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N-Acylpyrrolidin-2-ones:
Potential inhibitors of serine proteases and esterases
Paul W. Elsinghorst and Michael Gütschow
Pharmaceutical Institute, Poppelsdorf, Kreuzbergweg 26, University of Bonn, D-53115 Bonn, Germany
OMe
Investigating the methylation of amides 1 (Scheme 1) which were derived from -aminobutyric acid we
observed the interesting chemical behaviour of these substances. When heated in the presence of sodium
hydride the truncated esters 2 were formed, which led us to the assumption that N-acylpyrrolidin-2-ones 5
might be involved as intermediates. Another interesting fact is that N-acylpyrrolidin-2-ones 5 can easily be
prepared by means of oxalyl chloride and acids 3. The formed acyl chlorides 4 seem to be very reactive as
slight heating in the presence of diisopropyl ethyl amine for deprotonation gives N-acylpyrrolidin-2-ones 5 in
moderate to good yields.
OMe
MeO
Cl
_
4a
N
MeO
MeO
_
O
Cl
N
MeO
O
O
OMe
N
MeO
MeO
OEt
_
1a
5a
OMe
MeO
Once the amide nitrogen in compounds 1 or 4 gets deprotonated (Scheme 2) its nucleophilicity seems to be
high and the formed five-membered ring is energetically favoured. As the chlorine in acyl chlorides 4
represents a good leaving group the reaction demands conditions only slightly above room temperature. Instead
the amides 1, deprotonated by sodium hydride, require higher temperatures for further reaction as the ethylate
function is a rather weak leaving group.
O
_
O
_
EtO
N
MeO
O
O
O
OMe
Thus, the intermediatly released ethylate might attack the formed N-acylpyrrolidin-2-ones 5 to yield the finally
observed esters 2. As this step imitates the acylation of an active site serine residue in serine proteases or
esterases [2,3] we synthesized compounds 5a-d for evaluation as inhibitors of chymotrypsin and human
leukocyte elastase (HLE).
MeO
_
OEt
OEt
MeO
_
O
_
N
2a
O
H
N
1
Scheme 2 Formation of N-acylpyrrolidin-2-ones
O
OEt
O
OMe
The synthesis of compounds 5 was carried out on two different ways. The first and obvious one was to react the
corresponding acyl chlorides with pyrrolidin-2-one as reported in the literature [1]. In comparison (Table 1) we
synthesized the target compounds by means of the previously described route involving the preparation of 1,
the hydrolysis to form the -aminobutyric acid derivates 3 and the ring closure of the intermediate acyl
chlorides 4.
MeO
OEt
2
MeO
O
1a-5a
1c-5c
O
H
N
3
The formation of esters 2 using equimolar amounts of sodium hydride in dimethyl formamide at 80°C was
shown for substances 1a and 1b.
OH
O
OMe
MeO
H
N
4
MeO
O
O
Cl
R
OEt
N
H
O
i
O
1
O
R
OH
N
H
O
3
O
ii
R
Cl
N
H
O
4
1d-5d
1b-5b
iii
N
5
O
O
i
50% acetone, H2SO4 pH 1, reflux
ii
dichloromethane, dimethyl formamide (cat.), oxalyl cloride, rt
iii
Scheme 1 Target Compounds
dichloromethane, Hünig’s base, reflux, column chromatography
O
O
N
R
5
Scheme 3 New synthetic route to N-acylpyrrolidin-2-ones
Human leukocyte elastase
Ki (µM)
Compound
Chymotrypsin
Activity (%)
OMe
Compound
MeO
52
N
MeO
110 (@ 200µM)
Classical synthesis
New synthesis
-
53%
59%
87%
74%
-
37%
-
OMe
MeO
O
O
N
MeO
O
OMe
O
MeO
351
N
MeO
O
106 (@ 100µM)
OMe
MeO
O
N
MeO
O
504
N
O
O
121 (@ 50µM)
130 (@ 200µM)
N
O
N
O
no inhibition
O
121 (@ 50µM)
129 (@ 200µM)
Chymotrypsin Assay
The chymotrypsin assay was done by Sonja Kolp
using a UV spectrophotometer and a chromogenic
substrate.
HLE Assay
The HLE assay was done by Markus Pietsch using a
UV spectrophotometer and a chromogenic substrate.
Conditions:
Conditions:
25°C, 405nm
25°C, 405nm
O
O
N
O
O
Table 1 Comparison between classical and new synthetic route
As shown in Table 2, only compound 5a showed slight inhibition of HLE. Surprisingly all compounds raised
the chymotrypsin activity by up to 30%. Nevertheless the new route to N-acylpyrrolidin-2-ones seems to be an
attractive way as the classical preparation does not always succeed. (Attempts with phenylacetic acid led to
unusable product mixtures.)
References:
Substrate:
Enzyme:
Inhibitor:
Buffer:
DMSO:
200µM Suc-Ala-Ala-Pro-Phe-pNA
bovine chymotrypsin, 50ng/ml
as stated above
20mM Tris-HCl pH 8.4
150mM NaCl
6%
Table 2 Enzyme assays
Substrate:
Enzyme:
Inhibitor:
Buffer:
DMSO:
100µM MeO-Suc-Ala-Ala-Pro-Val-pNA
HLE, 25ng/ml
100µM
50mM Na2HPO4/NaH2PO4 pH 7.8,
500mM NaCl
1.5%
[1] Sasaki, H.; Mori, Y.; Nakamura, J.; Shibasaki, J. Synthesis and anticonvulsant activity of 1-acyl-2pyrrolidinone derivatives. J.Med.Chem. 1991, 34, 628-633
[2] Sykes, N.O.; Macdonald, S.J.F.; Page, M.I. Acylating Agents as Enzyme Inhibitors and Understanding
Their Reactivity for Drug Design. J.Med.Chem. 2002, 45, 2850-2856
[3] Powers, J.C.; Asgian, J.L.; Ekici, O.D.; James, K.E. Irreversible Inhibitors of Serine, Cysteine, and
Threonine Proteases Chem.Rev. 2002, 102, 4639-4750
Thanks to Markus Pietsch and Sonja Kolp who did the enzyme assays.