The SAR studies of novel CB2 selective agonists, benzimidazolone derivatives

Article abstract of DOI:10.1016/j.bmcl.2008.04.032

Benzimidazolone derivatives were discovered as novel CB2 selective agonists. Structure Activity Relationship (SAR) studies around them were examined to improve metabolic stability. Compound 39 exhibited excellent metabolic stability in human liver microsomes (HLM) and significant attenuation of the chronic colonic allodynia in the TNBS-treated rats by po administration.

Full text of DOI:10.1016/j.bmcl.2008.04.032

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Bioorganic & Medicinal Chemistry Letters 18 (2008) 3310–3314
The SAR studies of novel CB2 selective agonists,
benzimidazolone derivatives
Hirofumi Omura,a,* Makoto Kawai,a Akiko Shima,a Yasuhiro Iwata,a Fumitaka Ito,a
Tsutomu Masuda,a Atsuko Ohta,a Naoya Makita,a Kiyoyuki Omoto,a Hiromi Sugimoto,b
Akira Kikuchi,b Hiroshi Iwatab and Kazuo Andoa
aDiscovery Chemistry Research, Pfizer Global Research & Development, Nagoya Laboratories, Pfizer Japan Inc., 5-2 Taketoyo,
Aichi 470-2393, Japan
bDiscovery Biology Research, Pfizer Global Research & Development, Nagoya Laboratories, Pfizer Japan Inc., 5-2 Taketoyo,
Aichi 470-2393, Japan
Received 16 February 2008; revised 6 April 2008; accepted 11 April 2008
Available online 15 April 2008
Abstract—Benzimidazolone derivatives were discovered as novel CB2 selective agonists. Structure Activity Relationship (SAR)
studies around them were examined to improve metabolic stability. Compound 39 exhibited excellent metabolic stability in human
liver microsomes (HLM) and significant attenuation of the chronic colonic allodynia in the TNBS-treated rats by po administration.
Ó 2008 Elsevier Ltd. All rights reserved.
A human cannabinoid receptor 2 (hCB2)1,2 was cloned
in 1993 and has been known to express mainly in the
cells of immune systems. In contrast, CB1 receptor3 is
expressed principally in the central nervous system
(CNS) and responsible for the overt physiological effect
of cannabinoids. CB2 selective agonists4 represented by
naphthalene as the lipophilic moiety and morpholine
as the amine.
The benzimidazolone derivatives (8 and 9) were synthe-
sized in four steps starting from 2-fluoronitrobenzene (5)
(Scheme 1). Morpholinoethylamine was introduced by
SNAr reaction followed by reduction to give diamino-
benzene 6. The subsequent carbonylation with 1,10-car-
bonyldiimidazole (CDI) gave benzimidazolone 7. The
external urea formation was carried out via carbonate
intermediate using p-nitrophenyl chloroformate fol-
lowed by addition of 1-naphthylamine or its non-ani-
line12 analog, (R)-1,2,3,4-tetrahydro-1-naphthylamine.
This procedure is useful for the syntheses of various
5
AM1241 (2) and HU-3086 were reported to mediate
peripheral antinociception without CNS related side-ef-
fects.5,7 Therefore, CB2 receptor has a potential to be a
promising drug target. In this paper, we report the SAR
studies of novel CB2 selective agonists, 1,3-dihydro-2H-
benzimidazol-2-one (benzimidazolone) derivatives, for
the treatment of chronic colonic allodynia.8
The representative CB2 agonists (1–4),5,9–11 are listed in
Figure 1. These compounds have a couple of common
structural features such as an amine (e.g., piperidine or
morpholine) at 1-position and a lipophilic moiety at 3-
position on the indole or indazole cores. In order to find
novel CB2 agonists, we tried to replace the indole and
indazole cores with benzimidazolone and 3,4-dihydro-
2(1H)-quinazolinone. For initial studies, we selected
Keywords: Cannabinoid; CB2 agonist; Benzimidazolone; Metabolic
stability.
*
Figure 1. Structures of representative CB2 agonists.
Corresponding author. E-mail: omurah@gmail.com
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.04.032

H. Omura et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3310–3314
3311
CB1. In contrast, quinazolinone 13 was a partial agonist
and its non-aniline derivative, quinazolinone 14 was an
inverse agonist. Therefore, 9 was selected as a lead
compound.
A half-life of compound 9 in human liver microsomes
(HLM) was not calculated due to its rapid metabolism
(Table 1). The major metabolite of 9 in HLM was iden-
tified as a product oxidized on the tetrahydronaphtha-
lene ring. Therefore our effort was focused on the
optimization at tetrahydronaphthalene moiety at 1-posi-
tion on the benzimidazolone ring to address the meta-
bolic liability.
In order to improve metabolic stability, two general
strategies were applied; (1) structural changes around
metabolic sites to block metabolism, (2) reduction of
molecular lipophilicity.14 Table 2 shows the results of
metabolic stabilities of representative compounds pre-
pared using various amines according to Scheme 1.
Scheme 1. Reagents and conditions: (a) 4-(2-aminoethyl)morpholine,
K2CO3, THF, rt, 20 h, 97%; (b) H2 (4 atm), Pd/C, THF, rt, 5 h; (c)
CDI, THF, rt, 20 h, 83% (2 steps); (d) 4-nitrophenyl chloroformate,
triethylamine, dichloromethane, rt, 3 h; then 1-naphthylamine or (R)-
1,2,3,4-tetrahydro-1-naphthylamine, 81% or 67%, respectively (2
steps); (e) N-(2-chloroethyl)morpholine hydrochloride, sodium
hydride, DMF, 80 °C, 2 h, 51%; (f) lithium aluminum hydride, THF,
rt, 4 h; (g) CDI, THF, rt, 4 h, 49% (2 steps); (h) 4-nitrophenyl
chloroformate, triethylamine, dichloromethane, rt, 3 h; then 1-naph-
thylamine or (R)-1,2,3,4-tetrahydro-1-naphthylamine, 15% or 55%,
respectively (2 steps).
Removal of benzene ring remained metabolically unsta-
ble with decreased CB2 binding activity (15). On the
other hand, secondary amine-derived compounds repre-
sented by 16 did not have CB2 binding activity. This re-
sult suggested that the fixed conformation at 1-position
on the benzimidazolone ring of 15 presumably con-
trolled by hydrogen bonding between hydrogen on urea
and oxygen of benzimidazolone was necessary for CB2
binding activity.15 Therefore, further optimization was
carried out by using primary amine-derived compounds.
Reducing alkyl portion still remained metabolically
unstable as exemplified by 17 and 18.
benzimidazolone derivatives for optimization at 1- and
3-positions.13
Among compounds possessing hydroxyl groups, only 20
had improved metabolic stability (HLM T1/2 = 40 min)
though its potency was weak. Introduction of amino
acids-derived amides was effective to improve metabolic
stability (23 and 24). Especially 24 was the best com-
pound in terms of both CB2 binding activity and meta-
bolic stability. Through intensive studies of various alkyl
groups instead of tert-butyl group as exemplified by 25,
26, and 27, tert-butyl moiety was found to be the best to
show potent CB2 binding activity. Replacement of the
amide moiety of 24 with nitrile, tetrazole, and oxadiaz-
ole decreased half-lives in HLM, although they showed
The quinazolinone derivatives (13 and 14) were also syn-
thesized in four steps starting from 2-aminobenzonitrile
(10) (Scheme 1). Alkylation of 10 followed by reduction
gave diamine 11. The subsequent carbonylation and
urea formation process were the same process as benz-
imidazolone derivatives 8 and 9.
As a result, benzimidazolone 8 was found to have CB2
agonist activity (Table 1). Its non-aniline derivative,
benzimidazolone 9, also showed potent (0.7 nM) agonist
activity and 250-fold selectivity for CB2 receptor over
Table 1. Benzimidazolone and quinazolinone derivatives
a
hCB2 Ki (nM)
b
hCB2 EC50 (nM)
b
hCB2 Emax (%)
c
hCB1 Ki (nM)
Compound
HLM T1/2 (min)
8
9
8
7
8
95
107
28
NTd
1730
NTd
1510
11
0.7
22
NDf
NCe
4
13
14
41
9
ꢀ98g
NCe
a Measured as the Ki value for the displacement of tritiated CP55940 from membrane of human CB2 transfectant cell.
b The inhibition of forskolin-stimulated cAMP production by CB2 agonists was measured. Emax values represent the relative efficacy that is defined as
the ratio of the maximum response of test compound to the maximum response of 2-arachidonoylglycerol.
c Measured as the Ki value for the displacement of tritiated SR141716A from membrane of human CB1 transfectant cell.
d Not tested.
e Not calculated because the compound was not detected at 10 min after incubation in HLM.
f Not determined.
g % inhibition at 30 nM.

3312
H. Omura et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3310–3314
Table 2. Representative SAR data at 1-position
Compound
R1
R2
H
hCB2 Ki (nM)
hCB1 Ki (nM)
HLM T1/2 (min)
15
60
>10,000a
4
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
>1000a
232
74
NTb
NTb
>10,000a
2100
2
2
51
>10,000a
>10,000a
>10,000a
>10,000a
2720
2
484
15
40
5
25
3
20
20
35
17
NCc
NCc
3
5
>10,000a
>4040
67
>2500a
>2500a
5
NTb
NTb
>10,000a
1480
5
4
15
>10,000a
3
a IC50
.
b Not tested.
c Not calculated because the compound was not detected at 10 min after incubation in HLM.

H. Omura et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3310–3314
3313
potent CB2 binding activity working as bioisosteres of
the amide. Therefore, R1 was fixed with tert-leucinamide
for further modifications.
in poor metabolic stability while showing the most po-
tent CB2 binding activity.
To reduce molecular lipophilicity, introductions of
hydroxyl group into the potent compounds 31 and
35 were carried out (36–39). As a result, 39 showed
significant improvement of metabolic stability
(>120 min) while preserving potent CB2 binding
activity (8 nM).
Although the half-life of 24 was improved compared
to lead compound 9, it was still insufficient. One of
major metabolic parts of 24 in HLM was identified
as morpholine moiety by a metabolite identification
study. Therefore, the optimization at morpholine
group was examined in order to improve metabolic
stability (Table 3).
The profile of compound 3917 is summarized in Table 4.
It showed potent CB2 agonist activity and approxi-
mately 180-fold selectivity for CB2 over CB1. Com-
pound 39 exhibited a good PK profile (33% of
bioavailability in rat) and significantly attenuated the
chronic colonic allodynia in the TNBS-treated rats18
(3 mg/kg, po).
Removals of oxygen and/or nitrogen of the morpholine
group were attempted as structural changes around the
metabolic site to block metabolism. Eliminating amine
moiety (31 and 32) did not improve metabolic stability
while retaining potent CB2 binding activity. On the
other hand, 33 and 34 without ether moiety showed
HLM half-lives of >120 min suggesting that ether moi-
ety in morpholine of 24 was a major metabolic site,
although their CB2 binding activities were weak.16 Re-
moval of both ether and amine moieties (35) resulted
In summary, benzimidazolone derivatives were identi-
fied as novel CB2 agonists based on structural analogy
of existent CB2 agonists. The SAR study of benzimi-
dazolone derivative was focused on the improvement
Table 3. Representative SAR data at 3-position
Compound
R
hCB2 Ki (nM)
hCB1 Ki (nM)
HLM T1/2 (min)
logDa
31
4
1470
15
2.4
32
33
34
35
36
37
38
39
6
242
338
1
1320
37
2.1
1.5
1.5
3.4
1.6
1.9
2.2
2.2
NTb
>120
>120
10
NTb
69
92
3010
>120
84
144
196
8
>10,000c
>10,000c
1459
>120
>120
a At pH 7.4.
b Not tested.
c IC50
.

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H. Omura et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3310–3314
Table 4. The pharmacological profile of compound 39
Zvonok, A.; Cockayne, D. A.; Kwan, J.; Mata, H. P.;
Vanderah, T. W.; Lai, J.; Porreca, F.; Makriyannis, A.;
Malan, T. P., Jr. Proc. Natl. Acad. Sci. U.S.A. 2003, 100,
10529; (c) Makriyannis, A.; Deng, H. WO02/060447.
6. Hanus, L.; Breuer, A.; Tchilibon, S.; Shiloah, S.; Golden-
berg, D.; Horowitz, M.; Pertwee, R. G.; Ross, R. A.;
Mechoulam, R.; Fride, E. Proc. Natl. Acad. Sci. U.S.A.
1999, 96, 14228.
hCB2 Ki
8 nM
2 nM
118%
1459 nM
182
hCB2 EC50
hCB2 Emax
hCB1 Ki
Selectivity CB1/CB2
HLM T1/2
>120 min
33%
2.2
Bioavailability in rat
logDa
7. Malan, T. P., Jr.; Ibrahim, M. M.; Lai, J.; Vanderah, T.
W.; Makriyannis, A.; Porreca, F. Curr. Opin. Pharmacol.
2003, 3, 62.
a At pH 7.4.
8. Cannabinoids for pain therapeutics, see (a) Di Marzo, V.;
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Valenzano, K. J. Curr. Med. Chem. 2007, 14, 917.
9. Compound 1: D’Ambra, T. E.; Estep, K. G.; Bell, M. R.;
Eissenstat, M. A.; Josef, K. A.; Ward, S. J.; Haycock, D.
A.; Baizman, E. R.; Casiano, F. M.; Beglin, N. C.;
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of metabolic stability by structural changes around met-
abolic sites and/or the reduction of molecular lipophilic-
ity. The SAR study starting from 9 resulted in the
discovery of 39 as the best compound. 39 showed 180-
fold CB2 selectivity over CB1, excellent metabolic stabil-
ity in HLM, and oral activity in rat visceral allodynia.
10. Compound 3: (a) Hynes, J., Jr.; Leftheris, K.; Wu, H.;
Pandit, C.; Chen, P.; Norris, D. J.; Chen, B.-C.; Zhao, R.;
Kiener, P. A.; Chen, X.; Turk, L. A.; Patil-Koota, V.;
Gillooly, K. M.; Shuster, D. J.; McIntyre, K. W. Bioorg.
Med. Chem. Lett. 2002, 12, 2399; (b) Wrobleski, S. T.;
Chen, P.; Hynes, J., Jr.; Lin, S.; Norris, D. J.; Pandit, C.
R.; Spergel, S.; Wu, H.; Tokarski, J. S.; Chen, X.;
Gillooly, K. M.; Kiener, P. A.; McIntyre, K. W.; Patil-
koota, V.; Shuster, D. J.; Turk, L. A.; Yang, G.; Leftheris,
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Acknowledgments
We thank Mr. Masatoshi Masuda and Dr. Hiroyuki
Nishida for metabolites identification studies, and Ms.
Misaki Suzuki for ADME screening data. We are also
grateful to Ms. Katsuyo Ohashi for in vivo assay and
Dr. Hiroki Sone for useful discussions on this study.
11. Compound 4: Makriyannis, A.; Liu, Q. WO03/035005.
12. Toxicity of aniline was reported. see: (a) Famulok, M.;
Boche, G. Angew. Chem., Int. Ed. Engl. 1989, 28, 468; (b)
Marques, M. M.; Mourato, L. L. G.; Amorim, M. T.;
Santos, M. A.; Melchior, W. B., Jr.; Beland, F. A. Chem.
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