Selective inhibitors of the serine protease plasmin: Probing the S3 and S3′ subsites using a combinatorial library

Article abstract of DOI:10.1021/jm050488k

A combinatorial library of 400 serine protease inhibitors with the general structure Cbz-X_aa-Trp-cyclohexanone-Trp-Y_aa-OH has been constructed. The library was synthesized on the solid phase using mix-and-split synthesis, where 20 di

Full text of DOI:10.1021/jm050488k

6908
J. Med. Chem. 2005, 48, 6908-6917
Selective Inhibitors of the Serine Protease Plasmin: Probing the S3 and S3′
Subsites Using a Combinatorial Library
Fengtian Xue and Christopher T. Seto*
Department of Chemistry, Brown University, 324 Brook Street, Box H, Providence, Rhode Island, 02912
Received May 24, 2005
A combinatorial library of 400 serine protease inhibitors with the general structure Cbz-Xaa-
Trp-cyclohexanone-Trp-Yaa-OH has been constructed. The library was synthesized on the solid
phase using mix-and-split synthesis, where 20 different amino acids were incorporated at both
the Xaa and Yaa positions. These two positions correspond to the S3 and S3′ subsites of the
active site. Iterative deconvolution was used to identify hits from the library. The library was
screened against four serine proteases: plasmin, kallikrein, thrombin, and trypsin. Seven
inhibitors from the library that showed promising activities were resynthesized using solution-
phase methods. Four of these compounds were good inhibitors of plasmin with IC50 values in
the range of 2.7-3.6 µM. The most potent of these inhibitors showed >150-fold selectivity for
plasmin when compared to the other three serine proteases.
Introduction
Proteases regulate a host of physiological processes
including digestion, fertilization, growth, cellular migra-
tion, immunological defense, blood pressure, wound
healing, and remodeling of the extracellular matrix
(ECM).1 Plasmin is a serine protease that plays an
important role during ECM remodeling by degrading a
number of ECM components such as fibrin, fibronectin,
laminin, and proteoglycans.2 In addition to its direct role
in this process, plasmin also plays an indirect role by
activating the matrix metalloproteases (MMPs) MMP-
1, 3, and 9.3 These MMPs in turn regulate matrix
deposition and remodeling and also stimulate the
release of several bioactive growth factors. During the
past decade, a number of studies have shown that ECM
remodeling is one of key processes associated with
angiogenesis, tumor growth, and invasion.4,5 Therefore,
potent and selective inhibitors of plasmin may have
potential as chemotherapeutic agents that impede an-
giogenesis and block the rapid growth of primary tumors
and the spread of secondary metastases.
There are several endogenous inhibitors of plasmin,
including R-macroglobulin6 and R-antiplasmin,7 that
regulate its biological activity.8,9 In addition, a number
of small molecule inhibitors have been developed. For
example, ꢀ-aminocaproic acid10 and trans-4-aminocyclo-
hexane carboxylic acid11 are two inhibitors that have
seen use in the clinic. These compounds act by blocking
the lysine binding site of plasmin.12,13 Okada and co-
workers have developed a variety of synthetic inhibitors
that are targeted to plasmin’s active site that show good
potency and selectivity.14-21
The ketone in the cyclohexanone ring is designed to
react with the Ser or Cys active site nucleophile to give
a reversibly formed hemiketal or hemithioketal linkage.
In one study, we demonstrated the formation of such
an intermediate in the active site of the cysteine
protease papain.25 The amino acid side chains at the
Aaa and Baa positions of the inhibitor were designed to
bind in the S2 and S2′ subsites of the protease target.
We constructed a combinatorial library around the
general structure 1 in order to investigate the specificity
of these two subsites in several proteases.26 The library
was synthesized using solid-phase methods and made
use of compound 2 as a key synthon that incorporated
the ketone in protected form and that was amenable to
Fmoc-based peptide synthesis. These studies revealed
that for plasmin, Trp was preferred at both the S2 and
S2′ subsites.
In the present work, we describe the preparation and
screening of a second generation library of inhibitors
with the general structure 3 (Figure 1). The compounds
contain the centrally positioned Trp-cyclohexanone-Trp
fragment that emerged from screening the first genera-
tion library against plasmin.26 The Xaa and Yaa diversity
elements, which are targeted to the S3 and S3′ subsites,
are varied among 20 different amino acid side chains.
The library has been screened against plasmin and
three other related serine proteases: kallikrein, throm-
bin, and trypsin. This study has three major objectives.
They are (1) To improve the potency of the plasmin
inhibitors by extending noncovalent binding interactions
into the S3 and S3′ subsites; (2) to investigate the
specificity of the S3 and S3′ subsites in plasmin; and
Over the past several years our group has investi-
gated a series of cyclohexanone-based inhibitors 1 that
have been targeted to serine and cysteine proteases.22-24
* To whom correspondence should be addressed. Christopher_Seto@
brown.edu.
10.1021/jm050488k CCC: $30.25 © 2005 American Chemical Society
Published on Web 10/11/2005

Inhibitors of the Serine Protease Plasmin
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22 6909
different Cbz-protected amino acid to give compound 12.
The inhibitors were cleaved from the resin, and the
protecting groups on the amino acid side chains were
removed with TFA. Finally, the ketal was hydrolyzed
to the corresponding ketone with aqueous TFA to give
20 pools of inhibitors, each containing a mixture of 20
amino acids at the Yaa position, and a single defined
amino acid at the Xaa position. After biological evalua-
tion of this initial library, we performed a deconvolution
of the most active pool by individually synthesizing and
evaluating the 20 members in that pool. A similar solid-
phase synthesis procedure was used in the deconvolu-
tion process, except that the mix-and-spit step was
omitted.
Assay of the Library. Serine proteases are generally
classified into three different families based upon their
substrate specificity at the P1 position of peptide sub-
strates. Trypsin-like serine proteases prefer to bind
substrates with positively charged amino acids at the
P1 position such as Lys and Arg. Elastase-like enzymes
bind small hydrophobic residues such as Ala or Val,
while chymotrypsin-like proteases are specific for large
hydrophobic residues such as Phe, Tyr, or Leu. Design-
ing inhibitors that are specific for one particular pro-
tease within a family can be a challenging prospect since
one of the dominant features of substrate specificity for
all of the enzymes within that family is the identity of
the P1 residue.
We screened the initial library against four different
serine proteases: plasmin, kallikrein, thrombin, and
trypsin. All of these enzymes are trypsin-like proteases
that prefer to bind positively charged side chains at P1.
We purposely selected four enzymes that are in the
same family in order to investigate the potential of this
library to generate inhibitors that are specific for
plasmin over other closely related enzymes. We had a
reasonable hope of discovering inhibitors with high
specificity since the library was designed to maximize
interactions with the S2, S3, S2′ and S3′ binding sites.
Unlike most other protease inhibitors, this library does
not have a functional group that is targeted toward the
S1 subsite. In our previous studies we have found that,
for this class of inhibitors, plasmin prefers to bind Trp
at both the S2 and S2′ subsites.10 Since we have
incorporated Trp at both the P2 and P2′ positions in
this inhibitor library, we expected that the inhibitors
would have better activity against plasmin than the
other three protease.
The initial library was screened against the four
proteases with a total inhibitor concentration in each
pool of 5 µM. This corresponds to a concentration of 250
nM for each of the 20 inhibitors in a pool. The assays
were performed in 50 mM phosphate buffer at pH 7.4
using p-nitroanilide substrates, and initial rates were
measured by UV spectroscopy. The assays contained a
DMSO concentration of 10% in order to ensure the
solubility of all of the inhibitors.
Figure 1. General structure of the compounds in the library.
Xaa and Yaa are each one of 20 amino acids.
(3) to generate inhibitors that are highly selective for
plasmin over other related proteases. To accomplish this
third objective, we have focused our attention on por-
tions of the inhibitor that do not interact with the
primary recognition element of plasmin, which is the
S1 subsite.
Result and Discussion
Synthesis of the Cyclohexanone Fragment. Be-
fore we began construction of the library, we first
prepared the central cyclohexanone portion of the
inhibitors in a form that was suitably protected for solid-
phase peptide synthesis. The synthesis of compound 2
is shown in Scheme 1 and began with double depro-
tonation of ketoester 4 followed by alkylation of the more
reactive enolate with 4-bromo-1-butene to give alkene
5 as a mixture of ketone and enol tautomers. Protection
of the ketone with 1,3-propanediol in the presence of
TMSCl yielded ketal 6, in which the ester and butene
substituents on the cyclohexane ring are oriented in the
thermodynamically favored cis-1,3-diequitorial arrange-
ment.27 Saponification of the ethyl ester in 6 gave
carboxylic acid 7. This compound was treated with
diphenylphosphoryl azide and diisopropylethylamine
(DIEA) to generate the acyl azide, which upon heating
underwent the Curtius rearrangement. The resulting
isocyanate was trapped with potassium tert-butoxide to
give the corresponding Boc-protected amine 8. The Boc
protecting group was removed with TFA, and the
resulting amine was treated with FmocCl to yield Fmoc-
protected amine 9. Finally, oxidative cleavage of the
alkene with KMnO4 and NaIO4 gave protected amino
acid 2. The synthesis and characterization of compounds
2 and 9 have been reported previously.26
Design and Synthesis of the Library. We used the
mix-and-split strategy to prepare the library and incor-
porated 20 amino acids at both the Xaa and Yaa positions
in compound 3 (Figure 1) to give a total library size of
400 compounds.28,29 The initial library was prepared as
20 pools that each contained 20 inhibitors. The com-
pounds in each pool incorporated a single defined amino
acid at the Xaa position, and a randomized mixture of
all twenty amino acids at the Yaa position. We used the
iterative deconvolution strategy, which was developed
by Houghten,30 to identify active compounds.
The solid-phase synthesis of the library is shown in
Scheme 2. Twenty batches of Wang resin that each had
been preloaded with an Fmoc-protected amino acid were
mixed together to randomize the Yaa position of the
inhibitors (compound 10). Three cycles of Fmoc group
deprotection and peptide coupling reactions were per-
formed sequentially with Fmoc-Trp-OH, compound 2,
and Fmoc-Trp-OH to yield compound 11. The resin was
then separated into 20 equal portions, the Fmoc group
was removed, and each portion was coupled to a
As shown in Figure 2, none of the inhibitor pools
showed higher than 20% inhibition against kallikrein,
thrombin, or trypsin. By contrast, several of the pools
had good activity against plasmin including pools with
the Xaa position defined as Leu (33% inhibition), Phe
(43% inhibition), and Trp (73% inhibition). This result
suggests that plasmin prefers to bind aromatic and large

6910 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22
Xue and Seto
Scheme 1a
a Reagents: (a) (i) LDA (2 equiv), (ii) 4-bromo-1-butene; (b) 1,3-propanediol, TMSCl; (c) NaOH, MeOH; (d) (i) (PhO)2PON3, DIEA, (ii)
t-BuOK; (e) TFA; (f) FmocCl, DIEA; (g) NaIO4, KMnO4, NaHCO3. Only one of the two enantiomers is shown.
Scheme 2a
Figure 3. Assay of the Xaa ) Trp deconvolution against
a Reagents: (a) piperidine; (b) Fmoc-Trp-OH, HBTU, DIEA; (c)
2, HBTU, DIEA; (d) Cbz-Xaa-OH, HBTU, DIEA; (e) TFA; (f) TFA,
H2O. One of two diastereomers is shown for 11, 12, and 3.
plasmin. Each bar represents the assay of a single compound
with the amino acid at the Yaa position defined by the x-axis
of the plot.
trypsin-like serine proteases. Amino acids that are
particularly disfavored at the S3 subsite include Asp,
Glu, and Gly.
Upon the basis of the results from screening of the
initial library, we chose the Xaa ) Trp pool for decon-
volution. The 20 inhibitors in this pool were each
synthesized individually using the solid-phase strategy
shown in Scheme 2. The activities of these inhibitors
were examined at 5 µM concentration against plasmin
(Figure 3). Four of the compounds gave >70% inhibition
at this concentration, including those with Leu, Phe,
Trp, and Tyr at the Yaa position. This observation is
consistent with data reported by Tanaka,37 and indi-
cates that, within the context of these cyclohexanone-
based inhibitors, plasmin prefers to bind hydrophobic
and aromatic amino acids at both the S3′ and S3
subsites. Three other compounds gave good activity
(>60% inhibition) including those with Ala, Ile, and Val
at the Yaa position. Polar amino acids such as Orn and
His and conformationally constrained residues such as
Pro and Hyp (hydroxyproline) gave low activity and are
detrimental to binding in the S3′ subsite.
Resynthesis and Evaluation of Inhibitors 29-
35. The assay results from our library screens prompted
us to select seven inhibitors (compounds 29-35, Scheme
3) to resynthesize and evaluate more completely against
the proteases. We began the synthesis by removing the
Boc protecting group from ketal 8 and coupling the
resulting primary amine with Fmoc-Trp(Boc)-OH to give
compound 13 (Scheme 3). Fmoc deprotection and cou-
pling with Cbz-Trp(Boc)-OH yielded intermediate 14.
The alkene group in 14 was subjected to oxidative
Figure 2. Assay of 20 pools that each contains 20 inhibitors
against plasmin, trypsin, thrombin and kallikrein. Each bar
represents the activity of a pool that contains inhibitors in
which the Xaa position is defined by the amino acids on the
x-axis of the graph, and Yaa is a mixture of 20 different amino
acids. The data represent the average of two measurements.
The error in the assays is approximately (5%.
hydrophobic amino acid side chains in the S3 subsite.
It also confirms our supposition that the inhibitors
would be specific for plasmin over the other three

Inhibitors of the Serine Protease Plasmin
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22 6911
Table 2. Inhibition of Serine Proteases by Inhibitor 34a
Scheme 3a
serine protease
IC50 (µM)
plasmin
kallikrein
thrombin
trypsin
2.7 ( 0.2
>400b
>400b
>400b
a Mixture of two diastereomers. bNo inhibition observed at this
concentration.
Baa are both the side chain of Trp, show only a 2-fold
difference in potency.26 Inhibitors 29, 30, and 35, which
gave between 60% and 70% inhibition during the
deconvolution process (Figure 3), had IC50 values in the
range of 6.6-14.1 µM. Among the seven inhibitors in
this series, these compounds gave the lowest activity
in both the deconvolution step and in our examination
of the purified inhibitors. This correlation demonstrates
that the data that we obtained during the initial library
screening and deconvolution procedures using crude
inhibitors was reliable enough to predict the activity of
the purified compounds. Inhibitors 30 and 35 were the
weakest of the seven inhibitors by a significant margin.
Both of these compounds incorporate a â-branched
amino acid at the Yaa position, suggesting that â-branch-
ing is an unfavorable characteristic for binding in the
S3′ subsite of plasmin. By contrast, compounds 31-34
are all good inhibitors with IC50 values that range from
2.7 to 3.6 µM. This observation confirms our conclusion
from the deconvolution process that plasmin prefers to
bind large hydrophobic and aromatic side chains in the
S3′ pocket.
Compound 34, with an IC50 value of 2.7 µM, is the
best plasmin inhibitor that we have identified through
this library screening process. To examine the specificity
of this compound, we also examined its activity against
the other three trypsin-like serine proteases (Table 2).
The assay solutions contained 10% DMSO in order to
maintain solubility of the inhibitors. Under these condi-
tions the limit of solubility of compound 34 is ap-
proximately 400 µM. At this concentration we observed
no inhibition of kallikrein, thrombin, and trypsin, show-
ing that compound 34 has >150-fold selectivity for
plasmin over the other three enzymes, even though all
four of them are members of the same family of serine
proteases.
a Reagents: (a) TFA; (b) Fmoc-Trp(Boc)-OH, HBTU, DIEA; (c)
piperidine; (d) Cbz-Trp(Boc)-OH, HBTU, DIEA; (e) NaIO4, KMnO4,
NaHCO3; (f) H2N-Trp-Yaa-OMe where Yaa ) Ala (15), Ile (16), Leu
(17), Phe (18), Trp (29), Tyr(tBu) (20), or Val (21), HATU, DIEA;
(g) TFA; (h) TFA, H2O. One of two diastereomers is shown for
compounds 13-14 and 22-35.
Table 1. Inhibition of Plasmin by Inhibitors 29-35
inhibitora
Yaa
IC50 (µM)
29
30
31
32
33
34
35
Ala
Ile
Leu
Phe
Trp
Tyr
Val
6.6 ( 0.6
13.5 ( 1.8
3.2 ( 0.3
3.3 ( 0.3
3.6 ( 0.2
2.7 ( 0.2
14.1 ( 1.3
The data presented in Figure 2 show that none of the
compounds in the library have high activity against
kallikrein, thrombin, or trypsin. This observation is
reasonable, since the library is based around a core
structure that incorporates Trp at both P2 and P2′. Data
from the literature indicate that, in their S2 sub-
sites, kallikrein,31,32 thrombin,33,34 and trypsin35 prefer
to bind Phe, Pro, and Asn/Thr/His, respectively. At S2′,
kallikrein prefers Arg, trypsin prefers Trp/Phe,36 while
thrombin does not show a strong preference at this
position. Therefore, none of the inhibitors provide a
complete match to the binding preferences of these
proteases.
a Assayed as a mixture of two diastereomers.
cleavage with KMnO4 and NaIO4, and the resulting
carboxylic acid was coupled with seven different dipep-
tides with the general structure H2N-Trp-Yaa-OMe
(compounds 15-21) using HATU at 60 °C to generate
compounds 22-28. Finally, the protecting groups on the
amino acid side chains were removed with TFA, and
the ketal protecting group on the cyclohexanone core
was removed using aqueous TFA to give inhibitors 29-
35. These final inhibitors were purified to homogeneity
by reverse phase HPLC.
Compounds 29-35 were assayed against plasmin,
and the results are presented in Table 1. We evaluated
the inhibitors as mixtures of the two diastereomers that
are generated during their syntheses. In previous stud-
ies, we have found that similar pairs of diastereomers
have only modest differences in activity. For example,
the two diastereomers of compound 1, where Aaa and
At first glance, the structure of compound 34 is a little
surprising since it incorporates Trp residues in three
out of the four amino acid sites, with the fourth being
occupied by Tyr. It is a very hydrophobic structure,
which might suggest that its binding interactions with
an enzyme active site would be predominantly through
nonspecific hydrophobic interactions. However, if this

6912 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22
Xue and Seto
by monitoring the formation of p-nitroaniline at 405 nm from
30 to 120 s after mixing on a Perkin-Elmer 8452A diode array
UV-vis spectrometer. When measuring the IC50 of inhibitors,
the substrate concentrations were held constant at its KM
values, which were measured to be 167, 135, 45, and 122 µM
for the respective substrates plasmin, kallikrein, thrombin,
and trypsin. Data analysis was performed with the commercial
graphing package Grafit (Erithacus Software Ltd.).
were the case, then compound 34 should have little
selectivity for a particular active site and would likely
interact similarly with a number of different enzymes.
Since we observe high selectivity for plasmin over other
similar enzymes, we believe that 34 must be making
specific hydrogen bonding, aryl-aryl, and hydrophobic
interactions with amino acids that make up the S3, S2,
S2′, and S3′ subsites of plasmin.
Alkene 5. To a solution of diisopropylamine (8.42 mL, 6.06
g, 60.0 mmol) in THF (60 mL) was added n-butyllithium (23.5
mL, 58.8 mmol, 2.5 M in hexanes) at -78 °C under an
atmosphere of nitrogen. The temperature of the solution was
slowly increased to 0 °C and maintained at that temperature
for an additional 10 min. To this solution was slowly added
ketoester 4 (5.0 g, 29.4 mmol). After 15 min, 4-bromo-1-butene
(3.80 mL, 5.34 g, 44.0 mmol) was added dropwise. The reaction
was stirred at room temperature for 30 h and then quenched
with water. The THF was removed by rotary evaporation, and
the mixture was partitioned between EtOAc (500 mL) and 1
N HCl (250 mL). The organic layer was washed with 1 N HCl
(250 mL), saturated NaHCO3 (250 mL), and brine (250 mL).
It was then dried over MgSO4, and the resulting solution was
concentrated by rotary evaporation. The crude oil was purified
by flash chromatography (EtOAc:hexanes 1:18) to yield 5 as a
mixture of ketone and enol tautomers (5.16 g, 22.9 mmol,
78%): 1H NMR (300 MHz, CDCl3) δ 1.12-1.34 (m, 3.8H),
1.35-1.55 (m, 2.7H), 1.56-1.83 (m, 2.1H), 1.84-1.99 (m, 1.4H),
2.00-2.24 (m, 4.0H), 2.25-2.43 (m, 1.1H), 4.05-4.33 (m, 2.0H),
4.85-5.10 (m, 2H), 5.60-5.90 (m, 1H), 12.42 (s, 0.7H); 13C
NMR (75 MHz, CDCl3) δ 14.07, 14.12, 14.23, 20.0, 21.7, 22.8,
24.1, 27.1, 28.1, 28.9, 30.3, 30.8, 31.0, 31.1, 31.2, 33.6, 34.2,
37.9, 48.7, 50.1, 56.1, 57.9, 60.1, 60.8, 61.1, 97.7, 114.7, 114.79,
114.81, 138.1, 138.2, 138.4, 169.9, 170.0, 172.9, 174.6; HRMS-
FAB (M + Na+) calcd for C13H21O3 225.1491, found 225.1481.
Ketal 6. A solution of compound 5 (5.0 g, 22.2 mmol) in THF
(10 mL) was cooled in an ice bath. To this solution, 1,3-
propanediol (30 mL, 31.7 g, 417 mmol) and TMSCl (5.57 mL,
4.8 g, 44.4 mmol) were added. The reaction was stirred at room
temperature for 48 h and then partitioned between EtOAc (500
mL) and saturated NaHCO3 (400 mL). The organic layer was
washed with saturated NaHCO3 (400 mL) and brine (400 mL).
It was then dried over MgSO4, and the resulting solution was
concentrated by rotary evaporation. The crude oil was purified
by flash chromatography (EtOAc:hexanes 1:18) to yield 6 (6.0
g, 19.8 mmol, 89%): 1H NMR (300 MHz, CDCl3) δ 1.16-1.39
(m, 5H), 1.40-2.06 (m, 10H), 2.07-2.35 (m, 2H), 3.28-3.60
(br s, 1H), 3.75-3.96 (m, 3H), 4.00-4.28 (m, 3H), 4.86-5.10
(m, 2H), 5.60-5.90 (m, 1H); 13C NMR (75 MHz, CDCl3) δ 14.7,
20.4, 25.6, 25.9, 26.5, 27.1, 32.4, 59.5, 59.7, 60.5, 99.2, 114.5,
139.8, 173.0; HRMS-FAB (M + Na+) calcd for C16H26NaO4
305.1729, found 305.1740.
Carboxylic Acid 7. To a solution of compound 6 (6.0 g, 19.7
mmol) in MeOH (50 mL) was added 2 N aqueous NaOH (50
mL). The reaction was heated at reflux for 24 h and then cooled
to room temperature. The MeOH was removed by rotary
evaporation. The resulting aqueous solution was washed with
EtOAc (50 mL), the organic phase was discarded, and the
aqueous phase was partitioned between EtOAc (500 mL) and
1 N HCl (300 mL). The organic layer was washed with brine
(300 mL) and then dried over MgSO4. The solvent was removed
by rotary evaporation. The crude product was recrystallized
from a mixture of EtOAc and hexanes to yield carboxylic acid
7 as a white solid (4.53 g, 16.4 mmol, 83%): 1H NMR (300
MHz, CDCl3) δ 1.10-1.61 (m, 7H), 1.61-2.00 (m, 4H), 2.01-
2.23 (m, 1H), 2.24-3.08 (m, 2H), 3.68-4.10 (m, 4H), 4.80-
5.06 (m, 2H), 5.60-5.87 (m, 1H), 10.35-11.08 (br s, 1H); 13C
NMR (75 MHz, CDCl3) δ 19.2, 24.8, 25.2, 25.5, 32.0, 59.5, 59.7,
100.7, 115.4, 138.7, 174.7; HRMS-FAB (M + Na+) calcd for
C14H22NaO4 277.1416, found 277.1410.
The inhibitors shown in Table 1 have molecular
weights that are in the range of 1000 Da and are
peptidic in nature. Thus, it is unlikely that these specific
compounds would have significant bioavailability. How-
ever, since they are designed to control remodeling and
degradation of the extracellular matrix the inhibitors
do not need to penetrate through the cell membrane.
One way to improve their pharmacokinetic character-
istics would be to replace the amide bonds by appropri-
ate nonhydrolyzable analogues to decrease their sus-
ceptibility to hydrolysis by proteases.
Conclusions
In summary, we have prepared and screened a library
of inhibitors that was designed to probe the specificity
of the S3 and S3′ binding sites of plasmin. The central
portion of the inhibitors is made up of a Trp-cyclo-
hexanone-Trp core structure, which originated from our
previous investigations of the binding preferences of the
S2 and S2′ subsites. From our screening studies of this
library we conclude that both the S3 and S3′ subsites
prefer to bind large hydrophobic and aromatic side
chains. For these inhibitors, Trp and Tyr are the
preferred amino acids at the P3 and P3′ sites, respec-
tively. Both conformationally constrained and polar
amino acids are detrimental to binding at the P3
position, while â-branched amino acids are less favor-
able at P3′ than residues such as Tyr and Leu. The best
inhibitor that we have discovered from this library,
compound 34, has an IC50 value of 2.7 µM and very high
selectivity for plasmin over other related serine pro-
teases.
This work highlights a strategy for designing inhibi-
tors with high specificity for one particular member of
a family of enzymes. We have disregarded the primary
recognition element of the trypsin-like serine proteases,
which in this example is the S1 subsite that prefers to
bind positively charged residues. Instead, we have
concentrated our design and optimization efforts on
regions of the active site that are usually less important,
but also less homologous in their specificity, among this
family of enzymes. As a result we have been able to
develop an inhibitor with both good activity and high
specificity for our chosen target, plasmin.
Experimental Section
Enzyme Assays. IC50 values for inhibitors 29-35 were
measured for the serine proteases plasmin, kallikrein, throm-
bin, and trypsin using the chromogenic substrates D-Val-Leu-
Lys-pNA, d-Pro-Phe-Arg-pNA, D-Phe-Pip-Arg-pNA, and D-Phe-
Pip-Arg-pNA, respectively (pNA ) p-nitroanilide). Enzymes
and substrates were purchased from Sigma or Chromogenix
(distributor DiaPharma Group, Inc.) and used as received
without further purification. All proteases were assayed at 25
°C in a 50 mM sodium phosphate buffer (pH 7.4) with or
without inhibitors. A final concentration of 10% DMSO was
used in the assay mixtures to ensure solubility of the inhibi-
tors. Initial rates of the enzymatic reactions were determined
Carbamate 8. Carboxylic acid 7 (3.0, 10.8 mmol) was
dissolved in toluene (50 mL). To this solution were added DIEA
(2.3 mL, 1.7 g, 13.0 mmol) and (PhO)2PON3 (2.8 mL, 3.6 g,
13.0 mmol). The reaction was heated at 85 °C under nitrogen
for 16 h and then cooled to room temperature. To a separated

Inhibitors of the Serine Protease Plasmin
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22 6913
flask containing a solution of potassium tert-butoxide (2.4 g,
21.6 mmol) in THF (150 mL) at 0 °C was added the isocyanate
solution dropwise. The reaction was allowed to warm to room
temperature over 30 min, and then it was quenched with water
(30 mL). The THF was removed by rotary evaporation, and
the resulting material was partitioned between EtOAc (300
mL) and 1 N HCl (300 mL). The organic layer was washed
with 1 N HCl (200 mL), saturated NaHCO3 (200 mL), and
brine (200 mL). It was then dried over MgSO4, and the solvent
was removed by rotary evaporation. The crude oil was purified
by flash chromatography (EtOAc:hexanes 1:9) to give com-
pound 8 (3.2 g, 9.3 mmol, 86%): 1H NMR (300 MHz, CDCl3) δ
1.20-1.39 (m, 3H), 1.40-1.47 (m, 10H), 1.48-1.63 (m, 3H),
1.64-1.81 (m, 3H), 1.84-2.28 (m, 3H), 3.70-3.95 (m, 4H),
3.96-4.30 (br s, 1H), 4.72-4.92 (br s, 1H), 4.93-5.10 (m, 2H),
5.70-5.95 (m, 1H); 13C NMR (75 MHz, CDCl3) δ 19.3, 25.5,
28.8, 32.2, 59.26, 59.33, 79.4, 99.4, 115.0, 139.3, 156.4; HRMS-
FAB (M + Na+) calcd for C18H31NNaO4 348.2151, found
348.2158.
Amide 13. Compound 8 (1.0 g, 2.9 mmol) was dissolved in
CH2Cl2 (16 mL). To this solution was added TFA (8 mL). The
reaction was stirred at room temperature for 30 min. The
solvents were removed by rotary evaporation, and the residue
was diluted with EtOAc (200 mL). The organic layer was
washed with saturated aqueous Na2CO3 (100 mL) and brine
(100 mL) and then dried over Na2CO3. The solvent was
removed by rotary evaporation to give the corresponding
primary amine. The resulting amine was redissolved in DMF
(5 mL). To this solution were added Fmoc-Trp(Boc)-OH (2.3
g, 4.4 mmol), HBTU (1.7 g, 4.4 mmol), and DIEA (1.5 mL, 1.1
g, 8.8 mmol). The reaction was stirred at room temperature
for 1 h, and then the reaction mixture was partitioned between
EtOAc (250 mL) and 1 N HCl (150 mL). The organic layer was
washed with 1 N HCl (150 mL), saturated NaHCO3 (150 mL),
and brine (150 mL). It was then dried over MgSO4, and the
solvent was removed by rotary evaporation. The crude oil was
purified by flash chromatography (EtOAc:hexanes 2:1) to yield
13 as a mixture of two diastereomers (1.5 g, 2.0 mmol, 69%):
1H NMR (300 MHz, CDCl3) δ 1.30-1.50 (m, 7H), 1.51-1.70
(m, 10H), 1.82-2.23 (m, 3H), 2.97-3.50 (m, 2H), 3.52-3.89
(m, 4H), 4.20-4.68 (m, 4H), 4.90-5.15 (m, 2H), 5.60-6.18 (m,
2H), 7.28-7.66 (m, 9H), 7.67-7.92 (m, 3H), 8.04-8.30 (m, 1H);
13C NMR (75 MHz, CDCl3) δ 14.6, 18.9, 21.1, 23.1, 25.2, 25.4,
25.7, 28.4, 28.58, 28.60, 29.3, 30.0, 32.0, 32.12, 32.15, 35.1, 47.5,
47.6, 55.0, 55.6, 59.08, 59.13, 59.21, 59.24, 67.5, 77.7, 83.9, 99.0,
115.2, 115.7, 116.1, 119.5, 119.8, 120.4, 123.2, 123.3, 124.9,
125.08, 125.13, 125.5, 125.6, 127.5, 128.1, 130.2, 138.97,
139.04, 141.7, 144.18, 144.22, 149.9, 156.3, 170.0, 170.6;
HRMS-FAB (M + Na+) calcd for C44H51N3NaO7 756.3625,
found 756.3610.
Amide 14. Compound 13 (1.2 g, 1.6 mmol) was stirred in a
1:1 mixture of piperidine and DMF (20 mL) at room temper-
ature for 1 h. The solvents were removed by rotary evapora-
tion. The resulting residue was dried under vacuum for 12 h
to remove residual piperidine. The white solid was then
redissolved in DMF (5 mL). To this solution were added Cbz-
Trp(Boc)-OH (1.1 g, 2.4 mmol), HBTU (900 mg, 2.4 mmol), and
DIEA (840 µL, 624 mg, 4.8 mmol). The reaction was stirred
at room temperature for 2 h, and the mixture was partitioned
between EtOAc (250 mL) and 1 N HCl (150 mL). The organic
layer was washed with 1 N HCl (150 mL), saturated aqueous
NaHCO3 (150 mL), and brine (150 mL). It was then dried over
MgSO4 and concentrated. The crude oil was purified by flash
chromatography (EtOAc:hexanes 2:1) to yield compound 14 as
a mixture of two diastereomers (1.2 g, 1.28 mmol, 80%): 1H
NMR (300 MHz, CDCl3) δ 1.30-1.50 (m, 7H), 1.51-1.79 (m,
21H), 1.80-2.00 (m, 1H), 2.10-2.22 (m, 1H), 2.50-2.90 (m,
1H), 2.91-3.31 (m, 3H), 3.52-3.89 (m, 4H), 4.45-4.80 (m, 2H),
4.90-5.15 (m, 4H), 5.30-5.50 (m, 1H), 5.52-6.10 (m, 2H),
6.70-7.27 (m, 5H), 7.29-7.35 (m, 5H), 7.36-7.80 (m, 5H),
8.04-8.25 (m, 2H); 13C NMR (75 MHz, CDCl3) δ 0.4, 18.9, 25.4,
25.6, 28.3, 28.6, 32.2, 54.1, 55.2, 55.3, 59.1, 59.2, 67.5, 77.7,
83.6, 84.0, 84.1, 99.0, 115.1, 115.3, 115.4, 115.5, 115.6, 115.7,
119.3, 119.5, 120.0, 123.0, 123.1, 124.8, 124.9, 125.1, 125.8,
128.4, 128.6, 128.9, 130.6, 130.7, 135.9, 136.4, 139.1, 149.8,
149.9, 156.2, 169.8, 171.0; HRMS-FAB (M + Na+) calcd for
C53H65N5NaO10 954.4629, found 954.4650.
Boc-Trp-Ala-OMe 15. Boc-Trp-OH (500 mg, 1.6 mmol) was
dissolved in DMF (10 mL). To this solution were added H2N-
Ala-OMe (170 mg, 1.6 mmol), HBTU (758 mg, 2.0 mmol), and
DIEA (530 µL, 390 mg, 3.0 mmol). The reaction was stirred
at room temperature for 2 h, and then it was partitioned
between EtOAc (250 mL) and 1 N HCl (200 mL). The organic
layer was washed with 1 N HCl (200 mL), saturated NaHCO3
(200 mL), and brine (200 mL). The organic layer was dried
with MgSO4 and concentrated by rotary evaporation. The
crude oil was purified by flash chromatography (EtOAc:
hexanes 2:1) to yield dipeptide 15 (580 mg, 1.50 mmol, 94%):
1H NMR (400 MHz, CDCl3) δ 1.20-1.30 (d, J ) 7.2 Hz, 3H),
1.45 (s, 9H), 3.10-3.40 (m, 2H), 3.67 (s, 3H), 4.30-4.60 (t, J
) 6.8 Hz, 2H), 5.15-5.28 (br s, 1H), 6.35-6.58 (d, J ) 6.8 Hz,
1H), 7.00-7.16 (m, 2H), 7.17-7.23 (m, 1H), 7.30-7.45 (d, J )
8.0 Hz, 1H), 7.55-7.75 (d, J ) 7.6 Hz, 1H), 8.50 (br s, 1H); 13
C
NMR (100 MHz, CDCl3) δ 18.5, 28.5, 48.4, 52.6, 55.4, 75.3,
80.3, 110.6, 111.5, 119.1, 119.9, 122.4, 123.6, 127.8, 136.5,
155.7, 171.6, 173.7; HRMS-FAB (M + Na+) calcd for C20H27N3-
NaO5 412.1848, found 412.1829.
Boc-Trp-Ile-OMe 16. Dipeptide 16 was synthesized using
a procedure that was similar to that used to prepare dipeptide
15 (635 mg, 1.47 mmol, 92%): 1H NMR (400 MHz, CDCl3) δ
0.70-0.80 (d, J ) 6.8 Hz, 3H), 0.81-0.90 (t, J ) 7.2 Hz, 3H),
0.95-1.10 (m, 1H), 1.29-1.40 (m, 1H), 1.46 (s, 9H), 1.70-1.82
(br s, 1H), 3.15-3.38 (m, 2H), 3.64 (s, 3H), 4.30-4.60 (dd, J )
8.4, 5.2 Hz, 2H), 5.05-5.35 (br s, 1H), 6.30-6.41 (d, J ) 8.4
Hz, 1H), 7.00-7.18 (m, 2H), 7.17-7.23 (t, J ) 7.2 Hz, 1H),
7.30-7.45 (d, J ) 8.0 Hz, 1H), 7.55-7.75 (d, J ) 8.0 Hz, 1H),
8.37 (br s, 1H); 13C NMR (100 MHz, CDCl3) δ 11.8, 15.4, 25.3,
28.4, 28.6, 38.1, 52.2, 55.5, 56.8, 59.4, 69.6, 75.3, 80.4, 110.8,
111.4, 119.1, 119.9, 122.4, 123.5, 127.7, 136.5, 155.8, 171.7,
172.0; HRMS-FAB (M + Na+) calcd for C23H33N3NaO5
454.2318, found 454.2320.
Boc-Trp-Leu-OMe 17. Dipeptide 17 was synthesized using
a procedure that was similar to that used to prepare dipeptide
15 (648 mg, 1.50 mmol, 94%): 1H NMR (300 MHz, CDCl3) δ
0.80-0.90 (m, 6H), 1.30-1.60 (m, 12H), 3.10-3.37 (m, 2H),
3.65 (s, 3H), 4.32-4.62 (m, 2H), 5.10-5.28 (br s, 1H), 6.20-
6.30 (d, J ) 8.1 Hz, 1H), 7.00-7.22 (m, 3H), 7.32-7.45 (d, J )
7.8 Hz, 1H), 7.55-7.70 (d, J ) 7.8 Hz, 1H), 8.37 (br s, 1H); 13
C
NMR (100 MHz, CDCl3) δ 21.9, 22.7, 24.6, 28.1, 28.3, 41.6,
50.8, 52.2, 80.1, 95.7, 110.5, 111.2, 118.9, 119.7, 122.2, 123.4,
127.5, 136.3, 155.5, 171.5, 172.9; HRMS-FAB (M + Na+) calcd
for C23H33N3NaO5 454.2318, found 454.2310.
Boc-Trp-Phe-OMe 18. Dipeptide 18 was synthesized using
a procedure that was similar to that used to prepare dipeptide
15 (729 mg, 1.57 mmol, 98%): 1H NMR (400 MHz, CDCl3) δ
1.45 (s, 9H), 2.90-3.00 (d, J ) 5.6 Hz, 2H), 2.62-2.88 (m, 2H),
3.62 (s, 3H), 4.35-4.60 (br s, 1H), 4.65-4.82 (br s, 1H), 5.05-
5.32 (br s, 1H), 6.25-6.45 (d, J ) 6.0 Hz, 1H), 6.75-6.90 (dd,
J ) 8.0, 1.2 Hz, 2H), 6.95-7.10 (br s, 1H), 7.12-7.22 (m, 5H),
7.32-7.42 (d, J ) 8.0 Hz, 1H), 7.65-7.75 (d, J ) 7.6 Hz, 1H),
8.46 (br s, 1H); 13C NMR (100 MHz, CDCl3) δ 28.4, 38.1, 52.4,
53.5, 55.4, 59.4, 69.6, 75.2, 80.3, 110.5, 111.5, 119.1, 119.9,
122.4, 123.6, 127.2, 127.7, 128.7, 129.4, 129.6, 135.8, 136.5,
155.6, 159.7, 171.6, 173.2; HRMS-FAB (M + Na+) calcd for
C26H31N3NaO5 488.2161, found 488.2154.
Boc-Trp-Trp-OMe 19. Dipeptide 19 was synthesized using
a procedure that was similar to that used to prepare dipeptide
15 (716 mg, 1.42 mmol, 89%): 1H NMR (400 MHz, CDCl3) δ
1.41 (s, 9H), 1.50-1.70 (br s, 1H), 3.00-3.25 (m, 3H), 3.27-
3.40 (m, 1H), 3.62 (s, 3H), 4.35-4.60 (br s, 1H), 4.75-4.92 (br
s, 1H), 5.00-5.20 (br s, 1H), 6.15-6.35 (br s, 1H), 6.55-6.70
(s, 1H), 6.85-7.00 (br s, 2H), 7.15-7.21 (m, 2H), 7.22-7.227
(m, 2H), 7.29-7.42 (m, 2H), 7.60-7.80 (d, J ) 7.6 Hz, 1H),
7.80-8.00 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 27.9, 28.6,
52.6, 53.0, 55.4, 60.7, 80.2, 110.0, 110.8, 111.4, 111.5, 118.8,
119.4, 119.9, 120.1, 122.5, 123.2, 123.8, 127.7, 127.9, 136.3,
136.5, 145.6, 155.6, 171.4, 172.0; HRMS-FAB (M + Na+) calcd
for C28H32N4NaO5 527.2270, found 527.2268.

6914 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22
Xue and Seto
Boc-Trp-Tyr(tBu)-OMe 20. Dipeptide 20 was synthesized
using a procedure that was similar to that used to prepare
dipeptide 15 (790 mg, 1.47 mmol, 92%): 1H NMR (300 MHz,
CDCl3) δ 1.32 (s, 9H), 1.44 (s, 9H), 2.75-2.95 (m, 2H), 3.00-
3.20 (m, 1H), 3.27-3.43 (m, 1H), 3.60 (s, 3H), 4.35-4.50 (br s,
1H), 4.60-4.75 (m, 1H), 5.10-5.17 (br s, 1H), 6.20-6.35 (d, J
) 7.5 Hz, 1H), 6.60-6.80 (m, 4H), 6.80-7.00 (br s, 1H), 7.09-
7.21 (m, 2H), 7.29-7.42 (d, J ) 7.8 Hz, 1H), 7.60-7.70 (d, J )
7.7 Hz, 1H), 8.49 (s, 1H); 13C NMR (75 MHz, CDCl3) δ 28.5,
28.7, 29.2, 37.7, 52.6, 53.7, 55.6, 78.9, 80.5, 110.6, 111.7, 119.2,
120.1, 122.6, 123.9, 124.5, 127.9, 130.0, 130.9, 136.6, 154.6,
155.8, 171.6, 171.8; HRMS-FAB (M + Na+) calcd for C30H39N3-
NaO6 560.2737, found 560.2720.
Boc-Trp-Val-OMe 21. Dipeptide 21 was synthesized using
a procedure that was similar to that used to prepare dipeptide
15 (647 mg, 1.55 mmol, 97%): 1H NMR (300 MHz, CDCl3) δ
0.70-0.87 (m, 6H), 1.45 (s, 9H), 1.85-2.01 (m, 1H), 3.10-3.40
(m, 2H), 3.64 (s, 3H), 4.35-4.55 (m, 2H), 5.00-5.40 (br s, 1H),
6.25-6.45 (d, J ) 8.4 Hz, 1H), 7.00-7.25 (m, 3H), 7.30-7.40
(d, J ) 7.8 Hz, 1H), 7.65-7.72 (d, J ) 7.8 Hz, 1H), 8.44 (s,
1H); 13C NMR (75 MHz, CDCl3) δ 18.2, 19.1, 28.5, 28.7, 31.6,
52.4, 55.7, 80.5, 110.8, 111.6, 119.2, 120.0, 122.5, 123.7, 127.9,
136.7, 156.0, 172.1, 172.2; HRMS-FAB (M + Na+) calcd for
C22H31N3NaO5 440.2161, found 440.2152.
Compound 22. Compound 14 (100 mg, 107 µmol) was
dissolved in a 2:1 mixture of acetone and water (60 mL). To
this solution were added NaIO4 (114 mg, 535 µmol), KMnO4
(12 mg, 75 µmol), and NaHCO3 (10 mg, 107 µmol). The reaction
was stirred at room temperature for 6 h, and then the acetone
was removed by rotary evaporation. The remaining material
was partitioned between EtOAc (100 mL) and 1 N HCl (75
mL). The organic layer was washed with 1 N HCl (3 × 75 mL)
and brine (75 mL) and dried over MgSO4. The solvent was
removed by rotary evaporation to give the crude carboxylic
acid.
In a separate flask, dipeptide 15 (Boc-Trp-Ala-OMe, 50 mg,
120 µmol) was dissolved in CH2Cl2 (3 mL). To this solution
was added TFA (1.5 mL). The reaction was stirred at room
temperature for 20 min. The solvents were removed by rotary
evaporation, and the resulting residue was partitioned between
EtOAc (75 mL) and saturated Na2CO3 (75 mL). The organic
layer was washed with saturated Na2CO3 (75 mL) and then
dried with Na2CO3. The resulting solution was concentrated
by rotary evaporation to yield H2N-Trp-Ala-OMe as a white
solid.
The crude carboxylic acid (95 mg, 100 µmol) was redissolved
in DMF (5 mL). To this solution were added H2N-Trp-Ala-OMe
(35 mg, 120 µmol), HATU (57 mg, 150 µmol), and DIEA (55
µL, 39 mg, 300 µmol). The reaction was stirred at 50 °C for 4
h and then partitioned between EtOAc (50 mL) and 1 N HCl
(50 mL). The organic layer was washed with 1 N HCl (50 mL),
saturated aqueous NaHCO3 (50 mL), and brine (50 mL). It
was dried over MgSO4, and the solvent was removed by rotary
evaporation. The crude material was purified by flash chro-
matography (EtOAc:hexanes 4:1) to give peptide 22 as a
mixture of two diastereomers (54 mg, 45 µmol, 45%): 1H NMR
(300 MHz, CDCl3) δ 1.18-1.32 (m, 5H), 1.33-1.39 (m, 4H),
1.40-1.53 (m, 3H), 1.55-1.68 (m, 18H), 1.70-2.00 (m, 2H),
2.05-2.15 (m, 1H), 2.20-2.38 (m, 1H), 2.90-3.30 (m, 7H),
3.46-3.95 (m, 6H), 4.40-4.65 (m, 2H), 4.68-4.85 (m, 3H),
4.90-5.05 (m, 2H), 6.88-7.10 (m, 2H), 7.12-7.40 (m, 11H),
7.45-7.70 (m, 5H), 8.00-8.50 (m, 3H); 13C NMR (100 MHz,
CDCl3) δ 14.4, 16.8, 18.4, 18.5, 21.6, 22.9, 25.2, 28.1, 28.5, 30.0,
30.1, 31.9, 32.3, 34.4, 34.9, 39.0, 48.46, 48.52, 52.7, 53.9, 59.1,
59.4, 62.2, 67.4, 67.5, 69.6, 75.3, 84.0, 84.1, 84.2, 111.5, 115.2,
115.3, 115.6, 115.7, 118.9, 119.1, 119.3, 119.8, 122.3, 122.9,
123.0, 123.9, 124.7, 124.9, 125.0, 127.7, 127.8, 128.2, 128.5,
128.8, 130.5, 135.6, 135.8, 136.1, 136.2, 136.6, 136.7, 149.8,
149.9, 170.2, 170.4, 171.0, 171.2, 171.3, 171.4, 173.20, 173.25,
173.29; HRMS-FAB (M + Na+) calcd for C67H80N8NaO14
1243.5692, found 1243.5677.
0.91 (m, 6H), 1.10-1.50 (m, 12H), 1.54-1.66 (m, 18H), 1.70-
1.90 (m, 2H), 2.00-2.14 (m, 1H), 2.20-2.40 (m, 1H), 2.85-
3.30 (m, 6H), 3.45-4.00 (m, 7H), 4.40-4.68 (m, 2H), 4.70-
4.85 (m, 2H), 4.91-5.05 (m, 1H), 6.85-7.10 (m, 3H), 7.11-
7.20 (m, 4H), 7.20-7.30 (m, 7H), 7.45-7.68 (m, 5H), 8.00-
8.15 (m, 2H), 8.16-8.30 (m, 1H); 13C NMR (75 MHz, MeOH-
d4) δ -1.0, 10.7, 10.8, 13.5, 14.8, 14.9, 18.8, 19.9, 21.4, 22.7,
25.2, 25.3, 27.1, 27.4, 27.7, 27.9, 28.3, 31.7, 34.1, 34.7, 35.2,
37.5, 37.9, 47.1, 51.4, 51.5, 53.7, 54.3, 54.6, 55.5, 57.2, 58.9,
59.0, 60.6, 66.6, 83.8, 98.9, 109.7, 109.8, 111.2, 111.3, 115.1,
115.9, 116.3, 118.3, 118.5, 118.8, 119.1, 119.2, 121.4, 122.7,
123.5, 123.7, 124.5, 127.5, 127.8, 127.9, 128.4, 130.7, 130.9,
135.8, 137.0, 149.9, 150.0, 157.2, 171.5, 172.0, 172.1, 172.2,
172.9, 173.1, 173.2, 175.2; HRMS-FAB (M + Na+) calcd for
C70H86N8NaO14 1285.6161, found 1285.6126.
Compound 24. Compound 24 was synthesized using a
procedure that was similar to that used to prepare amide 22
(50 mg, 40 µmol, 40%): 1H NMR (400 MHz, MeOH-d4) δ 0.81-
0.95 (m, 6H), 1.20-1.38 (m, 6H), 1.40-1.50 (m, 3H), 1.57-
1.70 (m, 21H), 1.75-2.00 (m, 2H), 2.01-2.15 (m, 1H), 2.20-
2.40 (m, 1H), 2.85-3.05 (m, 1H), 3.05-3.25 (m, 4H), 3.25-
3.30 (m, 1H), 3.50-3.90 (m, 7H), 4.50-4.62 (m, 2H), 4.71-
4.85 (m, 3H), 4.90-5.00 (m, 2H), 6.90-7.10 (m, 3H), 7.13-
7.22 (m, 5H), 7.23-7.35 (m, 6H), 7.45-7.68 (m, 5H), 8.00-
8.20 (m, 2H); 13C NMR (75 MHz, MeOH-d4) δ 1.4, 14.5, 14.6,
18.9, 21.1, 21.5, 22.3, 23.0, 23.1, 23.6, 25.1, 25.3, 25.7, 28.0,
28.1, 28.6, 28.8, 30.1, 30.8, 32.0, 32.2, 32.4, 34.4, 34.9, 35.1,
41.6, 41.7, 51.4, 52.7, 54.0, 55.3, 59.3, 60.8, 62.5, 67.5, 77.6,
84.2, 98.7, 110.5, 111.7, 111.9, 115.3, 115.4, 115.7, 115.8, 119.0,
119.2, 119.5, 119.7, 119.9, 120.1, 122.5, 122.6, 123.1, 123.2,
124.0, 124.9, 125.0, 125.2, 127.7, 127.8, 128.2, 128.3, 128.6,
128.9, 130.5, 135.7, 135.9, 136.3, 136.6, 136.8, 149.9, 150.0,
156.3, 170.3, 171.4, 171.6, 171.8, 173.3, 173.4; HRMS-FAB
(M + Na+) calcd for C70H86N8NaO14 1285.6161, found 1285.3078.
Compound 25. Compound 25 was synthesized using a
procedure that was similar to that used to prepare amide 22
(57 mg, 44 µmol, 44%): 1H NMR (400 MHz, MeOH-d4) δ 1.20-
1.35 (m, 5H), 1.38-1.50 (m, 3H), 1.50-1.70 (m, 18H), 1.78-
1.90 (m, 1H), 2.20-2.40 (m, 1H), 2.85-3.30 (m, 8H), 3.35-
3.46 (m, 4H), 3.60-3.90 (m, 7H), 4.57-4.77 (m, 3H), 4.90-
4.95 (m, 1H), 4.99-5.10 (m, 1H), 6.85-7.12 (m, 6H), 7.13-
7.22 (m, 9H), 7.23-7.33 (m, 3H), 7.45-7.65 (m, 5H), 8.00-
8.15 (m, 2H), 8.16-8.30 (m, 1H); 13C NMR (75 MHz, MeOH-
d4) δ 0.6, 15.1, 17.3, 20.1, 20.7, 21.7, 23.1, 24.5, 26.6, 28.7, 28.8,
29.1, 29.4, 29.6, 29.8, 30.6, 31.0, 31.3, 33.2, 34.3, 39.0, 39.2,
39.5, 53.3, 53.4, 55.2, 55.7, 55.8, 56.1, 57.0, 57.3, 59.9, 60.5,
60.7, 62.2, 64.9, 68.2, 71.3, 76.6, 85.4, 100.5, 109.7, 111.4, 112.9,
116.7, 117.7, 118.0, 119.9, 120.0, 120.4, 120.7, 120.9, 122.9,
124.3, 125.1, 125.3, 126.0, 126.1, 128.4, 129.0, 129.4, 130.0,
130.9, 131.0, 131.6, 132.3, 132.4, 137.2, 137.3, 138.4, 138.5,
138.6, 151.5, 158.7, 158.8, 173.0, 173.2, 173.6, 174.3, 174.6,
174.7, 176.6; HRMS-FAB (M + Na+) calcd for C73H84NaN8O14
1319.6005, found 1319.6030.
Compound 26. Compound 26 was synthesized using a
procedure that was similar to that used to prepare amide 22
(61 mg, 46 µmol, 46%): 1H NMR (400 MHz, MeOH-d4) δ 1.10-
1.35 (m, 6H), 1.35-1.50 (m, 3H), 1.53-1.70 (m, 18H), 1.72-
1.90 (m, 1H), 1.95-2.05 (m, 1H), 2.10-2.28 (m, 1H), 2.85-
3.30 (m, 8H), 3.60-3.90 (m, 7H), 4.47-4.67 (m, 1H), 4.68-
4.85 (m, 3H), 4.90-5.02 (m, 2H), 6.85-7.12 (m, 6H), 7.12-
7.41 (m, 12H), 7.42-7.70 (m, 6H), 7.90-8.20 (m, 3H); 13C NMR
(75 MHz, MeOH-d4) δ 13.5, 18.7, 19.9, 21.4, 21.5, 22.7, 25.2,
27.4, 27.7, 29.0, 29.4, 31.5, 31.7, 34.0, 34.7, 36.0, 37.9, 49.1,
49.8, 51.8, 53.7, 53.8, 54.0, 54.2, 54.5, 55.5, 55.7, 58.9, 60.6,
66.6, 83.8, 98.9, 109.1, 109.8, 111.4, 113.5, 115.1, 116.0, 116.3,
118.1, 118.3, 118.5, 118.6, 118.9, 119.1, 119.2, 121.5, 121.8,
122.7, 123.7, 123.8, 124.5, 127.5, 127.7, 127.8, 127.9, 128.4,
130.6, 130.9, 135.8, 136.9, 137.0, 149.9, 150.0, 157.2, 166.5,
171.5, 172.1, 172.4, 172.5, 172.7, 172.8, 172.9, 173.0, 175.1;
HRMS-FAB (M + Na+) calcd for C75H85N9NaO14 1358.6114,
found 1358.6161.
Compound 23. Compound 23 was synthesized using a
procedure that was similar to that used to prepare amide 22
(50 mg, 40 µmol, 40%): 1H NMR (400 MHz, MeOH-d4) δ 0.78-
Compound 27. Compound 27 was synthesized using a
procedure that was similar to that used to prepare amide 22
(53 mg, 40 µmol, 40%): 1H NMR (400 MHz, MeOH-d4) δ 1.10-

Inhibitors of the Serine Protease Plasmin
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22 6915
1.49 (m, 10H), 1.55-1.70 (m, 19H), 1.75-1.95 (m, 1H), 2.00-
2.16 (m, 1H), 2.20-2.40 (m, 1H), 2.75-3.30 (m, 9H), 3.47-
3.91 (m, 7H), 4.48-4.80 (m, 4H), 4.91-5.02 (m, 2H), 6.56-
6.71 (m, 1H), 6.87-6.97 (m, 2H), 6.98-7.01 (m, 1H), 7.02-
7.08 (m, 1H), 7.09-7.12 (s, 1H), 7.13-7.35 (m, 10H), 7.45-
7.65 (m, 5H), 8.00-8.30 (m, 3H); 13C NMR (75 MHz, MeOH-
d4) δ 0.6, 15.1, 20.4, 23.1, 26.8, 29.1, 29.5, 29.8, 30.7, 31.0, 32.3,
33.2, 35.8, 37.6, 38.3, 39.5, 53.2, 53.3, 53.7, 55.3, 55.8, 56.0,
56.1, 57.1, 60.6, 60.7, 62.2, 68.3, 85.5, 100.5, 111.5, 112.9, 113.0,
116.8, 116.9, 117.1, 117.6, 118.0, 119.9, 120.3, 120.4, 120.8,
122.9, 123.0, 124.4, 125.1, 126.1, 129.0, 129.1, 129.2, 129.5,
129.6, 130.1, 132.0, 132.3, 132.5, 137.4, 138.7, 151.6, 151.7,
158.0, 158.1, 158.9, 165.5, 173.2, 173.7, 173.8, 174.4, 174.5,
176.7; HRMS-FAB (M + Na+) calcd for C77H92N8NaO15
1335.5954, found 1335.5980.
Compound 28. Compound 28 was synthesized using a
procedure that was similar to that used to prepare amide 22
(63 mg, 50 µmol, 50%): 1H NMR (400 MHz, MeOH-d4) δ 0.80-
0.95 (m, 6H), 1.14-1.35 (m, 6H), 1.36-1.97 (m, 4H), 1.52-
1.67 (m, 18H), 1.80-1.95 (m, 1H), 2.02-2.18 (m, 2H), 2.22-
2.42 (m, 1H), 2.83-3.40 (m, 7H), 3.55-3.87 (m, 7H), 4.21-
4.65 (m, 3H), 4.72-4.82 (m, 2H), 4.90-5.02 (m, 2H), 6.90-
7.10 (m, 3H), 7.11-7.35 (m, 11H), 7.40-7.70 (m, 6H), 8.00-
8.20 (m, 2H); 13C NMR (75 MHz, MeOH-d4) δ 0.6, 15.1, 19.1,
19.2, 20.0, 20.1, 20.4, 21.5, 23.1, 26.8, 29.1, 29.3, 29.5, 32.3,
32.6, 33.2, 35.7, 37.6, 39.5, 53.0, 53.1, 55.3, 55.9, 56.3, 57.1,
59.8, 59.9, 60.6, 62.2, 68.3, 70.5, 85.4, 100.5, 111.4, 111.5, 112.8,
112.9, 116.8, 117.6, 117.9, 119.9, 120.4, 120.7, 120.8, 123.0,
124.3, 125.2, 126.1, 129.1, 129.4, 129.5, 130.1, 132.3, 132.5,
137.4, 138.6, 151.6, 151.7, 158.8, 165.5, 168.1, 173.1, 173.6,
173.7, 173.8, 173.9, 174.4, 174.5, 174.8, 174.9, 175.1, 176.7;
HRMS-FAB (M + Na+) calcd for C69H84N8NaO14 1271.6005,
found 1271.6041.
Inhibitor 29. To peptide 22 (40 mg, 33 µmol) was added
an aqueous TFA solution (4 mL, 70%) at 0 °C. The reaction
was warmed to room temperature and stirred for an additional
12 h, and then it was concentrated by rotary evaporation. The
resulting residue was dried under vacuum for 24 h. The crude
material was purified by flash chromatography (MeOH in
CH2Cl2 2-6%) to yield inhibitor 29 as a mixture of two
diastereomers (24 mg, 25 µmol, 75%): 1H NMR (300 MHz,
DMSO-d6) δ 0.77-1.40 (m, 7H), 1.41-1.70 (m, 3H), 1.71-1.85
(m, 2H), 1.86-2.36 (m, 4H), 2.72-2.94 (m, 2H), 2.95-3.22 (m,
3H), 3.45-3.55 (m, 3H), 4.15-4.75 (m, 4H), 4.90-5.00 (m, 1H),
6.81-7.12 (m, 7H), 7.13-7.48 (m, 8H), 7.52-7.68 (m, 2H),
7.90-8.55 (m, 3H), 10.60-10.95 (m, 2H); 13C NMR (75 MHz,
DMSO-d6) δ 17.7, 18.0, 21.9, 24.1, 25.5, 26.0, 28.6, 29.0, 30.1,
31.3, 32.8, 33.5, 34.8, 35.2, 35.4, 42.6, 44.0, 48.4, 29.0, 52.7,
53.7, 54.2, 56.5, 58.1, 66.1, 66.4, 66.8, 67.9, 98.5, 110.6, 111.0,
111.1, 112.1, 119.0, 119.3, 119.4, 121.7, 124.5, 124.6, 125.8,
127.5, 128.1, 128.3, 128.5, 128.9, 129.1, 136.8, 136.9, 137.8,
140.1, 156.7, 158.7, 171.7, 172.4, 172.6, 172.7, 173.8, 174.9,
209.0; HRMS-FAB (M + Na+) calcd for C54H58N8NaO9
985.4224, found 985.4205.
Inhibitor 30. Inhibitor 30 was synthesized using a proce-
dure that was similar to that used to prepare inhibitor 29 (23
mg, 23 µmol, 70%): 1H NMR (300 MHz, DMSO-d6) δ 0.65-
0.95 (m, 6H), 1.05-1.48 (m, 6H), 1.49-1.85 (m, 6H), 1.86-
2.36 (m, 4H), 2.76-3.21 (m, 5H), 3.51-3.70 (m, 3H), 4.20-
4.40 (m, 3H), 4.55-4.85 (m, 2H), 4.90-5.00 (m, 1H), 6.85-
7.01 (m, 3H), 7.02-7.12 (m, 4H), 7.17-7.28 (m, 3H), 7.29-
7.37 (m, 5H), 7.50-7.71 (m, 3H), 7.90-8.30 (m, 3H), 10.67-
10.95 (m, 2H); 13C NMR (75 MHz, DMSO-d6) δ 12.0, 16.2, 24.1,
25.5, 25.6, 25.9, 28.5, 28.6, 29.0, 31.3, 33.4, 34.8, 35.2, 35.4,
37.2, 49.1, 52.5, 53.7, 54.2, 56.5, 57.2, 58.1, 66.1, 66.4, 67.9,
110.6, 110.9, 111.1, 112.1, 119.0, 119.3, 119.4, 121.7, 124.4,
124.6, 125.8, 127.5, 128.1, 128.2, 128.3, 128.5, 128.9, 129.1,
136.8, 136.9, 137.0, 137.8, 156.7, 171.7, 172.4, 172.5, 172.7,
172.9, 208.9; HRMS-FAB (M + Na+) calcd for C57H64N8NaO9
1027.4694, found 1027.4670.
1.85 (m, 2H), 1.86-2.35 (m, 4H), 2.78-3.20 (m, 5H), 3.55-
3.65 (m, 3H), 4.20-4.70 (m, 4H), 4.85-5.00 (m, 1H), 6.85-
7.12 (m, 7H), 7.13-7.38 (m, 8H), 7.40-7.66 (m, 3H), 7.90-
8.40 (m, 3H), 10.65-10.95 (m, 2H); 13C NMR (75 MHz, DMSO-
d6) δ 21.9, 22.1, 22.2, 23.6, 23.7, 24.1, 25.0, 25.1, 25.5, 25.9,
27.3, 28.6, 28.9, 30.1, 30.6, 31.3, 31.7, 32.8, 33.5, 34.7, 35.2,
35.4, 43.1, 45.6, 49.1, 51.1, 52.7, 53.7, 54.2, 56.5, 58.1, 66.1,
66.4, 67.9, 110.6, 111.0, 111.1, 112.1, 118.1, 118.6, 119.0, 119.3,
119.4, 121.7, 122.0, 124.4, 124.6, 125.4, 125.8, 127.5, 128.1,
128.3, 128.5, 128.8, 128.9, 129.0, 129.1, 136.8, 136.9, 137.0,
137.8, 140.0, 156.6, 171.7, 172.4, 172.6, 172.8, 172.9, 173.2,
173.7, 174.0, 174.9, 208.9; HRMS-FAB (M + Na+) calcd for
C57H64N8NaO9 1027.4694, found 1027.4674.
Inhibitor 32. Inhibitor 32 was synthesized using a proce-
dure that was similar to that used to prepare inhibitor 29 (24
mg, 23 µmol, 70%): 1H NMR (300 MHz, DMSO-d6) δ 1.00-
1.40 (m, 4H), 1.41-1.70 (m, 3H), 1.72-2.35 (m, 6H), 2.72, 3.20
(m, 6H), 3.50-3.67 (m, 3H), 4.18-4.83 (m, 4H), 4.85-4.99 (m,
1H), 6.80-7.12 (m, 7H), 7.13-7.37 (m, 10H), 3.38-7.77 (m,
3H), 7.80-8.50 (m, 3H), 10.65-10.95 (m, 2H); 13C NMR (75
MHz, DMSO-d6) δ 21.9, 24.0, 25.5, 25.9, 27.3, 28.6, 29.0, 30.2,
30.6, 31.3, 31.6, 32.6, 32.8, 33.5, 34.8, 35.2, 35.4, 37.4, 42.3,
42.5, 43.3, 43.6, 44.1, 44.5, 49.1, 52.7, 53.2, 53.7, 54.3, 54.5,
56.5, 58.1, 66.2, 66.4, 66.8, 67.9, 70.6, 75.1, 110.6, 111.0, 111.1,
111.5, 112.1, 112.5, 119.0, 119.3, 119.4, 121.7, 124.4, 124.6,
125.8, 127.4, 128.1, 128.3, 128.5, 129.1, 129.7, 129.9, 130.0,
130.5, 130.7, 136.8, 136.9, 137.0, 137.8, 137.9, 138.3, 140.0,
152.3, 156.7, 158.8, 159.3, 171.7, 172.4, 172.5, 172.6, 172.7,
173.6, 209.0; HRMS-FAB (M + Na+) calcd for C60H62N8NaO9
1061.4537, found 1061.4515.
Inhibitor 33. Inhibitor 33 was synthesized using a proce-
dure that was similar to that used to prepare inhibitor 29 (19
mg, 18 µmol, 53%): 1H NMR (300 MHz, DMSO-d6) δ 1.10-
1.28 (m, 2H), 1.29-1.48 (m, 2H), 1.51-1.70 (m, 2H), 1.71-
1.85 (m, 3H), 1.87-2.13 (m, 3H), 2.15-2.32 (m, 1H), 2.72-
2.96 (m, 2H), 2.97-3.25 (m, 4H), 3.50-3.70 (m, 4H), 4.20-
4.45 (m, 2H), 4.46-4.75 (m, 2H), 4.86-4.95 (m, 1H), 6.80-
7.13 (m, 9H), 7.14-7.38 (m, 9H), 7.39-7.69 (m, 4H), 7.85-
8.08 (m, 2H), 8.09-8.45 (m, 2H), 10.60-10.90 (m, 3H); 13C
NMR (75 MHz, DMSO-d6) δ 24.0, 25.4, 25.9, 27.3, 27.8, 28.6,
28.9, 30.1, 30.6, 32.7, 33.5, 34.8, 35.4, 43.6, 49.1, 52.7, 53.8,
53.9, 54.3, 56.5, 58.1, 66.1, 67.9, 110.1, 110.6, 111.0, 111.1,
112.1, 112.3, 118.8, 119.0, 119.3, 119.4, 121.7, 121.8, 124.4,
124.6, 127.5, 127.9, 128.1, 128.3, 128.5, 129.1, 130.4, 136.8,
136.9, 137.8, 159.3, 171.7, 172.4, 172.7, 172.8, 173.0, 209.0;
HRMS-FAB (M + Na+) calcd for C62H63N9NaO9 1100.4646,
found 1100.4619.
Inhibitor 34. Inhibitor 34 was synthesized using a proce-
dure that was similar to that used to prepare inhibitor 29 (21
mg, 20 µmol, 62%): 1H NMR (300 MHz, DMSO-d6) δ 1.09-
1.45 (m, 3H), 1.46-1.68 (m, 2H), 1.70-1.85 (m, 5H), 1.86-
2.10 (m, 3H), 2.15-2.35 (m, 1H), 2.72-2.97 (m, 3H), 2.98-
3.16 (m, 3H), 3.50-3.60 (m, 5H), 4.25-4.75 (m, 4H), 4.86-
4.99 (m, 1H), 6.50-6.80 (m, 2H), 6.85-7.12 (m, 9H), 7.13-
7.38 (m, 8H), 7.50-7.72 (m, 3H), 7.86-8.05 (m, 2H), 8.06-
8.20 (m, 1H), 8.25-8.36 (m, 1H), 10.65-10.90 (m, 2H); 13C
NMR (75 MHz, DMSO-d6) δ 24.0, 25.4, 26.0, 28.6, 29.0, 30.2,
30.6, 33.5, 34.8, 35.4, 36.8, 43.9, 49.1, 52.6, 53.7, 54.3, 54.6,
54.8, 56.5, 58.1, 66.1, 67.9, 110.6, 111.0, 111.1, 112.1, 115.9,
119.0, 119.3, 119.4, 121.7, 122.0, 124.1, 124.3, 124.5, 125.5,
127.5, 127.8, 128.1, 128.3, 128.5, 129.1, 130.9, 136.7, 136.8,
136.9, 137.8, 155.7, 156.7, 156.9, 158.5, 158.9, 171.7, 172.4,
172.6, 172.7, 172.8, 173.8, 208.9; HRMS-FAB (M + Na+) calcd
for C60H62N8NaO10 1077.4487, found 1077.4452.
Inhibitor 35. Inhibitor 35 was synthesized using a proce-
dure that was similar to that used to prepare inhibitor 29 (22
mg, 22 µmol, 67%): 1H NMR (300 MHz, DMSO-d6) δ 0.71-
0.99 (m, 6H), 1.00-1.17 (m, 1H), 1.18-1.34 (m, 2H), 1.35-
1.45 (m, 1H), 1.48-1.83 (m, 5H), 2.69-2.95 (m, 2H), 2.96-
3.22 (m, 3H), 3.50-3.70 (m, 4H), 4.10-4.42 (m, 3H), 4.55-
4.95 (m, 3H), 6.80-7.15 (m, 7H), 7.16-7.48 (m, 8H), 7.49-
7.70 (m, 3H), 7.89-8.30 (m, 3H), 10.60-10.92 (m, 2H); 13C
NMR (75 MHz, DMSO-d6) δ 19.1, 19.8, 24.1, 25.5, 26.0, 28.5,
28.6, 29.0, 30.6, 30.8, 33.4, 34.7, 35.4, 49.0, 52.5, 53.8, 54.2,
Inhibitor 31. Inhibitor 31 was synthesized using a proce-
dure that was similar to that used to prepare inhibitor 29 (24
mg, 24 µmol, 72%): 1H NMR (300 MHz, DMSO-d6) δ 0.72-
0.97 (m, 6H), 0.98-1.43 (m, 5H), 1.44-1.70 (m, 5H), 1.71-

6916 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22
Xue and Seto
(21) Enomoto, R.; Sugahara, C.; Komai, T.; Suzuki, C.; Kinoshita,
N.; Hosoda, A.; Yoshikawa, A.; Tsuda, Y.; Okada, Y.; Lee, E. The
Structure-Activity Relationship of Various YO Compounds,
Novel Plasmin Inhibitors, in the Apoptosis Induction. Biochim.
Biophys. Acta 2004, 1674, 291-298.
(22) Conroy, J. L.; Sanders, T. C.; Seto, C. T. Using the Electrostatic
Field Effect to Design a New Class of Inhibitors for Cysteine
Proteases. J. Am. Chem. Soc. 1997, 119, 4285-4291.
56.5, 58.1, 58.2, 66.1, 67.9, 110.6, 111.0, 111.1, 112.1, 119.0,
119.3, 119.4, 121.7, 124.4, 124.6, 128.1, 128.2, 128.3, 128.5,
129.1, 136.8, 136.9, 137.0, 137.8, 156.7, 171.7, 172.4, 172.7,
173.1, 208.9; HRMS-FAB (M + Na+) calcd for C56H62N8NaO9
1013.4537, found 1013.4573.
Acknowledgment. This research was supported by
the NIH NIGMS (Grant R01 GM057327).
(23) Sanders, T. C.; Seto, C. T. 4-Heterocyclohexanone-Based Inhibi-
tors of the Serine Protease Plasmin. J. Med. Chem. 1999, 42,
2969-2976.
(24) Abato, P.; Yuen, C. M.; Cubanski, J. Y.; Seto, C. T. Inhibitors of
Plasmin that Extend into Both the S and S′ Binding Sites:
Cooperative Interactions between S1 and S2. J. Org. Chem.
2002, 67, 1184-1191.
Supporting Information Available: 1H and 13C NMR
spectra for all new compounds. HPLC characterization for
compounds 29-35. This material is available free of charge
via the Internet at http://pubs.acs.org.
(25) Conroy, J. L.; Seto, C. T. Demonstration by 13C NMR Studies
that Tetrahydropyranone-Based Inhibitors Bind to Cysteine
Proteases by Reversible Formation of a Hemithioketal Adduct.
J. Org. Chem. 1998, 63, 2367-2370.
References
(26) Abato, P.; Conroy, J. L.; Seto, C. T. Combinatorial Library of
Serine and Cysteine Protease Inhibitors that Interact with Both
the S and S′ Binding Sites. J. Med. Chem. 1999, 42, 4001-4009.
(27) Conroy, J. L.; Abato, P.; Ghosh, M.; Austermuhle, M. I.; Kiefer,
M. R.; Seto, C. T. Synthesis of Cyclohexanone-Based Cathepsin
B Inhibitors that Interact with Both the S and S′ Binding Sites.
Tetrahedron Lett. 1998, 39, 8253-8256.
(28) (a) Furka, A.; Sebestyen, F.; Asgedom, M.; Dibo, G. General
Method for Rapid Synthesis of Multicomponent Peptide Mix-
tures. Int. J. Pept. Protein Res. 1991, 37, 487-493. (b) Lam, K.
S.; Salmon, S. E.; Hersh, E. M.; Hruby, V. J.; Kazmierski, W.
M.; Knapp, R. J. A New Type of Synthetic Peptide Library for
Identifying Ligand-Binding Activity. Nature 1991, 354, 82-84.
(c) Houghten, R. A.; Pinilla, C.; Blondelle, S. E.; Appel, J. R.;
Dooley, C. T.; Cuervo, J. H. Generation and Use of Synthetic
Peptide Combinatorial Libraries for Basic Research and Drug
Discovery. Nature, 1991, 354, 84-86.
(29) For other libraries that were constructed for screening against
proteases, see: (a) Campbell, D. A.; Bermak, J. C.; Burkoth, T.
S.; Patel, D. V. A Transition State Analogue Inhibitor Combi-
natorial Library. J. Am. Chem. Soc. 1995, 117, 5381-5382. (b)
Owens, R. A.; Gesellchen, P. D.; Houchins, B. J.; DiMarchi, R.
D. The Rapid Identification of HIV Protease Inhibitors through
the Synthesis and Screening of Defined Peptide Mixtures.
Biochem. Biophys. Res. Commun. 1991, 181, 402-408. (c) Kick,
E. K.; Ellman, J. A. Expedient Method for the Solid-Phase
Synthesis of Aspartic Protease Inhibitors Directed Toward the
Generation of Libraries. J. Med. Chem. 1995, 38, 1427-1430.
(d) Wang, J. T.; Li, S.; Wideburg, N.; Krafft, G. A.; Kempf, D. J.
Synthetic Chemical Diversity: Solid-Phase Synthesis of Librar-
ies of C2 Symmetric Inhibitors of HIV Protease Containing
Diamino Diol and Diamino Alcohol Cores. J. Med. Chem. 1995,
38, 2995-3002.
(30) Dooley, C. T.; Chung, N. N.; Wilkes, B. C.; Schiller, P. W.;
Bidlack, J. M.; Pasternak, G. W.; Houghten, R. A. An All
D-Amino Acid Opioid Peptide with Central Analgesic Activity
from a Combinatorial Library. Science 1994, 266, 2019-2022.
(31) (a) Fiedler, F. Effects of Secondary Interactions on the Kinetics
of Peptide and Peptide Ester Hydrolysis by Tissue Kallikrein
and Trypsin. Eur. J. Biochem. 1987, 163, 303-312. (b) Araujo-
Viel, M. S.; Juliano, M. A.; Oliveira, L.; Prado, E. S. Horse
Urinary Kallikrein, II. Effect of Subsite Interactions on Its
Catalytic Activity. Biol. Chem. Hoppe-Seyler 1988, 369, 397-
401.
(1) Leung, D.; Abbenante, G.; Fairlie, D. P. Protease Inhibitors:
Current Status and Future Prospects. J. Med. Chem. 2000, 43,
305-341.
(2) Mignatti, P.; Rifkin, D. B. Plasminogen Activators and Matrix
Metalloproteinases in Angiogenesis. Enzyme Protein 1996, 49,
117-137.
(3) Murphy, G.; Stanton, H.; Cowell, S.; Butler, G.; Knauper, V.;
Atkinson, S.; Gavrilovic, J. Mechanisms for Pro Matrix Metallo-
proteinase Activation. APMIS 1999, 107, 38-44.
(4) Sternlicht, M. D.; Werb, Z. How Matrix Metalloproteinases
Regulate Cell Behavior. Annu. Rev. Cell Dev. Biol. 2001, 17,
463-516.
(5) Nagase, H.; Woessner Jr, J. F. Matrix Metalloproteinases. J.
Biol. Chem. 1999, 274, 21491-21494.
(6) Iwamoto, M.; Abiko, Y. Plasminogen-Plasmin System. VI. Prepa-
ration of Alpha 2-Macroglobulin Antiplasmin from Human
Plasma. Biochim. Biophys. Acta 1970, 214, 402-410.
(7) Moroi, M.; Aoki, N. Isolation and Characterization of Alpha2-
Plamsin Inhibitor from Human Plasma. A Novel Proteinase
Inhibitor which Inhibits Activator-Induced Clot Lysis. J. Biol.
Chem. 1976, 251, 5956-5965.
(8) Markus, G. The Role of Hemostasis and Fibrinolysis in the
Metastatic Spread of Cancer. Semin. Thromb. Hemostasis 1984,
10, 61-70.
(9) Andreasen, P. A.; Egelund, R.; Petersen, H. H. The Plasminogen
Activation System in Tumor Growth, Invasion, and Metastasis.
Cell Mol. Life Sci. 2000, 20, 25-40.
(10) Okamoto, S. Plasmin and Antiplasmin-their Pathologic Physiol-
ogy. Keio J. Med. 1959, 8, 211-217.
(11) Okamoto, S.; Sato, S.; Tanaka, Y.; Okamoto, U. An Active
Stereoisomer (Trans-Form) of Amcha and its Antifibrinolytic
(Antiplasminic) Action in Vitro and in Vivo. Keio J. Med. 1964,
13, 177-185.
(12) Iwamoto, M. Plasminogen-Plasmin System IX. Specific Binding
of Tranexamic Acid to Plasmin. Thrombos. Diathes. Haemorrh.
1975, 33, 573-585.
(13) Sherry, S. Fibrinolysis, Thrombosis, and Hemostasis; Lea &
Febiger: Philadelphia, 1992; Chapter 1.
(14) Okada, Y.; Tsuda, Y.; Teno, N.; Wanaka, K.; Bohgaki, M.;
Hijikata-Okunomiya, A.; Naito, T.; Okamoto, S. Synthesis of
Active Center-Directed Peptide Inhibitors of Plamsin. Chem.
Pharm. Bull. 1988, 36, 1289-1297.
(15) Teno, N.; Wanaka, K.; Okada, Y.; Tsuda, Y.; Okamoto, U.;
Hijikata-Okunomiya, A.; Naito, T.; Okamoto, S. Development
of Active Center-Directed Inhibitors against Plasmin. Chem.
Pharm. Bull. 1991, 39, 2340-2346.
(16) Okada, Y.; Tsuda, Y.; Tada, M.; Wanaka, K.; Okamoto, U.;
Hijikata-Okunomiya, A.; Okamoto, S. Development of Potent and
Selective Plasmin and Plasma Kallikrein Inhibitors and Studies
on the Structure-Activity Relationship. Chem. Pharm. Bull.
2000, 48, 1964-1972.
(17) Okada, Y.; Matsumoto, Y.; Tsuda, Y.; Tada, M.; Wanaka, K.;
Hijikata-Okunomiya, A.; Okamoto, S. Development of Plasmin-
Selective Inhibitors and Studies of their Structure-Activity
Relationship. Chem. Pharm. Bull. 2000, 48, 184-193.
(18) Tsuda, Y.; Tada, M.; Wanaka, K.; Okamoto, U.; Hijikata-
Okunomiya, A.; Okamoto, S.; Okada, Y. Structure-Inhibitory
Activity Relationship of Plasmin and Plasma Kallikrein Inhibi-
tors. Chem. Pharm. Bull. 2001, 49, 1457-1463.
(19) Okada, Y.; Tsuda, Y.; Wanaka, K.; Tada, M.; Okamoto, U.;
Okamoto, S.; Hijikata-Okunomiya, A.; Bokonyi, G.; Szenda, B.;
Keri, G. Development of Plasmin and Plasma Kallikrein Selec-
tive Inhibitors and their Effect on M1 (Melanoma) and HT29
Cell Lines. Bioorg. Med. Chem. Lett. 2000, 10, 2217-2221.
(20) Lee, E.; Enomoto, R.; Takemura, K.; Tsuda, Y.; Okada, Y. A
Selective Plasmin Inhibitor, Trans-Aminomethylcyclohexane-
carbonyl-L-(O-Picolyl)Tyrosine-Octylamide (YO-2), Induces Thy-
mocyte Apoptosis. Biochem. Pharmacol. 2002, 63, 1315-1323.
(32) Pimenta, D. C.; Chao, J.; Chao, L.; Juliano, M. A.; Juliano, L.
Specificity of Human Tissue Kallikrein towards Substrates
Containing Phe-Phe Pair of Amino Acids. Biochem. J. 1999, 339,
473-479.
(33) (a) Hervio, L. S.; Coombs, G. S.; Bergstrom, R. C.; Trivedi, K.;
Corey, D. R.; Madison, E. L. Negative Selectivity and the
Evolution of Protease Cascades: The Specificity of Plasmin for
Peptide and Protein Substrates. Chem. Biol. 2000, 7, 443-452.
(b) Harris, J. L.; Backes, B. J.; Leonetti, F.; Mahrus, S.; Ellman,
J. A.; Craik, C. S. Rapid and General Profiling of Protease
Specificity by Using Combinatorial Fluorogenic Substrate Li-
braries. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 7754-7759. (c)
Backes, B. J.; Harris, J. L.; Leonetti, F.; Craik, C. S.; Ellman, J.
A. Synthesis of Positional-Scanning Libraries of Fluorogenic
Peptide Substrates to Define the Extended Substrate Specificity
of Plasmin and Thrombin. Nat. Biotechnol. 2000, 18, 187-193.
(d) Edwards, P. D.; Mauger, R. C.; Cottrell, K. M.; Morris, F. X.;
Pine, K. K.; Sylvester, M. A.; Scott, C. W.; Furlong, S. T.
Synthesis and Enzymatic Evaluation of a P1 Arginine Amino-
coumarin Substrate Library for Trypsin-Like Serine Proteases.
Bioorg. Med. Chem. Lett. 2000, 10, 2291-2294.
(34) Petrassi, H. M.; Williams, J. A.; Li, J. Tumanut, C.; Ek, J.; Nakai,
T.; Masick, B.; Backes, B.; Harris, J. L. A Strategy to Profile
Prime and Non-Prime Proteolytic Substrate Specificity. Bioorg.
Med. Chem. Lett. 2005, 15, 3162-3166.

Inhibitors of the Serine Protease Plasmin
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22 6917
(35) Furlong, S. T.; Mauger, R. C.; Strimpler, A. M.; Liu, Y.-P.; Morris,
F. X.; Edwards, P. D. Synthesis and Physical Character-
Protease from Schistosoma mansoni. Biochemistry 1994, 33,
4251-4257.
(37) Campos, I. T.; Silva, M. M.; Azzolini, S. S.; Souza, A. F.; Sampaio,
C. A. M.; Fritz, H.; Tanaka, A. S. Evaluation of Phage Display
System and Leech-Derived Tryptase Inhibitor as a Tool for
Understanding the Serine Proteinase Specificities. Arch. Bio-
chem. Biophys. 2004, 425, 87-94.
ization of
a P1 Arginine Combinatorial Library, and Its
Application to the Determination of the Substrate Specific-
ity of Serine Proteases. Bioorg. Med. Chem. 2002, 10, 3637-
3647.
(36) Schellenberger, V.; Turck, C. W.; Rutter, W. J. Role of the S′
Subsites in Serine Protease Catalysis. Active-Site Mapping of
Rat Chymotrypsin, Rat Trypsin, R-Lytic Protease, and Cercarial
JM050488K