Characterization of a novel and selective CB1 antagonist as a radioligand for receptor occupancy studies

Article abstract of DOI:10.1016/j.bmcl.2011.09.016

Obesity remains a significant public health issue leading to Type II diabetes and cardiovascular disease. CB1 antagonists have been shown to suppress appetite and reduce body weight in animal models as well as in humans. Evaluation of pre-clinical CB1 antagonists to establish relationships between in vitro affinity and in vivo efficacy parameters are enhanced by ex vivo receptor occupancy data. Synthesis and biological evaluation of a novel and highly selective radiolabeled CB1 antagonist is described. The radioligand was used to conduct ex vivo receptor occupancy studies.

Full text of DOI:10.1016/j.bmcl.2011.09.016

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6856–6860
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Characterization of a novel and selective CB1 antagonist as a radioligand
for receptor occupancy studies
⇑
Yifan Yang, Keith J. Miller , Yeheng Zhu, Yang Hong, Yuan Tian, Natesan Murugesan, Zhengxiang Gu,
Eva O’Tanyi, William J. Keim, Kenneth W. Rohrbach, Susan Johnghar, Kamelia Behnia,
Mary Ann Pelleymounter, Kenneth E. Carlson, William R. Ewing
Research & Development, Bristol-Myers Squibb Co., PO Box 4000, Princeton, New Jersey 08543, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 July 2011
Revised 2 September 2011
Accepted 6 September 2011
Available online 13 September 2011
Obesity remains a significant public health issue leading to Type II diabetes and cardiovascular disease.
CB1 antagonists have been shown to suppress appetite and reduce body weight in animal models as well
as in humans. Evaluation of pre-clinical CB1 antagonists to establish relationships between in vitro affin-
ity and in vivo efficacy parameters are enhanced by ex vivo receptor occupancy data. Synthesis and
biological evaluation of a novel and highly selective radiolabeled CB1 antagonist is described. The radio-
ligand was used to conduct ex vivo receptor occupancy studies.
Keywords:
Ó 2011 Elsevier Ltd. All rights reserved.
Cannabinoid
Radioligand
Receptor occupancy
Obesity
The biological effects of the endocannabinoids are primarily
mediated by the CB1 and CB2 receptors in the central nervous sys-
tem and periphery and may be modulated by additional proteins as
well.1 CB1 is highly expressed in the brain, with the highest levels
of expression in the hippocampus, cerebellum, substantia nigra,
basal ganglia, caudate putamen, and pre-frontal cortex as deter-
mined by receptor autoradiography and immunohistochemical
analysis.2 The CB1 receptor has been implicated in the regulation
of food intake and body weight.3 Indeed, antagonists to the CB1
receptor such as rimonabant, taranabant, and ibipinabant (SLV-
319) have proven to be clinically effective in the treatment of
obesity.4 BMS-725519 1 was identified as compound exhibiting
properties suitable for pre-clinical development for obesity (Fig. 1).
In support of studies to understand the relationships between
receptor affinity, protein binding and drug exposure on in vivo effi-
cacy, as well as to assist with human dose projections we wished to
characterize BMS-725519’s potential as a radioligand. The com-
pound’s excellent binding affinity coupled with well-balanced
physicochemical properties, including calculated LogP and protein
binding, made it a good candidate for radiolabeling and assessment
in receptor autoradiography/occupancy studies. While the CB1
radioligand [3H]SR141716A was commercially available, and used
by our group to assess receptor binding affinity and ex vivo
occupancy, we wished to characterize the properties of tritiated
BMS-725519 as an additional in vitro tool and potential PET ligand
option as compounds such as [18F]MK-9470 and MePPEP were not
commercially available to us. While it is always advisable to utilize
radioligands and test drugs from distinct chemical classes to
N
F
F
F
N
N
N
O
N
N
Cl
BMS-725519 1
3H-SR-141716 binding rCB1
3H-SR-141716 binding hCB1
12.4 nM
4.6 nM
3H-CP55940 binding hCB2 >10,000 nM
cLogP
2.37
% Protein binding (h, r)
95, 92
⇑
Figure 1. The structure, in vitro binding affinities, calculated LogP and in vitro
protein binding values for BMS-725519.
Corresponding author. Tel.: +1 609 818 5497; fax: +1 609 818 3239.
E-mail address: keith.miller@bms.com (K.J. Miller).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.016

Y. Yang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6856–6860
6857
OH
N
O
HCl
13
d
O
O
O
O
Br
+
NHNHSO2CH3
N2
Cl
b
c
e
OH
a
N
O
Cl
Cl
Cl
N
Cl
I
I
I
I
5
14
4
3
2
O
N
O
N
Cl
N
O
NH
NSO2CH3
NH
N
g
f
N
N
NH
N
i
h
O
O
Cl
Cl
Cl
Cl
I
I
I
I
6
7
8
9
I
Cl
N
N
NH
N
N
CF3
CF3
O
+
Cl
N
N
k
N
N
N N
N
N
j
N
O
CF3
O
N
N
N
N
Cl
Cl
I
16
Cl
Cl
10
I
I
12
11
l
Cl
O
N
CF3
HO
15
Scheme 1. Reagents and conditions: (a) (COCl)2, CH2Cl2, DMF(cat.); (b) diazomethyltrimethylsilane, CH3CN; (c) HBr/HOAc; (d) CH3SO2NHNH2, CDI, CH3CN; (e) compd 13,
KOtBu, DMF, À50 °C to rt; (f) NaOAc, rt; (g) DBU, 50 °C; (h) POCl3; (i) (i) NH2NH2; (ii) CDI; (j) compd 15, K2CO3, DMF, 80 °C; (k) NaI, TMS-Cl, CH3CN, 180oC, 180 °C, microwave;
(l) (i) BH3-THF; (ii) SOCl2, CH2Cl2.
minimize non-selective interactions, resource constraints necessi-
tated the use of unlabeled BMS-725519 as the test reagent for
our characterization of [3H]BMS-725519.
for the CB1 receptor in rat brain (Fig. 2) and a Kd = 3.95 nM and a
Bmax of 41 pmol/mg protein for the recombinantly expressed
human CB1 receptor.5 Rimonabant and ibipinabant inhibited
[3H]BMS-725519 binding to human recombinant CB1 receptor in
a concentration dependent manner with Ki values of 0.73 and
0.42 nM, respectively.6 CP-55,940 and BMS-725519 were tested
in competitive binding assays using [3H]BMS-725519 on rat cere-
The synthesis of di-iodo analog is illustrated in scheme 1. Com-
mercially available 4-chloro-3-iodobenzoic acid 2 was converted to
compound 5 in 3 steps by reacting first with oxalyl chloride, fol-
lowed by diazomethyltrimethylsilane and then HBr in acetic acid.
Compound 5 then was reacted with compound 14 which was
derived from 2-(pyridin-4-yl)acetic acid HCl salt to give compound
6. Compound 6 was then treated first with NaOAc to afford
compound 7 which was cyclized to form compound 8 under
DBU. Compound 8 was then refluxed in POCl3 to form compound
9. The triazolopyridazinone 10 was constructed by reacting com-
pound 9 with hydrazine first, followed by CDI. Compound 10 was
then coupled with compound 16 which was synthesized from
2-chloro-6-(trifluoromethyl)nicotinic acid in 2 steps to give com-
pound 11. The second iodo was introduced by reacting compound
11 with NaI and TMS-Cl in acetonitrile at 180 °C in microwave
reactor.
bellar tissue sections, yielding IC50 values of
4 and 37 nM,
respectively.7
In vitro autoradiography studies revealed that [3H]BMS-725519
produced selective labeling of distinct regions of the rat brain
including the cerebellum, hippocampus, striatum and frontal
cortex (Fig. 3).7 The labeling pattern matches results from previ-
ously published studies with [3H]CP-55940 and [3H]SR141716
(rimonabant).5 CB1 receptor autoradiographic labeling by
[3H]BMS-725519 was also characterized in wild-type and CB1
knockout mice. In wild-type animals the pattern of labeling was
equivalent to that seen in the rat while in the KO animals specific
labeling was absent (Fig. 4).7
[3H]BMS-725519 17 (Scheme 2) was tested against membrane
homogenates prepared from whole rat brain and CHO cells that
recombinantly-expressed human CB1 receptor.6 Saturation analy-
sis revealed a Kd = 29.1 nM and a Bmax of 2.16 pmol/mg protein
Ex vivo binding studies were conducted on rat brain sections
from animals treated with orally administered increasing doses
of BMS-725519 (0.03, 0.3, 3.0 and 30.0 mg/kg p.o.).8 As highlighted
in Figure 5 it was possible to determine the level of displacement

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Y. Yang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6856–6860
T
I
N
N
CF3
CF3
N
N
N
N
N
N
O
O
Pd/C
N
N
N
N
TEA/EtOH
Cl
Cl
17
16
T
I
Scheme 2. [3H]BMS-725519 17 was prepared by tritio-de-iodination of the di-iodinated analog 16 with carrier-free tritium gas in the presence of palladium/carbon, 10% at
room temperature for 3 h yielding a specific activity of 29.0 Ci/mmol. The crude products were purified by HPLC to more than 99% radiochemical purity.
of [3H]BMS-725519 labeling by increasing doses of BMS-725519 in
sections of rat brain, resulting in a determination of receptor
occupancy.
4000
TB
NSB
When coupled with measures of food intake, plasma drug levels
3000
SB
and brain drug levels, relationships between receptor occupancy
and efficacy can be determined using this radioligand.8,9 An
in vivo study evaluating doses of 0.03, 0.1, 0.3, 1.0, 3.0, 10.0, and
30.0 mg/kg was carried out to evaluate the relationships described
above 6 h after dosing (Table 1). As calculated free plasma and
brain drug levels increase there is a corresponding increase in both
the suppression of food intake (as measured by the number of food
pellets consumed) and receptor occupancy. The reduction in food
intake appears to reach a plateau at the 10 and 30 mg/kg doses
despite further increases in plasma and brain drug levels. Receptor
occupancy also seems to be maximal at these two doses indicating
that CB1 receptors may be fully occupied. While the absolute level
of occupancy has not reached 100%, it is likely that the receptor
occupancy at the highest doses corresponds to greater level of
actual occupancy, possibly approaching 100%, given the fact that
2000
1000
0
0
50
100
nM
Figure 2. Representative saturation binding experiment (total n = 2) using [3H]-
BMS-725519 and 40 g of rat brain membranes. TB = total binding; NSB = non
specific binding defined by addition of 1000-fold excess of unlabeled BMS-725519;
l
SB = specific binding.
Figure 3. Representative autoradiogram of a sagittal section of rat brain incubated with 40 nM [3H]-BMS-725519 (A) or section incubated with 40 nM [3H]-BMS-725519 and
M unlabelled BMS-725519 (B).
5
l
Figure 4. Ex vivo autoradiography of mouse brain sagittal sections of either wild-type (A) or CB1 knockout (B) animals.

Y. Yang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6856–6860
6859
Figure 5. Ex vivo autoradiography of rat brain sagittal sections following drug treatment. Rats were administered vehicle (A), 0.03 mpk (B), or 3 mpk (C) or 30 mpk (D) of
BMS-725519 and then processed for receptor autoradiography with [3H]BMS-725519.
Table 1
3. (a) Mechoulam, R.; Fride, E. Nature 2001, 389, 25; (b) Di Marzo, V.; Goparaju, S.
K.; Wang, L.; Liu, J.; Bátkai, S.; Járai, Z.; Fezza, F.; Miura, G. I.; Palmiter, R. D.;
Sugiura, T.; Kunos, G. Nature 2001, 410, 822.
Effects of BMS-725519 on receptor occupancy and efficacy, and the determination of
plasma and brain exposures
4. Pi-Sunyer, F. X.; Aronne, L. J.; Heshmati, H. M.; Devin, J.; Rosenstock, J.; RIO-
North America Study Group. JAMA 2006 295, 761.; (b) Addy, C.; Wright, H.; Van
Laere, K.; Gantz, I.; Erondu, N.; Musser, B. J.; Lu, K.; Yuan, J.; Sanabria-Bohórquez,
S. M.; Stoch, A.; Stevens, C.; Fong, T. M.; De Lepeleire, I.; Cilissen, C.; Cote, J.;
Rosko, K.; Gendrano, I. N., 3rd; Nguyen, A. M.; Gumbiner, B.; Rothenberg, P.; de
Hoon, J.; Bormans, G.; Depré, M.; Eng, W. S.; Ravussin, E.; Klein, S.; Blundell, J.;
Herman, G. A.; Burns, H. D.; Hargreaves, R. J.; Wagner, J.; Gottesdiener, K.;
Amatruda, J. M.; Heymsfield, S. B. Cell Metab. 2007, 7, 68; (c) Lange, J. H. M.;
Coolen, H. K. A. C.; van Stuivenberg, H. H.; Dijksman, J. A. R.; Herremans, A. H. J.;
Ronken, E.; Keizer, H. G.; Tipker, K.; McCreary, A. C.; Veerman, W.; Wals, H. C.;
Stork, B.; Verveer, P. C.; den Hartog, A. P.; de Jong, N. M. J.; Adolfs, T. J. P.;
Hoogendoorn, J.; Kruse, C. G. J. Med. Chem. 2004, 47, 627.
P.O. Dose (mg/kg)
Receptor occupancy (%)
Free plasma exposure
(nM)
Free brain exposure (nM)
Reduction in food intake
(%)
0.03 0.1
12.7 14.3 20.6 29.8 55.6 64.5 74.1
1.5 5.4 15.3 49.4 112 531 1047
0.3
1.0
3.0
10
30
0.93 2.6
6.4
18.6 19.9 99.7 308
0
0
22.3 23.7 41.5 67.4 72.5
5. (a) Horti, A. G.; Van Laere, K. Curr. Pharm. Res. 2008, 14, 3363; (b) Petitet, F.;
Marin, L.; Doble, A. NeuroReport 1996, 7, 789; (c) Burns, H. D.; Van Laere, K.;
Sanabria-Bohorquez, S.; Hamill, T. G.; Bormans, G.; Eng, W. S.; Gibson, R.; Ryan,
C.; Connolly, B.; Patel, S.; Krause, S.; Vanko, A.; Van Hecken, A.; Dupont, P.; De
Lepeliere, I.; Rothenberg, P.; Stoch, S. A.; Cote, J.; Hagmann, W. K.; Jewell, J. P.;
Lin, L. S.; Liu, P.; Goulet, M. T.; Gottesdiener, K.; Wagner, J. A.; de Hoon, J.;
Mortelmans, L.; Fong, T. M.; Hargreaves, R. J. Proc. Natl. Acad. Sci. U.S.A. 2007, 104,
9800; (d) Suter, T. M.; Chesterfield, A. K.; Bao, C.; Schaus, J. M.; Krushinski, J. H.;
Statnick, M. A.; Felder, C. C. Eur. J. Pharmacol. 2010, 649, 44.
6. Brain tissue or cells scraped from plates were homogenized manually by hand in
10 volumes of the cold buffer with 10 mM HEPES and 1 mM EGTA, pH 7.4,
containing complete protease inhibitor cocktail, 1 mM dithiothreitol, and 10%
sucrose. The homogenate was centrifuged at 1000 g for 10 min at 40 °C.
Supernatant was collected and centrifuged at 100,000 g for 20 min. The pellet
was re-suspended in buffer containing 20 mM HEPES, 1 mM EGTA, pH 7.4, 1 mM
DTT, and 1 mM MgCl2. Saturation analyses were carried out to determine the
dissociation constant, Kd of [3H]BMS-725519. Specifically, increasing
concentrations of the radioligand was added to the assay mixture and the
specific bound was measured using cold BMS-725519 as the non-specific. The
binding reaction was incubated for 90 min at room temperature and then
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.
dissociation of receptor-bound drug is known to occur during the
in vitro processing of tissue sections.
In summary we have developed a selective radioligand for
assessing CB1 receptor occupancy that is similar to other available
CB1 radioligands. As part of our evaluation we determined that
[3H]BMS-725519 could facilitate the development of an under-
standing of relationships of receptor affinity, drug exposure and
receptor occupancy in the brain to develop human dose projections
in support of pre-clinical development studies.
Acknowledgment
We greatly appreciate the support of Bristol Myers Squibb
Department of Analytical Sciences.
Supplementary data
7. Male Sprague-Dawley rats or wild-type or CB1 receptor knockout mice
(obtained from the laboratory of Andreas Zimmer) were sacrificed by
decapitation. Brains were collected, incubated in pre-chilled (À20 °C) 2-
methyl butane for 2–3 min and stored at À80 °C until use. The samples were
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.09.016.
sectioned to 20
lm at À20 °C using a cryostat (Jung Frigo-Cut, 2800E) and stored
References and notes
at À80 °C. For binding studies, the slide-mounted sections were brought to 22–
24 °C, dried, and pre-incubated in the binding buffer containing 25 mM HEPES
pH 7.4, 150 mM NaCl, 1 mM EDTA, 2 mM MgCl2, 0.25% BSA, 1 mM leupeptin for
2 min. The sections were then incubated in the same solution containing 2.1–
385 nM [3H]-BMS-725519 for 45 min at 22–24 °C. The adjacent sections were
1. Pertwee, R. G.; Howlett, A. C.; Abood, M. E.; Alexander, S. P.; Di Marzo, V.;
Elphick, M. R.; Greasley, P. J.; Hansen, H. S.; Kunos, G.; Mackie, K.; Mechoulam,
R.; Ross, R. A. Pharmacol. Rev. 2010, 62, 588.
2. (a) Herkenham, M.; Lynn, A. B.; Johnson, M. R.; Melvin, L. S.; de Costa, B. R.; Rice,
K. C. J. Neurosci. 1991, 11, 563; (b) Glass, M.; Dragunow, M.; Faull, R. L. M.
Neuroscience 1997, 77, 299.
incubated under the same conditions in the presence of 10 lM unlabelled BMS-
725519 to define nonspecific binding. Concentration-related displacements of
BMS-725519 and CP-55940 were assessed by incubating the compounds at a

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Y. Yang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6856–6860
concentration of 0.01–10,000 nM in the total binding buffer with 40 nM
[3H]BMS-725519. After incubation, the sections were rinsed in phosphate
(specific binding in vehicle treated) Â 100%. The data were reported as the
mean S.E.M.
buffer saline three times, 2 min each, and rinsed quickly in de-ionized H2O. The
sections were then placed in cassettes against a tritium-sensitive screen for 2–
3 days. The captured storage phosphor images were analyzed using OptiQuant
Acquisition and Analysis software (Perkin Elmer Life Science).
9. For feeding studies male Sprague Dawley rats (ꢀ225 g starting weight) were
obtained from Charles River Laboratories and housed on a 12:12 light/dark cycle
in the BMS vivarium. Animals were acclimated to the facility for 10 days prior to
use with standard rodent chow (Harlan Teklad) and water ad libitum. There
were five groups: Vehicle (10%DMAC, 10%EtOH, 10% Cremophor,70% water;
2 ml/kg; p.o.) and 7 doses of Test Agent (BMS-725519; 0.03, 0.1, 0.3, 1, 3, 10 and
30 mg/kg; p.o.). There were six rats in each group, and all animals were tested
simultaneously. Animals were trained to bar-press for food in an operant
chamber (Coulbourn Instruments, Allentown, Pa) equipped with a lever, a food
hopper, a water bottle with photocells and an infra-red activity monitor. The
training sequence was initiated by depriving the animal of food for 16 h. At the
end of that period, which coincided with the beginning of the dark cycle, the rat
was placed into an operant chamber with 5–45 mg pellets (Research Diets
purified food pellet) present in the feeding trough. Subjects could obtain
additional pellets by pressing a lever situated adjacent to the trough. During the
first 20 h of training one lever press was sufficient to deliver one pellet. During
the second 20 h, three lever presses were required to obtain a pellet. At all times
the animal had access to one water bottle situated on the opposite side of the
cage. Photobeams positioned around the opening to the water bottle were
broken by a nose-poke and served to provide a measure of licks taken by the
animal. All testing sessions began at the start of the dark-cycle, the time at
which nocturnal feeding begins. Animals were subject to two 20 h sessions with
an interval of 4 h between sessions. The first session served as a baseline
measure of feeding and animals were assigned to treatment groups in such a
way that there were no differences in the average number of pellets eaten
during the 20 h baseline period. Test compounds were delivered orally
immediately prior to the start of the second session. The second session
commenced with a 60 min lock-out period during which lever pressing did not
result in pellet delivery. At the end of this period, rats could obtain food reward
on an FR3 schedule for the remainder of the twenty hour session. Statistical
evaluation of pellets consumed at each time interval after the lock-out period (1,
2, 4, 8, 12 and 20 h) was conducted using one-way ANOVA. Group differences
were further assessed using Fishers PLSD as a post-hoc test.
8. For the determination of receptor occupancy and plasma/brain exposures, BMS-
725519 was orally administered to male Sprague-Dawley rats at 0.03, 0.1, 0.3,
1.0, 3.0, 10.0, and 30.0 mg/kg one hour prior to the dark cycle (six animals per
dose group). The vehicle used to make the dosing solution consisted of 10%
DMAC/10% cremophor/10% ethanol and 70% water and the dosing volume was
at 2 ml/kg. At 6 h post-dose, blood samples and brain tissues were collected
from three animals per dose group for determination of exposures, and three rat
brains were collected for the determination of CB1 receptor occupancy. Blood
samples were centrifuged at 4 °C to obtain serum. Brain tissue for exposure
determination was rinsed with ice-cold water and blotted dry to remove excess
water. After recording the weights, the brains were homogenized using three
volumes of water. The homogenized brains and the serum samples were stored
at À20 °C before analysis by LC–MS. Free drug concentrations were calculated
based on total drug levels  8% unbound drug determined from in vitro protein
binding studies (data in Fig. 1). Intact brains for the determination of receptor
occupancy were rapidly frozen and sectioned as described in Ref.7. The
radioligand binding procedures for receptor occupancy studies were similar to
the in vitro with a few changes to minimize the loss of receptor occupancy of
exogenously administered BMS-725519: The pre-incubation of the tissue
sections in buffer was shortened to 30 s, [3H]BMS-725519 incubation time
was 6 min at 40 nM, non-specific binding was determined by adding 5 lM
unlabelled BMS-725519 to the binding buffer, and post incubation washes were
three times at 30 s each. The binding buffer and washing buffer were pre-chilled
at 4 °C. Digital images were generated with a Cyclone storage phosphor-imaging
system as described earlier. Radioligand binding density in a defined brain
region (cerebellum) was measured as digital light units per millimeter squared
(DLU/mm2). All measurements were first subtracted by the background DLU/
mm2. For ex vivo binding studies, the percent of specific binding was calculated
as the following: percent specific binding = (specific binding in drug treated)/