5366
M. Ohmi et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5364–5368
Table 2
stability and in equilibrium solubility. The amide derivatives (15a–
15c) also had IC50 values less than 100 nM in the human TRPM8
calcium influx assays, but the metabolic stability and solubility of
these compounds were not improved compared to compound 13.
Similarly, the benzyl alcohol derivative 14, whilst having good
potency with an IC50 value of 42 nM, did not show improvements
in metabolic stability and solubility. Based on the SAR on the A-
region of the molecule (Fig. 2), we selected compound 13 as a lead
compound, and shifted our efforts to the SAR exploration on the
pyridine (B-region) and benzyl (C-region) moieties.
Initial SAR of the pyridine core (B-region)
Compd
R1
R2
hTRPM8
HLM Clint
(ml/min/kg)
IC50 (nM)a
In order to better understand the role of each region and substi-
tuent as pharmacophores, molecular overlay analysis was con-
ducted using the compound 13 and compd 306. As one can
imagine from the similarity in the two-dimensional structures of
the molecules, minimized conformers of the two molecules were
easily superimposed (Fig. 3). The trifluoromethyl group in com-
pound 13 overlapped well with the benzene moiety of the benzo-
thiophene ring in compd 306, indicating that these two moieties
behave as hydrophobic groups in the pharmacophore. Further-
more, the molecular overlay suggested that there would be space
for accommodating substituents on the benzyl moiety of com-
pound 13. With this hypothesis in mind, we set out to explore
the SAR in the B- and C-regions.
13
16
17
18
19
20
21
22
23
24
25
CF3
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
F
Me
CN
H
61
9859
201
23,782
707
122
213
736
<7
N.D.
<7
N.D.
>168
85
b
CN
Me
Ph
CF3
CF3
CF3
CF3
Cl
<7
N.D.
N.D.
N.D.
N.D.
2472
2983
1562
CF3
a
IC50 values based on inhibition of menthol (30
HEK293 cells.
l
M) induced Ca2+ influx in
b
N.D. = Not determined.
As shown in Table 2, the substitution effect of R1 on the pyridine
ring was significant when R2 was held constant as chlorine.
Lipophilic atoms or groups such as chloro (17) and phenyl (20)
were associated with strong antagonistic activity, while the hydro-
gen (16) and cyano (18) substituents reduced potency by 160- and
400-fold, respectively, compared with compound 13 bearing a
trifluoromethyl group. Methyl derivative 19 showed moderate
activity, with a 10-fold drop in potency compared to compound
13. These results suggested that lipophilicity, rather than electro-
negativity, plays a key role in the antagonistic activity on TRPM8,
which is in good accordance with the pharmacophore hypothesis
suggested by the molecular overlay discussed above.
Table 3
Initial SAR of benzyl core (C-region)
Compd
R5
hTRPM8
HLM Clint
(ml/min/kg)
IC50 (nM)a
The effect of R2 on the pyridine ring was then investigated with
R1 group being held constant as a trifluoromethyl group. The fluoro
21 and methyl 22 derivatives were 4- and 10-fold less potent,
respectively, than the chloro-substituted compound 13. R2 groups
such as cyano 23 and hydrogen 24 led to a drop in potency, with a
40-fold increase of the IC50 compared to compound 13. Compound
25, where the R1 and R2 groups were switched compared to com-
pound 13, had moderate activity, which suggested that there
may be some restriction to the amount of lipophilicity and steric
bulk that can be introduced at the R2 position.
13
26
61
<7
762
N.D.b
27
28
29
49
104
81
>168
<7
8419
Taken altogether, we concluded that the combination of
R1 = CF3 or Cl and R2 = Cl are optimum substituents for the
B-region, and shifted to the SAR exploration of the benzyl moiety
(C-region).
30
3720
<7
a
IC50 values based on inhibition of menthol (30
HEK293 cells.
l
M) induced Ca2+ influx in
b
N.D. = Not determined.
In an initial SAR study of the R5 substituent, the phenylpropyl
and cyclohexylmethyl derivatives (27 and 28) proved similar to
or more potent than the benzyl derivative 13 in the in vitro assay
of human calcium influx, but the phenethyl derivative 26 displayed
a 10-fold drop in potency (Table 3). Additionally, the metabolic sta-
bility of these compounds decreased, as shown by a high clearance
in HLM. Introduction of heteroaryl and heterocyclic moieties such
as pyridyl and morpholyl (29 and 30) also resulted in a moderate
activity, despite the improved metabolic stability. Therefore, as
for the optimization of group, we selected the benzyl scaffold for
R.5
Finally, we proceeded to optimize the substitution on the ben-
zyl moiety (C-region), focusing on the in vitro antagonistic activity,
Figure 3. Molecular overlay of 13 (brown) and compd 306 (green).8