5000
Y. Ogino et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4997–5001
Table 4
Biological properties of the representative compounds (2r, 2s, and 2u)
a
Compound
IC50 (nM)
Y2
Rat plasma (
l
M) levelsc
4 h
B/P ratiod
Y5
Y1
Y4
Y5b
2 h
2r
2s
2u
3.3
5.1
5.9
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
5.5
7.0
7.2
1.4
0.4
3.2
0.85
n.d.e
0.99
0.05
—
0.12
a
IC50 values are the mean of two or more independent assays.
b
c
Antagonist activity was determined by the inhibition of NPY-induced [Ca2+] increase in LMtkꢁ cells expressing the human Y5 receptor.
Compounds (10 mg/kg) were orally administered to rats (n = 3), and the plasma levels were measured at 2 and 4 h.
d
Compounds (10 mg/kg) were orally administered to rats (n = 3), and the plasma and brain levels were measured at 2 h. B/P means the ratio of the brain level (nmol/g) to
M) of the compounds.
plasma level (
l
e
Not detected.
prepared and its Y5 affinity was compared with that of 2k.
The 1-methylbenzimidazole derivative 14 almost completely
lost its potency, suggesting that the N–H group plays a key
role in the binding interaction between 14 and the Y5
receptor.
The above SAR study demonstrated that steric factors in the
substituents at the 5-position or at both the 5- and 6-positions of
the benzimidazole core ring were important for high Y5 binding
affinity. However, if there is a high lipophilicity associated with
compounds (clogD7.4: >5),10 this may worsen the aqueous solubil-
ity of compounds and influence their ADME aspects, which can re-
sult in problems during the drug development phase.11 Therefore,
we focused further modification on reducing the lipophilicity of
compounds, while the compounds are retaining high Y5 potency
(Table 2).
induced [Ca2+] increase in LKtkꢁ cells that expressed the human
Y5 receptor. These results suggested that compounds (2r, 2s, and
2u) are potent and subtype-selective Y5 receptor antagonists. To
examine the oral absorbability and brain penetrability, these com-
pounds were orally administered to rats at 10 mg/kg dosage. Plasma
levels were then measured at 2 and 4 h after dosing, and the brain
penetrability (B/P) was also examined at 2 h after oral administra-
tion. Even at 4 h after oral administration, compounds (2r and 2u)
showed moderate plasma exposures (ꢀ1
lM), suggesting that these
were indeed orally bioavailable Y5 antagonists in rats. With regard
to the brain penetrability, these compounds showed marginal brain
penetrability unlike that seen in the case of 2n (B/P = 0.55). The mar-
ginal brain exposures of the compounds (2r and 2u) suggested that
these would not show in vivo efficacy in rats (10 mg/kg po).13
In conclusion, structure–activity relationship studies were per-
formed in a class of novel benzimidazoles with the aim of identify-
ing potent, selective, and orally bioavailable NPY Y5 receptor
antagonists, leading to the discovery of the two potent compounds
(2r and 2u). These results were achieved by incorporating a hetero-
aromatic ring into the 6-position of the benzimidazole core part. At
the present time, further modifications of this class that focus on
improving brain penetrability are underway to identify in vivo po-
tent compounds.
Incorporation of
a methoxycarbonyl group (2n) into the
benzimidazole core ring was found to be well tolerated (IC50
:
7.7 nM), and this group clearly contributed to reducing the lipo-
philicity (clogD7.4: 2n = 4.1). In addition, as shown in Table 3, it
is noteworthy that an intravenous administration of 2n to rats
revealed a fairly good brain/plasma ratio (B/P = 0.55). This result
prompted us to further replace the methyl ester with ester bio-
isosters such as 1,3,4-oxadiazole and 1,2,4-oxadiazole moieties,
because ester bioisosters are thought to be metabolically more
stable than the ester moiety.10 In addition, theoretically, incor-
poration of these heteroaryl groups should not only reduce
the lipophilicity but also increase the aqueous solubility. As ex-
pected, compounds bearing a 5-methyl-1,3,4-oxadiazole (2r), 5-
methyl-1,2,4-oxadiazole (2s), or 2-methyltetrazole (2u) retained
the potent Y5 binding affinity, and had better metabolic stabil-
ity than 2n in the rat hepatocyte assay.12 In addition, these
compounds were more likely to be hydrophilic than 2n
(clogD7.4: 2r = 3.3, 2s = 3.7, 2u = 3.6, and 2n = 4.1). Comparison
of the aqueous solubility of the compounds (2r and 2u) with
that of the representative lipophilic compounds (2l and 2n) in
0.2 M phosphate buffer (pH 6.8) indicated that the heteroaryl
groups contributed to the aqueous solubility of these com-
References and notes
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A. O.; Whitebread, S.; Hofbauer, K. G.; Taber, R. I.; Branchek, T. A.; Weinshank,
R. L. Nature 1996, 382, 168; (b) Kanatani, A.; Ishihara, A.; Iwaasa, H.; Nakamura,
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pounds (2r: 0.94
2n: 0.60 g/mL).
lg/mL, 2u: 5.9 lg/mL, 2l: <0.01 lg/mL, and
l
To determine the importance of the trans-configuration of this
class of compounds in terms of the Y5 binding affinity, the cis-iso-
mers (15 and 16) of the potent compounds (2o and 2r) were eval-
uated as representatives of this class. As expected, these had far
less Y5 binding affinity, which provides some conformational in-
sight into the binding interaction of this class of compounds and
the Y5 receptor.
As seen in Table 4, further characterization of the representative
benzimidazoles (2r, 2s, and 2u) was carried out. In the binding as-
say, these compounds showed greater than 1000-fold subtype
selectivity over the other NPY receptors (Y1, Y2, and Y4). Moreover,
these exhibited potent antagonist activity by inhibiting NPY-