Pyrolo[1,2:4,5]-1,4-dioxopyrazino[1,2:1,6]pyrido[3,4-b]indoles: A Group of Urokinase Inhibitors, their Synthesis, and Stereochemistry-Dependent Activity

Article abstract of DOI:10.1002/cmdc.201100345

Antifibrinolytic agents are required during complex surgeries to decrease bleeding; their pro-thrombotic potency and efficacy in causing hemostasis has attracted much attention. To discover new inhibitors of urokinase with high selectivity for antifibrinolytic effects over pro-thrombotic effects, the 12-position of (5aS,12S,14aS)- and (5aS,12R,14aS)-5,14-dioxo-1,2,3,5,5a,6,11, 12,14,14a-decahydro-5H,14H-pyrolo[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]indoles were modified with L-Ala, L-Asp, L-Phe, L-Trp, L-Lys, L-Ser, Gly, and L-Leu to provide 16 (5aS,12S,14aS) and (5aS,12R,14aS) derivatives. In a murine bleeding model, the (5aS,12S,14aS) derivatives containing L-Ala, L-Asp, L-Phe, and L-Trp induced blood coagulation for the treated mice; they also stimulated thrombus formation in a rat thrombosis model, but the other derivatives inhibited thrombosis. The most potent compound, the L-Asp derivative, showed a good therapeutic window: the minimum effective dose for coagulation was <1nmolkg^-1, whereas at 10nmolkg^-1, no pro-thrombotic effect was observed. This type of coagulation action was correlated with a mechanism of urokinase inhibition, and these results could lead to the discovery of novel urokinase inhibitors. Stop the clot: Small molecular antifibrinolytic agents in clinical use such as tranexamic acid carry the risk of prompting thrombus formation. Herein we present an investigation of pyrolo[1,2:4,5]-1,4-dioxopyrazino[1,2:1,6]pyrido[3,4-b]indoles (DAPPP), some of which show strong hemostatic activities invivo. The most potent compound, 5c, has a good therapeutic window for antifibrinolytic therapy.

Full text of DOI:10.1002/cmdc.201100345

MED
DOI: 10.1002/cmdc.201100345
Pyrolo[1,2:4,5]-1,4-dioxopyrazino[1,2:1,6]pyrido[3,4-
b]indoles: A Group of Urokinase Inhibitors, their Synthesis,
and Stereochemistry-Dependent Activity
Jiawang Liu, Yuji Wang, Yifan Yang, Xueyun Jiang, Ming Zhao,* Wenjing Wang,
Guofeng Wu, Jianhui Wu, Meiqing Zheng, and Shiqi Peng*[a]
Antifibrinolytic agents are required during complex surgeries
to decrease bleeding; their pro-thrombotic potency and effica-
cy in causing hemostasis has attracted much attention. To dis-
cover new inhibitors of urokinase with high selectivity for anti-
fibrinolytic effects over pro-thrombotic effects, the 12-position
of (5aS,12S,14aS)- and (5aS,12R,14aS)-5,14-dioxo-1,2,3,5,5a,6,11,
12,14,14a-decahydro-5H,14H-pyrolo[1,2:4,5]pyrazino[1,2:1,6]py-
rido[3,4-b]indoles were modified with l-Ala, l-Asp, l-Phe, l-Trp,
l-Lys, l-Ser, Gly, and l-Leu to provide 16 (5aS,12S,14aS) and
(5aS,12R,14aS) derivatives. In a murine bleeding model, the
(5aS,12S,14aS) derivatives containing l-Ala, l-Asp, l-Phe, and l-
Trp induced blood coagulation for the treated mice; they also
stimulated thrombus formation in a rat thrombosis model, but
the other derivatives inhibited thrombosis. The most potent
compound, the l-Asp derivative, showed a good therapeutic
window: the minimum effective dose for coagulation was
<1 nmolkgÀ1, whereas at 10 nmolkgÀ1, no pro-thrombotic
effect was observed. This type of coagulation action was corre-
lated with a mechanism of urokinase inhibition, and these re-
sults could lead to the discovery of novel urokinase inhibitors.
Introduction
Antifibrinolytic agents have been widely and successfully used
to decrease blood loss caused by injuries as well as complex
surgeries.[1–3] Antifibrinolytic agents in current clinical use in-
clude polypeptides such as aprotinin (Trasylol, Bayer)[4,5] and
amino acids such as tranexamic acid and e-aminocaproic acid
(EACA, Figure 1).[6–8] These agents have different mechanisms
acid, the competitive inhibitor of plasminogen activation,
dose-dependently increases thrombus formation under the
same conditions.[11] Although small molecules like tranexamic
acid and EACA have elicited effects similar to those of aproti-
nin in some clinical studies, the potential increased risk of
thrombus formation should be highly concerning.[12–14] None-
theless, the significant advantages of small molecules, such as
low cost and oral bioavailability, have attracted the same
amount of interest in their use as is the case for larger poly-
peptide agents.[15–17]
We recently reported a novel antifibrinolytic small molecule
CIPPC (5d, the 12S isomer discussed below), which can be
considered an oligopeptide analogue.[18] It was discovered
from a combined study of molecular modeling and compound
library screening. CIPPC was shown to decrease rat tail bleed-
ing times in vivo (minimal effective dose: 1 nmolkgÀ1) and to
inhibit the action of urokinase plasminogen activator (u-PA;
also known as urokinase, or UK) in vitro. However, it also in-
creased thrombus formation in vivo (minimal effective dose:
10 nmolkgÀ1) in a dose-dependent manner. Cardiovascular
events such as deep-vein thrombosis, myocardial infarction,
pulmonary embolism, and stroke are serious health hazards,
while intravascular thrombosis is known to be among the
most prominent causes of morbidity and mortality.[19] There-
Figure 1. Structures of the polypeptide aprotinin (from PDB ID: 3LDJ), the
oligopeptide analogue series (5aS,12S,14aS)-5,14-dioxo-12-(2-[amino acid]-N-
ylethyl-1-yl)-1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo-
[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]indoles (DAPPP), and amino acids
tranexamic acid and e-aminocaproic acid (EACA).
of action and cannot be substituted with each other.[6,9] Due to
the close relationship between the coagulation and fibrinolysis
pathways, it is reasonable that antifibrinolytic agents were
generally thought to prompt thrombus formation.[9,10] Aproti-
nin, an inhibitor of both kallikrein and plasmin, has been
proven to inhibit thrombus formation in vivo, while tranexamic
[a] Dr. J. Liu, Dr. Y. Wang, Y. Yang, Dr. X. Jiang, Prof. Dr. M. Zhao, W. Wang,
Dr. G. Wu, Dr. J. Wu, M. Zheng, Prof. Dr. S. Peng
College of Pharmaceutical Sciences
Capital Medical University, Beijing 100069 (P.R. China)
E-mail: mingzhao@mail.bjmu.edu.cn
sqpeng@mail.bjmu.edu.cn
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Urokinase Inhibitors
fore, the pro-coagulant agents that lack pro-thrombotic activity
are needed.
acid methyl ester. The configuration of the C1 atom of 1 and
1’ was assigned by nuclear Overhauser effect (NOE) spectra. In
these tests compound 1 exhibited a positive NOE signal be-
tween 1-OCH3 and 3-OCH3, whereas 1’ exhibited positive NOE
signals between 1-H and 3-OCH3 as well as 3-H and 1-OCH3.
Therefore, the C1 atoms of 1 and 1’ were assigned the S and R
configurations, respectively. In the presence of thionyl chloride,
1 was treated successively with Fmoc-l-Pro and diisopropyla-
mine to effect an in situ cyclization and to provide cyclodipep-
tide analogue 2 (20% yield). Similarly, 1’ was converted into 2’
(40% yield). The configuration at C1 of 2 and 2’ was confirmed
by NOE experiments; compound 2 exhibited no positive NOE
signal between the methoxy group and 5a-H, whereas 2’ ex-
hibited a positive NOE signal between the 5a-H and the me-
thoxy group. Therefore, the C1 configurations for 2 and 2’ are
respectively S and R.
To increase the antifibrinolytic activity and to minimize the
adverse effects of CIPPC, we designed a series of CIPPC ana-
logues (DAPPP group, Figure 1) by changing the configuration
at C12 and by incorporating various functional groups on an
amino acid side chain. In the current compound series, nonpo-
lar (methyl and 2-methylpropyl) and polar (hydroxymethyl)
groups, acidic (carboxymethyl) and basic (4-aminobutyl-1-yl)
groups, and an aromatic (benzyl) group were used as substi-
tutes of the original indole-3-yl-methyl group on CIPPC. By
changing the configuration at C12, two sets of diastereomers,
C12S analogues (compounds 5a–h) and C12R analogues (com-
pounds 5’a–h) were generated. The eight pairs of diastereo-
mers (including CIPPC and its C12 diastereomer) were tested
on a time-resolved tail-bleeding model in mice and an arterio-
venous shunt thrombosis model in rats to identify their antifi-
brinolytic efficacy and thrombotic effects. Further observations
with a thrombus clot lysis assay and a thrombolytic assay
showed that compound 5c inhibits u-PA-mediated thromboly-
sis in vitro and in vivo.
para-Toluenesulfonic acid mediated hydrolysis of 2 provided
aldehyde 3 in 90% yield. Reductive alkylation of amino acid
methyl ester and 3 provided 4a–h (32–96% yield). The hydrol-
ysis of 4a–h provided (5aS,12S,14aS)-DAPPP (5a–h, 87–96%
yield). Similarly, para-toluenesulfonic acid promoted hydrolysis
of 2’ provided the (5aS,12R,14aS) isomer of 3 (3’, 92% yield);
reductive alkylation of amino acid methyl ester and 3’ provided
4’a–h (32–95% yield), and the hydrolysis of 4’a–h provided
(5aS,12R,14aS)-DAPPP (5’a–h, 86–96% yield).
Results and Discussion
Chemistry
A six-step reaction sequence illustrated in Scheme 1 was used
to synthesize the (5aS,12S,14aS) isomers 5a–h and the
(5aS,12R,14aS) isomers 5’a–h of DAPPP. Pictet–Spengler con-
densation of l-Trp-OCH3 and 1,1,3,3-tetramethoxypropane pro-
vided the (1S,3S) isomer (1, 60% yield) and (1R,3S) isomer (1’,
30% yield) of 1-(2,2-dimethoxyethyl)-b-carboline-3-carboxylic
Biological evaluation of DAPPP derivatives
In vivo evaluation of coagulation in a tail bleeding assay
The in vivo coagulation activities of 5a–h and 5’a–h were de-
termined by monitoring the tail bleeding times of compound-
treated mice. In this assay, ICR
mice were fed normal saline (NS,
negative
control),
aspirin
(165 mmolkgÀ1, positive control),
or each of 5a–h and 5’a–h
(0.1 mmolkgÀ1). Thirty minutes
after administration, mice were
placed in a tube holder with tails
protruding, and a 2-mm cut was
made on the tail. Flowing blood
was gently wiped away with a
tissue every 30 s until bleeding
ceased, and the observed bleed-
ing time was recorded (Table 1).
The tail bleeding times of mice
receiving 0.1 mmolkgÀ1 of each
of 5a–d are significantly shorter
than those of the mice receiving
NS, suggesting that 5a–d can
induce blood coagulation. On
the other hand, the tail bleeding
times
of
mice
receiving
Scheme 1. Synthesis of (5aS,12S,14aS)-DAPPP (5a–h) and (5aS,12R,14aS)-DAPPP (5’a–h): a) 1,1,3,3-tetramethoxy-
propane, HCl (5m), CH3OH, 458C, 48 h; b) Fmoc-l-Pro-OH, SOCl2, reflux, 5 h, Et2O, then CH2Cl2, 1, 08C, 0.5 h;
c) (iPr)2NH, RT, 24 h; d) acetone, p-TsOH, 458C, 1 h; e) Et3N, AA-OCH3, Na2SO4, RT, 3 h, then KBH4, CH3OH, 1 h;
f) NaOH (2m), RT, 2 h.
0.1 mmolkgÀ1 of each of 5e–h
and 5’a–h are equal to those of
the mice receiving NS, suggest-
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M. Zhao, S. Peng, et al.
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mice that received NS, but also that the tail bleeding times of
mice orally administered 5c steadily decreased with increasing
dose. This means that 5c dose-dependently inhibits bleeding
in treated mice, and even at a dose of 1 nmolkgÀ1, 5c still elic-
its blood coagulation.
Table 1. In vivo evaluation of coagulation effects of 5a–h and 5’a–h in
mice using a tail bleeding assay.[a]
Compd
Time [s]
Compd
Time [s]
NS
5a
5b
5c
5d
5e
5 f
280.9Æ25.5
179.7Æ29.5[b]
171.9Æ28.2[b]
154.9Æ28.9[b]
182.7Æ29.9[b]
277.8Æ27.9
266.8Æ29.0
267.3Æ30.1
269.0Æ28.5
Aspirin
5’a
5’b
5’c
5’d
5’e
5’f
313.1Æ29.7[c]
276.7Æ17.6
269.1Æ30.1
269.9Æ30.8
274.0Æ30.3
275.8Æ27.6
272.3Æ30.6
274.7Æ27.3
277.1Æ25.9
In vivo pro-thrombotic or antithrombotic effects
Pro-thrombotic and antithrombotic activities were determined
by monitoring the weight of the thrombus formed inside the
carotid artery of treated rats. In this thrombosis assay, Wistar
rats were fed NS, aspirin (165 mmolkgÀ1) as positive control, or
each of 5a–h and 5’a–h at 0.1 mmolkgÀ1, and the thrombus
weights were recorded. Table 2 shows that the thrombus
5g
5h
5g
5h
[a] Data represent the mean ÆSD tail bleeding time (n=10); NS=normal
saline (vehicle); doses used: aspirin: 165 mmolkgÀ1, 5a–h and 5’a–h:
0.1 mmolkgÀ1
; [b] Compared with the vehicle control (NS), p <0.01;
[c] Compared with the vehicle control (NS), p <0.05.
Table 2. In vivo evaluation of the pro-thrombotic and antithrombotic ac-
tivities of 5a–h and 5’a–h in rats.[a]
ing that 5e–h and 5’a–h cannot induce blood coagulation.
Among 5a–d and 5’a–d, only the former induced blood coag-
ulation, indicating that the blood coagulation activity of
DAPPPs depend on the configuration at C12. Among 5a–h,
only 5a–d induced blood coagulation, indicating that the
blood coagulation capacity of (5aS,12S,14aS)-DAPPP is influ-
enced by the side chain of the amino acid moiety.
Compd
Weight [mg]
Compd
Weight [mg]
NS
5a
5b
5c
5d
5e
5 f
26.22Æ2.03[b]
31.33Æ2.04[c]
34.14Æ2.03[c]
29.08Æ1.34[c]
34.84Æ2.84[c]
18.22Æ1.93[c]
24.62Æ1.83[c]
17.54Æ1.61[c]
24.20Æ1.41[c]
Aspirin
5’a
5’b
5’c
5’d
5’e
5’f
13.22Æ1.67
23.49Æ2.01
23.79Æ2.08
22.24Æ2.06
25.71Æ1.12
16.14Æ1.31
16.33Æ1.42
15.44Æ1.81
19.20Æ1.70
5g
5h
5g
5h
Dose-dependent blood coagulation in mice treated with 5c
To observe the dose-dependent effects of 5a–d in the in vivo
tail bleeding time assay, the most potent compound 5c was
selected as a representative, and oral doses of 100, 10, and
1 nmolkgÀ1 were used. Aspirin (165 mmolkgÀ1) was used as
positive control, NS was used as negative control, and tail
bleeding times were recorded and compared with those of
5’c, 5d, and 5’d. Figure 2 shows that not only the tail bleeding
times of mice orally administered compound 5c at 100, 10,
and 1 nmolkgÀ1 are significantly shorter than those of the
[a] Data represent the mean ÆSD weight of wet thrombus (n=12); NS=
normal saline (vehicle); doses used: aspirin: 165 mmolkgÀ1, 5a–h and
5’a–h: 0.1 mmolkgÀ1
. [b] Compared with 5a–h and 5’a–h, p <0.01;
[c] Compared with the corresponding 5’ derivative, p <0.01.
weight of rats orally administered 5a–d are significantly higher
than those of mice that received NS. This indicates that com-
pounds 5a–d stimulate thrombus formation in rats. Table 2
also shows that the thrombus weight of the rats orally admin-
istered 0.1 mmolkgÀ1 of each of 5e–h and 5’a–h are signifi-
cantly lower than those of rats that received NS. This indicates
that 5e–h and 5’a–h inhibit thrombus formation. The fact that
5a–d and 5’a–d have opposing actions suggests that the con-
figuration at C12 of DAPPP is critical for its antithrombotic and
pro-thrombotic activities on treated rats. Interestingly, the
most potent compound 5c in tail bleeding time assay is not
the most potent pro-thrombotic agent in the thrombosis
assay; it induces a mild pro-thrombotic effect at a dose of
0.1 mmolkgÀ1. This suggests that antifibrinolytic therapy with
5c within a certain dose range might not induce thrombosis.
Dose–effect relationship of 5c for thrombogenesis
To clarify the therapeutic window of 5c, its dose–effect rela-
tionship for pro-thrombotic activity was determined. In this
in vivo thrombogenesis assay, rats were orally administered 5c
at 1 mmolkgÀ1 and 100 and 10 nmolkgÀ1, or aspirin at
165 mmolkgÀ1 as positive control, or NS alone. The thrombus
weight of the rats is shown in Figure 3. The results show that
Figure 2. Tail bleeding time assay with mice orally administered 5c, 5’c, 5d,
5’d. Values represent the mean ÆSD, NS=vehicle, n=12. a) Compared with
NS p <0.05, with 100, 10, and 1 nmolkgÀ1 5c and 5d p <0.01; b) compared
with NS and 1 nmolkgÀ1 5c or 5d p <0.01, with 10 nmolkgÀ1 5c or 5d
p <0.05; c) compared with NS p <0.01, with 1 nmolkg 5c or 5d p <0.05;
d) compared with NS p <0.05; e) compared with NS p <0.01.
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Urokinase Inhibitors
Figure 4. In vitro thrombolytic activity of UK with and without 5c. Values
represent the mean ÆSD, NS=vehicle, [UK]=100 IUmLÀ1, n=8. a) Com-
pared with NS, as well as UK plus 10 and 100 nm 5c p <0.01, with UK plus
1 nm 5c and UK plus 100 nm 5’c p >0.05; b) compared with NS, as well as
UK plus 100 and 10 nm 5c p <0.01; c) compared with NS and UK plus
100 nm 5c p <0.01; d) compared with NS p >0.05.
Figure 3. Thrombus weights from rats orally administered 5c at 1000, 100,
and 10 nmolkgÀ1. Values represent the mean ÆSD, n=12. a) Compared with
NS, as well as 1000, 100, and 10 nmolkgÀ1 5c p <0.01; b) compared with NS
and 10 nmolkgÀ1 5c p <0.01; c) compared with NS p >0.05.
the thrombus weight of the rats given 1 mmolkgÀ1 5c is equal
to those from rats administered 5c at 100 nmolkgÀ1, suggest-
ing that doses above 100 nmolkgÀ1 have no greater effect on
thrombosis activity. Thrombus weights of rats given 5c at
10 nmolkgÀ1 are equal to those of rats given NS, suggesting
that 5c has no thrombosis activity at this dose. Therefore, 5c
is a weak thrombosis agent, and a safe dose against thrombus
formation would be no greater than 10 nmolkgÀ1.
were used as an indicator of this activity. Figure 5 shows that
the decreased thrombus weight from rats given UK at
20000 IUkgÀ1 is significantly greater than those from rats ad-
ministered the same amount of UK and pre-fed with 5c at 100,
10, and 1 nmolkgÀ1. This means that 5c antagonizes UK-in-
duced thrombolysis in vivo. Figure 5 also shows that the antag-
onism of 5c steadily increases with the increase in dose. This
means that 5c dose-dependently antagonizes the in vivo
thrombolytic activity of UK. The decreased thrombus weight
from rats given UK at 20000 IUkgÀ1, pre-fed with 5c at
100 nmolkgÀ1 is equal to that from rats given NS. This means
that 5c at 100 nmolkgÀ1 completely antagonizes the in vivo
thrombolytic action of UK. Figure 5 further indicates that the
decreased thrombus weight of rats administered UK
(20000 IUkgÀ1) pre-fed with 100 nmolkgÀ1 5’c is equal to that
of the rats given UK alone. This means that 5’c (100 nmolkgÀ1)
does not antagonize the in vivo thrombolytic action of UK.
Effect of 5c on the in vitro thrombus clot lysis activity of
urokinase
To identify the antagonism of 5a–d against urokinase (UK), the
in vitro thrombus clot lysis activity of UK (final concentration:
100 IUmLÀ1) was compared with those of UK (100 IUmLÀ1) plus
the representative 5c (final concentration: 100, 10, or 1 nm).
The decrease in thrombus weight was used to represent activi-
ty. Figure 4 shows that the decrease in thrombus weight elicit-
ed by UK alone is significantly greater than those
from UK plus 5c at 100, 10, and 1 nm. This means
that 5c antagonizes the in vitro lysis activity of UK.
Figure 4 also indicates that the decreases in throm-
bus weights of UK plus 5c steadily diminish with in-
creasing concentrations of 5c. This means that 5c
antagonizes the in vitro lysis activity of UK in a con-
centration-dependent manner. Figure 4 further indi-
cates that the decreased thrombus weight effected
by UK plus 100 nm 5c is effectively equal to that of
NS. This means that the in vitro lysis activity of UK
can be completely antagonized by 5c at 100 nm.
Effect of 5c on the in vivo thrombolytic activity of
urokinase
Figure 5. Effect of 5c on the in vivo thrombolytic activity of UK. Decreased thrombus
weight values represent the mean ÆSD, NS=vehicle, [UK]=20000 IUkgÀ1, n=12.
a) Compared with NS as well as UK plus 1, 10, and 100 nm 5c p <0.01, with UK plus
100 nm 5’c p >0.05; b) compared with NS, as well as UK plus 10 and 100 nm 5c
p <0.01; c) compared with NS, as well as UK plus 100 nm 5c p <0.01; d) compared with
To confirm the efficacy of 5a–d as in vivo inhibitors
of UK, the in vivo thrombolytic activity of UK was
compared with those of UK plus the representative
5c concentrations. The decrease in thrombus weights NS p >0.05.
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M. Zhao, S. Peng, et al.
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Effect of 5c on the interaction of urokinase and plasminogen
b]indoles (DAPPP), and compounds 5a–d showed strong he-
mostatic activities in a mouse tail bleeding assay, with the
most potent compound, 5c, demonstrating dose-dependent
effects. The coagulation potency of DAPPP depends on its con-
figuration at C12 and the substituent group. Compounds 5a–d
show pro-thrombotic effects in a rat thrombosis assay; howev-
er, 5c showed very mild activity; this indicates that 5c has a
good therapeutic window for antifibrinolytic therapy. Specifi-
cally, the minimal effective dose of 5c for hemostatic activity is
<1 nmolkgÀ1, whereas the safe dose of 5c against thrombus
formation is 10 nmolkgÀ1. Further investigation of 5c in vitro
and in vivo shows that inhibition of UK is involved in this coag-
ulation process, and the SDS-PAGE assays supplied direct evi-
dence that 5c inhibits the function of u-PA on plasminogen in
vitro.
It is well documented that both tissue-type plasminogen acti-
vator (t-PA) and u-PA (UK) catalyze the conversion of zymogen
plasminogen to plasmin and play a central role in the fibrino-
lytic processes. Whereas the primary role of t-PA is the dissolu-
tion of blood clots in vessels, u-PA mediates cell-related pro-
teolysis.[20,21] Our previous work suggested that the antithrom-
botic activity of pyrazino-pyridoindole-1,4-diones is a platelet-
mediated process.[22] In this context, the effect of compound
5c on the interaction between UK and plasminogen was moni-
tored by electrophoresis, and the results are shown in Figure 6.
SDS-PAGE of plasminogen alone (PLG, 5 mL, final concentration:
0.1 UmLÀ1) plus NS (10 mL) gives two bands of PLG. In electro-
phoresis of PLG (5 mL, 0.1 UmLÀ1) plus UK (5 mL, final concen-
tration: 100 UmLÀ1) and NS (5 mL), these PLG bands disap-
peared; PLG is completely degraded by UK. Electrophoresis of
PLG (5 mL, 0.1 UmLÀ1) plus UK (5 mL, 100 UmLÀ1) and 5c (5 mL,
2500 ngmLÀ1) again gives two PLG bands, indicating the action
of UK is completely antagonized by 5c. In electrophoresis of
PLG (5 mL, 0.1 UmLÀ1) plus UK (5 mL, 100 UmLÀ1) and 5c (5 mL)
in a series of final concentrations (2500, 1250, 250, 125, 25,
12.5, 2.5, 1.25, 0.25, 0.05, and 0.01 ngmLÀ1), the intensity of the
two PLG bands steadily decreases with decreasing 5c concen-
tration. Therefore, 5c concentration-dependently antagonizes
the action of UK.
Experimental Section
Chemistry
General: All chemicals were purchased from Sigma–Aldrich Co. (St.
Louis, MO, USA) and were purified when necessary. Column chro-
matography was performed using silica gel (200–300 mesh, Qing-
dao Haiyang Chemical Co., Qingdao, P. R. China). Purity of inter-
mediates (>95%) and products (>97%) were determined by TLC
analysis (Merck silica gel plates 60 F254, 0.25 mm layer thickness)
and HPLC analysis (Hewlett Packard HP Agilent 1050 system
equipped with a Waters C18 column 4.6ꢁ150 mm). Melting points
(mp) were determined in capillary
tubes on an electrothermal SM/
XMP apparatus and are uncorrect-
ed. Mass spectrometry (MS) was
performed on a Micromass Quattro
micro TM API bench-top mass
spectrometer (Waters Co.) using
electrospray ionization (ESI). Infra-
red spectra (IR) were run on a Shi-
madzu 8010M spectrophotometer,
using the KBr disk method.
1H NMR (500 Hz) and 13C NMR
(125 Hz) spectra were recorded on
a Bruker Advance 500 spectrome-
ter in CDCl3 with TMS as an inter-
nal standard, and chemical shifts
(d) are expressed in parts per mil-
Figure 6. SDS-PAGE of plasminogen (PLG, 5 mL, final concentration 0.1 UmLÀ1) plus NS (10 mL); PLG (5 mL,
0.1 UmLÀ1) plus UK (5 mL, final concentration 100 UmLÀ1) and NS (5 mL); PLG (5 mL, 0.1 UmLÀ1) plus UK (5 mL,
100 UmLÀ1) and 5 mL 5c (in a series of final concentrations as indicated); PLG (5 mL, 0.1 UmLÀ1) plus UK (5 mL,
100 UmLÀ1) and EACA (5 mL, final concentration 2500 ngmLÀ1); PLG (5 mL, 0.1 UmLÀ1) plus UK (5 mL, 100 UmLÀ1
and EACA (5 mL, 1250 ngmLÀ1).
lion (ppm). Optical rotations were
determined with a Jasco P-1020
polarimeter.
)
1-(2,2-Dimethoxyethyl)-1,2,3,4-
tetrahydrocarboline-3-carboxylic
acid methyl ester (1 and 1’): A
suspension of l-tryptophan methyl
ester (5.0 g, 24.5 mmol) and
Conclusions
1,1,3,3-tetramethoxypropane (6.0 mL, 23.6 mmol) in CH3OH (50 mL)
was adjusted to pH 1–2 with HCl (5m) and stirred at 458C for 48 h.
After evaporation of the solvent in vacuo, the residue was diluted
with water (50 mL) and extracted with EtOAc (3ꢁ30 mL). The com-
bined organic extracts were washed successively with 10% aq
Na2CO3 (3ꢁ30 mL) and saturated aq NaCl (2ꢁ30 mL), then dried
over with anhyd Na2SO4, filtered and concentrated in vacuo. Purifi-
As increasing attention has been given to current small-molec-
ular antifibrinolytic agents in current clinical use, such as tra-
nexamic acid, which carry a significant risk of prompting
thrombus formation, we carried out our investigation of new
u-PA inhibitors. In summary, we designed and synthesized a
group of pyrolo[1,2:4,5]-1,4-dioxopyrazino[1,2:1,6]pyrido[3,4-
2316
www.chemmedchem.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 2312 – 2322

Urokinase Inhibitors
cation by column chromatography (CHCl3/CH3OH, 30:1) gave 1
(3.2 g, 60%) and 1’ (1.6 g, 30%).
concentrated in vacuo to remove acetone, and purified by column
chromatography (CHCl3/CH3OH, 40:1). Recrystallization from ace-
tone gave 3 (158 mg, 90%): mp: 133–1358C; [a]2D0 = +176 (c=
0.71, CHCl3/CH3OH, 1:1); 1H NMR (500 MHz, CDCl3): d=10.22 (s,
1H), 9.86 (s, 1H), 7.52 (t, J=7.6 Hz, 1H), 7.35 (t, J=7.6 Hz, 1H), 7.25
(d, J=7.6 Hz, 1H), 7.06 (d, J=7.6 Hz, 1H), 5.83 (t, J=6.4 Hz, 1H),
5.12 (t, J=4.1 Hz, 1H), 4.77 (t, J=6.1 Hz, 1H), 4.42 (t, J=4.6 Hz,
1H), 3.90 (m, 1H), 3.65 (dd, J=4.6, 14.7 Hz, 1H), 2.88 (dd, J=11.0,
15.8 Hz, 1H), 2.52 (m, 2H), 1.92–2.21 ppm (m, 4H); IR (KBr): n˜ =
3340, 2937, 2835, 1726, 1684, 1643, 1460, 1332, 743 cmÀ1; MS
(ESI+): m/z: 338 [M+H]+; Anal. calcd for C19H19N3O3: C 67.64, H
5.68, N 12.46, found: C 67.85, H 5.53, N 12.69.
(1S,3S)-1-(2,2-Dimethoxyethyl)-1,2,3,4-tetrahydrocarboline-3-car-
boxylic acid methyl ester (1): 1H NMR (500 MHz, CDCl3): d=8.50
(s, 1H), 7.48 (d, J=7.9 Hz, 1H), 7.34 (d, J=7.9 Hz, 1H), 7.15 (m, J=
7.1 Hz, 2H), 4.66 (t, J=6.9 Hz, 1H), 4.38 (t, J=6.9 Hz, 1H), 4.14 (t,
J=4.0 Hz, 1H), 3.94 (dd, J=6.9, 2.9 Hz,1H), 3.74 (s, 3H), 3.47 (s,
3H), 3.40 (s, 3H), 3.13 (m, J=2.1 Hz, 1H), 2.96 (m, J=2.1 Hz, 1H),
2.41 (s, 1H), 2.32 (t, J=7.1 Hz, 1H), 2.22 ppm (t, J=4.0 Hz, 1H); MS
(ESI+): m/z: 319 [M+H]+.
(1R,3S)-1-(2,2-Dimethoxyethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-
b]indole-3-carboxylic acid methyl ester (1’): 1H NMR (500 MHz,
CDCl3): d=8.80 (s, 1H), 7.49 (d, J=7.2 Hz, 1H), 7.34 (d, J=7.8 Hz,
1H), 7.15 (m, J=7.1 Hz, 2H), 4.66 (q, J=3.2 Hz, 1H), 4.31 (m, J=
3.2 Hz, 1H), 3.94 (s, 3H), 3.82 (dd, J=7.0, 3.9 Hz,1H), 3.51 (s, 3H),
3.43 (s, 3H), 3.13 (m, J=2.1 Hz, 1H), 2.84 (m, J=2.1 Hz, 1H), 2.22 (t,
J=7.1 Hz, 1H), 2.12 (t, J=4.0 Hz, 1H), 1.96 ppm (s, 1H); MS (ESI+):
m/z: 319 [M+H]+.
(5aS,12R,14aS)-5,14-Dioxo-12-cabonylmethyl-1,2,3,5,5a,6,11,12,
14,14a-decahydro-5H,14H-pyrolo[1,2:4,5]pyrazino[1,2:1,6]pyrido-
[3,4-b]indole (3’): Compound 2’ (200 mg, 0.52 mmol) was reacted
using the same procedure described for the preparation of 2 to
give 3’ (162 mg, 92%): mp: 133–1358C; [a]2D0 = +176 (c=0.71,
1
CHCl3/CH3OH, 1:1); H NMR (500 MHz, CDCl3): d=10.26 (s, 1H), 9.83
(s, 1H), 7.54 (t, J=7.5 Hz, 1H), 7.33 (t, J=7.5 Hz, 1H), 7.27 (d, J=
7.5 Hz, 1H), 7.09 (d, J=7.5 Hz, 1H), 5.80 (t, J=6.2 Hz, 1H), 5.10 (t,
J=4.3 Hz, 1H), 4.70 (t, J=6.0 Hz, 1H), 4.35 (t, J=4.3 Hz, 1H), 3.95
(m, 1H), 3.60 (dd, J=4.3, 14.0 Hz, 1H), 2.82 (dd, J=11.2, 15.4 Hz,
1H), 2.55 (m, 2H), 1.90–2.23 ppm (m, 4H); IR (KBr): n˜ =3344, 2932,
2830, 1730, 1680, 1647, 1463, 1335, 748 cmÀ1; MS (ESI+): m/z: 338
[M+H]+; Anal. calcd for C19H19N3O3: C 67.64, H 5.68, N 12.46,
found: C 67.85, H 5.53, N 12.69.
(5aS,12S,14aS)-5,14-Dioxo-12-(2,2-dimethoxyethyl)-1,2,3,5,5a,6,
11,12,14,14a-decahydro-5H,14H-pyrolo[1,-2:4,5]pyrazino-
[1,2:1,6]pyrido[3,4-b]indole (2): Fmoc-l-Pro (5.0 g, 14.8 mmol) was
treated dropwise with SOCl2 (50 mL), and the reaction mixture was
held at reflux for 5 h. The mixture was concentrated in vacuo to
remove excess SOCl2. The residue was treated with Et2O (30 mL) to
provide Fmoc-protected prolinyl chloride (colorless powder), which
was treated with 1 (3.2 g, 10 mmol) in CH2Cl2 (50 mL) at 08C and
the mixture was stirred for 0.5 h. The reaction mixture was adjust-
ed to pH 9 with (iPr)2NH and stirred at RT for 24 h. The reaction
mixture was concentrated in vacuo, and the residue was purified
on column chromatography (CHCl3/CH3OH, 40:1) to give 2 (2.61 g,
40%): mp: 180–1838C; [a]D20 = +76 (c=0.35, CHCl3/CH3OH, 1:1);
1H NMR (500 MHz, CDCl3): d=10.14 (s, 1H), 8.67 (s, 1H), 7.50 (t, J=
7.5 Hz, 1H), 7.34 (t, J=7.5 Hz, 1H), 7.22 (d, J=7.5 Hz, 1H), 7.09 (d,
J=7.5 Hz, 1H), 5.82 (t, J=6.0 Hz, 1H), 4.76 (t, J=6.0 Hz, 1H), 4.41
(t, J=4.5 Hz, 1H), 4.14 (t, J=4.0 Hz, 1H), 3.92 (m, 1H), 3.64 (dd, J=
4.5, 15.0 Hz, 1H), 3.46 (s, 3H), 3.40 (s, 3H), 2.86 (dd, J=11.1,
16.0 Hz, 1H), 2.51 (m, 2H), 1.90–2.20 ppm (m, 5H); IR (KBr): n˜ =
3346, 2933, 2832, 1683, 1645, 1462, 1335, 744 cmÀ1; MS (ESI+): m/
z: 384 [M+H]+; Anal. calcd for C21H25N3O4: C 65.78, H 6.57, N
10.96, found: C 66.02, H 6.74, N 10.75.
(5aS,12S/R,14aS)-5,14-Dioxo-12-(2-amino-acid-methyl
ester-N-
ylethyl-1-yl)-1,2,3,5,-5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo-
[1,2:4,5]pyrazino[1,2:1,6]pyrido-[3,4-b]indoles (4a–h and 4’a–h):
A solution of 3 or 3’ (0.52 mmol) in CHCl3 (10 mL) was treated with
the appropriate amino acid methyl ester (0.52 mmol), Et3N (0.5 mL)
and anhyd Na2SO4 (3 g). The reaction mixture was stirred at RT for
3 h, then mixed with a solution of KBH4 (100 mg, 1.85 mmol) in
CH3OH (5 mL), and stirred for another 1 h, until TLC (CHCl3/CH3OH,
20:1) indicated complete disappearance of the starting material (3
or 3’). The reaction mixture was concentrated in vacuo, and the
residue was mixed with CHCl3 (10 mL) and deionized water
(10 mL). The two-phase solution was treated with HCl (1 mL, 5m)
to decompose excess KBH4, and adjusted to pH 9 with aq NH3
(5m). The CHCl3 phase was separated, and the aqueous phase was
extracted with CHCl3 (3ꢁ5 mL). The combined organic phases
were washed with saturated aq NaCl (20 mL), dried over with
anhyd Na2SO4, filtered and concentrated in vacuo. Purification by
column chromatography (CHCl3/CH3OH, 30:1) gave 4a–h or 4’a–h.
(5aS,12R,14aS)-5,14-Dioxo-12-(2,2-dimethoxyethyl)-1,2,3,5,5a,6,
11,12,14,14a-decahydro-5H,14H-pyrolo[1,-2:4,5]pyrazino-
[1,2:1,6]pyrido[3,4-b]indole (2’): Fmoc-l-Pro (5.0 g, 14.8 mmol)
and 1’ (3.2 g, 10 mmol) were reacted using the procedure as for
preparing 2 to give compound 2’ (1.30 g, 20%): mp: 205–2078C;
(5aS,12S,14aS)-5,14-Dioxo-12-(2-alanine-methyl ester-N-ylethyl-
1-yl)-1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo-
[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]indole (4a): Yield: 212 mg
1
[a]2D0 = +76 (c=0.33, CHCl3/CH3OH, 1:1); H NMR (500 MHz, CDCl3):
d=10.17 (s, 1H), 8.65 (s, 1H), 7.52 (t, J=7.6 Hz, 1H), 7.36 (t, J=
7.6 Hz, 1H), 7.23 (d, J=7.6 Hz, 1H), 7.11 (d, J=7.6 Hz, 1H), 5.80 (t,
J=6.2 Hz, 1H), 4.70 (t, J=6.2 Hz, 1H), 4.32 (t, J=4.6 Hz, 1H), 4.10
(t, J=4.2 Hz, 1H), 3.89 (m, 1H), 3.86 (s, 3H), 3.75 (s, 3H), 3.64 (dd,
J=4.6, 15.1 Hz, 1H), 2.84 (dd, J=11.0, 15.8 Hz, 1H), 2.54 (m, 2H),
1.92–2.26 ppm (m, 5H); IR (KBr): n˜ =3348, 2937, 2829, 1688, 1640,
1467, 1330, 749 cmÀ1; MS (ESI+): m/z: 384 [M+H]+; Anal. calcd for
C21H25N3O4: C 65.78, H 6.57, N 10.96, found: C 66.02, H 6.74, N
10.75.
1
(96%): H NMR (300 MHz, [D6]DMSO): d=10.55 (s, 1H), 7.45 (d, J=
7.9 Hz, 1H), 7.38 (d, J=7.9 Hz, 1H), 7.11 (t, J=7.4 Hz, 1H), 6.99 (t,
J=7.4 Hz, 1H), 5.74 (d, J=10.0 Hz, 1H), 4.31 (m, 2H), 3.67 (s, 3H),
3.62 (q, J=8.4 Hz, 1H), 3.46 (dt, J=2.4 Hz, 10.1 Hz, 1H), 3.21 (q, J=
6.5 Hz, 1H), 3.14 (dd, J=15.0, 3.92 Hz, 1H), 2.96 (m, 2H), 2.79 (s,
1H), 2.38 (s, 1H), 2.07 (m, 1H), 1.99 (m, 2H), 1.85 (s, 3H), 1.30 ppm
(d, J=6.9 Hz, 3H); IR (KBr): n˜ =3345, 2936, 2834, 1733, 1685, 1642,
1465, 1336, 1295, 748 cmÀ1; MS (ESI+): m/z: 425 [M+H]+.
(5aS,12S,14aS)-5,14-Dioxo-12-cabonylmethyl-1,2,3,5,5a,6,11,12,
14,14a-decahydro-5H,14H-pyrolo[1,2:4,5]pyrazino[1,2:1,6]pyrido-
[3,4-b]indole (3): A solution of 2 (200 mg, 0.52 mmol) in acetone
(15 mL) was treated with p-TsOH (20 mg) and then stirred at 458C
for 1 h, until TLC (CHCl3/CH3OH, 20:1) indicated complete disap-
pearance of 2. The reaction mixture was treated with Et3N (0.5 mL),
(5aS,12S,14aS)-5,14-Dioxo-12-(2-aspartic-acid-dimethyl ester-N-
ylethyl-1-yl)-1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo-
[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]indole (4b): Yield: 227 mg
(88%): H NMR (300 MHz, [D6]DMSO): d=10.71 (s, 1H), 7.47 (d, J=
7.9 Hz, 1H), 7.38 (d, J=8.0 Hz, 1H), 7.06 (t, J=7.0 Hz, 1H), 7.03 (t,
J=7.2 Hz, 1H), 5.69 (d, J=7.7 Hz, 1H), 4.34 (m, 2H), 3.67 (s, 3H),
1
ChemMedChem 2011, 6, 2312 – 2322
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
2317

M. Zhao, S. Peng, et al.
MED
3.65 (s, 3H), 3.62 (m, 2H), 3.46 (t, J=9.5 Hz, 2H), 3.14 (dd, J=4.1,
15.0 Hz, 2H), 2.96 (m, 2H), 2.68 (dd, J=8.8, 16.0 Hz, 1H), 2.28 (m,
2H), 2.11 (m, 1H), 1.93 ppm (m, 4H); IR (KBr): n˜ =3343, 2930, 2835,
1731, 1679, 1642, 1465, 1330, 1294, 741 cmÀ1; MS (ESI+): m/z: 483
[M+H]+.
(5aS,12S,14aS)-5,14-Dioxo-12-(2-leucine-methyl ester-N-ylethyl-
1-yl)-1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo-
[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]indole (4h): Yield: 228 mg
1
(94%): H NMR (300 MHz, [D6]DMSO): d=10.49 (s, 1H), 7.48 (d, J=
7.7 Hz, 1H), 7.41 (d, J=7.8 Hz, 1H), 7.14 (t, J=7.2 Hz, 1H), 7.06 (t,
J=7.4 Hz, 1H), 5.69 (dd, J=11.1, 2.6 Hz, 1H), 4.38 (t, J=5.2 Hz,
1H), 4.36 (dd, J=12.0, 8.0 Hz, 1H), 3.66 (s, 3H), 3.64 (m, 2H), 3.48
(dt, J=2.8, 7.0 Hz, 1H), 3.36 (t, J=6.6 Hz, 1H), 3.15 (dd, J=14.8,
4.3 Hz, 1H), 2.98 (t, J=11.7 Hz, 1H), 2.85 (t, J=9.5 Hz, 1H), 2.39 (s,
1H), 2.33 (m, 2H), 2.21 (dq, J=10.4, 2.6 Hz, 1H), 1.95 (m, 4H), 1.58
(m, 2H), 0.94 ppm (dd, J=12.0, 6.2 Hz, 6H); IR (KBr): n˜ =3346,
2934, 2835, 1731, 1685, 1640, 1465, 1337, 1293, 740 cmÀ1; MS
(ESI+): m/z: 467 [M+H]+.
(5aS,12S,14aS)-5,14-Dioxo-12-(2-phenylalanine-methyl ester-N-
ylethyl-1-yl)-1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo-
[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]indole (4c): Yield: 247 mg
1
(95%): H NMR (300 MHz, [D6]DMSO): d=10.63 (s, 1H), 7.49 (d, J=
7.8 Hz, 1H), 7.41 (d, J=8.0 Hz, 1H), 7.35 (m, 5H), 7.16 (t, J=7.6 Hz,
1H), 7.06 (t, J=7.6 Hz, 1H), 5.64 (dd, J=3.3, 11.0 Hz, 1H), 4.39 (m,
1H), 4.33 (dd, J=3.4, 11.2 Hz, 1H), 3.65 (s, 3H), 3.63 (m, 3H), 3.15
(m, 3H), 2.97 (m, 2H), 2.39 (m, 1H), 2.26 (m, 1H), 2.17 (m, 1H), 1.97
(m, 1H), 1.89 (m, 2H), 1.28 ppm (m, 2H); IR (KBr): n˜ =3344, 2935,
2834, 1731, 1683, 1643, 1466, 1337, 1295, 740 cmÀ1; MS (ESI+): m/
z: 501 [M+H]+.
(5aS,12R,14aS)-5,14-Dioxo-12-(2-alanine-methyl ester-N-ylethyl-
1-yl)-1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo-
[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]indole (4’a): Yield: 207 mg
1
(94%): H NMR (300 MHz, [D6]DMSO): d=10.50 (s, 1H), 7.43 (d, J=
7.8 Hz, 1H), 7.37 (d, J=7.8 Hz, 1H), 7.12 (t, J=7.4 Hz, 1H), 6.97 (t,
J=7.4 Hz, 1H), 5.82 (d, J=10.1 Hz, 1H), 4.39 (m, 2H), 3.69 (q, J=
8.2 Hz, 1H), 3.65 (s, 3H), 3.51 (dt, J=2.6, 9.8 Hz, 1H), 3.28 (q, J=
6.6 Hz, 1H), 3.14 (dd, J=3.9, 15.0 Hz, 1H), 2.87 (m, 2H), 2.70 (s,
1H), 2.31 (s, 1H), 1.98 (m, 1H), 1.94 (m, 2H), 1.80 (s, 3H), 1.27 ppm
(d, J=6.7 Hz, 3H); IR (KBr): n˜ =3343, 2934, 2831, 1730, 1681, 1645,
1462, 1332, 1297, 743 cmÀ1; MS (ESI+): m/z: 425 [M+H]+.
(5aS,12S,14aS)-5,14-Dioxo-12-(2-tryptophan-methyl
ester-N-
ylethyl-1-yl)-1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo-
[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]indole (4d): Yield: 263 mg
(94%): 1H NMR (300 MHz, [D6]DMSO): d=10.86 (s, 1H), 10.53 (s,
1H), 7.64 (d, J=7.9 Hz, 1H), 7.49 (d, J=8.0 Hz, 1H), 7.35 (m, 2H),
7.27 (s, 1H), 7.09 (m, 2H), 6.99 (m, 2H), 5.69 (d, J=8.1 Hz, 1H), 4.31
(m, 2H), 3.67 (s, 3H), 3.61 (m, 2H), 3.44 (m, 1H), 3.28 (dd, J=14.8,
5.5 Hz, 1H), 3.12 (m, 2H), 2.95 (m, 2H), 2.64 (m, 1H), 2.23 (m, 2H),
2.08 (m, 1H), 1.93 ppm (m, 4H); IR (KBr): n˜ =3346, 2935, 2837,
1730, 1678, 1644, 1459, 1337, 1295, 742 cmÀ1; MS (ESI+): m/z: 540
[M+H]+.
(5aS,12R,14aS)-5,14-Dioxo-12-(2-aspartic-acid-dimethyl ester-N-
ylethyl-1-yl)-1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo-
[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]indole (4’b): Yield: 224 mg
1
(87%): H NMR (300 MHz, [D6]DMSO): d=10.62 (s, 1H), 7.48 (d, J=
7.7 Hz, 1H), 7.36 (d, J=8.0 Hz, 1H), 7.09 (t, J=7.1 Hz, 1H), 7.05 (t,
J=7.1 Hz, 1H), 5.73 (d, J=7.5 Hz, 1H), 4.39 (m, 2H), 3.69 (s, 3H),
3.67 (m, 2H), 3.65 (s, 3H), 3.50 (t, J=9.4 Hz, 2H), 3.14 (dd, J=4.2,
15.0 Hz, 2H), 2.82 (m, 2H), 2.63 (dd, J=8.6, 15.5 Hz, 1H), 2.22 (m,
2H), 2.00 (m, 1H), 1.84 ppm (m, 4H); IR (KBr): n˜ =3340, 2934, 2831,
1735, 1676, 1640, 1462, 1334, 1291, 743 cmÀ1; MS (ESI+): m/z: 497
[M+H]+.
(5aS,12S,14aS)-5,14-Dioxo-12-(2-lysine-methyl ester-N-ylethyl-1-
yl)-1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo-
[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]indole (4e): Yield: 80 mg
1
(32%): H NMR (300 MHz, [D6]DMSO): d=10.62 (s, 1H), 7.49 (d, J=
7.6 Hz, 1H), 7.39 (d, J=7.6 Hz, 1H), 7.15 (t, J=7.6 Hz, 2H), 7.06 (t,
J=7.4 Hz, 1H), 5.77 (dd, J=3.2, 10.8 Hz, 1H), 4.39 (m, 1H), 4.36
(dd, J=3.8, 11.0 Hz, 1H), 3.68 (s, 3H), 3.64 (m, 2H), 3.15 (m, 2H),
2.95 (m, 2H), 2.35 (m, 2H), 2.31 (m, 2H), 2.21 (m, 3H), 1.97 (m, 2H),
1.85 (m, 2H), 1.66 (m, 2H), 1.57 (m, 2H), 1.29 ppm (m, 2H); IR (KBr):
(5aS,12R,14aS)-5,14-Dioxo-12-(2-phenylalanine-methyl ester-N-
ylethyl-1-yl)-1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo-
[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]indole (4’c): Yield: 242 mg
n˜ =3347, 2928, 2836, 1731, 1686, 1640, 1458, 1337, 1294, 741 cmÀ1
MS (ESI+): m/z: 482 [M+H]+.
;
1
(93%): H NMR (300 MHz, [D6]DMSO): d=10.55 (s, 1H), 7.51 (d, J=
7.9 Hz, 1H), 7.43 (d, J=8.1 Hz, 1H), 7.41 (m, 5H), 7.21 (t, J=7.7 Hz,
1H), 7.11 (t, J=7.7 Hz, 1H), 5.69 (dd, J=3.5, 10.7 Hz, 1H), 4.44 (m,
1H), 4.38 (dd, J=3.5, 10.7 Hz, 1H), 3.64 (s, 3H), 3.62 (m, 3H), 3.15
(m, 3H), 2.35 (m, 2H), 2.30 (m, 1H), 2.21 (m, 1H), 2.11 (m, 1H), 1.90
(m, 1H), 1.85 (m, 2H), 1.24 ppm (m, 2H); IR (KBr): n˜ =3342, 2933,
2837, 1735, 1680, 1646, 1462, 1332, 1291, 743 cmÀ1; MS (ESI+): m/
z: 501 [M+H]+.
(5aS,12S,14aS)-5,14-Dioxo-12-(2-serine-methyl ester-N-ylethyl-1-
yl)-1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo-
[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]indole (4 f): Yield: 213 mg
1
(93%): H NMR (300 MHz, [D6]DMSO): d=10.60 (s, 1H), 7.47 (d, J=
7.9 Hz, 1H), 7.39 (d, J=8.0 Hz, 1H), 7.12 (t, J=7.7 Hz, 1H), 6.99 (t,
J=7.7 Hz, 1H), 5.76 (dd, J=3.3, 10.0 Hz, 1H), 4.37 (m, 2H), 3.75 (m,
1H), 3.66 (m, 2H), 3.65 (s, 3H), 3.48 (m, 1H), 3.15 (m, 2H), 2.98 (m,
1H), 2.79 (s, 1H), 2.25 (m, 2H), 2.00 (m, 1H), 1.94 (m, 1H), 1.89 ppm
(m, 5H); IR (KBr): n˜ =3344, 2936, 2833, 1732, 1685, 1641, 1465,
1337, 1293, 746 cmÀ1; MS (ESI+): m/z: 441 [M+H]+.
(5aS,12R,14aS)-5,14-Dioxo-12-(2-tryptophan-methyl
ester-N-
ylethyl-1-yl)-1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo-
[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]indole (4’d): Yield: 261 mg
(93%): 1H NMR (300 MHz, [D6]DMSO): d=10.54 (s, 1H), 10.50 (s,
1H), 7.66 (d, J=7.7 Hz, 1H), 7.53 (d, J=8.1 Hz, 1H), 7.39 (m, 2H),
7.30 (s, 1H), 7.14 (m, 2H), 7.00 (m, 2H), 5.76 (d, J=8.0 Hz, 1H), 4.38
(m, 2H), 3.69 (s, 3H), 3.64 (m, 2H), 3.48 (m, 1H), 3.31 (dd, J=5.1,
14.0 Hz, 1H), 3.12 (m, 2H), 2.27 (m, 2H), 2.17 (m, 1H), 2.04 (m, 2H),
1.99 (m, 1H), 1.90 ppm (m, 4H); IR (KBr): n˜ =3342, 2933, 2834,
1735, 1673, 1640, 1462, 1334, 1291, 740 cmÀ1; MS (ESI+): m/z: 540
[M+H]+.
(5aS,12S,14aS)-5,14-Dioxo-12-(2-glycine-methyl ester-N-ylethyl-
1-yl)-1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo-
[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]indole (4g): Yield: 192 mg
1
(90%): H NMR (300 MHz, [D6]DMSO): d=10.58 (s, 1H), 7.47 (d, J=
7.9 Hz, 1H), 7.42 (d, J=8.0 Hz, 1H), 7.12 (t, J=7.4 Hz, 1H), 6.99 (t,
J=7.4 Hz, 1H), 5.82 (dd, J=4.4, 9.3 Hz, 1H), 4.36 (m, 1H), 3.67 (s,
3H), 3.66 (q, J=14.4 Hz, 1H), 3.46 (m, 1H), 3.25 (m, 2H), 3.14 (dd,
J=4.2, 14.6 Hz, 1H), 2.98 (m, 1H), 2.82 (m, 2H), 2.29 (m, 2H), 2.11
(m, 1H), 1.95 ppm (m, 5H); IR (KBr): n˜ =3344, 2935, 2834, 1731,
1677, 1642, 1458, 1337, 1293, 741 cmÀ1; MS (ESI+): m/z: 411 [M+
H]+.
(5aS,12R,14aS)-5,14-Dioxo-12-(2-lysine-methyl ester-N-ylethyl-1-
yl)-1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo-
[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]indole (4’e): Yield: 82 mg
1
(32%): H NMR (300 MHz, [D6]DMSO): d=10.66 (s, 1H), 7.47 (d, J=
2318
www.chemmedchem.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 2312 – 2322

Urokinase Inhibitors
7.5 Hz, 1H), 7.36 (d, J=7.5 Hz, 1H), 7.14 (t, J=7.5 Hz, 2H), 7.04 (t,
J=7.2 Hz, 1H), 5.82 (dd, J=3.4, 10.6 Hz, 1H), 4.42 (m, 1H), 4.40
(dd, J=3.6, 11.2 Hz, 1H), 3.65 (s, 3H), 3.62 (m, 2H), 3.15 (m, 2H),
2.70 (m, 2H), 2.31 (m, 2H), 2.27 (m, 2H), 2.16 (m, 3H), 1.92 (m, 2H),
1.80 (m, 2H), 1.62 (m, 2H), 1.53 (m, 2H), 1.25 ppm (m, 2H); IR (KBr):
6.9 Hz, 3H); 13C NMR (75 MHz, [D6]DMSO): d=174.0, 167.2, 164.4,
136.5, 133.9, 126.5, 121.6, 119.1, 118.2, 111.9, 106.0, 58.4, 57.8, 55.4,
47.7, 45.4, 43.5, 31.1, 29.9, 25.8, 21.9, 17.4 ppm; IR (KBr): n˜ =3346,
3133, 3001, 2932, 2835, 2712, 1682, 1641, 1462, 1331, 741 cmÀ1
;
MS (ESI+): m/z: 411 [M+H]+; Anal. calcd for C22H26N4O4: C 64.37, H
n˜ =3347, 2934, 2835, 1731, 1686, 1640, 1452, 1335, 1293, 742 cmÀ1
MS (ESI+): m/z: 482 [M+H]+.
;
6.38, N 13.65, found: C 64.16, H 6.22, N 13.43.
(5aS,12S,14aS)-5,14-Dioxo-12-(2-aspartic-acid-N-ylethyl-1-yl)-
1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo-
(5aS,12R,14aS)-5,14-Dioxo-12-(2-serine-methyl ester-N-ylethyl-1-
yl)-1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo-
[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]indole (5b): Yield: 227 mg
(96%): mp: 218–2228C; [a]2D0 = +77 (c=0.47, CHCl3/CH3OH, 1:1);
1H NMR (300 MHz, [D6]DMSO): d=11.2 (s, 2H), 10.69 (s, 1H), 7.45
(d, J=7.8 Hz, 1H), 7.36 (d, J=8.1 Hz, 1H), 7.09 (t, J=7.2 Hz, 1H),
7.00 (t, J=7.4 Hz, 1H), 5.67 (d, J=7.9 Hz, 1H), 4.32 (m, 2H), 3.65
(m, 2H), 3.44 (t, J=9.7 Hz, 2H), 3.11 (dd, J=15.2, 4 Hz, 2H), 2.98
(m, 1H), 2.69 (dd, J=16.3, 9.1 Hz, 1H), 2.26 (m, 2H), 2.08 (m, 2H),
1.91 ppm (m, 4H); 13C NMR (75 MHz, [D6]DMSO): d=172.7, 171.1,
167.3, 164.4, 136.4, 133.5, 126.4, 121.8, 119.2, 118.3, 111.9, 106.2,
79.6, 58.4, 57.5, 55.4, 47.6, 46.0, 45.5, 43.7, 36.0, 29.8, 21.9 ppm; MS
(ESI+): m/z: 455 [M+H]+; Anal. calcd for C23H26N4O6: C 60.78, H
5.77, N 12.33, found: C 60.96, H 5.92, N 12.55.
[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]indole (4’f): Yield: 218 mg
1
(95%): H NMR (300 MHz, [D6]DMSO): d=10.62 (s, 1H), 7.49 (d, J=
7.7 Hz, 1H), 7.36 (d, J=8.1 Hz, 1H), 7.10 (t, J=7.8 Hz, 1H), 6.96 (t,
J=7.6 Hz, 1H), 5.79 (dd, J=3.5, 10.1 Hz, 1H), 4.42 (m, 2H), 3.81 (m,
1H), 3.69 (m, 2H), 3.66 (s, 3H), 3.53 (m, 1H), 3.15 (m, 2H), 2.87 (m,
1H), 2.63 (s, 1H), 2.20 (m, 2H), 1.96 (m, 1H), 1.90 (m, 1H), 1.83 ppm
(m, 5H); IR (KBr): n˜ =3343, 2930, 2835, 1731, 1686, 1641, 1464,
1335, 1291, 740 cmÀ1; MS (ESI+): m/z: 441 [M+H]+.
(5aS,12R,14aS)-5,14-Dioxo-12-(2-glycine-methyl ester-N-ylethyl-
1-yl)-1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo-
[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]indole (4’g): Yield: 196 mg
1
(92%): H NMR (300 MHz, [D6]DMSO): d=10.60 (s, 1H), 7.50 (d, J=
(5aS,12S,14aS)-5,14-Dioxo-12-(2-phenylalanine-N-ylethyl-1-yl)-
1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo-
7.8 Hz, 1H), 7.45 (d, J=7.9 Hz, 1H), 7.15 (t, J=7.3 Hz, 1H), 6.94 (t,
J=7.3 Hz, 1H), 5.86 (dd, J=4.3, 9.1 Hz, 1H), 4.42 (m, 1H), 3.66 (s,
3H), 3.62 (q, J=14.1 Hz, 1H), 3.49 (m, 1H), 3.31 (m, 2H), 3.14 (dd,
J=4.3, 14.2 Hz, 1H), 2.87 (m, 1H), 2.70 (m, 2H), 2.21 (m, 2H), 2.02
(m, 1H), 1.90 ppm (m, 5H); IR (KBr): n˜ =3342, 2934, 2832, 1732,
1665, 1640, 1461, 1335, 1298, 741 cmÀ1; MS (ESI+): m/z: 411 [M+
H]+.
[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]indole (5c): Yield: 231 mg
(95%): mp: 186–1898C; [a]2D0 = +75 (c=0.53, CHCl3/CH3OH, 1:1);
1H NMR (300 MHz, [D6]DMSO): d=11.06 (s, 1H), 10.67 (s, 1H), 7.47
(d, J=7.8 Hz, 1H), 7.38 (d, J=8.1 Hz, 1H), 7.31 (m, 4H), 7.11 (t, J=
7.5 Hz, 2H), 7.02 (t, J=7.7 Hz, 1H), 5.63 (dd, J=11.2, 3.1 Hz, 1H),
4.37 (m, 1H), 4.30 (dd, J=11.5, 3.6 Hz, 1H), 3.61 (m, 3H), 3.12 (m,
3H), 2.98 (m, 2H), 2.37 (m, 1H), 2.28 (m, 1H), 2.18 (m, 1H), 1.99 (m,
1H), 1.88 (m, 2H), 1.25 ppm (m, 2H); 13C NMR (75 MHz, [D6]DMSO):
d=170.1, 167.5, 164.2, 136.4, 135.1, 133.0, 129.9, 129.0, 128.0,
126.5, 122.0, 119.4, 118.5, 111.8, 106.6, 60.5, 58.4, 55.4, 47.3, 45.4,
44.0, 35.5, 30.1, 29.8, 29.5 ppm; MS (ESI+): m/z: 487 [M+H]+; Anal.
calcd for C28H30N4O4: C 69.12, H 6.21, N 11.51, found: C 69.34, H
6.05, N 11.28.
(5aS,12R,14aS)-5,14-Dioxo-12-(2-leucine-methyl ester-N-ylethyl-
1-yl)-1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo-
[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]indole (4’h): Yield: 221 mg
1
(91%): H NMR (300 MHz, [D6]DMSO): d=10.52 (s, 1H), 7.51 (d, J=
7.8 Hz, 1H), 7.46 (d, J=7.9 Hz, 1H), 7.17 (t, J=7.3 Hz, 1H), 7.10 (t,
J=7.5 Hz, 1H), 5.72 (dd, J=11.0, 2.4 Hz, 1H), 4.43 (t, J=5.3 Hz,
1H), 4.39 (dd, J=12.1, 7.9 Hz, 1H), 3.67 (s, 3H), 3.65 (m, 2H), 3.51
(dt, J=7.1, 2.9 Hz, 1H), 3.42 (t, J=6.4 Hz, 1H), 3.15 (dd, J=14.5,
4.4 Hz, 1H), 2.86 (t, J=11.4 Hz, 1H), 2.72 (t, J=9.2 Hz, 1H), 2.33 (s,
1H), 2.27 (m, 2H), 2.19 (dq, J=10.0, 2.4 Hz, 1H), 1.90 (m, 4H), 1.51
(m, 2H), 0.95 ppm (dd, J=11.8, 6.0 Hz, 6H); IR (KBr): n˜ =3340, 2934,
2833, 1738, 1684, 1640, 1465, 1330, 1291, 740 cmÀ1; MS (ESI+): m/
z: 467 [M+H]+.
(5aS,12S,14aS)-5,14-Dioxo-12-(2-tryptophan-N-ylethyl-1-yl)-
1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo-
[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]indole (5d): Yield: 250 mg
(95%): mp: 230–2358C; [a]2D0 = +62 (c=0.39, CHCl3/CH3OH, 1:1);
1H NMR (300 MHz, [D6]DMSO): d=11.55 (s, 1H), 10.59 (s, 1H), 7.60
(d, J=7.9 Hz, 1H), 7.49 (d, J=8.1 Hz, 1H), 7.31 (m, 2H), 7.24 (s, 1H),
7.04 (m, 2H), 6.97 (m, 2H), 5.66 (d, J=8.3 Hz, 1H), 4.27 (m, 2H),
3.57 (m, 2H), 3.41 (m, 1H), 3.23 (dd, J=15.1, 5.3 Hz, 1H), 3.08 (m,
2H), 2.93 (m, 2H), 2.69 (m, 1H), 2.27 (m, 2H), 2.01 (m, 1H),
1.88 ppm (m, 5H); 13C NMR (75 MHz, [D6]DMSO): d=185.6, 167.2,
164.4, 136.7, 136.4, 133.6, 127.8, 126.4, 124.4, 121.8, 121.4, 119.3,
119.0, 118.8, 118.3, 111.8, 110.0, 106.1, 62.9, 58.4, 55.3, 47.7, 45.4,
44.4, 31.3, 29.9, 27.3, 25.8, 22.8, 22.0 ppm; MS (ESI+): m/z: 526
[M+H]+; Anal. calcd for C30H31N5O4: C 68.55, H 5.94, N 13.32,
found: C 68.32, H 5.79, N 13.08.
(5aS,12S/R,14aS)-5,14-Dioxo-12-(2-amino-acid-N-ylethyl-1-yl)-
1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo-
[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]indoles (5a–h and 5’a–h):
A solution of NaOH (300 mg, 7.5 mmol) in CH3OH (5 mL) at 08C
was treated with 4a–h or 4’a–h (0.5 mmol). The reaction mixture
was stirred at RT for 2 h, until TLC (CHCl3/CH3OH, 20:1) indicated
complete disappearance of 4a–h or 4’a–h. The reaction mixture
was adjusted to pH 3 with HCl (2m) and then concentrated in va-
cuo. The residue was purified by column chromatography (CHCl3/
CH3OH, 30:1) to give 5a–h or 5’a–h.
(5aS,12S,14aS)-5,14-Dioxo-12-(2-lysine-N-ylethyl-1-yl)-
(5aS,12S,14aS)-5,14-Dioxo-12-(2-alanine-N-ylethyl-1-yl)-
1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo-
1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo-
[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]indole (5e): Yield: 110 mg
(90%): mp: 220–2258C; [a]2D0 = +36 (c=0.39, CHCl3/CH3OH, 1:1);
1H NMR (300 MHz, [D6]DMSO): d=11.03 (s, 1H), 10.59 (s, 1H), 7.46
(d, J=7.5 Hz, 1H), 7.34 (d, J=7.5 Hz, 1H), 7.10 (t, J=7.5 Hz, 1H),
7.09 (t, J=7.6 Hz, 1H), 5.73 (dd, J=11.1, 3.0 Hz, 1H), 4.37 (m, 1H),
4.31 (dd, J=11.5, 3.6 Hz, 1H), 3.12 (m, 2H), 2.98 (m, 2H), 2.53 (m,
2H), 2.37 (m, 2H), 2.28 (m, 2H), 2.18 (m, 2H), 2.12 (s, 2H), 1.99 (m,
2H), 1.88 (m, 2H), 1.64 (m, 2H), 1.55 (m, 2H), 1.25 ppm (m, 2H);
13C NMR (75 MHz, [D6]DMSO): d=175.0, 169.3, 164.6, 136.4, 133.2,
[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]indole (5a): Yield: 195 mg
(95%): mp: 231–2358C; [a]D20 = +109 (c=0.35, CHCl3/CH3OH, 1:1);
1H NMR (300 MHz, [D6]DMSO): d=11.81 (s, 1H), 10.65 (s, 1H), 7.43
(d, J=7.8 Hz, 1H), 7.35 (d, J=8 Hz, 1H), 7.07 (t, J=7.2 Hz, 1H), 6.98
(t, J=7.3 Hz, 1H), 5.72 (d, J=10.3 Hz, 1H), 4.31 (m, 2H), 3.61 (q, J=
8.6 Hz, 1H), 3.44 (dt, J=10.1, 2.2 Hz, 1H), 3.23 (q, J=6.3 Hz, 1H),
3.11 (dd, J=15.1, 3.96 Hz, 1H), 2.95 (m, 2H), 2.77 (s, 1H), 2.37 (s,
1H), 2.05 (m, 1H), 1.97 (m, 2H), 1.84 (s, 3H), 1.28 ppm (d, J=
ChemMedChem 2011, 6, 2312 – 2322
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
2319

M. Zhao, S. Peng, et al.
MED
126.1, 121.0, 118.7, 118.0, 111.4, 106.3, 58.6, 57.1, 55.2, 47.2, 45.9,
43.0, 42.5, 34.8, 31.6, 31.0, 29.2, 25.1, 22.4, 21.3, 17.1 ppm; MS
(ESI+): m/z: 468 [M+H]+; Anal. calcd for C25H33N5O4: C 64.22, H
7.11, N 14.98, found: C 64.01, H 7.02, N 14.77.
118.2, 111.9, 105.9, 80.0, 58.4, 55.4, 47.5, 46.0, 45.4, 29.9, 25.9, 22.8,
21.8, 16.7 ppm; MS (ESI+): m/z: 411 [M+H]+; Anal. calcd for
C22H26N4O4: C 64.37, H 6.38, N 13.65, found: C 64.55, H 6.52, N
13.42.
(5aS,12S,14aS)-5,14-Dioxo-12-(2-serine-N-ylethyl-1-yl)-
1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo-
(5aS,12R,14aS)-5,14-Dioxo-12-(2-aspartic-acid-N-ylethyl-1-yl)-
1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo-
[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]indole (5 f): Yield: 186 mg
(87%): mp: 230–2358C; [a]2D0 = +83 (c=0.29, CHCl3/CH3OH, 1:1);
1H NMR (300 MHz, [D6]DMSO): d=11.67 (s, 1H), 10.61 (s, 1H), 7.43
(d, J=7.8 Hz, 1H), 7.35 (d, J=8.1 Hz, 1H), 7.07 (t, J=7.6 Hz, 1H),
6.98 (t, J=7.5 Hz, 1H), 5.71 (dd, J=10.2, 3.5 Hz, 1H), 4.32 (m, 1H),
3.71 (m, 1H), 3.61 (m, 2H), 3.44 (m, 1H), 3.12 (m, 2H), 2.95 (m, 1H),
2.75 (s, 1H), 2.28 (m, 2H), 2.09 (s, 1H), 2.04 (m, 1H), 1.97 (m, 1H),
1.85 (m, 5H); 13C NMR (75 MHz, [D6]DMSO): d=174.1, 167.1, 164.4,
136.5, 134.0, 126.5, 121.6, 119.1, 118.2, 111.9, 105.9, 64.5, 61.2, 58.4,
55.4, 47.8, 45.9, 44.6, 31.3, 29.8, 25.8, 23.0 ppm; MS (ESI+): m/z:
427 [M+H]+; Anal. calcd for C22H26N4O5: C 61.96, H 6.15, N 13.14,
found: C 62.19, H 6.00, N 12.92.
[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]indole (5’b): Yield: 222 mg
(95%): mp: 218–2228C; [a]2D0 =À75 (c=0.56, CHCl3/CH3OH, 1:1);
1H NMR (300 MHz, [D6]DMSO): d=11.6 (s, 1H), 7.42 (d, J=7.9 Hz,
1H), 7.35 (d, J=8.2 Hz, 1H), 7.06 (t, J=7.0 Hz, 1H), 6.97 (t, J=
7.4 Hz, 1H), 5.78 (s, 1H), 4.32 (m, 2H), 3.62 (q, J=10.4 Hz, 2H), 3.43
(t, J=8.8 Hz, 2H), 3.09 (dd, J=15.2, 4 Hz, 2H), 2.94 (m, 1H), 2.70
(m, 1H), 2.26 (m, 2H), 2.08 (m, 2H), 1.91 ppm (m, 4H); 13C NMR
(75 MHz, [D6]DMSO): d=174.5, 167.1, 164.5, 136.5, 134.1, 126.5,
121.5, 118.7, 118.1, 111.8, 105.9, 79.7, 58.2, 57.3, 55.1, 47.8, 46.1,
45.4, 29.8, 25.8, 23.8, 21.5 ppm; MS (ESI+): m/z: 469 [M+H]+; Anal.
calcd for C23H26N4O6: C 60.78, H 5.77, N 12.33, found: C 60.99, H
5.91, N 12.10.
(5aS,12S,14aS)-5,14-Dioxo-12-(2-glycine-N-ylethyl-1-yl)-
1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo-
(5aS,12R,14aS)-5,14-Dioxo-12-(2-phenylalanine-N-ylethyl-1-yl)-
1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo-
[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]indole (5g): Yield: 176 mg
(89%): mp: 233–2378C; [a]2D0 = +59 (c=0.37, CHCl3/CH3OH, 1:1);
1H NMR (300 MHz, [D6]DMSO): d=11.85 (s, 1H), 10.59 (s, 1H), 7.43
(d, J=7.8 Hz, 1H), 7.37 (d, J=8.1 Hz, 1H), 7.07 (t, J=7.2 Hz, 1H),
6.98 (t, J=7.2 Hz, 1H), 5.78 (dd, J=9.9, 4.3 Hz, 1H), 4.32 (m, 1H),
3.62 (q, J=14.8 Hz, 1H), 3.43 (m, 1H), 3.21 (m, 2H), 3.10 (dd, J=
15.1, 4.0 Hz, 1H), 2.95 (m, 1H), 2.85 (m, 2H), 2.26 (m, 2H), 2.05 (m,
1H), 1.91 ppm (m, 5H); 13C NMR (75 MHz, [D6]DMSO): d=173.2,
167.0, 164.4, 136.6, 133.9, 126.5, 121.5, 119.0, 118.1, 111.9, 106.0,
58.4, 55.4, 51.6, 47.6, 45.3, 31.5, 29.8, 25.9, 22.6, 21.9 ppm; MS
(ESI+): m/z: 397 [M+H]+; Anal. calcd for C21H24N4O4: C 63.62, H
6.10, N 14.13, found: C 63.41, H 5.93, N 13.90.
[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]indole (5’c): Yield: 232 mg
(96%): mp: 180–1838C; [a]2D0 =À24 (c=0.33, CHCl3/CH3OH, 1:1);
1H NMR (300 MHz, [D6]DMSO): d=11.32 (s, 1H), 7.44 (d, J=7.8 Hz,
1H), 7.33 (d, J=8.1 Hz, 1H), 7.27 (m, 3H), 7.20 (m, 1H), 7.09 (t, J=
7.5 Hz, 2H), 6.99 (t, J=7.5 Hz, 1H), 5.67 (dd, J=10.5, 2.9 Hz, 1H),
4.30 (m, 2H), 3.60 (m, 2H), 3.43 (m, 2H), 3.09 (m, 2H), 2.97 (m, 2H),
2.83 (m, 1H), 2.75 (m, 1H), 2.26 (m, 2H), 2.04 (m, 1H), 1.92 (m, 2H),
1.85 ppm (m, 2H); 13C NMR (75 MHz, [D6]DMSO): d=171.8, 167.2,
164.3, 138.0, 136.4, 133.6, 129.7, 128.7, 126.9, 126.5, 121.7, 119.2,
118.3, 111.8, 106.2, 62.8, 58.4, 55.4, 47.6, 45.4, 44.1, 37.4, 31.2, 29.9,
25.8, 21.9, 21.5 ppm; MS (ESI+): m/z: 487 [M+H]+; Anal. calcd for
C28H30N4O4: C 69.12, H 6.21, N 11.51, found: C 69.30, H 6.06, N
11.73.
(5aS,12S,14aS)-5,14-Dioxo-12-(2-leucine-N-ylethyl-1-yl)-
1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo-
(5aS,12R,14aS)-5,14-Dioxo-12-(2-tryptophan-N-ylethyl-1-yl)-
1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo-
[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]indole (5h): Yield: 199 mg
(88%): mp: 127–1308C; [a]2D0 = +83 (c=0.36, CHCl3/CH3OH, 1:1);
1H NMR (300 MHz, [D6]DMSO): d=11.40 (s, 1H), 10.60 (s, 1H), 7.44
(d, J=7.8 Hz, 1H), 7.35 (d, J=8.1 Hz, 1H), 7.09 (t, J=7.3 Hz, 1H),
7.00 (t, J=7.5 Hz, 1H), 5.66 (dd, J=11.0, 2.8 Hz, 1H), 4.34 (t, J=
5.0 Hz, 1H), 4.31 (dd, J=12.5, 8.2 Hz, 1H), 3.60 (m, 1H), 3.44 (dt,
J=7.2, 2.6 Hz, 1H), 3.32 (t, J=6.9 Hz, 1H), 3.11 (dd, J=15.1, 4.1 Hz,
1H), 2.96 (t, J=12.1 Hz, 1H), 2.82 (t, J=9.9 Hz, 1H), 2.35 (s, 1H),
2.29 (m, 1H), 2.18 (dq, J=10.7, 2.8 Hz, 1H), 1.90 (m, 4H), 1.76 (m,
2H), 1.53 (m, 2H), 0.90 ppm (dd, J=12.4 Hz, 6.5 Hz, 6H); 13C NMR
(75 MHz, [D6]DMSO): d=171.9, 167.3, 164.3, 136.4, 133.6, 126.4,
121.8, 119.2, 118.3, 111.8, 106.2, 60.7, 58.4, 58.1, 55.4, 47.6, 45.7,
45.4, 43.9, 30.9, 29.8, 25.7, 24.9, 23.3, 22.7 ppm; MS (ESI+): m/z:
453 [M+H]+; Anal. calcd for C25H32N4O4: C 66.35, H 7.13, N 12.38,
found: C 66.54, H 7.29, N 12.60.
[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]indole (5’d): Yield: 250 mg
(93%): mp: 148–1528C; [a]2D0 =À50 (c=0.52, CHCl3/CH3OH, 1:1);
1H NMR (300 MHz, [D6]DMSO): d=11.51 (s, 1H), 10.61 (s, 1H), 7.60
(d, J=7.9 Hz, 1H), 7.44 (d, J=8.1 Hz, 1H), 7.33 (dd, J=3.7 Hz,
8.1 Hz, 2H), 7.26 (d, J=1.8 Hz, 1H), 7.07 (m, 2H), 6.99 (t, J=7.6 Hz,
1H), 6.95 (t, J=7.6 Hz, 1H), 5.70 (dd, J=10.6, 3.2 Hz, 1H), 4.31 (m,
2H), 3.69 (t, J=6.1 Hz, 1H), 3.58 (m, 1H), 3.41 (m, 1H), 3.25 (dd, J=
15.3, 5.6 Hz, 1H), 3.17 (dd, J=15.2, 6.8 Hz, 1H), 3.10 (dd, J=15.1,
4.0 Hz, 1H), 2.94 (m, 2H), 2.86 (m, 1H), 2.32 (m, 1H), 2.23 (m, 1H),
2.07 (m, 1H), 1.92 (s, 3H), 1.81 ppm (m, 2H); 13C NMR (75 MHz,
[D6]DMSO): d=172.4, 167.3, 164.3, 136.7, 136.4, 133.6, 127.8, 126.5,
124.4, 121.7, 121.4, 119.2, 118.9, 118.7, 118.3, 111.8, 109.7, 106.2,
62.2, 58.4, 55.4, 47.6, 45.4, 44.1, 29.8, 27.3, 25.8 ppm; MS (ESI+): m/
z: 526 [M+H]+; Anal. calcd for C30H31N5O4: C 68.55, H 5.94, N
13.32, found: C 68.34, H 5.77, N 13.10.
(5aS,12R,14aS)-5,14-Dioxo-12-(2-alanine-N-ylethyl-1-yl)-
1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo-
(5aS,12R,14aS)-5,14-Dioxo-12-(2-lysine-N-ylethyl-1-yl)-
[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]indole (5’a): Yield: 195 mg
(95%): mp: 224–2288C; [a]2D0 =À67 (c=0.45, CHCl3/CH3OH, 1:1);
1H NMR (300 MHz, [D6]DMSO): d=12.00 (s, 1H), 10.62 (s, 1H), 7.43
(d, J=7.8 Hz, 1H), 7.37 (d, J=8 Hz, 1H), 7.07 (t, J=7.6 Hz, 1H), 6.98
(t, J=7.6 Hz, 1H), 5.83 (d, J=10.3 Hz, 1H), 4.34 (dd, J=11.7, 4.3 Hz,
2H), 3.64 (q, J=6.3 Hz, 2H), 3.43 (dt, J=2.6 Hz, 9.0 Hz, 1H), 3.26 (q,
J=6.6 Hz, 1H), 3.12 (dd, J=15.0, 3.8 Hz, 1H), 2.96 (t, J=12.6 Hz,
1H), 2.88 (s, 1H), 2.28 (t, J=5.2 Hz, 1H), 2.08 (s, 1H), 1.97 (m, 1H),
1.87 (s, 4H), 1.30 ppm (d, J=6.9 Hz, 3H); 13C NMR (75 MHz,
[D6]DMSO): d=173.6, 167.3, 164.3, 136.6, 133.8, 126.4, 121.5, 119.0,
1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo-
[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]indole (5’e): Yield: 112 mg
(90%): mp: 150–1548C; [a]2D0 =À79 (c=0.33, CHCl3/CH3OH, 1:1);
1H NMR (300 MHz, [D6]DMSO): d=10.97 (s, 1H), 10.55 (s, 1H), 7.43
(d, J=7.5 Hz, 1H), 7.31 (d, J=7.5 Hz, 1H), 7.12 (t, J=7.5 Hz, 1H),
7.07 (t, J=7.6 Hz, 1H), 5.72 (dd, J=11.1, 3.0 Hz, 1H), 4.38 (m, 1H),
4.34 (dd, J=11.5, 3.6 Hz, 1H), 3.11 (m, 2H), 2.96 (m, 2H), 2.40 (m,
2H), 2.30 (m, 2H), 2.26 (m, 2H), 2.14 (m, 2H), 2.01 (s, 2H), 1.98 (m,
2H), 1.87 (m, 2H), 1.62 (m, 2H), 1.53 (m, 2H), 1.24 ppm (m, 2H);
13C NMR (75 MHz, [D6]DMSO): d=175.2, 169.0, 164.3, 136.1, 133.0,
2320
www.chemmedchem.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 2312 – 2322

Urokinase Inhibitors
126.6, 121.3, 118.5, 118.4, 111.2, 106.0, 58.3, 57.6, 55.0, 49.9, 47.8,
43.2, 42.9, 34.3, 31.3, 31.1, 29.5, 25.4, 22.1, 21.0, 17.6 ppm; MS
(ESI+): m/z: 468 [M+H]+; Anal. calcd for C25H33N5O4: C 64.22, H
7.11, N 14.98, found: C 64.04, H 7.26, N 15.21.
in accordance with the requirements of the Animal Welfare Act
and according to the committee’s guide for the care and use of
laboratory animals.
In vivo tail bleeding time assay of oral 5a–h and 5’a–h: Male ICR
mice were housed in a 12 h light/12 h dark cycle at 21Æ28C for
one day before operation. Each of them was administered (oral
gavage) 0.6 mL of normal saline (NS) containing each of 5a–h and
5’a–h (0.1 mmolkgÀ1), or 0.6 mL NS containing aspirin
(165 mmolkgÀ1) or 0.6 mL NS alone. Thirty minutes after administra-
tion, mice were placed in a tube holder with tail protruding, and a
2 mm cut was made on the tail. Flowing blood was gently wiped
away with a tissue every 30 s until bleeding ceased, and the ob-
served bleeding time was recorded.
(5aS,12R,14aS)-5,14-Dioxo-12-(2-serine-N-ylethyl-1-yl)-
1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo-
[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]indole (5’f): Yield: 183 mg
(86%): mp: 224–2308C; [a]2D0 =À67 (c=0.36, CHCl3/CH3OH, 1:1);
1H NMR (300 MHz, [D6]DMSO): d=11.87 (s, 1H), 10.60 (s, 1H), 7.42
(d, J=7.8 Hz, 1H), 7.39 (d, J=8.1 Hz, 1H), 7.06 (t, J=7.1 Hz, 1H),
6.97 (t, J=7.5 Hz, 1H), 5.84 (dd, J=10.2, 3.6 Hz, 1H), 4.33 (m, 1H),
3.69 (dd, J=11.0, 4.8 Hz, 1H), 3.61 (m, 2H), 3.44 (m, 1H), 3.12 (m,
2H), 2.95 (m, 1H), 2.82 (m, 1H), 2.28 (m, 2H), 2.07 (s, 1H), 2.03 (m,
1H), 1.97 (m, 1H), 1.87 pp, (m, 5H); 13C NMR (75 MHz, [D6]DMSO):
d=173.7, 167.1, 164.4, 136.5, 133.9, 126.4, 121.5, 119.0, 118.2,
112.0, 105.9, 64.7, 61.7, 58.4, 55.4, 47.5, 45.4, 44.5, 31.5, 29.8, 25.9,
22.8 ppm; MS (ESI+): m/z: 427 [M+H]+; Anal. calcd for C22H26N4O5:
C 61.96, H 6.15, N 13.14, found: C 61.75, H 6.31, N 12.91.
In vivo thrombogenesis assay of oral 5a–h and 5’a–h: Male Wistar
rats were fed 0.1 mmolkgÀ1 of each of 5a–h and 5’a–h in NS or as-
pirin (165 mmolkgÀ1) in NS (0.6 mL) or NS alone, and then the rats
were anesthetized with sodium pentobarbital (80.0 mgkgÀ1, i.p.).
Thirty minutes later the right carotid artery and left jugular vein
were separated. A weighed 6 cm thread was inserted into a poly-
ethylene tube, which was filled with heparin sodium (50 IUmLÀ1 in
NS) and one end was inserted into the left jugular vein, while the
other was inserted into the right carotid artery. Blood flowed from
the right carotid artery to the left jugular vein through the polyeth-
ylene tube for 15 min. The thread was removed, and the weight of
the wet thrombus was recorded.
(5aS,12R,14aS)-5,14-Dioxo-12-(2-glycine-N-ylethyl-1-yl)-
1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo-
[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]indole (5’g): Yield: 174 mg
(88%): mp: 223–2278C; [a]2D0 =À72 (c=0.36, CHCl3/CH3OH, 1:1);
1H NMR (300 MHz, [D6]DMSO): d=11.82 (s, 1H), 10.62 (s, 1H), 7.41
(d, J=7.8 Hz, 1H), 7.36 (d, J=8.0 Hz, 1H), 7.06 (t, J=7.2 Hz, 1H),
6.99 (t, J=7.3 Hz, 1H), 5.74 (dd, J=9.9, 4.3 Hz, 1H), 4.33 (m, 1H),
3.62 (q, J=8.6 Hz, 1H), 3.45 (m, 1H), 3.23 (m, 2H), 3.12 (dd, J=
15.1, 4.0 Hz, 1H), 2.94 (m, 1H), 2.87 (m, 2H), 2.27 (m, 2H), 2.05 (m,
1H), 1.90 ppm (m, 5H); 13C NMR (75 MHz, [D6]DMSO): d=173.0,
167.2, 164.7, 136.4, 133.8, 126.6, 121.4, 119.1, 118.2, 111.7, 106.3,
58.5, 55.2, 51.7, 47.5, 45.2, 31.4, 29.9, 25.7, 22.8, 21.7 ppm; MS
(ESI+): m/z: 397 [M+H]+; Anal. calcd for C21H24N4O4: C 63.62, H
6.10, N 14.13, found: C 63.82, H 6.26, N 14.35.
In vitro thrombus clot lysis assay of UK with and without 5c: The cy-
lindrical thrombus prepared from rat blood was carefully removed
from the cylindrical thrombus-forming tube and suspended into an
incubation bottle filled with 8 mL distilled water for 1 h. The
thrombus was then removed from the bottle, weighed precisely to
record its initial weight, and suspended in another incubation
bottle filled with 8 mL NS or a solution of UK (100 IUmLÀ1, Sigma,
UK) in 8 mL NS or UK (100 IUmLÀ1) plus 5c (100, 10, or 1 nm) in
8 mL NS. The bottle was incubated at 378C and agitated on a rock-
ing-bed (70 rpm) for 1 h, and the thrombus was precisely weighed
to record its final weight. Subtracting the final weight from the ini-
tial weight, the decreased weight of the thrombus was obtained
and used to represent the in vitro thrombus lysis potency.
(5aS,12R,14aS)-5,14-Dioxo-12-(2-leucine-N-ylethyl-1-yl)-
1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo-
[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]indole (5’h): Yield: 201 mg
(89%): mp: 188–1918C; [a]2D0 =À85 (c=0.52, CHCl3/CH3OH, 1:1);
1H NMR (300 MHz, [D6]DMSO): d=11.4 (d, 1H), 10.62 (s, 1H), 7.45
(d, J=7.8 Hz, 1H), 7.35 (d, J=8.1 Hz, 1H), 7.09 (t, J=7.3 Hz, 1H),
7.00 (t, J=7.4 Hz, 1H), 5.74 (d, J=9.3 Hz, 1H), 4.34 (m, 1H), 3.60 (q,
J=8.6 Hz, 2H), 3.43 (dt, J=8.2, 2.2 Hz, 1H), 3.28 (q, J=6.5 Hz, 1H),
3.12 (dd, J=15.2, 3.9 Hz, 1H), 2.96 (t, J=17.1 Hz, 1H), 2.83 (t, J=
9.7 Hz, 1H), 2.34 (s, 1H), 2.26 (m, 1H), 2.11 (s, 1H), 1.90 (m, 4H),
1.71 (m, 2H), 1.52 (m, 2H), 0.90 ppm (dd, J=12.5 Hz, 6.5 Hz, 6H);
13C NMR (75 MHz, [D6]DMSO): d=173.0, 171.7, 167.6, 164.4, 136.5,
133.1, 126.4, 121.9, 119.3, 118.3, 111.9, 106.3, 60.9, 58.4, 55.4, 47.5,
46.0, 45.5, 43.7, 30.2, 29.8, 25.8, 24.8, 23.0, 19.0 ppm; MS (ESI+): m/
z: 453 [M+H]+; Anal. calcd for C25H32N4O4: C 66.35, H 7.13, N
12.38, found: C 66.56, H 7.00, N 12.15.
In vivo thrombolytic assay of UK on rats pretreated with oral 5c:
Male Wistar rats were fed with three doses (100, 10, and
1 nmolkgÀ1) of 5c in NS or NS (0.6 mL) alone. The rats were then
anesthetized with sodium pentobarbital (80.0 mgkgÀ1, i.p.). Thirty
minutes later the right carotid artery and left jugular vein were
separated. The cylindrical thrombus-supporting helix (15 circles,
pitch 1.2 mm, diameter 1.0 mm) was placed into the cylindrical
thrombus-forming tube, and this tube was then immediately filled
with arterial blood (0.2 mL) from the right carotid artery. After
15 min the cylindrical thrombus was carefully removed from the
thrombus-forming tube, weighed precisely to record its initial
weight, and then inserted into a polyethylene tube. The tube was
filled with heparin sodium (50 IUmLÀ1 in NS), and one of the ends
was inserted into the left jugular vein. Heparin sodium was inject-
ed via the other end of the polyethylene tube as the anticoagu-
Biology
General: Biological data were analyzed using an ANOVA test, and
p <0.05 was considered statistically significant.
Animals: Male imprinting control region (ICR) mice weighing 28–
31 g and male Wister rats weighing 250–300 g, purchased from
the Experimental Animal Center at Peking University Health Sci-
ence Center, were maintained at 218C with a natural day/night
cycle in a conventional animal colony. The assessments described
herein were performed according to a protocol reviewed and ap-
proved by the Ethics Committee of Capital Medical University. The
committee assures the welfare of animals in research is maintained
lant, followed by injection of a solution of UK (3 mL, 20000 IUkgÀ1
)
in NS. The blood was circulated through the polyethylene tube for
90 min, after which the cylindrical thrombus was carefully taken
out of the polyethylene tube, and its final weight was recorded
precisely. Subtracting the final weight from the initial weight, the
decreased weight of the thrombus was obtained and used to rep-
resent the in vivo thrombolytic potency.
ChemMedChem 2011, 6, 2312 – 2322
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
2321

M. Zhao, S. Peng, et al.
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acrylamide) of 4%, and contained 10% glycerol (GE healthcare,
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tration 0.1 UmLÀ1) and UK (5 mL, final concentration 100 UmLÀ1
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plus 5 mL NS or a solution of PLG (5 mL, final concentration
0.1 UmLÀ1) and UK (5 mL, final concentration 100 UmLÀ1) plus 5 mL
5c (in a series of final concentrations ranging from 0.01 to
2500 ngmLÀ1) was incubated at 378C for 30 min. The separation of
these solutions was performed at a constant current of ~100 mA,
starting at 90 V (increasing to ~120 V at the end of the run), and
lasting for 2 h until the bromophenol blue (as a tracer, Sigma, USA)
band had reached the bottom of the gel. Gels were stained in
methanol/acetic acid/water (45:10:45) with coomassie brilliant blue
R250 (0.1%, Amresco, USA) for 4 h. The gels were kept in metha-
nol/acetic acid/water (10:10:80) for ~14 h for destaining.
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Keywords: antifibrinolytic agents · hemostasis · medicinal
chemistry · peptidomimetics · urokinase antagonism
Received: July 13, 2011
Revised: September 7, 2011
Published online on September 23, 2011
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