL/min/kg)
37
19
Vss (L/kg)
1.2
1.0
c, d, e
O2N
a,b
T1/2, (h)
iv
X
O2N
Br
N
H
a
Vehicle for po dosing is 0.5% gelatin in 5% mannitol (aq) and for iv dosing is 20%
N,N-dimethylacetamide in saline.
N
H
14
15a; X = CH
15b; X = N
Compound 18c had reasonable oral bioavailability (F = 37%),
moderate clearance (CL = 19 mL/min/kg) and volume of distribu-
tion (Vss = 1.2 L/kg) and a half-life of 1 h.
H
N
f
Ar
O
N
X
In conclusion, we report a novel series of CB1 antagonists that
H2N
Ar
17a,b
were developed from
a benzimidazole-based screening hit.
N
Replacement of the benzimidazole scaffold with isosteric indole
and ‘inverted’ indole, and incorporation of a hydroxyl moiety into
the amide side-chain, led to structures with improved properties
including lower log P, enhanced aqueous solubility, and better sta-
bility to both human and mouse liver microsomes. In addition,
compound 18c demonstrated in vivo efficacy in a CB1 agonist in-
duced hypothermia model and was orally bioavailable in rat.
g
H
N
16a; X = CH
16b; X = N
N
R
Ar
O
N
18a-d
Scheme 3. Reagents and conditions: (a) 3-bromobenzyaldehyde or 2-bromo-5-
formylpyridine, NaOMe, MeOH; (b) TFA, Et3SiH, DCM; (c) NaH, THF; add CH3I; (d)
Ar-B(OH)2, 1 N Na2CO3 (aq), Pd(PPh3)4, dioxane, reflux; (e) raney Ni, H2 (1 atm),
THF; (f) trimethylacetylchloride, TEA, DCM; (g) 3-hydroxy-3-methylbutyric acid or
3-hydroxy-2,2-dimethylpropanoic acid, DIPEA, HATU, DCM.
Acknowledgements
We thank members of the ADME group at Pharmacopeia for met-
abolic stability testing and colleagues in the Chemistry, Molecular
Pharmacology and Pharmacology Departments at MSD Newhouse
for purity and structure determination, solubility measurements,
pharmacokinetic, bioanalytical and pharmacological testing.
were incorporated at the B-ring position as these substituents were
earlier demonstrated to be optimal for potency.
Four out of eight of the analogs in Table 2 met our requirement
for aqueous solubility (>10 lg/mL). Of these, compound 18c had
the best potency in the luciferase assay (5 nM), and it also had ade-
quate HLM stability (i.e., >50% remaining @ 0.5 h). In addition, this
compound is substantially less lipophilic than highly lipophilic CB1
antagonists such as Rimonabant (i.e., Rimonabant A log P = 7.02
versus compound 18c A log P = 3.60).3
References and notes
1. For recent reviews see: (a) Jagerovic, N.; Fernandez-Fernandez, C.; Goya, P. Curr.
Top. Med. Chem. 2008, 8, 205; (b) Lange, J. H. M.; Kruse, C. G. Drug Discovery
Today 2005, 10, 693. and references therein.
2. http://www.emea.europa.eu/htms/human/withdraw/withdraw.htm
3. A log P values calculated using ADME Profiler: Program for discreet compound
analysis and calculation of physicochemical properties, version 1.6.0;
Pharmacopeia Inc., 2004.
Because it had the best balance of potency, microsomal stability
and aqueous solubility, compound 18c was further profiled in vivo
including an in vivo efficacy model (i.e., ability to reverse WIN
55,212-2 induced mouse hypothermia)12 and rat pharmacokinetics
(PK). Compound 18c demonstrated a partial reversal of hypother-
4. (a) Lange, J. H. M.; van Stuivenberg, H. H.; Veerman, W.; Wals, H. C.; Stork, B.;
Coolen, H. K. A. C.; McCreary, A. C.; Adolfs, T. J. P.; Kruse, C. G. Bioorg. Med. Chem.
Lett. 2005, 15, 4794; (b) Debenhaum, J. S.; Madsen-Duggan, C. B.; Walsh, T. F.;
Wang, J.; Tong, X.; Doss, G. A.; Lao, J.; Fong, T. M.; Schaeffer, M.-T.; Xiao, J. C.;
Huang, C. R.-R. C.; Shen, C.-P.; Feng, Y.; Marsh, D. J.; Stribling, D. S.; Shearman, L.
P.; Strack, A. M.; MacIntyre, D. E.; Van der Ploeg, L. H. T.; Goulet, M. T. Bioorg.
Med. Chem. Lett. 2006, 16, 681; (c) Ellsworth, B. A.; Wang, Y.; Zhu, Y.;
Annapurna, P.; Gerritz, S. W.; Sun, C.; Carlson, K. E.; Kang, L.; Baska, R. A.; Yang,
Y.; Huang, Q.; Burford, N. T.; Cullen, M. J.; Johnghar, S.; Behnia, K.;
Pelleymounter, M. A.; Washburn, W. N.; Ewing, W. R. Bioorg. Med. Chem. Lett.
2007, 17, 3978.
mia at a dose of 30
at a dose of 100 mol/kg (compound dosed sc) resulting in an ED50
of ꢀ30 mol/kg. In comparison, Rimonabant demonstrated an ED50
of 0.8
mol/kg in this model (Table 3).13 The relatively weak activ-
lmol/kg and nearly full reversal of hypothermia
l
l
l
ity of 18c in the mouse hypothermia model is perhaps due to lower
affinity of this compound at the mouse CB1 receptor versus the
human CB1 receptor.14 Rat PK data for compound 18c is summa-
rized in Table 4.
5. (a) Baldwin, J. J.; Burbaum, J. J.; Henderson, I.; Ohlmeyer, M. H. J. J. Am. Chem.
Soc. 1995, 117, 5588; (b) Ohlmeyer, M. H. J.; Swanson, R. N.; Dillard, L. W.;

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Reader, J. C.; Asouline, G.; Kobayashi, R.; Wigler, M.; Still, W. C. Proc. Natl Acad.
Sci. USA 1993, 90, 10922; (c) Nestler, H. P.; Bartlett, P. A.; Still, W. C. J. Org. Chem.
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1 mM NADPH in a final assay volume of 0.1 mL. Incubations were for 30 min at
37 °C and were terminated by the addition of 0.2 ml of acetonitrile. Samples
were centrifuged at 2000g and analyzed by LC/MS. The percentage of
compound remaining at 30 min was calculated.
6. (a) Davies, A. M.; Keeling, D. J.; Steele, J.; Tomkinson, N. P.; Tinker, A. C. Curr.
Top. Med. Chem. 2005, 5, 421; (b) Alanine, A.; Nettekoven, M.; Roberts, E.;
Thomas, A. W. Comb. Chem. High Throughput Screening 2003, 6, 51; (c) Michne,
W. F. Pharm. News 1996, 3, 19.
7. (a) Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Delivery
Rev. 1997, 23, 3; (b) Egan, W. J.; Merz, K. M.; Baldwin, J. J. J. Med. Chem. 2000, 43,
3867; (c) Veber, D. F.; Johnson, S. R.; Cheng, H. Y.; Smith, B. R.; Ward, K. W., et al
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11. Aqueous solubilities were determined using a medium-throughput adaptation
of a shake-flask methodology. A 10 mM solution of the test compound in
DMSO was added to 0.05 M phosphate buffered saline pH 7.4 such that the
final concentration of DMSO was 2%. The resultant mixture was then vortex
mixed (1500 rpm) for 24 0.5 h at 21 2 °C. After mixing, the resultant
solution/suspension was filtered under vacuum using a filter plate (Millipore
Multiscreen HTS, 0.4 lM). The concentration of the compound in the filtrate
8. CB1 reporter assay. Chinese hamster ovary cells expressing the human CB1
receptor and containing a luciferase gene under the regulatory control of seven
AP1 response element repeats were grown at 37 °C and 5% CO2 in DMEM/F12
(Cellgro) containing 10% FBS (Fetalclone II, Hyclone Cat. #SH30066.03), 0.2 mg/
was determined by High Performance Liquid Chromatography (HPLC) running
a generic acid gradient method with UV detection at 230 nm. Peak areas from
analysis of the diluted filtrates were quantified by comparison to a calibration
line prepared by injecting onto the HPLC three different volumes of a 50 lM
mL Geneticin, 0.5 mg/mL Hygromycin, 1
l
g/mL Fungizone, 5 mL Penicillin/
solution of the test compound in DMSO. Solubilities were determined in
duplicate for each test compound and average values reported.
12. Fox, A.; Kesingland, A.; Gentry, C.; McNair, K.; Patel, S.; Urban, L.; James, I. Pain
2001, 92, 91.
13. WIN 55,212-2 induced hypothermia test. The antagonist or vehicle (5%
mulgofen in saline, 10 ml/kg) was administered sc 75 min before rectal
Streptomycin (Invitrogen Cat. #15070-063) and 2 mM GlutaMax (GIBCO Cat.
#35050-061). The cells were seeded into white 96-well plates at a density of
3 Â 104 cells per well in 100
lL/well DMEM/F12 without Phenol Red containing
Penicillin/Streptomycin and Fungizone and incubated at 37 °C overnight. Test
compounds were serially diluted in DMEM/F12 containing 3% bovine serum
albumin, and 10
appropriate wells of the cell plate. After 5 min, 10
DMEM/F12 without BSA were added to all wells of the assay plate. After 5 h at
37 °C, detection was performed by adding 100 L/well Bright-Glo, incubating the
l
L
of each compound dilution was transferred to the
temperature was measured. WIN 55,212-2 mesylate (10 lmol/kg, 10 ml/kg)
l
L of 10À6 M CP-55940 in
was administered sc 60 min prior to the rectal temperature measurement. The
60 min pre-treatment with WIN 55,212-2 mesylate corresponded to the
maximal hypothermia attained by this agonist. Rectal temperature was
measured using a metal probe with a Fluke 51 K/J thermometer. The probe
was covered in a lubricant (vaseline) and was inserted approximately 1.5 cm
into the rectum.– The highest temperature stable for 10 s was recorded.
Following completion of the test animals were humanely terminated.
l
assay plate at RT for a further 5 min and counting for 0.5 s/well in a Wallac
Microbeta Trilux.
9. The CB1 receptor affinity (displacement of specific binding of [3H]CP-55,940 in
commercially available membranes prepared from Sf9 cells expressing the
human CB1 receptor) of certain compounds was assessed and found to be in
good agreement with the data obtained in the luciferase-based CB1 reporter
assay. For example, compounds 8b, 12b and 17b had IC50 values of 20 nM,
13 nM and 8 nM, respectively, in the CB1 binding assay.
14. Compound 18c and other compounds from this series were demonstrated to
have significantly lower affinity at the mouse CB1 receptor versus the human
CB1 receptor. For example, compound 18c was found to be ꢀ14-fold less
potent at the mouse CB1 receptor than at the human CB1 receptor as
10. Liver microsome stability assays were performed as follows: Assay mixtures
typically contained human or mouse microsomes (300 nM cytochrome P450,
determined in
(unpublished data).
a mouse brain membrane binding displacement assay
BD Gentest, Woburn, MA), phosphate buffer (pH 7.4), 1 lM test compound,