1712
S. F. McHardy et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1711–1714
H3CO
N
N
Cl
N
CH3
OH
Cl
H3CO
N
N
N
H
N
OH
1
2-PAM
2
N
OH
N
O
OH
R
HO
H3C
O
H
N
O
O
N
N
)
n
O
OH
N
H
(
N
OH
O
R = CH3, DAM
R = H, MINA
3 (n
= 1-
3)
RS41A
Figure 1. Structure of existing AchE reactivators.
Herein we report the design, synthesis and in vitro evaluation of
a novel class of small molecule reactivators of GF-inhibited
hAChE.14 In all design iterations, several factors were taken into
consideration. A balanced affinity for GF-inhibited AChE vs. unin-
hibited AChE is critical; however oxime pKa has also been postu-
lated to play a critical role for an efficient reactivation process.15
Physicochemical property values (LogD, tPSA, MW, # hydrogen
bond donors/acceptors) were determined and structures adjusted
to maintain the desired range of values for CNS drug-like proper-
ties.16 Based on these studies we chose to focus on two chemical
series highlighted by the amide oxime analogs 9–22 (Fig. 2) and
heteroaryl keto-oxime analogs 27–43 (Fig. 3). Both structural ser-
ies allow for introduction of lipophilic amine templates, as well
as various functional groups to modulate oxime properties.
The synthesis of the lipophilic amide oxime series is detailed
below in Scheme 1. Treatment of primary amines with ethyl 2-
(hydroxyimino)acetate provided the amide oxime analogs 4 in
moderate yield (50–70%). The primary amines could also be trea-
ted with the 1,3-dioxin-4-one in toluene at 150 °C to produce the
desired b-ketoamides 5, which underwent subsequent oxime for-
mation with sodium nitrite (NaNO2), under cold, aqueous acidic
conditions, which cleanly afforded the desired b-ketoamide oximes
6 in low (20–40%) yields. Finally, the cyanoamide analogs 8 were
easily prepared in two steps via condensation with methyl-2-cya-
noacetate, followed by oxime formation under tert-butyl nitrite/so-
dium propoxide conditions.
were prepared from the corresponding dihydroisoquinoline17 and
aminotacrine18 primary amines, respectively.
A representative synthetic route used to prepare the heteroaryl
keto-oxime analogs is shown below in Scheme 2. Cyclic amino
alcohol derivatives such as 23 were treated under reductive amina-
tion conditions with aldehydes and sodium triacetoxyborohydride
to provide the tertiary amines 24 in good yields.19 The acetyl pyr-
idyl ethers 25 were prepared via palladium catalyzed coupling
reaction of alcohol 24 and 2-bromo-acetyl pyridines in the pres-
ence of Pd2(dba)3/BINAP, which produced the desired pyridyl ke-
tones in moderate yields.20 Finally, oxime formation was
accomplished under t-BuONO/KOt-Bu21 conditions to provide the
keto-oxime analogs 26 in low yields. Unfortunately, attempts to
optimize the reaction conditions, as well as employ alternative
conditions (i.e. TMSCl, NOCl)22 were unsuccessful.
The synthetic route highlighted in Scheme 2 was used to pre-
pare a number of analogs varying substitution on the piperidine
and pyrrolidine rings, as well as the position of the keto-oxime
on the pyridine or pyrimdine rings (Fig. 3).
In vitro screening for reactivation of GF-inhibited hAChE was
assayed by a robotic spectrophotometric assay using acetylthioch-
oline as substrate at pH 7.4 and 25 °C.23 The time-course of reacti-
vation of GF-inhibited hAChE was determined by adding various
concentrations of oxime to GF-inhibited hAChE. Samples of the
oxime/inhibited AChE mixture were removed at sequential times
to measure the oxime-induced recovery of AChE activity.
The synthesis route highlighted in Scheme 1 was used to pre-
pare the analogs of various lipophilic amine templates as shown
in Figure 2. The N-benzyl piperidine analogs 9–15 were prepared
from the commercially available primary amines or the corre-
sponding N-BOC protected piperidines. Analogs 16–18 and 19–22
Table 1 summarizes two data points collected on each analog:
(1) Corrected cumulative reactivation by 20 uM oxime and (2)
Oxime/hAChE equilibrium constant for inhibition of hAChE. In
our initial studies, we were gratified to see low levels of reactiva-
tion of GF-inhibited hAChE as a proof of concept for our new chem-
ical matter in our piperidine amide oxime analogs 9 and 10.
Furthermore, the analogs also possessed weak inhibition of native
enzyme. Incorporation of the b-ketoamide moiety (analog 12) not
only produced a significant increase in reactivation activity, but
also maintained weak inhibition of native hAChE. A similar result
was observed with the 3,4-dimethoxybenzyl analog 15. Addition
of the cyano group in compounds 13 and 14 resulted in a complete
loss of reactivation activity. Replacement of the N-benzyl piperi-
dine with the dihydroisoquinoline group delivered hAChE reactiva-
tors with very good reactivation levels and low inhibition of native
enzyme as long as the b-ketoamide moiety was present (17 and
18). Finally, incorporation of the tacrine group as represented by
compounds 19–22, provided compounds with substantial inhibi-
tion of native enzyme, yet only low levels of reactivation, regard-
less of amide oxime substitution or length of the amine tether.
Structure activity relationship (SAR) studies on the heteroaryl
keto-oxime series provided compounds with very good reactiva-
tion of GF-inhibited hAChE. Compounds 27–30 all possessed the
N-benzyl-4-piperidine moiety and only differed by the substitution
pattern of the ketooxime group on the pyridine ring, which had
9, n = 0, R1, R2 = H
R2
R2
10
,
n = 1, R1, R2 = H
11, n = 0, R1 = COCH3, R2 = H
12
N
R1
H
N
n
OH
(
)
,
n = 1, R1 = COCH3, R2 = H
13, n = 0, R1 = CN, R2 = H
14
N
O
,
n = 1, R1 = CN, R2 = H
15, n = 1, R1 = COCH3, R2 = OCH3
O
O
R1
16, X = Ph, R1 = H
H
N
N
OH
17
,
X = Ph, R1 = COCH3
N
18, X = H, R1 = COCH3
X
O
R1
19, n = 1, R1 = H
H
N
H
N
OH
20
,
n = 2, R1 = H
21, n = 1, R1 = CN
22
N
(
)
n
O
N
,
n = 4, R1 = H
Figure 2. Structure of prepared amide oxime analogs
McHardy, Stanton F.
