New Tetrahydropyrido-anellated Thieno-1,3-oxazin-4-ones
as Inhibitors of Acetylcholinesterase
and Leukocyte Elastase
Markus Pietsch and Michael Gütschow
Pharmaceutical Institute, Poppelsdorf, Kreuzbergweg 26, University of Bonn, D-53115 Bonn, Germany
1.2 x 105
slope or intercept x 1000
400
300
1/rate (min)
Human leukocyte elastase (HLE) is implicated in several inflammatory diseases, e.g. pulmonary emphysema
and cystic fibrosis, thus representing a particularly attractive target for the development of low molecular
weight inhibitors.1 A potent and well-studied class of HLE inhibitors are thieno-1,3-oxazin-4-ones.2 Inhibition
of acetylcholinesterase (AChE) is desirable due to its implication in some pathological processes, e.g. the
formation of amyloid plaques and neurofibrillary tangles. Inhibitors of the enzyme improve the cholinergic
deficit thus reducing the symptoms of Alzheimer’s disease.3 We have synthezised a series of thieno[2,3-d][1,3]oxazin-4-ones and investigated the inhibition of the serine proteinase HLE and the serine esterase
acetylcholinesterase (Electrophorus electricus) kinetically. Some tetrahydropyrido-anellated derivatives were
found to selectively inhibit acetylcholinesterase and exhibit IC50 values in the high nanomolar range.
200
100
0
-100
-0.002
formula
S
1
K i HLE (*M)
IC50 AChE (*M)
N
N
-1 -1
-4 -1
2
S
N
N
O
S
N
N
N
O
0.43 ± 0.03
108 ± 4
0.46
-1.24
53.9 ± 3.7
4.17 ± 0.11
24.9 ± 0.9
1.04
-0.98
15.2 ± 1.3
3.28 ± 0.19
18.9 ± 1.1
0.62
-0.99
O
4
S
N
N
0.55 ± 0.05
O
N
2
4
6
[5] (7M)
7.39 ± 0.23
111 ± 9
8.2
-1.16
O
X
N
N
O
a
1.29 ± 0.11
1.43 ± 0.06
321
87.6 ± 7.8
2.13 ± 0.13
80.2
4.59
-0.97
1.71
-0.79
S8
O
O
5
0
CN
N
S
-2
0.004
Fig. 3 left: Plot of the reciprocal rates versus the reciprocal substrate concentrations for the inhibition of
acetylcholinesterase from Electrophorus electricus by compound 5. Substrate was acetylthiocholine. Circles: [5] = 0;
squares: [5] = 2 7M; triangles: [5] = 4 7M; and rhombuses: [5] = 6 7M. Data were average values of duplicate
experiments. right: Plot of the slopes (circles) and the intercepts x 1000 (triangles) versus the concentration of 5. The
data were obtained from linear fits shown in the left figure. Linear regressions gave Ki = 1.35 ± 0.05 7M (slope vs. [5])
and Ki = 1.33 ± 0.11 7M (intercept x 1000 vs. [5]). The agreement of both Ki values with the IC50 value (see Tab. 1)
indicated noncompetitive inhibiton.
O
3
0
-1 -1
O
N
3 x 104
k on HLE (M s ) k off HLE (10 s ) logk OH- (M s )
530
O
0.002
6 x 104
1/[acetylthiocholine] (7M-1)
Tab. 1 Inhibition of acetylcholinesterase from Electrophorus electricus and HLE by thieno[2,3-d][1,3]oxazin-4-ones.
compd
0
9 x 104
S
X
NH2
O
morpholine, ethanol, 45 °C, 5 h
(60 - 90%)
O
O
O
O
6
S
N
N
N
O
b
X
O
S
7
N
N
290
O
0.013
c
2500
c
0.32
c
-1.49
c
O
S
8
N
N
220
O
0.0058
c
9500
c
0.55
c
-1.13
c
O
S
9
N
N
280
O
0.013
c
3600
c
0.45
c
-1.03
c
N
X = CH2, (44%)
1
CH2
2
N–CH3
3
N–CH2CH3
4
N–CH2CH(CH3)2
5
N–CH2C6H6
6
N–COC6H6
S
X
H
+
N
+
Cl
Cl
O
_
_
Cl
N
O
Cl
ethereal HCl,
1,2-dichloroethane,
reflux, 3.5 h
H2O, Na2CO3
X = N–R, (7 – 28%)
S
X
N
N
O
50% ethanol, Na2CO3
X = CH2, (84%)
O
O
= 4.11 ± 0.28 7M; b Kd = 6.28 ± 0.19 7M; c Data from Ref. 2.
Scheme 1 Synthesis of 2-dimethylaminothieno[2,3-d][1,3]oxazin-4-ones.
0.06
0.02
6 x 10-5
0.09
200
100
0 2
0.01
OD405 nm
0.02
1/rate (min)
0.04
rate (min-1)
OD412 nm
0.03
4
6 8 10 12
0
0.03
[5] (7M)
1
2
3
4
5
0
2
4
6
8
10
12
0
[5] (7M)
X
N
N
9
pIC50 or pKi
O
6
3
1
2
3
4
5
6
7
8
9
O
4
10 15 20 25
[5] (7M)
1000
2000
3000
4000
0
5
10
15
20
25
[5] (7M)
Fig. 4 left: Inhibition of HLE by compound 5 in 0.05 M phosphate buffer, 0.5 M NaCl, pH 7.8, 25 °C. Substrate was
MeOSuc-Ala-Ala-Pro-Val-pNA. Progress curves were analyzed by nonlinear regression according to an equation of a
slow-binding inhibition.4 Black: [5] = 0; violet: [5] = 2 7M; blue: [5] = 4 7M; green: [5] = 6 7M; brown: [5] = 8 7M;
and red: [5] = 10 7M. right: Plot of the final rates versus the concentration of 5. The data were obtained from fits of the
curves shown in the left figure and were average values of triplicate experiments. The solid line was drawn using the
best-fit parameters from a fit according to an equation of a competitive inhibition, which gave Ki(1+[S]/Km) = 4.15 ±
0.18 7M. The insert shows a plot of the initial rates versus the concentration of 5. The data were average values of
duplicate experiments. A fit according to an equation of a competitive inhibition gave Kd(1+[S]/Km) = 11.9 ± 0.8 7M.
4
pKi - HLE
N
N
5
1 Hlasta,
8
7
S
0
2 x 10-5
5
pIC50 - AChE
O
N
2 x 10-5
A series of nine thieno[2,3-d][1,3]oxazin-4-ones was investigated as inhibitors of HLE and acetylcholinesterase
from Electrophorus electricus. Cyclopentene- and cyclohexene- as well as benzo-anellated derivatives
inhibited HLE three to five orders of magnitude stronger than acetylcholinesterase. These compounds were
poor inhibitors of acetylcholinesterase and exhibited IC50 values in the high micromolar range. The exchange
of the cycloaliphatic and aromatic anellation pattern for a tetrahydropyrido moiety led to an enhancement of the
inhibitory potency against acetylcholinesterase. Among the basic pyrido compounds 2-5, introduction of a
bulky residue was favorable. Compound 4, the most active acetylcholinesterase inhibitor of this series,
inhibited the enzyme with IC50 = 550 nM. In contrast, inhibition of HLE was reduced up to three orders of
magnitude resulting in a reversed selectivity profile of the inhibitor. Investigations of the acetylcholinesterase
inhibition in the presence of different substrate concentrations were done exemplarily with compound 5. The
data indicated a noncompetitive mode of interaction resulting in an independence of the inhibition of the
substrate concentration.
R
O
4 x 10-5
0
time (s)
Fig. 1 left: Inhibition of acetylcholinesterase from Electrophorus electricus by compound 5 in 0.1 M phosphate buffer,
0.1 M NaCl, pH 7.3, 25 °C. Substrate was acetylthiocholine. Progress curves were analyzed by linear regression. Open
circles: [5] = 0; full circles: [5] = 2 7M; open squares: [5] = 4 7M; full squares: [5] = 6 7M; open triangles: [5] = 8 7M;
full triangles: [5] = 10 7M; and open rhombuses: [5] = 12 7M. right: Plot of the rates versus the concentration of 5. The
data were obtained from fits of the curves shown in the left figure and were average values of duplicate experiments. The
solid line was drawn using the best-fit parameters from a fit according to the equation vi = v0/(1+[I]/IC50), where vi and v0
were the rates in the presence and the absence of the inhibitor, respectively, and [I] was the concentration of the inhibitor.
Nonlinear regression gave IC50 = 1.29 ± 0.11 7M. The insert is a Dixon plot to show the linearity.
R
4 x 10-5
6 x 10-5
0
0
time (min)
S
0.06
0
0
initial rate (s-1)
0.12
300
final rate (s-1)
aK
d
compound
Fig. 2 pIC50 values and pKi values for the inhibition of acetylcholinesterase from Electrophorus electricus and HLE by
thieno[2,3-d][1,3]oxazin-4-ones.
References:
D. J., Pagani, E. D., Ann. Rep. Med. Chem. 1994, 29, 195-204.
2 Gütschow, M. et al., J. Med. Chem. 1999, 42, 5437-5447.
3 Sugimoto, H. et al., Curr. Med. Chem. 2000, 7, 303-339.
4 Morrison, J. F., Trends Biochem. Sci. 1982, 7, 102-105.
The authors thank the graduate college 677/1 “Structure and Molecular Interaction as a Basis for Drug Action” for support.