Towards rational design of cannabinoid receptor 1 (CB1) antagonists for peripheral selectivity

Article abstract of DOI:10.1016/j.bmcl.2011.08.032

CB1 receptor antagonists that are peripherally restricted were targeted. Compounds with permanent charge as well as compounds that have increased polar surface area were made and tested against CB1 for binding and activity. Sulfonamide and sulfamide with high polar surface area and good activity at CB1 were rationally designed and pharmacologically tested. Further optimization of these compounds and testing could lead to the development of a new class of therapeutics to treat disorders where the CB1 receptor system has been implicated.

Full text of DOI:10.1016/j.bmcl.2011.08.032

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5711–5714
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Towards rational design of cannabinoid receptor 1 (CB1) antagonists
for peripheral selectivity
Alan Fulp a, Katherine Bortoff a, Yanan Zhang c, Herbert Seltzman b, Rodney Snyder a, Rangan Maitra a,
⇑
a Pharmacology and Toxicology, RTI International, 3040 Cornwallis Rd., Research Triangle Park, NC 27709-2194, USA
b Organic and Medicinal Chemistry, RTI International, 3040 Cornwallis Rd., Research Triangle Park, NC 27709-2194, USA
c Analytical Chemistry and Pharmaceutics, RTI International, 3040 Cornwallis Rd., Research Triangle Park, NC 27709-2194, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
CB1 receptor antagonists that are peripherally restricted were targeted. Compounds with permanent
charge as well as compounds that have increased polar surface area were made and tested against CB1
for binding and activity. Sulfonamide and sulfamide with high polar surface area and good activity at
CB1 were rationally designed and pharmacologically tested. Further optimization of these compounds
and testing could lead to the development of a new class of therapeutics to treat disorders where the
CB1 receptor system has been implicated.
Received 22 June 2011
Revised 2 August 2011
Accepted 4 August 2011
Available online 11 August 2011
Keywords:
CB1
Ó 2011 Elsevier Ltd. All rights reserved.
Peripheral
Antagonist
Cannabinoid
Topological polar surface area
Cannabinoid receptors (CBRs) belong to the endocannabinoid
(EC) system, which consists of receptors, transporters, endocannab-
inoids, and the enzymes involved in synthesis and degradation of
endocannabinoids.1 To date, two different cannabinoid receptors
have been identified CB1 and CB2. Both CB1 and CB2 are G pro-
tein-coupled receptors (GPCRs) primarily activating inhibitory G
proteins (Gi/o).2,3 The EC system regulates many important physio-
logical processes and several components of the EC system are under
evaluation as targets to treat a diverse array of indications including
obesity, liver disease, diabetes, pain and inflammation.4
The CB1 receptor is prominently expressed in the central nervous
system (CNS) and also in peripheral tissues. It has emerged as an
important target to treat metabolic disorders including obesity
and diabetes. The first CB1 receptor selective drug developed for
medical use was rimonabant 1 (SR141716A), an inverse agonist/
antagonist. Rimonabant was designed to treat obesity and other re-
lated disorders that have both CNS and peripheral components.
However, rimonabant was withdrawn from European markets and
denied approval in the United States due to CNS-related side effects
including anxiety, depression and suicidal ideation.5 Subsequently,
development of other CB1 receptor antagonists taranabant, otena-
bant, and ibipinabant were halted due to regulatory concerns.6
A strategy to minimize CNS-related side effects noted with CB1
antagonists while maintaining beneficial effects of blocking the
receptor in peripheral tissues is to develop compounds that do
not cross the blood–brain barrier (BBB). This approach is being
pursued by several groups and some compounds have been re-
ported (Fig 1).7–11 However, most of these reported compounds
need to be fully characterized and their clinical efficacies remain
O
O
S
O
O
N
N
N
NH
NH
N
N
N
Cl
Cl
Cl
NC
Cl
Cl
1 (rimonabant)
2 (AM6545)
F
F
O
O
H2N
N
N
N
O
H2N
N
Cl
Cl
Cl
Cl
4 (Sanofi-aventis)
3 (URB447)
⇑
Corresponding author. Tel.: +1 919 541 6795; fax: +1 919 541 6499.
E-mail address: rmaitra@rti.org (R. Maitra).
Figure 1. Structure of CB1 antagonists
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.08.032

5712
A. Fulp et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5711–5714
O
N
unproven. Herein, we describe our efforts in developing CB1 antag-
onists selective for the periphery using two strategies. First, since
charged compounds do not normally cross the BBB unless trans-
ported by specific transporters,12 permanently charged com-
pounds, such as alkyl pyridinium salts and N-oxides, were
targeted. The second strategy was driven by topological polar sur-
face area (TPSA). The total surface area of polar atoms in a molecule
has been shown to correspond with passive transport through
membranes, where higher TPSA corresponds to lower penetration
into the CNS.13 Therefore, compounds with relatively high TPSAs,
such as sulfonamide and sulfamide, were targeted. In addition to
the significantly increased TPSA, these neutral sulfonamides and
sulfamides also have hydrogen available for hydrogen bonding,
offering the opportunity for further interaction with the receptor
and thus possibly leading to improved potency.
X
NH
NH2
O
N
OH
i
N
Cl
N
Cl
Cl
Cl
Cl
A
Cl
C
O
S
NH
O
X
O
N
NH
ii
C
N
Cl
Cl
Charged compounds were synthesized as described in Scheme
1. Acid A is readily available using a procedure developed in our
laboratories.14,15 The acid was then coupled by first making an acid
chloride using oxalyl chloride and a catalytic amount of DMF fol-
lowed by amide formation with the appropriate aminopyridine
and triethylamine; or by the use of standard BOP coupling condi-
tions.16 Alkyl pyridinium salts (6, 9 and 12) were made by reacting
B with methyl iodide in dichloromethane or methanol.17 Pyridine
N-oxides (7, 10 and 13) were readily obtained by reacting B with
m-CPBA in dichloromethane.18
E
Cl
O
S
NH
O
NH2
X
NH
O
N
iii
C
N
Cl
Cl
Sulfonamide and sulfamide compounds were synthesized by
the route described in Scheme 2. Acid A was coupled to diamines
via a method previously described by our group.16 The diastereo-
meric ratio of compound 19 could be enriched by careful column
chromatography. These amines were converted to the correspond-
ing sulfonamide compound (14, 17, 20, 22, 24 and 27) with meth-
anesulfonyl chloride and triethylamine in THF. The desired
sulfamides (15, 18, 21, 23, 26 and 28) were synthesized by reacting
F
Cl
Scheme 2. Reagents and conditions: (i) diamine, BOP, THF, rt; (ii) methanesulfonyl
chloride, Et3N, THF, rt, 16 h; (iii) sulfamide, dioxane, 85 °C, 16 h. X is used to
designate a spacer of any kind.
the appropriate amine with sulfamide at an elevated temperature,
as has been previously described.19
All compounds were purified on a Teledyne ISCO CombiFlash
Companion system using RediSepRf prepacked columns and char-
acterized by 1H NMR (300 MHz), TLC, and mass spectroscopy. All
compounds were evaluated for antagonism using a calcium mobi-
lization assay. Each compound was pharmacologically character-
ized for antagonism using a functional fluorescent CB1 activated
R
N
O
N
O
N
OH
N
H
i or ii
N
Cl
N
Cl
Cl
Gaq16-coupled intracellular calcium mobilization assay in CHO-
Cl
K1 cells as has been described in our previous publication.16
Briefly, the ability of each compound to antagonize the concentra-
tion-response of CP55940 at CB1 was measured.20 Antagonism was
noted by a right-ward shift of the CP55940 concentration-response
curve upon pre-incubation with the test agent. The EC50 values in
the presence and absence of an antagonist was used to determine
its apparent antagonist dissociation equilibrium constant (Ke).21
Further characterization of select compounds was performed using
radioligand displacement of [3H]1 and equilibrium dissociation
constant (Ki) values were determined.16 Selectivity of these com-
pounds at CB1 versus CB2 was also determined by obtaining Ki val-
ues at either receptor using displacement of [3H]CP55940 in
membranes of CHO-K1cells over-expressing either receptor. Data
reported are average values from 3 to 6 measurements.16
The pyridinium compounds were charged analogs of a reported
methylpyridine amide similar to 11.22 To date, only limited activity
has been observed with alkyl pyridinium salts and pyridine N-oxi-
des (Table 1). The parent pyridines (5, 8 and 11) were more potent
than their alkyl pyridinium salt or N-oxide analogues in all cases.
All pyridinium salt analogues made to date have Ke values of great-
Cl
A
Cl
B
R
N
O
N
I
N
H
iii
B
N
Cl
Cl
C
Cl
R
O
N
O
N
H
iv
B
N
N
Cl
Cl
er than 8
active with compounds 7 and 10 both having Ke values less than
M, making them interesting for future interrogation.
The initial CB1 antagonists with high TPSAs, sulfonamide 14 and
sulfamide 15, were both active at CB1 receptor (Table 2) and have
lM against CB1. The pyridine N-oxides were modestly
D
Cl
2
l
Scheme 1. Reagents and conditions: (i) (a) oxalyl chloride, CH2Cl2, DMF cat., rt, 2 h;
(b) aminopyridine, CH2Cl2, Et3N, rt; (ii) aminopyridine, BOP, i-Pr2EtN, DMF, rt, 16 h;
(iii) methyl iodide, CH2Cl2 or MeOH, 2–7 d; (iv) m-CPBA, CH2Cl2, 16 h.

A. Fulp et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5711–5714
5713
Table 1
Alkyl pyridinium salts and N-oxides derivatives 1–9 via Scheme 1
Table 2
Sulfonamide and sulfamide derivatives 14–28 via Scheme 2
O
N
O
N
R
R
NH
NH
N
N
Cl
Cl
Cl
Cl
Cl
Cl
Compound
R
Ke CB1 (
l
M)
Yielda (%)
90
Compound
R
Ke CB1 (
l
M)
tPSA
N
N
5
0.117
HN
14
0.207
0.304
101
127
O
S
O
6
7
>10
55
70
I
HN
O
15
N
1.39
O
S
NH2
O
NH2
8
0.384
8.41
91
61
99
19
16
17
>10
>10
73
N
H
N
S
I
9
N
N
101
O
O
10
11
1.20
H
N
NH2
O
S
18
9.43
3.76
127
73
O
O
0.980
NH2
N
19a
12
13
8.59
4.58
16
20
I
N
O
O
S
NH
20a
21a
22b
23b
24
0.113
0.106
0.030
0.093
5.34
101
127
101
127
105
101
N
O
O
O
S
a
H2N
NH
Yield of the final step only.
O
S
O
significantly higher TPSAs than rimonabant 1 (rimonabant’s TPSA
is 50 and the TPSAs for 14 and 15 are 101 and 127, respectively).
With compounds 14 and 15 in hand, attempts were made to im-
prove potency for these CB1 receptor antagonists while maintain-
ing high TPSAs. Analogs that contain a rigid element in the amide
functionality were targeted in hopes of improving potency. The ini-
tial partially constrained analogues 16–18 had little or no activity.
However, our laboratories had previously discovered that more po-
lar functionality could be tolerated if linkers of sufficient length are
used.16 Therefore, compounds 19–21, which have longer spacers
(X, Scheme 2), were targeted. Compounds 20 and 21, as a 1:1 cis/
trans mixture, were functionally potent (Ke ꢀ100 nM) and bound
CB1 with high affinity (Ki ꢀ10 nM).
NH
O
O
S
H2N
NH
N
HN
O
S
O
O
O
S
NH
Compounds 22 and 23, enriched in the cis- isomer (cis:trans
ꢀ7:1) compared to 20 and 21 and were demonstrated to be even
more potent.23 Compound 24 suggested that basic spacer groups
with sulfonamides were not tolerated. Basic groups at the terminus
of the linker were not tolerated (16, 19) either. Compounds 25–28
were prepared to study the effect of in the nature of the spacer on
activity. Unfortunately, aromatic groups used as spacers were
deemed detrimental for CB1 activity. Further exploration of con-
strained analogues is under way.
25
2.93
O
O
S
H2N
NH
26
27
0.376
2.83
127
101
H
N
O
S
O
Select compounds (Table 3) were chosen for further study in
radioligand displacement assays using radiolabeled SR141716
([3H]1). Several of these compounds demonstrated excellent Ki
(continued on next page)

5714
A. Fulp et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5711–5714
Table 2 (continued)
Acknowledgments
Compound
R
Ke CB1 (
l
M)
tPSA
127
The authors would like to thank David Perrey for graciously pro-
viding all of acid A that was used in this work. We would also like to
thank Anne Gilliam for her assistance in performing binding assays.
Patricia Reggio helped in our understanding of possible membrane
penetration issues faced by our charged compounds. We express
our gratitude to the NIDA drug supply program for providing radi-
olabeled probes. This research was funded by research grants
1R21AA019740-01 and 1R03AA017514-01 to R.M. from NIAAA.
H
N
O
S
O
28
4.20
NH2
a
Compounds isolated are approximately 1:1 mixture of cis and trans isomers.
Compounds are 7:1 mixture of cis/trans isomers. A 7:1 mixture of 15 was iso-
lated after careful column chromatography. Assignment of relative stereochemistry
was tentatively made by H1NMR. Further efforts to isolate pure cis- and trans-
isomers are ongoing.
b
References and notes
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Table 3
Radioligand displacement data for select compounds
No Ki (
l
M) CB1
Ki (
CP55940
l
M) CB1
Ki (
CP55940
l
M) CB2
CB2:CB1
CP55940
SR141716
5
7
8
9
0.013
0.061
0.026
0.786
0.056
0.294
0.102
1.698
0.158
0.049
0.055
0.135
0.036
0.107
1.74
4.52
4.01
2.27
0.78
1.01
0.90
2.31
0.96
1.79
31
15
39
1.3
4.9
21
16
17
27
17
14 0.060
15 0.020
20 0.011
21 0.021
22 0.008
23 0.015
values in the low nM range, with 22 having a Ki of 8 nM. Selectivity
against the CB2 receptor was determined by comparing the com-
pound displacement of radiolabeled CP55940 at CB1 and CB2
receptors. In general, most compounds were selective for CB1 over
CB2.
Charged compounds generally showed poor activity in the cal-
cium flux assay. However, an interesting piece of data was the good
affinity of compound 7 for CB1 versus 3H-SR141716 (Ki of 61 nM)
in contrast to its Ke = 1.39 lM for calcium flux. An explanation
for this disparity could be that binding of 7 induced a conforma-
tional change to the CB1 receptor, which in turn selectively abro-
gated signaling through the Gaq16 pathway coupled to calcium
12. Chen, R. Z.; Frassetto, A.; Lao, J. Z.; Huang, R. R.; Xiao, J. C.; Clements, M. J.;
Walsh, T. F.; Hale, J. J.; Wang, J.; Tong, X.; Fong, T. M. Eur. J. Pharmacol. 2008,
584, 338.
13. Clark, D. E.; Pickett, S. D. Drug Discovery Today 2000, 5, 49.
14. Seltzman, H. H.; Carroll, F. I.; Burgess, J. P.; Wyrick, C. D.; Burch, D. F. J. Labelled
Compd. Radiopharm. 2002, 45, 59.
15. Zhang, Y.; Burgess, J. P.; Brackeen, M.; Gilliam, A.; Mascarella, S. W.; Page, K.;
Seltzman, H. H.; Thomas, B. F. J. Med. Chem. 2008, 51, 3526.
16. Zhang, Y.; Gilliam, A.; Maitra, R.; Damaj, M. I.; Tajuba, J. M.; Seltzman, H. H.;
Thomas, B. F. J. Med. Chem. 2010, 53, 7048.
17. Huang, S.; Wong, J. C.; Leung, A. K.; Chan, Y. M.; Wong, L.; Fernendez, M. R.;
Miller, A. K.; Wu, W. Tetrahedron Lett. 2009, 50, 5018.
18. Fang, Y. Q.; Yuen, J.; Lautens, M. J. Org. Chem. 2007, 72, 5152.
19. Jones, C., Pass, M., Rudge, D. World Intellectual Property Organization Patent;
Patent Appl. PCT Int. Appl. WO 2,007,138,277, 2007.
20. Each compound was pharmacologically characterized using
a functional
mobilization. This interesting result needs further investigation
in the future.
fluorescent CB1 activated G q16-coupled intracellular calcium mobilization
a
assay in CHO-K1 cells as has been described in our previous publication and
apparent affinity (Ke) values were determined.16 Further characterization of
select compounds was performed using radioligand displacement of [3H]1 and
equilibrium dissociation constant (Ki) values were determined.16 Selectivity of
these compounds at CB1 versus CB2 was also determined by obtaining Ki values
at either receptor using displacement of [3H]CP55940 in membranes of CHO-
K1cells over-expressing either receptor. Data reported are average values from 3
to 6 measurements.
Two compounds were selected to advance into an in vitro mod-
el of brain penetration. The MDCK-mdr1 (Madin-Darby Canine Kid-
ney (MDCK) stably expressing mdr1 (multi-drug resistance gene
1)) cell line has been found to be useful in identifying compounds
that passively distribute into the CNS.24,25 When compounds 22
and 23 were tested in the MDCK-mdr1 transport assay they were
found to have less than 1% transport from apical to basal side of
the membrane.26 This would be consistent with little are no brain
penetration if no active transport was taking place. By contrast,
rimonabant and otenabant6 were used as control compounds and
these compounds were transported ꢀ15% and ꢀ90% across
MDCK-mdr1 cells, respectively.
In conclusion, two classes of CB1 antagonists were rationally
targeted for exclusion from the CNS. First, compounds with perma-
nent charge were synthesized and tested. However, these com-
pounds showed poor activity in the calcium flux assay. Charge at
other positions on the pyrazole ring is being explored. Compounds
with TPSAs greater than rimonabant have been synthesized. Polar
compounds with sufficient potency and selectivity have been iden-
tified. These compounds have poor in vitro permeability and ap-
pear to be more promising for further development and
refinement. Select compounds are being advanced into in vivo
experiments.
21. Kosterlitz, H. W.; Lees, G. M.; Wallis, D. I.; Watt, A. J. Br. J. Pharmacol. 1968, 34,
691P.
22. Cheng, L., Jonforsen, M., Schell, P. Patent Appl. PCT Int. Appl. WO 2,007,010,217,
2007.
23. Efforts to isolate diastereomerically pure compounds are ongoing.
24. Wang, Q.; Rager, J. D.; Weinstein, K.; Kardos, P. S.; Dobson, G. L.; Li, J.; Hidalgo, I.
J. Int. J. Pharm. 2005, 288, 349.
25. Garberg, P.; Ball, M.; Borg, N.; Cecchelli, R.; Fenart, L.; Hurst, R. D.; Lindmark, T.;
Mabondzo, A.; Nilsson, J. E.; Raub, T. J.; Stanimirovic, D.; Terasaki, T.; Oberg, J.
O.; Osterberg, T. Toxicol. In Vitro 2005, 19, 299.
26. MDCK-mdr1 cells were grown on Transwell type filters (Corning) for 4 days to
confluence. Compounds were added to the apical side at a concentration of
3.16 lM and incubated for 1 h at 37 °C. Compounds selected for MDCK-mdr1
cell assays were infused on an Applied Biosystems API-4000 mass
spectrometer to optimize for analysis using multiple reaction monitoring
(MRM). Flow injection analysis was also conducted to optimize for mass
spectrometer parameters. Samples from the apical and basolateral side of the
MDCK cell assay were dried under nitrogen on
a Turbovap LV. The
chromatography was conducted with an Agilent 1100 binary pump with a
flow rate of 0.5 mL/min. Mobile phase solvents were A, 0.1% formic acid in
water, and B, 0.1% formic acid in methanol. The initial solvent conditions were
10% B for 1 min, then a gradient was used by increasing to 95% B over 5 min,
then returning to initial conditions.