CB 1/2 dual agonists with 3-carbamoyl 2-pyridone derivatives as antipruritics: Reduction of CNS side effects by introducing polar functional groups

Article abstract of DOI:10.1016/j.bmcl.2012.02.054

Our lead compound 1 showed high affinity for both CB1 and CB2 receptors, suggesting the possibility of inducing psychoactive side effects through the CB1 receptor in the brain. To solve this issue, polar functional groups were introduced at the 3-position of the pyridone core of compound 1 to find CB1/2 dual agonists such as 17 and 20 which did not show any CNS side effects.

Full text of DOI:10.1016/j.bmcl.2012.02.054

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2894–2897
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
CB 1/2 dual agonists with 3-carbamoyl 2-pyridone derivatives as antipruritics:
Reduction of CNS side effects by introducing polar functional groups
⇑
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:
Our lead compound 1 showed high affinity for both CB1 and CB2 receptors, suggesting the possibility of
inducing psychoactive side effects through the CB1 receptor in the brain. To solve this issue, polar func-
tional groups were introduced at the 3-position of the pyridone core of compound 1 to find CB1/2 dual
agonists such as 17 and 20 which did not show any CNS side effects.
Received 23 January 2012
Revised 16 February 2012
Accepted 17 February 2012
Available online 3 March 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Cannabinoid
CB receptor agonist
GPCR
Antipruritics
Pruritus can be defined as an unpleasant cutaneous sensation
associated with the immediate desire to scratch. It may be con-
strued as part of the body’s defense mechanism by which we deal
with potentially dangerous organisms or stimuli. However, chronic
or strong scratching leads to the development of skin lesions and
the release of inflammatory mediators that induce or aggravate
pruritus resulting in further scratching. Pruritus is a common
symptom in dermatology and is induced by factors such as inflam-
mation, cancer, infection, psychiatric diseases, drug application,
and stress. There are many reports indicating the existence of an
interactive network between the skin and not only the peripheral
but also the central nervous system to regulate and respond to pru-
ritic stimuli.1 This means that specific sensory nerves and their
receptors are involved in the pathophysiology of pruritus. Recently,
among them, the cannabinoid (CB) receptor was reported to be
implicated in an anti-inflammatory and anti-nociceptive action be-
cause of its abundant distribution on skin nerve fibers and mast
cells.2 Furthermore, the cannabinoid agonist (HU-210) was re-
ported to attenuate histamine-induced itch by peripheral adminis-
tration.3 These findings suggest that the CB agonist could be useful
against the various pruritic diseases such as atopic dermatitis.
CB receptors are G-protein coupled receptors (GPCRs). Two
types of receptors, CB1 and CB2, have been identified in
mammalian tissues and were cloned.4,5 The CB1 receptors are
O
O
4
3
5
6
O
N
2
H
O
1 N
Ki(hCB1): 1.7 nM
Ki(hCB2): 0.3 nM
1
Figure 1. Structure of lead compound 1.
widely expressed in the brain with the highest density in the cells
of the basal ganglia, hippocampus and cerebellum.6 They are also
found in various peripheral tissues including the gastrointestinal
tract, pancreas, liver, kidney, prostate, testis, ovary, eye, lungs
and heart.7,8 In the CNS, the CB1 receptor is responsible for cogni-
tion, memory and sensory perception, while in the periphery, it
plays an important role in maintaining energy balance, metabo-
lism, nociception and cardiovascular health.9,10 On the other hand,
the CB2 receptor is mainly located in the periphery and is
associated with the cells of immune system11,12 and, to a far lesser
extent in the brain.13 Recent data indicate that the CB2 receptor is
responsible for the control of peripheral pain14 and inflamma-
tion.15 However, undesirable CNS side effects induced by
D
9-tetra-
hydrocannabinol (
D
9-THC), the major psychoactive component of
marijuana, are believed to be mainly mediated by the central
CB1 receptor.16 In order to avert central CB1 effects, research
efforts have been focused on highly selective CB2 agonists and
peripherally restricted CB1 or CB1/CB2 dual agonists.
⇑
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.2012.02.054

M. Odan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2894–2897
2895
O
O
O
O
O
R2
R3
R1
N
a, b
d
f
N
H
H
O
O
O
O
N
N
N
2
3: R1 = OEt
4: R1 = OH
1: R2 = OMe
5: R2 = OH
c
e
6-11, 12a, 13, 14, 15a, 16-18
12a, 15a: R3 = OMe
g
12, 15: R3 = OH
Scheme 1. Reagents and conditions: (a) cyclohexyl metylamine, toluene, reflux. (b) diethyl ethoxymethylene malonate, toluene, reflux. (c) aq. NaOH, THF. (d) Oxalyl chloride,
cat. DMF, THF followed by dimethyl glycine methyl ester hydrochloride, Et3N. (e) aq. NaOH, THF. (f) (COCl) 2, cat. DMF,THF followed byamines or hydrazines, Et3N. (g) aq.
NaOH, THF.
O
O
O
O
O
O
O O
S
H
HO
H2NHN
N
N
N
N
N
H
H
H
H
a
b
O
O
O
N
N
N
5
19
20
Scheme 2. Reagents and conditions: (a) NH2NH2 H2O, WSCD, HOBt, DMF. (b) MeSO2Cl, Et3N, THF.
Our lead compound 1 from our compound library showed high
affinity for both CB1 and CB2 receptors with Ki values of 1.7 and
0.3 nM, respectively (Fig. 1). To evaluate the CNS side effects of
our compounds, the psychoactivity scoring test17 was performed,
and compound 1 exhibited strong CNS side effects such as cata-
lepsy. Considering the CNS side effects by CB1 activation, exploring
CB2 selective agonists would be one option. Alternatively, the CB1/
2 dual agonist with poor brain penetration could eliminate the CNS
side effect, especially when the compounds are administered top-
ically. In this study, we adopted the latter approach by introducing
a polar functional group at the 3-position of the pyridone core
which restricts BBB permeability. Topological polar surface area
(tPSA)18 was utilized as the index to analyze the polarity of our
compounds. This knowledge led to the finding of the clinical can-
didate of S-444823 and S-777469. The SAR of these compounds
will be described in subsequent reports.19
The compounds used in this study were prepared as shown in
Schemes 1 and 2. Cyclooctanone (2) was treated with cyclohexyl
methylamine in refluxing toluene to give imine in situ, then diethyl
ethoxymethylene malonate was reacted with refluxing to give 3
with a 3-carbamoyl 2-pyridone skeleton.20 Hydrolysis of com-
pound 3 afforded the core compound 4. The carboxylic acid 4
was converted to acid chloride then condensed with dimethyl gly-
cine methyl ester to give lead compound 1. The key intermediate 5
was obtained by alkaline hydrolysis of 1 (Scheme 1). Compound 5
was converted to acid chloride as described above, then coupled
with several amines to give the amide derivatives 6–11, 12a, 13,
14, 15a, 16–18 (46–99%). Hydrolysis of esters (12a and 15a) affor-
ded the corresponding carboxylic acids 12 and 15 in 99% and 85%,
respectively.
so we next focused on the amide derivative with a polar functional
group at the chain terminal. As a clue of incorporation of the polar
group, compound 6, replacing by the partial structure of ananda-
mide,21 possessed affinities as high as 1. In addition, other amide
derivatives 7–9 with a terminal hydroxyl group also showed strong
affinities for CB1 receptors, while compound 10 with a terminal
amino group had decreased affinities. When the terminal carboxyl-
ate or amino functional group was converted to the corresponding
–CONH2 or acetamide, the affinities were comparable with the ter-
minal –OH analogues (11 and 13). Piperidine analogues (14, 15 and
17) with a functional group at the 4-position of the piperidine ring
also showed comparable affinity as found with open chain
analogues.
With some potent analogues in hand, we next examined the CNS
side effects responsible for CB1 affinity. To qualitatively estimate
the level of CB1-induced CNS side effects, we conducted the psycho-
activity scoring (PS) test using mice.17 The apparent behaviors of
mice (n = 3) at 15 min after intravenous injection were evaluated
with the score based on Table 2. PS (0 6 PS 6 10) represents the to-
tal of the average score at two doses (1.0 mg/kg and 0.1 mg/kg).
tPSA values were also calculated as physicochemical parameters
to estimate the polarities of each molecule. The relationships be-
tween PS and tPSA are shown in Table 3.
The lead compound 1, which has a tPSA value of 75.71 Å2, in-
duced spontaneous catalepsy by intravenous injection, exhibited
very strong CNS activity (PS = 9.5) as expected. Terminal alcohol
derivatives, such as 6 and 14, which have the same tPSA value
(89.95 Å2) improved the PS score, causing mild catalepsy
(PS = 6.7 and 3.6, respectively). The compounds with much higher
tPSA value (7–9) also reduced the CNS side effects (PS 63.6) in
spite of high affinities for CB1 receptors as the lead compound 1.
Compound 11, 13 and 16, with carboxamide derivatives at the
terminal position, have tPSA values over 100 Å2 and displayed a
mild CNS side effect. From these results, there seems to be a good
inverse relationship between PS and tPSA (Fig. 2) suggesting that
the introduction of a polar functional group may successfully re-
duce the CNS effects caused by CB1 receptor affinity.
The syntheses of 19 and 20 are outlined in Scheme 2. Com-
pound 19 was obtained by coupling compound 5 with hydrazine
(81%). Treatment of 19 with methanesulfonyl chloride provided
20 (80%).
The affinities for CB1/CB2 of the compounds 1–20 were evalu-
ated and the Ki values are shown in Table 1. Carboxylic acid 5
showed very weak affinities for both hCB1 and hCB2 receptors
compared with the corresponding methyl ester 1, suggesting that
carboxylic functionality is not tolerated to keep the affinities. This
trend was also found for other carboxylic acids, such as 12 and 15,
Encouraged by the above findings, we next introduced a
sulfonyl moiety as a more polar functional group. Sulfonamide
17 had slightly weak affinity for CB1 receptor compared with 16.

2896
M. Odan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2894–2897
Table 1
Table 2
Affinities for CB1 and CB2 receptor
The score for the level of CB1-induced side effects
O
O
Status
Score
R
O
Spasm, rigor
Catalepsy
Stretching the body without moving
Walking with lying on the belly
Moving sluggishly
5
4
3
2
1
0
N
H
N
Moving actively
Compd
R
Ki (hCB1) (nM)a
Ki (hCB2) (nM)a
MeO
HO
1
5
1.7
0.3
1351
349
Table 3
N
6
7
8
9
2.9
6.3
5.8
3.4
0.6
1.0
1.2
0.6
Psychoactive score (PS) and tPSA
HO
HO
a
tPSA (Å2)b
H
N
Compd
PS
1
6
7
8
9.5
6.7
3.6
2.5
1.9
2.2
0.2
3.6
0.2
0
75.71
89.95
98.74
H
N
HO
HO
98.74
H
N
9
107.97
107.61
121.6
O
11
13
14
16
17
19
20
H
N
89.95
10
11
242
4.7
81
N
O
112.81
115.89
104.53
124.68
H
N
3.0
0
2.5
N
H
O
a
See Ref. 17 for assay protocol.
Calculated by ChemDraw Ultra 9.0.7.
H
N
b
12
13
>5000
11
2823
6.0
HO
O
H
N
H2N
HO
10
9
8
7
6
5
4
3
2
1
0
14
15
8.0
1.1
N
O
HO
2970
377
N
O
H2N
16
17
18
4.9
39
3.0
4.4
128
N
H
N
S
O
O
N
O
O
S
O
70
90
110
130
tPSA
N
218
H
N
Figure 2. Relationship between psychoactive score (PS) and tPSA.
H
N
19
20
30
32
2.8
7.3
H2N
and showed no side effects (PS = 0). The representative compounds
(1, 6 and 20) were evaluated for their ability to penetrate the CNS
in rats. Compound 1 that showed strong catalepsy exhibited good
brain penetrability with brain/plasma ratio of 3.35. Compound 6,
by converting the ester moiety to a more polar functional group
exhibited a lower brain/plasma ratio (0.28). Compound 20 without
CNS side effects had a very low brain/plasma ratio (0.02). These re-
sults supported the relationship between PS and brain/plasma
ratio.
The selected compounds 17 and 20 without CNS side effects
were tested with our pruritic model induced by Compound 48/80
(Fig. 3).23 Painting at 1.0% of these compounds strongly inhibited
scratching with percent inhibition levels of 97% and 87%,
respectively.
O
S
O
H
N
N
H
a
See Ref. 22 for assay protocol.
Compound 18, with acyl sulfonamide introduced as the more polar
functional group, showed loss of affinities. In the case of hydra-
zides, 19 and 20 showed moderate affinities without improving
the affinity for CB1 receptors. Compound 17 with a high tPSA value
(115.89 Å2) showed no CNS side effect on injection to mice (PS = 0).
While hydrazide 19 represented medium PS (PS = 3.0), its
methanesulfonylated compound 20 increased tPSA (tPSA = 124.68)

M. Odan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2894–2897
2897
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50
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Glass, M.; Zimmer, A. Eur. J. Pharmacol. 2000, 396, 141.
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Marzo, V.; Pittman, Q. J.; Patel, K. D.; Sharkey, K. A. Science 2005, 310, 329.
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150
100
*
*
50
0
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Y.; Sugiura, T. J. Biol. Chem. 2005, 280(18), 18488.
17. Psychoactivity scoring test (Psychoactive score: PS): 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: Normal) was determined. The higher the score, the
more potent were the CNS side effects.
0
Control
Vehicle
17
Control
Vehicle
20
Figure 3. Inhibition scratching of compound 17 and 20 in the pruritic model
induced by Compound 48/80 in mice.
In summary, hypothesizing that addition of polar functional
groups would limit CNS exposure, we successfully decreased the
side effects by incorporating polar functional groups on the termi-
nal side chain of the 3-position on the lead compound 1. Com-
pounds 13, 16, 17 and 20 with higher tPSA values (tPSA >110)
displayed little psychoactive side effect. Compound 17 and 20, in
particular, showed no CNS side effects via intravenous administra-
tion in mice. Both strongly inhibited scratching induced by Com-
pound 48/80 in the pruritic model. This study showed that it is
possible to reduce the CB1-induced CNS side effects of CB1/2 dual
agonists to treat atopic disease.
18. Peter, E.; Bernhard, R.; Paul, S. J. Med. Chem. 2000, 43, 3714.
19. (a) Odan, M.; Ishizuka, N.; Hiramatsu, Y.; Inagaki, N.;Hashizume, H.; Fujii, Y.;
Mitsumori, S.; Morioka, Y.; Soga, M.; Deguchi, M.; Yasui, K.; Arimura, A. Bioorg.
Med. Chem. Lett. accepted for publication, doi:10.1016/j.bmcl.2012.02.050; (b)
Odan, M.; Ishizuka, N.; Hiramatsu, Y.; Inagaki, N.;Hashizume, H.; Fujii, Y.;
Mitsumori, S.; Morioka, Y.; Soga, M.; Deguchi, M.; Yasui, K.; Arimura, A. Bioorg.
Med. Chem. Lett., doi:10.1016/j.bmcl.2012.02.072.
20. Bondavalli, F.; Bruno, O.; Presti, E. L.; Menozzi, L.; Mosti, L. Synthesis 1999, 7,
1169.
21. Structure of 2-arachidonoylethanolamine (anandamide)
O
HO
N
H
Acknowledgments
22. 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 100,000g 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.
The authors thank Drs. Hiroki Sato and Yutaka Yoshida of this
manuscript for many helpful suggestions.
References and notes
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B. R.; Rice, K. C. Proc. Natl. Acad. Sci U.S.A. 1990, 87(5), 1932.
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R. G. Pharmacol. Rev. 2002, 54(2), 161.
23. In vivo assay (pruritic model): Crj:CD-1 (ICR) (Japan Charles River Lab.) mice
were used for scratching tests 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 mice. After fifteen
minutes, 50 l of pruritogen was injected intradermally into the back of the
a
l
l
mice. Thereafter, their behavior was videotaped to count the scratching
behavior for 30 min. The value of% inhibition was calculated by fitting the
equation:% inhibition = (B–A) – (C–A)/(B–A/100); scratching of the control
(n = 29) as A, scratching of vehicle (n = 6) as B, scratching of test mice (n = 6) as
C.
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