Identification of novel plasmin inhibitors possessing nitrile moiety as warhead

Article abstract of DOI:10.1016/j.bmcl.2011.08.121

Lysine-nitrile derivatives having a trisubstituted benzene, which belongs to a new chemical class, were prepared and tested for inhibitory activities against plasmin and the highly homologous plasma kallikrein and urokinase. The use of the novel chemotype in the development of plasmin inhibitors has been demonstrated by derivatives of compound 9.

Full text of DOI:10.1016/j.bmcl.2011.08.121

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6305–6309
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Bioorganic & Medicinal Chemistry Letters
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Identification of novel plasmin inhibitors possessing nitrile moiety as warhead
Naoki Teno a, , Keigo Gohda b, Keiko Wanaka c, Takuya Sueda a, Yuko Tsuda d
⇑
a Hiroshima International University, Faculty of Pharmaceutical Science, 5-1-1, Hirokoshingai, Kure, Hiroshima 737-0112, Japan
b Computer-aided Molecular Modeling Research Center, Kansai (CAMM-Kansai), 5-1-7, Ohmichidori, Nagata, Kobe 653-0833, Japan
c Kobe Research Projects on Thrombosis and Haemostasis, 3-15-18, Asahigaoka, Tarumi-ku, Kobe 655-0033, Japan
d Kobe Gakuin University, Faculty of Pharmaceutical Sciences, 1-1-3, Minatojima, Chuo-ku, Kobe 650-8586, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Lysine-nitrile derivatives having a trisubstituted benzene, which belongs to a new chemical class, were
prepared and tested for inhibitory activities against plasmin and the highly homologous plasma kallikrein
and urokinase. The use of the novel chemotype in the development of plasmin inhibitors has been dem-
onstrated by derivatives of compound 9.
Received 29 June 2011
Revised 26 August 2011
Accepted 29 August 2011
Available online 1 September 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Plasmin inhibitors
Nitrile warhead
Plasmin substrate
Plasma kallikrein
Urokinase
Plasmin is best known as the key molecule in the fibrinolytic
system and is critical for clot lysis,1 which directly degrades extra-
cellular matrix components including the glycoproteins fibronec-
tin, laminin, and elastin, as well as proteoglycans.2 Otherwise,
plasmin can initiate matrix metalloproteinase (MMP) activation
cascade. MMPs, themselves, are implicated in a complex cascade
of activation.3 Along with MMP, plasmin is suggested to be in-
volved in physiological processes including angiogenesis, tissue re-
pair, tumor invasion and metastasis.4–6 It seems likely that the
aging process is influenced by the activity of serum proteolytic en-
zymes, MMP-9 and plasmin.7 Thus, the research area on plasmin
has dynamically shifted from historical molecule in the fibrinolytic
system to a newly confirmed drug target. Although plasmin inhib-
itors should be now perceived as a promising new principle in the
treatment of diseases triggered by plasmin, few plasmin inhibitors
are available in recognized compounds, such as tranexamic acid
pharmaceuticals, or YO-2 and peptidic inhibitors possessing cyclic
ketone group as experimental tools.8–10
amine group at the P3 position, such that D-Ile-Phe-Lys-pNA was
identified as a specific substrate for plasmin .14,15
A selective peptidic substrate for a targeted protease provides
many potential approaches for developing a selective inhibitor
against the protease. Many chemotypes have been described as
peptidic and non-peptidic inhibitors with warhead of cysteine
and serine proteases.16,17 A number of cathepsin K inhibitors with
a nitrile group as warhead have been identified that covalently
bind to active site Cys25 of cathepsin K.18,19 Likewise, the dipepti-
dyl peptidase (DPP-IV) inhibitor Vildagliptin (LAF237) is clinically
used as an anti-diabetes drug also has a nitrile group that cova-
lently interacts with Ser630 in the active site of DPP-IV.20 Merck
recently reported that the electrophilicity and reactivity of
nitrile-containing inhibitors have an impact on the reversibility
of the enzyme–inhibitor complex.21 In addition, a survey of ni-
trile-containing pharmaceuticals and clinical candidates identified
several roles for the nitrile group, including an effect on the ADME
profile.22 Identification of the nitrile group as a warhead had a
significant impact on the development of cysteine and serine pro-
tease inhibitors. This accounts for our attempts to design new plas-
min inhibitors with nitrile group.
Up to the mid of 1980’s, only a few studies identified the sub-
strates for plasmin; however, they were not selective against other
serine proteases.11–13 Subsequently, some research focused on
increasing the selectivity against other members of this enzyme
family. From these studies it was discovered that plasmin exhib-
As the first step in tackling plasmin inhibitors possessing a
nitrile group, compound 1 was designed by replacing the scissile
ited a strong preference for hydrophobic
D
-amino acids and a free
amide bond of substrate (D-Ile-Phe-Lys-pNA) with the warhead
(electron deficient center), which was capable of reacting in a
reversible manner (Table 1). Compound 2 was also designed to elu-
cidate whether the nitrile group had an impact on the reactivity of
the enzyme–inhibitor complex formation. Both the peptide–nitrile
⇑
Corresponding author. Tel.: +81 823 73 8942; fax: +81 823 73 8981.
E-mail address: n-teno@ps.hirokoku-u.ac.jp (N. Teno).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.08.121

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N. Teno et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6305–6309
Table 1
Fig. 2a) which is a benzyl moiety. Incorporation of picolyl group
into R1 resulted in plasmin inhibitor 3 with no substantial change
in the selectivity against PK and UK (Table 2). Replacement of the
nitrile in 3 with an amide exhibited a reduced potency against
Inhibition of plasmin, plasma kallikrein and urokinase by compounds 1and 2
Compd nos.
Structures
IC50 (lM)
Plasmin
Plasma
Urokinase
plasmin (IC50 >1000 lM). This substantial difference of the activity
kallikrein
is another supportive evidence that the nitrile group works as the
warhead for plasmin. Molecular modeling supported that if the ni-
trile group was covalently bound to the active site serine, the pic-
olyl group would occupy the S3 subsite while the S2 subsite would
remain unoccupied (Fig. 3). In addition, modeling suggested that
the substitutent at the meta-position (P2 moiety as R2 in Fig. 2b)
of the hydrophobic group would point toward the S2 subsite.
Hence, derivatives of compound 3 were prepared to explore the
role of R2 for inhibitory activity against plasmin. As indicated in
Table 2, introduction of an aromatic ring into R2 lost their inhibi-
tory activity against plasmin (3 vs 4). Compound 5, which differs
from 4 by the replacement of p-fluorobenzene with 2-naphthyl,
showed increased potency against plasmin, whereas compound
6, the regioisomer of 5, lost its inhibitory activity. Compounds 5
and 6 increased the affinity for PK as well, whereas the indole
derivatives 7–9 completely lost the inhibitory activity against the
untargeted enzyme, PK. Inhibitors 7 and 8 were found to be potent
and selective to a similar level to that of compounds 3 and 5.
Replacement of t-butoxycarbonyl with a p-fluorobenzyl group
exhibited increased inhibitory activity for plasmin without sub-
stantial change in the selectivity against PK and UK (7 vs 9). As ex-
pected above from molecular modeling, these results suggested
that the P2 moiety could have an interaction with the S2 pocket
in plasmin. The structures of 9 and 10 imply that the picolyl group
in P3 moiety contributes to the increase in inhibitory activity
against plasmin while retaining selectivity against PK and UK.
Especially, since compound 3 possesses considerable inhibitory
activity against plasmin, the picolyl group as the P3 moiety seems
to be an essential asset for the scaffold of plasmin inhibitors re-
ported here. Molecular modeling of 9 reported here will be covered
in future publications.
1
2
78
38% at 1000
0% at 1000
15% at 1000
0% at 1000
D
-Ile-Phe-Lys-CN
-Ile-Phe-Lys-NH2
290
D
derivatives were prepared and tested for inhibitory activities
against plasmin and the highly homologous plasma kallikrein
(PK) and urokinase (UK).23 Compound 1 displayed potent plasmin
inhibition combined with significant selectivity for plasmin over
PK and UK. Compound 2 showed about a 3-fold decrease in the
inhibitory activity for the target enzyme and proved to be less ac-
tive for other enzymes. These results suggest that the nitrile group
is covalently bound to the Ser active site. Additionally, molecular
modeling of D-Ile-Phe-Lys substrate complexed with plasmin sup-
ported experimental data that the each amino acid in the inhibitor
sequence occupied well-recognized S1 subsite in plasmin and a
wider subsite, comprising S2 and S3,24 was also occupied by the
hydrophobic residues of D-Ile and Phe (Fig. 1). The initial investiga-
tion led to the development of the first nitrile-containing peptide-
derived plasmin inhibitor, which was the basis for the subsequent
development of nitrile-containing non-peptide-related plasmin
inhibitors.
Okada et al. discovered a non-peptidic plasmin inhibitor, YO-2.9
The inhibitor had tranexamic acid instead of Lys and occupied the
S1 subsite. Modeling studies suggested that the O-picolyl-Tyr moi-
ety in YO-2 assumes an extended conformation along with the wall
of S2 site.24 Molecular modeling of the substrate and YO-2 com-
plexed with plasmin suggested that a rigid spacer extended from
the N-terminus of the P1 residue, Lys, would be directed toward
the broad and hydrophobic space comprised by the S2 and S3
subsites; for example, in the case of R1 (P3 moiety as R1 in
Figure 1. D
-Ile-Phe-Lys substrate docked into the active site of plasmin.24
a
b
S1
S1
NH2
N
NH2
N
P1 moiety
O
P1 moiety
S2
S2
P2 moiety
R2
O
N
H
N
H
R1
R1
O
O
P3 moiety
P3 moiety
S3
S3
Figure 2. Design of new chemotypes for plasmin inhibitors.

N. Teno et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6305–6309
6307
Table 2
Inhibition of plasmin, plasma kallikrein and urokinase by compounds 3–10
Figure 3. Compound 3 docked into the active site of plasmin.24
The peptidic derivatives 1 and 2 were prepared by conventional
solution method.25 It has been previously reported that dipeptide
or tripeptide nitrile was shown to be prone to racemization due
of representative plasmin inhibitor 9 is illustrated in Scheme 1.
Derivative 12 was prepared from 1127 and picolyl chloride, which
was coupled with 3-{1-[(4-fluorophenyl)methyl]indol-3-yl}propa-
noic acid28 to yield 13. Nitrile 16 was synthesized from condensa-
to the increased acidity of the nitrile
a
-position.26 However, the
diastereomers of tripeptide nitrile 1 were not detected with re-
verse phase HPLC and 1H NMR. On the basis of this observation,
compounds 3–10 discussed here are prepared and the synthesis
tion of 14 and L-Lys(Boc)-NH2 following dehydration of the
resulting amide 15 with trifluoroacetic anhydride and Et3N.29
Derivative 16 was deprotected with 50% TFA in CH2Cl2 and the

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N. Teno et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6305–6309
O
H
O
O
N
O
H
N
O
O
N
H
N
a
b, c
O
O
O
O
O
O
O
HO
F
N
11
12
13
O
N
O
HN
O
O
H
N
N
d
e
OH
H
N
N
NH2
O
N
H
O
O
O
O
O
F
N
14
15
F
N
N
O
NH2
N
HN
N
O
H
N
f
g, h
O
H
N
N
H
N
O
N
O
H
O
O
F
F
N
16
9
N
Scheme 1. Reagents and conditions: (a) Picolyl chloride, KI, K2CO3, CH3CN, 70 °C, 12 h; (b) 4 N HCl/dioxane, rt, 1 h; (c) 3-{1-[(4-fluorophenyl)methyl]indol-3-yl}propanoic
acid, Et3N, HOAt, WSCDÁHCl, DMF, rt, 12 h; (d) LiOH, H2O, THF, MeOH, rt, 12 h; (e) H–Lys(Boc)–NH2, Et3N, HOAt, WSCDÁHCl, DMF, rt, 12 h; (f) (CF3CO)2O, Et3N, THF, 0 °C, 5 min;
(g) CF3COOH, CH2Cl2, rt, 1 h; (h) reverse phase HPLC.
11. Pierzchala, P. A.; Dorn, C. P.; Zimmerman, M. A. Biochem. J. 1979, 183, 555.
12. Weinsteina, M. J.; Doolittle, R. F. Biochim. Biophys. Acta 1972, 258, 577.
13. Kato, H.; Adachi, N.; Ohno, Y.; Iwanaga, S.; Takada, K.; Sakakibara, S. J. Biochem.
resulting compound was purified by preparative HPLC to provide
compound 9.
In summary, the new plasmin inhibitors reported here repre-
sent an important step in tackling chemical modification of pepti-
dyl derivatives. Evaluating how to develop the trisubstituted
benzene derivatives (compound 3) is a particularly attractive start-
ing point based on the ready availability of a wide range of starting
materials. The chemical modification of 3 was focused on the P2
and P3 moieties which contributed to the potency for plasmin
and the selectivity against other serine proteases. The capability
of the novel chemotypes in the development of plasmin inhibitors
is further demonstrated by derivatives represented by 9, and
appropriate moieties extending from the trisubstituted benzene,
which become engaged with key pockets in the active site of
plasmin.
1980, 88, 183.
14. Cs-Szabo, G.; Pozsgay, M.; Elodi, P. Thrombosis Res. 1980, 20, 199.
15. Okada, Y.; Tsuda, Y.; Teno, N.; Wanaka, K.; Sasaki, K.; Hijikata, A.; Naito, T.;
Okamoto, S. Int. J. Pept. Protein Res. 1986, 27, 79.
16. Lecaille, F.; Kaleta, J.; Brömme, D. Chem. Rev. 2002, 102, 4459.
17. Hedstrom, L. Chem. Rev. 2002, 102, 4501.
18. Black, W. C. Curr. Top. Med. Chem. 2010, 10, 745.
19. Teno, N.; Masuya, K. Curr. Top. Med. Chem. 2010, 10, 752.
20. Peters, J.-U. Curr. Top. Med. Chem. 2007, 7, 579.
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Okunomiya, A.; Okamoto, S. Chem. Pharm. Bull. 1993, 41, 1079.
24. Gohda, K.; Teno, N.; Wanaka, K.; Tsuda, Y. J. Enzyme Inhib. Med. Chem. accepted
for publication.
25. All compounds were characterized by 1H NMR and MS(ESI). Spectral data of
representative compounds, 1, 2, 7, 8 and 9: Compound 1: 1H NMR (DMSO-d6,
600 MHz): d 8.99 (1H, d), 8.76 (1H, d), 7.94 (2H, br s), 7.68 (2H, br s), 7.29–7.24
(5H, m), 4.69–4.65 (2H, m), 3.61 (1H, br s), 3.32 (1H, dd), 3.02 (1H, dd), 2.78–
2.74 (2H, m), 1.84–1.81 (1H, m), 1.74–1.72 (1H, m), 1.56–1.54 (3H, m), 1.41–
1.39 (2H, m), 1.05–0.95 (1H, m), 0.71–0.69 (1H, m), 0.63–0.59 (6H, m); MS (ESI)
m/z: 386 (M-1). Compound 2: 1H NMR (DMSO-d6, 600 MHz): d 8.58 (1H, d),
8.32 (1H, d), 7.90 (2H, br s), 7.65 (2H, br s), 7.32–7.08 (7H, m), 4.75–4.72 (1H,
m), 4.17–4.15 (1H, m), 3.60–3.57 (1H, m), 3.13–3.10 (1H, m), 2.75–2.51 (3H,
m), 1.68–1.62 (1H, m), 1.55–1.48 (3H, m), 1.30–1.27 (2H, m), 0.95–0.88 (1H,
m), 0.60–0.56 (7H, m); MS (ESI) m/z: 404 (M-1). Compound 7: 1H NMR (DMSO-
d6, 600 MHz): d 9.50 (1H, s), 8.99 (1H, d), 8.60 (2H, br s), 8.34 (1H, s), 8.00 (1H,
d), 7.66–7.57 (6H, m), 7.50 (1H, s), 7.32 (1H, t), 7.25 (1H, t), 7.12 (1H, d), 5.38
(2H, s), 4.92–4.91 (1H, m), 3.02–3.00 (2H, m), 2.87–2.84 (2H, m), 2.80–2.76
(2H, m), 1.90–1.86 (2H, m), 1.57 (9H, s), 1.57–1.54 (2H, m), 1.43–1.42 (2H, m);
MS (ESI) m/z: 625 (M+1). Compound 8: 1H NMR (DMSO-d6, 600 MHz): d 10.79
(1H, s), 9.43 (1H, s), 9.00 (1H, d), 8.62 (2H, br s), 8.35 (1H, s), 8.00 (1H, d), 7.61–
7.55 (7H, m), 7.32 (1H, d), 7.16 (1H, s), 7.13 (1H, d), 7.06 (1H, t), 6.97 (1H, t),
5.39 (2H, s), 4.94–4.90 (1H, m), 3.05–3.02 (2H, m), 2.82–2.78 (4H, m), 1.90–
1.87 (2H, m), 1.57–1.54 (2H, m), 1.47–1.41 (2H, m); MS (ESI) m/z: 525 (M+1).
Compound 9: 1H NMR (DMSO-d6, 600 MHz): d 9.41 (1H, s), 9.00 (1H, d), 8.57
(2H, br s), 8.37 (1H, s), 7.60–7.52 (7H, m), 7.40 (1H, d), 7.31 (1H, s), 7.20–7.18
(2H, m), 7.17–7.01 (4H, m), 5.35 (2H, s), 5.31 (2H, s), 4.93–4.91 (1H, m), 3.05–
Acknowledgments
We are grateful to Dr. Lawrence Lazarus and Dr. Tadamune
Otsubo for helpful critical reading of the manuscript.
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