Discovery of S-777469: An orally available CB2 agonist as an antipruritic agent

Article abstract of DOI:10.1016/j.bmcl.2012.02.072

The discovery of novel CB2 ligands based on the 3-carbamoyl-2-pyridone derivatives by adjusting the size of side chain at 1-, 5- and 6-position is reported. The structure-activity relationship around this template lead to the identification of S-777469 as a selective CB2 receptor agonist, which exhibited the significant inhibition of scratching induced by Compound 48/80 at 1.0 mg/kg po and 10 mg/kg po (55% and 61%, respectively).

Full text of DOI:10.1016/j.bmcl.2012.02.072

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2803–2806
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Bioorganic & Medicinal Chemistry Letters
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Discovery of S-777469: An orally available CB2 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 discovery of novel CB2 ligands based on the 3-carbamoyl-2-pyridone derivatives by adjusting the
size of side chain at 1-, 5- and 6-position is reported. The structure–activity relationship around this tem-
plate lead to the identification of S-777469 as a selective CB2 receptor agonist, which exhibited the sig-
nificant inhibition of scratching induced by Compound 48/80 at 1.0 mg/kg po and 10 mg/kg po (55% and
61%, respectively).
Received 23 January 2012
Revised 16 February 2012
Accepted 23 February 2012
Available online 3 March 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Cannabinoid
CB receptor agonist
GPCR
Antipruritics
CB1 is mainly expressed in neurons in the brain1,2 and is
responsible for most the psychoactive properties of cannabinoids.
CB2 is abundant in splenocytes and leukocytes3 and is responsible
for many of the immunomodulatory and anti-inflammatory effects
of cannabinoids.4 Because of the predominant peripheral distribu-
tion of the CB2 receptor, CB2 selective agonists are very attractive
therapeutic agents without CB1 mediated psychoactive effects.
Recently, various chemical scaffolds having high affinity and
selectivity for CB2 receptor have been reported. Preclinical data
support the effects of these cannabinoids in a wide range of pain
models, including neuropathic pain and chronic pain. In spite of
the high CB2 selectivity, most of these compounds exhibit CB1
mediated side effects at higher doses. For example, CB2 selective
ligand A-836339 (Abbott) possesses high hCB2 affinity (0.64 nM)
with 425-fold selectivity against hCB15 and does not display signif-
icant affinity at other GPCRs and ion channels. However, at high
doses, A-836339 displayed brain CB1 mediated decrease of sponta-
neous motor activity.6
the need to discover a safer CB agonist having high CB2 selectivity
and CNS exposure restriction.
Recently, we reported on CB receptor agonists having carboxylic
acid at the 3-position in the 3-carbamoyl-2-pyridone system
(Fig. 1). Among these compounds, compound 1 exhibited CB2
selectivity (Ki (hCB1): 793 nM, Ki (hCB2): 6.2 nM). However, in
spite of good selectivity for CB2 (>130) and the presence of carbox-
ylic acid which could limit BBB penetration, compound 1 displayed
CNS side effects in our psychoactive test (PS = 0.7).11 It seemed that
the high lipophilicity of compound 1 facilitated passage through
the BBB and could induce CNS side effects by activating CB1 recep-
tors. Therefore, we directed our attention to reducing the lipophil-
icity by using ClogP as an index to design an analogue of
compound 1 which would not show any CNS side effects.
The syntheses and structures of the compounds in this Letter
were presented in Scheme 1 and Table 1. As a general synthetic
scheme, amines were reacted with various ketones in refluxing tol-
uene to give the corresponding imines in situ, then diethyl
ethoxymethylene malonate was reacted with refluxing to give
3-carbamoyl-2-pyridones 1a–16a (24–98%).12 Hydrolysis of com-
pound 1a–16a afforded the core compound 1b–16b (47–98%).
Carboxylic acid 1b–16b were converted to acid chloride then con-
densed with the appropriate amino acid methyl esters to give com-
pound 1c–16c (66–97%). Compound 1–16 were obtained by
alkaline hydrolysis of the corresponding esters (75–98%).
Cannabinoid ligands often have highly lipophilic structures that
facilitate passage through the blood–brain barrier (BBB). One way
to limit CNS exposure is to add polar functional groups such as car-
boxylic acid. Ajulemic acid, CB1/CB2 dual agonist, exhibits analge-
sic and anti-inflammatory activity.7–9 However, some CNS side
effects were displayed. This compound may pass through the
BBB and activate the central CB1 receptor.10 These findings indicate
First, we investigated various Z groups to reduce ClogP. We uti-
lized various a-amino acids in the pyridone ring-constructing step
to introduce various Z groups (1–6). The binding affinities of these
compounds with different Z groups are summarized in Table 2.
⇑
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.2012.02.072

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M. Odan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2803–2806
O
O
4
3
5
HO
CO2H
O
N
H
2
S
O
OH
N
6
N
1
N
O
O
1
Ki(hCB1) / Ki(hCB2) (nM)]
793 / 6.2
A-836339
Ajulecim acid
CLogP: 7.56
CLogP: 5.76
IC50[cAMP(CB1) / cAMP(CB2) (nM)]
468 / 1.0
CLogP: 7.32
Figure 1. Structures of the lead compound 1.
O
O
R2
Y
Z
R2
R3
a, b
d
O
X
N
H
R1 NH2
R3
O
R2
O
N
R3
O
N
R1
R1
1a–16a: X = OEt
1b–16b: X = OH
1c–16c:Y = OMe
1–16:Y = OH
c
e
Scheme 1. Reagents and conditions : (a) toluene, reflux. (b) diethyl ethoxymethylene malonate, toluene, reflux. (c) aq NaOH, THF. (d) oxalyl chloride, cat. DMF, THF followed
by amino acid methyl ester, Et3N. (e) aq NaOH, THF.
Unsubstituted methylene derivative 2 showed decreased affin-
ities for CB receptors, especially for the CB2 receptor, compared
with compound 1. Substitution with small alkyl groups such as
Table 1
Structure of compounds in this manuscript
the gem-dimethyl group did not improve the affinities. Among
Compd
Z
R1
R2
R3
the spiro-derivatives, cyclohexylidene analogue 5 had high affini-
ties as compound 1. Cyclohexyl substituted congener 6 resulted
in reduction of affinities. These results suggest that a spiro-frame-
work and an appropriate ring size are crucial for the affinities for
CB receptors. In our psychoactive test, compound 5 did not show
CNS side effects (PS = 0).11 Judging from the ClogP value, com-
pound 5 had still high lipophilicity (ClogP = 6.20), so we next ex-
plored the possibility of further reducing the lipophilicity by
modification of other moieties.
To reduce the lipophilicity, less lipophilic R1 groups were intro-
duced. The binding affinities of the compounds having several side
chains at the 1-position of pyridone skeleton are summarized in
Table 3.
The compound substituted with n-butyl: 7 exhibited moderate
affinity and strong IC50 value for CB2 receptor. Interestingly, the
affinity for CB1 receptor was drastically reduced in comparison
with 5. However, the introduction of methoxyethyl moiety: 8 led
to marked reduction in affinities for CB2 receptor compared with
7. As another approach to reduce the lipophilicity, introduction of
a polar group such as tetrahydrofuranyl methyl: 9 and N-ethyl pyr-
rolidinyl methyl: 10 led to marked reduction in affinities for CB
receptors. In the case of a compound having a benzyl group with
slightly reduced ClogP, introduction of p-fluorobenzyl: 11 led to
moderate affinities for CB receptors compared with 5. In contrast,
p-cyano derivative 12 led to low affinities for CB receptors. From
these data, the 1-position of the pyridone skeleton was restricted
at the lipophilic site and did not tolerate the polar groups. However,
these results suggest that reduction of lipophilicity at the 1-position
contributes to improve the selectivity for CB2 against CB1.
To optimize the 5- and 6-substituents of the pyridone skeleton,
we directed our attention toward opening the 8-membered ring to
reduce lipophilicity. The CB receptors affinities of those com-
pounds (13–16) are shown in Table 4.
CH2
6
1
CH2
CH2
CH2
CH2
CH2
2
3
4
5
6
6
6
6
6
6
CH2
CH2
CH2
CH2
7
8
9
6
6
6
O
O
10
6
N

M. Odan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2803–2806
2805
Table 1 (continued)
160
Compd
Z
R1
R2
R3
140
120
100
80
CH2
CH2
*
*
11
12
6
6
* *
**
F
* *
60
CN
40
13
14
Me
Br
Et
20
0
n-Pr
Control Vehicle 0.3
1
3
10
10
30
Compound 15
(mg/kg)
Fexofenadine
(mg/kg)
15
16
Me
Et
Et
F
F
Figure 2. Evalution of 15 with the pruritic model of the ICR mice. Compound 15
was dosed after injection of Compound 48/80 as a pruritogen. n = 9–11 per group.
Data are expressed as a mean percentage of vehicle SEM and the results of two
experiments. ⁄P <0.05 versus vehicle by Welch test.
n-Pr
The n-butyl (13 and 14) or p-fluorobenzyl (15 and 16) group
was selected as the R1 substituent due to their favorable CB2 affin-
ity and the selectivity against CB1 receptor for optimization. For
analogues with the n-butyl group at the 1-position, ring opening
resulted in loss of affinities for CB receptors (13 and 14). However,
when the p-fluorobenzyl group was used as R1, compound 15
exhibited moderate potency for CB2 receptor and excellent selec-
tivity (>120). In addition, ClogP of compound 15 was dramatically
reduced to 4.25. The reduction of lipophilicity might avert the
undesired CNS side effects (PS = 0). Introducing the longer alkyl
chain 16 led to decreased affinities for CB receptors.
The selected compound 15 was further evaluated with the
in vivo animal model.15 The inhibition of scratching induced by
Compound 48/80 is shown in Figure 2. In this test, compound 15
was compared with Fexophenadine, which is a type of antihista-
mine. Fexophenadine was mild inhibition of scratching at 30 mg/
kg (39%). On the other hand, compound 15 revealed significant
inhibitions when dosed at 1.0 mg/kg (46%), 3.0 mg/kg (48%) and
10 mg/kg (59%).
Table 2
ClogP and CB receptor affinities of compound 1–6
Compd ClogP
Kib (hCB1) Kib (hCB2) IC50c (nM) CB1 IC50 (nM)c CB2
a
(nM)
(nM)
(cAMP)
(cAMP)
1
2
3
4
5
6
7.32
4.27
4.89
5.64
6.20
6.70
793
1056
1354
258
181
3062
6.2
177
349
56
6.7
111
468
—
—
113
76
>2000
1.0
—
—
14
7.0
51
a
b
c
Calculated by ChemDraw Ultra 9.0.7.
See Ref. 13 for assay protocol.
See Ref. 14 for assay protocol.
Table 3
ClogP and CB receptor affinities of compound 7–12
Compd ClogPa Kib (hCB1) Kib (hCB2) IC50 (nM)CB1 IC50 (nM) CB2
c
c
(nM)
(nM)
(cAMP)
(cAMP)
The data indicated that CB2 selective agonists had the antipru-
ritic effect. The pharmacokinetic properties of 15 were evaluated in
several species (Table 5). Compound 15 had moderate clearance
and absorption across species.
In summary, we discovered novel CB2 receptor agonists con-
taining a 3-carbamoyl-2-pyridone system with carboxylic acid at
the 3-position. Among them, we selected compound 15 (S-
777469) as a CB2 selective agonist for development. This com-
pound is considered to be a promising candidate for an orally ac-
tive antipruritic.
5
7
8
9
10
11
12
6.20
5.14
3.77
3.99
2.61
5.46
4.75
181
6.7
76
>2000
—
—
—
514
—
7.0
7.0
—
—
—
>5000
>5000
>5000
—
266
3952
56
294
247
2114
16
9.0
—
144
a
b
c
Calculated by ChemDraw Ultra 9.0.7.
See Ref. 13 for assay protocol.
See Ref. 14 for assay protocol.
Table 4
ClogP and CB receptor affinities of compound 13–16
Table 5
Pharmacokinetic Properties of 15
Compd ClogPa Kib (hCB1) Kib (hCB2) IC50c (nM) CB1 IC50c (nM) CB2
Clearance (mL/min/kg)a
t1/2 (h)
Vdss (L/Kg)
BAd (%)
b
c
Species
(nM)
(nM)
(cAMP)
(cAMP)
Rat
11.3
6.00
13.6
6.20
12.8
7.43
1.90
1.23
0.87
37.9
18.7
9.01
13
14
15
16
3.92
4.98
4.25
5.304
—
>5000
4607
348
382
36
—
—
>2000
—
—
—
24
—
Dogc
Monkeyd
>5000
263
a
b
c
iv dose at 3.0 mg/kg.
Oral dose at 10 mg/kg.
Beagle dog.
a
b
c
Calculated by ChemDraw Ultra 9.0.7.
See Ref. 13 for assay protocol.
See Ref. 14 for assay protocol.
d
Cynomolgus monkey.

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M. Odan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2803–2806
Normal) was determined. The higher the score, the more potent are the CNS
Acknowledgments
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12. Bondavalli, F.; Bruno, O.; Presti, E. L.; Menozzi, G.; Mosti, L. Synthesis 1999, 7,
1169–1174.
The authors thank Drs. Hiroki Sato and Yutaka Yoshida for many
helpful suggestions.
13. 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
References and notes
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a buffer for membrane suspensions
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14. 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
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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
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were used for the scratching test to investigate the antipruritic effect. Test
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11. 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 mg/kg
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:
compounds were dissolved in acetone (Sigma). Compound 48/80 as
pruritogen dissolved at 75 g/ml in isotonic saline (Otsuka Pharma.). Test
compounds were administered as forced oral dosages to mice. After 1 h, 40
a
l
ll
of pruritogen was injected intradermally into the back of mice. Thereafter,
their behavior was videotaped to count the scratching behavior for 30 min. The
% inhibition value was calculated with the equation: % inhibition = (B–A) – (C–
A)/(B–A/100); scratching of control (n = 29) as A, scratching of vehicle (n = 6) as
B, scratching of test mice (n = 6) as C.