Discovery of S-444823, a potent CB1/CB2 dual agonist as an antipruritic agent

Article abstract of DOI:10.1016/j.bmcl.2012.02.050

The optimization of a series of 3-carbamoyl 2-pyridone derivatives as CB agonists is reported. These efforts resulted in the discovery of 3-(2-(1-(cyclohexylmethyl)-2-oxo-1,2,5,6,7,8,9,10-octahydrocycloocta[b] pyridine-3-carboxamido)thiazol-4-yl)propanoic acid (21), a potent dual CB1/CB2 agonist without CNS side effects induced by CB1 receptor activation. It exhibited strong inhibition of scratching as a 1.0% acetone solution in the pruritic model.

Full text of DOI:10.1016/j.bmcl.2012.02.050

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2898–2901
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of S-444823, a potent CB1/CB2 dual agonist as an antipruritic agent
⇑
Masahide Odan , Natsuki Ishizuka, Yoshiharu Hiramatsu, Masanao Inagaki, Hiroshi Hashizume,
Yasuhiko Fujii, Susumu Mitsumori, Yasuhide Morioka, Masahiko Soga, Masashi Deguchi, Kiyoshi Yasui,
Akinori Arimura
Shionogi Pharmaceutical Research Center, Shionogi & Co., Ltd, 1-1, Futaba-cho 3-chome, Toyonaka, Osaka 561-0825, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The optimization of a series of 3-carbamoyl 2-pyridone derivatives as CB agonists is reported. These
efforts resulted in the discovery of 3-(2-(1-(cyclohexylmethyl)-2-oxo-1,2,5,6,7,8,9,10-octahydrocyclooc-
ta[b]pyridine-3-carboxamido)thiazol-4-yl)propanoic acid (21), a potent dual CB1/CB2 agonist without
CNS side effects induced by CB1 receptor activation. It exhibited strong inhibition of scratching as a
1.0% acetone solution in the pruritic model.
Received 23 January 2012
Revised 16 February 2012
Accepted 17 February 2012
Available online 23 February 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Cannabinoid
CB receptor agonist
GPCR
Antipruritics
Cannabinoid (CB) receptors are G-protein coupled receptors
(GPCRs). In mammalian tissues, two types of receptors, CB1 and
CB2, have been identified and cloned.1,2 CB1 receptor is mainly ex-
pressed in the central nervous system (CNS)3,4 and is responsible
for most of the psychoactive activities of the cannabinoids.5 CB2
receptor is mostly located in immune cells6 and is responsible for
many of the immunomodulatory and anti-inflammatory effects
of cannabinoids.7 Differences in cannabinoid receptor distribution,
their signaling mechanism and structural requirements for selec-
tive activation provide occasion for selective pharmacotherapeutic
targeting. Development of ligands acting on the CB receptor should
offer promising therapeutic agents for various disorders associated
with CB receptors. Some CB1 and CB1/CB2 dual agonists have dem-
onstrated potential clinical efficacy in treating pain, glaucoma,
depression and GI disorders.8,9 However, their agonists display
undesirable CB1 mediated CNS side effects such as dizziness, dry
mouth, tiredness/fatigue, muscle pain and palpitations.10,11 To
avoid unfavorable side effects on the CNS, polar functional groups
such as carboxylic acid were introduced to restrict blood–brain
barrier (BBB) permeability.12,13
previously reported. As shown in Figure 1, most CB ligands have
bulky side chains with a carbamoyl or carbonyl linker. With these
structural features in mind, we carried out a structure–activity
relationship (SAR) study focusing on the 3-position of 3-carbamoyl
2-pyridone of 1 to introduce a bulky group.
To explore the C-3 side chain, pyridone derivatives were pre-
pared from commercially available cyclooctanone (2) via a com-
mon intermediate 4 as shown in Scheme 1. Cyclooctanone (2)
was treated with cyclohexyl methylamine in refluxing toluene to
give imine in situ and diethyl ethoxymethylene malonate was re-
acted with reflux to give compound 3.15 Hydrolysis of compound
3 and subsequent coupling of the resulting carboxylic acid 4 with
amino acid alkyl esters afforded corresponding amide derivatives
1a, 5a–21a (50–99%), which were then deprotected to give carbox-
ylic acids with various X groups (5–21) in 66–99%.
The binding affinities of compounds 5–9 are summarized in Table
1. Substitution of the aliphatic group such as isopropyl: 5 and cyclo-
hexyl: 6 did not enhance affinities for CB receptors. On the other
hand, substitution of aromatic group 7 and 8 vastly improved activ-
ities for CB receptors except for cAMP of CB1. However, these com-
pounds proved to be partial agonists in cAMP assay. As a more
bulky group, benzyl derivative 9 displayed moderate affinities. From
these results, insertion of a phenyl ring seemed to enhance the affin-
ities for CB receptors in spite of having carboxylic acid.
Encouraged by these results, we explored some compounds
having an aromatic ring as a linker to carboxylic acid (Table 2).
Benzoic acid derivatives (10–12) exhibited weak affinity in the
binding assay. However, insertion of a methylene linker between
the phenyl ring and carboxylic acid resulted in enhancement of
In our previous paper,14 we reported the discovery of a potential
CB 1/2 agonist 1 having carboxylic acid on the 3-carbamoyl 2-pyri-
done core (Fig. 1). Compound 1 showed weak affinities for both
CB1 and CB2 receptors with a Ki value of 1354 and 349 nM, respec-
tively. As a clue to designing increased affinities for CB receptors,
we took into account the chemical structure of several CB ligands
⇑
Corresponding author.
E-mail address: masahide.oodan@shionogi.co.jp (M. Odan).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.02.050

M. Odan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2898–2901
2899
O
O
N
H
N
O
N
O
O
N
Cl
4
N
O
3
5
6
HO
N
H
2
O
1 N
WIN55212-2
SR144528
O
1
O
Ki(hCB1): 1354 nM
Ki(hCB2): 349 nM
H2N
N
N
H
N
H
O
N
O
N
H
O
O
O
OH
JTE-907
PF-03550096
Figure 1. Structures of lead compound 1 and reported CB receptor ligands.
O
O
O
O
O
R1
X
R1
a, b
d
O
N
N
2
3: R1 = OMe
4: R1 = OH
1a, 5a, 6a, 9a−21a : R1 = OMe
1, 5, 6, 9−21 : R1 = OH
7a: R1 = -tBu
c
e
f
7: R1 = -OH
8a: R1 = -OBn
8: R1 = -OH
g
Scheme 1. Reagents and conditions: (a) cyclohexyl metylamine, toluene, reflux. (b) Diethyl ethoxymethylene malonate, toluene, reflux. (c) Aqueous NaOH, THF. (d) Oxalyl
chloride, DMF, THF followed by amino acid alkylester hydrochloride, Et3N. (e) aq NaxOH, THF. (f) TFA. (g) H2, Pd/C, MeOH.
Table 1
Structures and CB receptor affinities of the 3-carbamoyl 2-pyridone derivatives with
a-amino acid introduced at the 3-position
O
X
N
H
O
N
Compd
X
Ki (hCB1) (nM)a
Ki (hCB2) (nM)a
IC50 (nM) CB1 (cAMP)b
IC50 (nM) CB2 (cAMP)b
HO
1
1354
349
—
—
O
O
HO
HO
5
6
1877
3062
116
111
—
—
>2000
51
O
(continued on next page)

2900
M. Odan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2898–2901
Table 1 (continued)
Compd
X
Ki (hCB1) (nM)a
Ki (hCB2) (nM)a
IC50 (nM) CB1 (cAMP)b
IC50 (nM) CB2 (cAMP)b
7
8
34
1.2
>2000
5.7
HO
O
O
O
69
3.0
>2000
297
4.4
HO
HO
9
738
33
126
a
See Ref. 15 for assay protocol.
See Ref. 16 for assay protocol.
b
Table 2
Structures and CB receptor affinities of the 3-carbamoyl 2-pyridone derivatives introduced an amino acid with an aromatic ring as a linker
O
X
N
H
O
N
Compd
X
Kia (hCB1) (nM)
Kia (hCB2) (nM)
IC50 (nM) CB1 (cAMP)b
IC50 (nM) CB2 (cAMP)b
10
2526
181
657
167
HO
HO
O
11
12
1557
370
614
107
611
291
268
38
O
O
HO
HO
13
548
46
—
—
O
O
14
15
2880
3987
120
74
997
180
96
HO
HO
>2000
O
O
HO
HO
16
17
—
923
—
—
S
>5000
>5000
>2000
nd
N
S
O
O
HO
18
>5000
885
>2000
800
N

M. Odan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2898–2901
2901
Table 2 (continued)
Compd
X
Kia (hCB1) (nM)
Kia (hCB2) (nM)
IC50 (nM) CB1 (cAMP)b
398
IC50 (nM) CB2 (cAMP)b
O
S
HO
19
20
425
22
11
N
O
HO
235
580
4.6
18
23
3.5
S
N
HO
S
21
454
15
O
N
a
See Ref. 16 for assay protocol.
See Ref. 17 for assay protocol.
b
References and notes
200
150
100
250
200
150
100
50
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0
Control Vehicle 19
Control Vehicle
21
Figure 2. Antipruritic effects of compounds 19 and 21. ⁄P <0.01, ⁄⁄P <0.05, control:
n = 29, vehicle: n = 5 (mice).
the activities (13–15). In a similar manner, while compounds 17
and 18 having a thiazole linker led to an essentially inactive com-
pound, the insertion of a methylene linker led to significant
improvement (19–21). Unfortunately, compound 20 was found to
be a partial agonist, which was not suitable for our research target.
On the basis of these results, we selected compounds 19 and 21
as CB1/2 dual agonists for further in vivo profiling. In order to inves-
tigate the CB1-induced CNS side effects, compounds 19 and 21 were
tested by in vivo psychoactive testing.18 These compounds were
administered by injection at 1.0 and 0.1 mg/kg into test mice and
no CNS side effects such as sedation or catalepsy were observed.
Next, these compounds were subjected to the painting test in our
pruritic mouse model (Fig. 2).19 Compounds 19 and 21 showed sig-
nificant antipruritic effects in mice at 1.0% painting, with the inhibi-
tion of 109% and 99%, respectively. The data indicated that CB 1/2
dual agonists having carboxylic acid had the efficacy for the anti-
pruritics without CNS side effects induced by CB1 agonistic activity.
In summary, we explored the SAR of 3-carbamoyl 2-pyridone
derivatives having carboxylic acid as CB receptor agonists. Optimi-
zation was focused on the substituent at the 3-position of pyridone.
Among them, compounds 19 and 21 showed potent affinities for CB
receptors and displayed the strong inhibition of scratching induced
by compound 48/80 without CNS side effects caused from activat-
ing CB1 receptor. These efforts led to the discovery of clinical can-
didate 21, S-444823, as the agent for atopic diseases.
15. Bondavalli, F.; Bruno, O.; Presti, E. L.; Menozzi, G.; Mosti, L. Synthesis 1999, 7,
1169.
16. Binding assay: CB receptor binding assay was carried out using the membrane;
recombinant human CB1 (hCB1), CB2 (hCB2), radioligand [3H]-CP55940.
Membrane fractions, used for the measurement of binding activity, were
prepared as reported elsewhere and stored in a deep freezer (À80 °C). In brief,
confluent cultures of the hCB1 and hCB2 cells were harvested. The harvested
cells were sonicated in a buffer for membrane suspensions (membrane buffer:
20 mM Tris–HCl pH 7.4, 2 mM EDTA, 0.25 M sucrose containing protease
inhibitor) on ice, and centrifuged at 3000 rpm for 10 min at 4 °C. The
supernatants were centrifuged at 100,000 g for 60 min at 4 °C. The pelleted
membrane fractions were homogenized in the membrane buffer, and stored in
a deep freezer (À80 °C). The Kd values of [3H]-CP55940 for each membrane
fraction were determined by Scatchard plot analysis.
17. Cyclic AMP assay method: The CHO cells expressing hCB1 or hCB2 were seeded
into 24-well plates. The cells were incubated at 37 °C for 20 min with
compounds in the cAMP assay buffer (Hanks’ solution with 20 mM HEPES,
0.1 mM IBMX, 0.2 mM Ro20-1724, 0.1% BSA). The cells were stimulated with
4 lM forskolin at 37 °C for 25 min (hCB1 cells) or 45 min (hCB2 cells). The
cAMP concentrations in the cells were measured using cAMP kits (CIS Bio
International).
18. Estimation of in vivo psychoactivity: In order to qualitatively estimate the level
of CB1 induced CNS side effects, test compounds were dissolved in MDAA and
PEG solution and intravenously injected into the tail of the ICR mice (0.1 and
1.0 mg/kg, n = 3 each). The apparent behavior of each mouse was observed at
15 min after injection and the score (5: Tonic convulsion; 4: Catalepsy; 3:
Prone position, Sedation; 2: Crawling; 1: Decrease in locomotor activity; 0:
Normal) was determined. The higher the score, the more potent are the CNS
side effects.
19. In vivo assay (pruritic model): Crj:CD-1 (ICR) (Japan Charles River Lab.) mice
were used for scratching test to investigate the antipruritic effect. Test
compounds were dissolved in acetone (Sigma). Compound 48/80 as
pruritogen was dissolved at 60 g/mL in isotonic saline (Otsuka Pharma.).
Test compounds were painted on the shaved back of the mice. After 15 min,
50 l of pruritogen was injected intradermally into the back of the mice. Their
behavior was then videotaped to count the scratching behavior for 30 min. The
inhibition value was calculated by the equation:
a
l
Acknowledgments
l
%
%
The authors thank Drs. Hiroki Sato and Yutaka Yoshida of this
manuscript for many helpful suggestions.
inhibition = (B À A) À (C À A)/(B À A/100); scratching of control (n = 29) as A,
scratching of vehicle (n = 6) as B, scratching of test mice (n = 6) as C.