Discovery of pyrazine carboxamide CB1 antagonists: The introduction of a hydroxyl group improves the pharmaceutical properties and in vivo efficacy of the series

Article abstract of DOI:10.1016/j.bmcl.2007.04.087

Structure-activity relationships for a series of pyrazine carboxamide CB1 antagonists are reported. Pharmaceutical properties of the series are improved via inclusion of hydroxyl-containing sidechains. This structural modification sufficiently improved ADME properties of an orally inactive series such that food intake reduction was achieved in rat feeding models. Compound 35 elicits a 46% reduction in food intake in ad libidum fed rats 4-h post-dose.

Full text of DOI:10.1016/j.bmcl.2007.04.087

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3978–3982
Discovery of pyrazine carboxamide CB1 antagonists:
The introduction of a hydroxyl group improves the
pharmaceutical properties and in vivo efficacy of the series
Bruce A. Ellsworth,* Ying Wang, Yeheng Zhu, Annapurna Pendri, Samuel W. Gerritz,
Chongqing Sun, Kenneth E. Carlson, Liya Kang, Rose A. Baska, Yifan Yang,
Qi Huang, Neil T. Burford, Mary Jane Cullen, Susan Johnghar, Kamelia Behnia,
Mary Ann Pelleymounter, William N. Washburn and William R. Ewing
Pharmaceutical Research Institute, Bristol Myers Squibb Co., PO Box 5400 Princeton, NJ 08543, USA
Received 4 April 2007; revised 24 April 2007; accepted 25 April 2007
Available online 29 April 2007
Abstract—Structure–activity relationships for a series of pyrazine carboxamide CB1 antagonists are reported. Pharmaceutical prop-
erties of the series are improved via inclusion of hydroxyl-containing sidechains. This structural modification sufficiently improved
ADME properties of an orally inactive series such that food intake reduction was achieved in rat feeding models. Compound 35
elicits a 46% reduction in food intake in ad libidum fed rats 4-h post-dose.
Ó 2007 Elsevier Ltd. All rights reserved.
Overweight and obese individuals, as characterized by
BMI > 25 and 30 kg/M2, respectively, constitute over
60% of the US population, and this fraction of the pop-
ulation has significantly increased over the last
20 years.1,2 Obesity is caused by an imbalance between
nutrient intake and energy expenditure,3 is a significant
risk factor for cardiovascular disease and diabetes, and
is associated with significantly reduced longevity.4 Feed-
ing behavior is known to be modulated through the can-
nabinoid receptor 1 (CB1); increased feeding activity
results upon activation of the receptor by its endogenous
agonists, the endocannabinoids.5 CB1 knockout mice
are lean, hypophagic, and resistant to diet-induced obes-
ity, demonstrating the receptor’s role in energy
homeostasis.6
blocks signaling of the receptor.7 In libidum-fed rats,
SR141716 administered at 10 mg/kg i.p. produces a
50% reduction in food intake upon acute administration
versus saline-treated control animals.8 Upon chronic
administration, animals initially display a fairly dra-
matic reduction in food intake which, over several days,
returns to levels that are only slightly less than those of
vehicle controls, resulting in an overall body weight
reduction.9 In the RIO clinical trials in North America
and Europe, 20 mg q.d. of SR141716 (rimonabant)
was shown to reduce the body weight of obese subjects
by ꢀ4.7% over placebo during the first year of treat-
ment. This weight loss was maintained over the second
year of treatment.10
The structures of many small molecule CB1 antagonists
have been recently reviewed.1,11 In general, the proper-
ties of the lead compounds do not deviate from a recent
analysis of GPCR ligands. In this analysis of lipid-bind-
ing GPCRs, a class that includes CB1, the authors find
that drugs developed to bind these receptors have a
mean calculated logP (c logP) of 5.5.12 A few published
medicinal chemistry reports focus on efforts to reduce
the logP of CB1 antagonists to improve pharmaceutical
properties. For example, Lange et al. describe an effort
to reduce the logP of clinical candidate SLV-319
through modifications of peripheral groups including
Pharmacological intervention to inhibit the actions of
the endocannabinoid-cannabinoid receptor system leads
to a reduction in food intake in vivo. The most clinically
advanced CB1 antagonist, SR141716 (Fig. 1), binds to
the human CB1 receptor with nanomolar affinity and
Keywords: Cannabinoid; CB1; GPCR; Obesity; Pyrazine; ADME;
Hypophagia; Antagonist.
*
Corresponding author. Tel.:+1 609 818 4965; fax: +1 609 818 3550;
e-mail: Bruce.Ellsworth@bms.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.04.087

B. A. Ellsworth et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3978–3982
3979
O
N
N
O
N
N
S
N
CF3
N
N
NH2
O
1 (Ki = 970 nM)
2 (Ki = 650 nM)
Cl
Cl
N
O
HN
N
N
HN N
N
N
N
N
O2N
O
O
N
X
3 (Ki = 2200 nM
)
X = Cl, SR141716
X = I, AM251
4 (Ki = 52 nM)
Figure 1. A comparison of SR1417167 and AM25116 with BMS HTS screening hits. hCB1 binding Kis are reported in parentheses.17
the sulfonamido-sidechain.13 Most of the CB1 antago-
nists in this report have a lower logP, however they sac-
rifice some in vitro and in vivo potency relative to the
starting point. To address a lead compound’s poor PK
properties, Debenhem et al. sought to modify the central
core to effect reductions in logP.14 As a consequence,
the in vivo efficacy of the lead compound improved
due to increases in plasma and brain exposure as well
as oral bioavailability despite a fivefold reduction in
CB1 binding affinity. A similar effort to improve phar-
maceutical properties of a lead compound is described
herein.
binding results for a series of alkyl pyrazine carboxa-
mides are shown in Table 1. While short-chain alkyl
groups demonstrated sub-micromolar potency, the
inclusion of an aromatic group with a 3-carbon linker
(entries 13 and 15) gave rise to greater binding potency
versus recombinant CB1. An ether linkage in 14 is detri-
mental to CB1 binding versus compound 13, but a ter-
tiary amine is well tolerated in the linker (entry 16).
Compound 13, with a Ki value of 18 nM and full-func-
tional CB1 antagonism in a GTP-cS assay,20 was chosen
for evaluation in our in vivo rat food intake model.
Compound 13 did not induce any reduction in food in-
take when administered orally at 30 mg/kg in food-de-
prived rats.21 In an oral PK study, this compound
exhibited low plasma and brain exposure, and slow
absorption (Table 3) when administered at 30 mg/kg as
a suspension in 0.5% Methocel and 1% Tween 80 to
male Sprague–Dawley rats. The plasma and brain con-
centrations observed are likely too low to elicit a robust
effect on food intake via CB1 inhibition.
High-throughput screening of the Bristol Myers Squibb
chemical collection for binding to the CB1 receptor re-
sulted in many compounds of interest, including com-
pounds 1–3, that were suggestive of known ligands for
type-1 GPCRs. The diaryl-substituted cores of these
leads (Fig. 1, boxed moieties) are similar to the diaryl-
substituted pyrazole of the known CB1 inverse agonist,
SR141716, a moiety proposed to be important in aro-
matic stacking interactions that stabilize the inactive
form of the receptor.15 Structural overlays of these hits
with SR141716 suggested that the 6-membered core ring
of compounds 1 and 3 may be preferable to a linear 6,6-
fused structure of compound 2. The inclusion of a car-
In an attempt to simultaneously improve efficacy and
oral exposure, we sought to lower c logP guided by both
the ‘Rule of 5’22 concepts and prior CB1 literature re-
ports (vide supra). Incorporation of polar amines such
as 3-aminopyrrolidin-2-one and piperizin-2-one yielded
analogs 33 and 34 which exhibited significantly reduced
c logP and CB1 affinity (Table 2). The reported finding
from a SAR study of the SR141716 pyrazole chemotype
suggested that hydroxylation may offer a more conser-
`
boxamide may be important vis-a-vis published reports
of the importance of an H-bond acceptor with Lys192
for the inverse-agonist activity of SR141716.15
Combining design elements from compounds 1 to 3 with
those from known literature compounds rapidly led to
the synthesis of pyrazine carboxamide 418 (Fig. 1). The
SAR of the carboxamide sidechain was elucidated by
preparing a library of alkyl carboxamides derived from
carboxylic acid A which was readily obtained by con-
densation of 2,3-diaminopropionic acid and 4,40-dim-
ethylbenzil.19 Following treatment of carboxylic acid A
with oxalyl chloride, the resulting acid chloride B
(X=Cl) (Scheme 1) was isolated and distributed into
reactor tubes containing PS-DIEA in methylene chlo-
ride, followed by addition of the amines. Final com-
pounds were purified via preparative RP-HPLC. CB1
O
OH
X
H2N
H2N
N
a
N
N
b
OH
O
O
50%
50-90%
N
A
B
Scheme 1. Synthesis of pyrazine carboxamides
B (X=NHR).
Reagents: (a) i—HCl (gas), MeOH; ii—4,40-dimethylbenzil, KOH,
MeOH; iii—LiOH, H2O/THF/CH3CN; (b) i—(COCl)2, DMF,
CH2Cl2; ii—RNH2, PS-DIEA, CH2Cl2.

3980
B. A. Ellsworth et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3978–3982
Table 1. SAR of pyrazine carboxamides B17
Compound
X = NHR
hCB1 Ki (nM)
SD (nM)
c logP
AM251
(see Fig. 1)
1-Aminopiperidine
3
53
0.5
13
6.9
5.5
4.3
4.8
5.1
6.3
6.1
5.3
6.9
5.2
6.8
6.3
7.1
7.0
7.6
7.0
4
5
Methylamine
Ethylamine
3848
2381
656
195
509
110
59
2636
397
86
6
7
i-Propylamine
i-Amylamine
Aniline
8
21
36
9
10
11
12
13
14
15
16
17
18
N-Methylbenzylamine
Cyclohexylmethylamine
Cyclopropylmethylamine
3-Phenylpropylamine
2-Phenoxyethylamine
4-Phenylbutyl-2-amine
3-(N-methyl-N-phenylamino)propylamine
3-(2,6-Dimethylphenoxy)-propyl-2-amine
1-Benzyloxybutyl-2-amine
20
11
178
18
31
6
631
5
322
2
25
39
10
5
49
203
Table 2. SAR of pyrazine carboxamides B (Scheme 1) with H-bond donor sidechains
vative means to increase polarity of the alkyl pyrazine
carboxamides.23 We hoped that the reduced logP of
hydroxylated pyrazine carboxamides might improve
pharmaceutical properties to produce compounds that
would display in vivo efficacy in the CB1 feeding behav-
ior study.
R
R
NH
O
O
H
HN
O
H
N
N
N
O
N
Figure 2. Proposed conformations for intramolecular H-bonded
pyrazine carboxamides.
Ethanolamine carboxamide 19 (Table 2) exhibited
reduced binding affinity as compared to the des-hydroxyl
derivative 6, though the 4-fold loss in affinity was moder-
ate given the difference in polarity (Dc logP = 0.9). The
hydroxyl group in aminocyclohexan-4-ol 27 and
piperidin-4-ol 28, however, resulted in compounds with
significantly reduced CB1 binding affinity. Upon recogni-
tion that intramolecular hydrogen bonding (Fig. 2) of the
hydroxyl group in compound 19 may minimize the impact
of the polar group within the hydrophobic CB1 binding
pocket, we focused on substituted ethanolamines.
CB1 binding affinity in pyrazine carboxamides is depen-
dent on the stereochemical configuration of the alkyl
sidechain as demonstrated by the ten-fold affinity differ-
ence for the enantiomeric pair 21 and 22. One isomer,
compound 21, is ꢀfourfold more potent than its des-
hydroxymethyl counterpart 8. In contrast, hydroxyl-
ation of SR141716 analogs substantially reduced CB1
binding affinity with no stereochemical preference for

B. A. Ellsworth et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3978–3982
3981
the functionalized alkyl sidechain.23 Benefits of an inter-
nal hydrogen bond were not limited to alcohols. The
hydroxymethyl group of compound 21 was replaced
with a primary carboxamide to give compound 31.
Intramolecular H-bonding of 31 to minimize unfavor-
able hydrophobic receptor interactions would account
for the twofold greater binding potency than that of
the alkyl derivative 8 despite a reduced c logP (Dc
logP = 1.4).
Cl
OH
HN
N
N
O
35
Cl
Figure 3. Bis-(4-chlorophenyl)pyrazine carboxamide (35).
Compound 2124 exhibited reduced lipophilicity (Dc
logP = 1.3) and similar CB1 affinity to the lead in vivo
candidate, 13.25 In contrast to compound 13, compound
21 displayed improved exposure, and a more favorable
brain/plasma ratio at 4 h when administered to rats at
10 mg/kg (Table 3). In addition, when administered oral-
ly at 10 mg/kg in the food deprived rat feeding model,21
compound 21 reduced feeding by 37% 2 h after adminis-
tration, by 32% 4 h after administration, and by 16%
24 h after administration (with no change in locomotor
activity versus vehicle-treated animals). Using a model
of ad libidum fed rats that are trained to operate a lever
to obtain food, a 10 mg/kg dose of compound 21 did not
produce a statistically significant change in food intake,
however a 30 mg/kg dose induced a 34% (p < 0.05)
cumulative reduction in food intake at 4 h versus vehi-
cle-treated control animals.26,27 These results suggested
that hydroxyl-containing pyrazine carboxamides pro-
vided the improvement in physicochemical properties
needed to drive in vivo efficacy.
that of the dimethyl analog 21. When administered at
10 mg/kg to rats trained to operate a lever to obtain
food, compound 35 demonstrated a 46% cumulative
reduction of food intake at 4 h versus vehicle-treated
control animals. Inclusion of a hydroxylic sidechain
potentially provides a means to further enhance solubil-
ity and oral bioavailability by incorporation of a pro-
drug moiety.29,30
We report a series of pyrazine carboxamides as CB1
receptor antagonists that were developed from high-
throughput screening lead structures. The incorporation
of a hydroxyl moiety provides physicochemical benefits,
including lowered c logP, that results in compounds
with enhanced oral exposures and in vivo efficacy in a
food intake model in rats. The hydroxyl group also pro-
vides an opportunity for prodrug strategies that provide
further improvements in physicochemical characteristics
such as aqueous solubility. Our efforts to increase the
in vitro and in vivo potency of this series while maintain-
ing low logP will be the subject of future
communications.
A limited exploration of SARs at the diaryl portion of
the pyrazine carboxamide core revealed that CB1 bind-
ing affinity was enhanced by substituting chlorine for
methyl substituents.18 A priori, the bis-(4-chlorophenyl)
series would be expected to be too lipophilic to exhibit
good PK properties; however, hydroxylation of the side-
chain to generate 3528 (Fig. 3) was anticipated to amelio-
rate this matter. The expected enhancement in affinity of
35 relative to 21 was maintained; CB1 Ki values of 14
and 45 nM, respectively. Moreover, the two compounds
display very similar PK properties (Table 3) despite c
logP of the dichloro analog 35 being ꢀ0.4 U higher than
Acknowledgments
We thank Drs. Robert Zahler and Marc Pfister for help-
ful comments during the preparation of this manuscript.
References and notes
1. Antel, J.; Gregory, P. C.; Nordheim, U. J. Med. Chem.
2006, 49, 4008.
2. Ogden, C. L.; Carroll, M. D.; Curtin, L. R.; McDowell,
M. A.; Tabak, C. J.; Flegal, K. M. JAMA 2006, 295, 1549.
3. Bray, G. A. J. Med. Chem. 2006, 49, 4001.
4. Olshansky, S. J.; Passaro, D. J.; Hershow, R. C.; Layden,
J.; Carnes, B. A.; Brody, J.; Hayflick, L.; Butler, R. N.;
Allison, D. B.; Ludwig, D. S. N. Engl. J. Med. 2005, 352,
1138.
Table 3. Measured PK parameters for pyrazine carboxamidesa
Compound p.o. dose
13b
21c
35c
30 mg/kg
(nM)
10 mg/kg
(nM)
10 mg/kg
(nM)
Time (h)
5. (a) Pagotto, U.; Marsicano, G.; Cota, D.; Lutz, B.;
Pasquali, R. Endocr. Rev. 2006, 27, 79; (b) Di Marzo, V.;
Bifulco, M.; De Petrocellis, L. Nat. Rev. Drug Disc. 2004,
3, 771; (c) Lambert, D. M.; Fowler, C. J. J. Med. Chem.
2005, 48, 1.
6. Cota, D.; Marsicano, G.; Tschop, M.; Grubler, Y.;
Flachskamm, C.; Schubert, M.; Auer, D.; Yassouridis,
A.; Thone-Reineke, C.; Ortmann, S.; Tomassoni, F.;
Cervino, C.; Nisoli, E.; Linthorst, A. C. E.; Pasquali, R.;
Lutz, B.; Stalla, G. K.; Pagotto, U. J. Clin. Invest. 2003,
112, 423.
0.5
1
23
35
89
84
74
245
148
105
218
257
187
157
2
4
Avg. Tmax (h)
2
256
34
1
541
306
3
1
736
491
3.1
AUC0ꢁ4h (nM h)
Brain concd 4 h (nM)
Brain/plasma at 4 h
0.4
a Tabular values are averages from n = 2 male Sprague–Dawley rats.
b Administered as a suspension in 0.5% Methocel/1% Tween 80/water;
compound 13 is insoluble in 10%EtOH/10% Cremaphor/water.
c Administered as a solution in 10% EtOH/10% Cremaphor/water.
7. (a) Rinaldi-Carmona, M.; Barth, F.; Heaulme, M.; Shire,
D.; Calandra, B.; Congy, C.; Martinez, S.; Maruani, J.;

3982
B. A. Ellsworth et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3978–3982
Neliat, G.; Caput, D.; Ferrara, P.; Soubrie, P.; Breliere, J.
Lebuis, A.-M.; Finkelstein, J. M.; Gour-Salin, B. J.; Chan,
T. H.; Leyland-Jones, B. R. J. Med. Chem. 1998, 41, 3062.
20. Compounds were diluted in 100% DMSO and then added
1:100 into 96-well plates containing 1 lg of CHO-CB1
membrane protein in assay buffer (20 mM Hepes, pH 7.4,
100 mM NaCl, 10 mM MgCl2, 10 lg/ml saponin, 1 mM
EDTA, 0.25% BSA, WGA-PVT beads 150 lg, cold GTPc-
S 10, and 60 lM GDP). After adding [35S]GTPc-S to all
the wells, the plates were incubated for 1 h at room
temperature. The binding reaction was terminated by
centrifuging at 1000 rpm for 5 min. The plates were then
counted on a Packard TopCount Scintillation Counter
immediately.
21. fasted rats were treated with test compounds and, after
30 min, food was introduced with quantitation of re-
feeding as reported in Carpino, P. A.; Griffith, D. A.;
Sakya, S.; Dow, R. L.; Black, S. C.; Hadcock, J. R.;
Iredale, P. A.; Scott, D. O.; Fichtner, M. W.; Rose, C. R.;
Day, R.; Dibrino, J.; Butler, M.; DeBartolo, D. B.;
Dutcher, D.; Gautreau, D.; Lizano, J. S.; O’Connor, R.
E.; Sands, M. A.; Kelly-Sullivan, D.; Ward, K. M. Bioorg.
Med. Chem. Lett. 2006, 16, 731–736.
C.; Le Fur, G. FEBS Lett. 1994, 350, 240; (b) Mokhopad-
hyay, S.; Howlett, A. C. Mol. Pharmacol. 2005, 67, 2016.
8. Colombo, G.; Agabio, R.; Diaz, G.; Lobina, C.; Reali, R.;
Gessa, G. L. Life Sci. 1998, 63, PL113.
9. Trillou, C. R.; Arnone, M.; Delgorge, C.; Gonalons, N.;
Keane, P.; Maffrand, J.-P.; Soubrie, P. Am. J. Physiol.
Regul. Integr. Comp. Physiol. 2003, 284, R345.
10. (a) Pi-Sunyer, F. X.; Aronne, L. J.; Heshmati, H. M.;
Devin, J.; Rosenstock, J. JAMA 2006, 295, 761; (b) Van
Gaal, L. F.; Rissanen, A. M.; Scheen, A. J.; Ziegler, O.;
Ro¨ssner, S. Lancet 2005, 365, 1389; (c) Bellocchio, L.;
Mancini, G.; Vicennati, V.; Pasquali, R.; Pagotto, U.
Curr. Opin. Pharmacol. 2006, 6, 586.
11. Muccioli, G. G.; Lambert, D. M. Expert Opin. Ther.
Patents 2006, 16, 1405.
12. Vieth, M.; Sutherland, J. J. J. Med. Chem. 2006, 49, 3451.
13. 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.
14. Debenham, 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.
22. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P.
J. Adv. Drug Delivery Rev. 1997, 23, 3.
23. Francisco, M. E. Y.; Seltzman, H. H.; Gilliam, A. F.;
Mitchell, R. A.; Rider, S. L.; Pertwee, R. G.; Stevenson, L.
A.; Thomas, B. F. J. Med. Chem. 2002, 45, 2708.
1
24. Compound 21: H NMR (CDCl3) d 9.33 (s, 1H), 7.91 (d,
15. (a) Shim, J.-Y.; Welsh, W. J.; Cartier, E.; Edwards, J. L.;
Howlett, A. C. J. Med. Chem. 2002, 45, 1447; (b)
McAllister, S. D.; Rizvi, G.; Anavi-Goffer, S.; Hurst, D.
P.; Barnett-Norris, J.; Lynch, D. L.; Reggio, P. H.;
Abood, M. E. J. Med. Chem. 2003, 46, 5139.
16. Lan, R.; Liu, Q.; Fan, P.; Lin, S.; Fernando, S. R.;
McCallion, D.; Pertwee, R.; Makriyannis, A. J. Med.
Chem. 1999, 42, 769.
1H, J = 8.2 Hz), 7.38 (dd, 2H, J = 4.5, 1.8 Hz), 7.35 (dd,
2H, J = 4.4 Hz, 2.4 Hz), 7.16 (d, 2H, J = 7.9 Hz), 7.11(d,
2H, J = 8.0 Hz), 4.31–4.26 (m, 1H), 3.81 (dd, 1H, J = 3.6,
11.1), 3.68 (dd, 1H, J = 4.0, 11.1), 2.38 (s, 3H), 2.36 (s,
3H), 1.75–1.67 (m, 1H), 1.60-1.45 (m, 2H), 0.98 (dd, 6H,
0.8, 6.5 Hz) 13C NMR (CDCl3) d 164.1, 155.2, 150.5,
141.2, 141.0, 139.4, 139.2, 135.2, 135.1, 129.7, 129.5, 129.2,
129.1, 66.4, 50.5, 40.2, 25.0, 23.1, 22.2, 21.4. HRMS anal.
calcd for C25H29N3O2 403.22598 found [M+H] 404.2326.
25. Rat brain prep CB1 data: compound 13 Ki = 80 nM,
compound 21 Ki = 35 nM, compound 35 Ki = 30 nM.
26. Rohrbach, K. W.; Han, S.; Gan, J.; O’Tanyi, E. J.; Zhang,
H.; Chi, C. L.; Taub, R.; Largent, B. L.; Cheng, D. Eur. J.
Pharmacol. 2005, 511, 31.
17. Method for hCB1 Ki determination: the CHO-CB1 cell
line was licensed from Euroscreen (cell line EC-110-C)
with a Bmax value of 18 pmol/mg protein (using [3H]-CP-
55940). Test compounds were serially diluted in DMSO
and added 1:100 to 96-well microtiter plates containing
3 lg of CHO-CB1 membrane protein and 2–5 nM [3H]-
CP-55940 in a Hepes-based binding buffer containing
0.25% BSA (pH 7.4). After 3 h at room temperature, the
binding reaction was terminated by transferring the
reaction mixtures onto GF/B filter plates using a Packard
Cell Harvester. The filter plates were then washed and the
contents of the plates were counted on a Packard
TopCount Scintillation Counter. Non-specific binding
was determined by the addition of 1000-fold excess of
cold CP-55940. Specific binding was calculated by sub-
tracting non-specific binding from total binding. CPM
were converted into % inhibition based on total and non-
specific binding and Ki values were calculated by the
Cheng–Prusoff correction (Cheng, Y. -C.; Prusoff, W. H.
Br. Pharmacol., 1973, 22, 3099–3108) using the parameters
of radioligand concentration and the Kd values determined
for each radioligand. Reported Ki values are averages of at
least three independent determinations.
18. During preparation and editorial review of this manu-
script, compound 4 was reported in a manuscript in press:
Bostrom, J.; Berggren, K.; Elebring, T.; Greasley, P. J.;
Wilstermann, M. Bioorg. Med. Chem. 2007. doi:10.1016/
j.bmc.2007.03.075, SARs for diaryl analogs of compound
4 are also reported.
19. (a) Karmas, G.; Spoerri, P. E. J. Am. Chem. Soc. 1956, 78,
2141; (b) Karmas, G.; Spoerri, P. E. J. Am. Chem. Soc.
1952, 74, 1580; (c) Jones, P.; Villeneuve, G. B.; Fei, C.;
DeMarte, J.; Haggarty, A. J.; Nwe, K. T.; Martin, D. A.;
27. Inoue, K.; Valdex, G. R.; Reyes, T. M.; Reinhardt, L. E.;
Tabarin, A.; Rivier, J.; Vale, W. W.; Sawchenko, P. E.;
Koob, G. F.; Zorrilla, E. P. J. Pharmacol. Exp. Ther.
2003, 305, 385.
28. Compound 35: 1H NMR d 9.38 (s, 1H), 7.85 (d, 1H,
J = 8.4 Hz), 7.61–7.31 (m, 8H), 4.33–4.28 (m, 1H), 3.83
(dd, 1H, J = 3.6, 11.1), 3.69 (dd, 1H, J = 4.7, 11.1), 1.75–
1.64 (m, 1H), 1.62–1.46 (m, 2H), 0.98 (dd, 6H, 2.0, 6.5 Hz)
13C NMR (CDCl3) d 163.5, 151.2, 149.4, 141.7, 141.0,
136.0, 135.9, 131.1, 131.0, 129.0, 128.9, 66.1, 50.4, 40.2,
25.1, 23.1, 22.3. HRMS anal. calcd for C23H23Cl2N3O2
443.11673 found [M+H] 444.1257.
29. Ettmayer, P.; Amidon, G. L.; Clement, B.; Testa, B. J.
Med. Chem. 2004, 47, 2392.
30. Compound 35 was converted to a phosphate ester via
reaction with POCl3 in THF at 0 °C, followed by workup
with aqueous HCl to give phosphate 36 that exhibited
0.43 mg/mL aqueous solubility at pH 6.5.
Cl
Cl
O
HN
PO3H2
N
N
O
36