4
222
G. P. Jadhav et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4219–4224
Table 2
3,7-disubstituted 1-hydroxypurine-2,6-diones 8 were prepared
from the O-benzyl precursor 6, obtained by cyclisation of imidazole
Activity of 3-substituted-1-hydroxy-7-phenethyl-3,7-dihydro-1H-purine-2,6-diones
8
1
34
3
2 2
(R = (CH ) Ph) with O-benzylhydroxylamine, by successive
2
alkylation with appropriate alkyl halides and hydrogenolysis of
products 7 (Scheme 1). Introduction of a methyl (8a), ethyl (8c),
n-pentyl (8d), isohexyl (8e), n-nonyl (8f), n-decyl (8g), or n-dodecyl
R in
IC50a
IC50a
(lM) (PARN)
Cmpd
(l
M) (Caf1)
3
group (8h) at N showed optimal activity for the isohexyl congener
8
e, although its activity was not improved with respect to the par-
ent compound 5j. However, replacement of the isohexyl group
with the 1-(N,N-dimethylamino)prop-3-yl group (8j) resulted in a
further affinity enhancement and afforded the most potent com-
pound in our series with activity at the submicromolar level.35
Again this potency gain can be rationalised based on docking stud-
ies, which suggest that the protonated (at physiological pH) 3°
8
8
8
8
a
b
c
CH
CH
CH
CH
3
2
2
2
9.3 ± 1.7
2.1 ± 0.3
12.2 ± 3.5
4.8 ± 1.2
n.d.
n.d.
n.d.
n.d.
(CH
2
)
2
3
Ph
CH
3
d
(CH
2
)
CH
3
8
e
1.7 ± 0.4
n.d.
8
8
8
8
f
CH
2
CH
2
CH
2
CH
2
(CH
(CH
(CH
Ph
2
2
2
)
)
)
7
CH
CH
3
9.9 ± 3.6
20.7 ± 6.7
29.5 ± 22.4
14.5 ± 0.9
n.d.
n.d.
n.d.
n.d.
amine group in 8j is H-bonded to the amide carbonyl O of Pro44
,
g
h
i
8
3
10CH
3
while maintaining most of the hydrophobic interactions of the
3
N -substituent compared with the isohexyl group in 8e (Fig. 2d).
According to our binding hypothesis (Fig. 2c) the N-hydroxy-
imide function, especially the C - and N-linked O atoms, present
8
j
0.59 ± 0.11
8.7 ± 0.3
23.9 ± 3.7
n.d.
6
8
k
in all compound of the series discussed thus far, is important for
2
+
polar interactions with the Mg ions coordinated in the active site
a
IC50 values were determined using a fluorescence-based biochemical assay as
of Caf1. Additionally, we propose that the N-hydroxyl function
H-bonds to the side-chain carboxyl of Asp . To probe this hypothesis
2
2
described. Also indicated are the standard errors of the means (n = 3). N.d., not
determined.
61
we tested compounds containing modified N-hydroxyimide sub-
structures (Table 3). To this end, a purine-2,6-dione derivative (9)
lacking the N-hydroxy group of compounds 5 and 8 was obtained
1
Table 3
from intermediate 3 (R = Bn) by cyclisation with urea under basic
Activity of 7-substituted 1-hydroxy-3,7-dihydro-1H-purine-2,6-diones with modified
N-hydroxyimide function
34
conditions (Scheme 1).
6-Hydroxy-1-phenethyl-1,4-dihydro-
5
H-imidazo[4,5-b]pyridin-5-one 13 was obtained from the
IC50a
(lM) (Caf1)
36
Cmpd
Structure
nitroimidazole precursor 10 (Scheme 2). A dichloromethyl group
was introduced into this compound at the 5-position using a
t
vicarious nucleophilic procedure with chloroform and KOBu ,
followed by hydrolysis of the dichloromethyl group to the
aldehyde and reduction of the nitro function to the aminoaldehyde
4
a
>1000
>1000
3
7
1
1. This intermediate was then cyclised with ethyl allyloxyac-
3
8
etate to the imadazopyridinone 12, which was deprotected to
afford target compound 13. Blocking of the N-hydroxyl function
with an allyl group in the synthesis intermediate 4a, as well as
removing the N-hydroxyl group in 9, abolished activity completely,
9
1
6
as expected. Furthermore, omission of the C -carbonyl oxygen in
the hydroxy-imidazopyridinone 13 reduced activity significantly,
pointing to an important contribution of this carbonyl group to
binding, as suggested by our docking studies.
3
15.1 ± 0.3
a
IC50 values were determined using a fluorescence-based biochemical assay as
described. Also indicated are the standard errors of the means (n = 3).
The selectivity of the compounds was determined by comparing
their activities against Caf1 and two related deadenylase enzymes:
the PARN ribonuclease, which has a conserved nuclease domain
similar to that of Caf1, and Ccr4, whose biological function is
related to Caf1, but which contains a dissimilar EEP-type nuclease
2
2
(
5g) was not productive. On the other hand, insertion of an O atom
into the linker (5i), or replacement of the phenyl group in 5j with a
-thienyl (5h) or 2-pyridyl (5k) group, retained most of the
activity.
We next turned our attention to the purine N -substituent in
the context of the optimal 7-phenethyl group (Table 2). Here we
envisaged that a comparatively bulky substituent may make
favourable contacts with the Caf1 active site pocket lined by
2
2
7
2
domain. Compounds with N substituents containing an aro-
matic group linked via a single carbon spacer (5a and 5c–e) selec-
tively inhibited Caf1 (Fig. 3; Table 1). By contrast, the more potent
3
7
compounds 5h, 5j and 5k with N substituents containing an aro-
matic group linked via a two-carbon spacer facilitating optimal
cation–p interactions displayed reduced selectivity and also inhib-
ited the related deadenylase PARN. Selectivity was not improved
4
3
44
112
116
residues Phe , Pro , Ser , and Leu
(Fig. 2c and d). The
t
Scheme 2. Synthesis of 6-hydroxy-1-phenethyl-1,4-dihydro-5H-imidazo[4,5-b]pyridin-5-one 13. Reagents and conditions: (a) CHCl
5 min, then CaCO , H O, 75 °C, 48 h, then H
78 °C to rt over 16 h (36%); (c) Pd(OAc) , PPh
3
, KOBu , THF–DMF (2:1), ꢀ100 °C,
1
ꢀ
3
2
2
, 10% (w/w) Pd(C), EtOAc–MeOH–AcOH (18:1:1), 55 psi, 18 h (60% over 3 steps); (b) ethyl allyloxyacetate, (Me
, HCOOH, EtOH–H O (8:2), 80 °C, 2 h (33%).
3 2
Si) NLi, THF,
2
3
2