Synthesis and evaluation of tripeptidic plasmin inhibitors with nitrile as warhead

Article abstract of DOI:10.1002/psc.2442

Plasmin is best known as the key molecule in the fibrinolytic system, which is critical for clot lysis and can initiate matrix metalloproteinase (MMP) activation cascade. Along with MMP, plasmin is suggested to be involved in physiological processes that are linked to the risk of carcinoma formation. Plasmin inhibitors could be perceived as a promising new principle in the treatment of diseases triggered by plasmin. On the basis of the peptidic sequence derived from the synthetic plasmin substrate, a series of peptidic plasmin inhibitors possessing nitrile as warhead were prepared and evaluated for their inhibitory activities against plasmin and other serine proteases, plasma kallikrein and urokinase. The most potent peptidic inhibitors with the nitrile warhead exhibit the potency toward plasmin (IC₅₀=7.7-11μM) and are characterized by their selectivity profile against plasma kallikrein and urokinase. The results and molecular modeling of the peptidic inhibitor complexed with plasmin reveal that the P2 residue makes favorable contacts with the open binding pocket comprising the S2 and S3 subsites of plasmin.

Full text of DOI:10.1002/psc.2442

Research Article
Received: 23 May 2012
Revised: 5 July 2012
Accepted: 16 July 2012
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/psc.2442
Synthesis and evaluation of tripeptidic
plasmin inhibitors with nitrile as warhead
Naoki Teno,a* Tadamune Otsubo,a Keigo Gohda,b Keiko Wanaka,c
Takuya Sueda,a Kiyoshi Ikeda,a Akiko Hijikata-Okunomiyad
and Yuko Tsudae
Plasmin is best known as the key molecule in the fibrinolytic system, which is critical for clot lysis and can initiate
matrix metalloproteinase (MMP) activation cascade. Along with MMP, plasmin is suggested to be involved in physiolog-
ical processes that are linked to the risk of carcinoma formation. Plasmin inhibitors could be perceived as a promising
new principle in the treatment of diseases triggered by plasmin. On the basis of the peptidic sequence derived from
the synthetic plasmin substrate, a series of peptidic plasmin inhibitors possessing nitrile as warhead were prepared
and evaluated for their inhibitory activities against plasmin and other serine proteases, plasma kallikrein and urokinase.
The most potent peptidic inhibitors with the nitrile warhead exhibit the potency toward plasmin (IC50 = 7.7–11 mM)
and are characterized by their selectivity profile against plasma kallikrein and urokinase. The results and molecular
modeling of the peptidic inhibitor complexed with plasmin reveal that the P2 residue makes favorable contacts with
the open binding pocket comprising the S2 and S3 subsites of plasmin. Copyright © 2012 European Peptide Society and
John Wiley & Sons, Ltd.
Supporting information can be found in the online version of this article.
Keywords: plasmin inhibitors; P2 hydrophobic group; nitrile; warhead; structure–activity relationship
the basic Arg or Lys as P1 residue in endogenous substrates
[13]. The trypsin-like serine proteases, such as plasmin, also
Introduction
have a preference for Lys as the P1 moiety [14]. These pre-
ferred residues were then successfully utilized for identifying
peptidic and non-peptidic inhibitors for the proteases as the
Plasmin, like trypsin, belongs to the family of serine proteases
that act to dissolve fibrin blood clots [1]. Apart from fibrinolysis,
plasmin proteolyses proteins, fibronectin, thrombospondin,
charged P1 group [13]. Of the various types of the trypsin-
laminin, and von Willebrand factor in various other systems
like serine protease inhibitors investigated, rivaroxaban for fac-
[2]. As an antifibrinolytic agent for reduction of perioperative
tor Xa (FXa) was developed as a new inhibitor with a non-basic
bleeding [3], the plasmin inhibitor aprotinin (Trasylol) was the
chlorothiophene as the P1 moiety instead of a basic residue like
most effective. The use of aprotinin retains the potential to
trigger myocardial infarction [4], vein graft hypercoagulation
Arg or Lys [15].
[5], and renal failure [6,7] in clinical trials. Otherwise, plasmin
can initiate the matrix metalloproteinase (MMP) activation cas-
*
Correspondence to: Naoki Teno, Hiroshima International University, Faculty
of Pharmaceutical Sciences, 5-1-1, Hirokoshingai, Kure, Hiroshima 737-0112,
Japan. E-mail: n-teno@ps.hirokoku-u.ac.jp
cade. Moreover, MMPs themselves are implicated in a complex
cascade of activation [8]. Along with MMP, plasmin is suggested
to be involved in physiological processes including angiogenesis,
tissue repair, tumor invasion, and metastasis [9–11]. Thus, the
research on plasmin has dynamically shifted from a historical
molecule in the fibrinolytic system to a newly confirmed drug
target. Hence, low molecular weight plasmin inhibitors have
become regarded as a potential therapy for the treatment of
various cancers and as antifibrinolytic agents without the ad-
verse effects described earlier. In addition, the available evidence
for physiological and pathophysiological roles of the serine
protease plasmin in inflammatory processes has been recently
reviewed [12].
a Hiroshima International University, Faculty of Pharmaceutical Sciences, 5-1-1,
Hirokoshingai, Kure, Hiroshima 737-0112, Japan
b Computer-aided Molecular Modeling Research Center, Kansai (CAMM-Kansai),
5-1-7, Ohmichidori, Nagata-ku, Kobe 653-0833, Japan
c
Kobe Research Projects on Thrombosis and Haemostasis, 3-15-18, Asahigaoka,
Tarumi-ku, Kobe 655-0033, Japan
d Kobe International University, Faculty of Rehabilitation, 9-1-6, Koyocho-naka,
Higashinada-ku, Kobe 658-0032, Japan
e Kobe Gakuin University, Faculty of Pharmaceutical Sciences, 1-1-3, Minatojima,
Chuo-ku, Kobe 650-8586, Japan
The investigation of plasmin inhibitors has been initiated
with the available information on subsite specificity of plasmin.
The characteristic feature of the trypsin-like serine proteases
is a deep S1 primary or specificity binding pocket with an
acidic Asp at the bottom, which forms a salt bridge with
Abbreviations used: MMP, matrix metalloproteinase; FXa, factor Xa; PK,
plasma kallikreine; UK, urokinase; TMEDA, tetramethylethylenediamine;
<Glu, pyroglutamic acid.
J. Pept. Sci. 2012
Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.

TENO ET AL.
Up to the mid-1980s, the enzymatic characteristics of plasmin
including the substrate specificity, especially of S2 and S3
subsites, remained unknown. Only a few studies to identify the
specific substrates of plasmin have been performed [16–18],
but these have not worked well in the development of plasmin
inhibitors. Since then, the optimization using a parallel synthesis
or systematic way became feasible as shown subsequently. It
has been recently reported that the extended substrate specificity
of plasmin has been investigated using positional-scanning
synthetic combinatorial libraries [19–21]. Using the parallel synthe-
sis approach allowed access to suitable P2 and P3 moieties of plas-
min inhibitors with a high degree of structural diversity. These
studies revealed that Trp was preferred at both the S2 and S3
subsites of plasmin [22,23]. Another important feature of the S3
subsite to be considered is that advantage may also be taken of
differing responses to a D-amino acid residue in P3 [24] or to
various side-chain extensions that utilize an additional unique
binding capacity in this region [25]. Indeed, on the basis of the
sequence of the specific substrate, D-Ile-Phe-Lys-pNA for plasmin,
D-Ile-Phe-Lys-CH2Cl was designed by replacing the scissile amide
bond of the substrate with the warhead (electron deficient center),
which was capable of reacting in an irreversible manner. The
irreversible warhead, an aldehyde, has been recently utilized as
plasmin inhibitor consisting of a tetrapeptide [26]. When the
irreversible inhibitors react with the enzyme, they permanently
deactivate the enzyme and inhibit a superfamily of cysteine
proteases because of the strong electron deficiency of the
warhead, leading to serious adverse effects. In contrast, the
irreversible inhibition of the kinase may be beneficial to produce
a sustained effect on cancer therapy [27].
Our efforts in this area have recently focused on the
optimization of plasmin inhibitors having a Lys residue as the
P1 moiety and the nitrile function as a reversible warhead in
the peptidic sequence D-Ile-Phe-Lys [28]. We have disclosed that
the S2 and S3 subsites in plasmin possess a wide and deep
binding site, indicating that the dimension of the binding site
defines the size and angle of the P2 and/or P3 moiety in the
inhibitors [29]. These results indicated that the optimization of
the P2 and P3 moieties of peptidic inhibitor (1) (Table 1) could
be utilized as valuable information on the development of non-
peptidic plasmin inhibitors against the targeted plasmin. Thus,
we then turned our attention to modification of the P3 and P2
on peptidic scaffold and started to exploit novel P1 residues.
phase for preparation and analysis was a combination of water
(A) and acetonitrile (B), both containing 0.1% TFA, and the flow
rate was 8 and 1 ml/min, respectively. TSK gel ODS columns
(21.5 ꢀ 300 mm for preparation and 4.6 ꢀ 150 mm for analysis,
TOSOH Corporation) were used.
Synthesis
Synthesis, yields and characterization data of all compounds
depicted in Schemes 1 and 2 are given in Supporting Information.
Compounds 2–9 and 27–29 listed in Tables 1–3 are also available
in Supporting Information.
Docking Studies
Plasmin coordinates used in this study were prepared from
micro-plasmin complex with streptokinase (PDB ID: 1BML)
structure. The preparation procedure was described in detail in
[29]. The docked conformation of compound 3 was deduced by
Monte Carlo multiple minimum method [30] with the BatchMin
molecular mechanics engine [31] with standard parameters. The
MMFF94s parameter was used as the force field [32]. The MMFF
and AMBER94 [33] atomic charges were loaded for inhibitor
and enzyme atoms, respectively. Manipulation of molecular
structures was done using the MAESTRO 9.0 molecular modeling
package (Schrodinger Inc., Portland, USA) on a single Pentium IV
central processing unit (3.0GHz) computer with Linux/CentOS4.0.
Inhibition of Plasmin, Plasma Kallikrein, and Urokinase
Human plasmin was purchased from Chromogenix (Sweden) and
human urokinase from Green Cross (Japan). Human plasma
kallikrein was purified from human plasma [34]. H-D-Val-Leu-Lys-
pNA (S-2251), H-D-Phe-Pro-Arg-pNA (S-2302), and <Glu-Gly-Arg-pNA
(S-2444) were purchased from Chromogenix (Sweden). Inhibitory
activities were determined by measuring the effect of the
inhibitor on the hydrolytic activities of enzyme on synthetic
peptide substrate. Plasmin activity was measured with 0.3 mM
S-2251, plasma kallikrein with 0.2 mM S-2302, and urokinase with
0.1 mM S-2444. The IC50 value was taken as the concentration of
inhibitor that reduced the absorbance at 405 nm by 50%
compared with the absorbance measured under the same
conditions without inhibitor.
Materials and Methods
Results and Discussion
General
Synthesis
All chemicals were purchased from Tokyo Chemical Industry Co.,
Ltd. Japan or Wako Pure Chemical Industries, Ltd. Japan and used
without further purification. All protected amino acids and the
coupling reagents were purchased from Watanabe Chemical
Industries, Ltd. 1 H NMR experiments were recorded on a JMTC-600
(JEOL Ltd.) 600 NMR spectrometer with CDCl3 or DMSO-d6 as
solvents. Chemical shifts are expressed in parts per million (ppm, d)
and referred to the solvent signal. High-resolution mass spectrome-
try (HRMS) spectra were recorded on The AccuTOF (JMS-T100LC)
equipped with an electrospray ion source (JEOL Ltd.). The
preparative HPLC system consisted of a Hitachi L-2130 System
Controller, a Hitachi In-Line Degasser, and a Hitachi L-2300
Absorbance Detector (Hitachi High-Technologies Corporation).
The absorbance detector was operated at 254 nm. The mobile
Compounds 1–9 listed in Tables 1 and 2 were prepared by
conventional solution method. Conversion of the protected
dipeptide or tripeptide-amides to the corresponding nitrile form
was carried out by trifluoroacetic anhydride and Et3N [35]
followed by treatment with 20% piperidine in DMF, yielding
crude compounds 1–9. The resulting compounds were purified
by preparative HPLC to provide compounds 1–9 (see Supporting
Information for experimental details).
Only a few studies that prepared the g-heterocyclic a-amino-
butanoic acid have been performed [36–47] but are not effective
for preparing our targeted amino acids. Looking at the successful
example reported by Roth and Thomas [48], key intermediates
12–14 for our targeted analogs 10 and 11 were prepared from
commonly and commercially available starting material as
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PEPTIDIC PLASMIN INHIBITORS POSSESSING NITRILE AS WARHEAD
Table 1. Inhibition of plasmin, plasma kallikrein, and urokinase by
compounds 1–5
NaI in acetone to yield 14, which was coupled with imidazole in
DMF to give 10. Azidation of 13 with NaN3 gave 15. [2 + 3]-Cyclo-
addition reaction between azide derivative 15 and TMS acetylene
occurred to give 16 under the conditions, CuI, TMEDA, Et3N, and
THF [49]. Then, TMS group of 16 was removed by TBAF [50] in the
presence of acetic acid to obtain 11. Subsequently, the
preparation for the desired products was accomplished by means
of 10 and 11 and outlined in Scheme 2. Compounds 10 and 11
were converted to amide derivatives 19 and 20 through Boc
protected 17 and 18, respectively. Protected dipeptide 22 was
prepared from 21. Condensation of 19 and 20 with 22 and
subsequent treatment with (CF3CO)2O and Et3N [35] yielded the
corresponding 25 and 26. Removal of Fmoc group with 20%
piperidine in DMF provided the desired products 27 and 28
(see Supporting Information for experimental details and yields).
Compound
nos.
R
IC50 (mM)
Plasmin
78
Plasma
kallikrein
Urokinase
>1000
(38%)a
>1000
(15%)a
1
2
3
4
Inhibitory Activity against Plasmin, Plasma Kallikrein, and
Urokinase
>1000
(7%)a
>1000
(9%)a
400
We proceeded to prepare a range of aromatic units to be incor-
porated as the P2 moiety while preserving P3 residue (D-Ile) and
P1 moiety (Lys-CN) of compound 1 and tested the inhibitory
activity against serine proteases [51] listed in Table 1. Change in
the ring system to 2 resulted in an analog that reduced potency
for plasmin. Extension by one more picolyl group with a polar
functional group on Phe as in compound 1 (compound 3)
showed an increased potency against plasmin. As indicated in
Figure 1, molecular modeling implied that the extended portion
of 3 reached deep into the S2 and S3 subsites, resulting in
an increase in inhibitory activity against plasmin. Compound 4,
which differs from 3 only by the replacement of a nitrogen atom
in the picolyl group with a carbon atom, had retained potent
action against plasmin. The increased hydrophobicity associated
with further extension of the P2 group (compound 5) revealed
inhibitory activity for plasmin at single-digit micromolar range.
Unlike the case of compounds 1 and 2, the P2 moieties of
compounds 3–5 were being tolerated for plasma kallikrein (PK)
but not for urokinase (UK). The potency and high selectivity
found for these P2 analogs confirm that the hydrophobicity of
the P2 moiety provides a new access to excellent substrate
analog of plasmin inhibitors. In respect to the interaction with
wide subsite comprising the S2 and S3 subsites, Saupe and
Steinmetzer [52] have also disclosed that the hydrophobic linker
>1000
(16%)a
11
12
210
250
>1000
(8%)a
>1000
(11%)a
5
7.7
320
aValues in parentheses are % inhibition at the concentration.
depicted in Scheme 1. Boc-L-Asp-OBut was acylated by ethyl
chloroformate, which was followed by sodium borohydride
reduction to Boc-L-homoserine-OBut (12). Mesylation of 12 was
used to obtain the common intermediate 13 with Et3N and
methanesulfonylchloride. Compound 13 was iodinated by using
Scheme 1. Reagents and conditions: (a) EtOCOCl, Et3N, THF, 0 ꢁC. (b) NaBH4, MeOH, 0 ꢁC. (c) MsCl, Et3N, THF, 0 ꢁC. (d) NaI, acetone, reflux. (e) Imidazole,
DMF. (f) NaN3, DMF, 50 ꢁC. (g) TMS acetylene, CuI, TMEDA, Et3N, THF, r.t. (h) TBAF, CH3COOH, THF, r.t.
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TENO ET AL.
Scheme 2. Reagents and conditions: (a) TFA/CH2Cl2, 0 ꢁC ! r.t., 2 h. (b) (Boc)2O, NaOH or Et3N, 0 ꢁC ! r.t., 12 h. (c) HOAt, NH4Cl, Et3N, WSCDꢂHCl, DMF,
r.t., 18 h. (d) Isobutyl chloroformate, Et3N, THF, NH3, ꢃ20 ꢁC, 0.5 h ! 0 ꢁC, 2 h. (e) Picolyl chlorideꢂHCl, K2CO3, KI, DMF, r.t., 12 h. (f) LiOHꢂH2O, THF/MeOH/
H2O, 0 ꢁC ! r.t., 3 h. (g) Fmoc-D-Ile-OSu, Et3N, CH3CN/H2O, 0 ꢁC ! r.t., 12 h. (h) WSCDꢂHCl, HOAt, Et3N, DMF, ꢃ20 ꢁC, 0.5 h ! 0 ꢁC, 12 h. (i) (CF3CO)2O, Et3N,
THF/DMF, 0 ꢁC, 5 min. (j) 20% Piperidine/DMF, r.t., 20 min. (k) Preparative HPLC.
Table 2. Inhibition of plasmin, plasma kallikrein, and urokinase by
compounds 3 and 6–9
Table 3. Inhibition of plasmin, plasma kallikrein, and urokinase by
compounds 3 and 27–29
Compound
nos.
R
IC50 (mM)
Compound nos.
R
IC50 (mM)
Plasmin
11
Plasma
kallikrein
Urokinase
Plasmin Plasma kallikrein Urokinase
3
6
7
D-Ile
Ile
11
440
320
210
>1000 (16%)a
>1000 (16%)a
>1000 (18%)a >1000 (23%)a
>1000 (24%)a >1000 (6%)a
3
210
Gly
8
1200
>1000 (10%)a >1000 (8%)a
1000 (11%)a >1000 (8%)a >1000 (13%)a
1000 (0%)a >1000 (6%)a >1000 (9%)a
1000 (0%)a >1000 (7%)a >500 (16%)a
27
28
9
1300
>1000 (27%)a >1000 (9%)a
aValues in parentheses are % inhibition at the concentration.
29
aValues in parentheses are % inhibition at the concentration.
between the P2 and P3 side chains are directed toward the large
open binding pocket of plasmin.
Compounds 6–9, as derivatives of compound 3, were prepared
to explore the role of D-Ile as the P3 moiety and tested the
inhibitory activity against plasmin. Derivatives 6–9 proved to be
less active but retained the selectivity against PK and UK. These
results revealed that plasmin preferred a branched alkyl group
with (R)-configuration (e.g., isobutyl as D-Ile) and the amino group
(3, 6, 7 vs 8, 9) at the P3 position. The observation is consistent
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PEPTIDIC PLASMIN INHIBITORS POSSESSING NITRILE AS WARHEAD
imidazole during synthetic process, unlike previous P1 residue
with a basic moiety. Likewise, the synthetic protocol depicted in
Scheme 1 is capable of responding appropriately to the prepara-
tion for non-natural amino acids having different side chain
lengths.
Acknowledgements
The work was supported by Grant-in-Aid for Scientific Research
(C, 24590154) from the Ministry of Education, Science, Sports
and Culture of Japan. We are grateful to Dr. Lawrence H. Lazarus
for critically reading the manuscript. We also thank Mr. Satoshi
Ohsawa for providing help in acquiring HRMS spectra.
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