A new strategy for the development of highly potent and selective plasmin inhibitors

Article abstract of DOI:10.1021/jm2011996

A new structure-based strategy for the design of potent and selective plasmin inhibitors was developed. These compounds could be prepared by cyclizations between the P3 and P2 amino acid residues of substrate-analogue inhibitors using metathesis or a copper-catalyzed azide alkyne cycloaddition in combination with standard peptide couplings. The most potent bis-triazole derivative 10 inhibits plasmin and plasma kallikrein with K_i of 0.77 and 2.4 nM, respectively, whereas it has poor activity against the related trypsin-like serine proteases thrombin, factor Xa, or activated protein C. Modeling experiments revealed that inhibitor 10 adopts a compact and rigid structure that fits well into the relatively open active site of plasmin and plasma kallikrein, while it is rejected from sterically demanding residues present in loops of the other enzymes. These results from modeling confirm the selectivity profile found for inhibitor 10 in enzyme kinetic studies. Such compounds might be useful lead structures for the development of new antifibrinolytic drugs for use in cardiac surgery with cardiopulmonary bypass or organ transplantations to reduce bleeding complications.

Full text of DOI:10.1021/jm2011996

Article
pubs.acs.org/jmc
A New Strategy for the Development of Highly Potent and Selective
Plasmin Inhibitors
Sebastian M. Saupe and Torsten Steinmetzer*
Department of Pharmacy, Institute of Pharmaceutical Chemistry, Philipps University Marburg, Marbacher Weg 6,
D-35032 Marburg, Germany
S
* Supporting Information
ABSTRACT: A new structure-based strategy for the design of potent
and selective plasmin inhibitors was developed. These compounds could
be prepared by cyclizations between the P3 and P2 amino acid residues of
substrate-analogue inhibitors using metathesis or a copper-catalyzed azide
alkyne cycloaddition in combination with standard peptide couplings.
The most potent bis-triazole derivative 10 inhibits plasmin and plasma
kallikrein with Ki of 0.77 and 2.4 nM, respectively, whereas it has poor
activity against the related trypsin-like serine proteases thrombin, factor
Xa, or activated protein C. Modeling experiments revealed that inhibitor
10 adopts a compact and rigid structure that fits well into the relatively
open active site of plasmin and plasma kallikrein, while it is rejected from
sterically demanding residues present in loops of the other enzymes. These results from modeling confirm the selectivity profile
found for inhibitor 10 in enzyme kinetic studies. Such compounds might be useful lead structures for the development of new
antifibrinolytic drugs for use in cardiac surgery with cardiopulmonary bypass or organ transplantations to reduce bleeding
complications.
inhibitors have been described so far. For instance, the Okada
group has developed several inhibitors with IC50 of
approximately 0.5 μM containing a trans-4-aminomethylcyclo-
hexanecarboxylic acid residue.3,4 A series of cyclic ketone-based
inhibitors with Ki > 1 μM has been described by Seto.5,6 Solid
phase peptide synthesis was recently used to prepare potent
peptidic arginal-derived plasmin inhibitors (see known
structures below).7
INTRODUCTION
■
The family of trypsin-like serine proteases contains more than
70 members, which are involved in many physiological and
pathological processes. Although all of them cleave their
substrates after the basic amino acids arginine or lysine, there
are big differences in their substrate specificities. Some of them,
like the clotting factor Xa, are highly specific and process only
very few natural substrates. Others, like the digestive protease
trypsin, are relatively unspecific and hydrolyze a variety of
different substrates. An additional nonspecific trypsin-like
serine protease is the fibrinolytic enzyme plasmin, an antagonist
of the clotting proteases, which is responsible for the degrada-
tion of fibrin clots in blood. Therefore, plasmin inhibitors can
be used for the treatment of hyperfibrinolysis, which may occur
during cardiac surgery with cardiopulmonary bypass or organ
transplantation. Aprotinin, a 58 amino acid long peptidic
plasmin inhibitor isolated from bovine lung, was clinically used
to reduce blood loss under these conditions over many years.
However, because of later reported side effects,1 such as an
increased number of heart attacks and kidney damage, aprotinin
was withdrawn from market in 2008. Presently, only tranexamic
acid or p-aminomethylbenzoic acid can be used as alternative
antifibrinolytics. However, both compounds inhibit only the
plasminogen activation but have no direct inhibitory effect on
already formed plasmin. Therefore, the development of new
effective and selective plasmin inhibitors as replacement for
aprotinin is of special therapeutic interest. In contrast to the
large number of potent inhibitors developed for the clotting
proteases thrombin and factor Xa,2 only a few synthetic plasmin
Received: September 9, 2011
Published: January 25, 2012
© 2012 American Chemical Society
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How is it possible to develop selective plasmin inhibitors,
although the trypsin-like serine proteases share a significant
sequence identity in their protease domains? A closer com-
parison of their primary structure reveals that in plasmin a loop
segment around the amino acid in position 99 is completely
missing. The direct connection between the plasmin residue 94
and the amino acid in position 101 is named as 94-shunt and a
unique structural feature of plasmin (Figure 1).8
In addition, a comparison of the crystal structures available for
more than 25 different trypsin-like serine proteases reveals that
the size of the amino acid in position 99 has a major influence on
possible P2 residues found in substrates and substrate-analogue
inhibitors of these enzymes (the terms P1, P2, and P3 of the
amino acid positions in substrates and the binding pockets S1, S2,
and S3 within the active site of the protease correspond to the
nomenclature of Schechter and Berger).9 For instance, factor Xa
containing a sterically demanding tyrosine in position 99
preferentially cleaves substrates with glycine in P2 position,10
whereas PK possessing a glycine 99 prefers more bulky P2 amino
acids, e.g., phenylalanine.11 This residue 99 also belongs to an
active site loop that spatially separates the proximal S2 from the
distal S3/S4 binding pocket in trypsin-like serine proteases.
Therefore, the missing segment 95−100 leads to a unique open
active site in the case of plasmin, due to a fusion of its proximal
and distal binding pockets.2 Most likely, this is the main cause for
plasmin being a relatively nonspecific protease that cleaves many
different substrates as long as they contain a basic amino acid in
P1 position. The structural differences between plasmin and the
other trypsin-like serine proteases around their 99-loop are shown
in Figure 2.
Various groups have developed substrate-analogue inhibitors of
trypsin-like serine proteases containing a decarboxylated arginine
mimetic as P1 residue, e.g., a 4-amidinobenzylamide. It was often
combined with a P2 residue in L-configuration and a D-amino acid
in P3 position,13,14 whereas a benzylsulfonyl moiety was used as
the P4 group.15,16 During the development of inhibitors for the
coagulation proteases thrombin and factor Xa the fibrinolytic
plasmin was normally an antitarget and should not be inhibited.
On the other hand, we have also found among these derivatives
several potent plasmin inhibitors, such as compound 1 (Figure 3).
However, all of these compounds suffered from relatively poor
selectivity; e.g., inhibitor 1 inhibits plasmin, PK, and aPC with
inhibition constants of <1 nM (Table 2).17 Such a low selectivity
could be a disadvantage for further drug development because it
may lead to more side effects.
Figure 1. ClustalW multiple sequence alignment of the protease domains
from physiologically relevant trypsin-like serine proteases around the amino
acid in position 99 (PK, plasma kallikrein; FXa, factor Xa; uPA, urokinase-
type plasminogen activator; aPC, activated protein C). The sequence
positions are labeled according to the chymotrypsinogen-numbering.
Figure 3. Structure of the linear nonspecific inhibitor 1.
Figure 2. Stereoview of plasmin (1bui.pdb, shown as gray surface) overlaid with the structures of relevant trypsin-like serine proteases. For clarity, only
the hairpin loops around their amino acid in position 99 are shown for the other enzymes (FIXa, 3lc3.pdb in red; FVIIa, 1klj, green; FXa, 2pr3, dark
blue; PK, 2any, brown; thrombin, 1k22, yellow; uPA, 1vja, black; aPC, 1aut, light blue). These loops protrude from the surface because it is completely
missing in plasmin. To clarify the active site, the substrate-analogue thrombin inhibitor melagatran (1k22.pdb) is shown with yellow carbon atoms.12
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Figure 4. Stereoview of the modeled structure of inhibitor 1 in plasmin. The P2 guanidino group binds to Glu60, whereas the P3 phenyl ring is
located above Trp215. Both side chains of the P2 and P3 amino acids are directed toward the enzyme surface and located relatively close to each
other. The distance between the two carbon atoms, which are exemplarily shown as green balls, is 4.9 Å.
a
On the basis of many crystal structures of substrate-analogue
inhibitors, which all bind in the form of an antiparallel β-sheet
to the trypsin-like serine proteases,10,15,16,18 it is known that the
P2 and P3 side chains point in the same direction toward the
enzyme surface, like in melagatran (Figure 2) or in the manually
modeled structure of inhibitor 1 in plasmin (Figure 4).
Therefore, we have assumed that it might be possible to design
potent plasmin inhibitors by appropriate cyclization between
these side chains. Such cyclized inhibitors should still be able to
bind to the relatively open binding pocket of plasmin. In addi-
tion, their rigid cyclic structure should induce a steric repulsion
from the 99-loop present in all other trypsin-like serine proteases,
which should improve the selectivity of this type of plasmin
inhibitor. In this manuscript we communicate our results
regarding the synthesis and enzyme kinetic characterization of
these new cyclic plasmin inhibitors.
Scheme 1. Synthesis of Inhibitor 6
RESULTS
■
Synthesis. In analogy to the acyclic reference compound 1
the 4-amidinobenzylamide anchor in the P1 position was
retained. In contrast, a nonsubstituted benzylsulfonyl group was
used as P4 residue for two reasons. On the basis of the model
shown in Figure 4, the carboxyl group is directed into the
solvent and not involved in specific contacts to plasmin.
Second, it is known from previous selectivity studies with
structurally related substrate-analogue urokinase18 and factor
Xa19 inhibitors that a 3-COOH-benzylsulfonyl residue in P4
position leads to a 3- to 5-fold reduced plasmin affinity com-
pared to analogues without carboxyl substitution. To simplify
the synthesis, we generally incorporated amino acids with
identical side chain functionalities in P2 and P3 positions,
which allowed a nondirectional cyclization with symmetrical
bridging groups.
Three different strategies were used for the cyclization. First,
inhibitors 2−7 were prepared by connecting the P2 and P3 side
chains via an appropriate linker moiety by amide bond for-
mation, as exemplarily shown for inhibitor 6 in Scheme 1. The
D- and L-3-aminomethylphenylalanine derivatives required for
the synthesis of intermediate 13 and its para-analogues required
for the preparation of inhibitors 3 and 4 were obtained by
hydrogenation of N-terminal protected cyanophenylalanines
(see Supporting Information).
a
(a) (i) 1 equiv of N,N′-1,4-piperazinediacetic acid, 2 equiv of PyBOP,
6 equiv of DIPEA, DMF, 0 °C; (ii) 3.2 equiv of 1 N NaOH, ethanol/
water, room temp, preparative HPLC, 37% yield (two steps); (b) 1
equiv of 4-Amba·2 HCl, 1 equiv of PyBOP, 4 equiv of DIPEA, DMF,
0 °C, preparative HPLC, 63% yield.
Meanwhile, metathesis is a well established method for the
design of macrocylic drugs, including peptidomimetic inhibitors
for the cysteine protease calpain,20,21 the HCV protease,22 or
the insulin-regulated aminopeptidase.23 Therefore, the addi-
tional inhibitor 8 and its hydrogenated analogue 9 were
synthesized by metathesis between O-allyl protected tyrosine
residues. A cleavage of the O-alkyl bond during the hydro-
genation in ethyl acetate was not observed (Scheme 2).
There exist several examples for the preparation of cyclized
peptidomimetics using a copper-catalyzed azide alkyne cyclo-
addition (CuAAC,24 also known as the copper-catalyzed
Huisgen cycloaddition).25,26 Therefore, a third cyclization strat-
egy via click chemistry by reaction of the bis-propargylglycine
intermediate 21 with various bis-azide derivatives was used for
the synthesis of inhibitors 10−12, which led to the formation of
rigid triazole rings in the P2 and P3 side chains (Scheme 3).
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a
The structures of the synthesized cyclic inhibitors are
summarized in Table 1.
Scheme 2. Synthesis of Inhibitor 9
Kinetic Measurements. A relatively weak inhibitory effect
against plasmin (Ki = 431 nM) was obtained for compound 2,
in which two glutamic acid side chains were cyclized by amide
bond formation with m-diaminoxylene. For comparison, we
also determined the inhibition constants against the related
trypsin-like serine proteases PK, thrombin, factor Xa, and aPC.
All Ki values were obtained from Dixon plots and are sum-
marized in Table 2.
Because of the higher potency found for the linear reference
inhibitor 1, which contains aromatic amino acid residues in P2
and P3 positions, we assumed that it might be beneficial to
replace the flexible Glu residues by more hydrophobic and rigid
amino acids. Therefore, inhibitors 3−7 containing the aromatic
3- or 4-aminomethylphenylalanine as P2 and/or P3 residues
were designed, which could be cyclized using p- and m-
phenylene or piperazinediacetic acid. Analogue 3 was already a
significantly more potent and selective plasmin inhibitor
compared to 2, whereas its better soluble piperazine analogue
4 has a similar specificity profile. Both analogues inhibit plasmin
with Ki < 10 nM. The plasmin inhibition could be further
enhanced in the cases of compounds 5−7, although inhibitors 6
and 7 possess reduced selectivity and inhibit various other
proteases with inhibition constants of <50 nM. Especially, the
potent inhibition of the clotting proteases thrombin and factor
Xa, which are the physiological antagonists of the fibrinolytic
plasmin, could be a disadvantage.
An improved selectivity was determined for compounds 8
and 9, obtained by metathesis. Similar to aprotinin, both
analogues retained some affinity against PK with inhibition
constants of <20 nM. It is noted that PK contains only a glycine
residue in position 99 (Figure 1). Most likely, its less demand-
ing 99-loop (Figure 2) leads to a reduced repulsion from the
cyclic ring structures of these inhibitors. Compounds 8 and 9
maintain high plasmin affinity with Ki < 3 nM. This is an
additional hint that the incorporation of hydrophobic phenyl
groups in the P2 and P3 side chains contributes to improved
plasmin binding.
The third cyclization strategy via click chemistry provided the
bis-triazole inhibitors 10−12. Of particular interest might be
the highly potent inhibitor 10 (Ki for plasmin of 0.77 nM),
which is a relatively weak inhibitor of thrombin, factor Xa, and
aPC. Its additional inhibitory effect on PK could also reduce the
liberation of bradykinin from high-molecular-weight kininogen,
which might result in a beneficial anti-inflammatory effect. This
was also discussed as an advantage of aprotinin (Ki for PK of
30 nM) compared to other antifibrinolytic drugs, such as
tranexamic acid, which has no inhibitory effect against PK.27,28
Docking of Inhibitor 10 in Plasmin. Because of its high
potency, inhibitor 10 was docked using FlexX into the active
site of plasmin to get an impression of its potential binding
mode. The coordinates of the protease domain of plasmin were
taken from the PDB entry 1bui.8 The shown structure of
inhibitor 10 (Figure 5) was selected based on similar backbone
interactions known from related complexes of noncyclized
substrate analogue inhibitors, e.g., with thrombin,29,30 factor
Xa,19 factor VIIa,31 trypsin,32 or urokinase.18
a
(a) (i) 0.05 equiv of Grubbs I 15, DCM, reflux; (ii) 5 equiv of 1 N
HCl/AcOH, room temp, preparative HPLC, 28% yield; (b) (i) 3 equiv
of Bzls-Cl, MeCN/water, pH 7−8; (ii) 19 equiv of 1 N NaOH, 1,4-
dioxane/water, 40 °C, 96% yield (two steps); (c) 10% Pd/C, EtOAc,
room temp, 73% yield; (d) 1.2 equiv of 4-Amba·2HCl, 1.2 equiv of
PyBOP, 2 equiv of DIPEA, DMF, 0 °C, preparative HPLC, 70% yield.
a
Scheme 3. Synthesis of Inhibitor 10
a
(a) 1 equiv of 20, 1 equiv of H-Ppg-OMe·HCl, 1.1 equiv of PyBOP, 3
equiv of DIPEA, DMF, 0 °C; (b) (i) 1 equiv of 1,3-bis(azidomethyl)-
benzene, 0.4 equiv of CuBr, 6 equiv of DIPEA, DMF/water,
microwave (120 °C, 5 min, 150 W); (ii) 9 equiv of 1 N NaOH,
DMF, room temp, 32% yield (two steps); (c) 1 equiv of isobutyl
chloroformate, 2 equiv of NMM, 1.5 equiv of 4-Amba·2 HCl, DMF,
−20 °C, preparative HPLC, 64% yield.
The benzamidine forms the characteristic salt bridge to
Asp189 and hydrogen-bonds to the carbonyl of Gly219 and the
side chain oxygen of Ser190, whereas the amide NH of the P1
residue binds to the carbonyl oxygen of Ser214. However, in
contrast to the noncyclized inhibitors only one hydrogen bond
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Table 1. Structures of the Synthesized Cyclic
Inhibitors
Table 2. Inhibition of Plasmin and Related Trypsin-like
Serine Proteases
a
Ki (nM)
compd
plasmin
PK
thrombin
FXa
aPC
0.61
1
0.81
431
9.4
9.0
6.9
4.1
1.1
2.5
1.1
0.77
2.2
8.9
0.075
25.4
868
493
136
25.3
17.4
9.4
667
42
2
2020
6520
3517
45.4
7.2
3430
9370
3472
152
21
5.1
3
12000
3510
564
4
5
6
48
7
26.8
137
42
223
8
220
784
206
1860
3123
90
9
19
510
4586
3000
8390
>6000
10
11
12
2.4
9490
6175
1180
10.1
67
is formed between the P3 backbone and Gly216. It seems that
the rigid cyclic structure prohibits the formation of the second
H-bond to the carbonyl oxygen of Gly216. Many docking
solutions were found with very similar conformations of the
P3−P2 ring structure, whereas the binding mode of the P4
benzylsulfonyl group seems to be more flexible. The sulfonyl
group of inhibitor 10 in the selected conformation is H-bonded
to the side chain of Gln192, which is additionally involved in an
interaction with the P2 carbonyl group. As expected, the
triazole rings and the phenyl linker between the P2 and P3 side
chains are directed toward the large open binding pocket of
plasmin. One triazole is located above the indole of Trp215,
and the second is placed nearly parallel to the imidazole of
His57, whereas the phenyl group is perpendicularly oriented
between both triazole rings.
The shown complex was also overlaid with the structures of
the other tested proteases. This resulted in a steric clash with
thrombin, factor Xa, and aPC, whereas the selected inhibitor
conformation seems to be well accepted in PK (see Figures
S1−S4 in Supporting Information). These results confirm the
found selectivity profile of inhibitor 10.
DISCUSSION
■
By use of different cyclization strategies, it was possible to
develop highly potent plasmin inhibitors with inhibition con-
stants of <3 nM. The described cyclization between
neighboring residues (i to i + 1) was possible because of the
different configurations of the P2 and P3 amino acids in this
substrate-analogue inhibitor type. This is quite different from
most other cyclized peptidomimetics consisting only of L-amino
acids, where the cyclization is often performed between the side
chains of an amino acid i and a residue in the third position
(i + 2)20−22,33 or between residues that are further away in the
peptide sequence. However, there also exist examples of a
cyclization between neighboring side chains, e.g., of several 14-
membered cycloisodityrosine derivatives, which were intro-
duced in peptide antibiotics with antitumor activity, prepared
by an Ullmann closure.34−36
The first cyclic inhibitor 2, obtained by connecting two
flexible glutamate side chains, had relatively weak potency,
missing any selectivity. In contrast, the replacement of the
glutamates by substituted phenylalanines immediately provided
compounds with enhanced potency and selectivity. Especially,
inhibitors 3 and 4 containing the largest 26-membered ring
structures possess a weak affinity against the other tested
a
The asterisk (∗) indicates that this inhibitor was obtained as a 1:2.1
(E/Z) mixture.
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Figure 5. Stereoview of the modeled structure of inhibitor 10 (with green carbon atoms) in plasmin. (a) Part a shows the plasmin in surface
representation for visualization of the overall binding mode. (b) Part b indicates the found binding interactions as yellow dashed lines. The residues
of the catalytic triad are provided with orange carbon atoms.
proteases. Reduction of the ring size to 24 atoms (5, 6) or 23
atoms (7) enhanced the affinity toward all enzymes but
reduced the selectivity. Further downsizing the cycle in the
cases of inhibitors 8 and 9 to 20 atoms, prepared by ring
closure metathesis, maintained the high potency as plasmin
inhibitors with Ki < 3 nM. A noncyclized analogue benzylsul-
fonyl-D-Tyr(All)-Tyr(All)-4-amidinobenzylamide has a signifi-
cantly reduced inhibitory potency against plasmin with an
inhibition constant of 65 nM. Therefore, we assume that a
significant contribution for the enhanced binding affinity is
caused by the rigidization of the inhibitor conformation in
solution, resulting in a beneficial entropic effect during binding
to plasmin. The smallest 17-membered ring containing
compounds were prepared by click chemistry, whereas
analogues 10 and 11 were found to be excellent plasmin
inhibitors with Ki of 0.77 and 2.2 nM, respectively. Both
compounds are also relatively potent PK inhibitors with
inhibition constants of ≤10 nM, whereas they have weak
affinity for thrombin, factor Xa, or aPC. However, these kinetic
data indicate that there is obviously no simple correlation
between the ring size of the inhibitors and their inhibitory
potency or selectivity.
The high plasmin affinity obtained for several analogues
demonstrates that the cyclization also can compensate for the
loss of the important guanidinomethyl group present in the P2
side chain of the linear analogue 1, which is most likely involved
in a salt bridge to the plasmin residue Glu60 (Figure 4). The
docking experiments exemplarily performed with compound 10
revealed that the inhibitor adopts a very compact and rigid
structure, which still enables an efficient binding to the open
active site of plasmin, whereas it decreases the affinity for
thrombin, factor Xa, and aPC.
CONCLUSION
■
These cyclized inhibitors might be of potential interest for the
development of novel antifibrinolytics. Some compounds were
found to be very selective plasmin inhibitors, while others
additionally inhibit PK with Ki ≤ 10 nM. An inhibitory effect on
PK could be an advantage for reducing systemic inflammatory
processes, which often occur in cardiac surgery with cardio-
pulmonary bypass that requires antifibrinolytic therapy. An
additional application could be the use of these compounds as
antidote to reduce excessive bleeding during fibrinolytic therapy
with plasmin37 or plasminogen activators. Without any doubt
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Intermediate 18. 17 (136 mg, 0.260 mmol, 1.0 equiv) was
dissolved in 10 mL of MeCN and 3 mL of water. Then Bzls-Cl
(148.3 mg, 0.778 mmol, 3.0 equiv) was added in several portions, and
the pH was adjusted with 1 N NaOH to 7−8. The solvent was
removed in vacuo after 4.5 h. The remaining residue was dissolved in
10 mL of 1,4-dioxane and 5 mL of water and treated with 5 mL of 1 N
NaOH. The mixture was stirred at 40 °C on the water bath for 1 h.
The solution was neutralized by addition of TFA, and the solvent was
removed in vacuo. The remaining residue was dissolved in a mixture of
5% KHSO4 and ethyl acetate, and the water layers were extracted
thrice with ethyl acetate. The combined organic phases were washed
twice with 5% KHSO4 and thrice with brine, dried over Na2SO4,
and filtered, and the solvent was removed in vacuo. Yield: 138 mg
(0.251 mmol, 96.4%) of a white amorphous solid. HPLC: 45.8 min,
start at 10% B (76.2%). MS (ESI, positive): calcd, 550.18; m/z, 551.5
[M + H]+, 573.5 [M + Na]+, 589.4 [M + K]+.
Intermediate 19. 18 (55 mg, 0.0999 mmol) was dissolved in
110 mL of ethyl acetate. After the addition of 5.8 mg of 10% Pd/C, the
mixture was hydrogenated at room temperature for 3 h. The catalyst
was removed by filtration, and the solvent was evaporated. Yield:
40 mg (0.0724 mmol, 72.5%) of a light gray amorphous solid. HPLC:
19.7 min, start at 40% B (purity, 95.9%). MS (ESI, positive): calcd,
552.19; m/z, 553.10 [M + H]+, 570.09 [M + NH4]+.
there may exist many alternative cyclization possibilities
between the P2 and P3 residues, as long as their side chains
contain suitable groups for coupling, either directly or with
appropriate linker segments. The strong potency and relatively
high selectivity found for these first analogues confirm that this
cyclization strategy provides a new access to excellent substrate-
analogue plasmin inhibitors.
EXPERIMENTAL SECTION
■
Analytical HPLC experiments were performed on a Shimadzu LC-10A
system (column, Nucleodur C18, 5 μm, 100 Å, 4.6 mm × 250 mm,
Machery-Nagel, Duren, Germany) with a linear gradient of acetonitrile
̈
(1%/min increase in concentration of acetonitrile, detection at
220 nm) containing 0.1% TFA at a flow rate of 1 mL/min. The
final inhibitors were purified to more than 95% purity (detection at
220 nm) by preparative HPLC using a Varian PrepStar model 218
gradient system (column, Nucleodur, 5 μm, 100 Å, 32 mm × 250 mm,
Macherey-Nagel, Duren, Germany) and a linear gradient of
̈
acetonitrile containing 0.1% TFA at a flow rate of 20 mL/min. All
inhibitors were finally obtained as TFA salts after lyophilization. The
molecular masses were determined using a QTrap 2000 ESI
1
spectrometer (Applied Biosystems). The H and 13C NMR spectra
Inhibitor 9. 19 (30 mg, 0.0543 mmol, 1.0 equiv) and 4-amidino-
benzylamine·2 HCl (14.5 mg, 0.0663 mmol, 1.2 equiv) were
suspended in 10 mL of DMF and stirred on an ice bath. The mixture
was treated with PyBOP (33.8 mg, 0.0654 mmol, 1.2 equiv) and
DIPEA (18.8 μL, 0.109 mmol, 2.0 equiv) and was stirred at room
temperature overnight. The solvent was removed in vacuo, and the
remaining residue was dissolved in 50% solvent B and filtered through
a 0.2 μm membrane filter. The filtrate was purified by preparative
HPLC (C18 column, start at 35% B). The product containing fractions
were combined and lyophilized. Yield: 30.1 mg (0.0377 mmol, 69.5%)
of a white lyophilized solid. HPLC: 13.9 min, start at 40% B (purity,
were recorded on a ECX-400 (Jeol Inc., U.S.) and are referenced to
internal solvent signals.
Boc-D-Tyr(All)-Tyr(All)-OMe (16). Boc-D-Tyr(All)-OH (2 g,
6.22 mmol, 1.0 equiv) and H-Tyr(All)-OMe (1.7 g, 6.22 mmol,
1.0 equiv) were dissolved in 50 mL of DMF and stirred on the ice
bath. After the addition of HBTU (2.36 g, 6.22 mmol, 1.0 equiv) and
DIPEA (3.25 mL, 18.7 mmol, 3.0 equiv), the solution was stirred for
2 h. The solvent was removed in vacuo, and the remaining residue was
dissolved in a mixture of a 5% KHSO4 and ethyl acetate. The organic
phase was washed thrice with 5% KHSO4, once with brine, thrice with
saturated aqueous NaHCO3, and thrice with brine, dried over Na2SO4,
and filtered and the solvent removed in vacuo. Yield: 3.32 g (6.16
mmol, 99.1%) as a yellow amorphous solid. HPLC: 52.1 min, start at
10% B (purity, 96.9%). 1H NMR (DMSO-d6, 400 MHz): δ = 8.36 (d,
J = 8.1 Hz, 1 H), 7.09 (d, J = 8.5 Hz, 2 H), 7.00 (d, J = 8.5 Hz, 2 H),
6.80 (d, J = 8.7 Hz, 2 H), 6.77 (d, J = 8.6 Hz, 2 H), 6.67 (d, J = 8.8 Hz,
1 H), 5.87−6.05 (m, 2 H), 5.12−5.38 (m, 4 H), 4.43−4.49 (m, 4 H),
3.60 (s, 3 H), 2.95 (dd, J = 13.7, 5.1 Hz, 2 H), 2.77 (dd, J = 13.8, 9.5
Hz, 2 H), 2.65 (s, 1 H), 2.59 (dd, J = 13.8, 3.9 Hz, 1 H), 1.25 ppm (s,
9 H). 13C NMR (DMSO-d6, 101 MHz): δ = 172.0, 171.7, 156.9, 156.7,
155.1, 133.9, 133.7, 130.1, 129.0, 117.2, 114.4, 114.1, 77.9, 68.1, 55.5,
53.5, 51.9, 36.7, 36.2, 28.1 ppm. MS (ESI, positive): calcd, 538.27;
m/z, 539.5 [M + H]+, 561.5 [M + Na]+, 1099 [2M + Na]+.
Intermediate 17. Boc-D-Tyr(All)-Tyr(All)-OMe (500 mg, 0.928
mmol, 1.0 equiv) was dissolved in 250 mL of dry DCM under an
atmosphere of argon, which was degassed before 30 min using
ultrasound. The mixture was flushed with argon on a water bath at
40 °C for an additional 30 min. After the addition of Grubbs I catalyst
(38 mg, 0.0464 mmol, 0.05 equiv), dissolved in 10 mL of degassed
DCM, the mixture was refluxed under an atmosphere of argon for 6 h
and stirred at room temperature overnight. The solvent was removed
in vacuo, and the dark red residue was dissolved in 40 mL of acetone,
adsorbed onto a small amount of silica gel 60, and evaporated. The
product was purified on silica gel 60 (column 3 cm × 40 cm) using
isohexane/TMBE (1/1, v/v) as eluent. The product containing
fractions were combined, and the solvent was evaporated. Yield:
310 mg (0.6068 mmol, 65.4%) as a white solid. HPLC: 43.3 min, start
at 10% B (purity, 59.2%). The white solid (294 mg, 0.575 mmol,
1.0 equiv) was dissolved in 575 μL of acetic acid and 2.9 mL of 1 N
HCl in AcOH. After 1 h, the solvent was removed in vacuo and the
remaining light yellow residue was dissolved in 40% solvent B and
purified by preparative HPLC (C18 column, start at 25% B). The
product containing fractions were combined and lyophilized. Yield:
136.4 mg (0.260 mmol, 28.0%) of a white lyophilized solid. HPLC:
30.7 min, start at 10% B (95.1%). MS (ESI, positive): calcd, 410.18;
m/z, 411.06 [M + H]+, 821.30 [2M + H]+.
1
100.0%). H NMR (DMSO-d6, 400 MHz): δ = 9.21 (br s, 2 H), 8.97
(br s, 2 H), 8.64 (t, J = 6.0 Hz, 1 H), 8.47 (d, J = 6.9 Hz, 1 H), 7.70
(d, J = 8.3 Hz, 2 H), 7.58 (d, J = 8.2 Hz, 1 H), 7.43 (d, J = 8.2 Hz,
2 H), 7.25−7.39 (m, 5 H), 6.88 (d, J = 8.5 Hz, 2 H), 6.77 (d, J =
8.5 Hz, 2 H), 6.59 (d, J = 8.5 Hz, 2 H), 6.49 (d, J = 8.5 Hz, 2 H),
4.38−4.49 (m, 2 H), 4.30−4.38 (m, 2 H), 4.21−4.29 (m, 2 H), 4.07−
4.14 (m, 2 H), 3.96−4.06 (m, 2 H), 2.76−2.87 (m, 2 H), 2.63−2.73
(m, 2 H), 1.72−1.92 ppm (m, 4 H). MS (ESI, positive): calcd, 683.82;
m/z, 684.43 [M + H]+. TLC: Rf = 0.79.
Bzls-D-Ppg-OH (20). H-D-Ppg-OH·HCl (500 mg, 3.34 mmol,
1.0 equiv) was suspended in 10 mL of dry DCM and treated with
TMS-Cl (912 μL, 7.35 mmol, 2.2 equiv) and DIPEA (1.861 mL,
10.7 mmol, 3.2 equiv). The mixture was refluxed for 1 h. Then
benzylsulfonyl chloride (705 mg, 3.70 mmol, 1.1 equiv) was added in
several portions within 35 min at 0 °C and the pH was maintained at
8−9 by addition of DIPEA (700 μL, 4.02 mmol, 1.2 equiv). The
mixture was stirred 1 h at 0 °C and at room temperature overnight.
The solvent was removed in vacuo, and the brown residue was
dissolved in water and the pH adjusted to 8−9 with 1 N NaOH. The
solution was extracted twice with EtOAc. The organic phases were
discarded. The aqueous layer was adjusted to pH 1 with 5% KHSO4
and extracted thrice with EtOAc. The combined organic phase was
washed twice with 5% KHSO4 and twice with brine. The organic layers
were dried over Na2SO4 and filtered, and the solvent was removed in
vacuo. Yield: 481 mg (1.80 mmol, 53.8%) of a brown oil. HPLC: 26.7
min, start at 10% B (purity, 81.0%). 1H NMR (DMSO-d6, 400 MHz):
δ = 13.06 (br s, 1 H), 7.61 (d, J = 8.3 Hz, 1 H), 7.27−7.43 (m, 5 H),
4.28−4.40 (m, 2 H), 3.93 (dt, J = 8.1, 6.5 Hz, 1 H), 2.92 (t, J = 2.6 Hz,
1 H), 2.48−2.52 ppm (m, 2 H). 13C NMR (DMSO-d6, 101 MHz): δ =
171.7, 131.0, 130.1, 128.3, 128.1, 80.1, 73.6, 58.7, 54.8, 22.8 ppm. MS
(ESI, positive): calcd, 267.06; m/z, 285 [M + NH4]+, 290 [M + Na]+.
Bzls-D-Ppg-Ppg-OMe (21). 20 (473 mg, 1.77 mmol, 1.0 equiv)
and H-Ppg-OMe·HCl (292 mg, 1.79 mmol, 1.0 equiv) were dissolved
in 23.5 mL of DMF and treated with PyBOP (1.02 g, 1.96 mmol, 1.1
equiv) and DIPEA (928 μL, 5.33 mmol, 3.0 equiv) at 0 °C, pH 8−9.
The mixture was stirred for 30 min at 0 °C and at room temperature
1177
dx.doi.org/10.1021/jm2011996 | J. Med. Chem. 2012, 55, 1171−1180

Journal of Medicinal Chemistry
Article
overnight. The solvent was removed in vacuo, and the remaining
residue was dissolved in EtOAc. The organic phase was washed thrice
with solutions of 5% KHSO4, once with brine, thrice with saturated
aqueous NaHCO3, and thrice with brine. The organic phase was dried
over Na2SO4 and filtered. The solvent was removed in vacuo, and the
product was crystallized from EtOAc. Yield: 236.7 mg (0.629 mmol,
35.5%) of a beige solid. HPLC: 35.6 min, start at 10% B (purity,
(DMSO-d6, 500 MHz): δ = 9.20 (br s, 2 H), 9.04 (br s, 2 H), 8.55
(t, J = 5.9 Hz, 1 H), 8.36 (d, J = 6.9 Hz, 1 H), 7.70 (d, J = 8.3 Hz,
2 H), 7.64 (d, J = 8.9 Hz, 2 H), 7.60 (s, 1 H), 7.37−7.46 (m, 5 H),
7.33−7.37 (m, 2 H), 7.28−7.31 (m, 3 H), 6.33 (s, 1 H), 5.51−5.57
(m, 2 H), 5.45−5.51 (m, 2 H), 4.49 (d, J = 13.7 Hz, 1 H), 4.38−4.43
(m, 1 H), 4.34 (d, J = 12.9 Hz, 3 H), 4.24−4.29 (m, 1 H), 3.16 (dd, J =
14.7, 10.5 Hz, 2 H), 2.95−3.03 ppm (m, 2 H). 13C NMR (DMSO-d6,
126 MHz): δ = 171.2, 170.3, 165.4, 145.7, 143.5, 142.1, 137.1, 137.1,
130.9, 130.1, 128.8, 128.2, 128.1, 128.0, 127.4, 127.3, 127.3, 126.4,
124.4, 123.0, 122.6, 58.2, 55.1, 54.0, 52.2, 52.2, 41.8, 29.3, 27.9 ppm.
MS (ESI, positive): calcd, 681.26; m/z, 341.58 [2M + H]2+/2, 682.08
[M + H]+. TLC: Rf = 0.43.
Molecular Modeling. Preparation of Plasmin. For the docking
process, we only used the coordinates of the protease domain of
plasmin taken from the microplasmin−staphylokinase−microplasmin
ternary complex irreversibly inhibited by H-Glu-Gly-Arg-chloromethyl
ketone (PDB entry 1bui). All water molecules and the covalent bonds
of the inhibitor to the plasmin residues His57 and Ser195 were
deleted, followed by addition of hydrogens and calculation of partial
charges (Gasteiger and Marsili) using SYBYL.39
Inhibitor Preparation and Docking. Ligand 10 was prepared using
the MOE builder function. For the docking process various con-
formers of the inhibitor40 were used, which were generated using
the conformational search function of MOE41 (conformational search
using LowModeMD42 and MM Iteration Limit 1000; all other
standard parameters were set on default). These conformers were
docked using FlexX.43 The active site of plasmin was defined by taking
all residues within 10 Å around H-Glu-Gly-Arg-chloromethyl ketone,
taken from the crystal structure. The 100 top-scoring poses were
retained and thoroughly inspected.
1
96.2%). H NMR (DMSO-d6, 400 MHz): δ = 8.83 (d, J = 7.7 Hz,
1 H), 7.67 (d, J = 8.8 Hz, 1 H), 7.32−7.41 (m, 5 H), 4.50 (q, J = 7.5
Hz, 1 H), 4.29 (d, J = 8.6 Hz, 2 H), 4.14 (q, J = 7.5 Hz, 1 H), 3.63 (s, 3
H), 2.98−3.06 (m, 1 H), 2.88−2.95 (m, 2 H), 2.64 (dd, J = 6.7, 2.3
Hz, 2 H), 1.73 ppm (s, 1 H). 13C NMR (DMSO-d6, 101 MHz): δ =
170.4, 169.8, 130.8, 130.0, 128.2, 127.9, 80.0, 79.6, 73.4, 58.6, 54.9,
52.2, 51.1, 23.3, 21.0 ppm. MS (ESI, positive): calcd, 376,11; m/z, 399
[M + Na]+, 775 [2M + Na]+.
1,3-Bis(azidomethyl)benzene (22). The synthesis was previously
ez et al.38 α,α-Dibromo-m-xylene (1.32 g,
́
described by Ramirez-Lop
́
5.0 mmol, 1.0 equiv) was dissolved in 30 mL of DMSO and treated
with sodium azide (810 mg, 12.5 mmol, 2.5 equiv). The mixture was
stirred for 2 h at room temperature. The yellow solution was quenched
with ice−water and extracted thrice with EtOAc. The combined
organic layers were washed twice with water and once with brine, dried
over Na2SO4, filtered, and the solvent was removed in vacuo. Yield:
880 mg (4.68 mmol, 93.5%) of a yellow oil. HPLC: 20.8 min, start at
40% B (purity, 96.0%). MS (EI): calcd, 188.08; m/z (%), 188 [M]+
(100), 146 [M − N3]+ (61), 104 [M − N6]+ (25), 91 [M − CH2 −
N6]+ (25), 77 [M − C2H4 − N6]+ (21), 118 [M − N5]+ (19), 189
[M + H]+ (14), 117 [C8H7N]+ (13), 78 [C6H6]+ (12), 131 [C8H7N2]+
(11), 147 [C8H9N3]+ (8), 105 [C8H9]+ (6).
Intermediate 23. 21 (150 mg, 0.399 mmol, 1.0 equiv), 1,3-
bis(azidomethyl)benzene 22 (63 mg, 0.399 mmol, 1.0 equiv), and CuBr
(23 mg, 0.159 mmol, 0.4 equiv) were dissolved in 50 mL of DMF and
1 mL of water by addition of DIPEA (416 μL, 2.39 mmol, 6.0 equiv).
The reaction was performed at 120 °C in a microwave (Discover,
CEM) for 5 min at 150 W (temperature priority). The solvent was
removed in vacuo, and the methyl ester was obtained as a crude
product. A small sample was purified by preparative HPLC for NMR
analysis. Oil. HPLC: 13.3 min, start at 30% B. MS (ESI, positive):
calcd, 564.19; m/z, 565.12 [M + H]+. 1H NMR (DMSO-d6, 500
MHz): δ = 8.56 (d, J = 6.9 Hz, 1 H), 7.79 (s, 1 H), 7.74 (d, J = 8.9 Hz,
1 H), 7.61 (s, 1 H), 7.40−7.47 (m, 5 H), 7.33−7.40 (m, 3 H), 6.39
(s, 1 H), 5.57 (d, J = 5.0 Hz, 2 H), 5.49 (s, 2 H), 4.55 (d, J = 13.7 Hz,
1 H), 4.47 (t, J = 7.5 Hz, 1 H), 4.31 (d, J = 13.7 Hz, 1 H), 4.23 (t, J =
8.2 Hz, 1 H), 3.64 (s, 3 H), 3.18 (dd, J = 14.3, 10.9 Hz, 1 H), 3.08 (dd,
J = 15.4, 2.0 Hz, 1 H), 2.98 (dd, J = 15.1, 11.0 Hz, 1 H), 2.82 ppm (dd,
J = 14.3, 2.6 Hz, 1 H). This procedure was repeated twice. The
combined products of these three reactions were dissolved in 30 mL of
DMF and treated with 3.6 mL of 1 N NaOH. The mixture was stirred
for 1 h at room temperature. The solvent was removed in vacuo. The
residue was suspended in a mixture of EtOAc and 5% KHSO4. The
water phase was extracted twice with EtOAc. The combined organic
phases were washed once with 5% KHSO4, thrice with brine, dried
over Na2SO4, and filtered, and the solvent was removed in vacuo.
Yield: 208 mg (0.378 mmol, 31.6%) of a slightly yellow, amorphous
solid. HPLC: 18.5 min, start at 20% B (purity, 85.3%). MS (ESI,
positive): calcd, 550.17; m/z, 551.22 [M + H]+, 573.12 [M + Na]+.
Inhibitor 10. 23 (98 mg, 0.178 mmol, 1.0 equiv) and NMM
(19.6 μL, 0.178 mmol, 1.0 equiv) were dissolved in 5 mL of DMF
at −20 °C and treated with isobutyl chloroformate (23.1 μL,
0.178 mmol, 1.0 equiv). The mixture was stirred for 10 min
at −15 °C and treated with 4-amidinobenzylamine·2HCl (59.3 mg,
0.267 mmol, 1.5 equiv) and NMM (19.6 μL, 0.178 mmol, 1.0 equiv).
The suspension was stirred at −20 °C for an additional hour and at
room temperature overnight. The solvent was removed in vacuo. The
remaining slightly yellow residue was dissolved in 35% solvent B and
purified by preparative HPLC (C18 column, start of the gradient at
20% B). The product containing fractions were combined, and the
solvent was partially removed in vacuo, followed by lyophilization of
the product. Yield: 78 mg (0.114 mmol, 64.3%) white lyophilized
solid. HPLC: 24.7 min, start at 10% B (purity, 96.1%). 1H NMR
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthesis procedures for all other inhibitors and analytical data,
information for enzyme kinetics, and also figures of the
modeled plasmin/inhibitor 10 complex overlaid with the X-ray
structures of the other tested proteases. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +49 6421 2825900. E-mail: steinmetzer@staff.
uni-marburg.de.
ACKNOWLEDGMENTS
■
We thank Peter Steinmetzer and Sven Letschert from The
Medicines Company (Leipzig) GmbH for Ki determinations.
We also thank Gerhard Klebe for providing us access to the
used modeling programs. Financial aid by The Medicines
Company (Leipzig) GmbH is acknowledged.
ABBREVIATIONS USED
■
AcOH, acetic acid; 4-Amba, 4-amidinobenzylamine; Bzls-Cl,
benzylsulfonyl chloride; DIPEA, N,N-diisopropylethylamine;
EtOAc, ethyl acetate; NMM, 4-methylmorpholine; Ppg,
propargylglycine; TMS-Cl, trimethylsilyl chloride
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