Evaluation of pyrrolin-2-one derivatives synthesized by a new practical method as inhibitors of plasminogen activator inhibitor-1 (PAI-1)

Article abstract of DOI:10.1016/j.bmcl.2009.11.102

We describe in this Letter a new synthetic method for pyrrolin-2-ones as potent plasminogen activator inhibitor-1 (PAI-1) inhibitors. Pyrrolin-2-one derivatives synthesized from N-2-oxoethylamides and aldehydes in aqueous NaOH by one-pot were evaluated for their PAI-1 inhibitory activity. Among these derivatives, compounds 16 and 18 were found to possess potent PAI-1 inhibitory activity (compound 16: IC₅₀: 0.69 μM, compound 18: IC₅₀: 0.65 μM).

Full text of DOI:10.1016/j.bmcl.2009.11.102

Bioorganic & Medicinal Chemistry Letters 20 (2010) 546–548
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Evaluation of pyrrolin-2-one derivatives synthesized by a new practical
method as inhibitors of plasminogen activator inhibitor-1 (PAI-1)
a
a
a
a
Hiroshi Miyazaki a, , Tsutomu Miyake , Yoshihiro Terakawa , Hiroshi Ohmizu , Tsuyoshi Ogiku ,
*
Akio Ohtani b
a Medicinal Chemistry Laboratory, Mitsubishi Tanabe Pharma Co., Ltd, 3-16-89, Kashima, Yodogawa, Osaka 532-8505, Japan
b Pharmacology Laboratory, Mitsubishi Tanabe Pharma Co., Ltd, 2-2-50, Kawagishi, Toda, Saitama 335-8505, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We describe in this Letter a new synthetic method for pyrrolin-2-ones as potent plasminogen activator
inhibitor-1 (PAI-1) inhibitors. Pyrrolin-2-one derivatives synthesized from N-2-oxoethylamides and alde-
hydes in aqueous NaOH by one-pot were evaluated for their PAI-1 inhibitory activity. Among these deriv-
Received 1 September 2009
Revised 14 November 2009
Accepted 19 November 2009
Available online 23 November 2009
atives, compounds 16 and 18 were found to possess potent PAI-1 inhibitory activity (compound 16: IC50
:
0.69 M, compound 18: IC50: 0.65 M).
l
l
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Plasminogen activator inhibitor-1
PAI-1 inhibitor
Antithrombotic agent
Pyrrolin-2-one
It is well known that plasminogen activator inhibitor-1 (PAI-1)
is a specific inhibitor of both tissue-type plasminogen activator and
urokinase-type plasminogen activator, and that PAI-1 plays an
important role in regulation of the fibrinolytic system.1 Elevated
levels of PAI-1 in plasma have been observed in patients with deep
vein thrombosis2 and unstable angina.3 Furthermore, a number of
animal studies have shown that PAI-1 is the factor which disturbs
fibrinolytic activity in both thrombotic and prethrombotic states.4
Thus, inhibition of PAI-1 activity or reduction of its production is
considered to shift the balance between thrombogenesis and
thrombolysis towards thrombolysis. In fact, an antibody against
PAI-1 has been shown to enhance clot lysis and decrease thrombus
growth in animal models of venous thrombosis5 and arterial
thrombosis.6 Recently, a number of small molecules have been
evaluated for their ability to inhibit PAI-1 or reduce its production
and for their potential to act as antithrombotic agents.7
synthetic method and evaluate the synthesized pyrrolin-2-ones
for their inhibitory activity towards PAI-1.
The conventional synthetic route of 1(T-1776Na) has some
drawbacks (Scheme 1). Indeed, construction of the pyrrolin-2-
one ring from the N-2-oxoethylamide 2 under basic conditions
produces in some cases side reactions. In addition, stability of the
pyrrolin-2-ones under preparation conditions remarkably changes
depending on the chemical properties of 3,4-diaryl group, and a
dimmer or the maleimide 3 are known to be obtained as major
by-products.9 Therefore, the synthesis of 1(T-1776Na) requires
construction of a pyrrolin-2-one ring from a phenacylamide under
acidic conditions (Ac2O) followed by Boc protection to introduce
the phenylmethylidene part, and a total of eight steps are needed.8
In order to overcome these problems, we anticipated that anion
intermediates, generated in situ during construction of the pyrro-
lin-2-one ring under basic conditions, could be trapped with an
aldehyde to directly afford the desired PAI-1 inhibitors.
We have previously reported the structure–activity relationship
(SAR) of a series of pyrrolin-2-one derivatives for PAI-1 inhibition
(Fig. 1)8 and showed that compound 1(T-1776Na) exhibited potent
inhibitory activity for PAI-1 (IC50: 9.6 lM), good antithrombotic
efficacy in an animal thrombosis model, and high selectivity for
PAI-1 over serine proteases and serpins. In the course of our search
for a superior antithrombotic agent, we have found a facile new
synthetic method for the pyrrolin-2-ones leading to a series of
PAI-1 inhibitors. Herein, we disclose the development of this new
* Corresponding author. Tel.: +81 6 6300 2578; fax: +81 6 6300 2564.
E-mail address: miyazaki.hiroshi@me.mt-pharma (H. Miyazaki).
Figure 1. Structures of pyrrolin-2-one derivatives as PAI-1 inhibitors and their
representative compound 1(T-1776Na).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.102

H. Miyazaki et al. / Bioorg. Med. Chem. Lett. 20 (2010) 546–548
547
Thus, anion intermediates could be trapped with an aldehyde,
which led us to achieve simple and practical synthesis of various
pyrrolin-2-ones as PAI-1 inhibitors. The pyrrolin-2-one derivatives
in entries 2–11 were designed to possess hydrophilic moieties at
R1–R3, and make up for the scarce solubility of the pyrrolin-2-
one scaffold (Fig. 2). These compounds are expected to have good
potential as antithrombotic agents.
Scheme 1. Synthesis of pyrrolin-2-ones under basic conditions.
Table 2
Inhibitory effect of pyrrolin-2-one derivatives against human PAI-1 activity
Compound
Inhibitory activity
Table 1
Reaction of N-2-oxoethylamides with aldehydesa
No effect at 15 lM
IC50; 9.6 lM
Entry
R1
R2
R3
Temp (°C)
Yieldc (%)
36% inhibition at 10
34% inhibition at 15
l
M
M
1
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
4
2-Furyl
5
6
7
50
rt
rt
rt
rt
rt
50
rt
rt
rt
rt
98 (9)
80 (1)
2b
3
2-Furyl
2-Furyl
2-Furyl
2-Furyl
2-Furyl
2-Furyl
2-Thienyl
2-MeOPh
2-MeOPh
93 (10)
71 (11)
83 (12)
77 (13)
30 (14)
70 (15)
69 (16)
72 (17)
83 (18)
4
5
6b
7
l
3,4-(MeO)2Ph
3,4-(MeO)2Ph
3,4-(MeO)2Ph
Ph
4-Pyridyl
8b
9b
10
11b
7
7
8
7
Ph
IC50; 1.2
l
M
M
a
Reactions were carried using N-2-oxoethylamide, aldehyde (1.0 equiv) and 2 N
NaOH aq (10 eqiuv) in MeOH.
b
Products were obtained as the corresponding carboxylic acid instead of the
ester residue.
c
Numbers of reaction products are shown in parentheses.
IC50; 1.5
l
The products of various reactions of N-2-oxoethylamides with
aldehydes in aqueous NaOH using MeOH as a solvent are shown in
Table 1. First, we examined the case where all substituents (R1–R3)
were phenyl. An anion intermediate of the pyrrolin-2-one could be
trapped with benzaldehyde, and only the Z-product was obtained
in 98% by one-pot synthesis (entry 1).10 Next, we applied this reac-
tion to 1(T-1776Na). In this case, a Z-product was also easily
obtained as a carboxylic acid by acidification of the reaction mixture
and filtration in 80% yield (entry 2). This Z-product could be quanti-
tativelyconverted to 1(T-1776Na). The reaction could also be used in
the case where R1–R3 were substituted or nonsubstituted heteroar-
omatic rings (entries 3–11). In these cases, the Z-products were eas-
ily obtained as crystals or solids by acidification, (in some cases,
extraction), and filtration. In all cases analytically pure compounds
were obtained in good yield without further purification (silica gel
chromatography or recrystallization).11
IC50; 7.8
l
M
M
IC50; 1.1
l
IC50; 0.69
l
M
IC50; 0.94
IC50; 0.65
l
M
M
l
Figure 2. Optimization of substituents in the pyrrolin-2-one.

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H. Miyazaki et al. / Bioorg. Med. Chem. Lett. 20 (2010) 546–548
B.; Van den Oord, J. J.; De Mol, M.; Mulligan, R. C.; Collen, D. J. Clin. Invest. 1993,
Biologic evaluation of the synthesized pyrrolin-2-ones was per-
92, 2756.
formed by measuring the inhibitory effect on the reaction of hu-
man PAI-1 with t-PA, according to our previously published
report8 (Table 2). In that report, we showed that 9 was inactive
against PAI-1 and that 1(T-1776Na), having a phenoxy acetic acid
group at R3 of the pyrrolin-2-one, had potent PAI-1 inhibitory
5. Levi, M.; Biemond, B. J.; Van Zonnenveld, A. J.; Ten Cate, J. W.; Pannekoek, H.
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Leahy, J. W.; Mac, M.; Matthews, D.; Prisbylla, M. P.; Sanderson, J.; Simon, R. J.;
Tesfai, Z.; Vicker, N.; Wang, S.; Webb, R. R.; Charlton, P. Bioorg. Med. Chem. Lett.
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J.; Ohtani, A.; Ohtani Bioorg. Med. Chem. Lett. 2008, 18, 6419.
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10. In the NOESY spectra (CDCl3), the vinyl proton of compound 9 at d 6.06
correlates with the protons of phenyl ring at R2, while it does not correlate with
the NH proton at d 7.91. On the other hand, the NH proton correlates with the
protons of phenyl ring at R3. These data show the stereochemistry of
compound 9 is Z.
activity (IC50: 9.6 lM). Various substituted pyrrolin-2-ones were
next examined. First, a furan ring was adopted at R2 of the pyrro-
lin-2-one as a hydrophilic equivalent to the benzene. Compounds
10, 11, having a furan or furan-2-carboxylic acid at R3 showed
weak PAI-1 inhibitory activity. However, in the case where the
hydrophilic group (nitrile and carboxylic acid) was located far from
the aromatic ring, the pyrrolin-2-ones exhibited good inhibitory
activity against PAI-1 (12: IC50: 1.2 lM, 13: IC50: 1.5 lM). Further-
more, to find PAI-1 inhibitors with decreased lipophilicity, 3,4-
dimethoxyphenyl substituents were introduced at R1. Although
14 did not inhibit PAI-1, 15 and 16 showed good PAI-1 inhibitory
activity. We speculate that this is due to the hydrophilic moiety
(carboxylic acid) in 15 and 16 being away from the aromatic ring
in R3. Finally, even when the 2-furyl or 2-thienyl at R2 was con-
verted into 2-methoxyphenyl, the inhibitory activity for PAI-1
was maintained. Compound 18 was found to be the most potent
inhibitor of PAI-1 with an IC50 value of 0.65 lM.
In summary, we found a practical new synthetic method of
pyrrolin-2-ones as potent PAI-1inhibitors. Using this new method,
a series of pyrrolin-2-one derivatives were synthesized and their
inhibitory activity for PAI-1 was evaluated. Some of the synthe-
sized compounds were designed to possess a hydrophilic moiety
at R1–R3, which led to the discovery of 1612 and 1813 with a PAI-
1 inhibitory activity ten times more potent than that of 1(T-
1776Na). Further studies on these promising compounds are
ongoing.
11. Typical procedure: 2 N NaOH aq (2.5 ml) was added to a mixture of N-2-
oxoethylamide (0.5 mmol) and aldehyde (0.5 mmol) in MeOH (5 ml) at room
temperature (16–25 °C). After the reaction mixture was stirred for 14 h at
room temperature, 2 N HCl aq (2.5 ml) was added at 0 °C. A precipitated solid
was collected by filtration, washed with water and then MeOH, and dried to
afford its products.
References and notes
12. Spectoscopic data of 16: 1H NMR (400 MHz, DMSO-d6) d: 12.38 (br, 1H), 10.60
(s, 1H), 7.78 (t, J = 3.1 Hz, 1H), 7.52 (d, J = 15.9 Hz, 1H), 7.24 (d, J = 3.1 Hz, 2H),
7.19 (dd, J = 8.4, 1.5 Hz, 1H), 7.09 (d, J = 3.6 Hz, 1H), 6.99 (d, J = 3.6 Hz, 1H), 6.95
(d, J = 8.4 Hz, 1H), 6.88 (d, J = 1.5 Hz, 1H), 6.62 (d, J = 15.9 Hz, 1H), 5.99 (s, 1H),
3.76 (s, 3H), 3.51 (s, 3H). IR (ATR) cmÀ1: 3279, 3103, 2835, 2479, 1681, 1637,
1600, 1256, 1146, 1024, 714, 625. MS (APCI): 450 [M+H]+.
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13. Spectoscopic data of 18: 1H NMR (400 MHz, DMSO-d6) d: 12.37 (br, 1H), 10.52
(s, 1H), 7.43–7.55 (m, 2H), 7.31–7.40 (m, 2H), 7.20–7.30 (m, 3H), 7.10–7.20 (m,
2H), 7.00–7.10 (m, 2H), 6.92 (d, J = 3.3 Hz, 1H), 6.61 (d, J = 15.9 Hz, 1H), 5.58 (s,
1H), 3.61 (s, 3H). IR (ATR) cmÀ1: 3262, 3071, 2836, 1686, 1645, 1274, 1253,
1215, 1022, 983, 760, 713, 693, 639. Anal Calcd for C25H19NO5: C, 72.63; H,
4.63; N, 3.39. Found: C, 72.35; H, 4.57; N, 3.40. MS (APCI): 414 [M+H]+.
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