Arylsulfonamide CB2 receptor agonists: SAR and optimization of CB2 selectivity

Article abstract of DOI:10.1016/j.bmcl.2008.01.042

A high-throughput screening campaign resulted in the discovery of a highly potent dual cannabinoid receptor 1 (CB1) and 2 (CB2) agonist. Following a thorough SAR exploration, a series of selective CB2 full agonists were identified.

Full text of DOI:10.1016/j.bmcl.2008.01.042

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1725–1729
Arylsulfonamide CB2 receptor agonists:
SAR and optimization of CB2 selectivity
b,
a
a
Monika Ermann,a,* Doris Riether, Edward R. Walker, Innocent F. Mushi,
*
James E. Jenkins,a Beatriz Noya-Marino,a Mark L. Brewer,a Malcolm G. Taylor,a
Patricia Amouzegh,a Stephen P. East,a Brian W. Dymock,a Mark J. Gemkow,c
Andreas F. Kahrs,c Andreas Ebneth,c Sabine Lo¨bbe,c Kathy O’Shea,b
Daw-Tsun Shihb and David Thomsonb
aEvotec (UK) Ltd., 114 Milton Park, Abingdon, Oxfordshire OX14 4SA, United Kingdom
bBoehringer Ingelheim Pharmaceuticals Inc., 900 Ridgebury Road, P.O. Box 368, Ridgefield, CT 06877, USA
cEvotec AG, Schnackenburgallee 114, 22525 Hamburg, Germany
Received 3 December 2007; revised 11 January 2008; accepted 12 January 2008
Available online 18 January 2008
Abstract—A high-throughput screening campaign resulted in the discovery of a highly potent dual cannabinoid receptor 1 (CB1)
and 2 (CB2) agonist. Following a thorough SAR exploration, a series of selective CB2 full agonists were identified.
ꢀ 2008 Elsevier Ltd. All rights reserved.
The therapeutic usage of cannabis can be dated back to
ancient dynasties of China and since then it has been
administered for the treatment of various indications
including lack of appetite, emesis, cramps, spasticity,
pain and rheumatism.1 The long history of cannabis
therapy has prompted the development of several phar-
maceutical agents, such as Marinolꢁ and Cesametꢁ.
Some of the physiological effects of these drugs are med-
iated by at least two G-protein coupled receptors, can-
nabinnoid receptor 1 (CB1) and 2 (CB2).2 The CB1
receptor is expressed predominantly in the central ner-
vous system and it regulates the release of neurotrans-
mitters from pre-synaptic neurons.3 CB1 is believed to
mediate most of the euphoric and other CNS effects of
cannabis. In contrast, the CB2 receptor is expressed al-
most exclusively in the periphery,4 particularly in cells
and tissues involved in immune response.5 Selective ago-
nists of CB2 have been shown to suppress inflammation
in vivo6 as well as inhibiting disease severity and spastic-
ity in an animal model of multiple sclerosis.7,8 Addition-
ally, CB2 agonists have been shown to inhibit
inflammatory and neuropathic pain and emesis.9–12 Po-
tent and selective inverse agonists acting on the CB2
receptor have also been reported and they are active in
inflammatory models.13
Development of selective CB2 receptor agonists may
avoid the undesirable psychoactive effects associated
with the CB1 receptor and could potentially be used
for the modulation of the inflammatory response as well
as for the treatment of pain. Several CB2 selective ago-
nists and modulators have been reported in the litera-
ture such as 114 and GW405833 (2)15 (Fig. 1) as well
as others.16–18 Recently, a novel class of pyrimidines
was described19 and the lead compound GW842166X
(3) has entered clinical trials for inflammatory pain.
The potential utility of selective CB2 receptor agonists
for the treatment of several therapeutic indications
encouraged us to screen our compound collection
against this target. This paper describes the identifica-
tion of an initial screening hit and the subsequent opti-
mization efforts, which resulted in a series of
compounds with excellent selectivity profiles.
Keywords: Arylsulfonamide; Cannabinoid receptor; CB1; CB2; Ago-
nists; Selectivity.
*
Corresponding authors. Tel.: +44 1235 441372 (M.E.), +1 203 791
6203 (D.R.); e-mail addresses: monika.ermann@evotec.com; doris.
riether@boehringer-ingelheim.com
From our high-throughput screening programme, the
commercially available arylsulfonamide 4 (Fig. 2) was
0960-894X/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.042

1726
M. Ermann et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1725–1729
potency, however 5 still lacked selectivity for CB2 over
O
O
O
CB1. In an effort to achieve selectivity, an extensive
SAR study of amide substitution was undertaken. The
synthesis of these compounds is outlined in Scheme 1,
route A.25 Starting from commercially available 4-meth-
ylbenzoic acid, treatment with chlorosulfonic acid pro-
vided the corresponding sulfonyl chloride. Reaction of
this sulfonyl chloride with piperidine was followed by
amide bond formation under standard conditions to
give amides 5–25 (Table 2).
N
O
O
N
HO
OH
O
O
Cl
Cl
1
2
CF3
O
Other fused aromatic amines such as isoquinoline 6 were
found to be tolerated in terms of potency on CB2,
although the pattern of substitution in these analogues
is important as demonstrated by a drop in potency
and efficacy for compound 7. Additionally, only modest
levels of selectivity could be achieved with these com-
pounds. Replacement of the fused system with a single
unsubstituted phenyl ring resulted in a loss of potency
however this could be regained by introducing substitu-
ents into the aromatic ring, preferably in the ortho-posi-
tion as illustrated by compound 9. It is noteworthy that
substitution in the para-position of the aromatic ring is
detrimental to activity as demonstrated by 12. Small po-
lar, electron-withdrawing groups in the ortho-position,
as exemplified by compounds 15 and 16, appear to be
beneficial for CB2 potency. Importantly, compounds
15 and 16 indicated that selectivity could be improved
to >50-fold.
Cl
N
NH
N
N
H
Cl
O
3
Figure 1. Structures of CB2-selective agonists.
O
O
S
N
N
O
O
4
Figure 2. High-throughput screening hit 4.
identified.20,21 Compound 4 demonstrated high binding
affinity for both the CB2 (Ki = 4 nM) and the CB1
(Ki = 3 nM) receptor, evaluated on the basis of
[3H]-CP-55940 binding to membranes of HEK cells
expressing human CB1 or CB2.22 The ability of 4 to
inhibit cyclic adenosine monophosphate (cAMP) pro-
duction was determined in a cellular assay employing
CB2 over-expressing CHO cells stimulated with for-
skolin and it was characterized as a potent full agonist
(<1 nM). In our CB1 cAMP assay, 4 was also character-
ized as a potent full agonist (Table 1).23,24 As we were
interested in CB2 selective agonists, we decided to focus
on the functional assay, rather than binding assay, to
drive the SAR.
Replacing the aromatic substituent with aliphatic
groups provided compounds with good CB2 potency,
e.g. 17, 18, 20, 21 although their selectivity over CB1
was still only modest. Disubstitution of the amide nitro-
gen, through introduction of a methyl group, e.g. 19 was
tolerated in the aliphatic series (unlike the aromatic ser-
ies (10)) although a drop in potency of about 10-fold
was observed. Nevertheless, this prompted an investiga-
tion into other tertiary amides and piperidine amide 22
suggested that selectivities of >200-fold could be
achieved. Confirmation of tertiary amides as being ben-
eficial to the selectivity profile was illustrated by the
introduction of the decahydroisoquinoline moiety as
indicated by 24. This compound has a CB2 EC50 of
1.5 nM with ꢀ4000-fold selectivity over CB1. The
trans-diastereoisomer 25 appeared to be the more potent
isomer.
In addition to a poor CB1/2 selectivity profile, com-
pound 4 also suffered from low chemical stability which
was attributed to ease of ester cleavage. Replacement of
the ester to give the amide analogue 5 improved the
chemical stability and maintained excellent CB2
With the optimized decahydroisoquinoline amide, the
SAR around the sulfonamide bond (Table 3) was ex-
plored to determine its effects on potency and selectivity.
To enable the rapid evaluation of this region of the mol-
ecule, the order of the synthetic steps was altered as out-
lined in Scheme 1, route B.25 This facilitated the
introduction of the sulfonamide substituent in the last
step. Replacement of the piperidine with primary
amines, compounds 26–28, generally resulted in a reduc-
tion in potency, which was most significant for aromatic
amine 26. In contrast, a range of secondary cyclic
amines are well tolerated in this position. Expansion
or reduction of the ring size (compounds 29–31) quickly
established that 4–7 membered systems provided
compounds with excellent CB2 potency and selectivity
Table 1. Cellular potency, efficacy and selectivity over CB1 for CB2
agonists
Compound
CB2 cAMP
EC50 [nM]
Efficacy
[%]
S
(CB1)a
2
3
4
0.65b
63c
44
95
99
n/a
>470
1
<1
a S (CB1) refers to selectivity (x-fold) over CB1 in cAMP agonism
assays.
b Literature data, see Ref. 15.
c Literature data from a reporter gene assay in yeast, see Ref. 19.

M. Ermann et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1725–1729
1727
Table 2. Amide SAR
O
O
S
O
N
N
1 R
R2
Compound
R1
R2
CB2 cAMP EC50 [nM]
Efficacy [%]
S (CB1)
5
8-Quinolinyl
1-Isoquinolinyl
H
H
H
H
H
4
100
98
45
74
89
—
80
117
91
76
92
96
58
86
86
73
85
85
82
95
93
7
25
6
1
7
2-Quinolinyl
Phenyl
195
139
5
n/a
6
8
9
2-Chlorophenyl
2-Chlorophenyl
3-Chlorophenyl
4-Chlorophenyl
2-Methoxyphenyl
2-Methylphenyl
2-Trifluoromethylphenyl
2-Methylsulfonylphenyl
1-Methylcyclohexane
Cyclohexyl
13
—
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Methyl
H
>20000
27
14100
21
154
2
31
—
H
H
9
H
1.5
96
H
H
6
66
H
18
14
119
5
n/a
24
H
Cyclohexyl
Methyl
H
38
(S)-1-Cyclohexylethyl
(R)-1-Cyclohexylethyl
Piperidine
2
45
H
18
82
6
>240
94
Decahydroquinoline
Decahydroisoquinoline
trans-Decahydroisoquinoline
1.5
1
4326
6894
O
O S
O
O S
N
O
(i)
(ii)
(iii, iv)
O
O S
CO2H
A
B
CO2H
N
N
N
1 R
5-25
R2
CO2H
Cl
O
O S
Cl
O
O S
N
O
O
O
(v)
(vi)
(vii)
N
N
3 R
R4
COCl
26-36
Scheme 1. Reagents and conditions: (i) chlorosulfonic acid, 65 ꢂC, 88%; (ii) piperidine, MeCN, 0 ꢂC to rt, 52%; (iii) SOCl2, DMF (cat.); (iv)
R1R2NH, DIPEA, CH2Cl2, ꢀ75% (two steps); (v) decahydroisoquinoline, MeCN, rt, 90%; (vi) chlorosulfonic acid, dichloroethane, 80 ꢂC, 90%; (vii)
R3R4NH, DIPEA, MeCN, rt, ꢀ70%.
profiles. Introduction of a heteroatom into the ring sys-
tem, such as in morpholine 32 or thiomorpholine 33 was
also well tolerated (potency and selectivity), however
addition of a hydroxyl group as in compound 35 re-
duced the potency. The basic centre in compound 36
was detrimental to activity.
thesis of compounds 37–45 proceeded by adaptation of
the routes illustrated in Scheme 1 (not shown);25 R3 and
R4 were fixed as pyrrolidine for this study. Substitution
in the 4-position was found to be essential for activity as
exemplified by the unsubstituted analogues 38 and 44
which are considerably less potent (EC50 > 20000 nM)
in the cAMP cellular assay. Replacement of the methyl
in 37 or 29 with an isopropyl group as shown with 39
and 41 gave compounds with improved potency and
similar selectivity profiles, however, further increasing
In parallel to the extensive amide and sulfonamide SAR
study, we also investigated the role of the substituent in
the 4-position (R5) of the phenyl core (Table 4). The syn-

1728
M. Ermann et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1725–1729
Table 3. Sulfonamide SAR
O
O
S
O
N
N
3 R
R4
Compound
R3
R4
CB2 cAMP EC50 [nM]
Efficacy [%]
S (CB1)
24
26
27
28
29
30
31
32
33
34
35
36
Piperidine
Phenyl
1.5
>20000
95
—
4326
—
H
H
H
Isopropyl
Isobutyl
2284
14
4
101
96
0.1
939
Pyrrolidine
Azetidine
94
94
2238
>1500
3276
2638
3247
7744
37
13
2
Homopiperidine
Morpholine
100
104
90
5
Thiomorpholine
Dioxothiomorpholine
4-Hydroxypiperidine
N-Isopropylpiperazine
1
2
99
86
215
588
45
n/a
Table 4. SAR of phenyl substitution
R5
O
O
N
S
N
O
1 R
R2
Compound
R5
R1
R2
CB2 cAMP EC50 [nM]
Efficacy [%]
S (CB1)
37
38
39
40
29
41
42
43
44
45
Methyl
H
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
H
H
H
H
21
>20000
81
—
86
74
94
101
—
93
—
89
181
—
Isopropyl
Chloro
Methyl
Isopropyl
Cyclohexyl
Chloro
H
4
60
85
3
Decahydroisoquinoline
Decahydroisoquinoline
Decahydroisoquinoline
Decahydroisoquinoline
4
0.4
2238
2864
—
>20000
4
520
—
trans-Decahydroisoquinoline
trans-Decahydroisoquinoline
>20000
3
Chloro
3768
the size and lipophilicity of the substituents as in com-
pound 42 resulted in a significant loss of potency. A
reduction in potency was also observed when an elec-
tron-donating substituent was introduced, such as a
methoxy group (data not shown). The effect of elec-
tron-withdrawing substituents were demonstrated by
the chloro-substituted analogues 40, 43 and 45 and gen-
erally resulted in compounds with good potency and
selectivity profiles.
ping binding sites can be proposed based on the muta-
genesis studies.26,27 Compound 29 demonstrated Ki’s
of 130 nM against [3H]-CP-55940 and 20 nM against
[3H]-WIN-55212-2. This suggests a better overlap be-
tween 29 and WIN-55212-2 within the binding pocket.
In a CB1 binding assay using [3H]-CP-55940 as a probe,
29 did not demonstrate any competition up to 5000 nM.
In conclusion, an SAR exploration of a non-selective,
chemically unstable high-throughput screening hit re-
sulted in the discovery of new series of highly selective
CB2 receptor agonists. Several compounds with selectiv-
ities, in functional agonism assays, of >2000-fold over
CB1 were identified. Further optimization and in vivo
evaluation of compounds from this chemical series will
be reported in due course.
Compound 29 was tested further for its affinity to the
human CB2 receptor in a competitive radiolabeled
ligand binding assay using either [3H]-CP-55940 or
[3H]-WIN-55212-2 as probes. Both probes were used
to evaluate whether this class of compounds adapt a
CP- or WIN-like binding mode as two partially overlap-

M. Ermann et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1725–1729
1729
20. Arylsulfonamide 4 was independently reported as a CB1
agonist during the course of our work: Lambeng, N.;
Lebon, F.; Christophe, B.; Burton, M.; De Ryck, M.;
References and notes
1. Voth, E. A.; Schwartz, R. H. Ann. Intern. Med. 1997, 126,
791.
´
2. Pacher, P.; Batkai, S.; Kunos, G. Pharmacol. Rev. 2006,
58, 389.
´ ´
Quere, L. Bioorg. Med. Chem. Lett. 2007, 17, 272.
21. Adolor Corp. WO2007/058960.
22. Human CB2 and CB1 receptor binding assays: CB2 and
CB1 membranes were isolated from HEK cells stably
transfected with the respective receptors. The membrane
preparation was bound to scintillation beads (Ysi-Poly-L-
lysine SPA beads, GE Healthcare) in assay buffer con-
taining 50 mM Tris, pH 7.5, 2.5 mM EDTA, 5 mM
MgCl2, 0.8% fatty acid free Bovine Serum Albumin.
Unbound membrane was removed by washing in assay
buffer. Membrane–bead mixture was added to 96-well
assay plates in the amounts of 2 lg membrane per well
(CB2) or 2.5 lg per well (CB1) and 1 mg SPA bead per
well. Compounds were added to the membrane–bead
mixture in dose-response concentrations ranging from
1 · 10À5 M to 1 · 10À10 M. The competition reaction was
initiated with the addition of [3H]-CP-55940 (Perkin-
Elmer Life and Analytical Sciences) at a final concentra-
tion of 4 nM (CB2) or 2.5 nM (CB1), or [3H]-WIN-55212-2
at 1.6 nM. IC50 values for each compound were calculated
as the concentration of compound that inhibits the specific
binding of the radioactively labeled ligand to the receptor
by 50% using the XLFit 4.1 four-parameter logistic model.
IC50 values were converted to inhibition constant (Ki)
values using the Cheng-Prusoff equation.
3. Howlett, A. C.; Breivogel, C. S.; Childers, S. R.; Dead-
wyler, S. A.; Hampson, R. E.; Porrino, L. J. Neurophar-
macology 2004, 47, 345.
4. Munro, S.; Thomas, K. L.; Abu-Shaar, M. Nature 1993,
365, 61.
5. Klein, T. W. Nat. Rev. Immunol. 2005, 5, 400.
6. Ashton, J. C. Curr. Opin. Invest. D. 2007, 8, 373.
7. Croxford, J. L.; Miller, S. D. J. Clin. Invest. 2003, 111,
1231.
8. Fernandez-Ruiz, J.; Romero, J.; Velasco, G.; Tolon, R.
M.; Ramos, J. A.; Guzman, M. Trends Pharmacol. Sci.
2007, 28, 39.
9. Whiteside, G. T.; Lee, G. P.; Valenzano, K. J. Curr. Med.
Chem. 2007, 14, 917.
10. Cheng, Y.; Hitchcock, S. A. Expert Opin. Invest. Drug
2007, 16, 951.
11. Guindon, J. Hohmann, A. G. Br. J. Pharmacol.
doi:10.1038/sj.bjp.0707531.
12. Yao, B. B.; Hsieh, G. C.; Frost, J. M.; Fan, Y.; Garrison,
T. R.; Daza, A. V.; Grayson, G. K.; Zhu, C. Z.; Pai, M.;
Chandran, P.; Salyers, A. K.; Wensink, E. J.; Honore, P.;
Sullivan, J. P.; Dart, M. J.; Meyer, M. D. Br. J.
Pharmacol. doi:10.1038/sj.bjp.0707568.
23. CB2 and CB1 cAMP assays: CHO cells expressing human
CB2 or CB1 (Euroscreen) were plated at a density of 5000
cells per well in 384-well plates and incubated overnight at
37 ꢂC. After removing the media, the cells were treated
with test compounds diluted in stimulation buffer con-
taining 1 mM IBMX, 0.25% BSA and 10 lM forskolin.
The assay was incubated for 30 min at 37 ꢂC. Cells were
lysed and the cAMP concentration was measured using
DiscoveRx XS+ cAMP kit, following the manufacturer’s
protocol. The maximal amount of cAMP produced by
forskolin compared to the level of cAMP inhibited by
200 nM CP-55940 is defined as 100%. The EC50 value of
each test compound was determined as the concentration
at which 50% of the forskolin-stimulated cAMP synthesis
was inhibited using a four-parameter logistic model.
24. Reproducibility of the cAMP assays is assessed using the
control compound CP-55940 which is tested twice on
every assay plate to rule out any plate artefacts. Efficacy at
CB1 and CB2 is expressed as a percentage relative to the
efficacy of CP-55940. Each compound is tested in triplicate
at least three times with individual dilutions from the
stock. The results reported in the table are the mean values
of the measurements and individual values for the
compounds do not differ by more than a factor three
from the mean.
13. Lavey, B. J.; Kozlowski, J. A.; Shankar, B. B.; Spitler, J.
M.; Zhou, G.; Yang, D. Y.; Shu, Y.; Wong, M. K.; Wong,
S. C.; Shih, N. Y.; Wu, J.; McCombie, S. W.; Rizvi, R.;
Wolin, R. L.; Lunn, C. A. Bioorg. Med. Chem. Lett. 2007,
17, 3760.
14. Pharmos Corp. WO2006/043260.
15. Valenzano, K. J.; Tafesse, L.; Lee, G.; Harrison, J. E.;
Boulet, J. M.; Gottshall, S. L.; Mark, L.; Pearson, M. S.;
Miller, W.; Shan, S.; Rabadi, L.; Rotshteyn, Y.; Chaffer,
S. M.; Turchin, P. I.; Elsemore, D. A.; Toth, M.;
Koetzner, L.; Whiteside, G. T. Neuropharmacology 2005,
48, 658.
16. Kai, H.; Morioka, Y.; Murashi, T.; Morita, K.; Shino-
nome, S.; Nakazato, H.; Kawamoto, K.; Hanasaki, K.;
Takahashi, F.; Mihara, S.; Arai, T.; Abe, K.; Okabe, H.;
Baba, T.; Yoshikawa, T.; Takenaka, H. Bioorg. Med.
Chem. Lett. 2007, 17, 4030.
´
17. Page, D.; Yang, H.; Brown, W.; Walpole, C.; Fleurent,
M.; Fyfe, M.; Gaudreault, F.; St-Onge, S. Bioorg. Med.
Chem. Lett. 2007, 17, 6183.
18. Ohta, H.; Ishizaka, T.; Tatsuzuki, M.; Yoshinaga, M.;
Iida, I.; Tomishima, Y.; Toda, Y.; Saito, S. Bioorg. Med.
Chem. Lett. 2007, 17, 6299.
19. Giblin, G. M. P.; O’Shaughnessy, C. T.; Naylor, A.;
Mitchell, W. L.; Eatherton, A. J.; Slingsby, B. P.;
Rawlings, D. A.; Goldsmith, P.; Brown, A. J.; Haslam,
C. P.; Clayton, N. M.; Wilson, A. W.; Chessell, I. P.;
Wittington, A. R.; Green, R. J. Med. Chem. 2007, 50,
2597.
1
25. All compounds described gave satisfactory H NMR and
LC-MS analytical data.
26. Reggio, P. H. Curr. Pharm. Des. 2003, 9, 1607.
27. Tuccinardi, T.; Ferrarini, P. L.; Manera, C.; Ortore, G.;
Saccomanni, G.; Martinelli, A. J. Med. Chem. 2006, 49, 984.