Structure-inhibitory activity relationship of plasmin and plasma kallikrein inhibitors

Article abstract of DOI:10.1248/cpb.49.1457

Based on the structure of Tra-Tyr(O-Pic)-octylamide, a portion of the octylamine was replaced with moieties bearing hydrophobic, basic or acidic groups. Replacement of the C-terminal residue with a moiety bearing a hydrophobic group gave the proper affinity of the inhibitor to both plasmin (PL) and plasma kallikrein (PK). While addition of a basic residue did not improve the affinity of the inhibitor, a carboxylic acid attached to the phenyl ring increased the PK selectivity of the inhibitor.

Full text of DOI:10.1248/cpb.49.1457

November 2001
Chem. Pharm. Bull. 49(11) 1457—1463 (2001)
1457
Structure–Inhibitory Activity Relationship of Plasmin and Plasma
Kallikrein Inhibitors1)
Yuko TSUDA,a,b Mayako TADA,a,c Keiko WANAKA,c Utako OKAMOTO,c Akiko HIJIKATA-OKUNOMIYA,d
Shosuke OKAMOTO,c and Yoshio OKADA*,a,b
Faculty of Pharmaceutical Sciences,a and High Technology Research Center,b Kobe Gakuin University, Nishi-ku, Kobe
651–2180, Japan, Kobe Research Projects on Thrombosis and Haemostasis,c 3–15–18 Asahigaoka, Tarumi-ku, Kobe
655–0033, Japan, and Faculty of Health Sciences, Kobe University School of Medicine,d Suma-ku, Kobe 654–0142, Japan.
Received June 27, 2001; accepted August 3, 2001
Based on the structure of Tra-Tyr(O-Pic)-octylamide, a portion of the octylamine was replaced with moi-
eties bearing hydrophobic, basic or acidic groups. Replacement of the C-terminal residue with a moiety bearing
a hydrophobic group gave the proper affinity of the inhibitor to both plasmin (PL) and plasma kallikrein (PK).
While addition of a basic residue did not improve the affinity of the inhibitor, a carboxylic acid attached to the
phenyl ring increased the PK selectivity of the inhibitor.
Key words enzyme-selective inhibitor; plasma kallikrein inhibitor; plasmin inhibitor; structure–inhibitory activity relationship
Plasmin (PL) (EC 3.4.21.7) is a serine protease, which fibrinolytic system occurs, as in thrombolytic therapy with
plays a central role in the fibrinolytic process. Fibrinolysis is streptokinase, t-PA or u-PA, e-aminocaproic acid (EACA)22)
a physiological response to prevent excessive Fn accumula- and trans-4-aminomethylcyclohexanecarboxylic acid (t-
tion in the circulating blood.2) The fibrinolytic system is a AMCHA),23) which are employed clinically as a PL inhibitor,
highly modulated enzyme system, in which a series of serine do not suffice to inhibit circulating PL, because they inhibit
proteases are involved. The generation of PL is strongly reg- PL by blocking the lysine binding sites (LBS) but not the cat-
ulated by two activators and three protease inhibitors. The alytic site. The active center-directed PL inhibitors might be
two major activators, the tissue-type PA (t-PA)3) and the uri- beneficial in controlling excessive bleeding that frequently
nary-type PA (u-PA, urokinase),4) are serine proteases which occurs in thrombolytic therapy and organ transplantation.
cleave a peptide bond in plasminogen, the zymogen form, to
Among the active center-directed inhibitors we developed,
generate PL on the biological surface. Two protease in- Tra-Tyr(O-2-bromobenzyloxycarbonyl)-octylamide (com-
hibitors include the PA inhibitors, which are called PAI,5) and pound I)24) specifically inhibited PL (IC50ϭ0.80 mM) and Tra-
the third is a PL inhibitor, namely a2-antiplasmin.6) Addi- Phe-4-carboxymethylanilide (compound II)25) specifically in-
tionally, the contribution of the contact system to the process hibited PK (IC50ϭ1.3 mM) (Fig. 1). In order to improve solu-
of plasminogen activation should be considered. Plasma bility, a picolyl (O-Pic) residue was incorporated into the
kallikrein (PK) (EC 3.4.21.34), which is one component of molecule as Tyr derivative to yield Tra-Tyr(O-Pic)-octy-
the contact system,7) probably mediates the activation of pro- lamide (compound III),26) which inhibited PL with an IC50
urokinase and accelerates the PL formation,8) while PK was value of 0.53 mM. Thus, this replacement did not reduce the
reported to activate the contact phase of coagulation.9)
inhibitory effect of peptide and increased the solubility of the
Although the primary function of PL is to remove intravas- peptide. From these results, it was deduced that we could de-
cularly formed thrombin by the degradation of Fn, PL has sign enzyme-selective inhibitors by combination of the Tra-
many other actions, such as the degradation of adhesive Tyr(O-Pic) sequence with various C-terminal residues. In this
macromolecules,10—13) the activation of growth factors,14,15) study, we present data on low molecular weight inhibitors
coagulation factor modification,16,17) the activation of t-PA18) based on the structure of compound III. The functional and
and u-PA,19) and so on. As a result, PL might function in structural roles of the C-terminal residue were investigated
pathologic phenomena, such as inflammation20) and tumor by replacement with moieties bearing hydrophobic, basic and
cells growth and metastasis.21) PK also has a broad activity acidic groups.
spectrum. PK might be involved in the induction of several
First of all, replacement of octylamine with aniline con-
pathologic phases including allergic rhinitis, asthma, arthritis taining various methylene moieties provided peptides 1—6.
and so on, due to kinin generation.9) However, detailed stud- The IC50 values of those peptides are summarized with com-
ies of the role of these enzymes remain to be performed.
parison with those of compound III in Table 1. In both PL
The regulation of PL and PK has been our interest in the and PK, systematic extension of the length by a methylene
regards of control of fibrinolytic actions as well as non-fibri- group (peptide 1—5) increased the inhibitory activity. Pep-
nolytic actions. We have focused our attention on the synthe- tide 5 (five methylene group) had an IC50 value of 0.58 and
sis of active center-directed PL and PK inhibitors. The first 0.88 mM, which were a 2.9-fold and a 1.9-fold increase in
aim of our study was to obtain a powerful new tool to explore affinity, respectively, compared with those of peptide 1 (with
the role of PL and PK in coagulation–fibrinolytic system. An one methylene group). It seems that the hydrophobic group
enzyme-selective inhibitor would be useful to discriminate enhanced the interaction between peptide and enzyme. How-
the functions of PL and PK in the fibrinolytic system. The ever, an insertion of phenyl ring reduced the selectivity of
second aim was to develop novel type of PL or PK inhibitor PL/PK; peptide 5 inhibited PK as well as PL, although com-
available for clinical therapy. When massive activation of the pound III inhibited PL a 60-fold more strongly than PK.
To whom correspondence should be addressed. e-mail: okada@pharm.kobegakuin.ac.jp
© 2001 Pharmaceutical Society of Japan

1458
Vol. 49, No. 11
Fig. 1. Structure and Inhibitory Activity of Compounds I—III
lectivity ratio was increased rather than being retained; the
selectivity of PL/PK (reciprocal of PL-to-PK IC50 ratio) of
peptide 10 was 86, while that of compound III was 56.
Third, the effect of an acidic group was investigated. Pep-
tide 13 and 14 were prepared and evaluated as shown in
Table 2. Among peptides 1, 12 and 13, peptide 13 was the
most potent PK inhibitor with an IC50 value of 1.3 mM, imply-
ing that incorporation of acidic group gave PK selectivity to
inhibitor. In contrast, peptide 14 lost affinity for PK
(IC50ϭ32 mM). Those findings indicated that the active-center
of PL would have a rather wide hydrophobic region, while
the active-center of PK would be a region consisting of both
aromatic and basic side chains. The property of the C-termi-
nal residue could regulate the enzyme selectivity ratio. On
the other hand, the amino group of the Tra moiety in peptides
1—14 would be critical to bind the peptide to the enzyme. In
fact, the study on X-ray crystal structures of trypsin–PKSI-
527 (compound II) complex revealed that the amino group of
the aminomethylcyclohexane moiety was hydrogen-bound to
the carboxyl oxygens of Asp189.27) We can expect that inter-
actions between these enzymes and the amino group of Tra
in peptides 1—14 are similar to that between trypsin and the
amino group of Tra in PKSI-527, because the amino acid se-
quences of enzymes in coagulation–fibrinolysis system are
highly homologous to that of trypsin. Indeed, peptides 1—14
had an inhibitory effect not only on fibrinolysis, but also on
amidolysis by PL with micromole range (Tables 1, 2). t-
AMCHA inhibited fibrinolytic and amidolytic actitivity of
PL with IC50 of 60 mM and Ki of 40 mM, respectively.28) The
inhibitory effect of peptides 1—14 on amidolysis was ap-
proximately 5000 times greater than that of t-AMCHA.
Fig. 2. Structure of Peptides 1—14
Next, the addition of basic group to C-terminal part was
examined. According to the previous structure–activity rela-
tionship (SAR) study, the presence of carboxyl group in C-
terminal part is not favorable for the inhibition of PL. The in-
troduction of a carboxyl group as in compound II contributed
to an increase of PK/PL selectivity. Instead of carboxyl
group, a series of peptides incorporated a basic group at the
C-terminus. The resulting peptides 7—12 and their IC50 val-
ues are summarized in Table 2. Peptide 10 inhibited PL with
an IC50 value of 3.8 mM; this affinity is one-seventh of that of
compound III. Furthermore, addition of basic group also re-
duced the affinity of the peptide with PK, resulting in an IC50
value of 330 mM (peptide 10), a loss of more than 10-fold rel-
ative to compound III. Thus, incorporation of a basic group
showed no improvement in affinity; however, the PL/PK se-
In conclusion, replacement of the C-terminal residue with
moiety bearing an aromatic or hydrophobic group increased
affinity of the inhibitor to both PL and PK. Incorporation of
an acidic group was not favorable for inhibition of PL, but
this modification yielded a PK specific inhibitor. In particu-

November 2001
1459
Table 1. The IC50 Values (mM) of Tra-Tyr(O-Pic)-NH
(CH2)n-CH3 (1—6) for Various Enzymes
PL
PK
Urokinase
Thrombin
S-2238
Trypsin
Peptide
n
S-2251
Fn
S-2302
S-2444
Fg
S-2238
1
2
3
4
5
6
1
2
3
4
5
6
1.7
1.4
0.51
0.38
0.39
0.18
0.23
0.35
0.36
1.7
1.7
1.2
60
42
40
480
300
130
Ͼ500
Ͼ200
Ͼ200
Ͼ400
Ͼ100
Ͼ100
Ͼ50
Ͼ20
Ͼ25
1.3
1.7
1.2
0.62
0.85
0.65
1.1
0.99
0.54
0.58
0.97
0.53
1.6
43
0.88
0.82
30
Ͼ200
Ͼ200
5.3
Ͼ12.5
Ͼ100
Compound III
–NH(CH2)7–CH3
Table 2. The IC50 Values (mM) of Tra-Tyr(O-Pic)-NH-X (7—14) for Various Enzymes
PL
PK
Urokinase
S-2444
Thrombin
Trypsin
S-2238
Peptide
X
S-2251
Fn
S-2302
S-2238
Fg
7
8
–(CH2)2–NH2
–(CH2)4–NH2
–(CH2)6–NH2
84
71
14
3.8
Ͼ1000
910
51
50
30
31
18
Ͼ1000
Ͼ1000
Ͼ1000
Ͼ1000
Ͼ1000
Ͼ1000
Ͼ1000
Ͼ1000
Ͼ1000
Ͼ1000
28
28
35
9
5.4
3.8
1.8
510
4.0
2.0
0.7
a)
10
11
–(CH2)7–NH2
–(CH2)8–NH2
2.4
1.1
330
170
12
CH2–NH2
1.6
1.1
28
27
Ͼ1000
Ͼ1000
1.0
13
14
CH2–COOH
–(CH2)7–COOHa)
–(CH2)7–CH3
9.6
7.4
1.3
32
30
52
29
5.3
Ͼ1000
Ͼ500
Ͼ400
Ͼ1000
Ͼ500
Ͼ100
3.8
4.2
1.1
5.5
4.6
Compound III
0.53
0.36
a) Cited from ref. 26).
which was collected by filtration and recrystallized from EtOH. Yield, mp,
[a]D25, elemental analysis and Rf value are summarized in Table 3.
General Procedure for Preparation of Boc-Tra-Tyr(O-Pic)-NH
(CH2)n-CH3 (n؍1—6) Boc-Tra-OH (0.26 g, 1.0 mmol) was added to the
corresponding amine component [prepared from the corresponding Boc-
Tyr(O-Pic)-NH-(CH2)n-CH3 derivative (1.0 mmol) and TFA (1.53 ml,
20 mmol) in the presence of anisole (0.15 ml, 1.4 mmol) as usual] in DMF
(30 ml) containing Et3N (0.34 ml, 2.4 mmol) and BOP reagent (0.53 g,
1.2 mmol). The reaction mixture was stirred at room temperature for 15 h.
After removal of the solvent, the residue was extracted with AcOEt. The ex-
tract was washed with 10% citric acid, 5% Na2CO3 and water, dried over
Na2SO4 and evaporated down. Diethyl ether was added to the residue to give
a white precipitate, which was collected by filtration and recrystallized from
EtOH. Yield, mp, [a]D25, elemental analysis and Rf values are summarized in
Table 4.
lar, the addition of carboxylic acid to the phenyl ring pro-
duced a selective PK inhibitor. Replacement with basic
residue did not improve the affinity of inhibitor. Thus, we de-
scribed data involving the active site of PL and PK by the ap-
plication of specific modifications in the peptide inhibitors.
We can emphasize that there is possibility to develop more
potent and selective PL or PK inhibitors by modification of
Tyr/Phe part and/or C-terminal part (Fig. 1). More potent PL
inhibitors than compound III are currently under study.
Experimental
Melting points were determined on a Yanagimoto micro-melting point ap-
paratus without correction. Optical rotations were measured with an auto-
matic polarimeter, model DIP-360 (Japan Spectroscopic Co.). Time of flight-
mass spectra (TOF-MS) were obtained on a KOMPACT MALDI IV mass
spectra (Kratos Analytical). Waters model 600E was used for HPLC analysis
and purification. Peptides were purified by reverse phase HPLC on a C18
semi-preparative column (20ϫ250 mm, Nakarai). The column was eluted in
40 min using a linear gradient from 10 to 50% acetonitrile in water, both
containing 0.05% TFA, at flow rate of 10 ml/min. The detection wavelength
was 220 nm. On TLC (Kieselgel G. Merck) Rf 1, Rf 2, Rf 3, Rf 4, Rf 5, Rf 6
and Rf 7 values refer to the solvent systems consisting of CHCl3–MeOH–
AcOH (90 : 8 : 2); CHCl3–MeOH–H2O (89 : 10 : 1); CHCl3–MeOH–H2O
(8 : 3 : 1, lower phase); n-BuOH–AcOH–H2O (4 : 1 : 5, upper phase); n-
BuOH–AcOH–pyridine–H2O (4 : 1 : 1 : 2); n-BuOH–AcOH–pyridine–H2O
(1 : 1 : 1 : 1); and AcOEt–n-hexane (1 : 1), respectively.
General Procedure for Preparation of H-Tra-Tyr(O-Pic)-NH
(CH2)n-CH3·TFA (n؍1—6) Boc-Tra-Tyr(O-Pic)-NH
(CH2)n-CH3
(0.30 mmol) was dissolved in TFA (1.28 ml, 17 mmol) containing anisole
(0.04 ml, 0.34 mmol) at 0 °C. The reaction mixture was stirred at room tem-
perature for 60 min. Diethyl ether was added to the solution to yield a white
precipitate, which was collected by filtration and dried over KOH pellets in
vacuo. Each product was lyophilized from water afford an amorphous pow-
der. Yield, mp, [a]D25, elemental analysis and Rf values are summarized in
Table 5.
General Procedure for Preparation of Fmoc-Tyr(O-Pic)-NH-(CH2)n-
NH-Boc (n؍2, 4, 6, 8) A solution of Boc-NH-(CH2)n-NH2 (5.5 mmol)29)
in DMF (50 ml) was added to Fmoc-Tyr(O-Pic)-OH (6.0 mmol) in DMF
(50 ml) containing BOP (2.6 g, 6.0 mmol), HOBt (0.81 g, 6.0 mmol) and
DIPEA (1.0 ml, 6.0 mmol) at 0 °C and the reaction mixture was stirred at the
same temperature for 5 min and was further stirred at room temperature
overnight. After removal of the solvent, the residue was extracted with
AcOEt. The extract was washed with 10% citric acid, 5% Na2CO3 and water,
dried over Na2SO4 and evaporated down. Diethyl ether was added to the
residue to give a white precipitate, which was collected by filtration and re-
crystallized from EtOH. Yield, mp, [a]D25, elemental analysis and Rf value
General Procedure for Preparation of Boc-Tyr(O-Pic)-NH
(CH2)n-
CH3 (n؍1—6) Boc-Tyr(O-Pic)-OH (1.9 g, 5.0 mmol) was added to the
corresponding amine component (5.0 mmol) in DMF (30 ml) containing
Et3N (1.5 ml, 11 mmol) and BOP reagent (2.7 g, 6.0 mmol). The reaction
mixture was stirred at room temperature for 15 h. After removal of the sol-
vent, the residue was extracted with AcOEt. The extract was washed with
10% citric acid, 5% Na2CO3 and water, dried over Na2SO4 and evaporated
down. Petroleum ether was added to the residue to give a white precipitate,

1460
Vol. 49, No. 11
Table 3. Yield, Melting Point, Optical Rotation, Rf Value and Elemental Analysis of Boc-Tyr(O-Pic)-NH
(CH2)n-CH3
Elemental analysis
Calcd (Found)
TLC
Yield
(%)
mp
(°C)
[a]D25
(MeOH)
Compound
Formula
Rf 1
Rf 7
C
H
N
nϭ1
ϭ2
ϭ3
ϭ4
ϭ5
ϭ6
50.9
84.0
90.0
58.0
95.0
33.0
165—169
114—116
130—142
153—156
162—165
164—167
ϩ44.4
(cϭ1.0)
ϩ39.3
(cϭ1.0)
ϩ37.7
(cϭ1.0)
ϩ36.6a)
(cϭ1.0)
ϩ41.4
(cϭ1.0)
ϩ39.3a)
(cϭ1.0)
C28H33N3O4
70.7
(70.8
71.1
(71.4
68.6
(68.5
69.5
(69.2
72.3
6.99
6.96
7.21
7.47
7.50
7.02
7.71
7.32
7.77
7.89
7.90
7.98
8.83
8.69)
8.58
8.07)
8.00
7.79)
7.84
7.63)
7.90
7.94)
7.66
7.61)
0.47
0.46
C29H35N3O4
C30H37N3O4·1.2H2O
C31H39N3O4
0.56
0.45
C32H41N3O4
0.27
0.58
(72.4
72.2
(72.5
C33H43N3O4·0.2H2O
a) DMF.
Table 4. Yield, Melting Point, Optical Rotation, Rf Value and Elemental Analysis of Boc-Tra-Tyr(O-Pic)-NH
(CH2)n-CH3
Elemental analysis
Calcd (Found)
TLC
Yield
(%)
mp
(°C)
[a]D25
(DMF)
Compound
Formula
Rf 1
Rf 7
C
H
N
nϭ1
ϭ2
ϭ3
ϭ4
ϭ5
ϭ6
61.9
81.2
61.1
64.1
41.2
63.4
175.185
181—183
167—170
161—170
155—157
172—176
ϩ23.7
(cϭ1.0)
ϩ13.5
(cϭ1.0)
ϩ18.6
(cϭ1.1)
ϩ22.3
(cϭ1.0)
ϩ23.9
C36H46N4O5·0.2H2O
C37H48N4O5·1.2H2O
C38H50N4O5·0.8H2O
C39H52N4O5·0.3H2O
C40H54N4O5
69.9
(69.9
68.3
(68.0
69.5
(69.4
70.7
(70.9
71.6
7.56
7.66
7.80
7.60
7.86
7.69
8.03
7.93
8.11
8.05
8.30
8.06
9.06
8.93)
8.60
8.55)
8.53
8.17)
8.45
8.42)
8.35
8.27)
8.19
7.97)
0.63
0.77
0.58
0.50
0.23
0.63
(cϭ1.0)
ϩ16.9
(cϭ1.0)
(71.3
70.9
(70.6
C41H56N4O5·0.5H2O
Table 5. Yield, Melting Point, Optical Rotation, Rf Values and Elemental Analysis of H-Tra-Tyr(O-Pic)-NH
(CH2)n-CH3
Elemental analysis
Calcd (Found)
TLC
Yield
(%)
mp
(°C)
[a]D25
(10% AcOH)
MS m/z:
[Calcd]
Compound
Formula
Rf 3
Rf 4
Rf 5
C
H
N
nϭ1
ϭ2
ϭ3
ϭ4
ϭ5
ϭ6
95.1
86.0
87.7
95.0
74.0
95.0
Amorphous
Amorphous
Amorphous
Amorphous
Amorphous
Amorphous
ϩ36.4
(cϭ1.0)
ϩ26.4
(cϭ1.0)
ϩ19.0
(cϭ1.0)
ϩ29.8
(cϭ1.0)
ϩ14.4
C31H38N4O3·
2TFA·0.2H2O
C32H40N4O3·
2.5TFA·1.2H2O
C33H42N4O3·
2TFA·0.8H2O
C34H44N4O3·
2.5TFA
C35H46N4O3·
2TFA
C36H48N4O3·
2.8TFA
69.9
(69.9
68.3
(68.0
69.5
(69.4
70.7
(70.9
71.6
7.56
7.66
7.80
7.60
7.86
7.69
8.03
7.93
8.11
8.05
8.30
8.06
9.06
8.93)
8.60
8.55)
8.53
8.17)
7.45
7.42)
8.35
8.27)
8.19
7.97)
515 [(MϩH)ϩ]
[515]
0.38
0.17
529 [(MϩH)ϩ]
[529]
0.43
0.50
0.44
0.66
0.65
543 [(MϩH)ϩ]
[543]
557 [(MϩH)ϩ]
[557]
0.48
571 [(MϩH)ϩ]
[571]
(cϭ1.0)
ϩ18.2
(cϭ1.0)
(71.3
70.9
(70.6
585 [(MϩH)ϩ]
[585]
are summarized in Table 6.
1.4 mmol) in DMF (20 ml) containing BOP (0.75 g, 1.7 mmol), HOBt
(0.23 g, 1.7 mmol) and DIPEA (0.30 ml, 1.7 mmol) at 0 °C. The reaction
mixture was stirred at room temperature overnight. After removal of the sol-
vents, the residue was extracted with AcOEt. The extract was washed with
10% citrtic acid, 5% Na2CO3 and water, dried over Na2SO4 and evaporated
to dryness. Diethyl ether was added to the residue to give crystals, which
were collected by filtration and recrystallized from EtOH. Yield, mp, [a]D25,
elemental analysis and Rf value are summarized in Table 7.
General Procedure for Preparation of Boc-Tra-Tyr(O-Pic)-NH-
(CH2)n-NH-Boc (n؍2, 4, 6, 8) Piperidine (8 ml) was added to the solution
of Fmoc-Tyr(O-Pic)-NH-(CH2)n-NH-Boc (1.5 mmol) in DMF (32 ml) and
stirred at room temperature for 90 min. After removal of the solvent, ether
was added to the residue to yield crystals [H-Tyr(O-Pic)-NH-(CH2)n-NH-
Boc], which were collected by filtration. A solution of H-Tyr(O-Pic)-NH-
(CH2)n-NH-Boc in DMF (20 ml) was added to Boc-Tra-OH (0.36 g,

November 2001
1461
Table 6. Yield, Melting Point, Optical Rotation, Rf Value and Elemental Analysis of Fmoc-Tyr(O-Pic)-NH-(CH2)n-NH-Boc
Elemental analysis
Calcd (Found)
TLC
Yield
(%)
mp
(°C)
[a]D25
(CHCl3)
Compound
Formula
Rf 1
C
H
N
nϭ2
ϭ4
ϭ6
ϭ8
57.8
38.6
76.7
61.4
148—150
124—126
117—119
123—125
Ϫ19.5a)
(cϭ1.0)
Ϫ9.7
(cϭ1.0)
Ϫ9.0
(cϭ1.0)
Ϫ9.4
(cϭ1.0)
C37H40N4O6·H2O
C39H44N4O6
69.8
(69.5
66.6
(66.6
70.5
(70.7
71.6
(71.5
6.33
6.32
6.79
6.60
7.18
6.91
7.27
7.04
8.80
8.67)
8.21
8.07)
7.83
7.96)
7.77
0.55
0.50
0.52
0.58
C41H48N4O6·1.2H2O
C43H52N4O6
7.82)
a) DMF.
Table 7. Yield, Melting Point, Optical Rotation, Rf Value and Elemental Analysis of Boc-Tra-Tyr(O-Pic)-NH-(CH2)n-NH-Boc
Elemental analysis
Calcd (Found)
TLC
Yield
(%)
mp
(°C)
[a]D25
(CHCl3 cϭ1.0)
Compound
Formula
Rf 1
C
H
N
nϭ2
ϭ4
ϭ6
ϭ8
66.4
20.7
77.0
77.0
161—163
141—142
132—134
144—146
Ϫ9.6a)
Ϫ4.8
Ϫ3.1
Ϫ2.6
C35H51N5O7 ·H2O
C37H55N5O7
62.6
(62.4
65.2
(65.3
66.0
(65.7
65.9
(65.7
7.95
7.72
8.13
7.90
8.38
8.62
8.64
8.73
10.4
10.5)
8.21
8.07)
7.83
7.96)
7.77
0.25
0.56
0.53
0.60
C39H59N5O7
C41H63N5O7·0.5H2O
7.82)
a) DMF.
Table 8. Yield, Melting Point, Optical Rotation, Rf Value and Elemental Analysis of H-Tra-Tyr(O-Pic)-NH-(CH2)n-NH2
Elemental analysis
Calcd (Found)
TLC
Yield
(%)
mp
(°C)
[a]D25
(10% AcOH)
MS m/z:
[Calcd]
Compound
Formula
Rf 4
Rf 7
C
H
N
nϭ2
ϭ4
ϭ6
ϭ8
39.7
88.0
93.2
85.3
Amorphous
Amorphous
Amorphous
Amorphous
ϩ6.26
(cϭ1.0)
Ϫ8.7
(cϭ1.0)
Ϫ9.0
(cϭ1.0)
Ϫ8.6
(cϭ1.0)
C25H35N5O3·
3TFA·H2O
C27H39N5O3·
3TFA·0.2H2O
C29H43N5O3·
3TFA
45.8
(45.4
47.4
(47.3
48.3
(48.7
48.1
(47.8
4.95
4.92
5.18
5.18
5.55
5.76
7.57
7.48
8.60
8.73)
8.38
8.53)
8.05
8.29)
7.57
7.67)
454 [(MϩH)ϩ]
[454]
0.15
482 [(MϩH)ϩ]
[482]
0.60
0.69
0.79
510 [(MϩH)ϩ]
[510]
C31H47N5O3·
3TFA·2.5H2O
538 [(MϩH)ϩ]
[538]
General Procedure for Preparation of H-Tra-Tyr(O-Pic)-NH-(CH2)n- (tubes 1 and 2) was removed by evaporation. Petroleum ether was added to
NH2·3TFA (n؍2, 4, 6, 8) Boc-Tra-Tyr(O-Pic)-NH(CH2)n-NH-Boc (0.76 the residue to give a powder: yield 4.7 g (42%), mp 75—76 °C, Rf1 0.50.
mmol) was dissolved in TFA (1.1 ml, 15 mmol) containing anisole (0.21 ml, Anal. Calcd for C12H18N2O2: C, 64.8; H, 8.16; N, 12.6. Found; C, 64.7; H,
1.9 mmol) at 0 °C and the reaction mixture was stirred at 0 °C for 10 min and
was further stirred at room temperature for 60 min. After removal of the sol-
8.27; N, 12.8.
[Fmoc-Tyr(O-Pic)]-4-(Boc-aminomethyl)anilide A mixed anhydride
vent, dry diethyl ether was added to the residue to afford a precipitate, which [prepared from Fmoc-Tyr(O-Pic)-OH (500 mg, 1.0 mmol), NMM (0.11 ml,
was collected by filtration. Yield, mp, [a]D25, elemental analysis and Rf val- 1.0 mmool) and isobutyl chloroformate (0.13 ml, 1.0 mmol) as usual] in
ues are summarized in Table 8.
4-(Boc-Aminomethyl)aniline
(5.0 g, 0.0225 mol) in dioxane (20 ml) was added over a period of 2.5 h to a
DMF (20 ml) was added to an ice-cooled solution of 4-(Boc-aminomethyl)-
aniline (0.45 g, 1.0 mmol) in THF–DMF (1 : 1, v/v, 10 ml each). The reaction
mixture was stirred at 0 °C for 1 h and at room temperature overnight. After
A solution of di-tert-butyldicarbonate
solution of 4-aminomethylaniline (6.1 g, 0.05 mol) in water (20 ml) contain- removal of the solvent, the residue was extracted with AcOEt. The extract
ing Et3N (3.5 ml, 0.025 mol). The mixture was allowed to stir for 22 h and was washed with 5% NaHCO3, 10% citric acid and water, dried over Na2SO4
the solvent was removed. AcOEt (40 ml) was added to the residue and the and evaporated down. Diethyl ether was added to the residue to give a white
insoluble product was removed by filtration. The filtrate was washed with precipitate, which was collected by filtration. The crude product in CHCl3
water (5ϫ40 ml) dried over Na2SO4 and evaporated to dryness. Petroleum (5 ml) was applied to a silica gel column (3ϫ18 cm), equilibrated and eluted
ether was added to the residue to afford a precipitate. The crude product in with CHCl3. Individual fractions (200 ml) were collected. The solvent of the
solvent (AcOEt–n-hexane, 1 : 1 v/v, 5 ml) was applied to a silica gel column
(3ϫ26 cm), equilibrated and eluted with solvent (AcOEt–n-hexane, 1 : 2 added to the residue to give a powder: yield 250 mg (36%), mp 140—
v/v). Individual fractions (200 ml) were collected. The solvent of the effluent
142 °C, [a]D25 ϩ11.2° (cϭ0.67, DMF), Rf 1 0.58, Rf 2 0.54. Anal. Calcd for
effluent (fractions 3—6) was removed by evaporation. Petroleum ether was

1462
Vol. 49, No. 11
C42H42N4O6: C, 72.2; H, 6.06; N, 8.20. Found; C, 71.9; H, 6.18; N, 7.91.
Assay Procedure The enzymes used were as follows: human PL and
[Boc-Tra-Tyr(O-Pic)]-4-(aminometyl)anilide A mixed anhydride [pre- PK (Chromogenix AB, Molndal), human urokinase (Green Cross Co.,
pared from Boc-Tra-OH (74 mg, 0.29 mmol), NMM (0.032 ml, 0.29 mmool) Osaka), bovine thronbin (Mochida Pharmaceutical, Tokyo) and trypsin
and isobutyl chloroformate (0.037 ml, 0.29 mmol) as usual] in THF (10 ml) (Sigma Chemical Co., St. Louis). Enzymatic activities of PL, PK, urokinase,
was added to an ice-cooled solution of [H-Tyr(O-Pic)]-4-(aminomethyl)- thrombin and trypsin were determined by the method described previously,
anilide {prepared from [Fmoc-Tyr(O-Pic)]-4-Boc-aminomethyl) anilide
using
D-Vla–Leu–Lys–pNA (S-2251), D-Pro–Phe–Arg–pNA (S-2302), ϽGlu–
(0.20 g, 0.29 mmol) and 20% piperidine/DMF} in DMF (10 ml). The reac-
Gly–Arg–pNA (S-2444) and D-Phe–Pip–Arg–pNA (S-2238), respectively. Fn
tion mixture was stirred at 0 °C for 1 h and at room temperature overnight. and Fg were used as substrates for PL and thrombin, respectively. IC50 val-
After removal of the solvent, the residue was extracted with AcOEt. The ex- ues were determined as follows: (1) Antiamidolytic assay30): the IC50 value
tract was washed with 5% NaHCO3, 10% citric acid and water, dried over was taken as the concentration of inhibitor which reduced the absorbance at
Na2SO4 and evaporated down. Diethyl ether was added to the residue to give 405 nm by 50% compared with the absorbance measured under the same
a white precipitate, which was collected by filtration. The crude product was conditions without inhibitor. (2) Antifibrinolytic assay30): the IC50 value was
recrystallized from AcOEt: yield 130 mg (63%), mp 186—188 °C, Rf 1 0.56,
taken as the concentration of inhibitor, which doubled the complete lysis
Rf 2 0.37. Anal. Calcd for C40H53N5O7·0.25H2O: C, 66.7; H, 7.48; N, 9.72. time compared to control samples without inhibitor. (3) Antifibrinolytic
Found; C, 66.6; H, 7.36; N, 9.71.
[H-Tra-Tyr(O-Pic)]-4-(aminomethyl)anilide·3TFA [Boc-Tra-Tyr(O-
assay: to a borate saline buffer (pH 7.4) was added solutions containing vari-
ous concentrations of the inhibitor to be tested (0.5 ml), 0.2% bovine Fg in
Pic)]-4-(Boc-aminomethyl)anilide (100 mg, 0.14 mmol) was dissolved in the above buffer (0.4 ml) and bovine thrombin 4 U/ml (0.1 ml). The assay
TFA (0.50 ml, 6.5 mmol) containing anisole (0.050 ml, 0.46 mmol) at 0 °C was carried out at 37 °C and the clotting time was measured. The IC50 value
and the reaction mixture was stirred at 0 °C for 10 min, and then further was taken as the concentration of inhibitor which doubled the clotting time
stirred at room temperature for 1 h. Anhydrous ether was added to the reac- compared to the controls without inhibitor.
tion mixture to afford a precipitate, which was collected by filtration. The
Acknowledgements The authors are grateful to Dr. Lawrence H.
Lazarus of the National Institute of Environmental Health Sciences (NIEHS)
for his kind help during the preparation of this manuscript.
crude peptide was applied to a column of Sephadex G-15 (2.2ϫ0.49 cm),
equilibrated and eluted with 3% AcOH. Individual fractions (13 g each)
were collected and the solvent of effluent (tubes 4—10) was removed by
lyophilization to give a white amorphous powder: yield 100 mg (83%), [a]D25
ϩ22.9° (cϭ0.86, 10% AcOH), Rf 5 0.10, Rf 6 0.62. TOF-MS m/z: 516
(MϩH)ϩ. Anal. Calcd for C30H37N5O3·3TFA·0.75H2O: C, 49.6; H, 4.80; N,
8.04. Found: C, 49.5; H, 4.60; N, 8.18.
References and Notes
1) The customary L-configuration for amino acid residues is omitted.
Abbreviations used in this report for amino acids, peptides and their
derivatives are those recommended by the IUPAC-IUB Commission
on Biochemical Nomenclature: Biochemistry, 5, 2485—2489 (1966);
6, 362—364 (1967); 11, 1726—1732 (1972). The following addi-
tional abbreviations are used: AcOEt, ethyl acetate; APAA, 4-car-
boxymethyanilide; Boc, tert-butyloxycarbonyl; BOP, benzotriazole-1-
yl-oxy-tris(dimethylamino)phosphoniumhexafluorophosphate; Bzl,
benzyl; DIPEA, N,N-diisopropylethylamine; DMF, N,N-dimethylfor-
mamide; Fg, fibrinogen; Fn, fibrin; Fmoc, 9-fluorenylmethoxycar-
bonyl; HOBt, 1-hydroxybenzotriazole; NMM, N-methylmorpholine;
pNA, p-nitroanilide; TFA, trifluoroacetic acid; THF, tetrahydrofuran;
Tos, p-toluensulfonyl; Tra, 4-aminomethylcyclohexanecarbonyl.
2) Hajjar K. A., Nachman R. L., “Hemostasis and Thrombosis; Basic
Principles and Clinical Practice,” 3rd Ed., eds. by Colman R. W., Hirsh
J., Marder V. J., Salzman E. W., J. B. Lippincott Company, Philadel-
phia, 1994, pp. 823—836.
Boc-Tyr(O-Pic)-APAA-OBzl A mixed anhydride of Boc-Tyr(O-Pic)-
OH [prepared from Boc-Tyr(O-Pic)-OH (1.5 g, 4.0 mmol), isobutyl chloro-
formate (0.55 ml, 4.0 mmol) and NMM (0.88 ml, 8.0 mmol) as usual] in
THF (10 ml) was added to a solution of H-APAA-OBzl. TosOH (1.52 g,
4.0 mmol), in DMF (10 ml) containing NMM (0.44 ml, 4.0 mmol) at 0 °C
and the reaction mixture was stirred at 6 °C overnight. After removal of the
solvent, the residue was extracted with AcOEt. The extract was washed with
5% Na2CO3 and water, dried over Na2SO4 and evaporated down. Petroleum
ether was added to the residue to give a white precipitate, which was col-
lected by filtration and recrystallized from EtOH: yield 940 mg (42%), mp
162—165 °C, [a]D25 ϩ5.7° (cϭ1.0, CHCl3), Rf 1 0.53. Anal. Calcd for
C35H37N3O6: C, 70.5; H, 6.26; N, 7.05. Found; C, 70.3; H, 6.14; N, 7.01.
Boc-Tra-Tyr(O-Pic)-APAA-OBzl A mixed anhydride of Boc-Tra-OH
[prepared from Boc-Tra-OH (0.33 g, 1.3 mmol), isobutyl chloroformate
(0.18 ml, 1.3 mmol) and Et3N (0.18 ml, 1.3 mmol) as usual] in THF (10 ml)
was added to a solution of H-Tyr(O-Pic)-APAA-OBzl [prepared from Boc-
Tyr(O-Pic)-APAA-OBzl (0.80 g, 1.3 mmol) and TFA (2.0 ml, 26 mmol) in
the presence of anisole (0.19 ml, 1.8 mmol) as usual] in DMF (30 ml) con-
taining Et3N (0.36 ml, 2.6 mmol) at 0 °C and the reaction mixture was stirred
at 6 °C overnight. After removal of the solvent, the residue was extracted
with AcOEt. The extract was washed with 5% Na2CO3 and water, dried over
Na2SO4 and evaporated down. Petroleum ether was added to the residue to
give a white precipitate, which was collected by filtration and recrystallized
from DMF and AcOEt: yield 0.59 mg (57%), mp 162—165 °C, [a]D25
ϩ23.7° (cϭ1.0, DMF), Rf 1 0.53. Anal. Calcd for C43H50N4O7: C, 70.3; H,
6.85; N, 7.62. Found; C, 70.1; H, 6.82; N, 7.84.
3) Ny T., Elgh F., Lund B., Proc. Natl. Acad. Sci. U.S.A., 81, 5355—5359
(1984).
4) Mannucci P. M., D’anelo A., “Basic and Clinical Aspects,” eds. by
Mannucci P. M., D’angelo A., Academic Press, London, 1982, pp. 1—
265.
5) Ny T., Sawdey M., Lawrence D., Millan J. L., Loskutoff D. J., Proc.
Natl. Acad. Sci. U.S.A., 83, 6776—6780 (1986).
6) Moroi M., Aoki N., J. Biol. Chem., 251, 5956—4965 (1976).
7) Dela Cadana R. A., Wachtfogel Y. T., Colman R. W., “Hemostasis and
Thrombosis; Basic Principles and Clinical Practice,” 3rd Ed., eds. by
Colman R. W., Hirsh J., Marder V. J., Salzman E. W., J. B. Lippincott
Company, Philadelphia, 1994, pp. 219—240.
8) Niewiarowski S., Thromb. Diath, Haemorrh., 3, 593—603 (1959).
9) Bhoola K. D., Figueroa C. D., Worthy K., Pharmacol. Rev., 44, 1—80
(1992).
Tra-Tyr(O-Pic)-APAA · 2TFA Boc-Tra-Tyr(O-Pic)-APAA-OBzl
(230 mg, 0.31 mmol) in DMF (10 ml) and MeOH (10 ml) was hydrogenated
for 2 h over a Pd catalyst. After removal of Pd and the solvent, ether was
added to the residue to yield crystals, which were collected by filtration. The
crude material in CHCl3 (3 ml) was applied to a silica gel column (3ϫ8 cm), 10) Coligan J. E., Slayter H. S., J. Biol. Chem., 259, 3944—3948 (1984).
equilibrated and eluted with CHCl3 (2.2 l), followed by CHCl3, MeOH and 11) Ott U., Odermatt E., Engel J., Furthmayr H., Timpl R., Eur. J.
H2O (16 : 3 : 1, lower phase, 0.80 l). The solvent of the latter effluent (200—
800 ml) was removed by evaporation to yield a powder, which was collected 12) Aplin J. D., Hughes R. C., Biochim. Biophys Acta, 694, 375—418
by filtration, yield 180 mg (80%), mp 198—200 °C, Rf 1 0.25. Boc-Tra-
(1982).
Biochem., 123, 63—72 (1982).
Tyr(O-Pic)-APAA (120 mg, 1.9 ml) was dissolved in TFA (0.30 ml, 13) Marder V. J., Sherry S., N. Engl. J. Med., 318, 1512—1520 (1988).
3.8 mmol) containing anisole (0.050 ml, 0.46 mmol) at 0 °C. The reaction 14) Sato Y., Rifkin D. B., J. Cell Biol., 109, 309—315 (1989).
mixture was stirred at 0 °C for 10 min and was further stirred at room tem- 15) Lyons R. M., Gentry L. E., Purchio A. F., Moses H. L., J. Cell Biol.,
perature for 1 h. Anhydrous ether was added to the reaction mixture to afford
110, 1361—1367 (1990).
a precipitate, which was collected by filtration. The crude peptide was ap- 16) Omar M. N., Mann K. G., J. Biol. Chem., 262, 9750—9755 (1987).
plied to a column of Sephadex G-15 (2.5ϫ53 cm), equilibrated and eluted 17) McKee P. A., Andersen J. C., Switzer M. E., Ann. N.Y. Acad. Sci., 240,
with 3% AcOH. Individual fractions (13 g each) were collected and the sol-
8—33 (1975).
vent of effluent (tubes 8—15) was removed by lyophilization to afford a 18) Ichinose A., Kisiel W., Fukihawa K., FEBS Lett., 175, 412—418
white amorphous powder: 150 mg (90%), [a]D25 ϩ36.6° (cϭ0.34, 10%
AcOH), Rf 1 0.25. Anal. Calcd for C30H37N5O3·2TFA·0.5H2O: C, 52.9; H,
5.12; N, 7.08. Found: C, 52.6; H, 4.83; N, 7.04.
(1984).
19) Kasai S., Arimura H., Nishida M., Suyama T., J. Biol. Chem., 260,
12382—12389 (1985).

November 2001
1463
20) Werb Z., Mainardi C. L., Vater C. A., Harris E. D., Engl. J. Med., 296,
1017—1023 (1977).
21) Andreasen P. A., Egelund R., Petersen H. H., Cell Mol. Life Sci., 20,
25—40 (2000).
26) Okada Y., Tsuda Y., Tada M., Wanaka K., Hijikata-Okunomiya A.,
Okamoto S., Chem. Pharm. Bull., 48, 1964—1972 (2000).
27) Tomoo K., Satoh K., Tsuda Y, Wanaka K., Okamoto S., Hijikata-
Okunomiya A., Okada Y., Ishida T., J. Biochem., 129, 455—460
(2001).
22) Okamoto S., Keio J. Med., 8, 211—247 (1959).
23) Okamoto S., Sato S., Tanaka Y., Okamoto U., Keio J. Med., 13, 177— 28) Okamoto S., Hijikata-Okunomiya A., Wanaka K., Okada Y., Okamoto
185 (1964). U., Semin. Thromb. Hemost., 23, 493—501 (1997).
24) Okada Y., Matsumoto Y., Tsuda Y., Tada M., Wanaka K., Hijikata- 29) Muller D., Zeltser I., Bitan G., Gilon C., J. Org. Chem., 62, 411—416
Okunomiya A., Okamoto S., Chem. Pharm. Bull., 48, 184—193
(2000).
25) Wanaka K., Okada Y., Tsuda Y., Oakmoto U., Hijikata-Okunomiya A.,
Okamoto S., Chem. Pharm. Bull., 40, 1814—1817 (1992).
(1997).
30) Okada Y., Tsuda Y., Teno N., Wanaka K., Bohgaki M., Hijikata-
Okunomiya A., Naito T., Okamoto S., Chem. Pharm. Bull., 36,
1289—1298 (1988).